Novel target genes of PsrA transcriptional regulator of Pseudomonas aeruginosa

FEMS MICROBIOLOGY LETTERS Volume 246, Issue 2, Pages 151-294 (15 May 2005)

1. Editorial board • EDITORIAL BOARD Pages iii-vi

2. Fluorescence in situ hybridisation (FISH) – the next generation • SHORT SURVEY Pages 151-158 Katrin Zwirglmaier

3. Biotin biosynthesis, transport and utilization in rhizobia • SHORT SURVEY Pages 159-165 Karina Guillén-Navarro, Sergio Encarnación and Michael F. Dunn

4. The Pseudomonas aeruginosa pirA gene encodes a second receptor for ferrienterobactin and synthetic catecholate analogues • SHORT COMMUNICATION Pages 167-174 Bart Ghysels, Urs Ochsner, Ute Möllman, Lothar Heinisch, Michael Vasil, Pierre Cornelis and Sandra Matthijs

5. Novel target genes of PsrA transcriptional regulator of Pseudomonas aeruginosa • SHORT COMMUNICATION Pages 175-181 Milan Kojic, Branko Jovcic, Alessandro Vindigni, Federico Odreman and Vittorio Venturi

6. Isolation and characterisation of the lipopolysaccharide from Acidiphilium strain GS18h/ATCC55963, a soil isolate of Indian copper mine • SHORT COMMUNICATION Pages 183-190 Rabindranath Bera, Abhijit Nayak, Asish Kumar Sen, Biswa Pronab Chowdhury and Ranjan Bhadra

7. Comprehensive analysis of classical and newly described staphylococcal superantigenic toxin genes in Staphylococcus aureus isolates • SHORT COMMUNICATION Pages 191-198 Katsuhiko Omoe, Dong-Liang Hu, Hiromi Takahashi-Omoe, Akio Nakane and Kunihiro Shinagawa

8. Passive immunisation of hamsters against Clostridium difficile infection using antibodies to surface layer proteins • SHORT COMMUNICATION Pages 199-205 Julie B. O’Brien, Matthew S. McCabe, Verónica Athié-Morales, George S.A. McDonald, Déirdre B. Ní Eidhin and Dermot P. Kelleher

9. Morphological and molecular taxonomy of Pythium longisporangium sp. nov. isolated from the Burgundian region of France • SHORT COMMUNICATION Pages 207-212 Bernard Paul, Kanak Bala, Sabine Gognies and Abdel Belarbi

10. Polymorphism and gene conversion of the 16S rRNA genes in the multiple rRNA operons of Vibrio parahaemolyticus • SHORT COMMUNICATION Pages 213-219 Narjol González-Escalona, Jaime Romero and Romilio T. Espejo

11. Induction of murine macrophage TNF-α synthesis by Mycobacterium avium is modulated through complement-dependent interaction via complement receptors 3 and 4 in relation to M. avium glycopeptidolipid • SHORT COMMUNICATION Pages 221-228 Vida R. Irani and Joel N. Maslow

12. Overexpression of a hydrogenase gene in Clostridium paraputrificum to enhance hydrogen gas production • SHORT COMMUNICATION Pages 229-234 Kenji Morimoto, Tetsuya Kimura, Kazuo Sakka and Kunio Ohmiya

13. RirA is the iron response regulator of the rhizobactin 1021 biosynthesis and transport genes in Sinorhizobium meliloti 2011 • SHORT COMMUNICATION Pages 235-242 Caroline Viguier, Páraic Ó Cuív, Paul Clarke and Michael O’Connell

14. Polymerase chain reaction for identification of aldoxime dehydratase in aldoxime- or nitrile-degrading microorganisms • SHORT COMMUNICATION Pages 243-249 Yasuo Kato, Satoshi Yoshida and Yasuhisa Asano

15. The gene encoding xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (xfp) is conserved among Bifidobacterium species within a more variable region of the genome and both are useful for strain identification • SHORT COMMUNICATION Pages 251-257 Xianhua Yin, James R. Chambers, Kathleen Barlow, Aaron S. Park and Roger Wheatcroft

16. The stabilization of housekeeping transcripts in Trypanosoma cruzi epimastigotes evidences a global regulation of RNA decay during stationary phase • SHORT COMMUNICATION Pages 259-264 Ana María Cevallos, Mariana Pérez-Escobar, Norma Espinosa, Juliana Herrera, Imelda López-Villaseñor and Roberto Hernández

17. Genotypic and phenotypic characterization of a biofilm-forming Serratia plymuthica isolate from a raw vegetable processing line • SHORT COMMUNICATION Pages 265-272 Rob Van Houdt, Pieter Moons, An Jansen, Kristof Vanoirbeek and Chris W. Michiels

18. Chemotypes significance of lichenized fungi by structural characterization of heteropolysaccharides from the genera Parmotrema and Rimelia • SHORT COMMUNICATION Pages 273-278 Elaine Rosechrer Carbonero, Caroline Grassi Mellinger, Sionara Eliasaro, Philip Albert James Gorin and Marcello Iacomini

19. Isolation of genes differentially expressed during the fruit body development of Pleurotus ostreatus by differential display of RAPD • SHORT COMMUNICATION Pages 279-284 Masahide Sunagawa and Yumi Magae

20. Author index Volume 246 • INDEX Pages 285-287

21. Subject index Volume 246 • INDEX Pages 289-294

Copyright © 2005 Federation of European Microbiological Societies

Volume 246, 2005

Chief EditorJ.A. Cole, School of Biosciences, University of Birmingham, Edgbaston, B15 2TT Birmingham, United Kingdom. Tel: +44-121-414 5440; Fax: +44-121-414

5925; E-mail: [email protected]

MiniReviews EditorsR.I. Aminov, Gut Immunology and Microbiology, Rowett Research Institute, Greenburn Road, Bucksburn, AB21 9SB Aberdeen, Scotland, United Kingdom.

Tel: +44-1224-716 643; Fax: +44-1224-716 687; E-mail: [email protected]

Phylogeny; Molecular ecology; Antibiotic resistance; Bacterial genetics; Intestinal microbiology and microbial genomics

I. Henderson, Bacterial Pathogenesis and Genomics Unit, Division of Immunity and Infection, The Medical School, University of Birmingham, Edgbaston,

B15 2TT Birmingham, United Kingdom. Tel: +44-121-414 4368; Fax: +44-121-414 3599; E-mail: [email protected]

Microbial pathogenesis; Gram-negative bacteria; Infection; Cellular microbiology; Autotransporter proteins; Protein secretion

R.C. Staples, Boyce Thompson Institute, Cornell University, Tower Road, NY 14850 Ithaca, United States of America. Tel: +1-607-257 4889; Fax: +1-607-

254 1242; E-mail: [email protected]

Development, physiology, cell biology and molecular biology of filamentous fungi including fungal pathogens of plants and animals

Editors and their specialist fields

BIOTECHNOLOGY

S. Casella, Dipartimento di Biotecnologie Agrarie, Agripolis, Universita di Padova, Via dell’Universita 16, 35020 Legnaro Padova, Italy.


Microbial physiology; Microbial biotechnology; Soil microbiology; Plant-bacteria interaction; Nitrogen metabolism

W. Kneifel, Department of Food Science and Technology, BOKU-University of Natural Resources and Applied Life Sciences, Muthgasse 18,

A-1190 Vienna, Austria. Tel: +43-1-36006-6290; Fax: +43-136006-6266; E-mail: [email protected]

Food fermentation; Lactic acid bacteria; Microbiological quality criteria of foods; Bacterial strain safety and virulence; product development and quality

assessment of functional foods (pro- and prebiotics); Food safety (hygiene issues)

D. Mattanovich, Institut fur Angewandte Mikrobiologie, Universitat fur Bodencultur Wien, Muthgasse 18, A-1190 Vienna, Austria. Tel: +43-1-360-066 569;

Fax: +43-1-369 7615; E-mail: [email protected]

Biotechnology, especially recombinant protein production with bacteria; Yeasts and filamentous fungi; Physiology of production strains; Metabolic

engineering

ENVIRONMENTAL MICROBIOLOGY; PLANT-MICROBE INTERACTIONS

E. Baggs, School of Biological Sciences, University of Aberdeen, Cruickshank Building, St Machar Drive, Aberdeen, AB24 3UU, United Kingdom. Tel: +44

(0)-122-427 2691; Fax: +44 (0)-122-427 2703 ; E-mail: [email protected]

Soil bacterial ecology, particularly in relation to nitrogen and carbon cycling; Functional genes involved in denitrification; Impacts of soil management,

pollution or climate change

C. Edwards, Division of Microbiology and Genomics, School of Biological Sciences, University of Liverpool, The Biosciences Building, L69 7ZB Liverpool,

United Kingdom. Tel: +44-151 795 4573; Fax: +44-151 795 4410; E-mail: [email protected]

Molecular ecology of micro-organisms; Novel methods for monitoring bacterial activity and biodiversity; Biogeochemical cycles (particularly methane cycling

bacteria); Bioremediation and environmental biotechnology; Extreme environments; Molecular methods

H-P.E. Kohler, Environmental Microbiology and Molecular Ecotoxicology, EAWAG, Ueberlandstrasse 133, CH-8600 Duebendorf, Switzerland. Tel: +41-1-

823 5521; Fax: +41-1-823 5547; E-mail: [email protected]

Microbial degradation and environmental fate of organic pollutants; Biochemistry of mono- and dioxygenases; Microbial transformation of chiral compounds

Y. Okon, Dept. of Plant Pathology & Microbiology, Faculty of Agricultural, Food & Environmental Quality Sciences, The Hebrew University of Jerusalem,

The Rehovot Campus, 76100 Rehovot, Israel. Tel: 972-8-948 9216; Fax: 972-8-946 6794; E-mail: [email protected]

Plant growth promoting bacterial-rhizosphere associations; Symbiotic and non-symbiotic biological nitrogen fixation; Physiology and ecology of Azospirillum

as a model system for rhizosphere studies

A. Oren, The Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel. Tel: +972-2-658 4951; Fax: +972-2-652 8008;

E-mail: [email protected]

Microbial ecology and physiology; Halophilic micro-organisms; Photosynthetic prokaryotes

EUKARYOTIC CELLS

L.F. Bisson, Dept of Viticulture and Enology, 1311 Haring Hall, University of California at Davis, One Shields Avenue, CA 95616-8749 Davis, United States

of America. Tel: +1-530-752 1717; Fax: +1-530-752 0382; E-mail: [email protected]

Molecular biology, genetics, biochemistry, physiology, ecology and applications of yeasts

R. Fischer, Applied Microbiology, University of Karlsruhe, Hertzstrasse 16, D-76187 Karlsruhe, Germany. Tel: 49-721-608-4630; Fax: 49-721-608-8932;


Cellular and molecular biology of filamentous fungi, especially polarized growth and development; Cytoskeleton, molecular motors and organelle movement;

Spore formation; Aspergillus nidulans

MICROBIOLOGY LETTERS

G.M. Gadd, Division of Environmental and Applied Biology, Biological Sciences Institute, School of Life Sciences, University of Dundee, DD1 4HN Dundee,

Scotland, United Kingdom. Tel: +44-1382-344 765; Fax: +44-1382-348 216; E-mail: [email protected]

Yeast and fungal physiology, ecology and differentiation; Metal-microbe interactions; Heavy metals and toxicology

N. Gunde-Cimerman, Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna Pot 111, 1000 Ljubljana, Slovenia. Tel: +386-1-423 3388;


Physiology, ecology and biodiversity of fungi, especially in extreme (hypersaline and cold) environments; Biotechnologically important fungi and production of

extracellular enzymes and secondary metabolites; Pathogenic fungi and medicinal mushrooms; Culture collections and strain preservation.

M. Jacquet, Institut de Genetique et Microbiologie, UMR8621 CNRS, Universite Paris-Sud, Bat 400, 91405 Orsay Cedex, France. Tel: +33-16915 7963;

Fax: +33-16915 4629; E-mail: [email protected]

Yeast molecular and cell biology; Signal transduction in fungus

B. Paul, Laboratoire des Sciences de la Vigne, Institut Jules Guyot, Universite de Bourgogne, BP 27877, 21078 Dijon, France. Tel: +33-380-396326; Fax:

+33-380-396326; E-mail: [email protected]

Mycology, in particular biological control of plant diseases; The genera Botrytis and Pythium; Aquatic phycomycetes

B.A. Prior, Department of Microbiology, University of Stellenbosch, Private Bag XI, 7602 Matieland, South Africa. Tel: +27-21-808 5856; Fax: +27-21-808


Stress responses by yeast to the environment; Microbial solute channels; Fungal biotechnology; Hemicellulose biodegradation by fungi

C. Remacle, Genetics of Microorganisms, Department of Life Sciences B22, University of Liege, Bld du Rectorat 27, B-4000 Liege, Belgium.

Tel: +32-4366 3812; Fax: +32-4366 3840; E-mail: [email protected]

Genetics and molecular biology of lower eukaryotes with emphasis on cell organelles; The function and biogenesis of mitochondria and chloroplasts

P. Schaap, Division of Cell and Developmental Biology, University of Dundee, MSI/WTB Complex, Dow Street, Dundee DD1 5EH, UK. Tel: +44 1382 348

078; Fax: +44 1382 345 386; E-mail: [email protected]

Cellular and developmental biology of social amoebae; Signal transduction, especially the role of cyclic nucleotide signalling pathways in the regulation of

developmental decisions, sporulation and responses to stress; Evolutionary relationships between eukaryote cyclic nucleotide signalling proteins and their

prokaryote ancestors

D.P. Wakelin, High Street (Kirtlands), WR12 7AL Broadway, Worcestershire, United Kingdom. Tel: +44-1386-852 747; E-mail: [email protected]

Parasitology; Helminthology; Host immunity; Intestinal immunity; Intestinal inflammation; Immunoepidemiology; Genetics of resistance

GENETICS AND MOLECULAR BIOLOGY

R.S. Buxton, Division of Mycobacterial Research, National Institute for Medical Research, The Ridgeway, Mill Hill, NW7 1AA London,

United Kingdom. Tel: 020 8816 2225; Fax: 020 8906 4477; E-mail: [email protected]

Mycobacteria, especially pathogenesis; Microbial genetics and molecular biology; Gene regulation; Two-component signal transduction

K. Forchhammer, Institut fur Mikrobiologie und Molekularbiologie, Justus-Liebig-Universitat, Heinrich-Buff-Ring 26-32, D-35392 Giessen,

Germany. Tel: +49-641-9935 545; Fax: +49-641-9935 549; E-mail: [email protected]

Physiology and molecular genetics of cyanobacteria; Microbial nitrogen control; Bacterial signal transduction through serine/threonine phosphorylation/

dephosphorylation

R.P. Gunsalus, Department of Microbiology and Molecular Genetics, 1602 MSB, University of California (UCLA), CA 90095 Los Angeles, United States of

America. Tel: +1-310-206 8201; Fax: +1-310-206 5231; E-mail: [email protected]

Molecular genetics; Microbial physiology; Methanogenesis; Anaerobic cell function; Electron transport; Metabolism

D.J. Jamieson, School of Life Sciences, Heriot-Watt University, Riccarton, EH14 4AS Edinburgh, Scotland, United Kingdom. Tel: +44-131-451 3644; Fax:

+44-131-451 3009; E-mail: [email protected]

Molecular biology; Genetics and biochemistry of yeasts

A. Klier, Dept des Biotechnologies, Unite de Biochemie Microbienne, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris, Cedex 15, France. Tel: +33-1-

44 27 6995; Fax: +33-1-44 27 6995; E-mail: [email protected]

Molecular biology, genetics, biochemistry and physiology of gram-positive bacteria

E. Ricca, Dipartimento di Fisiologia Generale ed Ambientale, Universita’ Federico II, Via Mezzocannone 16, 80134 Napoli, Italy. Tel: +39-81-253 4636; Fax:


Bacterial differentiation; Sporulation; Gene expression in gram-positives; Bacteria as vaccine vehicles and as probiotics; Display of molecules on bacterial

surfaces

W. Schumann, Institute of Genetics, Universitat Bayreuth, D-95440 Bayreuth, Germany. Tel: +49-921-552 708; Fax: +49-921-552 710;


Bacterial genetics, especially stress genes; Bacteriophages; Transposition

M.R. Soria, Professor of Biochemistry and Molecular Biology, Department of Experimental & Clinical Medicine ‘‘G. Salvatore’’, Magna Graecia University

School of Medicine, Via T.Campanella 115, 88100 Catanzaro, Italy. Tel: +39-961-770 880; Fax: +39-961-777 435; E-mail: [email protected]

Functional genomics of host-parasite interactions; Regulation of gene expression; Angiogenesis

GENOMICS AND BIOINFORMATICS

M.Y. Galperin, National Center for Biotechnology Information, National Library of Medicine, National Institute of Health, Building 38A, Room 507, Maryland

20894 Bethesda, United States of America. Tel: +1-301-435 5910; Fax: +1-301-435-7794; E-mail: [email protected]

Microbial genomics; Bio-informatics; Modelling of metabolic pathways; Evolution of metabolism

O.P. Kuipers, Dept. for Genetics, Rijksuniversiteit Groningen, Kerklaan 30, 9751 HN Laren, Netherlands. Tel: +31-50-3632093/2092;

Fax: +31-5-3632348; E-mail: [email protected]

Genetics and biotechnological applications of gram-positive bacteria (lactid acid bacteria, bacilli); Functional genomics; Bacteriocins;

Protein engineering

PATHOGENICITY INCLUDING VETERINARY MICROBIOLOGY

P.W. Andrew, Department of Infection, Immunity and Inflammation, University of Leicester, PO Box 138 (University Road), LE1 9HN Leicester, United

Kingdom. Tel: +44-116-252 2941; Fax: +44-116-252 5030; E-mail: [email protected]

Microbial pathogenicity; Intracellular parasites

M.J. Bidochka, Department of Biological Sciences, Brock University, Glenridge Ave 500, ON L2S 3A1 St. Catharines, Canada. Tel: +1-905-688 5550 ext

3392; Fax: +1-905-688 1855; E-mail: [email protected]

Microbial pathogenicity, especially pathogenic fungi; Microbial population genetics and phylogeography

T.H. Birkbeck, Division of Infection and Immunity, Institute of Biomedical & Life Sciences, Joseph Black Building, University of Glasgow,

G12 8QQ Glasgow, Scotland, United Kingdom. Tel: +44-141-330 5843; Fax: +44-141-330 4600; E-mail: [email protected]

Microbial toxins and pathogenicity in human and animal diseases; Immunochemistry; Fish disease

H.B. Deising, Faculty of Agriculture, Phytopathology and Plant Protection, Martin-Luther University Halle-Wittenberg, Ludwig-Wucherer Strasse 2, D-06099

Halle, Germany. Tel: +49-345-552 2660; Fax: +49-345-552 7120; E-mail: [email protected]

Fungal pathogenicity and virulence; Fungus-plant interactions, especially biochemistry and molecular biology of fungus-plant interactions; Fungal

morphogenesis, especially infection structure differentiation; Fungicide resistance

R. Delahay, Institute of Infection, Immunity & Inflammation, University of Nottingham, Floor C, West Block, Queen’s Medical Centre, Nottingham, NG7 2UH.

Tel: +44 115-924 9924 Ext 42449; Fax: +44 115-970 9923; E-mail: [email protected]

Microbial pathogenicity, especially enteric pathogens; enteropathogenic Escherichia coli; Helicobacter; Host-pathogen interaction; Bacterial virulence

secretion systems (Type III and IV in particular); Protein–protein interaction

M.C. Enright, Royal Society University Research Fellow, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, United Kingdom. Tel:

+44 1225-386871; Fax: +44 1225-386779; E-mail: [email protected]

Staphylococcus aureus; Streptococci; MRSA; molecular epidemiology evolution; antibiotic resistance; virulence; biochemistry; physiology and genetics

streptococci

J.-I. Flock, Department of Laboratory Medicine, Division of Clinical Bacteriology, Karolinska Institutet, Huddinge University Hospital F82, SE-141 86

Stockholm, Sweden. Tel: +46-8-5858 1169; Fax: +46-8-711 3918; Email: [email protected]

Genetics and virulence factors of Staphylococci; Adherence of gram-positive bacteria; Experimental infection models in animals;

Function of antibodies against surface structures of gram-positive bacteria; Microbial immunity and vaccines against gram-positive bacteria

K. Hantke, Mikrobiologie/Membranphysiologie, Universitat Tubingen, Auf der Morgenstelle 28, D-72076 Tubingen, Germany. Tel: +49-7071-297 4645; Fax:


Bacterial metal transport and regulation, especially iron, manganese and zinc; Functions of outer membrane and periplasmic proteins of gram-negative

bacteria; Colicins and microcins; Pathogenicity and iron

J.M. Ketley, Department of Genetics, University of Leicester, University Road, LE1 7RH Leicester, United Kingdom. Tel: +44-116-252 3434; Fax: +44-116-

252 3378; E-mail: [email protected]

Vibrio cholerae; Campylobacters; Pathogenic enteric bacteria; Pathogenesis; Microbial genomics; Gene regulation

R.Y.C. Lo, Department of Microbiology, University of Guelph, ON, N1G 2W1 Guelph, Canada. Tel: +1-519-824 4120; Fax: +1-519-837 1802;


Microbial pathogenicity; Bacterial genetics; Physiology and biochemistry

T. Mitchell, Division of Infection and Immunity, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ,

Scotland, United Kingdom, Tel: +44 141-330 4642; Fax: +44 141-330 3727; E-mail: [email protected]

Microbial pathogenicity, especially in Gram-positive bacteria and mainly streptococci; Bacterial protein toxins; Vaccine development; Bacterial virulence

gene expression; Genomic variation in bacterial pathogens; Use of bacterial microarrays

M. Mitsuyama, Department of Microbiology, Graduate School of Medicine (Rm203, Bldg D), Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, 606-8501

Kyoto, Japan. Tel: +81-75-753 4441; Fax: +81-75-753 4446; E-mail: [email protected]

Bacterial pathogenicity; Medical bacteriology; Intracellular bacteria; Immune response to infection

M. Schembri, School of Molecular and Microbial Sciences, University of Queensland, Building 76, QLD 4072 Brisbane, Australia. Tel: +61-7-3365 3306;


Microbial pathogenicity, especially gram-negative bacteria; Bacterial adhesins; Biofilms; Bacterial gene regulation and DNA microarrays; Bacterial display

systems; Vaccine development

S. Schwarz, Molecular Microbiology and Diagnostics, Institute for Animal Breeding, Federal Agricultural Research Centre (FAL), Holtystr. 10, D-31535

Neustadt-Mariensee, Germany. Tel: +49-5034-871-241; Fax: +49-5034-871-246; E-mail: [email protected]

Molecular biology of Staphylococci; Antibiotic resistance mechanisms; Mobile genetic elements and horizontal gene transfer; Pathogenicity; Molecular

epidemiology; Gram-positive cocci; Pasteurellaceae (Pasteurella, Mannheimia, Actinobacillus) and Enterobacteriaceae (Salmonella, Escherichia),

Bordetella

S. Smith, Department of Microbiology, Moyne Institute, Trinity College, 2 Dublin, Ireland. Tel: +353-1-6083713; Fax: +353-1-6799294;


Microbial pathogenicity; Gram-negative bacteria; Bacterial adhesion and invasion; Outer membrane proteins; Fimbriae and pili; Proteomics and genomics;

Bacterial gene regulation

A.H.M. van Vliet, Department of Gastroenterology and Hepatology (L-459), Erasmus MC, Dr. Molewaterplein 40, 3015 GD Rotterdam, Netherlands. Tel:

+31-10-463 5944; Fax: +31-10-463 2793; E-mail: [email protected]

Microbial pathogenesis and genetics, especially of Helicobacter and Campylobacter; Bacterial gene regulation; Microbial metal metabolism

W. Wade, Department of Microbiology, Dental Institute, King’s College London, Guy’s Hospital, Floor 28, Guy’s Tower, SE1 9RT London,

United Kingdom. Tel: +44-20-7188 3872; Fax: +44-20-7188 3871; E-mail: [email protected]

Clinical microbiology; Oral microbiology; Molecular microbial ecology; Molecular diagnostics; Bacterial systematics; Anaerobic bacteria

P.H. Williams, Department of Genetics, University of Leicester, University Road, LE1 7RH Leicester, United Kingdom. Tel: +44-116-252 3436; Fax: +44-

116-252 3378; E-mail: [email protected]

Molecular genetic and cell biological analysis of the pathogenesis of infectious diseases, especially the role of microbial iron uptake, both in infection and in

the survival, persistence and resuscitation of severely stressed micro-organisms; Virulence mechanisms of enteric pathogens

C. Winstanley, Division of Medical Microbiology, University of Liverpool, Duncan Building, Daulby Street, Liverpool L69 3GA, United Kingdom. Tel: +44-

151-706 4388; Fax: +44-151-706 5805; E-mail: [email protected]

Pathogenicity of and genetic variation amongst Gram-negative bacteria; Pseudomonas and Burkholderia; Pathogenicity islands

PHYSIOLOGY AND BIOCHEMISTRY

J.R. Andreesen, Institut fur Mikrobiologie, Martin-Luther-Universitat Halle-Wittenberg, Kurt-Mothes-Straße 3, D-06120 Halle, Germany. Tel: +49-345-552

6350; Fax: +49-345-552 7010; E-mail: [email protected]

Physiology and biochemistry of anaerobic bacteria; Metal(oids) involved in biochemical reactions (Mo, W, Se, Te, Zn) but not transport

R.A. Bonomo, Infectious Diseases Section, Louis Stokes Cleveland Veterans Affairs Medical Center, East Blvd 10701, Ohio 44106 Cleveland, United

States of America. Tel: +1-216-791-3800x4399; Fax: +1-216-231-3482; E-mail: [email protected]

Beta-lactamases; Resistance to beta-lactams; Mechanisms of antimicrobial resistance

R.A. Burne, Department of Oral Biology, College of Dentistry (Room D5-18), University of Florida, 1600 S.W. Archer Road, FL 32610 Gainesville, United

States of America. Tel: +1-352-392 4370; Fax: +1-352-392 7357; E-mail: [email protected]

Oral microbiology; Environmental regulation of bacterial gene expression; Stress tolerance; Biofilms; Streptococci

J.A. Cole, School of Biosciences, University of Birmingham, Edgbaston, B15 2TT Birmingham, United Kingdom. Tel: +44-121-414 5440; Fax: +44-121-414


Microbial physiology, especially the regulation of anaerobic metabolism of enteric bacteria; Nitrate and nitrite reduction by bacteria; Microbial pathogenicity

of gonococci; Bacterial cytochrome biosynthesis and electronic transfer pathways

C. Dahl, Institut fur Mikrobiologie und Biotechnologie, Rheinische Friedrich-Wilhelms Universitat Bonn, Meckenheimer Allee 168, vD-53115 Bonn,

Germany. Tel: +49-228-732 119; Fax: +49-228-737 576; E-mail: [email protected]

Physiology, biochemistry, molecular biology and genetics of anoxygenic phototropic bacteria; Microbial sulfur metabolism; Electron transport

A.M. George, Dept of Cell and Molecular Biology, University of Technology, Sydney, PO Box 123 (Broadway), NSW 2007 Sydney, Australia.


Molecular biology and biochemistry of multidrug resistance in bacteria and higher organisms; Bacterial resistance to antibiotics; Membrane transport; ABC

transporters

J.A. Gil, Dept de Microbiologıa, Facultad de Biologıa, Universidad de Leon, 24071 Leon, Spain. Tel: +34-987-291 503; Fax: +34-987-291 479;


Antibiotic biosynthesis and resistance; Actinomycetes and corynebacteria

D. Jahn, Institute for Microbiology, Technical University of Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany. Tel: +49-531-391 5804; Fax:


Bacterial biochemistry and bioenergetics; Enzyme mechanisms; Tetrapyrroles; Control of bacterial gene expression

W.J. Mitchell, School of Life Sciences, Heriot-Watt University, Riccarton, EH14 4AS Edinburgh, Scotland, United Kingdom. Tel: +44-131-451 3459; Fax:


Regulation of bacterial gene expression; Solute transport, particularly the bacterial phosphotransferase system

S. Mongkolsuk, Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, 10210 Bangkok, Thailand. Tel: (662) 574-0623 ext. 3816; Fax: (662)

574-2027; E-mail: [email protected]

Bacterial biochemistry, physiology and genetics of stresses and metals; Regulation of gene expression; Environmental microbiology;

Plantmicrobe interactions

M. Moracci, Institute of Protein Biochemistry -CNR, Via P. Castellino 111, 80131 Naples, Italy. Tel: +39 081 613 2271; Fax: +39 081 613 2277;


Physiology and biochemistry of hyperthermophilic Bacteria and Archaea; Control of gene expression in thermophilic Archaea; Biotechnological applications

of enzymes from extremophiles

S. Rimsky, Enzymologie et Cinetique Structurale, LBPA, UMR 8113, Ecole Normale Superieure de Cachan/CNRS, Universite Paris XI, Avenue du

President Wilson 61, 94235 Cachan Cedex, France. Tel: +33-1-4740 7676; Fax: +33-1-4740 7684; E-mail: [email protected]

Protein-DNA interaction; Bacterial chromatin organisation; Protein-protein interaction (non-membrane); DNA chemical/enzymatic reactivity

S. Silver, Department of Microbiology and Immunology, Room E-704, University of Illinois, S. Wolcott Avenue 835, IL-60612-7344 Chicago, United States

of America. Tel: +1-312-996 9608; Fax: +1-312-996 6415; E-mail: [email protected]

Bacterial membrane transport; Molecular genetics and biochemistry; Metal-resistance mechanisms; Gram-positive bacteria and pseudomonads

J. Simon, School of Biological Sciences, University of East Anglia, NR4 7TJ Norwich, United Kingdom. Tel: +44-1603-593 250; Fax: +44-1603-592 250;


Bacterial metabolism and bioenergetics, especially anaerobic respiration; Maturation of electron transport enzymes

A. Steinbuchel, Institut fur Molekulare Mikrobiologie und Biotechnologie, Westfalische Wilhelms-Universitat, Correnstraße 3, D-48149 Munster, Germany.


Metabolism and biotechnological production of biopolymers (Polyesters, Cyanophycin and other poly(amino acids)); Microbial degradation of rubber

B. Ward, ICMB, Darwin Building, Kings Buildings, University of Edinburgh, EH9 3JR Edinburgh, Scotland, United Kingdom. Tel: +44-131 650 5370;


Microbial physiology of gram-negative bacteria important in medicine or food; Campylobacter jejuni

A. Yokota, Laboratory of Microbial Resources and Ecology, Graduate School of Agriculture, Hokkaido University, 060-8589 Sapporo, Japan.


Energy metabolism in gram-positive bacteria and Escherichia coli; Lactic acid bacteria and bifidobacteria as probiotics

www.fems-microbiology.org

FEMS Microbiology Letters 246 (2005) 151–158

MiniReview

Fluorescence in situ hybridisation (FISH) – the next generation

Katrin Zwirglmaier *

Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK

Received 25 February 2005; received in revised form 8 April 2005; accepted 13 April 2005

First published online 27 April 2005

Edited by R.I. Aminov

Abstract

Fluorescence in situ hybridisation (FISH) has become one of the major techniques in environmental microbiology. The original

version of this technique often suffered from limited sensitivity due to low target copy number or target inaccessibility. In recent

years there have been several developments to amend this problem by increasing signal intensity. This review summarises various

approaches for signal amplification, focussing especially on two widely recognised varieties, tyramide signal amplification and mul-

tiply labelled polynucleotide probes. Furthermore, new applications for FISH are discussed, which arise from the increased sensi-

tivity of the method.

� 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Fluorescence in situ hybridisation; Tyramide signal amplification; Polynucleotide probes

1. Introduction

Fluorescence in situ hybridisation (FISH) allows the

visualisation of prokaryotic cells in their natural envi-

ronment. In short, cells are fixed (i.e., they are not viable

anymore and the status quo of their DNA and RNA is

preserved), permeabilised to facilitate access of the

probe to the target site and then hybridised with nucleicacid probes. The probes are either directly labelled with

a fluorochrome or the dye is introduced in a secondary

detection step. The samples can then be analysed by epi-

fluorescence or laser scanning microscopy or flow

cytometry. The classic FISH technique relied solely on

(usually 16S) rRNA as probe target. The rRNA imme-

diately suggests itself as the ideal target because it is

present in all living cells in relatively high copy numbers.Furthermore, since it is traditionally used as phyloge-

0378-1097/$22.00 � 2005 Federation of European Microbiological Societies

doi:10.1016/j.femsle.2005.04.015

* Tel.: +44 24765 22572; fax: +44 24765 23701.

E-mail address: [email protected].

netic marker a lot of sequence data is available for probe

design.

Since its origins some 20 years ago [1] this technique

has become an invaluable tool for environmental micro-

biologists and has spawned numerous variations. The

reasons for this popularity are obvious: (1) FISH allows

the detection of cells regardless of their culturability.

With as little as 0.3% of bacteria in soil and <0.1% inmarine water being culturable [2] FISH offers a glimpse

at the full bacterial biodiversity. (2) The possibility to

detect cells in situ allows an insight into the structure

of microbial communities and may help to unveil their

ecological function.

Despite these promising features the classic FISH

protocol suffers from some limitations. A major draw-

back is the often very low signal intensity. This canpartly be attributed to insufficient cell permeability

preventing access of the probes into the cells and to

the target site. The introduction of various chemical

and enzymological pre-treatments (reviewed in [2]) can

. Published by Elsevier B.V. All rights reserved.

mailto:[email protected]

152 K. Zwirglmaier / FEMS Microbiology Letters 246 (2005) 151–158

alleviate this problem to some extent. However, cell per-

meability always has to be carefully balanced against

cell integrity to avoid cell loss.

Apart from permeability issues, the main reason for

weak signals with the classic rRNA targeted FISH is

the low ribosome content found in slowly growing ormetabolically inactive cells in environmental samples.

Problems of this nature have led to a number of different

approaches for signal amplification being developed in

recent years. This improved sensitivity of the method

ultimately negated the dogma that FISH requires high

copy numbers of the target molecule and paved the

way for new applications, allowing microbiologists to

move away from 16S rRNA and instead target other nu-cleic acids present in lower copy numbers, such as

mRNA, plasmids or even single copy genes. This trend

is further aided by the increasing amount of available

sequence data gathered from multiple genome projects.

This review summarises recent developments in FISH

technology, focussing on different approaches for signal

amplification and discussing possible new applications

for FISH arising from these developments.

2. Different ways for increasing FISH signals

2.1. Multiple probes for one target organism

The number of probes binding to their target is limited

by the number of available targets (i.e., ribosomes).Therefore, one obvious solution to get better signals is

to use several probes targeting different regions of the

16S rRNA. In various studies [3–5] combinations of up

to five probes were used. Although this led to stronger

signals, the effect was not as pronounced as anticipated

and generally did not exceed a 2–3-fold amplification

compared to hybridisations with single probes. A further

complication is that it is usually difficult to design severalprobes with the same specificity.

The inverse strategy (using one probe that carries

multiple labels) has been shown to be unsuitable for sig-

nal amplification, since it leads to increased non-specific

binding and a poor signal to noise ratio [6].

2.2. Helper oligos

As well as low ribosome content the signal intensity

is also strongly affected by the accessibility of the target

sequence. The ribosomal RNA has a well-described

complex secondary and tertiary structure with many

loops and helices and embedded ribosomal proteins

leaving some stretches of the rRNA more accessible to

probes than others. Studies by Fuchs et al. [7,8] and

Behrens [9,10] resulted in an ‘‘accessibility map’’ ofthe rRNA, defining regions with strong or weak hybrid-

isation signals, thus allowing a more directed probe

design. Unfortunately, some of the highly variable

(and therefore valuable for specific probe design) re-

gions turned out to be rather inaccessible. This problem

was addressed with the introduction of so-called helper-

oligos [11]. These are unlabelled oligonucleotides which

bind in close vicinity to the target site of the labelledprobe and open the secondary structure of the rRNA,

which facilitates the binding of the labelled probe. A

combination of one (mono)labelled probe and up to

four unlabelled helper oligonucleotides resulted in up

to 25-fold signal amplification compared to the labelled

probe alone. However, it is essential that the helper oli-

gonucleotides have the same specificity as the probe,

which again can cause problems with probe design.

2.3. PNA probes

Another option to address the problem of target

accessibility is to use peptide nucleic acid (PNA) probes

[12–14]. Due to their uncharged peptide backbone these

probes have a much higher affinity to their hybridisation

target than DNA-oligonucleotides do. This allows thehybridisation parameters to be changed to very high

temperatures and low salt concentrations, which weak-

ens the secondary structure of the rRNA and therefore

increases target accessibility. PNA-FISH yields up to

5-fold stronger signals than the classic FISH using

DNA oligonucleotides. A practical drawback of this ap-

proach is the currently still rather high price for PNA

oligos.

2.4. Treatment with chloramphenicol to increase rRNA

content

Instead of amplifying the signal by introducing more

probes or multiple labels it is also possible to artificially

increase the number of target molecules to allow more

probes to bind. One way to do this is to incubate envi-ronmental samples with the antibiotic chloramphenicol

[15].

Chloramphenicol is an inhibitor of protein synthesis

and rRNA degradation. Cell division is also inhibited,

thus leading to an accumulation of rRNA in the cell.

In a study by Ouverney et al. [15] 93–99% of all

DAPI stained cells in a marine sample could be de-

tected by FISH after chloramphenicol treatment as op-posed to 75% in an untreated sample. This effect of

chloramphenicol is also interesting for a second reason,

as it indicates that rRNA is present initially, disproving

earlier assumptions that low fluorescent cell counts in

marine environments are due to high levels of dead

cells.

A possible limitation of this approach is a potential

shift in community composition after incubation withchloramphenicol, since the antibiotic does not affect

archaebacteria or eukaryotes.

K. Zwirglmaier / FEMS Microbiology Letters 246 (2005) 151–158 153

2.5. In situ PCR

Another way to increase the number of target mole-

cules and thereby the signal intensity is in situ PCR

[16]. Although strictly speaking not a FISH technique

it deserves to be mentioned in the context of signalamplification. PCR is carried out inside the cell using la-

belled nucleotides to allow later detection of the PCR

products.

While this method has the advantage to be applicable

not only to rRNA (rDNA), but any other gene or even

mRNA, it nevertheless still suffers from technical diffi-

culties. Vigorous enzymatic pre-treatment of the cells

is required to allow the Taq polymerase to access thecell, which subsequently can result in considerable efflux

of PCR product or cell loss.

2.6. Bacterial chromosomal painting (BCP)

This technique differs from the others in that it does

not rely on a single target gene but rather uses the whole

genome as a target. It is therefore closely related to solu-tion based genomic DNA:DNA reassociation [17] and

promises a higher resolution and possible differentiation

of closely related strains.

In the bacterial species concept strains are defined as

belonging to the same species if they show >97% identity

on rRNA level and 70% on the whole genome [18]. Con-

sequently, differentiation of strains with rRNA targeted

probes is at best difficult and often impossible, whereasit is still possible when the rest of the genome is consid-

ered [17].

In bacterial chromosomal painting (BCP), [19,20] the

whole genome of a target organism is used as a probe.

Fluorescently labelled probes are generated by nick

translation with the size of the probe fragments ranging

between 50 and 200 bp. Although promising, one of the

drawbacks of this technique is that hybridisation timesare unusually long (2 days). BCP has been shown to al-

low differentiation of Salmonella serotypes [19] and has

also been applied to marine samples [20].

2.7. Enzymatic signal amplification – TSA-FISH

A very dramatic increase of sensitivity can be

achieved by enzyme-mediated signal amplification.TSA-FISH (tyramide signal amplification) [21], also

known as CARD-FISH (catalysed reporter deposition)

[22] is based on the deposition of fluorescently labelled

tyramide by peroxidase activity. Horseradish peroxidase

(HRP) is introduced in the target cells either by using

HRP-labelled probes or via a secondary detection of

digoxigenin labelled probes and an HRP-coupled antidi-

goxigenin antibody. The enzyme then catalyses the oxi-disation of the fluorochrome labelled tyramide

substrate, leading to a deposition of the highly active

intermediates in the vicinity of the enzyme by covalent

binding to electron-rich protein moieties. The resulting

signals are up to 12 times stronger than in hybridisations

with traditional fluorescence labelled probes and 3–

4-fold stronger than in hybridisations with multiple

monolabelled fluorescent probes [23]. A drawback ofthis approach is the need for vigorous pre-treatment of

the cells using e.g., lysozyme and/or proteinase to enable

the relatively large HRP molecule to enter the cell. This

can lead to loss of some cell types in heterogenous envi-

ronmental samples. Cell integrity and cell wall perme-

ability thus have to be carefully balanced.

Despite these difficulties, this approach is becoming

increasingly popular and has recently been used to de-tect not only rRNA, but also plasmids [5], tmRNA

[24] and mRNA [25].

2.8. Polynucleotide probes and RING-FISH

Another approach to increase the sensitivity of FISH

is the use of polynucleotide probes. These probes can

range in length between 100 and several hundred basepairs. They are made of ssRNA [25–30] or dsDNA

[19,20,31], generated either by in vitro transcription

[25,28,29], nick translation [19,20] or PCR [31] and carry

multiple labels, either fluorescent dyes or digoxigenin/

biotin for a secondary detection. The signal amplifica-

tion achieved with these probes is based on both the

multiple labelling and the secondary structures formed

by the long probes. These secondary structures involvenot only intra-, but also intermolecular binding, which

results in a network of probes (see Fig. 1). The huge po-

tential of polynucleotide probes lies in the fact that a

network allows the incorporation of probe molecules,

which are not directly connected to the target site.

Therefore, the detectable fluorescence is no longer pro-

portional to the number of available targets. [32]. A

characteristic feature of in situ hybridisations with poly-nucleotide probes is the ring-shaped or halo-like appear-

ance of the fluorescence signal. This is especially true for

hybridisations with (intermediate–high concentrations

of) ssRNA [26–28] and dsDNA probes [31]. The halo,

which can be seen as an extracellular probe network an-

chored at the intracellular probe specific target site, is

usually not observed with very low probe concentrations

[25,29], hydrolysis of the probes to shorter fragmentsprior to the hybridisation [19,20] or excessive permeabil-

isation of the cell wall with lysozyme or other treatments

[27,33].

rRNA targeted polynucleotide probes have been ap-

plied in various studies in recent years [27,29,30,34].

However, due to the structure of the rRNA, with

patches of highly conserved sequence, they can only be

used at a group specific, rather than species specific level.Their special value therefore lies in the possibility to

target other, low copy nucleic acids.

Fig. 1. Schematic illustration of the formation of probe networks, which are the presumed origin of the ring-shaped hybridisation signal seen with

RING-FISH: (a) secondary structure of polynucleotide probe GAP-E targeting a 258 bp fragment of the E. coli glycerol aldehyde phosphate

dehydrogenase (GAPDH) gene, (b) simplified and schematised secondary structure, (c) denaturation step prior to hybridisation leads to linearised

probe molecules, (d) during hybridisation intra- and intermolecular renaturation of secondary structure leads to formation of probe network. The

network is anchored at the intracellular probe-specific target site, (e) interconnected probes accumulate mainly outside the target cell due to the

limited permeability of the cell wall, resulting in a ring-shaped hybridisation signal. The specificity of the signal is based on the intracellular anchor.

Probe networks not connected to an intracellular probe-specific target site will be washed away during post-hybridisation wash steps,

(f) epifluorescence micrograph of a RING-FISH signal using probe GAP-E targeting the GAPDH gene.


This has been implemented in the development of

RING-FISH (recognition of individual genes), a FISH

variety, which relies on the use of high concentrations

of polynucleotide probes and is characterised by a

ring-shaped fluorescence signal. RING-FISH has been

shown to allow the detection of plasmid encoded and

even chromosomal single copy genes [28,33].

3. Moving away from rRNA as target

Most of the above methods were developed with the

intention of improving detection of rRNA in cells with

low ribosome content. Due to its high copy number

rRNA has long been regarded as the only suitable mol-

ecule for bacterial FISH. However, with the develop-ment of potent signal amplification techniques,

especially TSA and polynucleotide probes, it became

possible to move away from the high copy (104–105 in

an actively growing cell) rRNA as the sole target for

fluorescence in situ hybridisation and instead target a

variety of other nucleic acids such as mRNA, tmRNA,

plasmids and chromosomal DNA, thereby opening the

door for new applications for FISH.

3.1. tmRNA

tmRNA, also called 10SaRNA or SsrA is a small sta-

ble RNA (length in Escherichia coli: 365 nt), which has

been shown to be involved in the degradation of trun-cated proteins. With copy numbers of approximately

103 in metabolically active cells it is slightly less abundant

than rRNA but still easily detectable with TSA [24].

Compared to rRNA it has the advantage of being more

accessible, since it is not complexed with ribosomal pro-

teins. tmRNA has so far been detected in all completely

sequenced bacterial genomes (in 17 of 20 phyla) and in

certain phage, mitochondrial and plastidial genomes,but not (yet) in archaeal or eukaryotic genomes.

3.2. Plasmids

The copy number of plasmids ranges (depending on

the type of plasmid) from 101 to 103 per cell. This


suggests them as a target for an improved sensitivity

FISH protocol. Also, as they are made of DNA, they

are more stable than mRNA, which, although present

in comparable copy numbers, requires special handling

to avoid degradation.

The polynucleotide probe based RING-FISH wasused to detect different types of plasmids with high,

medium and low copy numbers [28].

Various modifications of the protocol were necessary

to account for the decreased target copy number and

target type.

To allow hybridisation to the dsDNA, a denaturation

step prior to the hybridisation had to be introduced. The

RNA:DNA hybrid (polynucleotide RNA probe andDNA target) is thermodynamically weaker than the

RNA:RNA hybrid (RNA probe and rRNA target) in

conventional fluorescence in situ hybridisations, calling

for more relaxed hybridisation conditions. Finally, the

decreased target copy number decelerates the formation

of the signal amplifying probe network, requiring longer

hybridisation times.

The signal intensity was the same for high, mediumand low copy plasmids, but slightly weaker than for

rRNA targeted probes.

A TSA-FISH based approach was used for an indi-

rect detection of ColE1 related plasmids [5]. The RNA

II, a 555 nt transcript, which regulates plasmid replica-

tion by acting as primer for the DNA synthesis was

targeted with a combination of up to seven HRP la-

belled oligonucleotide probes resulting in strong sig-nals in cells containing the plasmid. This study

compared signal amplification gained from using mul-

tiple fluorescently monolabelled probes and tyramide

signal amplification. TSA was shown to be the supe-

rior technique.

3.3. mRNA

The detection of mRNA presents a special challenge

due to its inherent instability. However, in the context

of elucidating the role of individual species within an

ecosystem the prospect of being able to detect mRNA

in individual cells in situ is very attractive. In recent

years there have been various reports describing suc-

cessful detection of mRNA with FISH [25,35–37]. All

these studies employ digoxigenin labelled probes. Theywere detected via TSA or, in one early study [35], by a

colour reaction mediated by alkaline phosphatase.

Only one study used digoxigenin labelled oligonucleo-

tide probes [37], while the others were based on poly-

nucleotide transcript probes carrying multiple

digoxigenin labels. This approach combines the signal

amplification gained from introducing multiple labels

(via the multiply labelled polynucleotide probes) withthe amplification through enzyme mediated deposition

of fluorescent dye.

3.4. Genomic DNA

Detecting a chromosomal single copy gene in situ is

the ultimate challenge for signal amplification with

FISH. As with many other developments (such as

TSA, in situ PCR and mRNA detection) this has al-ready been a well-established technique for eukaryotic

cells, e.g. [38,39], before it was adapted for use in pro-

karyotes in the form of RING-FISH. While the

eukaryotic ‘‘chromosomal painting’’ uses probes with

a length of several kb, probe length for the prokaryotic

RING-FISH is only about 150–800 nt [28]. Character-

istic for RING-FISH is the use of multiply labelled

polynucleotide probes in a very high concentration(250 ng/ll), a denaturation step prior to the hybridisa-

tion and a rather long hybridisation period (up to

24 h).

It has been applied to detect the housekeeping gene

glycerol aldehyde 3-phosphate dehydrogenase (GAPDH)

inE. coli, a virulence factor in the plant pathogenXantho-

monas campestris, as well as a fragment of the RNA

polymerase gene rpoC1 in Synechococcus ([28] andZwirglmaier and Scanlan, unpublished).

3.5. Specificity of polynucleotide probes

As studies with plasmids, mRNA and genomic DNA

have shown, signal amplification with polynucleotide

probes, possibly further enhanced by TSA clearly has

the potential to detect any low copy nucleic acid target.One question that remains to be clarified is the specific-

ity of these probes. In contrast to oligonucleotide

probes, where a single mismatch can be discriminated,

polynucleotide probes clearly require more sequence dif-

ferences for a specific signal. Other factors, such as the

secondary structure of the probe and the fact that in

case of a network formation probably not the complete

probe binds to the target also influence specificity. Cur-rently there is only limited data about the threshold for

mismatch discrimination. Ludwig et al. [40] found the

cut-off point for positive/negative hybridisation signals

to be between 78% and 85% sequence identity using a

probe targeting the domain III of the 23S rRNA in

membrane based hybridisations. A more detailed study

of the same target region using FISH recently described

a cut-off point of 72–75% [33]. Similar conclusions canbe drawn from the study by Pernthaler [25], where a

probe targeting the mRNA of the pmoA gene (coding

for subunit A of the particulate methane monooxygen-

ase) was shown to detect methylotrophic symbionts of

Bathymodiolus azoricus with a similarity of around

80%, but not the more distantly related (64%) Methylo-

cystis echinoides.

Sequence alignment and conservativity profiles to de-tect highly variable regions in a target sequence should

therefore be an integral part of future probe design.


4. Monitoring bacterial activity

Currently the major question in microbial ecology of

who is out there can reliably be answered with FISH

technology. The next big question as to what they are

doing out there has been addressed more recently bycombining FISH with various other techniques.

Bacterial activity is linked to DNA synthesis. The

detection of newly synthesised DNA by monitoring

the uptake and incorporation of tritiated thymidine

[41] or, more recently, bromodioxyuridine BrdU [42–

44] is therefore a way to differentiate active from inactive

cells. Combining this technique with FISH consequently

allows an assertion of which parts of a bacterial commu-nity are metabolically active [42].

Information beyond a general assessment of cellular

activity can be gained from microautoradiography

(MAR). Radioactively labelled substrate is added to

an environmental sample and its uptake is monitored.

A subsequent phylogenetic identification of the cells by

FISH then gives a picture about the substrate utilisation

of different cells in a bacterial community and may allowconclusions about the underlying food-network. FISH-

MAR, also known as STAR-FISH (substrate tracking

autoradiography) has recently been applied in various

studies [15,45–48].

An even more detailed account of the activity of a

single microbial cell can be gained by studying gene

expression. The combination of mRNA and rRNA tar-

geted FISH [25] offers a true insight into the ‘‘blackbox’’ of complex biocommunities. Although currently

not yet a standard technique, future optimisations and

improvements could turn it into one of the core methods

for environmental microbiologists.

5. Conclusions and outlook

Fluorescence in situ hybridisation has seen some ma-

jor improvements since its development some 20 years

ago and is nowadays one of the chief techniques in envi-

ronmental microbiology. With the increased sensitivity

of the method and the ability to detect genes and mRNA

the questions that can be addressed with FISH are

changing. While originally developed to describe the

composition of a bacterial community, it is now alsopossible to look at the activity of individual members

and their ecological function. The correlation of phylog-

eny and physiology is becoming an ever more important

topic in our attempts to understand ecological systems.

The feasibility of in situ gene expression studies presents

a quantum leap in this context. The rapidly growing

amount of sequence data, with new bacterial genomes

being published almost weekly, further supports thesedevelopments, allowing comparative sequence analysis

and directed probe design for any given gene.

Apart from gene expression, combined rRNA and

DNA targeted FISH could unveil cases of horizontal

gene transfer. This would be of interest not only for evo-

lutionists, but also in the context of genetically modified

organisms and their potential impact on ecosystems.

Another new application for ultrasensitive FISHmight be the detection of viral infections. As with many

other developments (including the original FISH tech-

nique), this has already been applied to eukaryotic cells

[49,50], but would probably require some modification

and optimisation for use in prokaryotes.

With the problem of sensitivity solved, the next desir-

able step in the future of FISH technology would be an

efficient automation to increase the amount of samplesthat can be analysed, maybe by optimising existing flow

cytometry techniques or even a microarray based

approach.

References

[1] DeLong, E.F., Wickham, G.S. and Pace, N.R. (1989) Phyloge-

netic stains: ribosomal RNA-based probes for the identification of

single cells. Science 243, 1360–1363.

[2] Amann, R.I., Ludwig, W. and Schleifer, K.H. (1995) Phylogenetic

identification and in situ detection of individual microbial cells

without cultivation. Microbiol. Rev. 59, 143–169.

[3] Amann, R.I., Binder, B.J., Olson, R.J., Chisholm, S.W., Deve-

reux, R. and Stahl, D.A. (1990) Combination of 16S rRNA-

targeted oligonucleotide probes with flow cytometry for analyzing

mixed microbial populations. Appl. Environ. Microbiol. 56,

1919–1925.

[4] Lee, S.H., Malone, C. and Kemp, P.F. (1993) Use of multiple 16S

rRNA targeted fluorescent probes to increase signal strength and

measure cellular RNA from natural planktonic bacteria. Mar.

Ecol. Prog. Ser. 101, 193–201.

[5] Juretschko, S., Schonhuber, W., Kulakauskas, S., Ehrlich, D.S.,

Schleifer, K.H. and Amann, R. (1999) In situ detection of

Escherichia coli cells containing ColE1-related plasmids by

hybridization to regulatory RNA II. Syst. Appl. Microbiol. 22,

1–8.

[6] Wallner, G., Amann, R. and Beisker, W. (1993) Optimizing

fluorescent in situ hybridization with rRNA-targeted oligonu-

cleotide probes for flow cytometric identification of microorgan-

isms. Cytometry 14, 136–143.

[7] Fuchs, B.M., Wallner, G., Beisker, W., Schwippl, I., Ludwig, W.

and Amann, R. (1998) Flow cytometric analysis of the in situ

accessibility of Escherichia coli 16S rRNA for fluorescently

labeled oligonucleotide probes. Appl. Environ. Microbiol. 64,

4973–4982.

[8] Fuchs, B.M., Syutsubo, K., Ludwig, W. and Amann, R. (2001) In

situ accessibility of Escherichia coli 23S rRNA to fluorescently

labeled oligonucleotide probes. Appl. Environ. Microbiol. 67,

961–968.

[9] Behrens, S., Ruhland, C., Inacio, J., Huber, H., Fonseca, A.,

Spencer-Martins, I., Fuchs, B.M. and Amann, R. (2003) In

situ accessibility of small-subunit rRNA of members of the

domains Bacteria, Archaea, and Eucarya to Cy3-labeled

oligonucleotide probes. Appl. Environ. Microbiol. 69, 1748–

1758.

[10] Behrens, S., Fuchs, B.M., Mueller, F. and Amann, R. (2003) Is the

in situ accessibility of the 16S rRNA of Escherichia coli for Cy3-

labeled oligonucleotide probes predicted by a three-dimensional


structure model of the 30S ribosomal subunit? Appl. Environ.

Microbiol. 69, 4935–4941.

[11] Fuchs, B.M., Glockner, F.O., Wulf, J. and Amann, R. (2000)

Unlabeled helper oligonucleotides increase the in situ accessibility

to 16S rRNA of fluorescently labeled oligonucleotide probes.

Appl. Environ. Microbiol. 66, 3603–3607.

[12] Worden, A.Z., Chisholm, S.W. and Binder, B.J. (2000) In situ

hybridization of Prochlorococcus and Synechococcus (marine

cyanobacteria) spp. with RRNA-targeted peptide nucleic acid

probes. Appl. Environ. Microbiol. 66, 284–289.

[13] Perry-O�Keefe, H., Rigby, S., Oliveira, K., Sorensen, D., Stender,

H., Coull, J. and Hyldig-Nielsen, J.J. (2001) Identification of

indicator microorganisms using a standardized PNA FISH

method. J. Microbiol. Meth. 47, 281–292.

[14] Oliveira, K., Procop, G.W., Wilson, D., Coull, J. and Stender, H.

(2002) Rapid identification of Staphylococcus aureus directly from

blood cultures by fluorescence in situ hybridization with peptide

nucleic acid probes. J. Clin. Microbiol. 40, 247–251.

[15] Ouverney, C. and Fuhrman, J. (1997) Increase in fluorescence

intensity of 16S rRNA in situ hybridization in natural samples

treated with chloramphenicol. Appl. Environ. Microbiol. 63,

2735–2740.

[16] Hodson, R., Dustman, W., Garg, R. and Moran, M. (1995) In

situ PCR for visualization of microscale distribution of specific

genes and gene products in prokaryotic communities. Appl.

Environ. Microbiol. 61, 4074–4082.

[17] Grimont, P.A. (1988) Use of DNA reassociation in bacterial

classification. Can. J. Microbiol. 34, 541–546.

[18] Stackebrandt, E. and Goebel, B. (1994) Taxonomic note: a place

for DNA–DNA reassociation and 16S rRNA sequence analysis in

the present species definition in bacteriology. Int. J. Syst.

Bacteriol. 44, 846–849.

[19] Lanoil, B. and Giovannoni, S. (1997) Identification of bacterial

cells by chromosomal painting. Appl. Environ. Microbiol. 63,

1118–1123.

[20] Lanoil, B.D., Carlson, C.A. and Giovannoni, S.J. (2000) Bacterial

chromosomal painting for in situ monitoring of cultured marine

bacteria. Environ. Microbiol. 2, 654–665.

[21] Schonhuber, W., Fuchs, B., Juretschko, S. and Amann, R. (1997)

Improved sensitivity of whole-cell hybridization by the combina-

tion of horseradish peroxidase-labeled oligonucleotides and tyra-

mide signal amplification. Appl. Environ. Microbiol. 63, 3268–

3273.

[22] Pernthaler, A., Pernthaler, J. and Amann, R. (2002) Fluorescence

in situ hybridization and catalyzed reporter deposition for the

identification of marine bacteria. Appl. Environ. Microbiol. 68,

3094–3101.

[23] Lebaron, P., Catala, P., Fajon, C., Joux, F., Baudart, J. and

Bernard, L. (1997) A new sensitive, whole-cell hybridization

technique for detection of bacteria involving a biotinylated

oligonucleotide probe targeting rRNA and tyramide signal

amplification. Appl. Environ. Microbiol. 63, 3274–3278.

[24] Schonhuber, W., Le Bourhis, G., Tremblay, J., Amann, R. and

Kulakauskas, S. (2001) Utilization of tmRNA sequences for

bacterial identification. BMC Microbiol. 1, 20.

[25] Pernthaler, A. and Amann, R. (2004) Simultaneous fluorescence

in situ hybridization of mRNA and rRNA in environmental

bacteria. Appl. Environ. Microbiol. 70, 5426–5433.

[26] Stoffels, M., Ludwig, W. and Schleifer, K.H. (1999) rRNA probe-

based cell fishing of bacteria. Environ. Microbiol. 1, 259–271.

[27] Trebesius, K.H., Amann, R., Ludwig, W., Muhlegger, K. and

Schleifer, K.H. (1994) Identification of whole fixed bacterial cells

with nonradioactive 23S rRNA-targeted polynucleotide probes.


[28] Zwirglmaier, K., Ludwig, W. and Schleifer, K.H. (2004) Recog-

nition of individual genes in a single bacterial cell by fluorescence

in situ hybridization – RING-FISH. Mol. Microbiol. 51, 89–96.

[29] DeLong, E.F., Taylor, L.T., Marsh, T.L. and Preston, C.M.

(1999) Visualization and enumeration of marine planktonic

archaea and bacteria by using polyribonucleotide probes and

fluorescent in situ hybridization. Appl. Environ. Microbiol. 65,

5554–5563.

[30] Pernthaler, A., Preston, C.M., Pernthaler, J., DeLong, E.F. and

Amann, R. (2002) Comparison of fluorescently labeled oligonu-

cleotide and polynucleotide probes for the detection of pelagic

marine bacteria and archaea. Appl. Environ. Microbiol. 68, 661–

667.

[31] Zimmermann, J., Ludwig, W. and Schleifer, K.H. (2001) DNA

polynucleotide probes generated from representatives of the genus

Acinetobacter and their application in fluorescence in situ

hybridization of environmental samples. Syst. Appl. Microbiol.

24, 238–244.

[32] Zwirglmaier, K., Ludwig, W. and Schleifer, K.H. (2003)

Improved fluorescence in situ hybridization of individual micro-

bial cells using polynucleotide probes: the network hypothesis.

Syst. Appl. Microbiol. 26, 327–337.

[33] Fichtl, K. (2005) Polynucleotide probe based enrichment of

bacterial cells: development of probes for species of clinical

relevance. PhD thesis, Technical University Munich. Available

from: http://tumb1.biblio.tu-muenchen.de/publ/diss/karin.php.

[34] Karner, M.B., DeLong, E.F. and Karl, D.M. (2001) Archaeal

dominance in the mesopelagic zone of the Pacific Ocean. Nature

409, 507–510.

[35] Hahn, D., Amann, R. and Zeyer, J. (1993) Detection of mRNA in

Streptomyces cells by whole-cell hybridization with digoxigenin-

labeled probes. Appl. Environ. Microbiol. 59, 2753–2757.

[36] Wagner, M., Schmid, M., Juretschko, S., Trebesius, K.H., Bubert,

A., Goebel, W. and Schleifer, K.H. (1998) In situ detection of a

virulence factor mRNA and 16S rRNA in Listeria monocytogenes.

FEMS Microbiol. Lett. 160, 159–168.

[37] Bakermans, C. and Madsen, E.L. (2002) Detection in coal tar

waste-contaminated groundwater of mRNA transcripts related to

naphthalene dioxygenase by fluorescent in situ hybridization with

tyramide signal amplification. J. Microbiol. Meth. 50, 75–84.

[38] Rogan, P.K., Cazcarro, P.M. and Knoll, J.H. (2001) Sequence-

based design of single-copy genomic DNA probes for fluorescence

in situ hybridization. Genome Res. 11, 1086–1094.

[39] Sharma, A.K. and Sharma, A. (2001) Chromosome painting –

principles, strategies and scope. Meth. Cell Sci. 23, 1–5.

[40] Ludwig, W., Dorn, S., Springer, N., Kirchhof, G. and Schleifer,

K.H. (1994) PCR-based preparation of 23S rRNA-targeted

group-specific polynucleotide probes. Appl. Environ. Microbiol.

60, 3236–3244.

[41] Fuhrman, J.A. and Azam, F. (1982) Thymidine incorporation as

a measure of heterotrophic bacterioplankton production in

marine surface waters: evaluation and field results. Marine Biol.

66, 109–120.

[42] Pernthaler, A., Pernthaler, J., Schattenhofer, M. and Amann, R.

(2002) Identification of DNA-synthesizing bacterial cells in

coastal North Sea plankton. Appl. Environ. Microbiol. 68,

5728–5736.

[43] Steward, G.F. and Azam, F. (1999) Bromodeoxyuridine as an

alternative to 3H-thymidine for measuring bacterial productivity

in aquatic samples. Aq. Microbial Ecol. 19, 57–66.

[44] Urbach, E., Vergin, K.L. and Giovannoni, S.J. (1999) Immuno-

chemical detection and isolation of DNA from metabolically

active bacteria. Appl. Environ. Microbiol. 65, 1207–1213.

[45] Lee, N., Nielsen, P.H., Andreasen, K.H., Juretschko, S., Nielsen,

J.L., Schleifer, K.-H. and Wagner, M. (1999) Combination of

fluorescent in situ hybridization and microautoradiography – a

new tool for structure–function analyses in microbial ecology.


[46] Ouverney, C.C. and Fuhrman, J.A. (1999) Combined microau-

toradiography-16S rRNA probe technique for determination of

http://tumb1.biblio.tu-muenchen.de/publ/diss/karin.php


radioisotope uptake by specific microbial cell types in situ. Appl.


[47] Teira, E., Reinthaler, T., Pernthaler, A., Pernthaler, J. and

Herndl, G.J. (2004) Combining catalyzed reporter deposition-

fluorescence in situ hybridization and microautoradiography to

detect substrate utilization by bacteria and Archaea in the deep

ocean. Appl. Environ. Microbiol. 70, 4411–4414.

[48] Gray, N.D., Howarth, R., Pickup, R.W., Jones, J.G. and Head,

I.M. (2000) Use of combined microautoradiography and fluores-

cence in situ hybridization to determine carbon metabolism in

mixed natural communities of uncultured bacteria from the genus

Achromatium. Appl. Environ. Microbiol. 66, 4518–4522.

[49] Alonso, M.C., Cano, I., Castro, D., Perez-Prieto, S.I. and

Borrego, J.J. (2004) Development of an in situ hybridisation

procedure for the detection of sole aquabirnavirus in infected fish

cell cultures. J. Virol. Meth. 116, 133–138.

[50] Plummer, T.B., Sperry, A.C., Xu, H.S. and Lloyd, R.V. (1998) In

situ hybridization detection of low copy nucleic acid sequences

using catalyzed reporter deposition and its usefulness in clinical

human papillomavirus typing. Diagn. Mol. Pathol. 7, 76–84.



MiniReview

Biotin biosynthesis, transport and utilization in rhizobia

Karina Guillen-Navarro, Sergio Encarnacion, Michael F. Dunn *

Programa de Ingenierıa Metabolica, Centro de Ciencias Genomicas, Universidad Nacional Autonoma de Mexico, A.P. 565-A, Cuernavaca,

Morelos, Mexico



Edited by R.I. Aminov

Abstract

Biotin, a B-group vitamin, performs an essential metabolic function in all organisms. Rhizobia are a-proteobacteria with the

remarkable ability to form a nitrogen-fixing symbiosis in combination with a compatible legume host, a process in which the impor-

tance of biotin biosynthesis and/or transport has been demonstrated for some rhizobia–legume combinations. Rhizobia have also

been used to delimit the biosynthesis, metabolic effects and, more recently, transport of biotin. Molecular genetic analysis shows that

an orthodox biotin biosynthesis pathway occurs in some rhizobia while others appear to synthesize the vitamin using alternative

pathways. In addition to its well established function as a prosthetic group for biotin-dependent carboxylases, we are beginning

to delineate a role for biotin as a metabolic regulator in rhizobia.

� 2005 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.

Keywords: Biotin biosynthesis; Rhizobia; Rhizobia–legume symbiosis

1. Introduction

Root nodule bacteria, collectively known as rhizobia,

are a crucial component of the global nitrogen cycle be-

cause they reduce atmospheric nitrogen to ammonia in

symbiotic association with a compatible plant host and

thus reduce the need for synthetic nitrogen fertilizers.

Before establishing symbiosis, rhizobia must survive inthe soil awaiting the presence of a suitable host legume.

Infection of the host requires multiplication in the rhizo-

sphere as well as during early phases of the infection.

Mature, nitrogen-fixing intracellular rhizobia (bacter-

oids) require large amounts of energy and reductant de-

rived from the catabolism of plant-supplied organic

acids. Consequently, the metabolism and growth factor

0378-1097/$22.00 � 2005 Published by Elsevier B.V. on behalf of the Feder

doi:10.1016/j.femsle.2005.04.020

* Corresponding author. Tel.: +52 73 311 4662; fax: +52 73 317 5094.

E-mail address: [email protected] (M.F. Dunn).

requirements of rhizobia have long been studied (for re-

views, see [1–4]).

Biotin (vitamin H) has an essential metabolic func-

tion as the CO2-carrying prosthetic group of selected

carboxylases, decarboxylases and transcarboxylases

[5]. De novo biotin biosynthesis occurs in many pro-

karyotes while others are partly or totally dependent

on external sources. The purpose of this review is tosummarize what is known about biotin biosynthesis,

transport and utilization in rhizobia. Rhizobia are

the only prokaryotes in which novel regulatory roles

for biotin have been investigated. Biotin transport is

important for the establishment of symbiosis in some

rhizobia, and they are the only prokaryotes in which

genes encoding biotin transport proteins have been

identified. A new aspect of biotin biosynthesis in rhizo-bia is the probable presence of novel pathways in

some species.

ation of European Microbiological Societies.


160 K. Guillen-Navarro et al. / FEMS Microbiology Letters 246 (2005) 159–165

2. Biotin requirement of rhizobia

Early studies on biotin used Rhizobium leguminosa-

rum bv. trifolii to demonstrate that ‘‘heat-stable Rhizo-

bium growth factor’’ was identical to ‘‘coenzyme R’’

from Azotobacter and that both were, in fact, identicalto biotin [6]. Based on their growth response to biotin

in defined media, rhizobia may be grouped with

respect to their ability to biosynthesize the vitamin.

Biotin auxotrophs are incapable of biotin biosynthesis

and require external sources for growth [6–8]. An eco-

logically interesting example is provided by the non-

symbiotic Mesorhizobium loti strains isolated from

soils [9,10]. These isolates lack a 500 kb region of theirchromosome termed the ‘‘symbiosis island’’ which, in

addition to a variety of symbiosis-specific functions,

encodes the biosynthesis of thiamine, nicotinic acid

and biotin.

Biotin prototrophs synthesize biotin de novo and

show neither a growth nor a significant metabolic re-

sponse to exogenous biotin [6–8,11]. For example, Rhi-

zobium tropici CFN299 grows well in minimal mediumsubcultures in the absence of biotin and maintains a

high level of pyruvate carboylase activity and holo-en-

zyme protein regardless of biotin supplementation

[12,13].

Biotin bradytrophs synthesize biotin but either do

so inefficiently or only under certain growth conditions

[11,14]. A controversy exists as to whether Rhizobium

etli and Sinorhizobium meliloti fit into this class[11,12,15] or with the biotin auxotrophs [16]. It is

important to note that when S. meliloti Rm1021 was

grown in biotin-free medium, a concomitant several-

fold increase in biomass and extracellular biotin, de-

tected with an ELISA assay, were found, indicating

that this strain can produce the vitamin de novo

[15]. Growth studies show that S. meliloti strain

GR4B is a biotin bradytroph whose synthesis of bio-tin, detected with a standard bioassay, was dependent

upon growth conditions [11].

Wild-type R. etli strain CE3 behaves as a biotin aux-

otroph when serially subcultured in minimal medium,

where very low biotin-dependent carboxylase activities

and protein levels confirm the presence of a biotin

starved state. Growth is restored not only in the pres-

ence of exogenous biotin but also by supplementationwith thiamine, pimelic acid (a biotin precursor), fuma-

rate plus malate, cAMP, glutamate, proline, or oxygen

([12]; unpublished results). S. meliloti Rm1021 behaves

similarly to R. etli CE3 with respect to the ability of thi-

amine to prevent biotin auxotrophy [12]. Given that

both S. meliloti and R. etli lack genes homologous to

most or all of the orthodox biotin biosynthesis genes

(see Section 6), the challenge of providing an unequivo-cal demonstration of their ability to synthesize the vita-

min remains.

3. Biotin-dependent carboxylases and biotin–protein

ligase in rhizobia

Biotin and carbon dioxide are essential for the

growth of rhizobia [1,6,17]. Genome sequence and

biochemical analysis show that rhizobia contain thebiotin-dependent enzymes pyruvate carboxylase

(PYC), acetyl-CoA carboxylase (ACC), and two or

more acyl-CoA carboxylases, including propionyl-

CoA carboxylase (PCC) [18]. PYC is required for

growth on sugars or pyruvate and, although its inacti-

vation has no effect on nodulation and nitrogen fixa-

tion in S. meliloti, R. etli or R. tropici [13,19], it

would be interesting to determine whether it plays arole in rhizosphere competition, since sugars are ex-

creted to the rhizosphere by legume roots [20]. The

symbiotic phenotype of a rhizobial PCC mutant has

not been determined but inactivation of the S. meliloti

methylmalonyl-CoA mutase, which catalyzes the step

following that of PCC during propionyl-CoA degrada-

tion, does not affect symbiotic performance [21,22].

ACC has not been characterized but would be expectedto be essential for fatty acid synthesis [18] and thus

viability.

Apo-biotin-dependent carboxylases are converted to

their active holo-enzymes by biotin–protein ligase

(BPL) [5,23]. The BPLs of Bacillus and enteric bacteria

are called BirA�s (biotin regulators) because their N-

terminal helix-turn-helix motif binds to and represses

biotin operon transcription [23,24]. In these organisms,BirA catalyzes the conversion of biotin into biotinoyl-

AMP, which functions with BirA as a co-repressor:

when the intracellular concentration of biotin is elevated

(e.g., in biotin-supplemented cultures), more BirA-biot-

inoyl-AMP is formed and transcription is repressed.

The middle and C-terminal portion of BirA contain

the catalytic residues for ligating biotin to target

proteins [23].Genome sequence analysis of M. loti, B. japonicum,

S. meliloti (http://www.kazusa.or.jp/rhizobase) R. etli

(G. Davila, V. Gonzalez, R. Gomez and P. Bustos,

unpublished), and R. leguminosarum bv. viciae (http://

www.sanger.ac.uk/Projects/R_leguminosarum) shows

that their deduced BPL gene products lack the N-termi-

nus found in BirA�s and retain only the catalytic motifs

required for biotinylating apo-carboxylases. Thesemonofunctional BPLs are present in many prokaryotes

and all eukaryotes.

The fact that rhizobia contain multiple biotin-depen-

dent carboxylases raises the question of how biotin is

partitioned among them by a single BPL. Western blot-

ting experiments designed to follow the biotinylation of

the carboxylases in biotin-starved R. etli cells pulsed

with biotin suggest that the relative level of each apo-carboxylase determines the amount of holo-carboxylase

formed (M. Dunn, unpublished). It is not known if the

http://www.ncbi.nlm.hih.gov/genomes/MICROBES/Complete.html

http://www.kazusa.or.jp/rhizobase


K. Guillen-Navarro et al. / FEMS Microbiology Letters 246 (2005) 159–165 161

BPL has the same affinity for each of the different apo-

carboxylases.

4. Effect of biotin on gene expression

Biotin affects gene expression in eukaryotes [25] but

little information exists on biotin as a BirA-independent

regulatory molecule in prokaryotes. Proteome analysis

shows that biotin markedly alters global protein expres-

sion in R. etli [26] and S. meliloti [27,28]. In R. etli, how-

ever, most of the changes in protein expression caused

by biotin were similar to those observed with thiamine

supplementation or growth in complex medium [26].Thus most of the changes observed with biotin reflect

the general metabolic state of the cells rather than a spe-

cific effect of the vitamin, and so without appropriate

controls claims of biotin-dependent gene expression

must be interpreted with caution.

Gene fusion assays show that S. meliloti bhdA

(encoding b-hydroxybutyrate dehydrogenase), bioS

(putative biotin-responsive regulatory gene), and copC

(possible copper resistance gene) are induced in response

to culture biotin supplementation [27–29]. In contrast,

pcm (encoding L-isoaspartyl protein repair methyltrans-

ferase), sinI (homoserine lactone autoinducer syntha-

tase) and sinR (homoserine lactone autoinducer

transcriptional regulator) are repressed under these con-

ditions [27]. Proteome analysis revealed that proteins

whose levels decreased under biotin limitation includedthe gene product of the down-regulated copC mentioned

above, 50S ribosomal protein L7/L12, RNA polymerase

x subunit, periplasmic transporter substrate binding

proteins (two for sugars, one for amino acids) and 2-

keto-3-deoxy-6-phosphogluconate aldolase (part of the

Entner-Doudoroff pathway). As Heinz and Streit [27]

discuss in detail, there is some correlation between these

results and the physiological response of S. meliloti tobiotin. For instance, the upregulated BdhA participates

in the degradation of the carbon storage polymer poly-

b-hydroxybutyrate (PHB), consistent with the finding

that biotin supplementation prevents PHB accumula-

tion in S. meliloti [12,28]. Regulation of PHB degrada-

tion by biotin could prevent its accumulation in

bacteroids [3] and promote its accumulation and grad-

ual utilization in oligotrophic environments like soil.

5. Biotin and the rhizobia–legume symbiosis

The effect of biotin on symbiosis depends on the rhi-

zobia–legume combination in question, and many natu-

rally-occuring, symbiotically proficient rhizobia are

biotin auxotrophs. A M. loti R7A biotin auxotroph(bioA::Tn5) was indistinguishable from the wild-type in

colonizing the Lotus corniculatus rhizosphere. However,

the biotin phenotype of this mutant is leaky [14] perhaps

due to the presence of a second copy of bioA, as occurs

in M. loti MAFF303099 [30]. Studies with S. meliloti

and R. etli bioN and bioM biotin uptake mutants indi-

cate that high affinity uptake is required for efficient

nodulation of their respective legume hosts ([5,15]; K.Guillen-Navarro, submitted). Interestingly, S. meliloti

bioN mutants engineered for biotin overproduction by

the introduction of the E. coli bio operon were also

found to compete poorly with the wild-type in the alfalfa

rhizosphere, perhaps due to the reduced viability ob-

served in the overproducing strains [29].

Determining the absolute symbiotic requirement for

biotin in rhizobia is complicated by the fact that thevitamin is excreted from the roots of host plants

[15,31]. The very low bacteroid activities of biotin-

dependent carboxylases in the bradytroph R. etli CE3

indicate that little biotin is synthesized by, or available

to, the microsymbiont. In contrast, bacteroids of the

biotin prototroph R. tropici CFN299 from nodules

formed on the same host (bean) have high activities,

indicating de novo synthesis of the vitamin ([32]; M.Dunn, unpublished).

6. Biotin biosynthesis

Escherichia coli and Bacillus species are the model

organisms to which we owe most of our understanding

of biotin biosynthesis (Fig. 1). Bacillus spp. are able totake up (apparently by passive diffusion) and use pimelic

acid as a precursor of biotin. Pimelic acid is derived

through an unknown pathway which may involve the

postulated fatty acid synthase-like activities of BioX

and BioI [33]. Pimelic acid is activated to its CoA deriv-

ative by pimeloyl-CoA synthetase, the product of bioW

[34]. E. coli is unable to utilize pimelic acid as a biotin

precursor and instead synthesizes pimeloyl-CoA, possi-bly from acetyl-CoA [35], using BioH, a probable car-

boxyl esterase [36] and the yet uncharacterized BioC

[33]. bioW homologs do not occur in the sequenced

genomes of M. loti, B. japonicum, S. meliloti or

A. tumefaciens.

The M. loti gene encoding BioZ is part of the bioBF-

DAZ operon (Fig. 2) and shows similarity to b-ketoacid-acyl synthases. BioZ can functionally complement E.

coli bioH, but not bioC, mutants. Based on this, Sullivan

et al. [14] proposed that BioZ catalyzes both the conden-

sation of a thioester with an odd number of carbon

atoms to produce pimeoyl-ACP and its subsequent

transacetylation to pimeloyl-CoA. This hypothesis is

consistent with recent enzymological data on the E. coli

BioH [36].

Four enzymes convert pimeloyl-CoA to biotin,namely BioF (7-keto-8-aminopelargonic acid synthase),

BioA (7,8-diaminopelergonic acid aminotransferase),

C

O

CH2(CH2)4COOH

C

O

CH2(CH2)4COOH

HO

S CoA

CH2(CH2)4COOH

O NH2

H3C

CH2(CH2)4COOH

NH2

H3C

NH2

CH2(CH2)4COOH

HN

H3C

NH

O

CH2(CH2)4COOH

HN NH

O

S

Bacillus spp.

precursor

BioX, BioI Pimelic acid

ATP, CoA BioW

L-alanine

pimeloyl-CoA E. coli

precursor

BioC, BioH BioF

7-keto-8-aminoperlargonic acid(KAPA)

SAM BioA

7,8-diaminopelargonicacid (DAPA)

ATP, CO 2 BioD

d-Dethiobiotin

NADPH, SAM, Flavodoxin BioB

d-Biotin

KAPA synthase

DAPA synthase

Dethiobiotin synthetase

Biotin synthetase

pimeloyl-CoA synthase

At, Bj, Ml, Rla, Smb

At, Bj, Ml, Re, Rl, Sm, NGRc

At, Bj, Ml

At, Bj, Ml

Fig. 1. The orthodox biotin biosynthetic pathway derived largely from studies utilizing Bacillus spp. and E. coli. Where gene homologs encoding the

main biosynthesis pathway enzymes exist in rhizobia, they are designated by the following abbreviations: At, A. tumefaciens; Bj, B. japonicum; Ml,M.

loti, NGR, Rhizobium sp. NGR234; Re, R. etli; Rl, R. leguminosarum bv. viciae; Sm, S. meliloti. aHomolog with low sequence identity to other BioFs.bHomolog present in genome but does not complement an E. coli bioF mutant. cData obtained from a partial genome sequence [42].


BioD (dethiobiotin synthetase) and BioB (biotin synthe-tase) (Fig. 1). The physical arrangement of gene clusters

encoding these orthodox biotin biosynthetic enzymes

are presented in Fig. 2. In M. loti R7A, a functional bio-

BFDA operon was confirmed by complementation of E.

coli mutants inactivated in one of these genes [14]. Ent-

cheva and co-workers [5] used genome sequence analysis

and complementation tests with E. coli biotin mutants

to identify putative biotin biosynthesis genes in S. melil-

oti. bioF and bioA homologs, potentially encoding the

first two enzymes for the pimeloyl-CoA to biotin path-

way, were found, but only the bioF homolog could com-

plement the corresponding E. coli mutant. Homologs

for the last enzymes of the pathway (bioD and bioB)

were not found. Genes possibly encoding BioH and

BioZ, involved in pimeloyl-CoA synthesis, were alsofound but could not complement their respective E. coli

mutants (a bioI homolog was encountered but comple-

mentation was not tested since no homolog occurs in

E. coli). The genome sequence of R. etli CE3 contains

a bioA homolog on plasmid f, but no bioB, bioD or bioF

homologs (unpublished results), while that of R. legu-

minosarum bv. viciae contains bioA and bioF homologs

but lacks homologs for bioB and bioD.

7. Biotin transport

Active biotin uptake occurs in E. coli but the trans-

port system involved is not known [37]. An S. meliloti

B. subtilis

M. loti

A. tumefaciens

B. sphaericus

E. coli

B. japonicum

bioD bioA bioY bioB bioX bioW bioF

orf1 bioA bioF bioB bioC bio D bioH

bioB bioF bioD bioA panD bioA bioY dct A

bioB bioF bioD bioA bioA bioA bioY bioN bioM

panD bioYbioB bioF bioD bioA orf bioZ bioA

bioW bioA bioF bioD bioB bioI orf2

orf bioM bioN bioYR. etli / S. meliloti

Fig. 2. Biotin biosynthesis gene clusters in selected prokaryotes. Data were obtained from the literature cited in the text or by homology searches of

the following genome databases: A. tumefaciens, http://www.ncbi.nlm.hih.gov/genomes/MICROBES/Complete.html; B. japonicum, http://

www.kazusa.or.jp/rhizobase; R. etli, G. Davila, V. Gonzalez, R. Gomez and P. Bustos, unpublished. Contiguous arrows represent gene clusters

and spaces denote genes or clusters in other parts of the genome. The drawings are not to scale.


mutant inactivated in bioS, the biotin-upregulated genementioned in Section 4, has a higher level of biotin up-

take than the wild-type [38]. bioS encodes a LysR type

protein and its role in biotin uptake would appear to

be regulatory [28]. Both S. meliloti and R. etli contain

operons (bioMNB) encoding products involved in biotin

uptake or retention which are identically organized and

share high sequence identity ([5], Guillen-Navarro et al.,

submitted). Very similar operon exists in R. leguminosa-

rum bv. viciae and A. tumefaciens but have not been

characterized experimentally. The gene originally desig-

nated bioB in S. meliloti [5] does not resemble a biotin

synthase (the classical bioB product) but instead has

similarity to bioY, first implicated in biotin biosynthesis

in Bacillus sphaericus because of its proximity to other

genes involved in biotin biosynthesis [39]. We refer here

to the S. meliloti and R. etli ‘‘bioB’’ homologs as bioY.Sequence analysis and experimental data [5,40] suggest

that bioM and bioN are ABC-type transporters for bio-

tin and encode the ATPase and permease components,

respectively. BioY has six probable transmembrane do-

mains like those of transport permeases but constitutes

its own family in the Pfam database [40]. In S. meliloti,

uptake experiments with a high concentration of exter-

nal biotin (40 nM) showed that a bioM mutant was defi-cient in biotin retention but not uptake [5]. We used low

external biotin concentrations (10–100 pM) to show that

a R. etli bioM mutant had significantly reduced uptake

of biotin but was not defective in retaining it (Guillen-

Navarro, submitted). Overexpression of bioY in wild-

type S. meliloti Rm1021 allows better than wild-type

growth in medias supplemented with dethiobiotin. It

was suggested that BioY might play a role in convertingdethiobiotin to biotin by a mechanism distinct from that

of a classical biotin synthase [5]. However, because com-

mercially available dethiobiotin contains biotin as a con-taminant [41], extra copies of bioY may promote growth

in dethiobiotin-supplemented cultures by allowing more

efficient uptake of the contaminating biotin.

BlastN analysis was used to determine the presence of

homologs of bioB, bioD, bioF and bioA (the orthodox

biotin biosynthesis genes) and bioY (the putative high

affinity transport component) in 159 sequenced genomes

(including 37 incomplete genomes) in the GenBank andKEGG databases. We found that (i) nearly 16% of the

genomes contained only bioY, (ii) 39% lacked bioY

and contained all of the orthodox biosynthetic genes,

(iii) nearly 18% contained bioY and all of the orthodox

biosynthetic genes and (iv) the remainer contained bioY

plus one or two of the classical genes. The genomes

encoding all of the genes included those of A. tumefac-

iens and M. loti, which could benefit from possessingboth the orthodox biosynthetic route and high affinity

uptake capability, since both species colonize plant tis-

sues but also survive as saprophytes in soil.

8. Perspectives

Rhizobia make enlightening subjects for the study ofbiotin metabolism and utilization owing to characteris-

tics which differ from the standard model organisms

including (i) their ability to enter into symbiosis, which

has been disected at the molecular level and for which

the importance of biotin is dependent on the symbiotic

combination; (ii) the presence of multiple biotin-depen-

dent carboxylases; (iii) absence of BirA regulatory func-

tions; (iv) preliminary data indicating a metabolicregulatory function for biotin and (v) the apparent pres-

ence of novel biosynthetic pathways. We need to persue

http://www.ncbi.nlm.hih.gov/genomes/MICROBES/Complete.html




the work on possible novel biotin biosynthesis pathways

with a rigorous biochemical and physiological charac-

terization, including the use purified precursors to dem-

onstrate actual substrate/product relationships. The

application of global methodologies such as proteomics

and transcriptomics in rhizobia will allow further identi-fication of genes and gene products regulated by biotin.

Our knowledge of biotin uptake and the regulation of its

utilization can also be greatly expanded with rhizobia as

experimental organisms.

Acknowledgments

We apologize to the authors of papers which were not

cited because of the publishers space limitations. K.

G-N. was supported by graduate student fellowships

138526 from CONACyT and 202327 and 202363 from

DGAPA-UNAM. We thank G. Davila, V. Gonzalez,

R. Gomez and P. Bustos for access to the R. etli genome

sequence prior to publication.

References

[1] Allen, E.K. and Allen, O.N. (1950) Biochemical and symbiotic

properties of the rhizobia. Bacteriol. Rev. 14, 273–330.

[2] Dunn, M.F. (1998) Tricarboxylic acid cycle and anaplerotic

enzymes in rhizobia. FEMS Microbiol. Rev. 22, 105–123.

[3] Lodwig, E. and Poole, P. (2003) Metabolism of Rhizobium

bacteroids. Crit. Rev. Plant Sci. 22, 37–78.

[4] Streit, W. and Entcheva, P. (2003) Biotin in microbes, the genes

involved in its biosynthesis, its biochemical role and perspectives

for biotechnological production. Appl. Microbiol. Biotechnol. 61,

21–31.

[5] Entcheva, P., Phillips, D. and Streit, W. (2002) Functional

analysis of Sinorhizobium meliloti genes involved in biotin

synthesis and transport. Appl. Environ. Microbiol. 68, 2843–

2848.

[6] West, P.M. and Wilson, P.W. (1940) Biotin as a growth stimulant

for the root nodule bacteria. Enzymologia 8, 152–162.

[7] Graham, P.H. (1963) Vitamin requirements of root nodule

bacteria. J. Gen. Microbiol. 30, 245–248.

[8] Bunn, C.R., McNeill, J.J. and Elkan, G.H. (1970) Effect of biotin

on fatty acids and phospholipids of biotin-sensitive strains of

Rhizobium japonicum. J. Bacteriol. 102, 24–29.

[9] Sullivan, J.T. and Ronson, C.W. (1998) Evolution of rhizobia by

the acquisition of a 500-kb symbiosis island that integrates into a

phe-tRNA gene. Proc. Natl. Acad. Sci. USA 95, 5145–5149.

[10] Sullivan, J.T., Eardly, B.D., van Berkum, P. and Ronson, C.W.

(1996) Four unnamed species of nonsymbiotic rhizobia isolated

from the rhizosphere of Lotus corniculatus. Appl. Environ.

Microbiol. 62, 2818–2825.

[11] Sierra, S., Rodelas, B., Martınez-Toledo, M.V., Pozo, C. and

Gonzalez-Lopez, J. (1999) Production of B-group vitamins by two

Rhizobium strains in chemically defined media. J. Appl. Micro-

biol. 86, 851–858.

[12] Encarnacion, S., Dunn, M., Willms, K. and Mora, J. (1995)

Fermentative and aerobic metabolism in Rhizobium etli. J.

Bacteriol. 177, 3058–3066.

[13] Dunn, M.F., Encarnacion, S., Araıza, G., Vargas, M.C., Davalos,

A., Peralta, H., Mora, Y. and Mora, J. (1996) Pyruvate

carboxylase from Rhizobium etli: Mutant characterization, nucle-

otide sequence, and physiological role. J. Bacteriol. 178, 5960–

5970.

[14] Sullivan, J., Brown, S., Yocum, R. and Ronson, C. (2001) The bio

operon on the acquired symbiosis island of Mesorhizobium sp.

strain R7A includes a novel gene involved in pimeloyl-CoA

synthesis. Microbiologica 147, 1315–1322.

[15] Streit, W.R., Joseph, C.M. and Phillips, D.A. (1996) Biotin and

other water-soluble vitamins are key growth factors for alfalfa

root colonization by Rhizobium meliloti 1021. Mol. Plant-Microbe

Interact. 9, 330–338.

[16] Watson, R.J., Heys, R., Martin, T. and Savard, M. (2001)

Sinorhizobium meliloti cells require biotin and either cobalt or

methionine for growth. Appl. Environ. Microbiol. 67, 3767–3770.

[17] Lowe, R.H. and Evans, H.J. (1962) Carbon dioxide requirement

for growth of legume nodule bacteria. Soil Sci. 94, 351–356.

[18] Dunn, M.F., Araıza, G., Encarnacion, S., Finan, T.M. and Mora,

J. (2002) Characteristics and metabolic roles of biotin-dependent

enzymes in rhizobia In: Nitrogen Fixation: Global Perspectives

(Finan, T., O�Brian, M.R., Layzell, D.B., Vessey, J.K. and

Newton, W., Eds.), pp. 158–162. CABI, Wallingford, Oxon, UK.

[19] Dunn, M.F., Araıza, G. and Finan, T.M. (2001) Cloning and

characterization of the pyruvate carboxylase from Sinorhizobium

meliloti Rm1021. Arch. Microbiol. 176, 355–363.

[20] Rovira, A.D. (1960) Plant root exudates. Bot. Rev. 35, 35–59.

[21] Dunn, M.F., Araıza, G. and Mora, J. (2004) Biochemical

characterization of a Rhizobium etli monovalent cation-stimulated

acyl-coenzyme A carboxylase with a high substrate specificity

constant for propionyl-coenzyme A. Microbiologica 150, 399–

406.

[22] Charles, T.C. and Aneja, P. (1999) Methylmalonyl-CoA mutase

encoding gene of Sinorhizobium meliloti. Gene 226, 121–127.

[23] Cronan Jr, J.E. (1989) The E. coli bio operon: transcriptional

repression by an essential protein modification enzyme. Cell 58,

427–429.

[24] Bower, S., Perkins, J., Yocum, R.R, Serror, P., Sorokin, A.,

Rahaim, P., Howitt, C.L., Prasad, N., Ehrlich, S.D. and Pero, J.

(1995) Cloning and characterization of the Bacillus subtilis birA

gene encoding a repressor of the biotin operon. J. Bacteriol. 178,

2572–2575.

[25] Rodriguez-Melendez, R. and Zempleni, J. (2003) Regulation of

gene expression by biotin. J. Nutr. Biochem. 14, 680–690.

[26] Encarnacion, S., Guzman, Y., Dunn, M.F., Hernandez, M.,

Vargas, M.C. and Mora, J. (2003) Proteome analysis of aerobic

and fermentative metabolism in Rhizobium etli CE3. Proteomics

3, 1077–1085.

[27] Heinz, E.B. and Streit, W.R. (2003) Biotin limitation in Sinorhi-

zobium meliloti strain 1021 alters transcription and translation.


[28] Hofmann, K., Heinz, E.B., Charles, T.C., Hoppert, M., Liebl, W.

and Streit, W.R. (2000) Sinorhizobium meliloti strain 1021 bioS

and bdhA gene transcriptions are both affected by biotin available

in defined medium. FEMS Microbiol. Lett. 182, 41–44.

[29] Streit, W.R. and Phillips, D.A. (1996) Recombinant Rhizobium

meliloti strains with extra biotin synthesis capability. Appl.


[30] Kaneko, T., Nakamura, Y., Sato, S., Asamizu, E., Kato, T.,

Sasamoto, S., Watanabe, A., Idesawa, K., Ishikawa, A., Kawa-

shima, K., Kimura, T., Kishida, Y., Kiyokawa, C., Kohara, M.,

Matsumoto, M., Matsuno, A., Mochizuki, Y., Nakayama, S.,

Nakazaki, N., Shimpo, S., Sugimoto, M., Takeuchi, C., Yamada,

M. and Tabata, S. (2000) Complete genome structure of the

nitrogen-fixing symbiotic bacterium Mesorhizobium loti. DNA

Res. 7, 331–338.

[31] Rovira, A.D. and Harris, J.R. (1961) Plant root excretions in

relation to the rhizosphere effect. V. The exudation of B-group

vitamins. Plant Soil 14, 199–214.


[32] Dunn, M.F., Araıza, G., Cevallos, M.A. and Mora, J. (1997)

Regulation of pyruvate carboxylase in Rhizobium etli. FEMS

Microbiol. Lett. 157, 301–306.

[33] Lemoine, Y., Wach, A. and Jeltsch, J.-M. (1996) To be free or

not: the fate of pimelate in Bacillus sphaericus and in Escherichia

coli. Mol. Microbiol. 19, 645–647.

[34] Ploux, O., Soularue, P., Marquet, A., Gloeckler, R. and Lemoine,

Y. (1992) Investigation of the first step of biotin biosynthesis in

Bacillus sphaericus: purification and characterization of the

pimeloyl-CoA synthase, and uptake of pimelate. Biochem. J.

287, 685–690.

[35] Ifuku, O., Miyaoka, H., Koga, N., Kishimoto, J., Haze, S.,

Wachi, Y. and Kajiwara, M. (1994) Origin of carbon atoms of

biotin: 13C-NMR studies on biotin biosynthesis in Escherichia

coli. Eur. J. Biochem. 220, 585–591.

[36] Sanishvili, R., Yakunin, A., Laskowski, R., Skarina, T., Evd-

okimova, E., Doherty-Kirby, A., Lajoie, G., Thornton, T.,

Arrowsmith, C., Savchenko, A., Joachimiak, A. and Edwards,

A. (2003) Integrating structure, bioinformatics and enzymology to

discover function. BioH, a new carboxyl esterase from Escherichia

coli. J. Biol. Chem. 278, 26039–26045.

[37] Piffeteau, A. and Gaudry, M. (1985) Biotin uptake: influx, efflux

and countertransport in Escherichia coli K12. Biochim. Biophys.

Acta 816, 77–82.

[38] Streit, W.R. and Phillips, D.A. (1997) A biotin-regulated locus,

bioS, in a possible survival operon of Rhizobium meliloti. Mol.

Plant-Microbe Interact. 10, 933–937.

[39] Gloeckler, R., Ohsawa, I., Speck, D., Ledoux, C., Bernard, S.,

Zinsius, M., Villeval, D., Kisou, T., Kamogawa, K. and Lemoine,

Y. (1990) Cloning and characterization of the Bacillus sphaericus

genes controlling the bioconversion of pimelate into dethiobiotin.

Gene 87, 63–70.

[40] Rodinov, D.A., Mironov, A. and Gelfand, M. (2002)

Conservation of the biotin regulon and the BirA regula-

tory signal in Eubacteria and Archaea. Genome Res. 12,

1507–1516.

[41] Bowman, W.C. and DeMoll, E. (1993) Biosynthesis of biotin

from dethiobiotin by the biotin auxotroph Lactobacillus planta-

rum. J. Bacteriol. 175, 7702–7704.

[42] Viprey, V., Rosenthal, A., Broughton, W.J. and Perret, X. (2000)

Genetic snapshots of the Rhizobium species NGR234 genome.

Genome Biol. 1, 0014.1–0014.17.



The Pseudomonas aeruginosa pirA gene encodes a second receptorfor ferrienterobactin and synthetic catecholate analogues

Bart Ghysels a, Urs Ochsner b,1, Ute Mollman c, Lothar Heinisch c, Michael Vasil b,Pierre Cornelis a,*, Sandra Matthijs a

a Department of Molecular and Cellular Interactions, Laboratory of Microbial Interactions, Flanders Interuniversity Institute of Biotechnology (VIB6),

Vrije Universiteit Brussel, Brussels, Belgiumb University of Colorado Health Science Center, Denver, USA

c Hans Knoll Institut, Jena, Germany

Received 6 January 2005; received in revised form 1 April 2005; accepted 4 April 2005


Edited by S. Silver

Abstract

Actively secreted iron chelating agents termed siderophores play an important role in the virulence and rhizosphere competence

of fluorescent pseudomonads, including Pseudomonas aeruginosa which secretes a high affinity siderophore, pyoverdine, and the low

affinity siderophore, pyochelin. Uptake of the iron–siderophore complexes is an active process that requires specific outer membrane

located receptors, which are dependent of the inner membrane-associated protein TonB and two other inner membrane proteins,

ExbB and ExbC. P. aeruginosa is also capable of using a remarkable variety of heterologous siderophores as sources of iron, appar-

ently by expressing their cognate receptors. Illustrative of this feature are the 32 (of which 28 putative) siderophore receptor genes

observed in the P. aeruginosa PAO1 genome. However, except for a few (pyoverdine, pyochelin, enterobactin), the vast majority of

P. aeruginosa siderophore receptor genes still remain to be characterized. Ten synthetic iron chelators of catecholate type stimulated

growth of a pyoverdine/pyochelin deficient P. aeruginosa PAO1 mutant under condition of severe iron limitation. Null mutants of

the 32 putative TonB-dependent siderophore receptor encoding genes engineered in the same genetic background were screened for

obvious deficiencies in uptake of the synthetic siderophores, but none showed decreased growth stimulation in the presence of the

different siderophores. However, a double knock-out mutant of ferrienterobactin receptor encoding gene pfeA (PA 2688) and pirA

(PA0931) failed to be stimulated by 4 of the tested synthetic catecholate siderophores whose chemical structures resemble enterob-

actin. Ferric-enterobactin also failed to stimulate growth of the double pfeA–pirA mutant although, like its synthetic analogues, it

stimulated growth of the corresponding single mutants. Hence, we confirmed that pirA represents a second P. aeruginosa ferric-

enterobactin receptor. The example of these two enterobactin receptors probably illustrates a more general phenomenon of sider-

ophore receptor redundancy in P. aeruginosa.


Keywords: Pseudomonas aeruginosa; Enterobactin; Catecholate siderophores; pfeA; pirA; TonB-dependent receptors


doi:10.1016/j.femsle.2005.04.010

* Corresponding author. Tel.: 02 6291906; fax: 02 6291902.

E-mail address: [email protected] (P. Cornelis).1 Present address: Replidyne Inc., 1450 Infinite Drive, Louisville, CO

80027, USA.

1. Introduction

Pseudomonas aeruginosa is a Gram-negative bacte-

rium endowed with an extremely versatile metabolism,

reflected in its ability to colonize a wide variety of



168 B. Ghysels et al. / FEMS Microbiology Letters 246 (2005) 167–174

habitats, from mammalian host to the rhizosphere of

plants [1]. As for most organisms, iron is indispensable

for survival of P. aeruginosa. Unfortunately, iron, de-

spite being one of the most abundant elements in the

earth crust, is rarely freely accessible and its acquisi-

tion demands significant adaptations from micro-organisms. A common microbial strategy for iron

acquisition is the production of low-molecular mass

iron-chelating compounds named siderophores [2].

For Gram-negative bacteria, uptake of the ferrisidero-

phore complexes into the cell necessitates specialized

receptors acting as gated porin channels that recognize

and actively internalize the ferrisiderophore complexes

[2]. As a rule, ferrisiderophore receptors recognizeexclusively one iron–siderophore complex, but excep-

tions to this rule have been reported [3–5]. Since the

outer membrane is devoid of energy sources, all these

receptors rely on a conserved protein, called TonB,

and two other proteins, ExbB and ExbC, also located

in the inner membrane, to transduce energy generated

in the cytoplasmic membrane to the receptor protein

[2]. Although three TonB homologues have been de-scribed in P. aeruginosa, only TonB1 seems to be in-

volved in the uptake of ferrisiderophores [6]. Just

like outer-membrane porins, these receptor proteins

are shaped of a large C-terminal domain of 22 antipar-

allel b-strands, which form a membrane spanning

b-barrel [7]. What distinguishes TonB dependent

receptors from porins is an additional domain known

as �cork� or �plug� that blocks the b-barrel domainand by using energy transduced by TonB, allows selec-

tive uptake of siderophore/iron complexes [8].

P. aeruginosa secretes a high affinity siderophore

pyoverdine (PVD), and another siderophore, pyochelin

(PCH), which displays lower iron affinity compared to

PVD [9,10]. Besides producing endogenous sidero-

phores, P. aeruginosa has the capacity to take up and

utilize numerous siderophores secreted by other micro-organisms including those of other bacteria (aerobactin,

enterobactin and its precursor 2,3-dihydrobenzoic acid

and breakdown product N-(2,3-dihydrobenzoyl)-L-ser-

ine), pyoverdines/pseudobactin from other pseudomo-

nads, cepabactin, fungal siderophores (deferrioxamines,

dererrichrysin, deferrirubin, coprogen), synthetic chela-

tors, (e.g. nitrilotriacetic acid) and naturally occuring

chelators such as citrate and myo-inositol hexakisphos-phate [10]. Not surprisingly, the P. aeruginosa PAO1

genome [11] counts no less than 32 genes with ferrisider-

ophore receptor gene signature [9]. Only three of them

were previously matched with a siderophore ligand: fpvA

(ferri-PVD uptake) [12], fptA (ferric PCH uptake) [13]

and pfeA (ferrienterobactin uptake) [14]. Recently, we

described a gene, fpvB, which encodes a second receptor

for PVD [15]. The active transport of siderophore–ironcomplexes across the outer membrane of Gram-negative

bacteria has caught the interest of scientists exploring

possible novel concepts of anti-microbial drug delivery.

One possible approach to overcome the problem of resis-

tance in P. aeruginosa [16] lies in the synthesis of antibi-

otics conjugated with compounds active as siderophores

[17]. Two different carrier concepts are currently under

evaluation: making use of either a derivative of a naturalsiderophore or artificial synthetic siderophore entities

[17]. Tests on several Gram-negative species have dem-

onstrated the applicability of both type of conjugates,

exhibiting minimal inhibitory concentrations (MICs)

that are significantly lower (up to 100 times) compared

to the associated free drugs. However, drug conjugates

with synthetic siderophore analogues are often easier to

produce [17] and may be designed for application againsta broader range of species. Hence, while exploring the

properties of synthetic siderophore analogues for active

drug delivery it is important to map the receptors that

mediate their uptake. An ideal siderophore drug carrier

is taken up by several receptors in order to minimize

the chances of resistance development.

Many pathogenic bacteria, including P. aeruginosa,

have outer membrane receptors for heterologous trans-port of ferrienterobactin (FeEnt), a siderophore pro-

duced by enteric bacteria [14,18]. It has been

suggested that P. aeruginosa can take up enterobactin

via two distinct uptake systems, one of ‘‘high affinity’’

induced by enterobactin, the second of ‘‘low affinity’’

not induced by enterobactin [19]. The pfeA gene,

encoding the high affinity enterobactin receptor, has

been cloned and sequenced [14]. Synthetic analoguesthat mimic enterobactin, but change certain aspects

of its chemistry were previously used to determine the

structural feature of the siderophore that are important

to its transport. These studies have shown that the iron

binding centre contains the primary determinants of

the uptake reaction and that replacement of the natural

macrocyclic ring had little effect on ferrienterobactin

transport [20].We evaluated the siderophore properties of a number

of synthetic catecholate siderophore analogues on P.

aeruginosa and tried to map their receptors and con-

firmed earlier suggestions for the presence of two ferri-

enterobactin uptake systems in P. aeruginosa PAO1

and identified the second, low affinity ferric-enterobactin

uptake mediating receptor as the product of pirA. Since

both ferrienterobactin receptors are also involved in theuptake of several synthetic enterobactin analogues, they

represent good candidate drug carriers.

2. Materials and methods

2.1. Bacterial strains, media and growth conditions

The different P. aeruginosa mutations in putative fer-

risiderophore receptor genes used in this study are listed

Table 1

List of 36 P. aeruginosa PAO1 TonB-dependent receptors genes

Gene no. Gene name Identified ligand Gene no. Gene name

PA 2398 fpvA Ferripyoverdine PA 4675

PA 4168 fpvB Ferripyoverdine PA 1302

PA 4221 fptA Ferripyochelin PA 4837

PA 2688 pfeA Ferrienterobactin PA 2911

PA 0931* pirA Ferrienterobactin PA 1922 cirA

PA 4710 phuR Heme PA 2289

PA 3408 hasR Heme PA 0192

PA 1910 ufrA PA 0434

PA 0674* pigC PA 0781

PA 0470* fiuA PA 1365

PA 4514* piuA PA 1613

PA 1322* pfuA PA 2057

PA 3901 fecA PA 2089

PA 2466 optS PA 3268

PA 0151 PA 2335 optO

PA 4897 optI PA 4156

PA 4675 iutA PA 2590

PA 1302 hxuC PA 2070

List of 36 P. aeruginosa PAO1 TonB-dependent receptors genes for

which the corresponding knock-out mutants were engineered in an

unmarked allelic pvdD pchEF mutant.* Fur-regulated genes picked up by the SELEX technique [21].

B. Ghysels et al. / FEMS Microbiology Letters 246 (2005) 167–174 169

in Table 1. P. aeruginosa wild-type and mutants were

grown under conditions of good aeration at 37 �C either

in Casamino acid medium (CAA, low iron medium) or

LB medium. The ferrisiderophore growth stimulation

assays were performed in CAA supplemented with

10 lM of the iron chelator ethylenediamine dihydroxy-

phenylacetic acid (EDDHA) and 200 lM dipyridil (in

agar medium) or CAA with 5 lM EDDHA and100 lM dipyridil (in liquid medium) for iron-limiting

conditions. For ferrienterobactin stimulation assays, a

wild-type E. coli strain (MC4100) producing enterobac-

tin was grown in CAA medium plus 0.2% glucose during

48 h. The supernatant was collected after centrifugation

and filter-sterilized. Another E. coli strain, H6876, an

entC derivative of MC4100 was grown under the same

conditions. This strain is unable to produce enterobac-tin. The supernatants of both E. coli strains (15% V/V)

were added to LB-agar plates containing EDDHA and

dipyridil as described in 2.1.

2.2. Siderophores utilization assay

The catecholate synthetic siderophore analogues are

shown in Fig. 1. Petri dishes of CAA solid agar mediumcontaining 10 lM EDDHA + 200 lM dipyridil were

used. Two hundred microlitres of a 105 CFU/ml cell sus-

pension of the mutant was spread on the medium sur-

face. A sterile paper disc impregnated with 5 ll of

2 mM siderophore solution was placed on top of the

agar plate. Siderophore usage was detected, after 1 day

of incubation at 37 �C, as a halo of growth around the

filter disc. For the utilization of enterobactin, LB-ED-

DHA-dipyridil agar plates were used which contained

15% (V/V) filter-sterilized supernatants of E. coli

MC4100 (enterobactin producer) and H6876 (entC mu-

tant of MC4100, unable to produce enterobactin). On

these plates, 0.1 ml of dilutions of P. aeruginosa cells

(from 108 to 103 CFU per ml) was inoculated and theplates incubated overnight at 37 �C.

2.3. Liquid growth stimulation assays

For more accurate analysis, growth was assessed in

microtiter plates using a Bio-Screen C incubator (Life

Technologies�). Briefly, the following protocol was

used: pre-cultures (2–3 ml) were grown overnight inCAA medium. The next day the pre-cultures were used

to inoculate in a 1:100 ratio 3 ml cultures in CAA med-

ium, which were grown till OD600 = 0.5. Serial dilutions

in CAA were performed to reach a final 1:5000 dilution.

The following parameters were programmed to be exe-

cuted by the apparatus: each well contains: 295 ll of

(CAA + 5 lM EDDHA + 100 lM 2-2 0dipyridil) with

5 ll of 2 mM of the to be tested siderophores and 5 llof DMSO for control wells (solvent used to dissolve

the siderophores); shaking for 30 s every 3 min, absor-

bance measured every 20 min at 600 nm and tempera-

ture at 37 �C.

3. Results

3.1. Mutants in putative ferrisiderophore receptor genes in

P. aeruginosa PAO1

A siderophore-free background was created in P.

aeruginosa PAO1 by making unmarked deletions in

pvdD (pyoverdine biosynthesis) and pchEF (pyochelin

biosynthesis) [15]. Candidate siderophore receptor

genes of P. aeruginosa PAO1 were originally pickedup by a cycle selection procedure to identify iron re-

pressed genes that are directly regulated by the Ferric

Uptake Regulator (Fur) [21,22]. Five of these genes

were found to be similar to known siderophore recep-

tor genes (Table 1). More candidate siderophore recep-

tor genes were counted in the completed P. aeruginosa

PAO1 genome sequence (http://www.pseudomo-

nas.com) [9,11]. No less than 36 ORFs carry the signa-ture of TonB dependent receptor encoding genes. Four

of them are the previously identified ferrisiderophore

receptor genes for respectively ferri-pyoverdine (fpvA,

PA2398; fpvB, PA4168), ferri-pyochelin (fptA,

PA4221) and ferrienterobactin (pfeA, PA2688). Also

included are the TonB dependent receptors involved

in haem-uptake encoded by phuR (PA4710) and hasR

(PA3408) [23]. In the earlier created siderophore-freemutant (pvdD pchEF) of P. aeruginosa PAO1, �candi-date� siderophore receptor genes, 36 in total, were

http://www.pseudomonas.com


Fig. 1. Structures of enterobactin and the Tris-catecholate synthetic siderophores used in this study.


knocked-out by allelic exchange with interrupted cop-

ies of their gemonic alleles [15]. All mutants were ana-

lyzed by PCR in order to confirm the presence of theunmarked deletion [15].

Since P. aeruginosa can take up enterobactin via two

distinct uptake systems [19], an additional mutant was

engineered with simultaneously knocked-out pfeA

(PA2688), the ferrienterobactin receptor, and pirA


(PA0931), the candidate ferrisiderophore receptor gene

within the PAO1 genome with the highest similarity to

pfeA (72% similarity between their translation

products).

3.2. Utilization of synthetic catecholates by the different

TonB-dependent mutants

The P. aeruginosa PAO1 pvdD pchEF mutant car-

ries deletions in genes for PVD and PCH synthesis

and is therefore unable to grow in an iron restricted

situation created by the presence of both a ferric iron

chelator (10 lM EDDHA) and ferrous iron chelator

(200 lM 2-2 0-dipyridil). We aimed to select syntheticsiderophore analogues that are capable of stimulating

growth of this siderophore deficient mutant in presence

of EDDHA and dipyridil, which, in other words func-

tion as xeno-siderophores that can be assimilated by

P. aeruginosa. Ten ‘‘catecholate’’ compounds were

used [24] (see Table 2). The ‘‘catecholates’’ represent

either the free forms (compounds 3, 4 and 10) and

the protected forms (compounds 1, 2 and 5–9). Therewere 2 types of protected forms, form ‘‘a’’ as aliphatic

acyloxy group (compound 5), form ‘‘b’’ as heterocyclic

benzoxazine residue (compounds 6 and 8) and mixed

forms of both (compounds 1, 2 and 7). The basic

structures for the catecholates were either linear (com-

pounds 1–7) or tripodal (compounds 8–10). We as-

sume, that the protected forms can split off to the

free catecholates under physiological conditions sinceobviously only these structures can form iron com-

plexes. Additionally it should be mentioned that the

antibiotic conjugates of the protected catecholates are

active as antibacterials via uptake by ferrisiderophore

transport pathways [24–27].

Table 2

Summary of the results of growth stimulation tests

Catecholate

compound

Siderophore pvdD pchEF pvdD pchEF

pfeA pirA pfeA pirA

1 HKI 9824013 ++ ++ ++ ++

2 HKI 9824014 ++ ++ ++ ++

3 HKI 9824030 ++ ++ ++ ++

4 HKI 9824043 ++ ++ ++ ++

5 HKI 9824080 ++ ++ ++ ++

6 HKI 9924127 ++ ++ ++ ++

7 HKI 9824032 ++ ++ ++ �8 HKI 10024023 ++ ++ ++ �9 HKI 10024024 ++ ++ + �10 HKI 10024025 ++ ++ + �Summary of the results of growth stimulation tests peformed with the

synthetic siderophore analogues on the pvdD pchEF siderophore pro-

duction deficient background strain, and the mutants in pfeA, pirA and

the double pfeA pirA knock-out, all created in the pvdD pchEF back-

ground. Strong growth stimulation (++), weak/delayed growth stim-

ulation (+), no growth stimulation (�). The catecholate synthetic

siderophore structures are presented in Fig. 1.

All 10 catecholate compounds stimulated the

growth of the siderophore-deficient P. aeruginosa

PAO1 pvdD pchEF mutant under these conditions of

extreme iron limitation. Although each of the putative

TonB-dependent receptor genes of P. aeruginosa PAO1

had been inactivated, none of the 36 single mutantsfailed to be stimulated by any of the selected synthetic

siderophores, suggestive of a redundancy in sidero-

phore uptake systems in P. aeruginosa. Interestingly,

the synthetic enterobactin analogues 7–10 stimulated

single knock-out mutants of pfeA (PA2688), the high

affinity enterobactin receptor gene, and PA0931 (pirA)

its closest homologue within P. aeruginosa PAO1, but

failed to stimulate a mutant with both genes inacti-vated (Table 2).

3.3. Growth stimulation by enterobactin

Since we did not have purified enterobactin, we

looked at the growth stimulation conferred by the addi-

tion of cell-free supernatant from a culture of wild-type

E. coli MC4100 (enterobactin producer) and an entC

derivative from the same strain (unable to produce ente-

robactin) grown under iron-limiting conditions. As

shown in Fig. 2, the supernatant from MC4100 stimu-

lated the growth of the P. aeruginosa pvdD pchEF mu-

tant (Fig. 2(a)), pvdD pchEF pfeA (Fig. 2(b)) and pvdD

pchEF pirA (Fig. 2(c)), but not of the mutant pvdD

pchEF pfeA pirA (Fig. 2(d)). As could be expected, the

supernatant from the entC mutant could not stimulatethe growth of any of these P. aeruginosa strains (results

not shown). This observation confirms that PA0931

(pirA) serves as second ferrienterobactin receptor next

to pfeA.

3.4. Growth kinetics of pfeA and pirA in response to

stimulation by catecholates

The previous growth stimulation tests were per-

formed with siderophore impregnated filter-discs on

CAA-agar medium containing EDDHA and dipyridil.

In order to determine a hierarchical order between the

PfeA and PirA receptors in affinity for the different

enterobactin-like ligands, we kinetically measured

growth responses of the pfeA and pirA mutants to-

wards the different synthetic enterobactin analoguesin EDDHA- and dipyridil-containing liquid CAA cul-

tures. When compared to the pvdD pchEF strain and

the single pfeA mutant in the same genetic back-

ground, a delayed growth response of the pirA mutant

was observed towards compounds 9 (data not shown)

and 10 (Fig. 3) but not to 7 (Fig. 3) or 8 (result not

shown). In contrast to ferrienterobactin which is taken

up preferentially by PfeA [14,19] the iron complexeswith the synthetic siderophore analogues 9 and 10

are more efficiently taken up by PirA.

Gro

wth

OD

600n

m

(a) (b)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0 8 16 24 36 40Hours

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0 8 16 24 36 40Hours

rom

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0 8 16 24 36 400 8 16 24 36 40Hours

0.0

0.1

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0.4

0.5

0.6

0.0

0.1

0.2

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0 8 16 24 36 40Hours

Fig. 3. Growth stimulation of the pvdD pchEF (d), pvdD pchEF pfeA (s), pvdD pchEF pirA (m), and the pvdD pchEF pfeA pirA (n) mutants by

compound 7 (left) and compound 10 (right) in the presence of EDDHA and dipyridil. The values on the Y axis correspond to the OD at 600 nm. Only

one representative growth curve out of three separate experiments is shown.

Fig. 2. Growth of P. aeruginosa pvdD pchEF (a), pvdD pchEF pfeA (b), pvdD pchEF pirA (c) and pvdD pchEF pfeA pirA (d) in LB-agar plates

containing EDDHA, dipyridil and 15% (V/V) of filter-sterilized culture supernatant of wild-type E. coli MC4100 (producer of enterobactin). From

left to right, starting from the top, 107, 106, 105, 104, 103 and 102 P. aeruginosa cells. No growth was observed when the plates contained 15% (V/V)

supernatant from an E. coli entC mutant which does not produce enterobactin (results not shown).


4. Discussion

P. aeruginosa has the ability to use siderophores se-

creted by other species in order to fulfil its needs for iron[9,10]. This capacity of xenosiderophore usage illustrates

the importance of iron acquisition in microbial ecology.

Although, more than thousand different siderophore

compounds have been identified to date, they are usually

constructed with the same basic elements consisting of

catecholates, hydroxamates or carboxylates, preferen-tially in a tri-bidentate iron complexing conformation.

This inspired researchers to create novel, synthetic


siderophore compounds based on these naturally con-

served themes. Shared chemo-structural motifs between

synthetic and natural siderophores, allow their ferrisi-

derophore complexes to be taken up by the same cog-

nate receptors. Catecholate derivatives were generated

with high siderophore activities in strains of P. aerugin-osa and E. coli [25]. b-Lactam conjugates of these sidero-

phores showed enhanced antibacterial activities which

could be attributed to the active iron uptake routes used

by the conjugates to penetrate the bacterial cells [24–27].

The 10 synthetic catecholate siderophores used in this

study stimulated the growth of a siderophore-negative

P. aeruginosa under conditions of strong iron limitation,

indicating that these siderophores had sufficient ironbinding affinity to displace iron from EDDHA and

dipyridil and could be assimilated by the cell. The fact

that the growth of all siderophore-negative mutants with

a single receptor gene inactivation could be stimulated

by the 10 compounds suggests the presence of at least

two receptors for a given ferrisiderophore. Such receptor

redundancy has interesting implications for the use of

synthetic xenosiderophore analogues as drug carriers.Indeed, when a siderophore-drug conjugate can pene-

trate the cell via several independent receptors, the risk

of resistance development is significantly reduced.

Receptor redundancy, on the other hand, complicates

the mapping of receptor genes by a knock-out approach

since the dysfunctional receptor phenotype can be

masked by another receptor recognizing the same li-

gand. We recently demonstrated the applicability ofthe idea that receptor pairs with high sequence similarity

mediate the uptake of the same ligand [15], providing a

rational base for engineering multiple receptors knock-

out mutants. The pfeA gene, encoding the high affinity

enterobactin receptor, has been cloned and sequenced

[14]. Nonetheless, PfeA-deficient mutants display

growth, albeit reduced, in an enterobactin supple-

mented, iron-restricted minimal medium [19,28]. Thebest candidate for a second ferrienterobactin receptor

is the product of PA0931, dubbed pirA, which displays

substantial similarity with pfeA. With the receptor mu-

tants engineered in a pyoverdine and pyochelin-free

background, we unambiguously confirmed that P. aeru-

ginosa indeed counts two ferrienterobactin transporting

receptors, PfeA and PirA. Therefore the situation in P.

aeruginosa is similar to the situation in Salmonella enter-

ica where two receptors, FepA and IroN mediate the

transport of ferrienterobactin [29]. Interestingly, we

could not detect any difference in growth stimulation

by ferrienterobactin of the pfeA or pirA mutant (Fig.

2), which seems to be in contradiction with the results

obtained before [19,28]. This could be due to the differ-

ence of genetic background since we used a mutant of P.

aeruginosa which is unable to produce either pyoverdineof pyochelin. Growth kinetics of the mutants suggested

that two of the synthetic enterobactin analogues tested

are preferentially transported by the PirA receptor in

contrast to enterobactin for which PfeA acts as the high

affinity receptor. It is therefore likely that PirA trans-

ports another yet unknown natural siderophore different

from enterobactin as its primary substrate. Another

enterobactin-like siderophore, bacillibactin, is producedby the Gram-positive Bacillus subtilis [30] and it would

be interesting to look at the transport of this ferrisider-

ophore using the same set of mutants described in this

study. Another interesting question for the future is to

understand why only compounds 7–10 are taken by

fepA and pirA like enterobactin. It has to be mentioned

that compounds 8–10 are tripodal while compound 7 is

the only linear catecholate analogue which is taken upby these two receptors. Also, it would be interesting to

discover which TonB-dependent receptors mediate the

transport of compounds 1–6 since their growth stimula-

tion properties are not affected by the fepA pirA

mutations.

Acknowledgements

Support for these studies was provided in part by a

grant from the National Institutes of Allergy and Infec-

tious Diseases (AI15940) to Michael Vasil and of the

VUB OZR to Bart Ghysels. We thank Dr. Klaus Han-

tke for providing the E. coli MC4100 and H6876 strains.

References

[1] Goldberg, J. (1999) Pseudomonas: global bacteria. Trends Micro-

biol. 8, 55–57.

[2] Andrews, S.C., Robinson, A.K. and Rodrıguez-Quinones, F.

(2001) Bacterial iron homeostasis. FEMS Microbiol. Rev. 27,

215–237.

[3] Meyer, J.M., Geoffroy, V.A., Baysse, C., Cornelis, P., Barelmann,

I., Taraz, K. and Budzikiewicz, H. (2002) Siderophore-mediated

iron uptake in fluorescent Pseudomonas: characterization of the

pyoverdine-receptor binding site of three cross-reacting pyover-

dines. Arch. Biochem. Biophys. 397, 179–183.

[4] Meyer, J.M., Stintzi, A. and Poole, K. (1999) The ferripyoverdine

receptor FpvA of Pseudomonas aeruginosa PAO1 recognizes the

ferripyoverdines of P. aeruginosa PAO1 and P. fluorescens ATCC

13525. FEMS Microbiol. Lett. 170, 145–150.

[5] De Chial, M., Ghysels, B., Beatson, S.A., Geoffroy, V., Meyer,

J.M., Pattery, T., Baysse, C., Chablain, P., Parsons, Y.N.,

Winstanley, C., Cordwell, S.J. and Cornelis, P. (2003) Identifica-

tion of type II and type III pyoverdine receptors from Pseudo-

monas aeruginosa. Microbiology 149, 821–831.

[6] Zhao, Q. and Poole, K. (2002) Mutational analysis of the TonB1

energy coupler of Pseudomonas aeruginosa. J. Bacteriol. 184,

1503–1513.

[7] Koebnik, R., Locher, K.P. and Van Gelder, P. (2000) Structure

and function of bacterial outer membrane proteins: barrels in a

nutshell. Mol. Microbiol. 37, 239–253.

[8] Ferguson, A.D. and Deisenhofer, J. (2002) TonB-dependent

receptors-structural perspectives. Biochim. Biophys. Acta 1565,

318–332.


[9] Cornelis, P. and Mathhijs, S. (2002) Diversity of siderophore-

mediated iron uptake systems in fluorescent pseudomonads: Not

only pyoverdines. Environ. Microbiol. 4, 787–798.

[10] Poole, K. and McKay, G.A. (2003) Iron acquisition and its

control in Pseudomonas aeruginosa: many roads lead to Rome.

Front. Biosci. 8, D661–D686.

[11] Stover, C.K. et al. (2000) Complete genome sequence of Pseudo-

monas aeruginosa PA01, an opportunistic pathogen. Nature 406,

959–964.

[12] Poole, K., Neshat, S., Krebes, K. and Heinrichs, D.E. (1993)

Cloning and nucleotide sequence analysis of the ferripyoverdine

receptor gene fpvA of Pseudomonas aeruginosa. J. Bacteriol. 175,

4597–4604.

[13] Ankenbauer, R.G. and Quan, H.N. (1994) FptA, the Fe(III)-

pyochelin receptor of Pseudomonas aeruginosa: a phenolate

siderophore receptor homologous to hydroxamate siderophore

receptors. J. Bacteriol. 176, 307–319.

[14] Dean, C.R. and Poole, K. (1993) Cloning and characterization of

the ferrienterobactin receptor gene (pfeA) of Pseudomonas aeru-

ginosa. J. Bacteriol. 175, 317–324.

[15] Ghysels, B., Dieu, B.T., Beatson, S.A., Pirnay, J.P., Ochsner,

U.A., Vasil, M.L. and Cornelis, P. (2004) FpvB, an alternative

type I ferripyoverdine receptor of Pseudomonas aeruginosa.

Microbiology 150, 1671–1680.

[16] Poole, K. and Srikumar, R. (2001) Multidrug efflux in Pseudo-

monas aeruginosa: components, mechanisms and clinical signifi-

cance. Curr. Top. Med. Chem. 1, 59–71.

[17] Budzikiewicz, H. (2001) Siderophore-antibiotic conjugates used as

trojan horses against Pseudomonas aeruginosa. Curr. Top. Med.

Chem. 1, 73–82.

[18] Raymond, K.N., Dertz, E.A. and Kim, S.S. (2003) Enterobactin,

an archetype for microbial iron transport. Proc. Natl. Acad. Sci.

USA 100, 3584–3588.

[19] Poole, K., Young, L. and Neshat, S. (1990) Enterobactin-

mediated iron transport in Pseudomonas aeruginosa. J. Bacteriol.

172, 6991–6996.

[20] Ecker, D.J., Matzanke, B.F. and Raymond, K.N. (1986) Recog-

nition and transport of ferrienterobactin in Escherichia coli. J.

Bacteriol. 167, 666–673.

[21] Ochsner, U.A. and Vasil, M.L. (1996) Gene repression by the ferric

uptake regulator in Pseudomonas aeruginosa: cycle selection of

iron-regulated genes. Proc. Natl. Acad. Sci. USA 93, 4409–4414.

[22] Vasil, M.L. and Ochsner, U.A. (1999) The response of Pseudo-

monas aeruginosa to iron: genetics, biochemistry and virulence.

Mol. Microbiol. 34, 399–413.

[23] Ochsner, U.A., Johnson, Z. and Vasil, M.L. (2000) Genetics and

regulation of two distinct haem-uptake systems, phu and has, in

Pseudomonas aeruginosa. Microbiology 146, 185–198.

[24] Heinisch, L., Gebhardt, P., Heidersbach, R., Reissbrodt, R. and

Mollmann, U. (2002) New synthetic catecholate-type sidero-

phores with triamine backbone. BioMetals 15, 133–144.

[25] Heinisch, L., Wittmann, S., Stoiber, T., Scherlitz-Hofmann, I.,

Ankel-Fuchs, D. and Mollmann, U. (2003) Synthesis and

biological activity of tris- and tetrakiscatecholate siderophores

based on poly-aza alkanoic acids or alkylbenzoic acids and their

conjugates with b-lactam antibiotics. Arzneimittelforschung 53,

188–195.

[26] Heinisch, L., Wittmann, S., Stoiber, T., Berg, A., Ankel-Fuchs, D.

and Mollmann, U. (2002) Highly antibacterial active aminoacyl-

penicillin conjugates with bis-catecholate siderophores based on

secondary diamino acids and related compounds. J. Med. Chem.

45, 3032–3040.

[27] Wittmann, S., Scherlitz-Hofmann, I., Mollmann, U., Ankel-

Fuchs, D. and Heinisch, L. (2000) 8-acyloxy-1,3-benzoxazine-2,4-

diones as siderophore components for antibiotics. Arzneimittelf-

orschung/Drug Research 50, 752–757.

[28] Dean, C.R., Neshat, S. and Poole, K. (1996) PfeR, an enterob-

actin-responsive activator of ferrienterobactin receptor gene

expression in Pseudomonas aeruginosa. J. Bacteriol. 178, 5361–

5369.

[29] Rabsch, W., Methner, U., Voigt, W., Tschape, H., Reissbrodt, R.

and Williams, P.H. (2003) Role of receptor proteins for enterob-

actin and 2,3-dihydroxybenzoylserine in virulence of Salmonella

enterica. Infect. Immun. 71, 6953–6961.

[30] May, J.J., Wendrich, T.M. and Marahiel, M.A. (2001) The dhb

operon of Bacillus subtilis encodes the biosynthetic template for

the catecholic siderophore 2,3-dihydroxybenzoate-glycine-threo-

nine trimeric ester bacillibactin. J. Biol. Chem. 276, 7209–7217.



Novel target genes of PsrA transcriptional regulatorof Pseudomonas aeruginosa

Milan Kojic a,b,*, Branko Jovcic a, Alessandro Vindigni b,Federico Odreman b, Vittorio Venturi b

a Laboratory for Molecular Genetics of Industrial Microorganisms, Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a,

11010 Belgrade, Serbia and Montenegrob International Centre for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, 34012 Trieste, Italy

Received 23 February 2005; received in revised form 31 March 2005; accepted 4 April 2005


Edited by S. Silver

Abstract

The PsrA transcriptional regulator is involved in stationary phase induced transcriptional regulation of rpoS and in negative

auto-regulation in Pseudomonas aeruginosa. This study was designed to determine whether other loci were regulated by PsrA in

P. aeruginosa. Computer search was performed of the PsrA binding motif (G/CAAAC N2–4 GTTTG/C) against the P. aeruginosa

genome sequence. Four of 14 analysed promoters responded to and bound PsrA; (i) divergent promoters controlling PA2952/

PA2951 and PA2953, (ii) promoter of PA0506 and (iii) upstream region of PA3571. Promoters PA0506 and PA2952–PA2951 were

regulated negatively whereas promoters of PA2953 and PA3571 were regulated positively by PsrA. Two dimensional sodium dode-

cyl sulphate polyacrylamide gel electrophoresis (2D SDS-PAGE) analysis on total proteins from P. aeruginosa PAO1 and psrA

knock-out derivative was also performed resulting in the identification of 11 protein spots which were differentially regulated. These

studies have indicated PsrA as a global regulator.


Keywords: PsrA regulon; Stationary phase

1. Introduction

In their natural environment, bacteria are often chal-

lenged by constantly changing nutrient availability and

by exposure to various forms of physical stress, including

osmotic, oxidative and temperature shock. Exposure to

starvation and stresses leads to reduction or cessation

of growth, known as stationary phase, resulting in a ma-jor switch of gene expression that allows the cell to cope

with the new conditions [1]. A very simple and effective


doi:10.1016/j.femsle.2005.04.003

* Corresponding author. Tel.: +381 11 3975 960; fax: +381 11 3975

808.

E-mail address: [email protected] (M. Kojic).

mechanism employed by bacteria to bring about such a

major switch in gene expression is the use of alternative

sigma factors that alter RNA polymerase core specificity

[2]. The central regulator during stationary phase in

Pseudomonas spp., as in other Gram-negative bacteria,

is the stationary phase RpoS alternative sigma factor

[1,3,4]. In Escherichia coli, RpoS regulates more than

100 genes involved in cell survival, cross-protectionagainst various stresses and in virulence [2]. Similarly

in Pseudomonas aeruginosa RpoS regulates a large set

of genes as recently demonstrated using a microarray

transcriptome analysis [5]. The levels of RpoS within

a bacterial cell are carefully controlled and increase

considerably at the onset of stationary phase. The



176 M. Kojic et al. / FEMS Microbiology Letters 246 (2005) 175–181

regulatory mechanisms governing this control have been

extensively studied in E. coli revealing that regulation

takes place at the level of transcription, translation and

post-translational level all responding to various envi-

ronmental stimuli [1]. Regulation has also been studied

to a lesser extent in the fluorescent pseudomonads high-lighting that unlike in E. coli, transcriptional regulation

plays a major role [6]. The global two-component

GacA/GacS system and the N-acyl homoserine lactone

dependent quorum sensing systems are involved in the

regulation of rpoS; the precise mechanisms of these reg-

ulatory controls are unknown and their effect is rather

marginal only mildly affecting rpoS transcription. We

have shown that a TetR family regulator, designatedPsrA, plays a major role in positively regulating rpoS

transcription at the entry of P. aeruginosa into stationary

phase [7,8]. psrA knock-out mutants displayed 90%

reduction in rpoS promoter activity and 50% in protein

levels. DNA-binding studies showed that PsrA binds

specifically to the rpoS promoter at a sequence �35 to

�59 which contains a palindromic motif C/GAAAC

N2–4 GTTTG/C. In addition, PsrA negatively autoregu-lates its own expression through binding to a similar

sequence in its own promoter [8].

In this study, we identified four new genes, involved

in response to stationary phase, regulated by PsrA tran-

scriptional regulator.


2.1. Strains, plasmids, media and chemicals

The strains used in this study included E. coli DH5a[9], E. coli pRK2013 [10] and P. aeruginosa PAO1

(Holloway collection). P. aeruginosa PAO1 and its

psrA and rpoS knock-out mutants have been described

previously [7,11]. E. coli and P. aeruginosa strains weregrown in LB medium [12] at 37 �C. The following anti-

biotic concentrations were used: ampicillin, 100 lg/ml

(E. coli); kanamycin, 100 lg/ml (E. coli) and 300

lg/ml (PAO1); tetracycline, 15 lg/ml (E. coli) and

500 lg/ml (PAO1); gentamicin, 100 lg/ml (PAO1).

The plasmids used in this study are listed in Table A

(Supplementary data). The plasmid transcriptional fu-

sions were constructed as follows. Primers (Table B,Supplementary data) were designed in a way to ampl-

ify promoter regions starting from ATG and ending up

to 700 bp upstream. Amplified DNA fragments from

total genomic PAO1 DNA were treated with BamHI

and KpnI restriction enzymes and cloned in pBlue-

scriptKS (or SK) digested with the same restriction en-

zymes or directly cloned into pBluescriptKS digested

with SmaI, resulting in pBPA constructs (Table A,Supplementary data). pBPA constructs were sequenced

and fragments were then transferred into promoter

probe vector pMP220 using different restriction en-

zymes (BamHI/BglII and KpnI, XbaI and KpnI, EcoRI

and KpnI) to yield pMPA constructs (Table A, Supple-

mentary data).

2.2. Computer analysis

The genome of P. aeruginosa PAO1 ([13] http://

www.pseudomonas.com) was searched with the FIND-

PATTERNS (GCG) program using the PsrA binding

motif (G/CAAAC N2–4 GTTTG/C) derived from align-

ment of PsrA binding sequence in two promoters known

to be regulated by PsrA. However, to investigate the

importance of the spacing between the two motifs andto identify a maximum number of candidate genes, we

purposely expanded the spacing range to 2–4 nt of the

palindromic sequence.

2.3. Preparation of total cell proteins and 2-D gel

electrophoresis

Total cell proteins were prepared from overnightcultures of P. aeruginosa PAO1 and PAO1 psrA::Tn5.

Briefly, 6 mg of wet weight pellet was resuspended in

500 ll of solution [7 M urea, 2 M thiourea, 40 mM

dithiothreitol (DTT), 2% w/v 3-[(3-Cholamidopropyl)-

dimethylammonio]-1-propanesulfonate (CHAPS)] and

sonicated 4 · 15 s on ice. For the 2-D analysis, 20 ll(240 lg of total proteins) of sonicated sample was

mixed with 230 ll of the same solution to which1.5 ll of IPG buffer was added before loading. Two-

dimensional gel electrophoresis was performed using

immobilized pH gradient (pH 3–10 NL, IPG buffers)

13 cm long strips (Amersham Pharmacia Biotech).

Strips were rehydrated with entire protein sample for

2 h at room temperature under dry strip-cover fluid

(Amersham Pharmacia Biotech). Isoelectric focusing

was conducted using IPGphor Isoelectric Focusing Sys-tem (Amersham Pharmacia Biotech) at 20 �C. Proteinswere focused for 2 h at 1 kV, 5 h at 5 kV, 3 h at 1 kV,

for a total of 30 kV. IPG strips were equilibrated in

50 mM Tris–HCl pH 8.8, 6 M urea, 30% v/v glycerol,

2% w/v SDS and 15 mM DTT for 20 min at room tem-

perature. Strips were embedded on top of 15 · 15 cm,

12.5% SDS–PAGE gel for the second dimension.

Broad Range Prestained Protein Marker (6–175 kDa)purchased from BioLabs was loaded on the same gel

at one end of the strip. Protein spots were visualized

by Coomassie staining.

2.4. Mass spectrometric sequencing and protein

identification

The selected protein spots were cut out from the Coo-massie Brilliant blue-stained gels and placed in a silicon-

ized microcentrifuge tubes that had been rinsed with



M. Kojic et al. / FEMS Microbiology Letters 246 (2005) 175–181 177

ethanol, water and ethanol. An internal sequence analy-

sis of the protein spots was performed by using an elec-

tronspray ionization mass spectrometer (LCQ DECA

XP, ThermoFinnigam). The bands were digested with

trypsin, and the resulting peptides were extracted with

water and 60% acetonitrile–1% trifluoroacetic acid.The fragments were then analyzed by mass spectros-

copy, and the proteins were identified by analysis of

the peptides and by using the annotated P. aeruginosa

genome (www.pseudomonas.com).

2.5. Electrophoretic mobility shift

DNA mobility shift assays with purified His6-PsrAwere performed with modified previously described pro-

cedure [8]. Fragments carrying the promoters of genes

coding for acyl CoA dehydrogenase, electron transfer

protein, MmsR and MFS transporter were purified from

the plasmid constructs with BamHI–KpnI restriction en-

zymes. Purified DNA (0.1 pmol) was labelled at its Bam-

HI site with the Klenow fragment of DNA polymerase

and [a-32P]dCTP. Radiolebeled fragments (1000 cpm)and various quantities of purified His6-PsrA (PsrA pro-

tein with six histidine residues at N terminus) (from 0 to

150 ng) were incubated for 30 min at room temperature

in 10 ll reaction mixtures containing 1 · binding buffer

(20 mM HEPES-KOH pH 7.9, 20% v/v glycerol,

0.2 mM ethylendiaminetetraacetic acid disodium salt

(EDTA); 0.1 M KCl, 0.5 mM phenylmethanesulphonyl

fluoride (PMSF), 1 mM DTT), 10 lg of bovine serumalbumin (carrier protein), 400 ng of salmon sperm

(non-specific competitor) DNA and 1.5 mM MgCl2.

Supershifting was performed by incubating the reaction

mixtures with anti-PsrA antibodies for an additional

15 min at room temperature. Samples were than loaded

onto a non-denaturing 4.5% polyacrylamide 0.5 · TBE

(44.5 mM Tris, 44.5 mM boric acid, 0.5 mM EDTA)

�3% v/v glycerol gel, which was prerun for 1 h at110 V at room temperature, the samples were also run

at 110 V.

2.6. Recombinant DNA techniques

Digestion with restriction enzymes, agarose gel elec-

trophoresis, purification of DNA fragments, ligation

with T4 DNA ligase, end filling with the Klenow frag-ment of DNA polymerase, transformation of E. coli

and SDS–PAGE analysis were performed as previously

described [14]. Analytical amounts of plasmids were iso-

lated by procedure described by Birnboim [15], whereas

preparative amounts were purified with Qiagen columns

(Qiagen, Hilden, D). Total DNA from Pseudomonas was

isolated by Sarkosyl-pronase lysis [16]. Triparenatal

matings from E. coli to Pseudomonas were performedwith an E. coli (pRK2013) helper strain as previously

described [10].

2.7. Reporter gene fusion assays

Transcriptional fusions of all promoters possibly reg-

ulated by PsrA were made using pMP220 which harbors

a promoterless b-galactosidase lacZ gene. b-galactosi-dase activity was determined by method essentially de-scribed by Miller [12] with the modifications of Stachel

[17]. Miller units were defined as OD420 · 1000/

OD600 · T(min) · V(ml). All measurements were done

in triplicate and the mean value is given.

3. Results

3.1. Genomic analysis of the P. aeruginosa PAO1

chromosome for sequences representing potential PsrA

binding promoters

Having previously established that the TetR family

regulator PsrA was an important positive transcrip-

tional regulator of rpoS and a negative auto-regulator

via specific DNA-binding to a region of rpoS and psrA

promoters [6–8], it was of interest to determine whether

PsrA was transcriptionally regulating other loci in the P.

aeruginosa genome. The P. aeruginosa PAO1 genome

was therefore subjected to a degenerate pattern search

using the PsrA binding consensus sequences. The subse-

quence used to search the P. aeruginosa genome was

SAAAC N2–4 GTTTS where S was C or G and 2–4

was the spacing between the two palindromic motifs.This search resulted in the identification of the previ-

ously reported psrA and rpoS binding sites and in 16

new possible PsrA-binding sites distributed randomly

on the chromosome of P. aeruginosa PAO1 (Table 1).

Regions including 600 bp downstream of the potential

PsrA binding sites were examined for the presence of

open reading frames (ORFs) (Fig. 1 and Table 1). Fig.

1 illustrates the specific region in the chromosome wherethese putative binding sites were located with respect to

which ORF and Table 1 shows the precise location of

the putative binding site, the putative DNA-binding se-

quence and the possible downstream ORF that this

PsrA-site might be regulating.

3.2. Gene expression analysis of putative PsrA regulated

promoters identified by comparative genome analysis

In order to determine whether the putative PsrA

binding sites identified using a comparative genome

analysis search (see above) represented transcriptionally

regulated PsrA-dependent promoters, we tested 14 of

them by cloning with adjacent DNA into the lacZ

wide-host range pMP220 promoter probe vector. These

14 putative binding sites were located in or near inter-genic regions and what was believed to be a complete

promoter of a putative ORF. Of these, four putative


Table 1

Predicted binding sites for PsrA in the Pseudomonas aeruginosa PAO1 genome and b-galactosidase activity of these promoters

Binding site PA number of

downstream

genes

Position on PAO1

chromosome

Gene/protein Distance from

ATG

b-galactosidase activity (MU)

WT psrA

mutant

rpoS

mutant

Fold

change

GAAAC CC GTTTC PA0413 453497–453508 pilL 618 1282 1368 1315 NS

CAAAC GCCT GTTTG PA0506 564778–564791 Acyl CoA dehydrogenase 123 4305 13,545 3912 3.15

GAAAC TGAA GTTTC PA0806 883112–883125 Hypothetical 652 2965 2830 3201 NS

GAAAC GTAT GTTTC PA1394 1515759–1515772 Hypothetical 661 2130 2270 2050 NS

GAAAC CG GTTTC PA2258 2487773–2487784 ptxR 491 1069 1171 1120 NS

GAAAC CG GTTTC PA2259 2487784–2487773 ptxS 72 1030 1213 1153 NS

GAAAC CG GTTTC PA2260 2488926–2488937 Hypothetical 13 1224 1228 1280 NS

CAAAC TCC GTTTG PA2673 3021019–3021031 hplV 91 1256 1320 1304 NS

CAAAC AAAC GTTTG PA2952 3312671–3312684 etfB 202 4800 9940 4950 2.1

CAAAC GTTT GTTTG PA2953 3312684–3312671 Electron transfer

flavoprotein–ubiquinone

oxidoreductase

106 6650 5520 6470 0.83

GAAAC GTAT GTTTC PA3006 3367686–3367699 psrA 16 3890 24,378 4015 6.3

CAAAC ACTT GTTTG 3367699–3367712

GAAAC CGGG GTTTC PA3571 4003410–4003423 mmsR 309 3230 2357 3206 0.72

GAAAC CAGC GTTTC PA3595 4029672–4029685 MFS transporter 73 1268 1231 1259 NS

CAAAC TTCC GTTTG PA3622 4059323–4059336 rpoS 411 28,789 4190 27,630 0.15

GAAAC GCCC GTTTC PA4420 4955753–4955766 Hypothetical 183 2465 2716 2640 NS

GAAAC CG GTTTC PA4963 5572071–5572082 Hypothetical 143 3933 3753 3345 NS

NS – not significant, fold change – ratio of promoter activities (MU) in psrA mutant versus WT.

PA0505

PA0506 PA0507

PsrA-BS

123bp

PA0506

PsrA-BS

mmsRmmsA

309bp

mmsB

PA2952PA2953

PA3006PA3007

PA3571

PA3595

PA3622

PsrA-BS73bp

PA3594 PA3595 PA3596

PsrA-BS

rpoS

411bp

nlpD

fdxA

pcm

lexA

PsrA-BS

psrA

16bp 191bp

PA2953

PsrA-BS

etfB

106bp202bp

etfA

Fig. 1. Location of the putative PsrA binding sites for six promoters

regulated by PsrA and for MFS transporter in the P. aeruginosa PAO1

genome (for more details see Table 1). These sites were found using a

degenerate pattern search against the PAO1 genome. The position of

the putative binding site is given as well as its distance to the nearest

translation start codon of an annotated ORF. The PA number refers

to the possible ORF that PsrA might be transcriptionally regulating

(see text for further details). PsrA-BS – PsrA binding site.


binding sites were not tested (PA0099, PA1318, PA5372,

PA5451) since they were very distant from the annotated

translational start codons and were not in an intergenic

region and thus were most probably not located in puta-

tive gene promoters. Of the 14 tested putative gene pro-

moters, 4 were shown to be regulated by PsrA since

transcriptional fusions were behaving in a PsrA depen-

dent manner (Table 1). These were the promoter ofgenes PA0506 encoding a probable acyl-CoA dehydro-

genase, of operon PA2952–PA2951 encoding an elec-

tron transfer flavoprotein b-subunit and a subunit

respectively, of PA2953 encoding an electron transfer

flavoprotein–ubiquinone oxidoreductase and of

PA3571 encoding the transcriptional regulator MmsR.

Two of the promoters (PA0506 and PA2952–PA2951)

were regulated negatively whereas promoters ofPA2953 and PA3571 were regulated positively by PsrA.

3.3. Identification of PsrA regulated proteins

In order to characterise PsrA regulated genes more

fully in P. aeruginosa PAO1 we compared the protein

expression pattern in stationary phase of the wild type

strain PAO1 versus the PAO1psrA::Tn5 mutant bytwo dimensional (2D) SDS–PAGE gel electrophoresis.

Total protein extracts and analysis was performed in

triplicate as described in Section 2. The 2D, analysis, re-

vealed differences in protein levels between PAO1 and

PAO1 psrA::Tn5 mutant in all three experiments in 11

protein spots (Fig. 2). These 11 protein spots were se-

lected for further analysis; proteins present in spots 1,

2, 3, 5, 6, 7, 8, 9 and 10 (electron transfer flavoprotein

Fig. 2. Comparative 2-D gel analysis of total proteins of P. aeruginosa PAO1 (panel A) and P. aeruginosa PAO1 psrA::Tn5 (panel B). Numbers of

encircled protein spots refer to those represented in Table 2. kDa, kilo Daltons.


b-subunit; acyl-CoA-dehydrogenase; neomycin-kana-

mycin phosphotransferase from transposon Tn5; fatty

acid oxidation complex b-subunit; acyl-CoA-dehydroge-

nase; isocitrate dehydrogenase and elongation factor Tu;

DnaK protein; fatty acid oxidation complex a-subunitand GroEL, respectively) were over-expressed in

PAO1 psrA::Tn5 mutant, in contrast spots 4 (caraba-

mate kinase) and 11 (conserved hypothetical protein)

were more expressed in P. aeruginosa PAO1. Peptide

mass fingerprinting of tryptic digested fragments was

performed on all the 11 protein spots. Each protein spot

resulted in the identification of one protein with the only

exception of spot 7 which represented two proteins (iso-citrate dehydrogenase and elongation factor Tu). Pro-

tein spots numbered 2 and 6 contained the same

protein, annotated as PA0506, an acyl-CoA dehydroge-

nase of the same nominal mass of 66 kDa, but different

pI value, 5.62 (which correspond to calculated pI value

from aminoacid sequence) for spot 2 and about 4 for

spot 6. The difference in pI value could be the result of

post-translational modifications. The encoding genefor PA0506 contained a functional PsrA binding site

in its gene promoter as previously demonstrated (see

above). Spot number 3, present only in the PAO1

psrA::Tn5 mutant, was the neomycin–kanamycin phos-

photransferase from transposon Tn5. Protein spot num-

ber 1 represented protein PA2952 encoding an electron

Fig. 3. Retardation of the movement of a DNA fragment containing

dehydrogenase (BamHI-KpnI fragment of 429 bp) [A] electron transfer fl

oxidoreductase (BamHI-KpnI fragment of 377 bp) [B], mmsR BamHI-Kpn

fragment of 251 bp) [D] by purified PsrA protein. The amounts of PsrA prot

was used with anti-PsrA antibodies (lane 5). A 100-fold excess amount of the

added, except for mmsR promoter (100-fold excess amount of the same un

DNA, lane 6 and 7, respectively).

transfer flavoprotein b-subunit of which the gene, etfB,

contained a functional PsrA binding site and was shown

to be regulated by PsrA (see above). Interestingly, spots

5 and 9 were proteins PA3013 and PA3014 encoded by

faoA and faoB which are organized in an operon in-volved in fatty acid metabolism. The promoters of all

the genes encoding for the identified proteins in this

analysis were cloned in the lacZ promoter probe vector

pMP220 (as described in Section 2) and the expression

was determined in strain PAO1, PAO1psrA::Tn5 and

PAO1rpoS::Tn5. The b-galactosidase activities as ex-

pected for the two promoters previously identified using

a comparative genome search for PsrA binding sites (seeabove) display PsrA dependent expression. All other

gene promoter activities were comparable when ob-

tained in strain PAO1 and the psrA knock-out mutant.

The promoter activities were also tested in PAO1-

rpoS::Tn5 as PsrA is a positive transcriptional regulator

of rpoS; all promoters displayed comparable activities in

PAO1rpoS::Tn5 when compared to wild type PAO1.

3.4. Protein–DNA binding studies of PsrA regulated

promoters

In order to establish whether the identified PsrA-reg-

ulated promoters could bind PsrA, mobility shift assays

with the (i) etfBA promoter, PA2592/2591 (ii) the pro-

promoters (composed of complete intergenic region) of acyl CoA

avoprotein b-subunit and electron transfer flavoprotein-ubiquinone

I fragment of 417 bp) [C] and MFS transporter gene (BamHI-KpnI

ein used were 0, 50, 100 and 150 ng (lines 1–4, respectively) and 150 ng

same (lane 6) and psrA promoter (lane 7) unlabeled DNA fragment was

labeled DNA and 10-fold excess amount of unlabeled psrA promoter


moter of PA0506 (a probable acyl-CoA dehydrogenase),

and (iii) the mmsR promoter (PA3571), were performed.

As a control experiment the promoter of PA3595

(encoding a probable major facilitator superfamily,

MFS, transporter) was also used since it contained a

putative PsrA binding region however transcriptionalstudies showed that PsrA had no effect on its transcrip-

tion (Table 1). The three promoters which displayed

PsrA dependent expression were retarded and thus

shown to bind PsrA, and the shift was not observed in

the presence of excess unlabeled fragment (Fig. 3). A

supershift was detected in the presence of anti-PsrA

antibodies (Fig. 3). These results confirmed that these

gene promoters are regulated by PsrA. The promoterof PA3595 showed no retardation (Fig. 3) confirming

the transcriptional fusion data that PsrA was not in-

volved in its regulation.

4. Discussion

In this study, several new loci have been found whichare regulated at the transcriptional level by the TetR

family regulator PsrA of P. aeruginosa. PsrA has been

originally identified as a positive transcriptional regula-

tor of the stationary phase rpoS sigma factor, activating

transcription at the onset of stationary phase [7]. In

addition it was demonstrated that PsrA acts as a strong

negative autoregulator and the binding site in rpoS and

psrA promoters has been determined and was shown tobe well conserved [8]. Searching for the PsrA binding

motif in the P. aeruginosa genome revealed 18 putative

binding sites (Table 1 and Fig. 1), however only 4 of

the 14 tested were responding and could bind to PsrA

as determined with transcriptional fusions and pro-

tein–DNA gel retardation assays (Fig. 1, Fig. 3 and Ta-

ble 1). The search for the PsrA binding site was

performed using the consensus, SAAAC N2–4 GTTTS,it cannot be excluded that PsrA can bind to variants

of this sequence and therefore using this genome search

we did not find other functional PsrA binding sites.

Alternatively, of the 10 gene promoters tested which

contained a putative PsrA binding site but did not dis-

play any PsrA dependence, it cannot be excluded that

in some other environmental/growth condition these

promoters could become PsrA-dependent. ComparingPsrA binding motifs of promoters confirmed to be regu-

lated with PsrA indicate that the functional binding site

was C/GAAAC N4 GTTTG/C and that spacing of four

nucleotides was important between the two conserved

palindromic motifs. Interestingly, the promoter of

PA3595, which encodes a major facilitator superfamily

(MFS) transporter, contained a perfect GAAAC N4

GTTTC consensus, however we found that it was notregulated and does not bind PsrA in vitro (Table 1,

Fig. 3). It could be possible that other sequences are re-

quired outside this palindrome or possibly other factors

are required for PsrA recognition in certain gene pro-

moters. In summary, we have identified four new loci

which are directly regulated by PsrA in addition to the

already known rpoS and psrA promoters. All these pro-

moters have been shown to be able to bind PsrA andhave a very well conserved palindromic DNA sequence.

In order to identify other PsrA regulated loci, we also

performed total 2-D protein analysis of P. aeruginosa

versus P. aeruginosa psrA::Tn5 and could identify 11

protein spots, out of approximately 300, which were dif-

ferentially regulated in psrA::Tn5 mutant; two spots

were more expressed, eight were less and one was not

detectable in PAO1 wild type comparing to PAO1psrA::Tn5 (Fig. 2). The fact that RpoS and PsrA were

not identified using this approach indicates that there

are probably more proteins which are differentially ex-

pressed and were not detected here under these experi-

mental conditions. Interestingly however, three spots

represented proteins of which the encoding gene had a

PsrA-binding site as found in the comparative genome

search and as demonstrated with transcriptional fusionstudies and DNA-binding assays (see above). One of

these, PA0506 encoding an acyl-CoA dehydrogenase,

was detected twice probably due to having different pI

values possibly because of post-translational modifica-

tions. Of the remaining proteins which were differen-

tially expressed, the gene promoter was tested for PsrA

dependent transcriptional expression. Surprisingly all

promoters, with the exception of PA0506 and PA2592which contained a PsrA binding site, did not display

PsrA dependent transcription in stationary phase in P.

aeruginosa. The reason for this is not known, however

the fact that these protein spots were observed to be dif-

ferentially regulated in three independent experiments.

It could be that PsrA affected the levels of some of these

proteins through post-transcriptional and/or post-trans-

lational levels of control either directly and/or indirectly.PsrA has been shown to regulate rpoS expression in re-

sponse to stationary phase [6]. A stress encountered by

bacteria in stationary phase is starvation for energy-

yielding carbon source resulting in the induction of the

starvation-stress response [18,19]. Upon induction of

this response, numerous structural and physiological

changes in the cellular envelope occur in starved cells

of Gram-negative enteric bacteria. These include in-creased lipopolysaccharide in the outer membrane, a

shift from phosphatidylglycerol to diphosphatidylglyc-

erol in the inner membrane, decrease in the relative

amounts of long-chain monounsaturated fatty acid

and increased thickness and cross-linking of the peptido-

glycan as well as expanded attachment of the murein

layer to the outer membrane [20]. Degradation of these

fatty acids through b-oxidation, mediated by acyl-CoA-dehydrogenases, would generate acetyl-CoA to feed the

tricarboxylic acid (TCA) cycle, yielding C-compound


intermediates and electron/H+ ion donors for energy

production. This enzyme also catalyses a,b-dehydroge-nation of acyl-CoA esters and transfers electrons to

an electron transfer flavoprotein via the same mecha-

nism. The acyl-CoA-dehydrogenases (PA0506), the elec-

tron transfer flavoprotein (PA2951/PA2952) andelectron transfer flavoprotein-ubiquinone oxidoreduc-

tase (PA2953) were shown here to be all regulated by

PsrA in response to stationary phase and could there-

fore be part of the same cascade in this process in P.

aeruginosa linking up these gene products for the first

time.

In summary, we have identified new loci regulated by

the TetR family regulator PsrA, 4 of which have a func-tional PsrA box in their gene promoter. PsrA could

therefore play an important role in the adaptation to

stationary phase.

Acknowledgements

We are grateful to Rodolfo Garcia Carlos for help inpreparing 2-D SDS–PAGE electrophoresis and for

offering us the use of his laboratory facilities. We would

also like to thank Kristian Vlahovicek for computer

assistance. This work was funded by the ICGEB Collab-

orative Research Program, grant CRP/YUG02-01, and

partially supported by Ministry for Science and Envi-

ronmental Protection of Serbia, grant No. 1442.

Appendix A. Supplementary data

Supplementary data associated with this article can

be found, in the online version at doi:10.1016/j.fem-

sle.2005.04.003 [21].

References

[1] Hengge-Aronis, R. (2002) Signal transduction and regulatory

mechanisms involved in control of the sigma(S) (RpoS)

subunit of RNA polymerase. Microbiol. Mol. Biol. Rev. 66,

373–395.

[2] Ishihama, A. (2000) Functional modulation of Escherichia coli

RNA polymerase. Annu. Rev. Microbiol. 54, 499–518.

[3] Jorgensen, F., Bally, M., Chapon-Herve, V., Michel, G., Laz-

dunski, A., Williams, P. and Stewart, G.S. (1999) RpoS-depen-

dent stress tolerance in Pseudomonas aeruginosa. Microbiology

145, 835–844.

[4] Suh, S.J., Silo-Suh, L., Woods, D.E., Hassett, D.J., West, S.E.

and Ohman, D.E. (1999) Effect of rpoS mutation on the stress

response and expression of virulence factors in Pseudomonas

aeruginosa. J. Bacteriol. 181, 3890–3897.

[5] Schuster, M., Hawkins, A.C., Harwood, C.S. and Greenberg,

E.P. (2004) The Pseudomonas aeruginosa RpoS regulon and its

relationship to quorum sensing. Mol. Microbiol. 51, 973–985.

[6] Venturi, V. (2003) Control of rpoS transcription in Escherichia

coli and Pseudomonas: why so different?. Mol. Microbiol. 49, 1–9.

[7] Kojic, M. and Venturi, V. (2001) Regulation of rpoS gene

expression in Pseudomonas: involvement of a TetR family

regulator. J. Bacteriol. 183, 3712–3720.

[8] Kojic, M., Aguilar, C. and Venturi, V. (2002) TetR family

member psrA directly binds the Pseudomonas rpoS and psrA

promoters. J. Bacteriol. 184, 2324–2330.

[9] Hanahan, D. (1983) Studies on transformation of Escherichia coli

with plasmids. J. Mol. Biol. 166, 557–580.

[10] Figurski, D.H. and Helinski, D.R. (1979) Replication of an

origin-containing derivative of plasmid RK2 dependent on a

plasmid function provided in trans. Proc. Natl. Acad. Sci. USA

76, 1648–1652.

[11] Whiteley, M., Parsek, M.R. and Greenberg, E.P. (2000) Regula-

tion of quorum sensing by RpoS in Pseudomonas aeruginosa. J.

Bacteriol. 182, 4356–4360.

[12] Miller, J.H. (1972) Experiments in Molecular Genetics. Cold

Spring Harbor Laboratory, Cold Spring Harbor, NY.

[13] Stover, C.K., Pham, X.Q., Erwin, A.L., Mizoguchi, S.D.,

Warrener, P., Hickey, M.J., Brinkman, F.S., Hufnagle, W.O.,

Kowalik, D.J., Lagrou, M., Garber, R.L., Goltry, L., Tolentino,

E., Westbrock-Wadman, S., Yuan, Y., Brody, L.L., Coulter,

S.N., Folger, K.R., Kas, A., Larbig, K., Lim, R., Smith, K.,

Spencer, D., Wong, G.K., Wu, Z., Paulsen, I.T., Reizer, J., Saier,

M.H., Hancock, R.E., Lory, S. and Olson, M.V. (2000) Complete

genome sequence of Pseudomonas aeruginosa PA01, an opportu-

nistic pathogen. Nature 406, 959–964.

[14] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular

Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor

Laboratory, Cold Spring Harbor, NY.

[15] Birnboim, H.C. (1983) A rapid alkaline extraction method for the

isolation of plasmid DNA. Meth. Enzymol. 100, 243–255.

[16] Better, M., Lewis, B., Corbin, D., Ditta, G. and Helinski, D.R.

(1983) Structural relationships among Rhizobium meliloti symbi-

otic promoters. Cell 35, 479–485.

[17] Stachel, S.E., An, G., Flores, C. and Nester, E.W. (1985) A Tn3

lacZ transposon for the random generation of b-galactosidasegene fusions: application to the analysis of gene expression in

Agrobacterium. EMBO J. 4, 891–898.

[18] Spector, M.P., DiRusso, C.C., Pallen, M.J., Garcia del Portillo,

F., Dougan, G. and Finlay, B.B. (1999) The medium-/long-chain

fatty acyl-CoA dehydrogenase (fadF) gene of Salmonella typhimu-

rium is a phase 1 starvation-stress response (SSR) locus. Micro-

biology 145 (Pt 1), 15–31.

[19] Spector, M.P., Garcia del Portillo, F., Bearson, S.M., Mahmud,

A., Magut, M., Finlay, B.B., Dougan, G., Foster, J.W. and

Pallen, M.J. (1999) The rpoS-dependent starvation-stress response

locus stiA encodes a nitrate reductase (narZYWV) required for

carbon-starvation-inducible thermotolerance and acid tolerance

in Salmonella typhimurium. Microbiology 145 (Pt 11), 3035–3045.

[20] Huisman, G.W., Siegele, D.A., Zambrano, M.M. and Kolter, R.

(1996)Morphological and physiological changes during stationary

phase in Escherichia coli and Salmonella. In: Cellular and

Molecular Biology (Neidhart, F.C., et al., Eds.), 2nd ed., pp.

1672–1682. American Society for Microbiology, Washington, DC.

[21] Spaink, H.P., Okker, R.J.H., Wijffelmann, C.A., Pees, E. and

Lugtemberg, B.J.J. (1987) Promoter in the nodulation region of

the Rhizobium leguminosarum Sym plasmid pRL1JI. Plant. Mol.

Biol. 9, 27–39.

http://dx.doi.org/10.1016/j.femsle.2005.04.003

http://dx.doi.org/10.1016/j.femsle.2005.04.003



Isolation and characterisation of the lipopolysaccharide fromAcidiphilium strain GS18h/ATCC55963, a soil isolate

of Indian copper mine

Rabindranath Bera a, Abhijit Nayak c, Asish Kumar Sen b,Biswa Pronab Chowdhury b, Ranjan Bhadra a,*

a Department of Cellular Biochemistry, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, Indiab Department of Organic Chemistry, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India

c Division of Health and Education for Disabled, Pandit Sunderlal Sharma Central Institute of Vocational Education, Bhopal 46201, India

Received 28 February 2005; accepted 4 April 2005


Edited by K. Hantke

Abstract

The lipopolysaccharide (LPS) of the Gram-negative Acidiphilium strain GS18h/ATCC55963, a new soil isolate, exhibited

very low endotoxic activity as determined by Limulus gelation activity, lethal toxicity in galactosamine (GalN) sensitised mice,

and level of tumor necrosis factor alpha (TNFa) in the blood serum of BALB/c mice. Analysis of the LPS, specially of lipid

A which usually accounts for the toxicity, revealed the latter to contain glucosamine and phosphate besides fatty acids, of

which 14:0(3-OH), 18:0(3-OH), 18:1 and 19:0(cyclo) are the major components, while 12:0, 16:0, 19:1, 20:0(3-OH) and

20:1(3-OH) are present in small amounts. The 14:0(3-OH) and 18:0(3-OH) fatty acids are amide-linked, whereas the rest

are ester bound. Glucose, galactose, mannose, rhamnose, heptose, galacturonic acid and 3-deoxy-D-manno-oct-2-ulosonic acid

(Kdo) were present in the polysaccharide part of this LPS. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–

PAGE) of the LPS showed a macromolecular heterogeneity distinctly different from those of Escherichia coli or Salmonella.

The toxicity of this LPS being extremely low attributed to fatty acid composition of its lipid A, promises potential therapeutic

application.


Keywords: Acidiphilium ATCC55963; Lipopolysaccharide; Lipid A; Lethal toxicity

1. Introduction

As a major constituent of the outer leaflet of the cell

wall lipid bilayer [1], Gram-negative bacterial LPS vary

widely in composition as reflected in their multiple sero-


doi:10.1016/j.femsle.2005.04.001

* Corresponding author. Fax: +91 33 2473 5197/0284.

E-mail address: [email protected] (R. Bhadra).

type [2,3]. The hydrophilic polysaccharide and lipophiliclipid A moieties are the components of the LPS mole-

cule. Lipid A expresses numerous biological activities

such as antitumor activity as also protection against

X-irradiation and bacterial infection in higher verte-

brates [4], but it is not acceptable clinically since it acts

as an agent for endotoxic shock induced death, a human

health problem yet to be solved. The toxicity of lipid A

depends on the type of hexosamine present, the degree



184 R. Bera et al. / FEMS Microbiology Letters 246 (2005) 183–190

of phosphorylation, the presence of phosphate substitu-

ents and, most notably, the chemical structure, chain

length as well as the number and location of fatty acyl

groups [5,6]. Purified lipid A reported from few bacterial

sources [7,8] has very little toxicity and has notably dif-

ferent fatty acid composition (in respect of chain length,type of linkage, unsaturation and fatty acid hydroxyl-

ation) as also the degree of amino sugar phosphoryla-

tion. So lipid A may exist as a toxic or a non-toxic

entity depending on the constituents present.

Studies on the compositional analysis of the LPS

from Acidiphilium species [9–11] are limited. These spe-

cies have been suggested to be related to the acidophilic

Thiobacillus, the LPS of which is known to be non-toxicor weakly toxic [12]. The biological activity of Acidiphi-

lium LPS therefore may have interesting feature and va-

lue like the LPS of Thiobacillus. It is significant that the

LPS of the photobacteria Rhodobacter capsulatus and

Rhodopseudomonas sphaeroides were found to be non-

toxic and antagonistic to toxic LPS [12], which led to

the development of a preventive for endotoxemia [13].

We were interested by the finding that a new acidophilicGram-negative isolate, placed phylogenetically along

with Acidiphilium cryptum and Acidiphilium symbioticum

[14], has been characterised from the copper mine area

of Ghatshila, India, and submitted to the patent depos-

itory of ATCC and also to the national depository at

IMTECH, Chandigarh, (India) (designated as Acidiphi-

lium ATCC55963 and Acidiphilium strain GS18h,

respectively). It was therefore important to investigatethe LPS from this strain, described as Acidiphilium

ATCC55963 throughout this study. This LPS was found

to be nearly devoid of toxicity as described in a patent

[15] giving some preliminary observations. In this study

we have tested this LPS in vitro for its endotoxic activity

and studied for its biological activity in murine model,

which may be indicative of its clinical usefulness. To find

an explanation for its very low toxicity, the detailedcompositional analysis of this LPS was made by deter-

mining the constituent sugars and characterising the

chemical nature and linkage of the fatty acids present.


2.1. Bacterial strain and culture condition

In a typical culture, Acidiphilium ATCC55963 was

grown in a complex medium containing (NH4)2SO4

(15 mM), MgSO4 (2 mM), K2HPO4 (1.4 mM), KCl

(1.3 mM), glucose (5.5 mM) and yeast extract (0.01%),

under aerobic condition at pH 3 (adjusted with 1 N

H2SO4) and at 30 �C under constant stirring for 72 h.

The bacteria were then harvested by centrifugation at6000g (4 �C, 30 min) and washed thrice with the same

culture medium without glucose and yeast extract.

2.2. Extraction of the lipopolysaccharide and preparation

of lipid A

The LPS was extracted from wet cells instead of dry

cells by a conventional hot phenol–water method

[16,17]. The lyophilised crude LPS was suspended inpyrogen free water (2% w/v) to form a fine suspension

of LPS, and subjected to repeated (thrice) ultra centrifu-

gation at 105,000g (at 4 �C for 4 h) for the removal of

RNA and DNA. Finally the pellet was suspended in

pyrogen free water and treated with chilled 90% ethanol

(4 times the volume of the suspension) and kept at 4 �Cfor 24 h to get the LPS as a precipitate, which was col-

lected by centrifugation (5000g, at 4 �C for 10 min). Thiswas further purified to remove the endotoxin protein by

the ‘‘phenol re-extracted LPS’’ procedure by treating

with triethylamine, phenol, and deoxycholate (DOC)

as per a reported method [18]. The absorbance of the

LPS solution was measured at 260 nm to determine

the presence of any contaminating nucleic acid. To re-

move the phospholipids, the LPS was washed thrice

with chloroform:methanol:water (16:8:1).Lipid A was released from the pure LPS by hydroly-

sis in aqueous 1% acetic acid at 100 �C for 90 min [19].

The precipitated lipid A was recovered by centrifugation

(5000g, 30 min) and washed thrice with hot water. The

supernatant of the hydrolysate (degraded oligosaccha-

ride) was concentrated and freeze-dried. The precipi-

tated material was then lyophilised and further

purified by an established procedure [20]. Briefly, thecrude lipid was suspended in a two-phase solvent chlo-

roform/methanol/water (10:5:6) and centrifuged. The

lower organic layer was recovered, filtered using ultra-

free-filter vessels with Durapore membrane (pore diam-

eter 0.45 lm, Millipore), and evaporated to dryness.

This material was used for fatty acid analysis.

2.3. Sodium dodecyl sulfate polyacrylamide gel

electrophoresis

Sodium dodecyl sulfate polyacrylamide gel electro-

phoresis (SDS–PAGE) was performed by incorporating

4 M urea in a 13% separating gel, containing 0.1% (w/v)

SDS with the buffer system of Laemmli [21]. Electropho-

resis was conducted with a constant current of 35 mA

for 5 h. After SDS–PAGE, the gel was fixed and thenoxidised with periodate; LPS bands were visualised by

silver staining method as described previously [22].

2.4. Lethal toxicity of LPS in galactosamine-treated mice

Lethal toxicity test was performed in galactosamine-

sensitised mice as described previously [23]. Briefly, a

group of six BALB/c mice, 8–12 weeks old and weighing20 ± 2 g, were injected intraperitoneally with a mixture

of D-galactosamine (GalN; 18 mg/mouse; Sigma) and

R. Bera et al. / FEMS Microbiology Letters 246 (2005) 183–190 185

various concentrations of LPS in 0.2 ml pyrogen free

saline. Another group containing six naıve mice, which

received only 0.2 ml pyrogen free saline without any

LPS, was used as control. The survivability of the mice

was recorded up to a period of 72 h after the administra-

tion of LPS, and the lethal toxicity was expressed as100% loss of survivability.

2.5. Limulus amebocyte lysate test

The pyrogenicity of Acidiphilium ATCC55963 LPS

was examined by studying its gel forming activity with

Limulus amebocyte lysate (Endosafen Charleston,

USA) and the test was performed as previously de-scribed [24]. Briefly, the LPS solution and the reference

positive standard (E. coli serotype 0111:B4, Sigma) were

diluted serially with pyrogen free water; heparin (Sigma)

was used as negative control. The test solution was then

added to an equal volume of the lysate and the mixture

incubated at 37 �C for 1 h according to the manufac-

turer�s instructions. Formation of a gel or flocculation

in the tube indicated a positive result.

2.6. Measurement of TNFa levels in serum of LPS treated

mice

Female BALB/c mice (8–12 weeks old, weighing

20 ± 2 g) were subjected to i.p. injection with different

doses of E. coli (serotype 0111:B4) and Acidiphilium

ATCC55963 LPS in a total of 0.2 ml solution in pyrogenfree saline, taking a group of 5 mice for each dose. An-

other group of 5 naıve mice, which received only 0.2 ml

pyrogen free saline without any LPS, was used as con-

trol. All the mice from each group were sacrificed and

bled by cardiac puncture 90 min after treatment. The

blood samples pooled from each group were allowed

to clot for collecting the serum. The serum was centri-

fuged (5000g for 5 min) and collected to store at�70 �C until use. The immunoactive TNFa in serum

samples were analysed with a commercial ELISA kit

(Quantikine, R&D Systems, Minneapolis, MN, USA).

2.7. Compositional analysis

Neutral sugars were liberated from purified LPS and

polysaccharide by hydrolysis with 2 M trifluroaceticacid at 120 �C for 2 h. The hydrolysate was evaporated

to dryness, then reduced with NaBH4 and acetylated

with acetic anhydride:pyridine (1:1, v/v) at room tem-

perature for 12 h or by heating on a boiling water bath

for 45 min. The resulting alditol acetate derivatives [25]

were analysed by gas liquid chromatography (GLC)

and GLC–mass spectrometry, using a Hewlett–Packard

gas chromatograph (model 6890 plus, equipped with aflame ionisation detector) and a Jeol mass spectrometer

(Model JMS-AX500). Glucosamine (GlcN) was released

by hydrolysis in 4 N HCl at 100 �C for 10 h and deter-

mined by the same method as used for neutral sugars.

3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) was deter-

mined by the thiobarbituric acid method [26]. Phospho-

rus was determined colorimetrically by ashing procedure

as described previously [27]. Galacturonic acid wasdetermined by a colorimetric method [28] and by

GLC–mass spectrometry after derivatisation of the car-

boxylic group to methyl ester.

Fatty acids were determined as methyl ester deriva-

tives by GLC comparison with authentic standards

and also by the analysis of both electron impact (EI)

and chemical ionisation (CI) mass spectra. GC–MS-EI

was done on Hewlett–Packard 5890 Series II gas chro-matograph equipped with a DB-1 column

(30 m · 0.25 mm · 0.25 lm) and connected to a mass

selective detector (MSD model HP 5970) using the tem-

perature program as follows: 80 �C! 2 min ! 20 �C/min ! 160 �C! 2 min ! 2 �C/min ! 200 �C ! 10 �C/min ! 250 �C! 11 min, injector temp: 250 �C and

detector temp: 280 �C. Chemical ionisation mass spec-

trometry was done on an instrument from Agilent Tech-nologies (6890N Network GC system followed by

detection on 5973 Network MSD). The column and

temperature program were the same as mentioned be-

fore. Ammonia was used as the reagent gas for chemical

ionisation mass spectrometry. For quantitative fatty

acid analysis, methyl ester of tetradecanoic acid was

used as an internal standard as the fatty acid was absent

in this LPS. Total fatty acids were liberated from LPS bytrans-esterification with 2 N HCl in methanol at 85 �Cfor 16 h in nitrogen filled sealed glass tubes [29]. Ester-

bound fatty acids were liberated by treatment of LPS

or lipid A with sodium methylate (0.25 N CH3ONa) at

37 �C for 16 h [30]. After cleavage of the ester-bound

fatty acids, amide linked fatty acids were determined

by the method described above for total fatty acids

[29]. Identification of hydroxylated fatty acids was con-firmed by GC–MS analysis of the trimethylsilylated

(TMS) derivatives.

3. Results

3.1. Isolation and purification of the LPS

The yield of wet bacterial cells was 2 g per liter for

Acidiphilium ATCC55963 grown in mineral-glucose-

yeast extract medium. As acetone dried cells of acido-

philic species could not be dispersed homogeneously in

water, the wet cells were subjected to hot phenol–water

extraction for preparing the LPS effectively. The yield of

the purified LPS was 90 mg from 20 g wet cells, about

2.2% of the total cell mass on dry weight basis. SDS–PAGE analysis of the purified LPS did not show any

band which could be lighted up with Coomassie blue,


indicating it to be free of associated proteins (sometimes

termed as endotoxin proteins). Absorption at 260 nm

was found to be insignificant, suggesting the absence

of nucleic acids in the purified LPS.

3.2. SDS–polyacrylamide gel electrophoresis analysis

SDS–PAGE analysis was performed to determine the

structural characteristic of the Acidiphilium ATCC55963

LPS and to compare it with those of E. coli serotype

0111:B4 and Salmonella typhimurium (Sigma, USA).

As shown in Fig. 1, both E. coli and Salmonella LPS

were resolved into a large number of bands in stepladder

pattern, corresponding to various polysaccharide chainlengths anchored to the core lipid-A and characteristic

of smooth type LPS. The Acidiphilium ATCC55963

LPS was different from the above two LPS. It showed

condensed laddering bands starting from the middle of

the ladder of the other two LPS and extending upto

the low molecular weight region. At the bottom portion,

the laddering was not very distinct for Acidiphilium

ATCC55963 LPS. Thus the results clearly indicated thatit is of S- or SR-type, but not truly R-type as proposed

for other Acidiphilium species where the stepladder pat-

tern of resolution was absent there [9].

Fig. 1. Silver stained SDS–PAGE patterns with lipopolysaccharides

(LPS) isolated from E. coli serotype 0111:B4 (lane 1), Salmonella

typhimurium (lane 2) Acidiphilium ATCC55963 (lane 3).

3.3. Endotoxic properties of the LPS from Acidiphilium

ATCC55963

3.3.1. Lethal toxicity and pyrogenicity

The lethal toxicity of the new LPS was tested using

standard D-galactosamine-sensitised BALB/c mouse asthe test animal and compared with the reference toxic

LPS from E. coli serotype 0111:B4. The survival of the

mouse was recorded at 24, 48, and 72 h after the admin-

istration of the LPS and the results are shown in Table 1.

In this study, E. coli LPS exhibited 100% lethality

(LD100) and 50% (LD50) lethality at 80 and 50 ng/

mouse, respectively. On the other hand, the lethality of

Acidiphilium ATCC55963 LPS was not observed below500 lg per mouse (Table 1) and there was no loss of sur-

vivability up to a dose of 400 lg per mouse. A dose of

1000 g per mouse had to be administered to obtain

100% lethality. This indicated that the lethal toxicity

of Acidiphilium ATCC55963 LPS to BALB/c mice is ex-

tremely low, even negligible, and the LPS may be con-

sidered as non-toxic compared to E. coli LPS.

Similarly, the results obtained in the LAL assay showedthat the lowest concentration of LPS that produced a

positive test was 0.01 ng/ml for E. coli and >10 ng/ml

for Acidiphilium ATCC55963. So the toxic potency of

the new LPS was more than 1000 times less than that

of the E. coli LPS.

3.3.2. TNFa production after LPS administration in naive

BALB/c mice

Since serum TNFa levels peaked at 1–2 h after inject-

ing the toxic LPS [31] into mice, immunoreactive TNFawas measured at 90 min after administering Acidiphilium

ATCC55963 LPS. In mice treated with LPS, the circula-

tory level of TNFa, the primary mediator of LPS toxic-

ity, increased in a dose dependent manner (Table 2). In

saline treated control serum, the level of TNFa detected

Table 1

Lethal toxicity of Acidiphilium ATCC55963 and reference E. coli LPS

in galactosamine-sensitised BALB/c mice

Dose of LPS (lg/mouse) No. of dead mice/

No. of tested mice

% of lethality

after 72 h

Acidiphilium ATCC55963 24 h 48 h 72 h

10 0/6 0/6 0/6 0

100 0/6 0/6 0/6 0

200 0/6 0/6 0/6 0

400 0/6 0/6 0/6 0

500 1/6 2/6 2/6 33.33

750 2/6 4/6 4/6 66.66

1000 4/6 6/6 6/6 100

E.coli 0111:B4

0.01 0/6 0/6 0/6 0

0.02 1/6 1/6 1/6 16.66

0.04 2/6 2/6 2/6 33.33

0.05 2/6 3/6 3/6 50

0.08 4/6 6/6 6/6 100

Table 2

Serum TNFa and lethality induced by E. coli and Acidiphilium

ATCC55963 LPS in BALB/c mousea

Inducer Dose

(lg/mouse)

TNFa(ng/ml)

Survivors/

Total (%)b

E. coli 0111:B4 LPS 10 8.30 ± 1.10 20/20 (100)

25 18.16 ± 1.75 20/20 (100)

50 26.65 ± 3.25 15/20 (75)

100 42.25 ± 2.54 8/20 (40)

200 48.56 ± 4.25 0/20 (0)

Acidiphilium

ATCC55963 LPS

10 3.42 ± 0.75 20/20 (100)

100 10.33 ± 1.15 20/20 (100)

1000 26.85 ± 2.40 20/20 (100)

1500 27.75 ± 1.30 20/20 (100)

Control saline 0.65 ± 0.25 10/10 (100)

a Blood samples were collected from each group at 90 min after

treatment. Tabulated values represent means of four experiments

(n = 5 for each group).b Cumulative mortality followed over 72 h after both type LPS

administration.

Table 3

Chemical composition of LPS, lipid A and polysaccharide part of

Acidiphilium ATCC55963

Component Amount (nmol mg�1)

LPS Polysaccharide moiety Lipid A moiety

Glc 275 745 –a

Gal 280 772 –

Man 398 1095 –

Rha 136 355 –

Hep 67 186 –

Kdo 356 995 –

GalA 325 890 –

GlcN 545 – 984

Phosphorus 775 568 332

Ester-linked fa

12:0 25 – 42

16:0 32 – 56

18:1 234 – 372

19:1 42 – 65

19:1(cyclo) 145 – 218

20:0(3-OH) 28 – 56

20:1(3-OH) 62 – 90

Amide-linked fa

14:0(3-OH) 145 – 240

18:0(3-OH) 592 – 1056

Abbreviations are used as: Glc, glucose; Gal, galactose; Man, man-

nose; Rha, rhamnose; GlcN, glucosamine; GalA, galacturonic acid;

Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; Hep, heptose.a None. fa, fatty acids. The position of OH group is indicated.


was negligible. The results showed that the level of

TNFa induced by E. coli LPS was always higher than

that of the newly identified pure LPS.

3.4. Compositional analysis of the LPS

The constituent carbohydrates and the fatty acids

along with their linkage pattern present in the LPS ofAcidiphilium ATCC55963 have been included in Table

3. As determined by GLC and GLC–mass spectrometry

as alditol acetate derivatives, the constituent sugars in-

clude glucose (Glc), galactose (Gal), mannose (Man),

rhamnose (Rha), glucosamine (GlcN) and a minor

amount of heptose. Glucosamine is present only in lipid

A and usually is the main carbohydrate backbone of this

lipid in most of the Gram-negative bacterial LPS,though galactosamine is present in place of glucosamine

in some Acidiphilium species [9]. Analysis of lipid A, ob-

tained after treating the LPS with 1% aqueous acetic

acid, indicated that the O-specific polysaccharide core

moiety was linked to lipid A via a Kdo molecule. Kdo

and heptose are characteristic components of core oligo-

saccharides in LPS and are useful markers of Gram-neg-

ative bacterial cell wall constituents. Glucose, galactose,mannose and rhamnose are common monosaccharides

of other Acidiphilium species like A. cryptum and A.

symbioticum [9]. Based on the GLC profile of fatty acids

(Fig. 2), the major fatty acids identified were 18:0(3-

OH), 18:1, 14:0(3-OH) and 19:0(cyclo). Other fatty acids

present in small amounts were 12:0, 16:0, 19:1, 20:0(3-

OH) and 20:1(3-OH). Though the mass fragmentation

patterns of 19:0(cyclo) and 19:1 are indistinguishable,the presence of both in this LPS (Fig. 2) could be ascer-

tained from the difference in their retention times. The

ester-linked and amide-bound fatty acids in lipid A are

present in almost the same molar ratio as in the LPS

(Table 3).

4. Discussion

The analysis of LPS establishes that the architecturalelements of the molecule are: (i) the lipophilic lipid A

consisting of a phosphorylated disaccharide linked with

hydroxylated or non-hydroxylated fatty acids, (ii) the

core oligosaccharide short in length and with Kdo,

and (iii) the outer-most O-specific side chain having re-

peated units of specific oligosaccharides displaying the

chemical and serological determinants for the identifica-

tion of the species and strains [2,3,32]. The LPS of Acid-iphilium ATCC55963 contains all the usual constituents

of Gram-negative bacterial LPS, but is distinctly differ-

ent from the fatty acid profile of the earlier reported li-

pid A of A. crypticum and A. symbioticum [9]. Thus,

except for 12:0 and 14:0(3-OH), all the remaining fatty

acids in Acidiphilium ATCC55963 LPS were different.

Again, instead of 14:0(3-OH) as major constituent as

in LPS of A. crypticum and A. symbioticum, 18:0(3-OH) was the highest (about 40% of total fatty acid) con-

tributor in Acidiphilium ATCC55963 LPS. The cellular

fatty acid 18:1, reported by other investigators from

other Acidiphilium species [10,11], was also present in

Fig. 2. Fatty acids profile of Acidiphilium ATCC55963 LPS. Fatty acid methyl esters were obtained by methanolysis as procedure described in

Section 2 and converted to TMS derivative. Separation was performed on a DB-1 column connected to HP 5890 Series II gas chromatograph

equipped with mass selective detector HP5970 using temperature program as follows: 80 �C! 2 min! 20 �C/min! 160 �C! 2 min! 2 �C/min! 200 �C! 10 �C/min! 250 �C! 11 min, injector temp: 250 �C and detector temp: 280 �C. Unidentified peaks and peaks of substances not

belonging to fatty acid methyl esters or artifacts are not marked.


this LPS. Acidiphilium ATCC55963 LPS or lipid A con-tains two long chain b-hydroxy fatty acids, 20:0(3-OH)

and 20:1(3-OH), besides 18:0(3-OH) and 3-hydroxy tet-

radecanoic acid. Two other fatty acids, 19:0(cyclo) and

19:1, which are not common as the constituents of lipid

A, were also detected. It should be mentioned here that

fatty acids 19:1 and 19:0(cyclo) were recently found in

Mesorhizobium huakuii LPS [33].

The compositional diversity of any LPS is related toits biological activity, and endotoxicity is imparted to

LPS by its lipid A moiety [5,6]. The types of fatty acids

present, their linkages, and the state of sugar phosphor-

ylation in lipid A determine its toxicity. However the

presence of low amounts of LPS in the body fluid pro-

tects the host by enhancing resistance to infection and

malignancy through the release of immunomodulators

[34]. An appropriate control of the LPS response istherefore a central element in preserving the fine balance

between its harmful effect and toxicity. In order to ex-

plore the potentiality of the natural LPS as immunolog-

ical adjuvant, the type and amount of LPS present in the

host body fluid are of critical importance. The composi-

tion of Acidiphilium ATCC55963 LPS, specially the type

of fatty acids present, is strikingly different from those of

E. coli or Salmonella LPS. The lower activity of the lipid

A possessing relatively longer fatty acids was evidencedin Salmonella minnesota type lipid A using chemically

synthesised material [35,36], as well as in lipid A from

Porphyromonas gingivalis [30]. Indeed, the Acidiphilium

ATCC55963 LPS was found to contain mostly long

chain fatty acids that are either b-hydroxylated or unsat-

urated. In galactosamine-sensitised mouse, it was about

10,000 times less toxic than E. coli LPS. The chemical

nature, unsaturation and chain length of the fatty acidsin lipid A of Acidiphilium ATCC55963 LPS are com-

mensurate with such low or non-toxic activity. The fatty

acid compositions of two extensively studied non-toxic

LPS from R. sphaeroides and R. capsulatus are however

considerably different, since 3-oxotetradecanoic acid, 3-

hydroxytetradecanoic acid, 3-hydroxydecanoic acid and

7-tetradecenoic acid were reported as their major con-

tributory fatty acids [12].The host response to LPS is not induced directly but

mediated by immunomodulators, and TNFa is consid-

ered the principal mediator of LPS toxicity. In an in vivo

system, production of TNFa by E. coli and Acidiphilium

ATCC55963 LPS was compared. Dose dependent re-

lease was noticed in both the cases, but at each dose

the extent of release was lower for Acidiphilium

ATCC55963 LPS. Despite being able to induce compa-


rable levels of TNFa, no endotoxic shock was induced

by Acidiphilium ATCC55963 LPS in BALB/c mice under

comparable conditions. In all experiments, cumulative

mortality was followed over 72 h after treatment,

although 75% of the deaths occurred within 24 h. So it

was difficult to correlate the lethality with the level ofserum TNFa, since no loss of survivability was observed

in the case of Acidiphilium ATCC55963 LPS treated

mouse. The composition of lipid A or LPS is crucial

for the toxicity as evidenced by Salmonella LPS. Salmo-

nella monophosphoryl lipid A (MPL) induced high level

of TNFa comparable to Salmonella LPS and diphos-

phoryl lipid A, but endotoxic shock and subsequent

death was induced only by the last two [31]. Thereforethe level of serum of TNFa is not the only determinant

for lethal toxicity, as Acidiphilium ATCC55963 LPS re-

mained non-lethal (inspite of producing comparable le-

vel of serum TNFa). If the induction of serum TNFawithout harming the host is of any therapeutic signifi-

cance then Acidiphilium ATCC55963 LPS may be con-

sidered as a candidate. Further investigation on this

LPS for detailed structure determination to elucidatethe structure–activity relationship in the mammalian

host will undoubtedly establish its novel biological prop-

erties as a non-toxic LPS and work on this aspect is cur-

rently going on.

Acknowledgements

We thank our Director Prof. Siddhartha Roy for his

patronization of this study. We are indebted to Dr. Co-

lin Goding, editor Pigment Cell Research and Dr. Basu-

dev Achari of our institute for critically reviewing the

manuscript. We also acknowledge Dr. P.C. Banerjee of

our institute for his kind cooperation in maintaining the

strain. Thanks are due to DBT, Government of India

for financial assistance (R. Bera).

References

[1] Nikaido, H. and Vaara, M. (1985) Molecular basis of bacterial

outer membrane permeability. Microbiol. Rev. 49, 1–32.

[2] Luderitz, O., Jann, K. and Wheat, R. (1968) Somatic and capsular

antigens of Gram-negative bacteria In: Comprehensive Biochem-

istry: Extracellular and Supporting Structures (Florkin, M. and

Stotz, E.H., Eds.), Vol. 26A, pp. 105–227. Elsevier Publishing

Company, Amsterdam.

[3] Galanos, C., Freudenberg, M.A., Luderitz, O., Rietschel, E.T.

and Westphal, O. (1979) Chemical, physiological and biological

properties of bacterial lipopolysaccharides. Prog. Clin. Biol. Res.

29, 321–332.

[4] Nowotny, A. (1983) Beneficial Effects of Endotoxins. Plenum

Publishing Corp., New York.

[5] Qureshi, N., Takayama, K. and Ribi, E. (1982) Purification and

Structural determination of nontoxic lipid A obtained from the

lipoplysaccharide of Salmonella typhimurium. J. Biol. Chem. 257,

11808–11815.

[6] Takayama, K., Qureshi, N., Ribi, E. and Cantrell, J.L. (1984)

Separation and Characterization of toxic and nontoxic formed of

lipid A. Rev. Infect. Dis. 6, 439–443.

[7] Golenbock, D.T., Hampton, R.Y., Qureshi, N., Takayama, K.

and Raetz, C.R.H. (1991) Lipid A-like molecules that antagonize

the effects of endotoxins on human monocytes. J. Biol. Chem.

266, 19490–19498.

[8] Henricson, B.E., Perera, P.Y., Qureshi, N., Takayama, K. and

Vogel, S.N. (1992) Rhodopseudomonas sphaeroides lipid A derived

block in vitro induction of tumor necrosis factor and endotoxin

Tolerance by smooth lipopolysaccharide and monophosphoryl

lipid A. Infect. Immun. 60, 4285–4290.

[9] Basu, S., Das, S. and Banerjee, P.C. (1994) Lipopolysaccharides

of the acidophilic heterotrophic bacteria Acidiphilium cryptum

and Acidiphilium symbioticum. FEMS Microbiol. Lett. 118, 65–

70.

[10] Kishimoto, N., Kosako, Y. and Tano, T. (1991) Acidobacterium

capsulatum gen. Nov. sp. Nov.: an acidophilic chemorgano-

trophic bacterium containing menaquinone from acidic mineral

environment. Curr. Microbiol. 22, 1–7.

[11] Urakami, T., Tamaoka, J., Suzuki, K-I. and Komagata, K. (1989)

Acidomonas gen. Nov., incorporating Acetocacter methanolicus

as Acidomonas methanolica comb. Nov.. Int. J. Syst. Bacteriol.

39, 50–55.

[12] Mayer, H., Bhat, R., Masoud, H., Radziejewska-Lebrecht, J.,

Widemann, C. and Kraus, J.H. (1989) Bacterial lipopolysaccha-

rides. Pure Appl. Chem. 61, 1271–1282.

[13] Christ, W.J., Asano, O., Robidoux, A.L., Perez, M., Wang, Y.,

Dubuc, G.R., Gavin, W.E., Hawkins, L.D., McGuinness, P.D.

and Mullarkey, M.A., et al. (1995) E5531, a pure endotoxin

antagonist of high potency. Science 268, 80–83.

[14] Banerjee, P.C., Ray, M.K., Koch, C., Bhattacharyya, S., Shivaji,

S. and Stackebrandt, E. (1996) Molecular characterization of two

Acidophilic heterotrophic bacteria isolated from a copper mine of

India. Sys. Appl. Microbiol. 19, 78–82.

[15] Bhadra, R., Nayak, A., Bandyopadhyay, P.C. and Basu, S.

(1998) Process for preparing nontoxic lipopolysaccharides from

Acidiphilium species. United States Patent. Patent number:

5,846,789.

[16] Westphal, O., Luderitz, O. and Bister, F. (1952) Uber die

extraction von bacterien mit phenol wasser. Naturforsh. Z. 7b,

148–155.

[17] Westphal, O. and Jann, K. (1965) Bacterial lipopolysaccharides –

extraction with phenol–water and further applications of the

procedure. Meth. Carbohydr. Chem. 5, 83–91.

[18] Hirschefeld, M., Ying, M., Weis, J.H., Vogel, S.N. and Weis, J.J.

(2000) Cutting edge: repurification of lipoplysaccharide eliminates

signaling through both human and murine toll like receptor 2. J.

Immunol. 165, 618–622.

[19] Luderitz, O., Westphal, O., Staub, A.M. and Nikaido, H. (1971)

In: Microbiol Toxins (Weinbaum, G., Kadis, S. and Ajl, S.J.,

Eds.), Vol. 4, p. 145. Academic Press, New York.

[20] Qureshi, N., Kattashov, I., Walker, K., Doroshenko, V., Cotter,

R.J., Takayama, K., Sievert, T.R., Rice, P.A., Lin, J.S.L. and

Golenbock, D.T. (1997) Structure of the monophosphoryl lipid A

moiety obtained from the lipopolysaccharide of Chlamydia

trachomatis. J. Biol. Chem. 272, 10594–10600.

[21] Laemmli, U.K. (1970) Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature 227, 680–685.

[22] Tsai, C.M. and Frasch, C.E. (1982) A sensitive silver stain for

detecting lipopolysaccharide in polyacrylamide gels. Anal. Bio-

chem. 119, 115–119.

[23] Galanos, C., Freudenberg, M.A. and Reutler, W. (1979) Galac-

tosamine-induced sensitization to the lethal effects of endotoxin.

Proc. Natl. Acad. Sci. USA 76, 5939–5943.


[24] Ogawa, T. (1994) Immunobiological properties of chemically

defined lipid A from lipopolysaccharide of Porphyromonas

(Bacteroides) gingivalis. Eur. J. Biochem. 219, 737–742.

[25] Albershein, P., Nevins, D.J., English, P.D. and Karr, A. (1967) A

method for the analysis of sugars in plant cell-wall polysaccha-

rides by gas–liquid chromatography. Carbohydr. Res. 5, 340–345.

[26] Brade, H., Galanos, C. and Luderitz, O. (1983) Differential

determination of the 3-deoxy-D-manno-octulosonic acid residues

in lipopolysaccharides of Salmonella Minnesota rough mutants.

Eur. J. Biochem. 131, 195–200.

[27] Ames, B.N. (1966) Assay of inorganic phosphate, total phosphate

and phosphatases. Meth. Enzymol. 8, 115–118.

[28] Blumenkrantz, N. and Asboe-Hansen, G. (1973) New method for

quantitative determination of uronic acids. Anal. Biochem. 54,

484–489.

[29] Rietschel, E.T., Hase, S., King, M., Redmond, J. and Lehmann,

V. (1977) Chemical structure of lipid A. Microbiology 177, 262–

268.

[30] Wollenweber, H.-W. and Rietschel, E.T. (1990) Analysis of

lipopolysaccharide (lipid A) fatty acids. J. Microbiol. Meth. 11,

195–211.

[31] Kiener, P.A., Marek, F., Rodgers, G., Lin, P-F., Warr, G. and

Desiderio, J. (1988) Induction of tumor necrosis factor, IFN-c,and acute lethality in mice by toxic and non-toxic forms of lipid

A. J. Immunol. 141, 870–874.

[32] Lugtenberg, B. and van Alphen, L. (1983) Molecular arachitec-

ture and functioning of the outer membrane of Escherichia coli

and other Gram-negative bacteria. Biochem. Biophys. Acta 737,

51–115.

[33] Choma, A. (1999) Fatty acid composition of Mesorhizobium

huakuii lipopolysaccharides. Identification of 27-oxooctocosanoic

acid. FEMS Microbiol. Lett. 177, 257–262.

[34] Ulmer, A.J., Rietschel, E.Th., Zahringer, U. and Heine, H. (2002)

Lipopolysaccharide; structure, bioactivity, receptors, and signal

transduction. Trends Glycosci. Glycotechnol. 14, 53–68.

[35] Galanos, C., Luderitz, O., Freudenberg,M., Brade, L., Schade, U.,

Rietschel, E.Th., Kusumoto, S. and Shiba, T. (1986) Biological

activity of synthetic heptaacyl lipid A representing a component of

Salmonella minnesota R595 lipid A. Eur. J. Biochem. 160, 55–59.

[36] Tanamoto, K., Lida, T., Haishima, Y. and Azumi, S. (2001)

Endotoxic properties of lipid A from Comamonas testosteroni.




Comprehensive analysis of classical and newlydescribed staphylococcal superantigenic toxin genes

in Staphylococcus aureus isolates

Katsuhiko Omoe a,*, Dong-Liang Hu b, Hiromi Takahashi-Omoe c,Akio Nakane b, Kunihiro Shinagawa a

a Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka, Iwate 020-8550, Japanb Department of Bacteriology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan

c Chemical Management Center, National Institute of Technology and Evaluation, 2-49-10 Nishihara, Shibuya-ku, Tokyo 151-0066, Japan



Edited by S. Schwarz

Abstract

We describe a comprehensive detection system for 18 kinds of classical and newly described staphylococcal superantigenic toxin

genes using four sets of multiplex PCR. Superantigenic toxin genotyping of Staphylococcus aureus for 69 food poisoning isolates and

97 healthy human nasal swab isolates revealed 32 superantigenic toxin genotypes and showed that many S. aureus isolates harbored

multiple toxin genes. Analysis of the relationship between toxin genotypes and toxin genes encoding profiles of mobile genetic ele-

ments suggests its possible role in determining superantigenic toxin genotypes in S. aureus as combinations of toxin gene-encoding

mobile genetic elements.


Keywords: Staphylococcus aureus; Enterotoxin; Multiplex PCR; Genotyping; Mobile genetic elements

1. Introduction

Staphylococcal enterotoxins (SEs) are emetic toxins,

and staphylococcal food poisoning resulting from the

consumption of food contaminated with SEs is one of

the most common food-borne illnesses [1]. In addition,

SEs and the SE-related toxin, toxic shock syndrome tox-

in-1 (TSST-1), are members of the superantigenic toxin

family and have the ability to stimulate large popula-tions of T cells having a particular Vb element in their

T-cell receptors (TCR). This stimulation subsequently

leads to a massive proliferation of T cells and the uncon-


doi:10.1016/j.femsle.2005.04.007


E-mail address: [email protected] (K. Omoe).

trolled release of proinflammatory cytokines, whichcause life-threatening TSS [2–4]. SEs have been divided

into five serological types (SEA though to SEE) based

on their antigenicity [1]. In recent years, new types of

SEs (SEG, SEH, SEI, SEJ, SEK, SEL, SEM, SEN,

SEO, SEP, SEQ, SElR and SEU) have been reported

[2,5–11]. Several attempts to detect superantigenic toxin,

SE and TSST-1 genes in S. aureus isolates have been

made; these studies have shown that multiple superanti-genic toxin genes are commonly found among S. aureus

isolates [12–17]. These newly described SEs have been

designated as members of the SE family based on their

sequence similarity with classical SEs. The International

Nomenclature Committee for Staphylococcal Superanti-

gen Nomenclature (INCSSN) has recommended that



192 K. Omoe et al. / FEMS Microbiology Letters 246 (2005) 191–198

only staphylococcal superantigens that induce emesis

following oral administration in a monkey model should

be designated as SE while other related toxins that either

lack emetic properties in this model or have not been

tested should be designated staphylococcal entero-

toxin-like (SEl) superantigens [18]. Based on this recom-mendation from INCSSN, the toxins SEJ, SEK, SEL,

SEM, SEN, SEO, SEP, SEQ, and SEU should be re-

named SElJ, SElK, SElL, SElM, SElN, SElO, SElP,

SElQ, and SElU, respectively.

On the other hand, it has been known that certain SE

(SEl) and TSST-1 genes are associated with mobile ge-

netic elements such as pathogenicity islands, prophages,

and plasmids [6,7,9,19–23]. These facts imply that super-antigenic toxin genes are transferred horizontally be-

tween staphylococcal strains. There is a possibility that

these mobile genetic elements have played an important

role in the evolution of S. aureus as a pathogen. To date,

there is a need for a method to comprehensively detect

and identify the large family of superantigenic toxin

genes; such a method would be a powerful tool for evo-

lutionary analysis of the pathogenicity of S. aureus, aswell as for diagnostic and epidemiological purposes.

Here, we report fine superantigenic toxin genotyping

of S. aureus isolates using a multiplex PCR system that

is capable of detecting 18 kinds of staphylococcal super-

antigenic toxin genes. The analysis between superanti-

genic toxin genotypes and toxin genes encoding

profiles of mobile genetic elements provides a hypothesis

on possible role for determination of superantigenic tox-in genotypes in S. aureus.


2.1. Bacterial strains and culture media

A total of 177 S. aureus samples were used in thisstudy. Of these, 11 strains were reference strains, includ-

ing full genome sequencing strains (N315; DDBJ/Gen-

Bank/EMBL BA000018, Mu50; BA000017, MW2;

BA000033) (Table 1). Sixty-nine isolates were obtained

Table 1

Staphylococcal superantigenic toxin genotypes of Staphylococcus aureus refe

Strain Superantigenic toxin genotype

N315 sec, seg, sei, sell, selm, seln, selo

Mu50 sea, sec, seg, sei, sell, selm, seln

MW2 sea, sec, seh, selk, sell, selq

RN4220 no SE gene

196E sea, sed, selj, selr

S6 sea, seb, selk, selq

FRI-361 sec2, sed, seg, sei, selj, sell, selm

FRI-326 see, selq

FRI-569 seh

834 sec, seg, sei, sell, selm, seln, selo

Saga 1 seg, sei, selm, seln, selo, selp

from 30 food poisoning outbreaks diagnosed by 10 local

government laboratories in Japan from 1990 to 2002; S.

aureus isolates were isolated from patient feces, patient

vomit, or the foods involved, and collected from the lab-

oratories. Among these 30 food poisoning outbreaks, 6

were diagnosed as SE-unidentified, meaning that all S.aureus isolates from each outbreak were negative for

production of SEA to SED by commercial SET-RPLA

kit (DENKA Seiken Co. Ltd., Tokyo, Japan). In this

study, we included 21 isolates obtained from these SE-

unidentified outbreaks. In addition to the 69 food poi-

soning isolates, 97 isolates were obtained from nasal

swabs of healthy humans in Japan from 2000 to 2004.

Bacterial cultures were grown in brain heart infusion(BHI) broth prior to purification of genomic DNA.

2.2. DNA purification

Total DNA of S. aureus was purified with the QIA-

amp DNA purification kit (Qiagen GmbH, Hilden, Ger-

many) according to the manufacturer�s instructions. Theconcentration of DNA solution was determined accord-ing to A260 values.

2.3. Primers

The nucleotide sequences of all PCR primers used in

this study and their respective amplified products are

listed in Table 2. The primer sets used to detect selj, selk,

sell, selm, seln, selo, selp, selq and selr genes were de-signed according to published nucleotide sequences

[6,7,9,20–23]. These primer sets were designed to anneal

to unique regions and generate amplicons that would al-

low identification of each se gene based on the molecular

weight of its PCR product (Table 2). The primer sets

used to detect tst-1 and sea to see were those described

by Becker et al. [12]. The primer sets used to detect

seg, seh and sei were described by Omoe et al. [17]. Asan internal positive control, we used primers that are

specific to S. aureus to amplify femA and femB genes

[16,24]. To construct a multiplex PCR system, four sets

(Set 1; sea, seb, sec, sed, see, femB: Set 2; seg, seh, sei,

rence strains

References

, selp, tst-1 Kuroda et al. [7]

, selo, tst-1 Kuroda et al. [7]

Baba et al. [19]

Novick [29]

Omoe et al. [9,17]

Omoe et al. [17]

, seln, selo, selr Omoe et al. [17]

Omoe et al. [17]

Su and Wong [11]

tst-1 Nakane et al. [30]

Omoe et al. [17]

Table 2

Nucleotide sequences and predicted size of PCR products for the staphylococcal superantigen-specific oligonucleotide primers

Gene Primer Oligonucleotides sequence (5 0–30) PCR product (bp) PCR set References

sea SEA-3 CCTTTGGAAACGGTTAAAACG 127 1 [12]

SEA-4 TCTGAACCTTCCCATCAAAAAC

seb SEB-1 TCGCATCAAACTGACAAACG 477 1 [12]

SEB-4 GCAGGTACTCTATAAGTGCCTGC

sec SEC-3 CTCAAGAACTAGACATAAAAGCTAGG 271 1 [12]

SEC-4 TCAAAATCGGATTAACATTATCC

sed SED-3 CTAGTTTGGTAATATCTCCTTTAAACG 319 1 [12]

SED-4 TTAATGCTATATCTTATAGGGTAAACATC

see SEE-3 CAGTACCTATAGATAAAGTTAAAACAAGC 178 1 [12]

SEE-2 TAACTTACCGTGGACCCTTC

seg SEG-1 AAGTAGACATTTTTGGCGTTCC 287 2 [17]

SEG-2 AGAACCATCAAACTCGTATAGC

seh SEH-1 GTCTATATGGAGGTACAACACT 213 2 [17]

SEH-2 GACCTTTACTTATTTCGCTGTC

sei SEI-1 GGTGATATTGGTGTAGGTAAC 454 2 [17]

SEI-2 ATCCATATTCTTTGCCTTTACCAG

selj SEJ-1 ATAGCATCAGAACTGTTGTTCCG 152 2 This study

SEJ-2 CTTTCTGAATTTTACCACCAAAGG

selk SEK-1 TAGGTGTCTCTAATAATGCCA 293 3 This study

SEK-2 TAGATATTCGTTAGTAGCTG

sell SEL-1 TAACGGCGATGTAGGTCCAGG 383 4 This study

SEL-2 CATCTATTTCTTGTGCGGTAAC

selm SEM-1 GGATAATTCGACAGTAACAG 379 3 This study

SEM-2 TCCTGCATTAAATCCAGAAC

seln SEN-1 TATGTTAATGCTGAAGTAGAC 282 4 This study

SEN-2 ATTTCCAAAATACAGTCCATA

selo SEO-1 TGTGTAAGAAGTCAAGTGTAG 214 3 This study

SEO-2 TCTTTAGAAATCGCTGATGA

selp SEP-3 TGATTTATTAGTAGACCTTGG 396 2 This study

SEP-4 ATAACCAACCGAATCACCAG

selq SEQ-1 AATCTCTGGGTCAATGGTAAGC 122 4 This study

SEQ-2 TTGTATTCGTTTTGTAGGTATTTTCG

selr SER-1 GGATAAAGCGGTAATAGCAG 166 4 This study

SER-4 GTATTCCAAACACATCTAAC

tst1 TST-3 AAGCCCTTTGTTGCTTGCG 447 3 [12]

TST-6 ATCGAACTTTGGCCCATACTTT

femA femA1 AAAAAAGCACATAACAAGCG 134 2, 3 [16]

FemA2 GATAAAGAAGAAACCAGCAG

femB femB1 TTACAGAGTTAACTGTTACC 651 1, 4 [24]

FemB2 ATACAAATCCAGCACGCTCT

K. Omoe et al. / FEMS Microbiology Letters 246 (2005) 191–198 193

selj, selp, femA: Set 3; selk, selm, selo, tst-1, femA: Set 4;

sell, seln, selq, selr, femB) of 10· primer master mixes

(containing 2 lM each primer) were prepared.

2.4. Uniplex PCR and sequencing analysis

To evaluate the specificity of the newly designed pri-

mer sets for detecting selj, selk, sell, selm, seln, selo, selp,selq and selr genes, uniplex PCR using each primer pair

was performed. The amplification was performed in an

automated thermalcycler with a hot bonnet (Takara

PCR Thermal Cycler MP). The reaction mixture

(50 ll) for uniplex PCR contained 0.4 lM of each pri-

mer, 2 mM MgCl2, 200 lM each of dGTP, dATP, dTTP

and dCTP (Takara Syuzo Co., Kyoto, Japan), 0.5U of

TaKaRa EX Taq DNA polymerase (Takara), and 5 llof 10· buffer (Takara). Thermal cycles of 94 �C for

30 s, 55 �C for 30 s, and 72 �C for 60 s were repeated

30 times. The DNA fragments obtained from uniplex

PCR were subcloned to pGEM-easy vector (Promega,

Madison, WI) and subjected to nucleotide sequencing

analysis using an ABI3100-avant automatic DNA se-

quencer (Applied Biosystems, Foster City, CA).

2.5. Multiplex PCR

Multiplex PCR of each primer set was performed

with QIAGEN Multiplex PCR Kit (QIAGEN) accord-

ing to manufacturer�s instructions. Each reaction mix

(50 ll) consisted of 25 ll of 2· QIAGEN Multiplex

PCR Master Mix (containing QIAGEN HotStartTaq

DNA polymerase, QIAGEN multiplex PCR buffer,

and dNTP mix), 0.2 lM of each primer, and 10–

100 ng of template DNA. DNA amplification was car-ried out with the following thermal cycling profile:

an initial denaturation of DNA and QIAGEN


HotStartTaq DNA polymerase activation at 95 �C for

15 min was followed by 35 cycles of amplification

(95 �C for 30 s, 57 �C for 90 s, and 72 �C for 90 s), end-

ing with a final extension at 72 �C for 10 min. PCR

products were resolved by electrophoresis in 3% NuSi-

eve 3:1 agarose gel (Cambrex Bio Science Rockland,Inc., Rockland, ME) in 0.5· TBE (Tris-boric acid-

EDTA) buffer, stained by 0.5 lg/ml of EtBr, and visual-

ized on a transilluminator.

3. Results and discussion

3.1. Development of multiplex PCR system for detection

of se and tst-1 genes

First of all, we tried amplifying target DNA of newly

designed PCR primers for selj, selk, sell, selm, seln, selo,

selp, selq and selr genes. Uniplex PCR using each primer

set with total DNA of reference S. aureus strains was

performed: S. aureus 196E total DNA for selj and selr;

S. aureus MW2 for selk and selq; and S. aureus N315for sell, selm, seln, selo and selp. The sizes of PCR prod-

ucts obtained by these uniplex PCRs were identical to

those predicted from the design of the primers (data

not shown). Then, these PCR products were subcloned

Fig. 1. Detection of staphylococcal superantigenic toxin genes by multiplex

and MW2 were amplified with 4 sets of multiplex PCR. SE gene negative refe

molecular size marker HaeIII digested /X174; 1, N315; 2, Mu50; 3, MW2; 4,

of S. aureus 196E, S6, FRI 326, FRI 569, N315. (b) Total DNAs from S. aure

M, molecular size marker HaeIII digested /X174; 1, 9, mixture of total DNA

361; 5, FRI-362, 6, FRI-569; 7, 834; 8, Saga1; 10, Milli-Q water (negative c

to pGEM-easy vector and subjected to nucleotide

sequencing analysis. The DNA sequences of these clones

of se genes were almost exactly identical to the published

DNA sequences of the respective se genes. These results

showed that the newly designed PCR primer sets could

amplify respective se genes with specificity.The combinations of primer sets and reaction condi-

tions for the multiplex PCR were optimized to ensure

that all PCR products of target genes were satisfactorily

amplified. We ultimately constructed four optimized

multiple primer sets, as described in Section 2. Fig. 1

shows the results of multiplex PCR when total DNAs

of reference S. aureus strains were used as a templates.

As a positive control, a mixture of total DNA of S. aur-eus 196E, S6, FRI-326, FRI-569 and N315 was used.

Reliable amplification of PCR products was observed

in all multiplex PCR reactions using the four primer

sets. The sizes of the PCR products obtained from the

positive control and the reference strains corresponded

to their predicted sizes (Table 2). Furthermore, the toxin

gene genotypes of all reference strains determined by

multiplex PCR were exactly identical to the toxin genegenotypes determined by full genome sequencing

(N315, Mu50 and MW2) or southern blot analysis

(196E, S6, FRI-361, FRI-326, FRI-569, 834 and Saga1)

(Table 1). When Milli-Q water was used as a negative

PCR. (a) Total DNA from full genome sequenced strains N315, Mu50

rence strain RN4220 was also included as a negative control. Lanes: M,

RN4220, 5, Milli-Q water (negative control); 6, mixture of total DNA

us reference strains were amplified with 4 sets of multiplex PCR. Lanes:

of S. aureus 196E, S6, FRI 326, FRI 569, N315; 2, 196E; 3, S6; 4, FRI-

ontrol).


control instead of template genomic DNA, no PCR

products were observed in any of the four sets of multi-

plex PCR.

3.2. Superantigenic toxin gene genotyping of S. aureus

isolates from food poisoning outbreaks and healthy human

nasal swabs using multiplex PCR

A total of 166 S. aureus isolates were subjected to

superantigenic toxin gene genotyping analysis. A total

of 32 superantigenic toxin genotypes were observed

among the 166 isolates (Table 3). All of the 166 isolates

tested harbored the femA and femB genes. Of the 69 iso-

lates that originated in food poisoning, all isolates werediagnosed as positive for se genes. Thirteen SE-geno-

types were observed in food poisoning-related isolates.

Table 3

S. aureus superantigenic toxin genotypes and relationship with mobile genet

S. aureus superantigenic toxin genotypes Prevalenc

SFPa

(n = 69)

S. aureus harboring classical superantigenic toxin genes 4 (5.8)

sea 4 (5.8)

seb

S. aureus harboring classical and new superantigenic toxin genes 44 (63.8)

sea, sec, sell

sea, seg, tst-1

sea, seb, selk, selq 2 (2.9)

sea, sed, selj, selr 2 (2.9)

sea, seh, selk, selq 5 (7.3)

sea, seb, seh, selk, selq 21 (30.4)

sea, seg, sei, seln, tst-1 2 (2.9)

sea, seg, sei, selm, seln, selo 4 (5.8)

seb, seh 4 (5.8)

seb, selp 4 (5.8)

seb, selk, selq

seb, selk, selq, selp

seb, seg, sei, selm, seln, selo

sec, seg, sei, sell, selm, seln, selo

sec, seg, sei, sell, selm, seln, selo, tst-1

sed, seg, sei, selj, selm, seln, selo, selp, selr

tst-1, seg

tst-1, seg, seh, seln

tst-1, seg, sei, seln

tst-1, seg, sei, selk, selm, seln, selo

S. aureus harboring new superantigenic toxin genes 21 (30.4)

seg, sei, selm, seln

seg, sei, selm, seln, selo 7 (10.2)

seg, sei, selm, seln, selo, selp 8 (11.6)

seg, sei, selj, selm, seln, selo, selr 3 (4.4)

seg, sei, selk, selm, seln, selo, selq

seh, selk, selq

selj, selr 3 (4.4)

selm, selo

seln

selp

S. aureus harboring no superantigenic toxin gene

a Isolates from Staphylococcal food poisoning outbreaks.

Forty (58%) isolates were associated with the sea gene,

and the majority of these isolates possessed other se

genes. Among the 97 healthy human isolates, only 77

isolates (79.4%) were diagnosed as se-positive. A total

of 25 genotypes were observed in healthy human iso-

lates. In contrast to the trend in food poisoning-relatedisolates, there were only 8 (8.3%) sea-associated isolates.

Twenty-one isolates from 6 SE-unidentified food poi-

soning outbreaks possessed newly identified se genes

(seg, sei, selj, selm, seln, selo, selp, or selr). The superan-

tigenic toxin genotypes of isolates within each outbreak

were the same (2 outbreaks: seg, sei, selm, seln, selo; 2

outbreaks: seg, sei, selm, seln, selo, selp; 1 outbreak:

seg, sei, selj, selm, seln, selo, selr; 1 outbreak: selj, selr).However, it is difficult to conclude that these newly iden-

tified SEs were responsible for these food poisoning

ic elements

e (%) Suspected genomic islands and plasmids

Human nasal

swab (n = 97)

Total

(n = 166)

3 (3.1) 7 (4.2)

1 (1.0) 5 (3.0) /Sa3mu

2 (2.1) 2 (1.2)

46 (47.4) 90 (54.2)

3 (3.1) 3 (1.8) /Sa3mu, Type II mSa31 (1.0) 1 (0.6) /Sa3mu + seg, tst-1

2 (1.2) /Sa3mu, mSa1(SaPI3) Or /Sa3mw + seb

2 (1.2) /Sa3mu, pIB485

2 (2.1) 7 (4.2) /Sa3mw + seh, or /Sa3mu +

seh, selk, selq

21 (12.7) /Sa3mu, mSa1(SaPI3) + seh or

/Sa3mw + seb, seh

2 (1.2) /Sa3mu + seg, sei, seln, tst-1

1 (1.0) 5 (3.0) /Sa3mu, Type I mSab4 (3.1)

7 (7.2) 11 (6.6) /Sa3n + seb

2 (3.4) 2 (1.6) mSa1 (SaPI3)

2 (2.1) 2 (1.2) / Sa3n, mSa1 (SaPI3)

6 (6.2) 6 (3.6) Type I mSab + seb

2 (2.1) 2 (1.2) Type II mSa3, Type I mSab2 (2.1) 2 (1.2) Type I mSa4 or mSa2, Type I mSab9 (9.3) 9 (5.4) /Sa3n, Type I mSab, pIB4851 (1.0) 1 (0.6)

1 (1.0) 1 (0.6)

6 (10.2) 6 (4.7)

1 (1.0) 1 (0.6) mSa1 (SaPI1), Type I mSab28 (28.9) 49 (29.5)

1 (1.0) 1 (0.6)

16 (16.5) 23 (13.9) Type I mSab3 (3.1) 11 (6.6) /Sa3n, Type I mSab

3 (1.8) Type I mSab, pF51 (1.7) 1 (0.8) Type I mSab + selk, selq

2 (3.4) 2 (1.2)

3 (1.8) pF5

1 (1.0) 1 (0.6)

1 (1.0) 1 (0.6)

3 (3.1) 3 (1.8) /Sa3n20 (20.6) 20 (12.1)


outbreaks. The emetic activity of the newly described

SEs has not been proved, except in the cases of SEG

and SEI [5]. To confirm the relationship between these

newly identified SEs and food poisoning, it is important

to demonstrate the emetic activity of these newly de-

scribed SEs using an experimental primate model. Re-cent studies have shown that specific non-primate

animal models, such as the ferret and the house musk

shrew, respond to SEs and exhibit emetic reactions

[25,26]. However, as recommended by INCSSN, the pri-

mate model is still the gold standard for estimating the

emetic activity of SEs. Moreover, detection of superan-

tigenic toxin genes in S. aureus isolates does not imply

expression of these genes by these isolates. Omoe et al.[17] have shown that seg- and sei-harboring S. aureus

isolates produce very low levels of SEG and SEI in vitro,

although transcription of mRNA of SEG and SEI in

these isolates was proven by reverse-transcriptase PCR

analysis. Demonstration of toxin production at levels

that are sufficient to cause diseases by strains harboring

these se genes is also needed. It has been reported that

the production of specific SEs may depend on the hostenvironment and may play a role in the adaptation of

S. aureus to the host [27]. SE production should be as-

sessed in vitro, in vivo and in food, using an immunolog-

ical detection method such as ELISA to confirm the

relationship between newly identified SEs and diseases.

3.3. The relationship between superantigenic toxin

genotypes and toxin gene-encoding mobile genetic

elements

In the present study, we have shown that there are

many superantigenic toxin genotypes in S. aureus iso-

lated from food poisoning outbreaks or healthy human

nasal swabs. It has been known that almost all superan-

tigenic toxin genes are associated with mobile genetic

elements such as genomic islands (pathogenicity islands,prophages and staphylococcal cassette chromosomes)

and plasmids. Thus, we analyzed the relationship be-

tween superantigenic toxin genotypes obtained in this

study and known superantigenic toxin gene-encoding

mobile genetic elements. Table 3 summarizes the rela-

tionship between superantigenic toxin genotypes and

known mobile genetic elements. One half (16/32) of

superantigenic toxin genotypes observed in this studycould be considered as combinations of known superan-

tigenic toxin gene-encoding profiles of genomic islands

or plasmids. For example, genotype sea, sec, sell could

be a combination of /Sa3mu (sea) and Type II mSa3(sec, sell), and genotype sed, seg, sei, selj, selm, seln, selp,

selr could be a combination of pIB485 (sed, selj, selr),

Type I mSab (seg, sei, selm, seln, selo) and /Sa3n (selp).

Of the remaining 16 genotypes, 7 could be considered ascombinations of known mobile genetic elements plus

particular se genes. For example, genotype sea, seh, selk,

selq could be a combination of /Sa3mw (sea, selk, selq)

plus seh or /Sa3mu (sea) plus seh, selk, selq. The

remaining 9 genotypes, such as ‘‘seb, seh’’, ‘‘seh, selk,

selq’’, ‘‘tst-1, seg, seh, seln’’, did not follow the rule of

known superantigenic toxin gene profiles of mobile ge-

netic elements. In these genotypes, we observed severalgene combinations that could be considered incomplete

Type I mSab, such as ‘‘seg, sei, selm, seln’’, ‘‘seg, sei,

seln’’, ‘‘selm, selo’’, and ‘‘seln’’. Becker et al. [13] re-

ported the prevalence of Type I mSab-related SE genes,

and showed that a substantial number of isolates were

found to harbor only one or two of the selm, seln, and

selo genes. These results suggest the possibility of the

existence of SE-encoding variants within the genomic is-land Type I mSab, or the existence of new types of mo-

bile genetic elements encoding seg, sei, selm, seln, or

selo genes. There is also the possibility of existence of

many new types of toxin-gene-encoding mobile genetic

elements. As shown above, it seems that the se genotype

of S. aureus may be determined by mobile genetic ele-

ments it harbors. To prove this hypothesis, an effort to

explore new types of mobile genetic element is needed,as well as detailed characterization of SE-encoding

genomic islands. Previously, Baba et al. [19] mentioned

that genomic island allotyping would be a useful ap-

proach to S. aureus genotyping and that this process

would enable the prediction of the pathogenic capability

of an S. aureus clinical strain. Our multiplex PCR sys-

tem for detecting superantigenic toxin genes will be use-

ful in determining genomic island allotypes. Recently,Sergeev et al. [28] reported a PCR-based microarray as-

say system for simultaneous detection of SE genes. This

microarray system would be a powerful method for

detecting several types of se genes simultaneously. How-

ever, equipment for microarrays is expensive, and

microarrays are not widely used in common laboratories

at present. By contrast, our PCR-based superantigenic

toxin gene detection system could be performed easilyin commonly equipped clinical laboratories.

In conclusion, the newly developedmultiplex PCR sys-

tem for comprehensive detection and identification of

staphylococcal superantigenic toxin genes described here

is a potentially powerful tool for diagnosis and epidemio-

logical study ofS. aureus.The data presented here suggest

the system�s potential role in determining superantigenic

toxin genotypes as combinations of toxin gene-encodingmobile genetic elements, such as genomic islands and

plasmids. Further exploration and characterization of

new types of mobile genetic elements are needed.

Acknowledgements

This work was partly supported by grants-in-aids forscientific research from the Japan Society for the Promo-

tion of Science (Grants 15580272 and 16380205).


We thank Dr. Keichi Hiramatsu (at Juntendo Uni-

versity) for kindly providing the S. aureus strains used

in this work.

References

[1] Bergdoll, M.S. (1989) Staphylococcus aureus In: Foodbone

Bacterial Pathogens (Doyle, M.P., Ed.), pp. 463–523. Marcel

Dekker, Inc., New York, NY.

[2] McCormick, J.K., Yarwood, J.M. and Schlievert, P.M. (2001)

Toxic shock syndrome and bacterial superantigens: an update.

Annu. Rev. Microbiol. 55, 77–104.

[3] Uchiyama, T., Kamagata, Y., Yan, X.-J., Kohno, M., Yoshik-

awa, M., Fujikawa, H., Igarashi, H., Okubo, M., Awano, F.,

Saito-Taki, T. and Nakano, M. (1987) Study of the biological

activities of toxic shock syndrome toxin-1. II. Induction of the

proliferative response and the interleukin 2 production by T cells

from human peripheral blood mononuclear cells stimulated with

the toxin. Clin. Exp. Immunol. 68, 638–647.

[4] Uchiyama, T., Yan, X.-J., Imanishi, K. and Yagi, J. (1994)

Bacterial superantigens – Mechanism of T cell activation by

superantigens and their role in the pathogenesis of infectious

diseases. Microbiol. Immunol. 38, 245–256.

[5] Munson, S.H., Tremaine, M.T., Beteley, M.J. and Welch, R.A.

(1998) Identification and characterization of staphylococcal

enterotoxin type G and I from Staphylococcus aureus. Infect.

Immun. 66, 3337–3348.

[6] Jarraud, S., Peyrat, M.A., Lim, A., Tristan, A., Bes, M., Mougel,

C., Etienne, J., Vandenesch, F., Bonneville, M. and Lina, G.

(2001) egc, a highly prevalent operon of enterotoxin gene, forms a

putative nursery of superantigens in Staphylococcus aureus. J.

Immunol. 166, 669–677.

[7] Kuroda, M., Ohta, T., Uchiyama, I., Baba, T., Yuzawa, H.,

Kobayashi, I., Cui, L., Oguchi, A., Aoki, K., Nagai, Y., Lian, J.,

Ito, T., Kanamori, M., Matsumaru, H., Maruyama, A., Mura-

kami, H., Hosoyama, A., Mizutani-Ui, Y., Takahashi, N.K.,

Sawano, T., Inoue, R., Kaito, C., Sekimizu, K., Hirakawa, H.,

Kuhara, S., Goto, S., Yabuzaki, J., Kanehisa, M., Yamashita, A.,

Oshima, K., Furuya, K., Yoshino, C., Shiba, T., Hattori, M.,

Ogasawara, N., Hayashi, H. and Hiramatsu, K. (2001) Whole

genome sequencing of methicillin-resistant Staphylococcus aureus.

Lancet 357, 1225–1240.

[8] Letertre, C., Perelle, S., Dilasser, F. and Fach, P. (2003)

Identification of a new putative enterotoxin SEU encoded by

the egc cluster of S taphylococcus aureus. J. Appl. Microbiol. 95,

38–43.

[9] Omoe, K., Hu, D.-L., Takahashi-Omoe, H., Nakane, A.

and Shinagawa, K. (2003) Identification and characteriza-

tion of a new staphylococcal enterotoxin-related putative

toxin encoded by two kinds of plasmids. Infect. Immun.

71, 6088–6094.

[10] Omoe, K., Imanishi, K., Hu, D.-L., Kato, H., Takahashi-Omoe,

H., Nakane, A., Uchiyama, T. and Shinagawa, K. (2004)

Biological properties of staphylococcal enterotoxin-like toxin

type R. Infect. Immun. 72, 3664–3667.

[11] Su, Y.-C. and Wong, A.C.L. (1995) Identification and purification

of a new staphylococcal enterotoxin, H. Appl. Environ. Micro-

biol. 61, 1438–1443.

[12] Becker, K., Roth, R. and Peters, G. (1998) Rapid and specific

detection of toxigenic Staphylococcus aureus: Use of two multi-

plex PCR enzyme immunoassays for amplification and hybrid-

ization of staphylococcal enterotoxin genes, exfoliative toxin

genes, and toxic shock syndrome toxin1 gene. J. Clin. Microbiol.

36, 2548–2553.

[13] Becker, K., Friedrich, A.W., Peters, G. and von Eiff, C. (2004)

Systematic survey on the prevalence of genes coding for staph-

ylococcal enterotoxins SElM, SElO, and SElN. Mol. Nutr. Food

Res. 48, 488–495.

[14] Chen, T.-R., Chiou, C.-S. and Tsen, H.-Y. (2004) Use of novel

PCR primers specific to the genes of staphylococcal enterotoxin

G, H, I for the survey of Staphylococcus aureus strains isolated

from food-poisoning cases and food samples in Taiwan. Int. J.

Food Microbiol. 92, 189–197.

[15] Løvseth, A., Loncarevic, S. and Berdal, K.G. (2004) Modified

multiplex PCR method for detection of pyrogenic exotoxin

genes in staphylococcal isolates. J. Clin. Microbiol. 42, 3869–

3872.

[16] Mehrotra, M., Wang, G. and Johnson, W.M. (2000) Multiplex

PCR for detection for Staphylococcus aureus enterotoxins, exfo-

liative toxins, toxic shock syndrome toxin 1, and methicillin

resistance. J. Clin. Microbiol. 38, 1032–1035.

[17] Omoe, K., Ishikawa, M., Shimoda, Y., Hu, D.-L., Ueda, S. and

Shinagawa, K. (2002) Detection of seg, seh, and sei genes in

Staphylococcus aureus isolates and determination of the entero-

toxin productivities of S. aureus isolates harboring seg, seh, and

sei genes. J. Clin. Microbiol. 40, 857–862.

[18] Lina, G., Bohach, G.A., Nair, S.P., Hiramatsu, K., Jouvin-

Marche, E. and Mariuzza, R. (2004) Standard nomenclature for

the superantigens expressed by Staphylococcus. J. Infect. Dis. 189,

2334–2336.

[19] Baba, T., Takeuchi, F., Kuroda, M., Yuzawa, H., Aoki, K.,

Oguchi, A., Nagai, Y., Iwama, N., Asano, K., Naimi, T., Kuroda,

H., Cui, L., Yamamoto, K. and Hiramatsu, K. (2002) Genome

and virulence determinants of high virulence community-acquired

MRSA. Lancet 359, 1819–1827.

[20] Lindsay, J.A., Ruzin, A., Ross, H.F., Kurepina, N. and Novick,

R.P. (1998) The gene for toxic shock toxin is carried by a family of

mobile pathogenicity islands in Staphylococcus aureus. Mol.

Microbiol. 29, 527–543.

[21] Yarwood, J.M., McCormick, J.K., Paustian, M.L., Orwin, P.M.,

Kapur, V. and Schlievert, P.M. (2002) Characterization and

expression analysis of Staphylococcus aureus pathogenicity island

3. Implications for the evolution of staphylococcal pathogenicity

islands. J. Biol. Chem. 277, 13138–13147.

[22] Zhang, S., Iandolo, J.J. and Stewart, G.C. (1998) The enterotoxin

D plasmid of Staphylococcus aureus encodes a second enterotoxin

determinant (sej). FEMS Microbiol. Lett. 168, 227–233.

[23] Fitzgerald, J.R., Monday, S.R., Foster, T.J., Bohach, G.A.,

Hartigan, P.J., Meaney, W.J. and Smyth, C.J. (2001) Character-

ization of a putative pathogenicity island from bovine Staphylo-

coccus aureus encoding multiple superantigens. J. Bacteriol. 183,

63–70.

[24] Perez-Roth, E., Claverie-Martın, F., Villar, J. and Mendez-

Alvarez, S. (2001) Multiplex PCR for simultaneous identification

of Staphylococcus aureus and detection of methicillin and mup-

irocin resistance. J. Clin. Microbiol. 39, 4037–4041.

[25] Hu, D.-L., Omoe, K., Shimoda, Y., Nakane, A. and Shinagawa,

K. (2003) Induction of emetic response to staphylococcal entero-

toxins in the house musk shrew (Suncus murinus). Infect. Immun.

71, 567–570.

[26] Wright, A., Andrews, P.L.R. and Titball, R.W. (2000) Induc-

tion of emetic, pyrexic, and behavioral effects of Staphylococcus

aureus enterotoxin B in the ferret. Infect. Immun. 68, 2386–

2389.

[27] Banks, M.C., Kamel, N.S., Zabriskie, J.B., Larone, D.H., Ursea,

D. and Posnett, D.N. (2003) Staphylococcus aureus express unique

superantigens depending on the tissue source. J. Infect. Dis. 187,

77–86.

[28] Sergeev, N., Volokhov, D., Chizhikov, V. and Rasooly, A. (2004)

Simultaneous analysis of multiple staphylococcal enterotoxin


genes by an oligonucleotide microarray assay. J. Clin. Microbiol.

42, 2134–2143.

[29] Novick, R.P. (1991) Genetic systems in staphylococci. Methods

Enzymol. 204, 587–636.

[30] Nakane, A., Okamoto, M., Asano, M., Kohanawa, M. and

Minagawa, T. (1995) Endogenous gamma interferon, tumor

necrosis factor, and interleukin-6 in Staphylococcus aureus infec-

tion in mice. Infect. Immun. 63, 1165–1172.



Passive immunisation of hamsters against Clostridiumdifficile infection using antibodies to surface layer proteins

Julie B. O�Brien a,*, Matthew S. McCabe a, Veronica Athie-Morales a,George S.A. McDonald b, Deirdre B. Nı Eidhin a, Dermot P. Kelleher a

a Department of Clinical Medicine, Trinity College Dublin and Dublin Molecular Medicine Centre, St. James�s Hospital, Dublin, Irelandb Department of Histopathology and Morbid Anatomy, Trinity College Dublin, Ireland



Edited by J-I. Flock

Abstract

Clostridium difficile is a major cause of antibiotic-associated diarrhoea and the primary cause of psedomembraneous colitis in

hospitalised patients. We assessed the protective effect of anti-surface layer protein (SLP) antibodies on C. difficile infection in a

lethal hamster challenge model. Post-challenge survival was significantly prolonged in the anti-SLP treated group compared with

control groups (P = 0.0281 and P = 0.0283). The potential mechanism of action of the antiserum was shown to be through enhance-

ment of C. difficile phagocytosis. This report indicates that anti-SLP antibodies can modulate the course of C. difficile infection and

may therefore merit closer investigation for use as constituents of multi-component vaccines against C. difficile associated diarrhoea.


Keywords: Clostridium difficile; Diarrhoea; Surface layer proteins; Hamster model

1. Introduction

Clostridium difficile is a Gram-positive, spore-form-ing, anaerobic bacterium that is recognised as the pri-

mary cause of pseudomembraneous colitis and a

frequent cause of antibiotic-associated diarrhoea [1].

Following disruption of the normal bowel flora by anti-

biotic therapy, C. difficile colonises the gut, resulting in a

spectrum of disease ranging from asymptomatic carriage

to fulminant colitis [1]. C. difficile-associated diarrhoea

(CDAD) is a worldwide problem with major incidencein the elderly and hospitalised populations. A recent

prospective study estimated the annual cost of managing


doi:10.1016/j.femsle.2005.04.005

* Corresponding author. Fax: +353 1 4542043.

E-mail address: [email protected] (J.B. O�Brien).

CDAD in the United States of America at over US $1.1

billion [2]. The human and economic impacts highlight

the need for preventive approaches against CDAD.Two toxins secreted by the bacterium (toxins A and

B) mediate the pathogenesis of disease [1], and an IgG

response against toxin A is correlated with recovery in

humans [3]. Purified inactivated toxins and antibodies

against the toxins have been shown to protect against

CDAD in the hamster model [4]. However, these vac-

cines have not been shown to eradicate infection and

consequently permit persistence of the pool of asymp-tomatic carriers.

Adhesion to the intestinal epithelium is considered an

important primary step for gut colonisation by C. diffi-

cile. Direct binding of the bacterium to Caco-2 and

HT-29 colonic epithelial cell lines, as well as to primary



200 J.B. O’Brien et al. / FEMS Microbiology Letters 246 (2005) 199–205

intestinal epithelial cells, has recently been demonstrated

[5,6]. Two surface layer proteins (SLPs) termed high-

and low-molecular weight (MW) SLPs, form a crystal-

line regular array that covers the surface of the bacterium

[7]. Based on their location, it has been proposed that

SLPs are involved in binding of C. difficile to the intes-tinal epithelium [8]. Native forms of SLPs and a recom-

binant high-MW SLP are able to bind directly to human

gastrointestinal tissue sections [9]. Moreover, antisera

against whole bacteria and the high-MW SLP can re-

duce binding of C. difficile to the gastric epithelial cell

lines HT-29 and Hep-2, respectively [6,9]. SLPs have

been reported to elicit a strong IgG response in patients

infected with C. difficile in some studies [10,11] and highIgM anti-SLP levels have been associated with a reduced

risk of recurrent CDAD in humans [11]. These data sup-

port the use of SLPs as potential candidates for a vac-

cine against CDAD.

We tested the protective capacity of anti-SLP serum

against CDAD in a lethal hamster challenge model,

and showed the ability of high-specificity, high-titre

anti-C. difficile SLP serum to delay the progression ofCDAD but not to prevent death following onset of

CDAD. The acute lethal outcome obtained in the ham-

ster does not directly parallel clinical presentation in hu-

mans, which results in severe disease (fulminant colitis

with ileus, toxic megacolon, perforation and death) in

only 3% of patients [1,12]. This model therefore provides

an extremely stringent test for any protective effect

against CDAD.


2.1. Culture of C. difficile and preparation of SLPs

C. difficile (PCR Ribotype 1; toxin A and B posi-

tive; clindamycin resistant; PHLS UK referenceR13537, Anaerobe Reference Unit, Public Health

Laboratory, University Hospital of Wales) isolated

from a patient with CDAD was used for preparation

of SLPs and hamster challenges. SLPs were purified

from cultures grown anaerobically at 37 �C in BHI/

0.5% thioglycolate broth. Cultures were harvested

and crude SLP extracts made with 8 M urea comple-

mented with protease inhibitors (Complete�, Roche)[7]. The crude SLPs were further purified by anion ex-

change chromatography [13]. Briefly, the crude SLP

preparation was dialysed into 20 mM Tris–HCl pH

8.5 start buffer and applied to an anion exchange col-

umn attached to an AKTA FPLC system (MonoQ

HR 10/10 column, GE Healthcare). The pure SLPs

were eluted with a linear gradient of 0–0.3 M NaCl

at a flow rate of 4 ml/min. Peak fractions correspond-ing to pure SLPs were analysed on 12% SDS–PAGE

gels stained with Coomassie blue.

2.2. Antibody production and immunoblotting

Anti-SLP serum was raised in one New Zealand

White rabbit using 100 lg of purified SLPs at weeks 0,

2 and 4 in Freund�s complete and incomplete adjuvant,

respectively. For immunoblotting, 5 lg of a crude S-layer preparation was separated by SDS–PAGE, elec-

troblotted onto PVDF membrane, and incubated with

a range of dilutions (1:1,000, 1:5,000, 1:10,000,

1:15,000) of pre-immune or immune rabbit antiserum

followed by anti-rabbit HRP. Membranes were devel-

oped using ECL detection (Amersham Biosciences).

2.3. Agglutination assay

Twofold serial dilutions (1:2 to 1:4, 096) of anti-SLP

serum were prepared in duplicate in 96 well U-bottom

microtitre plates in PBS in 50 ll final volumes. C. diffi-

cile suspension was adjusted to an OD600 = 1, and 50

ll added to antiserum dilutions. PBS and rabbit pre-

immune serum served as negative controls. Plates were

incubated for 24 h at 4 �C and the degree of agglutina-tion scored. Endpoint titres were defined as the recipro-

cal of the highest dilution of serum causing strong

agglutination.

2.4. Hamster model of C. difficile infection

Female Golden Syrian hamsters (Charles River Lab-

oratories, UK), 6 to 7 weeks old with an average mass of134.6 g, were used for passive immunisation and chal-

lenge studies. Hamsters were assigned to treatment

groups on the basis of mass so that each group had sim-

ilar average mass. Animals were fed a standard labora-

tory diet ad libitum and caged individually in isolator

cages fitted with disposable air filters to prevent cross-

contamination among animals. Autoclaved food, bed-

ding, cages and filters were used. All animal procedureswere conducted under protocols approved by the Irish

Department of Health and Children and the Trinity

College BioResources Unit Committee.

Hamsters used for model optimisation were screened

for C. difficile carriage by culturing the bacterium from

their faeces. No C. difficile was found by this method

and all animal work thereafter was carried out under

identical conditions. Following infection, C. difficile iso-lated from perianal swabs was identified by culture in an

anaerobic environment. Presumptive C. difficile re-iso-

lated from hamsters in a preliminary experiment was

checked by immunoblotting with the rabbit anti-SLP

serum and invariably reacted identically to the infecting

type (data not shown).

Hamsters were given a 2 mg dose of clindamycin-HCl

(Sigma) orogastrically to predispose them to C. difficile

infection and challenged with 105 CFU 4 h later. For

experimental hamsters (n = 8), the challenge inoculum

J.B. O’Brien et al. / FEMS Microbiology Letters 246 (2005) 199–205 201

was pre-incubated with 100 ll of anti-SLP serum for 30

min at 37 �C and additional 100 ll antiserum doses were

given orogastrically at – 7, 6, 17 and 24 h of infection.

Antiserum was co-administered with 0.1 M sodium car-

bonate buffer, pH 9.6, to neutralise gastric acid. Control

hamsters (n = 8) were treated identically, but were givenan irrelevant rabbit antiserum raised against the malt-

ose-binding protein of Escherichia coli (anti-MBP ser-

um). A C. difficile only group, which received no

antiserum, was also included (n = 4).

From the day after infection, hamsters were observed

two-hourly in a blinded fashion by three individual

observers for 72 h, and four times a day at regular inter-

vals thereafter. Grading was as follows: 0, normal; 1,loose faeces or wet perianum, activity close to normal;

2, reduced activity, still responding to stimuli, tender

abdomen; 3, hunched, inactive, tender abdomen, loss

of balance, ruffled fur. Hamsters were sacrificed at grade

3. Time of sacrifice or last time seen alive (whichever was

earlier) was considered the endpoint.

To confirm C. difficile as the causative agent of dis-

ease, perianal swabs (no formed faeces due to diar-rhoea) were taken in a random order from a

representative number of symptomatic hamsters

(n = 5) and cultured anaerobically for four days on

blood agar plates containing 50 lg/ml clindamycin.

Caecum and colon samples were taken from a repre-

sentative number of animals (one hamster per group)

to confirm the typical epithelial damage seen in

CDAD. Tissues were fixed in 10% formalin andstained with haematoxylin and eosin.

2.5. C. difficile phagocytosis assay

C. difficile cultured on Columbia blood agar plates

was resuspended into 1 ml of RPMI-1640 medium (Gib-

coBRL) containing 5 lM 5-(and -6)-carboxyfluorescein

diacetate, succinimidyl ester (CFSE) (Molecular Probes)and stained for 10 min at 37 �C in the dark. The stained

bacteria were washed with RPMI/50% FCS and bacte-

rial concentration determined using OD600.

Human monocytic THP-1 cells were grown in RPMI

medium supplemented with 10% FCS, 2 mM L-gluta-

mine, 100 lg/ml penicillin and 100 U/ml streptomycin

at 37 �C in 5% CO2. THP-1 cells (1 · 106 cells per well

in 24-well plates) were induced to differentiate with100 nM PMA in complete medium for 72 h. Prior to

exposure to C. difficile, THP-1 cells were washed twice

with RPMI/10% FCS, mixed with the CFSE-C. difficile

(MOI of 400) and incubated at 37 �C in 5% CO2 for 2 h.

Cells were washed three times with PBS to remove de-

tached THP-1s and non-phagocytosed C. difficile.

THP-1s were detached with 250 ll per well of pre-cooleddetaching buffer (5 mM EDTA, 0.5% FCS in HBSS) for20 min at 200 rpm at 4 �C and washed three times with

pre-cooled PBS.

Green fluorescent emission of the phagocytosed C.

difficile was assessed by flow cytometry analysis. Single

THP-1 cells were gated using FSC and SSC. Events

(30,000) were acquired using a FACSCalibur flow

cytometer and data was analysed with Cell Quest soft-

ware (Becton Dickinson). The fluorescence of cell-surface attached C. difficile was quenched immediately

prior to acquisition by addition of 0.8 mg/ml crystal

violet.

The effects of anti-SLP and anti-MBP sera (12.5%) on

phagocytosis were assessed by co-incubation with the C.

difficile and THP-1 cells. To assess opsonising activity

due to complement, the anti-SLP serum was heat-trea-

ted at 56 �C for 30 min to inactivate complement. E. coliDH5a (pKFW408) expressing a modified GFP was used

as a positive control for phagocytosis (Fig. 3(d)).

2.6. Statistical analysis

Differences in mean survival time and mean time to

first symptoms between experimental and control

hamsters were tested for significance by a non-para-metric two-tailed Mann Whitney test (GraphPad

InStat). A P-value <0.05 was considered statistically

significant.

3. Results

3.1. SLP purification, antiserum production and

agglutination of C. difficile by anti-SLP

Based on their location on the outer bacterium sur-

face and their in vitro capacity to bind to human gastro-

intestinal tissues (9), SLPs represent a strong vaccine

candidate for targeting C. difficile colonisation and

CDAD. To develop an anti-SLP serum for passive

immunisation experiments, we initially purified SLPsto homogeneity from a crude S-layer preparation by an-

ion exchange chromatography (Fig. 1(a)). Pure SLPs

were then used to raise rabbit polyclonal antibodies,

which reacted strongly with both the high- and low-

MW SLPs by immunoblotting against a crude SLP

preparation (Fig. 1(b)). Pre-immune rabbit serum

showed no reactivity within a wide range of concentra-

tions (Fig. 1(b) and data not shown). Anti-SLP serumdid not recognise toxins as demonstrated by immuno-

blotting against a total C. difficile lysate (Fig. 1(b)).

The anti-MBP serum (diluted 1:100) showed no reactiv-

ity against a total C. difficile lysate by immunoblotting

(data not shown).

To corroborate that the anti-SLP serum was capable

of recognising SLPs on the surface of intact whole

C. difficile, we conducted in vitro agglutination experi-ments using twofold serial dilutions of anti-SLP or pre-

immune antiserum. The anti-SLP antibodies readily

Fig. 1. (a) Coomassie blue stained 12% SDS–PAGE gel showing purified C. difficile surface layer proteins (SLPs). MWmarker (lane 1), crude S-layer

preparation made with 8 M urea (lane 2), anion exchange purified SLPs (lane 3). (b) Immunoblot of the crude S-layer preparation probed with pre-

immune rabbit serum 1:15,000 (lane 1) and anti-SLP rabbit serum 1:15,000 (lane 2). C. difficile lysate probed with anti-SLP rabbit serum 1:15,000

(lane 3).


agglutinated whole C. difficile cells, with a titre of 512 as

compared to a titre of 8 for the pre-immune serum.

3.2. Passive immunisation with anti-SLP prolonged

survival in C. difficile infected hamsters

We tested the ability of passively administeredanti-SLP serum to protect against CDAD in a lethal

hamster challenge model. All animals were predisposed

1 2 30

50

100

150

200

250*

anti-

SLP

seru

m

anti-

MB

Pse

rum

untr

eate

d

post

-hc

lale

ng

us erv

ilav,h

Fig. 2. Passive immunisation with anti-SLP serum prolongs survival in

C. difficile-infected hamsters. Following clindamycin treatment ham-

sters were given anti-SLP serum (group 1, n = 8) or anti-MBP serum

(group 2, n = 8) or no serum (group 3, n = 4). All groups received 105

C. difficile. T0 = time of administration of bacteria. Dots (�) representindividual hamsters, bars represent median values for each group.

Post-challenge survival was significantly prolonged in experimental

hamsters compared to hamsters treated with anti-MBP serum

(*P = 0.0281) and untreated hamsters (*P = 0.0283). Differences in

means were calculated using a non-parametric two-tailed Mann

Whitney test.

to C. difficile infection by clindamycin treatment, fol-

lowed by passive immunisation with anti-SLP serum,

anti-MBP serum or no serum. All three groups were

then challenged with C. difficile (105 CFU). Hamsters

from all three groups developed diarrhoea within two

to five days post-challenge, showing no significant differ-

ence in time to first symptoms (data not shown). How-ever, post-challenge survival (Fig. 2) was significantly

prolonged in animals treated with anti-SLP serum

(group 1) as compared to animals treated with anti-

MBP serum (group 2) (P = 0.0281) and untreated ani-

mals (group 3) (P = 0.0283) (Table 1). Seven out of eight

(group 2) and three out of four (group 3) control ham-

sters as compared to two out of eight (group 1) anti-

SLP treated hamsters had died at 72 h post-challenge.The pathology was confirmed as CDAD by the

typical epithelial damage observed on histological

examination of caecum and colon sections. C. difficile

was confirmed as the causative agent of the pathology

by isolation from perianal swabs. Since all hamsters,

independently of the treatment group, were sampled

Table 1

Post-challenge survival time of hamsters passively immunised with

anti-SLP serum and challenged with 105 C. difficile

Groupa Median post-challenge survival (h)b Range (h)

1 156.9 62.5, 221.5

2 76.6 55.2, 143.0

3 69.6 55.3, 126.4

a Group 1 received anti-SLP serum plus 105 C. difficile, group 2

received anti-MBP serum plus 105 C. difficile, group 3 received 105 C.

difficile only.b Post-challenge survival time was measured from time of adminis-

tration of C. difficile.


at the same advanced stage of the infection and since

volume samples are physically impossible to standard-

ise, C. difficile counts in perianal swabs were not

determined. Since all hamsters eventually died from

CDAD, severe inflammation was observed to the same

Fig. 3. Anti-SLP serum enhances C. difficile phagocytosis by PMA differentia

(grey histogram), CFSE-C. difficile (open histogram). (b) Crystal violet q

histogram), plus 1.6 mg/ml crystal violet (open histogram, dashed line), plu

difficile (grey histogram). (c) C. difficile phagocytosis by THP-1s. THP-1s alon

E. coli DH5a (pKFW408) expressing a modified GFP was used as a pos

histogram). (e) In the presence of 12.5% anti-SLP serum (open histogram) o

(grey histogram). (f) In the presence of anti-SLP serum (open histogram) an

alone (grey histogram). Graphs are representative of at least three independ

extent in histological sections from all groups (data

not shown). The present data show that although pas-

sive immunisation with anti-SLP serum was unable to

delay the onset of CDAD and prevent animal death,

it did significantly prolong survival.

ted THP-1 cells. (a) CFSE staining of C. difficile. Unstained C. difficile

uenching of CFSE-C. difficile fluorescence. CFSE-C. difficile (open

s 0.8 mg/ml crystal violet (open histogram, dotted line), unstained C.

e (grey histogram), THP-1s and CFSE-C. difficile (open histogram). (d)

itive control for phagocytosis (open histogram), THP-1s alone (grey

r 12.5% anti-MBP serum (open histogram, dashed line), THP-1s alone

d heat-treated anti-SLP serum (open histogram, dotted line), THP-1s

ent experiments.


3.3. Anti-SLP enhanced C. difficile phagocytosis

We considered that the effect of the anti-SLP serum

on post-challenge survival of the hamsters might be

mediated by increasing phagocytosis of C. difficile. To

investigate this possibility, phagocytosis of fluorescentstained C. difficile by differentiated THP-1 monocytes

was assessed in the presence and absence of anti-SLP

serum. Using this technique, 100% of C. difficile were

consistently stained with CFSE (Fig. 3(a)). CFSE-C. dif-

ficile fluorescence could be readily quenched with crystal

violet (Fig. 3(b)) to eliminate fluorescence from cell-

surface attached C. difficile. The levels of phagocytosis

of CFSE-C. difficile were low (17.97%) (Fig. 3(c)). E. coliDH5a (pKFW408) expressing a modified GFP was used

as a positive control for phagocytosis (Fig. 3(d)). The

addition of the anti-SLP serum resulted in markedly in-

creased phagocytosis of C. difficile (69.19%) as com-

pared to the anti-MBP serum (Fig. 3(e)). To eliminate

the possibility that this increase was due to complement,

the anti-SLP serum was heat-treated to inactivate com-

plement before the phagocytosis assay. No major contri-bution of complement to the phagocytosis was found

(Fig. 3(f)). These results suggest that the anti-SLP anti-

bodies may exert at least some of their protective effect

in vivo by enhancing phagocytosis of C. difficile.

4. Discussion

C. difficile toxins are known to mediate many of the

pathogenic features of CDAD. Previous studies have

shown protection of hamsters against CDAD using anti-

toxin antibodies through neutralisation of enterotoxicity

[4,14]. In one of these studies, administration of mouse

anti-toxin ascites protected 100% of hamsters from

death [4]. Although the role of anti-toxin immunity in

protection from CDAD in the hamster model is clear,vaccines based on toxins are unlikely to prevent coloni-

sation, and carriage and transmission of C. difficile will

therefore remain a persistent threat. Hence, a more com-

plete approach against CDAD should consider not only

the inhibition of toxicity but also the prevention of bac-

terial colonisation. The SLPs of C. difficile are potential

colonisation agents thought to be involved in bacteria–

host interactions [5,6,9]. It has been shown that anti-SLP antibodies decrease the direct binding of C. difficile

to human gastrointestinal tissue sections in vitro [9]. We

tested whether anti-SLP antibodies, assessed indepen-

dently of the toxins, could have a protective effect

against CDAD in vivo. Here we demonstrate that pas-

sive immunisation using anti-SLP antibodies signifi-

cantly delays progress of C. difficile infection in a

lethal hamster challenge model. To the authors� knowl-edge, this report identifies anti-SLP antibodies as poten-

tially playing a role in the modulation of disease

progression in an in vivo model of C. difficile infection

for the first time.

The recognition and interaction of the anti-SLP anti-

bodies with the SLPs on intact C. difficile could be hin-

dered by spatial and temporal constrains inherent to the

in vivo setting, allowing for the presence of free bacteria.To minimise this possibility in our system, the C. difficile

inoculum was pre-incubated with the respective antisera

prior to infection, an approach that has been success-

fully used elsewhere in passive immunisation for gastro-

intestinal pathogens [15].

The mechanism of action of the anti-SLP serum is

most likely multifactorial. Following C. difficile coloni-

sation of the gut epithelium and subsequent toxin pro-duction, the epithelial barrier is severely disrupted

resulting in an influx of phagocytic cells. Despite the

presence of phagocytic cells, C. difficile can persist in this

inflammatory exudate [16]. Using an in vitro assay, we

demonstrated a marked increase in phagocytosis of C.

difficile in the presence of the anti-SLP serum. These re-

sults are in agreement with Dailey et al. [16], who

showed that C. difficile requires opsonisation for signif-icant phagocytosis to occur. These data suggest that the

prolonged survival of anti-SLP treated hamsters might

be explained through an increase in phagocytosis of C.

difficile at damaged mucosal surfaces. However, as

shown in vitro, the anti-SLP serum might also inhibit

colonisation by agglutinating C. difficile in vivo and

therefore inhibiting binding of the bacterium to the epi-

thelium. A correlation between antibody-mediatedagglutination of C. difficile and protection against death

has previously been demonstrated in a hamster study

[17].

Hamsters are highly sensitive to the C. difficile toxins

and once their systemic action occurs the animals even-

tually die. In our study, all hamsters showed a similar

time to first symptoms and eventually died, indicating

that independently of treatment all were exposed tothe enterotoxins. Hence, the prolonged survival in the

anti-SLP treated group most likely reflects exposure to

lower toxin concentrations from a lower bacterial load

achieved by increased phagocytosis.

Based on our study it seems possible that the develop-

ment of a strong, specific mucosal immune response

against the SLPs has the potential to result in improved

long-term protection and elimination of bacterial car-riage. Although the antiserum used in this study was

IgG, it was delivered directly to the mucosal surface,

thereby mimicking a mucosal immune response. This

approach has been successfully used with IgG and IgY

anti-toxin antibodies in the hamster model of CDAD

[4,18].

In conclusion, our findings point to a role for SLPs in

the design of effective vaccines against CDAD. We pro-pose SLPs as candidate components of a multi-compo-

nent vaccine against C. difficile. In this study we used


a C. difficile strain from the most commonly occurring

ribotype, Ribotype 1, which accounts for over half of

all the hospital isolates from England and Wales [19].

Sequencing of SLPs from three clinical isolates from

Ribotype 1 from different C. difficile episodes revealed

100% sequence identity (manuscript in preparation).However, given the well-known heterogeneity among

SLPs from different C. difficile strains [7,8], an effective

vaccine offering cross-protection between strains should

contain multiple SLPs. An active immunisation regimen

based on the SLPs combined with toxin neutralisation

could provide both a preventative and therapeutic vac-

cine strategy by reducing bacterial colonisation and car-

riage, as well as toxin-mediated pathology.

Acknowledgements

This work was supported by an Advanced Technolo-

gies Research Programme (Grant No. 01/165) fromEnterprise Ireland. Thanks to Dr. Kenneth F. Whelan,

Institute of Molecular Medicine, for providing E. coli

DH5a (pKFW408).

References

[1] Kelly, C.P. and LaMont, J.T. (1998) Clostridium difficile infection.

Annu. Rev. Med. 49, 375–390.

[2] Kyne, L., Hamel, M.B., Polavaram, R. and Kelly, C. (2002)

Health care costs and mortality associated with nosocomial

diarrhea due to Clostridium difficile. Clin. Infect. Dis. 34, 346–353.

[3] Kyne, L., Warny, M., Qamar, A. and Kelly, C.P. (2001)

Association between antibody response to toxin A and protection

against recurrent Clostridium difficile diarrhoea. Lancet 357, 189–

193.

[4] Giannasca, P.J., Zhang, Z., Lei, W., Boden, J.A., Giel, M.A.,

Monath, T.P. and J.R., W.D. (1999) Serum antitoxin antibodies

mediate systemic and mucosal protection from Clostridium

difficile disease in hamsters. Infect. Immun. 67, 527–538.

[5] Cerquetti, M., Serafino, A., Sebastianelli, A. and Mastrantonio,

P. (2002) Binding of Clostridium difficile to Caco-2 epithelial cell

line and to extracellular matrix proteins. FEMS Immunol. Med.

Microbiol. 32, 211–218.

[6] Drudy, D., O�Donoghue, D.P., Baird, A., Fenelon, L. and

O�Farrelly, C. (2001) Flow cytometric analysis of Clostridium

difficile adherence to human intestinal epithelial cells. J. Med.

Microbiol. 50, 526–534.

[7] Cerquetti, M., Molinari, A., Sebastianelli, A., Diociaiuti, M.,

Petruzzelli, R., Capo, C. and Mastrantonio, P. (2000) Character-

isation of surface layer proteins from different Clostridium difficile

clinical isolates. Microb. Pathog. 28, 363–372.

[8] McCoubrey, J. and Poxton, I.R. (2001) Variation in the surface

layer proteins of Clostridium difficile. FEMS Immunol. Med.

Microbiol. 31, 131–135.

[9] Calabi, E., Calabi, F., Phillips, A.D. and Fairweather, N.F. (2002)

Binding of Clostridium difficile surface layer proteins to gastro-

intestinal tissues. Infect. Immun. 70, 5770–5778.

[10] Pantosti, A., Cerquetti, M., Viti, F., Ortisi, G. and Mastrantonio,

P. (1989) Immunoblot analysis of serum immunoglobulin G

response to surface proteins of Clostridium difficile in patients

with antibiotic-associated diarrhea. J. Clin. Microbiol. 27, 2594–

2597.

[11] Drudy, D., Calabi, E., Kyne, L., Sougioultzis, S., Kelly, E.,

Fairweather, N. and Kelly, C.P. (2004) Human antibody response

to surface layer proteins in Clostridium difficile infection. FEMS

Immunol. Med. Microbiol. 41, 237–242.

[12] Borriello, S.P., Ketley, J.M., Mitchell, T.J., Barclay, F.E., Welch,

A.R., Price, A.B. and Stephen, J. (1987) Clostridium difficile – a

spectrum of virulence and analysis of putative virulence determi-

nants in the hamster model of antibiotic-associated colitis. J. Med.

Microbiol. 24, 53–64.

[13] Takeoka, A., Takumi, K., Koga, T. and Kawata, T. (1991)

Purification and characterisation of S layer proteins from Clos-

tridium difficile GAI 0714. J. Gen. Microbiol. 137, 261–267.

[14] Lyerly, D.M., Bostwick, E.F., Binion, S.B. and Wilkins, T.D.

(1991) Passive immunization of hamsters against disease caused

by Clostridium difficile by use of bovine immunoglobulin G

concentrate. Infect. Immun. 59, 2215–2218.

[15] Blanchard, T.G., Czinn, S.J., Maurer, R., Thomas, W.D., Soman,

G. and Nedrud, J.G. (1995) Urease-specific monoclonal antibod-

ies prevent Helicobacter felis infection in mice. Infect. Immun. 63,

1394–1399.

[16] Dailey, D.C., Kaiser, A. and Schloemer, R.H. (1987)

Factors influencing the phagocytosis of Clostridium difficile

by human polymorphonuclear leukocytes. Infect. Immun. 55,

1541–1546.

[17] Torres, J.F., Lyerly, D.M., Hill, J.E. and Monath, T.P. (1995)

Evaluation of formalin-inactivated Clostridium difficile vaccines

administered by parenteral and mucosal routes of immunization

in hamsters. Infect. Immun. 63, 4619–4627.

[18] Kink, J.A. and Williams, J.A. (1998) Antibodies to recombinant

Clostridium difficile toxins A and B are an effective treatment and

prevent relapse of C. difficile-associated disease in a hamster

model of infection. Infect. Immun. 66, 2018–2025.

[19] Brazier, J.S. (1998) The epidemiology and typing of Clostridium

difficile. J. Antimicrob. Chemother. 41 (Suppl. C), 47–57.



Morphological and molecular taxonomy of Pythium longisporangiumsp. nov. isolated from the Burgundian region of France

Bernard Paul a,*, Kanak Bala a, Sabine Gognies b, Abdel Belarbi b

a Laboratoire des Sciences de la Vigne, Institut Jules Guyot, Universite de Bourgogne, B.P. 27877, 21078 Dijon, Franceb Laboratoire de Microbiologie Generale et Moleculaire, Universite de Reims, Europol Agro, B.P. 1039, 51687 Reims cedex 2, France

Received 16 March 2005; received in revised form 6 April 2005; accepted 7 April 2005


Edited by R.C. Staples

Abstract

During the course of an investigation on the Pythiaceous oomycetes occurring in the Burgundian vineyards, some species of

Pythium possessing mainly hypogynous antheridia were found. These had been classified as oomycetes belonging to the ‘‘Pythium

rostratum’’ group for a long time. Three of these isolates, having similar structures and growth, are very closely related to a recently

described species, Pythium bifurcatum Paul. A close look at these, however, underlines some fundamental differences with the latter.

Not all of them produce zoospores but have very large sporangia. The type specimen is F-1200 (B 76a) which is a medium-slow

growing saprophyte. The sequence of the ITS region of the rDNA also shows a very close relationship with P. bifurcatum. On

the basis of morphological and molecular analysis, we now describe this species as Pythium longisporangium sp. nov. Morphological

features of this new species, the sequences of the ITS region of its nuclear ribosomal DNA, and its comparison with related species

are discussed.


Keywords: Pythium longisporangium; Sporangia; Oogonia; Antheridia; Oospores; ITS region; rRNA

1. Introduction

The genus Pythium is a very common oomycete

found all over the world. In 1990, it was known to have

more then 130 species [1]. The first author of this man-

uscript has also added more then 20 species to this genussince that date. Many of the isolates belonging to this

genus are currently being studied for their structural

and molecular diversity. Some of these are also being

studied for their mycoparasitic activities on fungal

pathogens like Botrytis cinerea [2]. Most of the members

of the genus Pythium live in soil or aquatic environments

as saprophytes, however, some of them are known to be


doi:10.1016/j.femsle.2005.04.004

* Corresponding author. Tel./fax: +33 3 80396326.

E-mail address: [email protected] (B. Paul).

very destructive plant pathogens, inflicting serious eco-

nomic losses of crops by destroying seed, storage or-

gans, roots, and other plant tissues [3]. Although, the

majority of these organisms produce biflagellate zoo-

spores, they are no longer considered as ‘‘aquatic fungi’’

and unlike most of the eumycetes, the members of thisgroup remain diploid throughout their life cycles with

meiosis occurring in the gametangia before fertilization.

The entire group of the oomycetes are now supposed to

be closer to algae (Phaeophyta and Chrysophyta) and

higher plants. These are now classified in the Kingdom

Stramenopila, one of the eukaryotic Kingdoms which

includes water molds and brown algae. The position of

the oomycetes as a unique lineage of stramenopileeukaryotes, unrelated to true fungi but closely related

to heterokont (brown) algae, has been well established



208 B. Paul et al. / FEMS Microbiology Letters 246 (2005) 207–212

using molecular phylogenies that are based on ribo-

somal RNA (rRNA) sequences [4–6].

The taxonomy of the genus Pythium was mainly

based on the morphological descriptions like the size

and shape of oogonia, antheridia, and sporangia. Keys

provided by Middleton [7], Plaats-Niterink [8] andWaterhouse [9] are all based on these morphological

characters and are still indispensable. However these

are now being supplemented with molecular characteris-

tics. Comparative studies of the internal transcribed

spacer (ITS) regions of the ribosomal RNA genes

(rDNA) have become a useful tool in fungal taxonomy

as these regions evolve sufficiently rapidly to distinguish

different species within a genus [10,11]. The ITS se-quence data can provide valuable information on

‘‘new’’ or undescribed taxa, as sequence diversity may

support the erection of new species [12].

The morphology of Pythium longisporangium is typi-

cal for the genus Pythium. It is a slow growing oomy-

cete, having large spherical, globose, to cylindrical

sporangia; smooth-walled oogonia and mostly hypogy-

nous antheridia. Sexual structures are readily formedin water and on solid media within a week. The morpho-

logical details of the new species together with the se-

quences of its ITS region of the ribosomal nuclear

DNA, comparison of morphological and molecular

characteristics with related species are discussed in this

article.


2.1. Fungal and oomyceteous material

P. longisporangium (Type strain, F-1201/B76a) was

isolated from soil samples taken in a vineyard in Mar-

sannay situated in the outskirts of the French city of Di-

jon in the Burgundian region by using the usual baitingtechniques [8,13]. It occurred thrice out of 65 such sam-

ples and was purified by repeated washing with sterile

distilled water and sub-culturing on solid media like po-

tato carrot agar (PCA) and corn meal agar (CMA). All

these isolates are maintained at ‘‘Institut Jules Guyot’’,

in Dijon, France. P. longisporangium was identified with

the help of keys provided by Middleton [7], Plaats-Nit-

erink [8] and Waterhouse [9] and also by its ITS se-quences using the BLAST search.

2.2. DNA isolation and PCR

All three isolates of P. longisporangium were grown in

PDB (potato dextrose broth) The culture conditions,

DNA isolation and the PCR of the ITS of the ribosomal

nuclear DNA was done using the procedures describedearlier [14,15]. Universal primers ITS1 (TCC GTA

GGT GAA CCT GCG G) and ITS4 (TCC TCC GCT

TAT TGA TAT GC) were synthesised and the DNA se-

quence was realised by Oligo Express (Paris). ITS1 is at

the 3 0 end of the 18S rDNA gene and ITS4 is at the 5 0

end of the 28S rDNA gene. The sequences obtained

were compared with the ITS1 sequences of related spe-

cies of Pythium: Pythium bifurcatum (Genbank accessionnumber AY083935), Pythium longandrum (AY039713),

P. sp. strain F-74 (AY455695), Pythium hypogynum

(AY455804),Pythium terrestris (AY039714), andPythium

segnitium (AY149173). Pythium sp. strain F-1216

(AY455697), Pythium canariense (AY06561) and Pythium

rostratum (AJ233456). The sequence of the ITS

region of the nuclear ribosomal DNA of P. longisporan-

gium (F-1200) has been deposited in Genbank.

3. Results

3.1. Morphological descriptions

P. longisporangium PAUL sp. nov. (Figs. 1–3).

Sporangia globosa, subglobosa, intercalaria, interdumterminalia, 25–50 lm diam., zoosporae non-observata.

Oogonia laevia, globosa, terminalia raro intercalaria,

17–36 lm diam, Antheridia hypogynata vel monoclinata.

cellulae antheridiales inflatae interdum bifurcatae, unam

vel duas in singulo oogonia. Oogonia continentia unam,

interdum duas oosporas pleroticas 16–35 lm diam, globo-

sas, 1.5–2 lm crassi tunicatas. Interdum oogonia et anthe-

ridia oriuntur ex appressorio. Incrementum radiale

quotiadianum 11 mm 25 �C in agaro Solani tuberosi et

Dauci carotae (PCA). Holotypus in herbario Universita-

tus Bourgogne conservatus (F-1200).

Etymology: The oomycete is being named as P. lon-

gisporangium because of the form of its sporangia which

often are pyriform to lemoniform and are formed in

abundance.

Mycelium hyaline, well branched. Main hyphae upto 6–8 lm wide. Colonies on PCA are submerged and

show a narrow chrysanthemal pattern. Average radial

growth of the oomycete at 25 �C on PCA is 11 mm/

day. It grows well in water on hemp-seed halves and

produces asexual and sexual structures at room temper-

atures (18–25 �C).Sporangia (Fig. 1, (a)–(d)) are globose to somewhat

cylindrical, oval, and at times peanut shaped mostlycatenulate and intercalary (Fig. 1(b)); measuring 15–55

lm in diameter (av. 32.6 lm) and up to 65 lm in length.

These structures are densely granulated and the larger

ones have a clear hyaline central zone which is sparsely

granulated (Fig. 1, (c)). Zoospores were not observed

despite repeated flooding of the cultures by distilled

water, tap water or pond water. The sporangia germi-

nate directly through germ tubes to produce a newmycelium (Fig. 1, (a, d)).

Fig. 1. (a)–(d): Sporangia of Pythium longisporangium. (a, c): terminal sporangia, (b): intercalary sporangia; (d): sporangia germinating through a

germ tube; (e)–(h): antheridia and oogonia of Pythium longisporangium. (e): Hypogynous antheridia with bulbous antheridial cells; (f): antheridia

showing intercalary antheridial cells; (g): intercalary oogonia with hypogynous antheridia and two bulbous antheridial cells; (h): fertilization. All

figures bar = 40 lm.

B. Paul et al. / FEMS Microbiology Letters 246 (2005) 207–212 209

The oomycete reproduces sexually by forming anthe-

ridia and oogonia plentifully in water cultures on hemp-seed halves and also on solid media like PCA. Oogonia

are smooth walled, spherical, terminal, sub-terminal and

intercalary (Fig. 1, (e)–(h)), measuring 17–36 lm in

diameter (av. 19.5 lm) and filled with dense, coarsely

granulated protoplasm.

All the oogonia are supplied with antheridia which

are usually hypogynous (Fig. 1, (e)–(h)) or monoclinous

sessile (Fig. 1, (c)). Each oogonium is supplied by 1–3antheridial cells which are at times borne in a catenulate

fashion on one antheridial branch (Fig. 1, (f), 2, (c, d)).

Very rarely diclinous antheridia are also present (Fig. 2,

(a, b)). After fertilization the oogonia is found attachedwith one or two balloon-shaped antheridial cells (Fig. 1,

(h), 2, (c, d, f)).

Oospores are plerotic or nearly so (Fig. 2, (e)–(h)),

spherical, usually one per oogonium (Fig. 2, (e)), occa-

sionally two per oogonium (Fig. 2, (f)) and rarely three

in a single oogonium (Fig. 2, (g)). In the intercalary

oogonia these can be aplerotic (Fig. 2, (h)); smooth-

walled, measuring 12–22 lm in diameter (av. 18.1 lm).The oospore wall is relatively thin, measuring 1–1.5

lm in thickness.

Fig. 2. Sexual reproduction of Pythium longisporangium. (a, b): Oogonia provided with diclinous antheridia; (c): oogonia provided with hypogynous

antheridia,antheridial branch bearing intercalary antheridial cells; (d): oogonia provided with hypogynous antheridia and bulbous antheridial cells;

(e): plerotic, single oospores; (f): two oospores per oogonium; (g): three oospores per oogonium; (h): intercalary oogonia having a single aplerotic

oospore. (a, c) bar = 50 lm, (b, d, e–h) bar = 25 lm.


3.2. Internal transcribed spacer region

The ITS region of the nuclear ribosomal DNA of P.

longisporangium (Genbank accession number

AY455693) is comprised of 890 bases which are:

1 ccacacctaa aaactctcca cgtgaactgt ttgtatcaga ttagcgc-

caa gattttcgtg

61 cgtgtttgtg gtatcactat gtattcgtac gtggtgttag caag-

cattgt atggagcttg121 gctgatcgaa ggtcggtgcg caccttgtgt gtgtattggc tgat-

taacct tttaaaccct

181 ttcaataaat actgattata ctgtaaggac gaaagtcttt

gcttttatct agataacaac

241 tttcagcagt ggatgtctag gctcgcacat cgatgaagaa

cgctgcgaac tgcgatacgt301 aatgcgaatt gcagaattca gtgagtcatc gaaattttga acg-

catattg cactttcggg

361 ttatacctgg aagtatgtct gtatcagtgt ccgtacatca

aacttgcctc tttttgtcgg

421 tgtagtccga ttgagagtat ggcagacgtg aggtgtctcg

cgactcgtat atcattgtgt

481 gtgtaaatcg taagagatac atacataagg tagtatataa

cttgttgcga gtccctttaa541 aacgacacga tctttctatt tgctttctat ggagcgtcta

tttcgaacgc ggtggtcctc

601 ggatcgcttg cagtcggcag cgacttcagt gaagacatag

tgaagaaacc tctattcgcg

Fig. 3. CLUSTAL W, multiple sequence alignment of ITS1 regions of the rDNA of Pythium longisporangium, P. bifurcatum, P. longandrum, P.

hypogynum, P. terrestris, P. segnitium, P. rostratum, P. canariense, P. sp. (F-74), and P. sp. (F-1216).

B. Paul et al. / FEMS Microbiology Letters 246 (2005) 207–212 211

661 gtacgttagg cttcggctcg acaatgttgc gttctagtgt

gtggactccg ttttcgcttt

721 gaggtgtact gttcggttgt gggtttgagc cttggtattg ctttgt-

tagt agagatatgt

781 cgatatttct gtagtttgat tctgcataca cgcaagtgtgtgtgggtaga gagtatctat

841 ttgggaaatt ttgtactgcg tgcgctttcg agtgtgtgta

tgtatctcaa

1–236 = ITS1 complete sequence, 237–395 = 5.8 S

gene (in bold) complete sequence, 396–890 = ITS 2 com-plete sequence.

Table 1

Comparison of morphological features of Pythium longisporangium

and P. bifurcatum

Characters Pythium longisporangium Pythium bifurcatum

Sporangia Intercalary and catenulate,

15–55 lm (av. 32.6 lm)

Intercalary and

terminal, 25–50 lm(av. 35 lm)

Oogonia

diameter

13–23 lm (av. 19.5 lm) 17–36 lm(av. 26.7 lm)

Antheridia Hypogynous, monoclinous sessile,

diclinous, antheridial cells at times

catenulate

Hypogynous,

monoclinous sessile

Oospore Usually one, frequently 2 and

rarely 3 per oogonium,

12–22 lm (av. 18.1 lm).

Usually 1, rarely 2,

per oogonium,

16–35 lm(av. 24.9 lm)


The comparison of the ITS1 sequences of P. longisp-

orangium and related species is given in Fig. 3 in the

form of CLUSTAL multiple alignments.

4. Discussion

P. longisporangium (F-1200) is a slow growing speciesthat falls within the category of species having spherical

non-proliferating, catenulate sporangia, smooth walled,

globose oogonia which are provided by hypogynous

antheridia. These characters bring this species very close

to a recently-described species, P. bifurcatum PAUL

from northern France. However the ITS sequences of

these species are very different, and a close look at the

morphological characters shows that they are distinctbut closely related (Table 1). The presence of triple

oospores and intercalary chained-antheridial cells are

unique for the taxon.

The ITS region of the nuclear ribosomal DNA of P.

longisporangium is comprised of 890 bases and a BLAST

search gives the closest resemblance of this oomycete

with P. bifurcatum having 99.6% similarity (Genbank

accession number AY083935); 96.2% with P. longan-

drum AY039713), 94.9% with an un-described species

P. sp strain F-74, AY455695) 84.6% with P. terrestris

(AY039714), P. hypogynum (AY455804), and another

un-described P. sp. Strain F-1216 (AY455697). Other

species with hypogynous type of antheridia that comes

close to P. longisporangium but having little resem-

blances as far as the ITS sequences concerned are:

P. segnitium (AY149173) 74.8% similarities, P. canar-iense (AY06561) having 60.7% similarity and P. rostra-

tum (AJ233456) having only 57.6% similarity with the

ITS 1 of P. longisporangium.

The morphological and molecular characteristics

indicate that the closest relative of P. longisporangium

is P. bifurcatum, a new species recently described from

northern France. However there are enough structural

as well as molecular differences between the two (Table

1) to justify the creation of a new taxon.

References

[1] Dick, M.W. (1990) Keys to Pythium. University of Reading Press,

Reading, UK.

[2] Paul, B. (1999) Pythium periplocum, an aggressive mycoparasite of

Botrytis cinerea causing the grey mould disease of grape-vine.

FEMS Microbiology Letters 227, 277–280.

[3] Hendrix, F.F. and Campbell, W. (1973) Pythiums as plant

pathogens. Annual Review of Phytopathology 11, 78–98.

[4] Kumar, C. and Rzhetsky, A. (1996) Evolutionary relationships of

eukaryotic kingdoms. Journal of Molecular Evolution 42, 183–

193.

[5] Van de Peer, Y. and De Wachter, R. (1997) Evolutionary

relationships among the eukaryotic crown taxa taking into

account site-to-site rate variation in 18S rRNA. Journal of

Molecular Evolution 45, 619–630.

[6] Paquin, B., Laforest, M.J., Forget, L., Roewer, I., Wang, Z.,

Longcore, J. and Lang, B.F. (1997) The fungal mitochondrial

genome project: evolution of fungal mitochondrial genomes and

their gene expression. Current Genetics 31, 380–395.

[7] Middleton, J.T. (1943) The taxonomy, host range and geographic

distribution of the genus Pythium. Mem. Torrey bot. Club. 20, 1–

171.

[8] Plaats-Niterink, A.J. Van der (1981) Monograph of the genus

Pythium. Studies in Mycology, Centraalbureau voor Schimmel-

cultures, Baarn 21, 1–242.

[9] Waterhouse, G.M. (1967) Key to Pythium Pringsheim. Mycolog-

ical Papers 109, 1–15.

[10] White, T.J., Bruns, T., Lee, S. and Taylor, J. (1990) Amplification

and direct sequencing of fungal ribosomal RNA genes for

phylogenetics. In: PCR Protocols: A Guide to Methods and

Applications (Innis, M.A., Gelfand, D.H., Sninsky, J.J. and

White, T.J., Eds.), pp. 315–322.

[11] Lee, S.B. and Taylor, J.W. (1992) Phylogeny of five fungus like

protoctistan Phytophthora species, inferred from the internal

transcribed spacer of ribosomal DNA. Molecular Biology and

Evolution 9, 636–653.

[12] Cooke Del, Jung, T., Williams, N.A., Schubert, R., Bahnweg, G.,

Oßwald, W. and Duncan, J.N. (1999) Molecular evidence

supports Phytophthora quercina as a new species. Mycological

Research 103, 799–804.

[13] Nechwatal, Jan. and Oßwald, W. (2003) Pythium montanum sp.

Nov., a new species from a spruce stand in the Bavarian Alps.

Mycological Progress 2 (1), 73–80.

[14] Paul, B., Galland, D. and Masih, I. (1999) Pythium prolatum

isolated from soil in the burgundy region: A new record for

Europe. FEMS Microbiology Letters 173, 69–75.

[15] Paul, B. (2001) ITS region of the rDNA of Pythium longandrum, a

new species; its taxonomy and its comparison with related species.

FEMS Microbiology Letters 202 (2), 239–242.



Polymorphism and gene conversion of the 16S rRNA genesin the multiple rRNA operons of Vibrio parahaemolyticus

Narjol Gonzalez-Escalona, Jaime Romero, Romilio T. Espejo *

Laboratorio de Biotecnologıa, Instituto de Nutricion y Tecnologıa de los Alimentos, Universidad de Chile, El Lıbano 5524, Macul, Santiago, Chile

Received 3 September 2004; received in revised form 13 January 2005; accepted 8 April 2005


Edited by J.M. Ketley

Abstract

The genome sequence of a strain of Vibrio parahaemolyticus holds 11 copies of rRNA operons (rrn) with identical 16S rRNA

genes (rrs). Conversely, the species type strain contains two rrs classes differing in 10 nucleotide sites within a short segment of

25 bp. Furthermore, we show here that the sequence of this particular segment largely differs between some strains of this species.

We also show that of the eleven rrn operons in the species type strain, seven contain one rrs class and four the other, indicating gene

conversion. Our results support the hypothesis that the rrs differences observed between strains of this species were caused by lateral

transfer of an rrs segment and subsequent conversion.


Keywords: Gene conversion; Polymorphism; rrn operons; Vibrio parahaemolyticus; 16S rRNA

1. Introduction

Vibrio parahaemolyticus is a natural inhabitant of

coastal waters and one of the major seafood-borne gas-troenteritis-causing bacteria. Since 1996 an increasing

number of V. parahaemolyticus infections caused by

strains belonging to a clonal complex have been ob-

served throughout the world [1]. We have recently found

that a strain of this complex caused two large diarrhoea

outbreaks in Chile, in 1998 and 2004 [2]. The genome of

one of these strains, RIMD2210633 (VpKX), consists of

two circular chromosomes with 11 copies of rRNAoperons (rrn), 10 on chromosome 1 and 1 on chromo-

some 2 [3]. Analysis of the reported sequence [3] shows

that this strain contains almost identical 16S rRNA

genes (rrs) in their 11 rRNA operons. However, like


doi:10.1016/j.femsle.2005.04.009

* Corresponding author. Tel.: +56 2 6781426; fax: +56 2 2214030.

E-mail address: [email protected] (R.T. Espejo).

many strains which have their genome sequenced [4],

most strains of the genus Vibrio show detectable se-

quence differences between their multiple rrs. The type

strain of the V. parahaemolyticus species, ATCC 17802(VpD), contains two rrs classes, differing in 10 nucleo-

tide sites of a 25 bp sequence that encodes a variable

stem loop of the 16S rRNA, including nucleotides

440–496 (Escherichia coli numbering) [5]. Furthermore,

the sequence of each of the segments in the two rrs clas-

ses of the type strain differ in 7 and 10 nucleotide sites

from the corresponding segment in the VpKX strain

(see sequences for VpD1, VpD2 and VpKX in Fig. 1).The presence of two different rrs classes in the genome

of the type strain and the difference between both rrs

classes of this strain with that in the pandemic strain

do not have a simple explanation.

The large number of mismatches together with the

compensating changes observed between the two rrs

segments found in VpD, implies that their divergence



Fig. 1. Polymorphism and sequence differences of the rrs in four V. parahaemolyticus strains. (a) Polyacrylamide gel electrophoresis of the

amplification products of the rrs from position 27 to 1492 (E. coli numbering). Ld corresponds to the molecular marker ladder, arrows indicate

product size in base pairs. (b) Polyacrylamide gel electrophoresis of the amplification products of the rrs from position 357 to 518 (E. coli numbering).

(c) Nucleotide sequences of the rrs from position 406 to 496 (E. coli numbering), sites showing mismatches are indicated in bold within a shadowed

box. The strains are: VpD, species type ATCC 17802T; VpI, serotype O4:K12 WP-1 or RIMD2210086; VpAQ serotype O3:K6 (1991) AQ4673 or

RIMD2210856; and VpKX serotype O3:K6 (1996) KXV237 or RIMD2210633.

214 N. Gonzalez-Escalona et al. / FEMS Microbiology Letters 246 (2005) 213–219

is relatively ancient and it is difficult to accept that they

evolved in the same cell. Hence, these segments probably

evolved in different strains and one of the two versions

was probably acquired by lateral transfer. However, it

would be expected that after transfer the rrs multigene

family would probably be homogenized. In most bacte-

rial species, members of the rrs multigene family are

homogenized to evolve in a concerted fashion [6,7].Without concerted evolution, mutations would accumu-

late in individual rrs at a similar rate to that observed

between species [7], causing high polymorphism among

repeated genes of the same family. It is believed that

in prokaryotes the homogenization involves gene con-

version [7], a process that causes a segment of DNA to

be copied onto another segment of DNA, probably by

nonreciprocal recombination between genes in the rrn

operons [8]. Homogenization may occur by conversion

of the incorporated segment to the autochthonous ver-

sion or, alternatively, by conversion of the autochtho-

nous segments to the laterally transferred version. To

explore the possible existence of rrs conversion in

VpD, we determined the number of rrs containing each

segment sequence. Since the possibility that two or more

rrs might independently acquire the same sequence seg-ment by random mutation is practically zero, the pres-

ence of both sequences in more than one gene would

be due to gene conversion. Our results can be best ex-

plained by the occurrence of rrs conversion among the

multiple operons of V. parahaemolyticus. The evidence

for gene conversion supports the hypothesis that differ-

ences between rrs of close phylogenetically related

strains may arise by lateral transfer of a segment even

though they contained multiple rrn operons.


2.1. Bacterial strains and media

The V. parahaemolyticus strains, RIMD 2210856

(VpAQ), 2210633 (VpKX) and 2210086 (VpI) were di-

rectly obtained from the Research Institute for Micro-

bial Diseases, Osaka University, Japan (RIMD)

respective culture collection. V. parahaemolyticus strainATCC17802T (VpD) was directly obtained from the

American Type Culture Collection, Manasas, VA. Bac-

terial strains were grown in Marine Broth (Difco) at

37 �C. The identification of these cultures was confirmed

subsequently by the determination of 16S–23S rDNA

spacer patterns.

2.2. DNA extraction, PCR amplification

Bacterial DNAs were extracted from overnight cul-

tures [12] and PCR amplifications were performed as pre-

viously described [9]. Primers employed for the different

N. Gonzalez-Escalona et al. / FEMS Microbiology Letters 246 (2005) 213–219 215

amplification protocols were: Eubac27F (5 0-AGAGTTT-

GATCCTGGCTCAG-3 0) and 1492R (5 0-GGTTACCT-

TGTTACGACTT-3 0) to amplify 16S rDNA, and

primers 357F (5 0 CTCCTACGGGAGGCAGCA-3 0)

and 518R (5 0-CGTATTACCGCGGCTGCTGG-3 0) to

amplify the shorter fragment containing the variable re-gion G1F (5 0-GAAGTCGTAACAAGG-3 0) and L1R

(5 0-AAGGCATCCACCGT-3 0) to amplify the 16S–23S

rDNA genes spacer [10]. PCR products were electropho-

resed and visualized as previously described [11].

2.3. Cloning and sequencing of rrs together with adjacent

spacer

Primers 357F and L1R were employed to amplify the

fragments containing both the 16S rDNA fragment and

the spacer. PCR products were purified using the Wiz-

ard system as indicated by the manufacturer (Promega)

and later cloned into pGEMT Easy Vector Systems

according to manufacturer�s instructions (Promega).

Plasmid DNA was obtained by a rapid alkaline extrac-

tion miniprep [12]. For analysis of the size of the spacersand the class of rrs by heteroduplex assay, the plasmid

DNA was diluted 1:100 (vol./vol.) in sterile distilled

water and 15 ll were used for PCR amplification of

either the 16S rDNA or the spacer, as described above.

The size of the spacer was subsequently determined by

polyacrylamide gel electrophoresis. The heteroduplex

assay was performed as described [13], except that the

electrophoresis was conducted at 150 V. For sequencing,plasmids were purified with E.Z.N.A. Plasmid Miniprep

Kit I (Omega Bio-tek) and the cloned segments were se-

quenced on an ABI 3100 Genetic Analyzer using Big

Dye Terminator Cycle Sequencing V2.0 Ready Reac-

tions Kit and recommended protocols with primers

M13F, 518R, G1F and L1R. DNA sequences were in-

spected individually and manually assembled. The align-

ments and sequence similarities were obtained usingBioEdit [14].

2.4. Pulsed-field gel electrophoresis and fragment analysis

Bacterial genomic DNA in agarose plugs was pre-

pared as described [15], and digested with the restriction

enzyme I-CeuI (New England Biolabs) for 16 h at 37 �C,using 50 U/plug. Electrophoresis was performed on aCHEF DRII System (BioRad,), using a 1% low melting

point agarose (Promega) gel in 0.5· TBE buffer

(0.45 mM Tris–borate, 1 mM EDTA-Na, pH 8.0). The

pulsed time employed was 6–60 s ramp time at 200 V

for 24 h, at a constant temperature of 14 �C. After elec-

trophoresis, the gel was stained with ethidium bromide

for 30 min and photographed. The observed bands were

excised from the gel with sterile razors and a slice ofeach band was then melted at 65 �C in 10 times its vol-

ume of 1· TE (10 mM Tris–Cl, 1 mM EDTA-Na, pH

8.0). Twelve liters of the solution containing DNA from

each band was then used for PCR, as described above

except that only 20 cycles were performed. Analysis of

both the size of the spacers and the class of rrs by het-

eroduplex assay was performed as described above.

2.5. Nucleotide sequences

Sequences have been deposited in GenBank under the

Accession Nos. AY298793, AY298798, AY298799–

AY298808, and AY527386–AY52735388.

3. Results

3.1. rrs polymorphism in the multi rrn operons of

V. parahaemolyticus strains

The polymorphism in the repeated rrs of the V.

parahaemolyticus species type strain was originally ob-

served by the formation of heteroduplexes after PCR

amplification of the rrs [5]. The presence of heterodu-plexes after PCR amplification of a single isolate with

multiple rrs occurs when these genes exhibit differences

in the nucleotide sequences. In polymorphic strains,

hybrids between synthesized copies with different se-

quences are formed, which show a retarded electropho-

retic migration in polyacrylamide gels. This assay was

employed for assessment of polymorphism in other

V. parahaemolyticus strains, including the pandemicstrain O3:K6 (VpKX), whose genome sequence shows

identical rrs genes [3]. Fig. 1(a) shows the results ob-

tained after PCR amplification of the 16S rRNA gene

from the species type strain VpD, the sequenced pan-

demic clone VpKX and two other non-pandemic

strains; VpAQ, an O3:K6 isolate obtained in 1991,

and VpI, an O4:K12 isolate found in 1968. After

amplification of the 16S rRNA gene, heteroduplexeswith retarded migration were observed for strains

VpD and VpAQ, but not for VpKX and VpI. The het-

eroduplex nature of the bands with retarded migration

was confirmed as previously described [13] (results not

shown).

Cloning and sequencing of the rrs in VpD has shown

the presence of two classes, called 1 and 2, which differ

in 10 nucleotide positions within a 25 bp segment in avariable stem loop of the 16S rRNA, including nucleo-

tides 440–496 (E. coli numbering) [5].To examine if the

polymophism observed in VpAQ occurred in this same

region, a shorter fragment of 161 bp encompassing the

variable segment was amplified by PCR and checked

for formation of heteroduplexes (Fig. 1(b)). The pres-

ence of extra bands observed above the main amplifica-

tion products in VpAQ and VpD indicated that thepolymorphism was in the same rrs region. The presence

of two bands above the main band in VpD is probably


due to the differential migration of the reciprocal hy-

brids formed in this strain. Two different heterodu-

plexes, composed by plus and minus complementary

strands of each amplicon with different sequence, are

formed after annealing in the last PCR cycle. Although

the extent of dissimilarity in this hybrid pair is the same,non-paired regions may form distinct structural confor-

mations in each hybrid, decreasing the mobility to differ-

ent extents [5,13,16]. The sequences of the rrs variable

region in the different strains were determined after

amplification and cloning as described below. Fig. 1(c)

shows that the sequences found in both VpKX and

VpI are identical but they greatly differ from the two se-

quences found in the species type strain VpD and fromone of the two sequences found in VpAQ.

3.2. Operons with each rrs sequence in the species type

strain

To explore if a new sequence may be enforced in

every repeated gene, we looked for evidence of gene con-

version. Homogenization of the rrs requires that one ofthe versions convert the others. The occurrence of rrs

gene conversion was explored in VpD. The presence of

Fig. 2. Separation of the genomic fragments containing rrn operons and

parahaemolyticus strain ATCC 17802. (a) Pulsed-field gel electrophoresis of

indicated on the left side; the class of rrs and the approximate size of the space

the right side. (b) Polyacrylamide gel electrophoresis of the rrs–rrl spacer regio

on top of each lane indicates the analyzed band. VpD corresponds to the

ATCC17802T. Ld corresponds to a 100 bp molecular size marker. (c) Polyac

and annealing of the amplification products of the variable region of the rr

amplification products of clones containing rrs class 1, class 2 and with thems

and 2 in the last lanes to the right correspond to the self-annealing products fr

both sequence versions in more than one operon would

indicate gene conversion because the possibility that two

or more rrs might acquire the same segment sequence by

random mutation is practically zero. The sequence of

the variable segment of the rrs in the different operons

of VpD was determined by analysis of restriction frag-ments obtained by cleavage of the genomic DNA with

the restriction enzyme I-CeuI. This enzyme cleaves a

19-bp sequence in the 23S rRNA gene [17] and allows

for the separation of the rrn operons by gel electropho-

resis. Fig. 2(a) shows the result of the pulsed-field gel

electrophoresis with the resolved bands numbered from

1 to 8. The sequence of rrs (class 1 or 2) is shown to the

right of each band. The rrs class was defined by a hetero-duplex assay based on the retarded migration of the hy-

brids formed between rrs segments of different

nucleotide sequence. For this assay, a 161 bp segment

containing the rrs variable region was amplified from

each fragment and the product was hybridized with

those obtained from recombinant plasmids containing

rrs of either class 1 or 2 [5]. Analysis of the hybridization

products by gel electrophoresis shows exclusivelyhomoduplexes when amplicons are of the same class,

and homoduplex plus heteroduplexes with retarded

characterization of their rrs genes and rrs–rrl spacer regions in V.

the DNA digested with I-CeuI. The number assigned to each band is

rs (between parenthesis), observed in each DNA band, are indicated on

ns PCR amplified as described in Materials and Methods. The number

amplification product from the whole DNA of V. parahaemolyticus

rylamide gel electrophoresis of the products formed after denaturation

s contained in each band (indicated above every three lanes) with the

elves (indicated as 1, 2 and - above each lane, respectively). Numbers 1

om class 1 and 2 rrs clones, respectively. VpD and Ld as indicated in b.

Fig. 3. Schematic representation of the different putative operons identified both in the clones of the PCR amplification products and in the

restriction fragments. Nucleotides in polymorphic sites are in bold. Each operon is identified by the class of rrs (1 or 2) and the size of its neighbor

spacer in bp (first column on the left). The scheme shows the sequence of the variable region of the rrs followed by the tRNAs present in its adjacent

spacer. Pulsed field electrophoresis bands that may contain the operon are identified on the right side. The uncertainty is caused by the inability to

distinguish between spacers of almost equal size by gel electrophoresis, i.e., 706 and 669. ND, not detected among the pulsed field gel electrophoresis

bands.


migration when the amplicons are of a different class.Fig. 2(c) shows the result of the heteroduplex assay for

each band. For some bands the presence of rrs of a sin-

gle sequence class is straightforward, as for the presence

of sequence class 1 in bands 1, 2 and 8 or class 2 in band

6 observed in this figure. Band 5 shows the presence of

heteroduplexes when hybridized with amplicons from

the clones of either class 1 or 2 and with itself. This kind

of result is expected when the band contains both rrs

classes. Heteroduplexes observed in minor proportion

after self-annealing of the amplicons of some bands,

e.g., band 7, are likely due to contamination in the gel

with other restriction fragments.

To define the number of different operons in each

band we determined the size of the 16S–23S rDNA

spacer regions by PCR amplification. The size of the

spacers allows for the placement of the operons of thisstrain into six groups [18]. Fig. 2(b) shows the spacers

observed after amplification of each band; the observa-

tion of more than one spacer in some bands (bands 1,

5 and 7) may be due to either the co-migration of two

restriction fragments or to the presence of two operons.

Fragments with two operons may be generated when

two neighbour operons are in opposite direction. The

sizes of the spacers found in each band are shown be-tween parenthesis in Fig. 2(a). Altogether, the determi-

nation of the size of the spacer and class of rrs

allowed for the identification of 11 putative rRNA oper-

ons, seven containing rrs 1 and four rrs 2.

The class of rrs was also determined on a complemen-

tary approach, independent of the location of the CeuI

restriction sites. This entailed the amplification and

cloning of about three-fourths of the rrs genes togetherwith their entire neighbouring spacers. The operons in

44 clones of the product were examined for both the size

of the spacer and the class of rrs. They were initially sep-

arated into four groups according to the estimated size

of the 16S–23S rDNA intergenic spacers by gel electro-

phoresis [18]. According to both the estimated size of the

spacer and the class of the rrs, six groups of clones were

distinguished. At least four clones from each group weresequenced. These sequences permitted the identification

of 8 groups of clones; six groups containing rrs with se-quence class 1 and two with sequence class 2. Fig. 3

shows a scheme of the rrs class and the size of the spacer

in the different groups. The sequence of the variable re-

gion of the rrs and the tRNAs deduced from the spacer

sequences are schematically shown on the right side. As

previously described by Maeda et al. [18], the sequences

we found allowed us to distinguish six groups of spacers

although only four bands are observed after polyacryl-amide electrophoresis.

3.3. Gene conversion in the 16S–23S rDNA spacer regions

To explore gene conversion in other regions of the rrn

operons, the sequence of the spacer in the different oper-

ons was analysed. The analysis of the spacer sequences

in VpD showed conserved sequence blocks in each rrn

operon corresponding to the 40 sites next to the 5 0 end

and the 208 sites next to the 3 0. Though these blocks

are highly conserved, a few polymorphic sites suggestive

of gene conversion were observed. Within the 40 sites

next to the 5 0 end, there is a polymorphic site with T

in two putative operons and A in the other six. Within

the 208 sites next to the 3 0 end there is a polymorphic

site with T in two putative operons and G in the othersix (results not shown).

4. Discussion

The high polymorphism observed between the multi-

ple rrs in VpD and VpAQ in a particular segment and

the differences between rrs of different strains in thissame segment is extraordinary. The segments with the

large changes found within the same strain are unlikely

to be due to accumulation of point mutations. It seems

more probable that each segment evolved independently

in different strains and that they came together by lateral

transfer. This transfer might have occurred via replace-

ment of a segment as proposed by Wang and Zhang

[19] in their simplified complexity hypothesis. Interest-ingly, despite their approximate 40% dissimilarity, the


different versions of the 25 bp segment in the four V.

parahaemolyticus strains fully match with those in the

16S rRNA of strains of the genus Vibrio or with non-

classified marine Vibrionaceae, exclusively. Most isolates

containing any of the sequence versions found in V.

parahaemolyticus corresponded to the species V. vulnifi-cus, V. coralliilyticus, V. alginolyticus, V. fischeri, and V.

ponticus. Being an autochthonous marine bacterium, V.

parahaemolyticus is probably subjected to a high level of

recombination with the diverse, closely related bacterial

strains populating the seawater. Seawater is a particular

habitat where vibrios are exposed to high levels of gene

transfer by transduction [20]; two bacteriophage classes

that could potentially transfer rrs genes among Vibriorelated strains have been reported. These comprise

filamentous phages [21], including one that is able to

integrate into the host chromosome of V. parahaemolyt-

icus [22,23] and T4-related broad host range phages [24].

However, lateral transfer would probably change only

one of the multiple rrs. Changing more than one or

every rrs in the genome would require gene conversion.

Our analysis of the rrn operons showed the presence ofat least 7 operons containing rrs with sequence class 1

and class 4 with sequence class 2. Considering the num-

ber of different nucleotides together with compensatory

mutations required for the generation of the two rrs

classes, it is very unlikely that the redundant sequences

appeared independently in more than one operon.

Although the occurrence of reciprocal double exchanges

between sister chromosomes, as described by Segall andRoth [25] may generate redundancy of the sequences, it

seems more likely that the redundancy described above

was caused by gene conversion. The repeated mis-

matches found in the spacer regions are also suggestive

of the occurrence of gene conversion. If gene conversion

occurs at a higher rate than gene differentiation, the rrs

would become identical, as was observed in VpI and

VpKX. However, besides having identical segments intheir multiple rrs, these two strains contain a sequence

segment very different from that observed in the species

type strain. Therefore, it seems probable that the ob-

served differences between strains may have originated

by lateral transfer of the variable segment followed by

gene conversion of other rrs in the genome. A similar

mechanism for the polymorphism and redundancy ob-

served in the intervening sequences of the multiple rrl

copies present in Salmonella typhimurium and Salmo-

nella typhi has been postulated by Mattatall et al.

[26,27].

Acknowledgments

N. Gonzalez-Escalona acknowledges a scholarshipDr. Abraham Stekel from INTA-Nestle. This work

was supported in part by FONDECYT Grant 1040805.

References

[1] Chowdhury, N.R., Chakraborty, S., Ramamurthy, T., Nishibu-

chi, M., Yamasaki, S., Takeda, Y. and Nair, G.B. (2000)

Molecular evidence of clonal Vibrio parahaemolyticus pandemic

strains. Emerg. Infect. Dis. 6, 631–636.

[2] Gonzalez-Escalona, N., Cachicas, V., Acevedo, C., Rioseco,

M.L., Vergara, J.A., Cabello, F., Romero, J. and Espejo, R.T.

(2005) Vibrio parahaemolyticus diarrhea, Chile, 1998 and 2004.

Emerg. Infect. Dis. 11, 129–131.

[3] Makino, K., Oshima, K., Kurokawa, K., Yokoyama, K., Uda,

T., Tagomori, K., Iijima, Y., Najima, M., Nakano, M., Yamash-

ita, A., Kubota, Y., Kimura, S., Yasunaga, T., Honda, T.,

Shinagawa, H., Hattori, M. and Iida, T. (2003) Genome sequence

of Vibrio parahaemolyticus: a pathogenic mechanism distinct from

that of V cholerae. Lancet 361, 743–749.

[4] Acinas, S.G., Marcelino, L.A., Klepac-Ceraj, V. and Polz, M.F.

(2004) Divergence and redundancy of 16S rRNA sequences in

genomes with multiple rrn operons. J. Bacteriol. 186, 2629–2635.

[5] Moreno, C., Romero, J. and Espejo, R.T. (2002) Polymorphism

in repeated 16S rRNA genes is a common property of type strains

and environmental isolates of the genus Vibrio. Microbiology 148,

1233–1239.

[6] Elder Jr., J.F. and Turner, B.J. (1995) Concerted evolution of

repetitive DNA sequences in eukaryotes. Q. Rev. Biol. 70, 297–

320.

[7] Liao, D. (2000) Gene conversion drives within genic sequences:

concerted evolution of ribosomal RNA genes in bacteria and

archaea. J. Mol. Evol. 51, 305–317.

[8] Lan, R. and Reeves, P.R. (1998) Recombination between rRNA

operons created most of the ribotype variation observed in the

seventh pandemic clone of Vibrio cholerae. Microbiology 144,

1213–1221.

[9] Espejo, R.T. and Romero, J. (1997) Bacterial community in

copper sulfide ores inoculated and leached with solution from a

commercial-scale copper leaching plant. Appl. Environ. Micro-

biol. 63, 1344–1348.

[10] Jensen, M.A., Webster, J.A. and Straus, N. (1993) Rapid

identification of bacteria on the basis of polymerase chain

reaction-amplified ribosomal DNA spacer polymorphisms. Appl.


[11] Pizarro, J., Jedlicki, E., Orellana, O., Romero, J. and Espejo, R.T.

(1996) Bacterial populations in samples of bioleached copper ore

as revealed by analysis of DNA obtained before and after

cultivation. Appl. Environ. Microbiol. 62, 1323–1328.

[12] Sambrook, J. and Russell, R.G. (2001) Molecular Cloning. A

Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory

Press, Cold Spring Harbor, NY.

[13] Espejo, R.T., Feijoo, C.G., Romero, J. and Vasquez, M. (1998)

PAGE analysis of the heteroduplexes formed between PCR-

amplified 16S rRNA genes: estimation of sequence similarity and

rDNA complexity. Microbiology 144, 1611–1617.

[14] Hall, T.A. (1999) BioEdit: a user-friendly biological sequence

alignment editor and analysis program for Windows 95/98/NT.

Nucleic Acids Symp. Ser. 41, 95–98.

[15] Iida, T., Suthienkul, O., Park, K.S., Tang, G.Q., Yamamoto,

R.K., Ishibashi, M., Yamamoto, K. and Honda, T. (1997)

Evidence for genetic linkage between the ure and trh genes in

Vibrio parahaemolyticus. J. Med. Microbiol. 46, 639–645.

[16] Jensen, M.A and Straus, N. (1993) Effect of PCR conditions on

the formation of heteroduplex and single-stranded DNA products

in the amplification of bacterial ribosomal DNA spacer regions.

PCR Methods Appl. 3, 186–194.

[17] Liu, S.L. and Sanderson, K.E. (1995) I-CeuI reveals conservation

of the genome of independent strains of Salmonella typhimurium.

J. Bacteriol. 177, 3355–3357.


[18] Maeda, T., Takada, N., Furushita, M. and Shiba, T. (2000)

Structural variation in the 16S–23S rRNA intergenic spacers of

Vibrio parahaemolyticus. FEMS Microbiol. Lett. 192, 73–77.

[19] Wang, Y. and Zhang, Z. (2000) Comparative sequence analyses

reveal frequent occurrence of short segments containing an

abnormally high number of non-random base variations in

bacterial rRNA genes. Microbiology 146, 2845–2854.

[20] Jiang, S.C and Paul, J.H. (1998) Gene transfer by transduction in

themarine environment. Appl. Environ.Microbiol. 64, 2780–2787.

[21] Chang, B., Taniguchi, H., Miyamoto, H. and Yoshida, S. (1998)

Filamentous bacteriophages of Vibrio parahaemolyticus as a

possible clue to genetic transmission. J. Bacteriol. 180, 5094–5101.

[22] Iida, T., Makino, K., Nasu, H., Yokoyama, K., Tagomori, K.,

Hattori, A., Okuno, T., Shinagawa, H. and Honda, T. (2002)

Filamentous bacteriophages of vibrios are integrated into the dif-

like site of the host chromosome. J. Bacteriol. 184, 4933–4935.

[23] Nasu, H., Iida, T., Sugahara, T., Yamaichi, Y., Park, K.S.,

Yokoyama, K., Makino, K., Shinagawa, H. and Honda, T. (2000)

A filamentous phage associated with recent pandemic Vibrio

parahaemolyticus O3:K6 strains. J. Clin. Microbiol. 38, 2156–

2161.

[24] Miller, E.S., Heidelberg, J.F., Eisen, J.A., Nelson, W.C., Durkin,

A.S., Ciecko, A., Feldblyum, T.V., White, O., Paulsen, I.T.,

Nierman, W.C., Lee, J., Szczypinski, B. and Fraser, C.M. (2003)

Complete genome sequence of the broad-host-range vibriophage

KVP40: comparative genomics of a T4-related bacteriophage. J.

Bacteriol. 185, 5220–5233.

[25] Segall, A.M and Roth, J.R. (1994) Approaches to half-tetrad

analysis in bacteria: recombination between repeated, inverse-

order chromosomal sequences. Genetics 136, 27–39.

[26] Mattatall, N.R., Daines, D.A., Liu, S.L. and Sanderson, K.E.

(1996) Salmonella typhi contains identical intervening sequences in

all seven rrl genes. J. Bacteriol. 178, 5323–5326.

[27] Mattatall, N.R. and Sanderson, K.E. (1996) Salmonella typhimu-

rium LT2 possesses three distinct 23S rRNA intervening

sequences. J. Bacteriol. 178, 2272–2278.



Induction of murine macrophage TNF-a synthesis by Mycobacteriumavium is modulated through complement-dependent interaction

via complement receptors 3 and 4 in relation to M. aviumglycopeptidolipid

Vida R. Irani a, Joel N. Maslow a,b,*

a Division of Infectious Diseases, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United Statesb Section of Infectious Diseases, VA Medical Center (151), Medical Research Building, 3900 Woodland Ave., Philadelphia, PA 19104, United States

Received 16 November 2004; received in revised form 17 February 2005; accepted 8 April 2005


Edited by A.H.M. van Vliet

Abstract

We studied whether complement receptor (CR) mediated Mycobacterium avium interaction modulated macrophage TNF-aexpression. Compared to control conditions, infections performed with C3-depletion yielded significantly higher TNF-a levels.

Blockage of the CR4 iC3b site yielded increases in TNF-a for all morphotypic variants of a virulent serovar-8 strain (smooth trans-

parent (SmT), smooth opaque (SmO), serovar-specific glycopeptidolipid (ssGPL) deficient knockout mutant) whereas CR3 blockage

increased TNF-a only for SmT and ssGPL-deficient strains. Thus, complement-mediated binding of M. avium to CR3 and CR4 was

shown to modulate TNF-a expression. The differential activation of morphotypic and isogenic variants of a single strain provides an

excellent model system to delineate signaling pathways.


Keywords: Mycobacterium avium; GPL; TNF-a; Macrophage; Complement receptor; Serum proteins

1. Introduction

Mycobacterium avium, a prevalent opportunistic

pathogen of immunocompromised patients, resides

and replicates inside the macrophages of the infected

host. Upon contact of M. avium with the host macro-

phage, reactions such as production of reactive oxygen

and nitrogen intermediates, release of pro-inflammatory(TNF-a, IL-6), and anti-inflammatory cytokines (IL-10)

are triggered [1–3]. It is widely accepted that the level of

macrophage TNF-a expression has important conse-

quences for host immunity [2,4]. In vivo, TNF-a induc-


doi:10.1016/j.femsle.2005.04.008


E-mail address: [email protected] (J.N. Maslow).

tion by M. avium correlates with increased granuloma

formation, resulting in rapid containment and clearance

of the infectious focus [5,6], whereas inhibition of TNF-

a expression by virulent strains of Mycobacterium tuber-

culosis or M. avium is associated with increased bacterial

survival [4,7,8].

Mycobacteria can bind to a number of macrophage

receptors such as the complement receptor types 1, 3and 4 (CR1, CR3, CR4), CD14, mannose receptor

(MR), transferrin receptor, and Fc receptors to gain en-

try into the macrophage [9,10]. CR3 (CD11b/CD18) in

particular, maintains intimate connections with the actin

cytoskeleton and is crucial to the uptake of intracellular

pathogens, including mycobacteria [11–13]. Opsonin-

mediated uptake is partially controlled by the bacterium



222 V.R. Irani, J.N. Maslow / FEMS Microbiology Letters 246 (2005) 221–228

since M. avium has been shown to recruit complement

fragment C2a to form a C3 convertase and generate ac-

tive C3 breakdown products [14]. Receptor preference at

the time of bacterial entry is considered to determine

subsequent intracellular processing by the infected host

cell. In fact, utilization of CRs by the intracellularpathogens Leishmania donovani, Histoplasma capsula-

tum, Mycobacterium leprae, and Legionella pneumophila

to enter the macrophage, allows these organisms to

avoid phagocytic pathways that induce the respiratory

burst [15,16].

A link between receptor usage and early signal trans-

duction events has been recently demonstrated for M.

avium strain 2151 SmO. This strain utilizes CD14 tomanipulate TNF-a synthesis via the extracellular sig-

nal-regulated kinase (ERK) mitogen-activated protein

kinase (MAPK) pathway [17]. While it has been demon-

strated by Aderem and Underhill [18] and Forsberg

et al. [19] that TNF-a regulates CR3-mediated MAPK

activation, respiratory burst, and phagocytosis, the con-

verse i.e., a role for CR3 in regulating TNF-a has never

been studied although work done by Bohlson et al. [20]suggests that complement-mediated interactions may

not impact TNF-a activation.

Non-virulent strains of M. avium stimulate more

TNF-a compared to virulent strains [21], however, the

bacterial surface structures modulating TNF-a activa-

tion have not been identified. The glycopeptidolipids

(GPL) represent the most abundant cell wall component

of M. avium and are situated on the outermost surfacesof the cell [22], thus making them likely ligands to medi-

ate bacterial–host interactions. Studies have suggested a

role for serovar-specific GPL (ssGPL) in the pathogene-

sis of M. avium infection as highly antigenic molecules

affecting host immune function. These data have relied

on comparison of strains representing different serovars

or have used purified and/or chemically modified GPL

and GPL components [23].In a previous study, we disrupted the M. avium

rhamnosyltransferase (rtfA) gene via homologous

recombination to construct isogenic mutants devoid of

serovar-8 specific GPL using an allelic exchange vector

incorporating a temperature-sensitive plasmid origin of

replication and sacB [24]. In this study, we investigate

whether serum C3, and macrophage receptors such as

CR3, and CR4 are involved in M. avium–macrophageinteraction and whether they modulate TNF-a induction

among M. avium strains differing in ssGPL expression.


2.1. Bacterial strains and reagents

M. avium 920A6 is a serovar-8 bloodstream isolate

cultured from a patient with AIDS that exists as smooth

opaque (SmO) and smooth transparent (SmT) morpho-

types [25,26]. 213R.4 is a ssGPL-null (serovar-null)

strain of 920A6 SmO generated by allelic exchange

mutagenesis for the rtfA gene [24]. Strain 233R.1 created

by transformation of 213R.4 with wild-type rtfA gene

incorporated into an integrative vector, contains onlya single-copy of rtfA, and demonstrates a pattern of

ssGPL and nsGPL similar to the parent wild-type strain

[24].

M. avium was grown in Middlebrook 7H9 sucrose

broth or 7H11 agar (Difco Laboratories, Detroit, MI)

supplemented with 10% oleic acid dextrose complex

(OADC) at 37 �C. Bacteria were grown to an optical

density at 600 nm of 0.4, diluted with an equal volumeof 10% glycerol, frozen on dry ice–ethanol, and stored

as 1 ml aliquots at �80 �C. One aliquot of frozen bacte-

ria of each type was serially diluted to determine base-

line colony forming units (cfu) and to confirm colony

morphotype. Prior to infections, bacteria were thawed

on ice and repeatedly passaged through a 27-gauge syr-

inge to disengage any clumps.

2.2. Macrophage infections and TNF-a measurements

The murine macrophage cell line, J774A.1 (ATCC,

TIB-67) was used in this study and propagated in RPMI

medium containing 10% fetal bovine serum (FBS, Sigma,

St. Louis, MO) without added antibiotics. To rule out

variability in results concerning the involvement of

serum proteins, only one source of FBS was usedthroughout the study to generate heat-inactivated and

complement-depleted serum. Heat-inactivated serum

was generated by heating FBS at 56 �C for 30 min. In

studies where C3-depleted serum was used, FBS was

incubated with fractionated C3 antiserum (Sigma, St.

Louis, MO) at a ratio of 8:1 (FBS:antiserum) for 30

min at 37 �C. To investigate the role of serum proteins

and serum opsonins in TNF-a synthesis, J774A.1 cellswere grown to confluence in RPMI with 10% heat-inac-

tivated FBS or 10% C3-depleted serum as indicated

above.

To derive murine bone-marrow derived macrophages

(BMDMs), bone marrow cells from femurs were cul-

tured (5 · 106 cells ml�1, 37 �C, 5% CO2), in complete

DMEM medium containing 10% fetal calf serum, 2

mM L-glutamine, penicillin (100 units ml�1), streptomy-cin (100 lg ml�1), and 30% (vol/vol) L-929 cell-condi-

tioned medium, to provide macrophage growth factor.

To eliminate contaminating fibroblasts, nonadherent

bone marrow cells were transferred after 24 h to nontis-

sue culture petri dishes and grown in L929 cell-condi-

tioned complete DMEM medium for 7 days. Adherent

BMDMs were lifted by incubation in PBS (20 min, 4

�C, 5% CO2) and used for this study.Endotoxin-free anti-CR3 Mac-1 and anti-CR4

monoclonal antibodies (moAb) M1/70 and HL3 against

0

50

100

150

200

250

300

350

Control SmO SmT 213R.4

M. avium 920A6 strains

TN

F-α

[pg

/ml]

0

50

100

150

200

250

300

350

400

control SmO 213R.4 233R.1M. avium 920A6 strains

TN

F-α

[pg

/ml]

0

100

200

300

400

500

600



TN

F-α

[pg

/ml]

(a)

(b)

(c)

Fig. 1. TNF-a induction in J774A.1 cells following infection with the

serovar-8 920A6 strains: (a) following the infection of J774A.1 murine

macrophage cell line in serum-containing culture media at a MOI of

5:1 with M. avium 920A6 SmO and SmT (serovar-8) and 213R.4

(serovar null, [24]), levels of TNF-a in supernatants were measured; (b)

TNF-a levels were measured following the infection of J774A.1 murine

macrophage cell line in serum-containing culture media at a MOI of

5:1 with M. avium 920A6 SmO, 213R.4 (serovar null, [24]), and strain

233R.1 (complemented serovar-null strain, [24]); (c) following the

infection of murine bone marrow derived macrophages (BMDMs) line

in serum-containing culture media at a MOI of 5:1 with M. avium

920A6 SmO and SmT (serovar-8) and 213R.4 (serovar null, [24]), levels

of TNF-a in supernatants were measured. Control = uninfected

J774A.1 (a), (b), or BMDMs (c). TNF-a measurements were done in

triplicates (three independent wells/test condition) and expressed as the

means ± SD. The results are representative of three separate

experiments.

0

200

400

600

800

1000



TN

F-α

[pg/

ml]

serum∆-inactivated serum


serovar-8 strains in RPMI media supplemented with 10% heat-

inactivated serum. Following the infection of J774A.1 murine macro-

phage cell line in heat-inactivated serum-containing culture media at a

MOI of 5:1 with M. avium 920A6 SmO and SmT (serovar-8) and

213R.4 (serovar null), levels of TNF-a in supernatants were measured.

Control = uninfected J774A.1 macrophage cells. TNF-ameasurements

were done in triplicates (three independent wells/test condition) and

expressed as the means ± SD. The results are representative of three

separate experiments.


overnight incubation of J774A.1 cells in medium sup-

plemented with heat-treated FBS did not yield maxi-

mal increases in TNF-a expression, suggesting that

residual bound serum proteins remained (data not

shown). Experiments performed with murine BMDMs

using shorter incubation periods in serum-free condi-

tions demonstrated results identical to the J774A.1

cells (data not shown), thus validating our use of this

cell line as a model of infection.

3.3. Increased TNF-a synthesis in heat-treated serum

conditions relates to loss of C3

It has been previously reviewed that C3 breakdown

components, C3b and iC3b bind to the mycobacterial

surface to initiate macrophage uptake via CR1, CR3,

and CR4 [12]. We next sought to determine whether

complement-mediated opsonization could be responsi-

ble for differences in TNF-a expression observed be-

tween serum and heat-inactivated serum conditions.As observed in Fig. 3, use of C3-depleted serum was

associated with increased levels of TNF-a for 920A6

SmO (�2-fold increase, p < 0.05), SmT (�8-fold in-

crease, p < 0.05), and 213R.4 (4-fold increase, p < 0.05)

relative to infections performed in the presence of nor-

mal serum. Between-strain comparisons in C3-depleted

serum were not significant. Similar to heat-treated ser-

um, we found that prolonged incubation of J774A.1cells with multiple changes of medium was necessary

to fully eliminate C3 (data not shown).

3.4. Opsonization of M. avium via CR3 and CR4 is

necessary to downregulate TNF-a synthesis

The observation that depletion of serum C3 resulted

in increased TNF-a expression for SmO, SmT, andserovar-null infected cells suggested that for all three

serovar-8 strains, the loss of opsonic interaction of

0

200

400

600

800

1000



TN

F-α

[pg/

ml]

serumM 1/70 anti-CR3 moAb


serovar-8 strains. J774A.1 cells were blocked for 1 h with moAb M1/70

(15 lg ml�1) directed against the I domain of CD11b (CR3). Cells were

washed twice prior to infection of J774A.1 murine macrophage cell line

at a MOI of 5:1 with M. avium 920A6 SmO and SmT (serovar-8) and






0

200

400

600

800

1000

Control SmO SmT 213R.4M. avium 920A6 strains

TN

F-α

[pg/

ml]

serumHL3 anti-CR4 moAb


serovar-8 strains. J774A.1 cells were blocked for 1 h with moAb HL3

(15 lg ml�1) directed against the I domain of CD11c (CR4). Cells were

washed twice prior to infection of J774A.1 murine macrophage cell line

at a MOI of 5:1 with M. avium 920A6 SmO and SmT (serovar-8) and






0

200

400

600

800

1000

1200

1400

SmO SmT 213R.4M. avium 920A6 strains

TN

F-α

[pg

/ml]

serumanti-C3 serum


serovar-8 strains in RPMI media supplemented with C3-depleted

serum. Following the infection of J774A.1 murine macrophage cell line

in C3-depleted serum-containing culture media at a MOI of 5:1 with

M. avium 920A6 SmO and SmT (serovar-8) and 213R.4 (serovar null),

levels of TNF-a in supernatants were measured. TNF-a measurements




V.R. Irani, J.N. Maslow / FEMS Microbiology Letters 246 (2005) 221–228 225

M. avium via iC3b to macrophage receptors CR3 and/or

CR4 resulted in higher TNF-a levels. Interaction

through CR1 was not considered since murine macro-

phages do not express this receptor [29].Although CR3 has consistently been implicated as a

key macrophage receptor for M. avium uptake, its role

in TNF-a activation has not been studied. We, therefore

first assessed the role of CR3 in TNF-amodulation. The

I-domain of CD11b contains the iC3b opsonic binding

site [10]. To confirm that opsonic binding of M. avium

to CD11b suppressed TNF-a induction, bacterial infec-

tions were performed while blocking the iC3b bindingsite with anti-CD11b moAb M1/70. Blockage increased

TNF-a levels 4-fold for 920A-6 SmT and serovar-null

strains (Fig. 4, p < 0.05). In contrast, the SmO strain

demonstrated no difference in TNF-a induction when

CR3 is blocked. The increase in TNF-a was not due

to activation of CR3 by moAb M1/70 since control wells

containing moAb alone yielded TNF-a levels no differ-

ent than control wells containing medium alone (Fig. 4).Since all strains manifested increases in TNF-a

expression under opsonin-free conditions (Figs. 2 and

3) and CR3 blockade did not cause an increase in

TNF-a for SmO strains, this would suggest that for

the SmO strain, regulation of TNF-a expression was

mediated via CR4. We therefore investigated the role

of CR4 in TNF-a activation. Bacterial infections were

performed while blocking the iC3b binding site ofCR4 with anti-CD11c moAb HL3. Blockage of the

I-domain of CD11c with moAb HL3 significantly in-

creased TNF-a levels for all 920A6 strains (Fig. 5,

p > 0.05 for all intra-strain pair wise comparisons), com-

parable to levels obtained during heat-inactivated and

C3-depleted serum conditions. Between-strain compari-

sons were not statistically different (p > 0.05). To rule

out non-specific Fc-mediated activation in TNF-ainduction, control antibodies were used at 15 lg ml�1.The level of host TNF-a induction was not statistically

significant (date not shown, p > 0.05).

4. Discussion

Like M. tuberculosis, M. avium can survive within

macrophages and evade the host immune response.


Because TNF-a appears to be involved in host immunity,

early bacterial down-regulation of TNF-a expression in

infected macrophages may be an important mechanism

for intracellular survival of virulent M. avium. One of

the goals of this research was to determine if serum pro-

teins, and receptors on the infected macrophage affectTNF-a expression and thus trigger pathways early on

during M. avium infection that could ultimately decide

the fate of the invading bacterium. Also, we wanted

to determine if strains differing in GPL expression differ-

entially induced TNF-a during the early phase of

M. avium–macrophage interaction and if possible, con-

struct consistent hypotheses and future experiments to

understand the variability in cytokine expression amongthe isogenic M. avium strains.

The role of serum proteins, in particular iC3b, to pre-

ventM. avium induced TNF-a expression is suggested by

our observation that M. avium infection of murine mac-

rophages in the presence of heat-inactivated serum re-

sulted in significantly higher levels of TNF-a than its

whole, active serum counterparts. Support for C3 opson-

ization in regulating TNF-a expression was demon-strated by our use of C3-depleted serum, where murine

macrophage cells infected with M. avium wt and

ssGPL-null strains produced higher levels of TNF-a.The complement-mediated opsonic interaction between

the mycobacterium and the infected macrophage

via the CR3 and/or CR4 could be advantageous to the

bacterium by avoidance of potentially harmful reactions,

such as TNF-a production, which could lead to increasedbacterial survival. Our results are in apparent contrast

with the study of Bohlson et al. [20] that demonstrated

no difference in TNF-a induction of BMDM macro-

phages derived from C3 +/+ and C3 �/� C57BL/6 mice

and J774A.1 macrophages. There are two important dif-

ferences between studies. First, the study by Bohlson

et al. studied two serovar-2 strains of M. avium,

TMC724 and 2-151, whereas our study investigated a vir-ulent serovar-8 strain and highlights the possibility of

GPL-related differences in the activation of macrophage

signaling pathways to affect the host immune response.

Moreover, unpublished results from our laboratory using

TMC724 have also demonstrated a lack of difference in

TNF-a induction in conditions varying in the presence

of complement. Second, Bohlson et al. studied TNF-ainduction of cells propagated in the presence of FBS.Prior to M. avium infection, cells were washed and then

incubated with C3-deficient medium for 2 h. We have

shown here that such short incubation times in C3-defi-

cient medium are insufficient to demonstrate differences

between conditions.

Intracellular pathogens, including mycobacteria,

bind CR1, CR3, CR4 as a first step in the invasion

of mammalian cells [10], linking the receptors cyto-plasmic domains to the actin cytoskeleton and proxi-

mal components of the cell signaling pathways [30].

While this process is regulated by TNF-a, we show

for the first time that C3 opsonization of M. avium

with subsequent binding to host macrophage receptors

CR3 and CR4 is necessary to decrease TNF-a secre-

tion by macrophages.

In addition to differences observed in TNF-a syn-thesis by M. avium infected J774A.1 cells during vari-

ous culture conditions, we also noted differences in

TNF-a induction among the three isogenic M. avium

serovar-8 strains. Effects were similar for the murine

macrophage cell line J774A.1 and the BMDMs demon-

strating the utility of this cell line to model in vivo

infections. We noted that the serovar-null mutant acti-

vated lower levels of TNF-a compared to the wt par-ent SmO strain under control conditions and that the

serovar-null strain complemented with wild-type

ssGPL induced macrophage TNF-a to levels identical

to the wild type M. avium parent strain. Absence of

M. avium ssGPL could be the reason for host TNF-

a suppression by the serovar-null strain since prior

studies have demonstrated the role of ssGPL as a po-

tent immunogen and its ability to trigger higher levelsof TNF-a [3]. Our results confirm the involvement of

M. avium ssGPL in macrophage TNF-a expression.

We also noted that the wt SmT strain activated lower

levels of TNF-a compared to wt SmO strain under

control (normal serum) conditions, this data is in

agreement with previous research [21]. One possible

explanation could be due to the differences in the

non-specific (ns) GPL/ssGPL ratios between the wtSmO and SmT strains. However, the full difference

in the expression profiles between SmO and SmT mor-

photypes is unknown, and thus we cannot rule out

other bacterial factors modulating host cell signaling

pathways. The latter may be likely since SmT morpho-

types typically express greater levels of ssGPL than

SmO strains [31].

Regulation of TNF-a induction mediated throughCR-interaction differed among strains. Wild-type

SmO parent strain and the derived serovar-null mu-

tant varied as to CR utilization responsible for regula-

tion of TNF-a levels. While the iC3b domain of CR4

was used by wt SmO for down-regulation of TNF-a,the serovar-null mutant was able to utilize the iC3b

binding domains of both CR3 and CR4 to downregu-

late TNF-a synthesis. Since these are isogenic strains,the differences in receptor binding and TNF-a modu-

lation between the parent SmO and the ssGPL-null

mutant are attributable to the absence of terminal

serovar specific sugars in the mutant strain. We have

demonstrated that the presence of M. avium ssGPL

triggers higher levels of host TNF-a. Whether altera-

tions in TNF-a expression correlate with survival is

unknown and is being investigated.Receptor preference has been shown to be crucial for

bacterial survival [15,16]. Little is known about the

V.R. Irani, J.N. Maslow / FEMS Microbiology Letters 246 (2005) 221–228 227

involvement of CR4 or whether it is preferred over CR3

during the M. avium infection process. In this study, we

have shown that the receptor preference depends on the

type of infecting M. avium strain. Preliminary results on

simultaneous blockade of the iC3b domains of CR3 and

CR4 suggests that on M. avium infection, modulation ofTNF-a synthesis via these macrophage receptors occurs

via independent MAPK p38 and p42/44 pathways (data

not shown), thus raising the possibility that these

M. avium strains could trigger different host MAPK sig-

naling pathways which could ultimately result in a dif-

ferent intracellular fate for each infecting strain.

In summary, serum protein C3, macrophage recep-

tors CR3, CR4, and M. avium ssGPL are involved inmodulation of TNF-a induction during the early stages

of M. avium–macrophage interaction. This is the first re-

ported study that demonstrates the involvement of CR3

and CR4 in suppression of TNF-a synthesis during M.

avium–macrophage interaction.

Acknowledgements

Y. Patterson and L. Buxbaum are gratefully

acknowledged for the gift of J774A.1 cells and BMPMs,

respectively. Paul M. Nealen is gratefully acknowledged

for assistance in statistical analyses. Thomas Glaze and

Andrea Rossi are acknowledged for technical assistance.

Support for this study was provided through Merit

Review and VISN 4 grant from the Veterans Affairs,and University Foundation Grant from the University

of Pennsylvania, 5-UO1-AI32783, P30-AI-045008-06 to

JNM.

References

[1] Appelberg, R., Sarmento, A. and Castro, A. (1995) Tumour

necrosis factor-alpha (TNF-alpha) in the host resistance to

mycobacteria of distinct virulence. Clin. Exp. Immunol. 101,

308–313.

[2] Eriks, I.S. and Emerson, C.L. (1997) Temporal effect of tumor

necrosis factor alpha on murine macrophages infection with

Mycobacterium avium. Infect. Immun. 65, 2100–2106.

[3] Barrow, W.W., Davis, T.L., Wright, E.L., Labrousse, V.,

Bachelet, M. and Rastogi, N. (1995) Immunomodulatory spec-

trum of lipids associated with Mycobacterium avium serovar 8.

Infect. Immun. 63, 126–133.

[4] Weiss, D., Evanson, O., Moritz, A., Deng, M. and Abrahamsen,

M. (2002) Differential responses of bovine macrophages to

Mycobacterium avium subsp. paratuberculosis and Mycobacterium

avium subsp. avium. Infect. Immun. 70, 5556–61.

[5] Ehlers, S., Kutsch, S., Ehlers, E., Benini, J. and Pfeffer, K. (2000)

Lethal granuloma disintegration in mycobacteria-infected TNFR

p55 �/� mice is dependent on T cells and IL-12. J. Immunol. 165,

483–92.

[6] Smith, D., Hansch, H., Bancroft, G. and Ehlers, S. (1997) T-cell-

independent granuloma formation, in response to Mycobacterium

avium: role of tumour necrosis factor-alpha and interferon-

gamma. Immunol. 92, 413–21.

[7] Greenwell-Wild, T., Vazquez, N., Sim, D., Schito, M., Chatterjee,

D., Orenstein, J. and Wahl, S. (2002) Mycobacterium avium

infection and modulation of human macrophage gene expression.

J. Immunol. 169, 6286–97.

[8] Blumenthal, A., Ehlers, S., Ernst, M., Flad, H-D. and Reiling, N.

(2002) Control of mycobacterial replication in human macro-

phages: role of extracellular signal-regulated kinases 1 and 2 and

p38 mitogen-activated protein kinase pathways. Infect. Immun.

70, 4961–4967.

[9] Roecklein, J.A., Swartz, R.P. and Yeager, H. (1992) Nonopsonic

uptake of Mycobacterium avium complex by human monocytes

and alveolar macrophages. J. Lab. Clin. Med. 119, 772–781.

[10] Ernst, J.D. (1998) Macrophage receptors for Mycobacterium

tuberculosis. Infect. Immun. 66, 1277–1281.

[11] Schlesinger, L.S., Bellinger-Kawahara, C.G., Payne, N.R. and

Horwitz, M.A. (1990) Phagocytosis of Mycobacterium tuberculo-

sis is mediated by human monocyte complement receptors and

complement component C3. J. Immunol. 144, 2771–80.

[12] Velasco-Velazquez, M., Barrera, D., Gonzalez-Arenas, A.,

Rosales, C. and Agramonte-Hevia, J. (2003) Macrophages–

Mycobacterium tuberculosis interactions – role of complement

receptor 3. Microb. Pathog. 35, 125–31.

[13] Agramonte-Hevia, J., Gonzalez-Arenas, A., Barrera, D. and

Velasco-Velazquez, M. (2002) Gram-negative bacteria and phag-

ocytic cell interaction mediated by complement receptor 3. FEMS

Immunol. Med. Microbiol. 34, 255–66.

[14] Schorey, J.S., Carroll, M.C. and Brown, E.J. (1997) A macro-

phage invasion mechanism of pathogenic mycobacteria. Science

277, 1091–1093.

[15] Wilson, M. and Pearson, R. (1988) Roles of CR3 and mannose

receptors in the attachment and ingestion of Leishmania donovani

by human mononuclear phagocytes. Infect. Immun. 56, 363–369.

[16] Wright, S. and Silverstein, S. (1983) Receptors for C3b and C3bi

promote phagocytosis but not the release of toxic oxygen from

human phagocytes. J. Exp. Med. 158, 2016–2023.

[17] Reiling, N., Blumenthal, A., Flad, H-D., Ernst, M. and Ehlers, S.

(2001) Mycobacteria-induced TNF-a and IL-10 formation by

human macrophages is differentially regulated at the level of

mitogen-activated protein kinase activity. J. Immunol. 167, 3339–

3345.

[18] Aderem, A. and Underhill, D.M. (1999) Mechanisms of phago-

cytosis in macrophages. Annu. Rev. Immunol. 17, 593–623.

[19] Forsberg, M., Lofgren, R., Zheng, L. and Stendahl, O. (2001)

Tumor necrosis factor-alpha potentiates CR3-induced respiratory

burst by activating p38 MAP kinase in human neutrophils.

Immunol. 103, 465–472.

[20] Bohlson, S.S., Strasser, J.A., Bower, J.J. and Schorey, J.S. (2001)

Role of complement in Mycobacterium avium pathogenesis: In

vivo and in vitro analyses of the host response to infection in the

absence of complement component C3. Infect. Immun. 69, 7729–

7735.

[21] Faldt, J., Dahlgren, C. and Ridell, M. (2002) Difference in

neutrophil cytokine production induced by pathogenic and non-

pathogenic mycobacteria. APMIS 100, 593–600.

[22] Chatterjee, D. and Khoo, K.-H. (2001) The surface glycopeptid-

olipids of mycobacteria:structures and biological properties. Cell

Mol. Life Sci. 58, 2018–2042.

[23] Newman, G.W., Gan, H.X., McCarthy Jr, P.L. and Remold,

H.G. (1991) Survival of human macrophages infected with

Mycobacterium avium-intracellulare correlates with increased

production of tumor necrosis factor-a and IL-6. J. Immunol.

147, 3942–3948.

[24] Irani, V., Lee, S., Eckstein, E., Inamine, J.M., Belisle, J.T. and

Maslow, J. (2004) Utilization of a ts-sacB selection system for the

generation of a Mycobacterium avium serovar-8 specific glyco-

peptidolipid allelic exchange mutant. Ann. Clin. Microbiol.

Antimicrob. 3, 18.


[25] Arbeit, R.D., Slutsky, A., Barber, T.W., Maslow, J.N., Niemczyk,

S., Falkinham, J.O.I., O�Conner, G.T. and Von Reyn, C.F. (1993)

Genetic diversity among strains of Mycobacterium avium causing

monoclonal and polyclonal bacteremia in patients with AIDS. J.

Infect. Dis. 167, 1384–1390.

[26] Lee, B-Y. et al. (1991) Prevalence of serum antibody to the type-

specific glycopeptidolipid antigens of Mycobacterium avium in

human immunodeficiency virus-positive and -negative individu-

als. J. Clin. Microbiol. 29, 1026–1029.

[27] Bermudez, L.E., Young, L.S. and Enkel, H. (1991) Interaction of

Mycobacterium avium complex with human macrophages: role of

membrane receptors and serum proteins. Infect. Immun. 59,

1697–1702.

[28] Moehring, J.M. and Solotorovsky, M.R. (1965) Relationship of

colonial morphology to virulence for chickens of Mycobacterium

avium and the nonphotochromogens. Am. Rev. Respir. Dis. 92,

704–713.

[29] Martin, B.K. and Weis, J.H. (1993) Murine macrophages lack

expression of the Cr2-145 (CR2) and Cr2-190 (CR1) gene

products. Eur. J. Immunol. 23, 3037–3042.

[30] Gahmberg, C.G. (1997) CD11/CD18 integrins and intracel-

lular adhesion molecules. Curr. Opin. Cell. Biol. 9, 643–

650.

[31] Belisle, J. and Brennan, P. (1994) Molecular basis of colony

morphology in Mycobacterium avium. Res. Microbiol. 145, 237–

242.



Overexpression of a hydrogenase gene in Clostridiumparaputrificum to enhance hydrogen gas production

Kenji Morimoto a, Tetsuya Kimura b, Kazuo Sakka b,*, Kunio Ohmiya c

a Rare Sugar Research Center, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japanb Faculty of Bioresources, Mie University, Tsu 514-8507, Japan

c Agricultural High-Tech Research Center, Meijo University, Tenpaku, Nagoya 468-8502, Japan



Edited by R.P. Gunsalus

Abstract

A [Fe]-hydrogenase gene (hydA) was cloned from Clostridium paraputrificum M-21 in Escherichia coli using a conserved DNA

sequence of clostridial hydrogenase genes amplified by PCR as the probe. The hydA gene consisted of an open reading frame of

1749 bp encoding 582 amino acids with an estimated molecular mass of 64,560 Da. It was ligated into a shuttle vector, pJIR751,

originally constructed for Clostridium perfringens and E. coli, and expressed in C. paraputrificum. Hydrogen gas productivity of

the recombinant increased up to 1.7-fold compared with the wild-type. In the recombinant, overexpression of hydA abolished lactic

acid production and increased acetic acid production by over-oxidation of NADH, which is required for reduction of pyruvic acid to

lactic acid in the wild-type.

� 2005 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.

Keywords: Clostridium paraputrificum; Hydrogenase; Hydrogen gas production

1. Introduction

Since hydrogen gas is an idealistic clean energy mate-

rial that does not generate carbon dioxide gas after com-

bustion, its sustainable production from biomass is in

demand worldwide [1]. To compensate for environmen-

tal problems by reducing carbon dioxide gas generationand overcome shortages of fossil energy in the future,

utilization of abundant biomasses such as chitin, a ma-

jor marine waste, is expected. Gaseous hydrogen is

widely produced by many microorganisms, but is virtu-

ally absent from higher organisms. Anaerobic microor-

ganisms are involved in hydrogen production,

especially photosynthetic microorganisms, and faculta-

0378-1097/$22.00 � 2005 Published by Elsevier B.V. on behalf of the Feder

doi:10.1016/j.femsle.2005.04.014


E-mail address: [email protected] (K. Sakka).

tive and obligatory anaerobic bacteria have been re-

ported to produce hydrogen gas from soluble and

insoluble biomass such as agricultural by-products and

marine wastes [1–11]. Some chitin-degrading bacteria

such as Aeromonas [12], Serratia [13], and Bacillus [14]

have been reported, although hydrogen gas productivity

has not been characterized in detail in these microorgan-isms. Anaerobic bacteria generally have the ability to

produce hydrogen gas during catabolism of carbohy-

drates and [Fe]-hydrogenases (EC 1.12.7.2) are known

to release hydrogen gas from the reduced form of ferre-

doxin in Clostridium and Desulfovibrio species (Fig. 1)

[15–18].

It is well known that many clostridial species evolve

hydrogen gas as a fermentation product during growth.Information about hydrogenase genes and their

products has been reported from a few clostridia, for

ation of European Microbiological Societies.


glucose

NADH

Fd

pyruvate

H2

acetyl-CoA

butyrateacetate

lactate

1

2

Fd

ATP

CO2

H2

ATP

Fig. 1. Outline of biological hydrogen gas production from glucose by

fermentative microorganisms: 1, ferredoxin–NAD+ reductase (EC

1.18.1.3); 2, ferredoxin hydrogenase (EC 1.12.7.2).

230 K. Morimoto et al. / FEMS Microbiology Letters 246 (2005) 229–234

example, [Fe]-hydrogenase I of Clostridium pasteuria-

num [15,16], hydrogenase A of Clostridium perfringens

[17], and hydrogenase A of Clostridium acetobutylicum

P262 [18]. A number of clostridial species are also

known to degrade and ferment various biomass poly-

mers such as polysaccharides and proteins to obtain en-ergy and reducing powers such as the proton/electron

and reduced compounds in cells. Since anaerobic bacte-

ria possess a mechanism to remove excessive reducing

powers as hydrogen gas using hydrogenase, it is possible

that they produce huge amounts of hydrogen gas during

growth on biomass materials.

Clostridium paraputrificum M-21 was isolated and

characterized as a chitin-degrading hydrogen-producinganaerobe in our laboratory [8,9]. Two chitinase and two

b-N-acetylglucosaminidase genes of this bacterium were

characterized along with their translated products [19–

22]. In our preceding paper [23], we reported the con-

struction of the host–vector system of C. paraputrificum

M-21, allowing us to improve its biomass-degrading and

hydrogen gas-producing abilities.

In the present study, we isolated the hydA geneencoding a [Fe]-hydrogenase from C. paraputrificum

M-21, which was isolated from soil as a chitin-degrading

hydrogen-producing anaerobe, and studied the effect of

hydA overexpression on the production of hydrogen gas.

As a result, we found that the recombinant clone over-

expressing the hydA gene produced 1.7 times as much

hydrogen gas as the parental clone, along with a drastic

reduction in lactic acid production.


2.1. Bacterial strains, plasmids, and growth conditions

C. paraputrificum M-21 was isolated and character-

ized as a chitin-degrading hydrogen-producing bacte-rium, was described previously [8]. Plasmid pJIR751

was obtained from Dr. J.I. Rood (Monash University,

Australia) [24] and used to overexpress the hydA gene.

C. paraputrificum was grown anaerobically at 45 �C in

modified GS medium (pH 6.5) supplemented with 1%

N-acetylglucosamine (GlcNAc). Cultivations were con-

ducted in test tubes under static condition or in a 1-ljar fermenter (B.E. Marubishi Lab., Tokyo) containing

500 ml of the medium with agitation at 250 rpm. Re-

combinant C. paraputrificum was cultivated under the

same conditions but with erythromycin (10 lg/ml).

The following plasmids were used as the cloning and

sequencing vectors for Escherichia coli: pT7Blue (Nova-

gen, Madison, WI), pBluescript II KS (�) and pBlue-

script II KS (+) (Stratagene, La Jolla, CA), andCharomid 9-28 (Nippon Gene, Tokyo). E. coli XL1-

Blue and DH5a were grown aerobically at 37 �C in Lur-

ia–Berrani (LB) medium supplemented with ampicillin

(100 lg/ml) and IPTG (50 lg/ml) when necessary.

2.2. Cloning of the C. paraputrificum hydA gene

Chromosomal DNA of C. paraputrificum M-21 wasisolated according to the procedure of Silhavy et al.

[25], partially digested with EcoRI, and separated on

0.4% Agarose H gel (Nippon Gene). DNA fragments

with appropriate sizes were recovered from the agarose

gel using the GeneClean (Bio101, La Jolla, CA) proce-

dure, and ligated into the EcoRI site of Charomid 9-

28. Ligation and transformation of E. coli DH5a were

carried out according to the protocol of the Charomidcloning kit. A pair of PCR primers were designed

according to the sequences highly conserved in the

[Fe]-hydrogenase genes from Clostridium and Desulf-

ovibrio species to obtain a partial region of a hydroge-

nase gene from C. paraputrificum M-21. The following

forward and reverse primers were used: 5 0-

TTYGGNGCNGAYATGACNATHATGGARGA-3 0

and 5 0-CANCCNCCNKGRCANGCCATNACYTC-3 0, respectively. A 700-bp PCR fragment amplified

from C. paraputrificum chromosomal DNA was li-

gated into pT7Blue and introduced into E. coli XL1-

Blue. The cloned DNA fragment was then amplified

and labeled with digoxigenin-11 dUTP (Roche Diag-

nostics GmbH, Penzberg) by PCR with the primers de-

scribed above. The labeled DNA fragment was used as

a probe for colony hybridization to clone the full-length [Fe]-hydrogenase gene (hydA) from the C.

paraputrificum genome library.

2.3. DNA sequencing

Nucleotide sequencing was carried out on a LICOR

model 4000L automated DNA sequencer (Lincoln,

Neb.), with appropriate dye primers and a series of sub-clones. Nucleotide sequence data was analyzed with

GENETYX computer software (Software Development

K. Morimoto et al. / FEMS Microbiology Letters 246 (2005) 229–234 231

Co. Ltd., Tokyo, Japan). Homology searches in DDBJ

were carried out with the BLAST program.

2.4. Construction of pJIR751–hyd

For overexpression of hydA in C. paraputrificum M-21, the hydA gene was subcloned into an E. coli–C. per-

fringens shuttle vector, pJIR751, as follows: plasmid

pHYD101 containing the full-length hydA gene was di-

gested with XbaI and SpeI then a 2.3-kbp DNA frag-

ment containing hydA was ligated into pJIR751, which

had been digested with XbaI in advance, yielding

pJIR751–hyd.

2.5. Electroporation of C. paraputrofocum M-21

C. paraputrificum M-21 was transformed with

pJIR751–hyd according to the electroporation proce-

dure described previously [23].

2.6. Analysis of hydrogen gas production

The total amount of gas produced by the wild-type

and recombinant strains from a 500-ml culture in a 1-l

jar fermenter was measured with a wet gas meter (W-

NK Da-0.5A, Shinagawa, Co., Tokyo) connected to

the jar fermenter by rubber tubing during cultivation.

The absorbance was measured at 600 nm for evaluating

bacterial cell growth with a double-beam spectropho-

tometer (UV-150-02, Shimadzu Co., Kyoto). The com-position of the fermentation gas was analyzed using a

gas chromatography system (model GC-323 equipped

with Molecular Sieve 5A and Porapak Q columns and

a TCD detector; GL Sciences Inc., Tokyo). Separation

was carried out at 50 �C using argon gas as the carrier

gas. Organic acids produced were analyzed using an

HPLC system, GL Sciences EZ Chrom Elite analyzer

equipped with a Shodex Rspak KC-811 column and aGL Sciences UV 620 detector. Separation was con-

ducted at 40 �C using 1 mM HClO4 as an eluent and

ST3-R as a regent at a flow rate of 1.0 ml/min.

2.7. Nucleotide sequence accession number

The nucleotide sequence reported in this paper is

available in the DDBJ, EMBL, and GenBank nucleotidesequence databases under accession number AB159510.

3. Results

3.1. Cloning and sequencing of the hydA gene from

C. paraputrificum M-21

Employing PCR and colony hybridization methods,

a 9.0-kbp DNA fragment expected to contain the full-

length hydA gene and its flanking region was cloned

from C. paraputrificum M-21 using Charomid 9-28.

Sequencing of the inserted DNA fragment identified

an open reading frame of 1749 bp, which encoded a no-

vel hydrogenase (HydA) of 582 amino acids with a pre-

dicted molecular mass of 64,560 Da. The predictedmolecular mass was in good agreement with that of

many clostridial [Fe]-hydrogenases [15,17,18]. A puta-

tive ribosomal-binding site (GGAGG) and �35 and

�10 regions (TTGAAC and AAAAAT with a 18-bp

spacing) were located upstream of the hydA ATG start

codon. Transcription of the hydA gene was expected to

end in rho-factor dependence, because there was no

clear stem-loop structure downstream of the stop codon.Comparisons of the amino acid sequence of HydA

with entries in the DDBJ database indicated that this en-

zyme is highly homologous with some clostridial [Fe]-

hydrogenases (EC 1.12.7.2) as expected; for example,

HydA of C. acetobutylicum P262 (sequence identity

75.1%) [18], HydI of C. pasteurianum W5 (69.4%) [15],

HydA of C. perfringens NTCT8237 (71.3%) [17], and

HydA of Clostridium thermocellum (47.6%, DDBJ acces-sion no. AAD33071). Fig. 2 shows alignment of amino

acid sequences of clostridial hydrogenases. In addition,

C. paraputrificum HydA showed a certain similarity to

some large subunits of [Fe]-hydrogenases in Desulfovib-

rio species such as Desulfovibrio vulgaris subsp. oxami-

cus Monticell (sequence identity 45.4%) [26] and D.

vulgaris subsp. vulgaris str. Hildenborough (45.1%) [27].

3.2. Hydrogen gas production by C. paraputrificum M-21

carrying multiple copies of hydA

When C. paraputrificum M-21 was cultivated in the

fermenter containing 500 ml of GS medium (pH 6.5)

with 1% GlcNAc as the carbon source at 45 �C, the totalvolume of fermentation gas evolved was about 2 l per li-

tre of medium. The ratio of H2 to CO2 was 2:1 and thehydrogen gas yield was 1.4 mol/mol GlcNAc (Fig. 3(a)).

Plasmid pJIR751–hyd contained the hydA structural

gene along with its flanking region containing the possi-

ble promoter region. This plasmid was introduced and

expressed in C. paraputrificum M-21. Although the ratio

of H2 to CO2 in the fermentation gas of the wild-type

and recombinant strains was constantly 2:1, the total

volume of fermentation gas produced by the recombi-nant was about 3.5 l per litre of the medium; hydrogen

gas yield was 2.4 mol/mol GlcNAc, 1.7-fold higher than

that of the wild type (Fig. 3(b)). These results suggested

that enforced hydrogenase activity accelerated oxidation

of ferredoxin to release hydrogen gas. The composition

of organic acids produced by the recombinant and host

was determined and compared (Table 1). The amount of

acetic acid remarkably increased in parallel with an in-crease in hydrogen gas evolution, while on the contrary,

the amount of lactic acid drastically decreased (Table 1).

Fig. 2. Alignment of [Fe]-hydrogenases of C. paraputrificum (C. par), C. acetobutylicum P262 (C. ace), C. pasteurianum W5 (C. pas), C. perfringens

NTCT8237 (C. per), and C. thermocellum (C. the). Amino acids which are conserved in all sequences are highlighted. A His residue and 19 Cys

residues responsible for holding Fe–S clusters are shown with sharp signs (#). –, gap left to improve alignment. Numbers refer to amino acid residues

at the start of the respective lines; all sequences are numbered from Met-1 of the peptide.


An increase in acetic acid with an increase in hydrogen

gas evolution seems to be reasonable since their produc-

tions result from the same metabolic pathway (Fig. 1).

The production of lactic acid was negligible in the

hydrogenase-fortified recombinant (Table 1), indicatingthat the conversion of pyruvic acid to lactic acid was al-

most shut down. The growth rate of the recombinant

clone was identical to that of the host and the trans-

formant containing pJIR751–hyd as judged by measure-

ment of absorbance at 600 nm (Fig. 3).

4. Discussion

In our previous papers, we reported the hydrogen gas

productivity of C. paraputricum M-21 from chitinous

materials [8,9] and suggested that the enhancement of

hydrogenase activity using genetic engineering would

improve hydrogen gas production. Kaji et al. [17] re-

ported that disruption of the hydA gene encoding a

[Fe]-hydrogenase by homologous recombination in C.

perfringens strain 13 abolished its hydrogen gas produc-

tion from glucose, suggesting that this gene was respon-

sible for hydrogen gas production. Therefore, we cloned

a C. paraputrificum M-21 [Fe]-hydrogenase gene that

was a homologue of the C. perfringens hydA gene.

[Fe]-hydrogenases belong to a category of metal-con-

taining hydrogenases including [Ni–Fe] and [Ni–Fe–Se]

hydrogenases [28]. The amino acid sequence ofC. paraputrificum M-21 HydA showed strong similarity

to that of clostridial [Fe] hydrogenases, particularly that

from C. acetobutylicum P262, and moderate similarity to

Table 1

Composition of organic acids produced from GlcNAc by C. parapu-

trificum M-21

Plasmid Organic acid (mM)

Lactic

acid

Acetic

acid

Butyric

acid

Formic

acid

Propionic

acid

None 29.3 38.1 12.7 4.87 0.03

pJIR751 28.6 33.9 14.4 5.13 0.04

pJIR751–hyd 0.42 51.8 13.1 5.90 0.02

Fig. 3. Enhanced hydrogen gas production by the overexpression of

the hydA gene in C. paraputrificum. Non-transformant cells (a) and

recombinant cells harboring pJIR751–hyd (b) were cultivated in 500

ml of GS medium containing 1% GlcNAc.

K. Morimoto et al. / FEMS Microbiology Letters 246 (2005) 229–234 233

those of a large subunit of hydrogenases from Desulf-

ovibrio species; no sequence similarity with those of

[Ni–Fe] or [Ni–Fe–Se] hydrogenases was seen. The

three-dimensional structure of a [Fe]-hydrogenase

(HydI) of C. pasteurianum was previously reported

[16]. In HydI and some other clostridial hydrogenases,

19 cysteine residues and a histidine residue are conserved

as shown in Fig. 2, and these residues are known to fas-ten one [2Fe–2S] cluster, three [4Fe–4S] clusters, and one

H cluster that functions as an active center [16].

Hydrogen gas production has been discussed in a

number of studies using various clostridia, especially in

C. acetobutylicum. Sugar metabolism of this bacterium

is composed of two phases: an acidogenesis phase and

solventogenesis phase [18]. During acidogenesis in clos-

tridia, a large amount of electron flow is directed tohydrogen gas production while sugars are converted to

organic acids such as acetic acid (Fig. 1). Kim et al.

[29] reported that a significant decrease in the rate of

hydrogen gas production correlated with the shift of

metabolic phase from acidogenesis to solventogenesis.

The metabolic pathway in acidogenic Clostridium has

several possible end products, including butyrate, ace-

tate, lactate, CO2, and H2 (Fig. 1). Acetate and butyratefermentation on glucose by typical acidogenic Clostrid-

ium species ideally proceeds according to the following

equation [30]:

Glucose ! 0.8 butyrateþ 0.4 acetate þ 2 CO2 þ 2.4 H2

Although C. paraputrificum M-21 also produced anenormous volume of hydrogen gas during the exponen-

tial growth phase [8,9], the yield of hydrogen gas from 1

mol of glucose did not reach 2.4 mol (1.4 mol). When C.

paraputrificum M-21 was cultured with 1% GlcNAc, not

only acetic and butyric acids but also lactic acid was

produced from GlcNAc (Table 1). Since hydrogen gas

is not coproduced with lactic acid (Glucose ! 2 lactic

acid), the production of lactic acid would reduce theyield of hydrogen gas production. On the other hand,

when the C. paraputrificum M-21 recombinant over-

expressing hydA was cultured with 1% GlcNAc, the re-

combinant produced higher amounts of acetic acid

and negligible amounts of lactic acid in the culture fluid

compared with the host organism: the yield of acetic

acid, lactic acid and butyric acid from 1 mol of glucose

was calculated as 0.93, 0.01, and 0.24 mol, respectively.Improvement of hydrogen gas production in the recom-

binant was caused by reduction in the amount of lactic

acid and enhancement in the amount of acetic acid. It

seems apparent that the enhanced hydrogenase activity

caused over-oxidation of NADH to NAD+, and conse-

quently the depletion of NADH to reduce pyruvic acid

to lactic acid (Fig. 1). However, the production of buty-

ric acid was not affected by the overexpression of hydA.Further improvement of hydrogen gas production might

be achieved by the inhibition of electron flow to butyric

acid by the disruption of the gene responsible for butyric

acid production.

In conclusion, the hydA-expressing recombinant of C.

paraputrificum M-21 produced an increased amount of

hydrogen gas from GlcNAc, along with increased acetic

acid production and reduced production of lactic acid.Further studies are necessary to further improve the

hydrogen gas productivity of C. paraputrificum M-21

by gene disruption leading to the inhibition of butyric

acid production.

Acknowledgements

This work was supported in part by a Grant-in-Aid

for University and Society Collaboration (Grant No.

12794004), the Ministry of Education, Culture, Sports,


Science, and Technology of Japan, and by the NEDO

project ‘‘High efficiency bioenergy conversion prohect

development of high efficiency hydrogen–methane fer-

mentation process using organic wastes’’.

References

[1] Hawkes, F.R., Dinsdale, R., Hawkes, D.L. and Hussy, I. (2002)

Susustainable fermentative hydrogen production: challenges for

process optimization. Int. J. Hydrogen Energy 27, 1339–1347.

[2] Miyake, J., Mao, X. and Kawamura, S. (1984) Photoproduction

of hydrogen from glucose by a co-culture of a photosynthetic

bacterium and Clostridium butyricum. J. Ferment. Technol. 62,

531–535.

[3] Yokoi, H., Ohkawara, T., Hirose, J., Hayashi, S. and Takasaki,

Y. (1995) Characteristics of hydrogen production by aciduric

Enterobacter aerogenes strain HO-39. J. Ferment. Bioeng. 80,

571–574.

[4] Ueno, Y., Haruta, S., Ishii, M. and Igarashi, Y. (2001) Microbial

community in anaerobic hydrogen-producing microflora enriched

from sludge compost. Appl. Microbiol. Biotechnol. 57, 555–562.

[5] Pel, R., Wessels, G., Aalfs, H. and Gottschal, J.C. (1989) Chitin

degradation by Clostridium sp. strain 9.1 in mixed cultures with

saccharolytic and sulphate-reducing bacteria. FEMS Microbiol.

Ecol. 62, 191–200.

[6] Taguchi, F., Mizukami, N., Yamada, K., Hasegawa, K. and

Saito-Taki, T. (1995) Direct conversion of cellulosic materials to

hydrogen by Clostridium sp. strain no. 2. Enzyme Microb.

Technol. 17, 147–150.

[7] Yokoi, H., Tokushige, T., Hirose, J., Hayashi, S. and Takasaki,

Y. (1997) Hydrogen production by immobilized cells of aciduric

Enterobacter aerogenes strain HO-39. J. Ferment. Bioeng. 83,

481–484.

[8] Evvyernie, D., Yamazaki, S., Morimoto, K., Karita, S., Kimura,

T., Sakka, S. and Ohmiya, K. (2000) Identification and charac-

terization of Clostridium paraputrificum M-21, a chitinolytic,

mesophilic and hydrogen-producing bacterium. J. Biosci. Bioeng.

89, 596–601.

[9] Evvyernie, D., Morimoto, K., Karita, S., Kimura, T., Sakka, K.

and Ohmiya, K. (2001) Conversion of chitinous wastes to

hydrogen gas by Clostridium paraputrificum M-21. J. Biosci.

Bioeng. 91, 339–343.

[10] Hussy, I., Hawkes, F.R., Dinsdale, R. and Hawkes, D.L. (2003)

Continuous fermentative hydrogen production from a wheat

starch co-product by mixed microflora. Biotechnol. Bioeng. 84,

619–626.

[11] Lin, C.Y. and Lay, C.H. (2004) Carbon/nitrogen-ratio effect on

fermentative hydrpgen production by mixed microflora. Int. J.

Hydrogen Energy 29, 41–45.

[12] Ueda, M., Shiro, M., Kawaguchi, T. and Arai, M. (1996)

Expression of the chitinase III gene of Aeromonas sp. No. 10S-

24 in Escherichia coli. Biosci. Biotechnol. Biochem. 60, 1195–1197.

[13] Suzuki, K., Sugawara, N., Suzuki, M., Uchiyama, T., Katouno,

F., Nikaidou, N. and Watanabe, T. (2002) Chitinases A, B, and

C1 of Serratia marcescens 2170 produced by recombinant

Escherichia coli: enzymatic properties and synergism on chitin

degradation. Biosci. Biotechnol. Biochem. 66, 1075–1083.

[14] Alam, M.M., Nikaido, N., Tanaka, H. and Watanabe, T. (1995)

Cloning and sequencing of chiC gene of Bacillus circulans WL-12

and relationship of its product to some other chitinases and

chitinase-like proteins. J. Ferment. Bioeng. 80, 454–461.

[15] Meyer, J. and Gagnon, J. (1991) Primary structure of hydroge-

nase I from Clostridium pasteurianum. Biochemistry 30, 9697–

9704.

[16] Peters, J.W., Lanzilotta, W.N., Lemon, B.J. and Seefeldt, L.C.

(1998) X-ray crystal structure of the Fe only hydrogenase (CpI)

from Clostridium pasteurianum to 1.8 angstrom resolution.

Science 282, 1853–1858.

[17] Kaji, M., Taniguchi, Y., Matsushita, O., Katayama, S., Miyata,

S., Morita, S. and Okabe, A. (1999) The hydA gene encoding the

H2-evolving hydrogenase of Clostridium perfringens: molecular

characterization and expression of the gene. FEMS Microbiol.

Lett. 181, 329–336.

[18] Santangelo, J.D., Durre, P. and Woods, R.D. (1995) Charac-

terization and expression of the hydrogenase-encoding gene

from Clostridium acetobutylicum P262. Microbiology 141, 171–

180.

[19] Morimoto, K., Karita, S., Kimura, T., Sakka, K. and Ohmiya, K.

(1997) Cloning, sequencing, and expression of the gene encoding

Clostridium paraputrificum chitinase ChiB and analysis of the

functions of novel cadherin-like domains and a chitin-binding

domain. J. Bacteriol. 179, 7306–7314.

[20] Morimoto, K., Karita, S., Kimura, T., Sakka, K. and Ohmiya, K.

(1999) Sequencing, expression, and transcription analysis of the

Clostridium paraputrificum chiA gene encoding chitinase ChiA.

Appl. Microbiol. Biotechnol. 51, 340–347.

[21] Li, H., Morimoto, K., Katagiri, N., Kimura, T., Sakka, K., Lun,

S. and Ohmiya, K. (2002) A novel b-N-acetylglucosaminidase of

Clostridium paraputrificum M-21 with high activity on chitobiose.

Appl. Microbiol. Biotechnol. 60, 420–427.

[22] Li, H., Morimoto, K., Kimura, T., Sakka, K. and Ohmiya, K.

(2003) A new type of b-N-acetylglucosaminidase from hydrogen-

producing Clostridium paraputrificum M-21. J. Biosci. Bioeng. 96,

268–274.

[23] Sakka, K., Kawase, M., Baba, D., Morimoto, K., Karita, S.,

Kimura, T. and Ohmiya, K. (2003) Electrotransformation of

Clostridium paraputrificum M-21 with some plasmids. J. Biosci.

Bioeng. 96, 304–306.

[24] Sloan, J., Warner, T.A., Scott, P., Bannam, T.I., Berryman, D.I.

and Rood, J.I. (1992) Construction of a sequenced Clostridium

perfringens–Escherichia coli shuttle plasmid. Plasmid 27, 207–219.

[25] Silhavy, T.J., Berman, M.L. and Enquist, L.W. (1984) Experi-

ments with Gene Fusions. Cold Spring Harbor Laboratory, Cold

Spring Harbor, New York.

[26] Voordouw, G., Strang, J.D. and Wilson, F.R. (1989) Organiza-

tion of the genes encoding [Fe] hydrogenase in Desulfovibrio

vulgaris subsp. oxamicus Monticello. J. Bacteriol. 171, 3881–3889.

[27] Voordouw, G. and Brenner, S. (1985) Nucleotide sequence of the

gene encoding the hydrogenase from Desulfovibrio vulgaris

(Hildenborough). Eur. J. Biochem. 148, 515–520.

[28] Vignais, P.M. and Colbeau, A. (2004) Molecular biology of

microbial hydrogenases. Curr. Issues Mol. Biol. 6, 159–188.

[29] Kim, B.H., Bellows, P., Datta, R. and Zeikus, J.G. (1984) Control

of carbon and electron flow in Clostridium acetobutylicum

fermentations: utilization of carbon monoxide to inhibit hydrogen

production and enhance butanol yield. Appl. Environ. Microbiol.

48, 764–770.

[30] Rogers, P. and Gottschalk, G. (1993) Biochemistry and regulation

of acid and solvent production in Clostridia In: The Clostridia and

Biotechnology (Woods, D.R., Ed.), pp. 25–50. Butterworth-

Heinemann, Stoneham, MA.



RirA is the iron response regulator of the rhizobactin1021 biosynthesis and transport genes in Sinorhizobium meliloti 2011

Caroline Viguier, Paraic O Cuıv, Paul Clarke, Michael O�Connell *

School of Biotechnology, National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland

Received 3 March 2005; received in revised form 24 March 2005; accepted 12 April 2005


Edited by K. Hantke

Abstract

The genes encoding the biosynthesis and transport of rhizobactin 1021, a siderophore produced by Sinorhizobium meliloti, are

negatively regulated by iron. Mutagenesis of rirA, the rhizobial iron regulator, resulted in abolition of the iron responsive regulation

of the biosynthesis and transport genes. Bioassay analysis revealed that the siderophore is produced in the presence of iron in a rirA

mutant. RNA analysis and GFP fusions supported the conclusion that RirA is the mediator of iron-responsive transcriptional

repression of the two transcripts encoding the biosynthesis and transport genes. RirA in S. meliloti appears to fulfil the role often

observed for Fur in other bacterial species. The regulator was found to mediate the iron-responsive expression of two additional

genes, smc02726 and dppA1, repressing the former while activating the latter. The rirA mutant nodulated the host plant Medicago

sativa (alfalfa) and fixed nitrogen as effectively as the wild type.


Keywords: Siderophore; Iron response; RirA; Fur

1. Introduction

Most bacteria possess a variety of mechanisms that

enable them to obtain iron from the environment. To

ensure that the various mechanisms are employed effec-tively and in a manner that does not result in oversupply

of iron, there is a need for coordinated regulation [1].

This is frequently achieved by regulating gene expression

at the transcriptional level. In the presence of adequate

amounts of iron, the transcriptional repressor Fur binds

ferrous iron and binds to DNA at a Fur box, preventing

transcription [2]. However, the binding of ferrous iron to

Fur is relatively weak and under iron-deplete conditionsthe metal does not bind the regulator preferentially, in

which case Fur does not bind DNA and transcription


doi:10.1016/j.femsle.2005.04.012


E-mail address: [email protected] (M. O�Connell).

can occur, often under the control of transcriptional

activators [3,4]. Fur has been found widely in bacteria

[5–7]. However, in some rhizobia, while there is a fur

homologue present, the encoded protein appears to

function in the regulation of manganese acquisitionand not in iron acquisition [8–10].

Rhizobia are found free-living in soil and also as

endosymbionts of legumes, where they induce the for-

mation of nitrogen fixing nodules. Rhizobia infect their

host plants in a species-specific manner; for example

Sinorhizobium meliloti is the endosymbiont of Medicago

sativa (alfalfa). In all cases, the functioning nodule con-

tains an abundance of iron-containing proteins, includ-ing nitrogenase, the central enzyme in nitrogen

fixation. Consequently, the acquisition of iron must be

essential for an effective nitrogen fixing symbiosis. Fur-

thermore, in many soils, free-living rhizobia would be

likely to encounter competition from other microbes



236 C. Viguier et al. / FEMS Microbiology Letters 246 (2005) 235–242

for iron. The bacteria must employ efficient mechanisms

to satisfy the iron requirement in these diverse environ-

ments. Some strains of S. meliloti produce the sidero-

phore rhizobactin 1021, which probably contributes to

their competitive ability while growing under free-living

conditions in soil. However, the siderophore is not pro-duced in mature nodules [11] and the mechanism by

which the bacteria satisfy their iron requirement in sym-

biosis is not as yet clearly understood.

Rhizobactin 1021 is a citrate hydroxamate sidero-

phore, biosynthesis of the core structure being encoded

by six genes, rhbABCDEF, which are contiguous in a

single operon with the gene encoding the novel permease

RhtX [12]. The gene encoding the outer membranereceptor RhtA is expressed on a separate transcript.

rhtA and the operon encoding the biosynthesis genes

have been shown previously to be iron-regulated [11].

Chao et al. [10] isolated a fur mutant of S. meliloti and

showed by microarray analysis that in the fur mutant

the rhizobactin 1021 biosynthesis genes and rhtA were

not upregulated, in comparison with the wild type, un-

der iron-replete conditions. Contrary to expectation,they observed that the genes were downregulated,

although it was suggested that this unexpected result

may be due to the effect of Fur regulation on the SitA-

BCD transport system, which transports manganese pri-

marily and would influence metal homeostasis.

However, the observation that the fur mutation does

not result in upregulation of the genes involved in rhi-

zobactin 1021 production and utilisation implies thatan alternative iron response regulator to Fur is present

in S. meliloti, a conclusion that concurs with that ob-

served for Rhizobium leguminosarum [13]. In contrast,

a role for Fur in iron-responsive regulation has been

established in another member of the rhizobia, Brady-

rhizobium japonicum [8].

A novel iron response regulator (RirA) has been re-

ported in R. leguminosarum and has been shown to reg-ulate at least eight operons including operons involved

in the production and utilisation of vicibactin [13]. In

view of the evidence that Fur is not the iron response

regulator for siderophore production and utilisation in

S. meliloti, it was of interest to determine if RirA fulfils

the role in this species. Here, we report that RirA is the

regulator controlling the iron response of genes involved

in the biosynthesis and transport of rhizobactin 1021and, in addition, other iron-related genes in S. meliloti.


2.1. Bacterial strains and growth conditions

The bacterial strains and plasmids used are describedin Table 1. S. meliloti was cultured on TY medium [19]

at 30 �C and Escherichia coli on LB [14] at 37 �C. Anti-

biotics were used at the following concentrations: for S.

meliloti, kanamycin at 100 lg · ml�1, gentamicin at

30 lg · ml�1, tetracycline at 10 lg · ml�1 and strepto-

mycin at 1 mg · ml�1; for E. coli, kanamycin at

30 lg · ml�1, gentamicin at 20 lg · ml�1, tetracycline

at 20 lg · ml�1, ampicillin at 100 lg · ml�1 and strepto-mycin at 100 lg · ml�1.

2.2. Construction of mutants and plasmid fusions

To construct the mutant S. meliloti 2011rirA2 carry-

ing a cassette encoding resistance to kanamycin inserted

in the rirA gene (smc00785 in the S. meliloti genome

available at http://bioinfo.genopole-toulouse.prd.fr/annotation/iANT/bacteria/rhime/), a region approxi-

mately 2 kb in length containing the gene and its flank-

ing sequences was amplified by PCR from genomic

DNA using the forward and reverse primers onto which

sites for XhoI and SpeI were added (Table 1). The frag-

ment was ligated to the pCR2.1 vector and then sub-

cloned into the vector pJQ200ks, which had been

restricted with XhoI and SpeI. This construct was thenrestricted with NcoI, which made a single cut within

the rirA gene into which a cassette encoding kanamycin

resistance, could be inserted by ligation. The cassette

was obtained as an NcoI fragment by PCR amplifica-

tion using plasmid pUC4K as a source (Table 1). The

pJQ200ks derivative carrying the rirA gene with the

kanamycin resistance gene insertion was mobilised into

S. meliloti 2011 by triparental mating with pRK600.Selection was made for integration of the narrow host

range plasmid into the S. meliloti genome by plating

on medium containing gentamicin. After purification

of the transconjugant, a second recombination and alle-

lic replacement were selected on medium containing 5%

sucrose and kanamycin. Gentamicin sensitivity was

checked and Southern hybridisation was used to con-

firm the loss of the vector and correct insertion of thecassette. The mutation was named 2011rirA2.

Construction of the pOTCV1 plasmid in which the

promoter for the rhtXrhbABCDEF operon is fused to

GFP was undertaken as follows: the promoter region

upstream of rhtX was amplified as a HindIII/PstI frag-

ment from S. meliloti genomic DNA by PCR, using

the forward and reverse primers described in Table 1.

The amplified product was cleaned using the PCR prod-uct purification kit (Eppendorf), restricted with HindIII

and PstI and ligated to the vector pOT1, which had been

restricted with the same enzymes.

2.3. Molecular biology techniques

Genomic DNA was prepared by the method of

Meade et al. [15]. RNA was prepared using RNA Whiz(Ambion) as directed by the manufacturers. Plasmid

DNA was isolated by the alkaline lysis method [20].

http://bioinfo.genopole-toulouse.prd.fr/annotation/iANT/bacteria/rhime/

http://bioinfo.genopole-toulouse.prd.fr/annotation/iANT/bacteria/rhime/

Table 1

Bacterial strains, plasmid and primers

Strain/plasmids/genes Relevant characteristic(s)/primers Source/reference

Strains

Escherichia coli

DH5a endA1 gyrA96 thi-1 hsdR17 supE44 relA1 recA1 DlacU169 (/80DlacZ DM15) [14]

Sinorhizobium meliloti

2011 Wild type, Strr, Nod+, Fix+ [15]

2011rhbA62 Tn5lac insertion in rhbA [10]

2011rirA2 Kanamycin resistance insertion in rirA This study

Plasmids

pJQ200ks Gmr mob sacB [16]

pRK600 Tra Cmr [17]

pCR2.1 Apr Invitrogen

pOT1 Gmr [18]

pOTCV1 Gmr with rhtXrhbABCDEF promoter region cloned upstream gfp This study

Primers 50 ! 3 0

rirA (mutation) rirAM-F: CTCGAG TCG CCG AGG CCC ATT CCT TCT This study

rirAM-R A: CTAGT GAA GTC GGC TGT AAA CGG TAT GCG

Kanamycin resistance cassette (rirA mutation) kanNcoI-F: CCATGG GAC GTT GTA AAA CGA CGG CCA GTG This study

kanNcoI-R: CCATGG GGA AAC AGC TAT GAC CAT GAT TAC G

pOTCV1 construction pOTCV1-F: CCCAAGCTTCCCTGGAGGCGTCCTATCGCC This study

pOTCV1-R: AAACTGCAGGGCAACATTGTCTGACGATAAACATG

16S rRNA 16S rRNA-F: TCT TTC CCC CGA AGG GCT C16S This study

rRNA-R: ACT TGA GAG TTT GAT CCT GGC

rhbA rhbA-F: ATG CCG GCC GAT TTA GCC This study

rhbA-R: TCG CGT CTT TCC TGT CGG

rhtA rhtA-F: CTATGGAATTGGCAACTACTC This study

rhtA:R: CGATGATCTCAACGGCAAGC

rirA rirA-F: GCG TCT GAC GAA GCA AAC C This study

rirA-R: TAC CGT CTC GAC CAG GCC

dppA1 dppA1-F: CAC TAC TCT CTT GGC AGC G This study

dppA1-R: ACG GCT GTA AAC GGT ATG CG

smc02726 smc02726-F: ATGCTCAACCGGCATCATCGCCTGGC This study

smc02726-R: CGCGACGATCTTCTTCAGCACGGTCG

C. Viguier et al. / FEMS Microbiology Letters 246 (2005) 235–242 237

Restriction, ligation and Southern hybridisation were

carried out by standard procedures [14]. Transformation

was by the method of Inoue et al. [21] and conjugation

by the method of O�Connell et al. [22]. Primers were ob-

tained from MWG Biotech (Milton Keynes) and PCRs

were undertaken using a Thermo Hybaid PCR Express

thermal cycler.

2.4. Fluorescence detection and microscopy

Green fluorescent protein activity was detected qual-

itatively using a UV microscope to view cells from cul-

tures grown in TY broth (iron-replete conditions) or

made iron-deplete with 200 lM 2,2 0-dipyridyl. For

quantitative measurements, 100 ll of culture was trans-

ferred to a microtitre plate and fluorescence was evalu-ated with a luminescence spectrometer LB 50 at

490 nm excitation and 520 nm emission. Fluorescence

was calculated according to Tang et al. [23].

2.5. Real time (quantitative) PCR

RNA was treated with DNAse prior to the RT-reac-

tion. For the RT-reaction, 2 lg of RNA was incubated

with a mix of random primers for 10 min at 95 �C before

addition of reverse transcriptase and incubation for 1 h

at 37 �C. The reaction was completed by incubation at

72 �C for 10 min.A 2 ll aliquot from the RT reaction was used for

PCR. Each reaction contained 12.5 ll of a SYBR Green

PCR Master mix in a final volume of 25 ll. Primers, as

described in Table 1, were added at a concentration of

0.4 lmol. PCR reactions were heated to 95 �C for

10 min and then for 50 cycles with steps of 95 �C for

20 s, 56 �C for 30 s and 72 �C for 30 s. The evolution

of the fluorescent intensity of each dye was recordedcontinuously by the Rotor Gene 3000 multiplex system

(Corbett Research). Data were normalised using the del-

ta–delta CT method with respect to 16S rRNA as the


housekeeping gene. Samples for which the RT reaction

was omitted were used as negative controls. The gener-

ation of specific PCR products was confirmed by melt-

ing curve analysis and gel electrophoresis.

2.6. Siderophore detection by bioassay

Production of siderophore under iron-replete condi-

tions was determined by concentration of the culture

supernatant and detection of the presence of the sidero-

phore by bioassay, as previously described [11].

2.7. Plant nodulation and nitrogen fixation assays

Symbiotic properties of the wild type and mutant

strains were assayed as previously described [11].

Fig. 1. Siderophore plate bioassay (a) iron-replete conditions, S.

meliloti 2011 siderophore preparation. (b) Iron-deplete conditions, S.

meliloti 2011 siderophore preparation. (c) Iron-replete conditions,

S. meliloti 2011rirA2 siderophore preparation. Haloes of growth are

arrowed.

3. Results

3.1. Identification and mutagenesis of rirASm

In view of the observation that Fur is not the iron re-

sponse regulator in S. meliloti [10], it was of interest to

identify the regulator that mediates the response to iron

stress that has clearly been observed for a number of

genes [11]. The protein encoded by smc00785 on the

chromosome of S. meliloti (as annotated on the genome

referenced above) shows 84% identity to the sequence of

RirA, the novel iron response regulator identified in R.

leguminosarum. This gene was mutated, constructing

the mutant S. meliloti 2011rirA2 as described in Section

2 and the mutant was phenotypically characterised with

respect to the production of rhizobactin 1021. Superna-

tants were prepared by growing the wild type and mu-

tant strains in TY medium in the presence and absence

of iron.

The supernatants were then analysed for the presenceof the siderophore using the plate bioassay with the non-

producing mutant S. meliloti 2011rhbA62 as an indica-

tor. In contrast to the wild type, the mutant produced

siderophore when grown in the presence of iron indicat-

ing that the smc00785 mutation resulted in the abolition

of the iron regulated repression of rhizobactin 1021 bio-

synthesis (Fig. 1).

Fig. 2. The regulon encoding rhizobactin 1021 biosynthesis, transport an

promoter of the rhtXrhbABCDEF operon that was cloned in pOTCV1.

To determine whether the mutation was affecting

the expression of genes involved in symbiosis, for

example genes that may function in iron acquisition

within the plant root nodule, alfalfa plants were in-

fected with the mutant and wild type strains. Theplants nodulated and fixed nitrogen, as measured by

the acetylene reduction technique, with no difference

being observed between the performance of the wild

type and the mutant.

3.2. RirA negatively regulates expression from the

promoter upstream from the rhtXrhbABCDEF operon

To determine if RirA was acting on the promoter of

the rhizobactin 1021 biosynthesis gene cluster (Fig. 2), a

sequence extending 138 base pairs upstream from the

ribosome-binding site of rhtX, and known to contain

the promoter for the operon, was cloned in the promoter

probe vector pOT1, to form pOTCV1, allowing expres-

sion from the promoter to be monitored by the level of

GFP activity. The plasmid was introduced into the S.

d activation. The striped region indicates the region containing the


meliloti 2011rirA2 mutant and the S. meliloti 2011 wild

type and cultures were analysed for levels of GFP activ-

ity when grown under iron-replete and iron-deplete con-

ditions. Microscopic analysis indicated that the

promoter was repressed under iron-replete conditions

in the wild type but not in the mutant. Levels of fluores-cence normalised with the strain containing the empty

vector were calculated to have a 9-fold increase in

S. meliloti 2011rirA2 pOTCV1 (Fig. 3) compared to

S. meliloti 2011 pOTCV1.

3.3. RirA regulates other iron responsive genes, in

addition to those involved in rhizobactin 1021 biosynthesis

and can activate, as well as repress, gene expression

Using ribonuclease protection assays we had previ-

ously analysed the abundance of RNA transcripts in

response to iron for genes involved in rhizobactin

1021 production and utilisation [11]. Here we report

the use of real-time RT PCR as the technique of

choice to detect transcripts, using the 16S ribosomal

RNA gene as a housekeeping gene, to assess the rela-tive abundance of the transcripts of interest. Initially

we confirmed that the biosynthesis gene rhbA and

the gene encoding the rhizobactin 1021 outer mem-

brane receptor, rhtA, are iron-regulated, demonstrating

the correlation of results obtained by the real-time RT

PCR and ribonuclease protection assays. Subse-

quently, we assessed the level of expression of these

Fig. 3. Cultures of S. meliloti 2011 [pOTCV1] (A and B) and S. meliloti 2011r

the bacteria (A and C) and UV light to detect green fluorescent protein (B a

genes in the S. meliloti 2011rirA2 mutant by compar-

ison with the wild type. In agreement with the results

obtained using the promoter probe plasmid, we de-

tected the transcripts under iron-replete conditions in

the mutant but not in the wild type (Fig. 4).

We investigated the expression of the gene encoding aputative iron transporter, smc02726. It was determined

that the gene is iron responsive and that it is expressed

in the rirA2 mutant but not in the wild type under iron

replete conditions, implying that RirA is a repressor of

the gene (Fig. 4). Furthermore, the iron-responsive

expression of the smc02726 gene was observed in

S. meliloti Rm818, a strain that is cured of the pSymA

megaplasmid and that therefore lacks rhrA, the geneencoding the AraC-like activator of rhizobactin 1021

biosynthesis and transport genes. This result is signifi-

cant in that it decouples the iron-responsive activity of

RirA from any effect of RhrA.

Interestingly, we observed that RirA appears to act as

an activator in the case of dppA1, the homologue of a

gene encoding an ABC transporter of d-aminolevulinic

acid, a precursor of heme, which was previously charac-terised in R. leguminosarum [24], and which is located

adjacent to rirA on the S. meliloti chromosome. Under

iron-replete conditions, dppA1 is expressed in the wild

type but the level of expression is significantly reduced

in the rirA2 mutant (Fig. 4).

Finally, we determined that rirA is itself downregu-

lated under iron deplete conditions.

irA2 [pOTCV1] (C and D) under bright light to confirm the presence of

nd D). Magnification 1000·.

Fig. 4. In vivo analysis of iron and RirA responsive genes by Real-Time PCR. (a) Iron regulation of rhbA, rhtA, smc02726 and dppA1 in the wild

type. (b) Iron regulation of rirA in the wild type. (c) Comparisons of rhbA, rhtA and Smc02726 expression in the wild type and the rirA2 mutant

under iron-replete conditions. (d) Comparison of dppA1 expression in the wild type and the rirA2 mutant under iron-replete conditions.


4. Discussion

Rhizobactin 1021 is the only siderophore producedby S. meliloti 2011, although an analysis of the genome

sequence (strain 1021 for which the genome sequence

was determined is a derivative of strain 2011) suggests

that the organism possesses a number of additional

mechanisms by which it can obtain iron. The genes

smc02889 and smc02726, for example, are two candi-

dates that would be predicted to function in iron trans-

port. The Fur homologue in S. meliloti has beeninvestigated regarding its role in iron-responsive gene

expression and it was concluded that it is the regulator

of the sitABCD operon, that functions primarily in man-

ganese acquisition, but it is not the general regulator of

the iron response [9,10]. Here we report that RirA is the

iron response regulator of rhizobactin 1021 biosynthesis

and transport genes, as well as other iron responsive

genes. RirA is the regulator of iron responsive genes inR. leguminosarum, including the genes encoding vicibac-

tin production and transport [13]. In contrast, in Brady-

rhizobium japonicum it has been discovered that an

additional protein, Irr, functions along with Fur in the

iron response [25]. There is no obvious reason why some

rhizobia have recruited RirA as an alternative to Fur as

the general iron response regulator.

Rhizobactin 1021 biosynthesis and transport are neg-atively regulated by iron and positively regulated by the

AraC-like activator RhrA. Under iron-replete condi-

tions but in the absence of RirA, expression of both

the rhtXrhbABCDEF operon and rhtA are de-repressed.

It is not clear however whether RirA mediates this effect

by directly affecting the individual promoters, or indi-

rectly by modulating the activity of RhrA. It is likelythat RhrA acts as a �local� regulator of the rhizobactin

1021 regulon and no other iron acquisition systems have

been detected that are affected either positively or nega-

tively by RhrA (O Cuıv and O� Connell, unpublishedobservation). In this study the expression of smc02726,

which encodes a protein (designated ShmR) that is a

hemin-binding iron regulated outer membrane protein

from S. meliloti 242 [26], was shown to be RirA-regulated. The fact that the iron-responsive regulation

of smc02726 is maintained in a pSymA cured strain indi-

cates that RirA can function independently of RhrA and

implies that RirA likely acts as a global regulator of iron

responsive genes.

In addition to our observation that RirA acts as a

negative regulator of gene expression in response to

the presence of iron, we also observed that it could actas a positive regulator in the case of the dppA1 gene. Re-

cently, Delany et al. [27] reported that Fur could act as

an activator of putative virulence genes in Neisseria

meningitidis. In E. coli, Fur is known to indirectly regu-

late gene expression in a positive manner through its ef-

fect on the expression of small RNAs [28]. RirA may be

acting through DNA binding or it may be acting indi-

rectly to positively regulate activity. Under iron-repleteconditions, the cell down-regulates the expression of

high affinity iron acquisition systems, and depends on

lower affinity iron acquisition systems to satisfy its iron

demands. By extension, under iron-deplete conditions,

the cell down-regulates the synthesis of many iron-

containing proteins that place an increased iron burden


on the cell. DppA1 was characterised as a member of a

heme precursor transporter system in R. leguminosarum

[24] and may function in a similar manner in S. meliloti.

The properties of heme enable it to function as a pros-

thetic group for many haemoproteins, however under

iron-deplete conditions it is likely that many of theseproteins are down-regulated as the cell switches to a

�low iron mode�. The dppA1 gene is located adjacent to

rirA and is transcribed in the same direction. As both

genes are positively regulated in the presence of iron it

is possible that they are regulated in a coordinated

manner.

Plants inoculated with the rirA2 mutant formed effec-

tive nitrogen fixing nodules indicating that the activa-tion properties of the regulator are not involved in

regulating the availability of iron during the nitrogen

fixing stage of symbiosis. Indeed, the mechanism by

which the bacterium acquires iron in symbiosis remains

to be elucidated, as does the regulator or signal that may

be controlling such a mechanism.

Acknowledgement

This work was supported by Science Foundation Ire-

land and Enterprise Ireland.

References

[1] Crosa, J.H. (1997) Signal transduction and transcriptional and

posttranscriptional control of iron-regulated genes in bacteria.

Microbiol. Mol. Biol. Rev. 61, 319–336.

[2] Escolar, L., Perez-Martin, J. and De Lorenzo, V. (1999) Opening

the iron box, transcriptional metalloregulation by the Fur protein.

J. Bacteriol. 181, 6223–6229.

[3] Beaumont, F.C., Kang, H.Y., Brickman, T.J. and Armstrong,

S.K. (1998) Identification and characterization of alcR, a gene

encoding an AraC-like regulator of alcaligin siderophore biosyn-

thesis and transport in Bordetella pertussis and Bordetella

bronchiseptica. J. Bacteriol. 180, 862–870.

[4] Heinrichs, D.E. and Poole, K. (1996) PchR, a regulator of

ferripyochelin receptor gene (fptA) expression in Pseudomonas

aeruginosa, functions both as an activator and as a repressor. J.

Bacteriol. 178, 2586–2592.

[5] De Lorenzo, V., Wee, S., Herrero, M. and Neilands, J.B. (1987)

Operator sequences of the aerobactin operon of plasmid ColV-

K30 binding the ferric uptake regulation (fur) repressor. J.

Bacteriol. 169, 2624–2630.

[6] Prince, R.W., Cox, C.D. and Vasil, M.L. (1993) Coordinate

regulation of siderophore and exotoxin A production, molecular

cloning and sequencing of the Pseudomonas aeruginosa fur gene. J.

Bacteriol. 175, 2589–2598.

[7] Baichoo, N., Wang, T., Ye, R. and Helmann, J.D. (2002) Global

analysis of the Bacillus subtilis Fur regulon and the iron starvation

stimulon. Mol. Microbiol. 45, 1613–1629.

[8] Diaz-Mireles, E., Wexler, M., Sawers, G., Bellini, D., Todd, J.D.

and Johnston, A.W.B. (2004) The Fur-like protein Mur of

Rhizobium leguminosarum is a Mn(II)-responsive transcriptional

regulator. Microbiology 150, 1447–1456.

[9] Platero, R., Peixoto, L., O�Brian, M.R. and Fabiano, E. (2004)

Fur is involved in manganese-dependent regulation of mntA (sitA)

expression in Sinorhizobium meliloti. Appl. Environ. Microbiol.

70, 4349–4355.

[10] Chao, T.C., Becker, A., Buhrmester, J., Puhler, A. and Weidner,

S. (2004) The Sinorhizobium meliloti fur gene regulates, with

dependence on Mn(II), transcription of the sitABCD operon,

encoding a metal-type transporter. J. Bacteriol. 186, 3609–3620.

[11] Lynch, D., O�Brien, J., Welch, T., Clarke, P., O Cuıv, P., Crosa,

J.H. and O�Connell, M. (2001) Genetic organisation of the region

encoding regulation, biosynthesis and transport of rhizobactin

1021, a siderophore produced by Sinorhizobium meliloti. J.

Bacteriol. 183, 2576–2585.

[12] O Cuıv, P., Clarke, P., Lynch, D. and O�Connell, M. (2004)

Identification of rhtX and fptX, novel genes encoding proteins

that show homology and function in the utilization of the

siderophores rhizobactin 1021 by Sinorhizobium meliloti and

pyochelin by Pseudomonas aeruginosa. J. Bacteriol. 186, 2996–

3005.

[13] Todd, J.D., Wexler, M., Sawers, G., Yeoman, K.H., Poole, P.S.

and Johnston, A.W.B. (2002) RirA, an iron-responsive regulator

in the symbiotic bacterium Rhizobium leguminosarum. Microbiol-

ogy 148, 4059–4071.


Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, NY.

[15] Meade, H.M., Long, S.R., Ruvkun, G.B., Brown, S.E. and

Ausubel, F.M. (1982) Physical and genetic characterisation of

symbiotic and auxotrophic mutants of Rhizobium meliloti induced

by transposon Tn5 mutagenesis. J. Bacteriol. 149, 114–122.

[16] Quandt, J. and Hynes, M.F. (1993) Versatile suicide vectors which

allow direct selection for gene replacement in Gram-negative

bacteria. Gene 127, 15–21.

[17] Finan, T.M., Kunkel, B., De Vos, G.F. and Signer, E.R. (1986)

Second symbiotic megaplasmid in Rhizobium meliloti carrying

exopolysaccharide and thiamine synthesis genes. J. Bacteriol. 167,

66–72.

[18] Allaway, D., Schofield, N.A., Leonard, M.E., Gilardoni, L.,

Finan, T.M. and Poole, P.S. (2001) Use of differential fluorescence

induction and optical trapping to isolate environmentally induced

genes. Environ. Microbiol. 3, 397–406.

[19] Beringer, J.E. (1974) R factor transfer in Rhizobium leguminosa-

rum. J. Gen. Microbiol. 84, 188–198.

[20] Birnboim, H. and Doly, J. (1979) A rapid alkaline extraction

procedure for screening recombinant plasmid DNA. Nucl. Acids

Res. 7, 1513–1523.

[21] Inoue, H., Nojima, H. and Okayama, H. (1990) High efficiency

transformation of Escherichia coli with plasmids. Gene 96, 23–28.

[22] O�Connell, M., Hynes, M.F. and Puehler, A. (1987) Incompat-

ibility between a Rhizobium Sym plasmid and a Ri plasmid of

Agrobacterium. Plasmid 18, 156–163.

[23] Tang, X., Lu, B.F. and Pan, S.Q. (1999) A bifunctional

transposon mini-Tn5gfp-km which can be used to select for

promoter fusions and report gene expression levels in Agrobac-

terium tumefaciens. FEMS Microbiol. Lett. 179, 37–42.

[24] Carter, R.A., Yeoman, K.H., Klein, A., Hosie, A.H., Sawers, G.,

Poole, P.S. and Johnston, A.W.B. (2002) dpp genes of Rhizobium

leguminosarum specify uptake of d-aminolevulinic acid. Mol.

Plant Microbe Interact. 15, 69–74.

[25] Hamza, I., Qi, Z., King, N.D. and O�Brian, M.R. (2000) Fur-

independent regulation of iron metabolism by Irr in Bradyrhiz-

obium japonicum. Microbiology 146, 669–676.

[26] Battistoni, F., Platero, R., Duran, R., Cervenansky, C., Batti-

stoni, J., Arias, A. and Fabiano, E. (2002) Identification of an

iron-regulated, hemin-binding outer membrane protein in Sino-

rhizobium meliloti. Appl. Environ. Microbiol. 68, 5877–5881.


[27] Delany, I., Rappuoli, R. and Scarlato, V. (2004) Fur functions as

an activator and as a repressor of putative virulence genes in

Neisseria meningitidis. Mol. Microbiol. 52, 1081–1090.

[28] Masse, E. and Gottesman, S. (2002) A small RNA regulates

expression of genes involved in iron metabolism in Escherichia

coli. Proc. Natl. Acad. Sci. USA 99, 4620–4625.



Polymerase chain reaction for identification of aldoximedehydratase in aldoxime- or nitrile-degrading microorganisms

Yasuo Kato, Satoshi Yoshida, Yasuhisa Asano *

Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama 939-0398, Japan



Edited by H-P.E. Kohler

Abstract

We developed a molecular screening procedure using Southern hybridization and polymerase chain reaction (PCR) to identify

aldoxime dehydratase (Oxd) encoding genes (oxds) among 14 aldoxime- or nitrile-degrading microorganisms. When an oxd gene of

Rhodococcus erythropolis N-771 was used as a probe, positive hybridization signals were seen with the chromosomal DNA of eight

strains, suggesting that these strains have similar oxd genes to R. erythoropolis N-771. By analyzing the PCR-amplified fragments

with degenerate consensus primers, the occurrence of homologous Oxd coexisting with Fe-containing NHase in the active eight

strains was demonstrated coinciding with the results of Southern hybridization. Whole length of oxd gene was cloned as an example

from one of the positive strains, Pseudomonas sp. K-9, sequenced, and expressed in E. coli. Analysis of the primary structure of the

protein (OxdK) encoded by the oxd gene of Pseudomonas sp. K-9 led to identify an Oxd having a new primary structure. Thus, the

PCR-based analysis of oxd gene is a useful tool to detect and analyze the ‘‘aldoxime-nitrile pathway’’ in nature, since Oxd is the key

enzyme for the pathway.


Keywords: Aldoxime dehydratase; Nitrile hydratase; PCR; Aldoxime-nitrile pathway; Screening

1. Introduction

Nitrile compounds, which are extensively used in the

chemical industry, are degraded by microorganisms to

carboxylic acids by nitrilase (Nit; EC 3.5.5.1) or by com-

bination of nitrile hydratase (NHase; EC 4.2.1.84) and

amidase (Ami; EC 3.5.1.4) [1–3]. Starting from the pio-

neering research for the first isolation of NHase from R.

rhodochrous J-1 [4,5], the enzyme has been extensively

studied and used for manufacturing acrylamide, nicotin-amide, and 5-cyanovaleroamide [1,6]. NHases are classi-

fied into two groups based on the prosthetic metal


doi:10.1016/j.femsle.2005.04.011


E-mail address: [email protected] (Y. Asano).

group, non-heme Fe [NHase(Fe)] or non-corrinoid Co

[NHase(Co)] [1–3]. Despite its important uses, however,the physiological function of NHase in nature remains

unclear. We have isolated several microbial aldoxime

degraders [1], which can convert aldoximes, such as pyr-

idine-3-aldoxime and phenylacetaldoxime as a model for

aryl- and arylalkyl-aldoximes, respectively, to the corre-

sponding carboxylic acids, from soil samples. The

metabolism occurs via intermediate nitrile and involves

a combination of enzymes including a novel heme-con-taining enzyme, aldoxime dehydratase (Oxd; EC

4.99.1.-), and nitrile-hydrolyzing enzymes, such as

NHase and Ami, and/or Nit (1, 5, 7): the pathway could

be named as ‘‘aldoxime-nitrile pathway’’ (Fig. 1). From

some aldoxime- or nitrile-degraders, Oxds were purified



R N

H

OH

R C N R CO

OHNitrile Carboxylic acid

Nitrilase

Nitrilehydratase R C

ONH2

Aldoximedehydratase

AldoximeAmidase

Amide

H2O

Fig. 1. The aldoxime-nitrile pathway in microorganisms.

244 Y. Kato et al. / FEMS Microbiology Letters 246 (2005) 243–249

and characterized and the genes (oxd) were cloned, se-

quenced and overexpressed in E. coli [8–11]. The oxd

genes were linked with genes for Nit and NHase/Ami

in the genome of the strains [8–11], confirming the genetic

relationship of the pathway. Oxds were used for the

enzymatic synthesis of nitriles from the corresponding

aldoximes [1,12]. Since Oxd is located upstream of the

‘‘aldoxime-nitrile pathway’’ (Fig. 1), the enzyme can be-

come the key enzyme for the pathway. It is of our inter-

est to accumulate the genetic information of thepathway from different sources in order to know diver-

sities and evolution of the pathway in nature. The aim

of this study is to develop a molecular screening proto-

col based on Southern hybridization and PCR to rapidly

identify genes coding Oxds in several aldoxime- and ni-

trile-degrading microorganisms, and to study the use of

the method as a tool to detect and analyze the pathway.


2.1. Materials

Restriction enzymes and DNA-modifying enzymes

were purchased from Takara (Tokyo, Japan), Toyobo

(Osaka, Japan), New England Biolabs. (Beverly, MA,USA), Roche (Mannheim, Germany), and MBI Fer-

mentas (Vilnius, Lithuania) and used according to the

manufacturers� protocols. All other chemicals were from

commercial sources and used without further

purification.

2.2. Bacterial strains, plasmids and culture conditions

Strains used for screening oxd gene were from culture

collections (TPU) of our own laboratory [7] and were

grown at 30 �C in TGY medium consisted of 0.5% yeast

extract (Nippon Seiyaku, Tokyo, Japan), 0.5% Bacto

Tryptone (Difco, Detroit, WI, USA), 0.1% of K2H-

PO4, and 0.1% D-glucose, pH 7.0. The E. coli strains,

JM109 {recA1 endA1 gyrA96 thi hsdR17 supE44 relA1

D(lac-proAB)/F 0[traD36 proAB+ lacIq lacZ M15]} andBL21 Stare (DE3) {F� ompT hsdSB ðr�B ; m�

B Þ gal dcm

rne131 (DE3)}, were used as hosts. Plasmids pT7-Blue

(Novagen, Madison, WI, USA) and pRSETB (Invitro-

gen, Carlsbad, CA, USA) were used as cloning and

expression vectors, respectively. Recombinant E. coli

cells were cultured in a Luria–Bertani (LB) medium

(1% Bacto Tryptone, 0.5% Bacto yeast extract (Difco),

and 1% NaCl, pH 7.5) containing 100 lg ml�1 of ampi-

cillin. Pseudomonas sp. K-9 [13] was used as the source

of the DNA to clone whole oxd gene. The partial

sequencing of the 16S rDNA fragment (1.6 kbp) of the

strain showed 100% identity with that of Pseudomonas

synxantha DSM 13080 (GenBank accession no.AF267911).

2.3. General recombinant DNA techniques

The plasmid DNA was isolated by a PI-100 Auto-

matic Plasmid Isolation System (Kurabo, Osaka, Ja-

pan). The other general procedures were performed as

described by Sambrook et al. [14]. The nucleotide se-quence was determined with an ABI PRISM 310 auto-

mated sequencer (Applied Biosystems, Foster City,

CA, USA) using the dideoxy chain termination method.

A homology search was performed with the programs

FASTA [15] and BLAST [16], and the ClustalW method

[17] was used to align the sequence.

2.4. Southern hybridization

Chromosomal DNAs of the strains were extracted

by the method as described by Saito and Miura [18]

and digested with EcoRI followed by fractionation

with a 0.7% agarose gel. The digested DNAs were

blotted onto a nylon membrane, GeneScreen Pluse

(Dupont, Boston, MA, USA), and the membrane

was hybridized with the oxd gene of R. erythropolis

N-771 [11] and Bacillus sp. OxB-1 [9], labelled with

the digoxigenin (DIG) system (Roche), at 37–42 �Caccording to the procedure recommended by the man-

ufacturer. Optimum conditions for stringency washing

of the blotted DNA were sought by increasing the

washing temperature (60–68 �C) or by raising the con-

centrations (0.1–2·) of SSC [14] in a washing solution.

The membrane was visualized with alkaline phospha-tase-conjugated anti-DIG and nitro blue tetrazolium

(NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP)

reagents (Roche).

Y. Kato et al. / FEMS Microbiology Letters 246 (2005) 243–249 245

2.5. PCR amplification method

One colony of the strains grown on a TGY agar plate

was picked-up with a sterile pipette tip and transferred it

directly to a PCR reaction mixture (50 ll) comprising

50 pmol each of primers, 500 lM dNTPs, 5 ll of 10·PCR buffer and 1–2.5 units of various DNA polymer-

ases, such as Taq (Takara), ExTaq (Takara), Blend

Taq (Toyobo), Pwo (Roche), KOD-Plus (Toyobo),

and Vent (New England Biolabs.) polymerases. The

PCR reaction was performed with a PCT 200 thermocy-

cler (MJ Research, Watertown, MA, USA) with the fol-

lowing program: 35 cycles of denaturation at 96 �C for

0.5 min, primer annealing at 50–55 �C for 0.5 min andextension at 72 �C for 1 min. The fragment was purified

with Qiaquick gel extraction kit (Qiagen, Valencia, CA,

USA) and ligated with pT7Bule vector for sequencing.

To amplify genes coding consensus regions for a-sub-unit (nha1) of NHase(Co) and NHase(Fe), the primer

pairs [19] NHCo1 [5 0-GTCGTGGCGAAGGCCTGG-

3 0]/NHCo2 [5 0-GTCGCCGATCATCGAGTC-3 0] and

NHFe1 [5 0- CCCGACGGTTACGTCGAG-3 0]/NHFe2[5 0-CCATGTAGCGAGTTTCGGCG-3 0], were used,

respectively.

2.6. Cloning of whole oxd gene from Pseudomonas sp.K-9

by an inverse PCR and expression of the gene in E. coli

Five micrograms of genomic DNA of Pseudomonas

sp. K-9 was digested with BamHI and the digestedDNA was electrophoresed through a 0.7% agarose

gel. Appropriate fragments (2.5–3.0 kb), which ex-

pected to contain oxd gene based on the results of

Southern hybridization with the PCR-amplified oxd

gene as a probe, were extracted from gel and purified.

The fragment was self-circularized as described by

Sambrook et al. [14]. The PCR was performed in

reactions (25 ll) containing 0.001–0.1 lg of circular-ized DNA, 50 pmol each of the primer InvR2

[5 0-GCGGTCGCGCATCGAGCCCCAATAACC-3 0]

and InvF2 [5 0-TCGGTGGAGAAACTCGAACGC-

TGGACCGAA-3 0], 500 lM dNTPs, 1· PCR buffer,

and 2.5 units of Vent polymerase. The PCR reaction

was 35 cycles of denaturation at 95 �C for 0.5 min,

primer annealing at 55 �C for 0.5 min, and exten-

sion at 72 �C for 6 min. The amplified band, ex-tracted from an agarose gel, was ligated with

pT7Blue vector and used for further sequencing.

A 1.0-kb NdeI-HindIII fragment containing the

whole oxd gene was PCR-amplified by Vent polymer-

ase using the primers K9OxdKNde (5 0-GCTCACA-

TATGAATCTGCAATC-3 0; the restriction site is

underlined) and K9OxdKHindR (5 0-AGGGAAGCT-

TTCAGGCGGGGCATACT-3 0) and the genomicDNA of Pseudomonas sp. K-9 as a template, then

subjected to enzyme digestion with NdeI and HindIII.

The fragment was cloned into the same site of

pRSETB to give pOxdKInt and the plasmid was used

to transform E. coli BL21 Stare (DE3). A 1% aliquot

of the overnight culture of E. coli BL21 Stare (DE3)/

pOxdKInt was added into 8 ml of LB medium con-

taining 100 lg ml�1 of ampicillin in a test tube(18 · 170 mm) and incubated with shaking (200 rpm)

at 37 �C for 3–4 h. When the optical density at

610 nm of the medium reached 1.0, isopropyl b-D-thio-galactopyranoside (IPTG) was added as an inducer to

a final concentration of 1 mM, and the culture was

further incubated at 20 �C for 60 h. The cells were

harvested by centrifugation (3500g, 10 min) at appro-

priate intervals, suspended in 0.1 M potassium phos-phate buffer (KPB, pH 7.0), and disrupted by

ultrasonication as described [8]. The Oxd activity in

a cell-free extract obtained by centrifugation

(15,000g, 10 min) was measured according to our pre-

vious report [1,7,9,11]. The electronic absorbance spec-

tra of the cell-free extract were recorded on a JASCO

V-530 spectrophotometer (JASCO, Tokyo, Japan).

3. Results

3.1. Screening for microorganisms carrying oxd genes by

Southern hybridization

The existence of oxd genes among 14 aldoxime- or ni-

trile-degraders shown in Table 1 was examined bySouthern hybridization with the oxd gene from R. ery-

thropolis N-771 and Bacillus sp. OxB-1 as probes. It

had been suggested that the strains had ‘‘aldoxime-

nitrile pathway’’ by activity measurement [7]: i.e., the

activities of Oxd and nitrile-degrading enzymes, NHase

and Ami and/or Nit, were detected in the strains. By

using R. erythropolis N-771oxd gene as a probe, the po-

sitive hybridization signals were found with the chromo-somal DNA of six strains, i.e., R. erythropolis JCM

3201, Rhodococcus sp. NCIBM 11215, B. butanicum

ATCC 21196, R. erythropolis BG 13, R. erythropolis

BG 16, and Pseudomonas sp. K-9, in addition to control

strains R. globerulus A-4 [10] and Rhodococcus sp. N-771

[11], even after stringency washing of the hybridized

membrane with 0.5· SSC containing 0.1% SDS at

68 �C for 30 min. The result suggests that these strainshave similar oxd gene to that of R. erythropolis N-771.

The positive signals were seen mainly with the genome

of the strains having NHase. Although various condi-

tions for hybridization and stringency washing were

examined, the other NHase-containing strains, R. rho-

dochrous NCIMB 11216, R. rhodochrous J-1 and Rhodo-

coccus sp. YH3-3 did not show any hybridization signals

with the Rhodococcus oxd gene despite the detection ofOxd activity in the strains [7]. The oxd gene of Bacillus

sp. OxB-1 did not hybridize with the genome of any

Table 1

Summary of activity measurement of aldoxime dehydratase, nitrile hydratase, and nitrilase, and Southern hybridization and PCR-amplification

analysis of their genes, in several aldoxime- or nitrile-degrading microorganisms

TPU no. Strain Oxd NHase(Fe) NHase(Co) Nit

Aa Hb Pc Aa Pd Aa Pe Aa Pf

3207 Rhodococcus erythropolis JCM 3201 + + + + + – – – NE

3311 Rhodococcus rhodochrous J-1 + – – – – + + + NE

3451 Rhodococcus sp. NCIBM 11215 + + + – + – – + NE

3452 Rhodococcus sp. NCIMB 11216 + + + – – – – + NE

3453 Rhodococcus sp. YH3-3 + – – – – + – – NE

3466 Rhodococcus sp. AK32 + – – – – – – + NE

5710 Brevibacterium butanicum ATCC 21196 + + + + + – – – NE

6007 Rhodococcus erythropolis BG 13 + + + * + * – – NE

6008 Rhodococcus erythropolis BG 16 + + + * + * – – NE

6015 Corynebacterium sp. C5 + – – – – – – + NE

7177 Pseudomonas sp. K-9 + + + * + * – – NE

3383 Rhodococcus globerulus A-4g + + + + + – – – NE

3467 Rhodococcus erythropolis N-771g + + + + + – – – NE

5563 Bacillus sp. OxB-1g + – + – – – – + NE

a ‘‘A’’ denotes activity measurement. Aldoxime dehydratase (Oxd), nitrile hydratase (NHase), and nitrilase (Nit) activities were measured in each

strain by our previous study [7]. Prosthetic metal group of NHase was suggested by an enzyme purification or a gene analysis. An asterisk (*)

indicates that the strain showed NHase activity but its metal group was not identified.b ‘‘H’’ denotes Southern hybridization. Positive (+) and no (�) hybridization signals were seen by Southern hybridization with the genomic DNA

of each strain by using oxd from R. erythropolis N-771 as a probe.c ‘‘P’’ denotes PCR amplification. A plus (+) indicates that the estimated length of PCR product was amplified with primers OxB4-S3/OxB4-AS2.d ‘‘P’’ denotes PCR amplification. A plus (+) indicates that the estimated length of PCR product was amplified with primers NHFe1/NHFe2.e ‘‘P’’ denotes PCR amplification. A plus (+) indicates that the estimated length of PCR product was amplified with primers NHCo1/NHCo2.f NE – not examined.g The strains were used as control: Oxd, NHase and Nit of the strains had been isolated, characterized, and their genes were cloned and sequenced

in our previous research [8–11].


strains including ones having Nit used in this study un-

der the examined conditions, suggesting that Bacillus

oxd gene had low similarities with oxds of the strains

tested.

3.2. PCR-based analysis of oxd genes

In order to know the genetic information of oxd iden-tified in the strains, we amplified oxd gene from the

strains by using PCR with degenerated consensus prim-

ers under various PCR conditions. The primers OxB4-S3

(5 0-CAYGRHTAYTGGGGHKCRATGCGCGA-3 0)

and OxB4-AS2 (5 0-ACCGADACYTCRTGSYA) used

were designed based on the conserved sequences among

the known Oxds, H(G/E)YMG(S/A)MRE/D and

HEVSV(F/S/L), respectively. A strong band of the ex-pected size (450 bp) was seen on an agarose gel when

Blend-Taq polymerase (Toyobo, Osaka) was used at

an annealing temperature of 55 �C. The PCR product

was amplified from the eight strains including the con-

trol strains (Table 1), all of which showed positive

Southern hybridization signals with R. erythropolis

N-771oxd gene. In addition, PCR product was also ob-

tained from Bacillus sp. OxB-1 that has Oxd and Nit[1,7]. As shown in Fig. 2, the comparison of amino acid

sequences deduced from the amplified genes from the 6

positive strains suggest the existence of similar (80.6%

identities) Oxds in the strains to those linked with NHa-

se(Fe) found in the control strains, R. globerulus A-4 [10]

and R. erythropolis N-771 [11]. The primers are shown

to be suitable for amplifying the oxd gene from the

microorganisms having NHase(Fe). We have previously

clarified that these strains had Oxd activities [7] but this

study gave us genetic information of the enzymes for

the first time. Under the conditions tested, no PCRproduct was obtained from the other strains except

Bacillus sp. OxB-1 that has Oxd and Nit and/or NHase

[1,7].

3.3. PCR amplification of genes coding for NHase

In order to confirm genetically the co-existence of

NHase in the positive strains, genes coding for NHase-(Co) and NHase(Fe) were amplified by PCR with the

primer pairs NHCo1/NHCo2 and NHFe1/NHFe2,

respectively, which were designed based on the con-

served sequences of a-subunit (nha1) of NHase(Co)

(PDGYVE/AETRYM) and NHase(Fe) (VVAKAW/

DSMIGD), respectively, as reported by Duran et al.

[19]. As shown in Table 1 and Fig. 3, NHFe1/NHFe2

primers allowed the amplification of a 400-bp DNAfragment from the positive six strains and proteins en-

coded by the fragments showed similarities (63.7% iden-

tities) to the known NHases (Fe) from P. chlororaphis

1 10 20 30 40 50 60 70 80

BG13,BG16 HGYWGAMRERFPISQTDWMQASGELRVVAGDPAVGGRVVVRGHDNIALIRSGQDWADAEADERSLYLDEILPTLQSGMDF

OxdRG, Bb21196 HGYWGSMRERFPISQTDWMQASGELRVVAGDPAVGGRVVVRGHDNIALIRSGQDWADAEADERSLYLDEILPTLQSGMDF

OxdRE, Re3201 HGYWGSMRERFPISQTDWMQASGELRVIAGDPAVGGRVVVRGHDNIALIRSGQDWADAEADERSLYLDEILPTLQSGMDF

Rs11215 HGYWGSMRERFPISRTDWTHASGELRVVAGDPAAGGRVVVRGHDNIALIRSGQDWADAEADERSLYLDEILPTLQSGMGF

K-9 HGYWGSMRDRFPISQTDWMKPTSELQVIAGDPAKGGRVVVLGHGNLTLIRSGQDWADAEAEERSLYLDEILPTLQDGMDF

***** ** ***** * * ** * ***** ****** ** * ************* ************** ** *

90 100 110 120 130 140 150

BG13, BG16 LRDNGPAVGCYSNRFVRNIDIDGNFLDLSYNIGHWASLDQLERWSESHPTHLRIFTTFFRVAEGLSKLRLYHEVSV

OxdRG, Bb21196 LRDNGPAVGCYSNRFVRNIDIDGNFLDLSYNIGHWASLDQLERWSESHPTHLRIFTTFFRVAEGLSKLRLYHEVSV

OxdRE, Re3201 LRDNGPAVGCYSNRFVRNIDIDGNFLDLSYNIGHWASLDQLERWSESHPTHLRIFTTFFRVAAGLSKLRLYHEVSV

Rs11215 LRDNGPAVGCYSNRFVRNIDIDGNFLDLSYNIGHWASLDQLERWSESHPTHLRIFTTFFRVAEGLSKLRLYHEVSV

K-9 LRDNGQPLGCYSNRFVRNIDLDGNFLDVSYNIGHWRSVEKLERWTESHPTHLRIFVTFFRVAAGLKKLRLYHEVSV

***** ************ ****** ******* * *************** ****** ** **********

Fig. 2. Amino acid sequence comparison of Oxds deduced from the PCR-amplified fragments from R. erythropolis BG 13 (BG13), R. erythropolis

BG 16 (BG16), R. globerulus A-4 (OxdRG), B. butanicum ATCC 21196 (Bb21196), Rhodococcus sp. N-771 (OxdRE), R. erythropolis JCM 3201

(Re3201), Rhodococcus sp. NCIBM 11215 (Rs11215), and Pseudomonas sp. K-9 (K-9). Identical amino acids are indicated by asterisks. The residues

used for designing primers for inverse PCR are underlined.


B23 (accession no. D90216), Brevibacterium sp. R312

(B37806), and Acinetobacter sp. ADP1 (CR543861)including control strains, R. globerulus A-4 [10] and R.

erythropolis N-771 (S04472). There is no report on a

detection of NHase activity in Rhodococcus sp. NCIMB

11215, which shows Oxd and Nit activities [7], but we

could suggest here the occurrence of NHase(Fe) in the

strain for the first time. No fragment was amplified with

NHCo1/NHCo2 primers except NHase(Co)-containing

R. rhodochrous J-1. We did not amplify Nit gene byPCR in this study since we could not identify consensus

sequences among the reported Nits.

1 10 20

Bb21196, Re3201 PDGYVEGWKKTFEEDFSPRRGAELVAR

Rs11215, BG16, A-4

BG13, N-771, 312 PDGYVEGWKKTFEEDFSPRRGAELVAR

K-9 PQGYVEQLTQLMEHGWSPENGARVVAK

B23 PEGYVEQLTQLMAHDWSPENGARVVAK

ADP PDGYVEGWKKTFEEDFSPRRGAELVAR

* **** ** ** ** *

70 80 90

Bb21196, Re3201 LKNVIVCSLCSCTAWPILGLPPTWYKS

Rs11215, BG16, A-4

BG13, N-771, 312 LKNVIVCSLCSCTAWPILGLPPTWYKS

K-9 LKNVIVCSLCSCTNWPVLGLPPEWYKG

B23 VKNVIVCSLCSCTNWPVLGLPPEWYKG

ADP LKNVIVCSLCSCTAWPILGLPPTWYKS

************* ** ***** ***

Fig. 3. Amino acid sequence comparison of a-subunit of NHases deduced f

Rhodococcus sp. N-771 (N-771), Pseudomonas sp. K-9 (K-9), B. butanicum

NCIBM 11215 (Rs11215), R. erythropolis BG 16 (BG16), and R. erythro

chlororaphis B23 (B23), Brevibacterium sp. R312 (312), and Acinetobacter sp

3.4. Cloning of whole oxd gene from Pseudomonas sp.

K-9 by an inverse PCR and expression of the enzyme in

E. coli

To show the effectiveness to identify oxd gene as a key

enzyme of the ‘‘aldoxime-nitrile pathway’’, we cloned

the whole oxd gene as a typical example from one of

the positive strain, Pseudomonas sp. K-9, which had

been isolated as a glutaronitrile-degrader [13], by an in-

verse PCR approach [14]. The primers, InvR2 andInvF2, used were designed based on the identified oxd

sequence of the strain by PCR, GYWGSMRDR and

30 40 50 60

AWTDPDFRQLLLTDGTAAVAQYGYLGPQGEYIVAVEDTPT

AWTDPEFRQLLLTDGTAAVAQYGYLGPQGEYIVAVEDTPT

AWVDPQFRALLLKDGTAACAQFGYTGPQGEYIVALEDTPQ

AWVDPQFRALLLKDGTAACAQFGYTGPQGEYIVALEDTPG

AWTDPDFRQLLLTDGTAAVAQYGYLGPQGEYIVAVEDTPT

* ** ** *** ***** ** ** ********* ****

100 110 120 130

FEYRARVVREPRKVLSEMGTEIASDVEIRVYDTTAETRYM


FEFRARLVREGRTVLRELGTELPNDMVVKVWDTSAESRYL

FEFRARLVREGRTVLRELGTELPSDTVIKVWDTSAESRYL


** *** *** * ** * *** * ** ** **

rom the amplified genes by PCR from R. erythropolis BG 13 (BG13),

ATCC 21196 (Bb21196), R. globerulus A-4 (A-4), Rhodococcus sp.

polis JCM 3201 (Re3201), and with the known NHase(Fe) from P.

. ADP1 (ADP1). Identical amino acids are indicated by asterisks.

1 10 20 30 40 50 60 70 80 90 100 110 120OxdRE MESAIGEHLQCPRTLTRRVPDTYTPPFPMWVGRADDALQQVVMGYLGVQFRDEDQRPAALQAMRDIVAGFDLPDGPAHHDLTHHIDNQGYENLIVVGYWKDVSSQHRWSTSTPIASWWESEDR-LOxdRG MESAIGEHLQCPRTLTRRVPDTYTPPFPMWVGRADDTLHQVVMGYLGVQFRGEDQRPAALRAMRDIVAGFDLPDGPAHHDLTHHIDNQGYENLIVVGYWKDVSSQHRWSTSPPVSSWWESEDR-LOxdK MESAIDTHLKCPRTLSRRVPDEYQPPFAMWMARADEHLEQVVMAYFGVQYRGEAQRAAALQAMRHIVESFSLADGPQTHDLTHHTDNSGFDNLIVVGYWKDPAAHCRWLRSAPVNAWWASEDR-LOxdA MESAIDTHLKCPRTLSRRVPEEYQPPFPMWVARADEQLQQVVMGYLGVQYRGEAQREAALQAMRHIVSSFSLPDGPQTHDLTHHTDSSGFDNLMVVGYWKDPAAHCRWLS-AEVNDWWTSQDR-LOxdB ----------------KNMPENHNPQANAWTAEFPPEMSYVVFAQIGIQSK---SLDHAAEHLGMMKKSFDLRTGPKHVDRALHQGADGYQDSIFLAYWDEPETFKSWVADPEVQKWWSGKKIDE

130 140 150 160 170 180 190 200 210 220 230 240 250OxdRE SDGLGFFREIVAPRAEQFETLYAFQED-LPGVGAVMDGISGEINEHGYWGSMRERFPISQTDWMQAS--GELRVIAGDPAVGGRVVVR-GHDNIALIRSGQDWADAEADERSLYLDEILPTLQSGOxdRG SDGLGFFREIVAPRAEQFETLYAFQDD-LPGVGAVMDGVSGEINEHGYWGSMRERFPISQTDWMQAS--GELRVVAGDPAVGGRVVVR-GHDNIALIRSGQDWADAEADERSLYLDEILPTLQSGOxdK NDGLGYFREISAPRAEQFETLYAFQDN-LPGVGAVMDRISGEIEEHGYWGSMRDRFPISQTDWMKPT--SELQVIAGDPAKGGRVVVL-GHGNLTLIRSGQDWADAEAEERSLYLDEILPTLQDGOxdA GEGLGYFREISAPRAEQFETLYAFQRDNLPGVGAVMDSTSGEIEEHGYWGSMRDRFPISQT-WMKPT--NELQVVAGDPAKGGRVVIM-GHDNIALIRSGQDWADAEAEERSLYLDEILPTLQDGOxdB NSPIGYWSEVTTIPIDHFETLHSGENY-DNGVSHFVP--IKHTEVHEYWGAMRDRMPVSASSDLESPLGLQLPEPIVRESFGKRLKVT-APDNICLIRTAQNWSKCGSGERETYIGLVEPTLIKA

260 270 280 290 300 310 320 330 340 350 360 370 OxdRE MDFLRDNGPAVGCYSNRFVRNIDIDGNFLDLSYNIGHWASLDQLERWSESHPTHLRIFTTFFRVAAG---LSKLRLYHEVSVFDAADQLYEYINCHPGTGMLRDAVTIAEHOxdRG MDFLRDNGPAVGCYSNRFVRNIDIDGNFLDLSYNIGHWASLDQLERWSESHPTHLRIFTTFFRVAEG---LSKLRLYHEVSVFDAADQLYEYINCHPGTGMLRDAVITAEHOxdK MDFLRDNGQPLGCYSNRFVRNIDLDGNFLDVSYNIGHWRSVEKLERWTESHPTHLRIFVTFFRVAAG---LKKLRLYHEVSVSDAKSQIFGYINCHPQTGMLRDAQVSPAOxdA MDFLRDNGQPLGCYSNRFVRNIDLDGNFLDVSYNIGHWRSLEKLERWAESHPTHLRIFVTFFRVAAG---LKKLRLYHEVSVSDAKSQVFEYINCHPHTGMLRDAVVAPTOxdB NTFLRENASETGCISSKLVYEQTHDGEIVDKSCVIGYYLSMGHLERWTHDHPTHKAIYGTFYEMLKRHDFKTELALWHEVSVLQSKDIELIYVNCHPSTGFLPFFEVTEIQEPLLKSPSVRI

Fig. 4. Amino acid sequence comparison of Oxds from Rhodococcus sp. N-771 (OxdRE), R. globerulus A-4 (OxdRG), Pseudomonas sp. K-9 (OxdK),

P. chlororaphis B23 (OxdA), and Bacillus sp. OxB-1 (OxdB). Residues in boxes indicate identical sequences among the Oxds.


SVEKLERWTE, respectively (Fig. 2). By sequencing

the amplified fragment, whole oxd gene sequence ofPseudomonas sp. K-9 was identified. Fig. 4 shows the

amino acid sequence similarities of a polypeptide en-

coded by the oxd gene with the known Oxds. It showed

identity with the Oxds of P. chlororaphis B23 (OxdA)

[20], R. erythropolis N-771 (OxdRE) [11], R. globerulus

A-4 (OxdRG) [10], and Bacillus sp. OxB-1 (OxdB) [8]

at 90.3%, 76.9%, 76.0%, and 32.7%, respectively. The se-

quence data have been submitted to DDBJ/EMBL/Gen-Bank databases under accession no. AB193508. A

plasmid, pOxdKInt, was constructed to express the

oxd gene in E. coli under the control of T7 promoter.

The protein was expressed in E. coli BL21 Star (DE3)/

pOxdKInt as we did for OxdRE [11]. The cell-free ex-

tract of the recombinant strain had an absorbance max-

imum at 410 nm, a characteristic Soret band for Oxds

[8–11], and exhibited a stoichiometric dehydration activ-ity of Z-phenylacetaldoxime into phenylacetonitrile

(320 U (lmol min�1) l�1 culture). Although further

investigations on the detailed properties of OxdK are

in progress, it is evidently clear that the enzyme is an

Oxd having a new primary structure and we tentatively

named it as OxdK.

4. Discussion

Here, we newly identified similar Oxd and NHase in

the 6 microbial nitrile- or aldoxime-degraders. The

PCR-based method shown in this study is quite useful

for the rapid identification of Oxds from various micro-

organisms without isolating Oxd protein. Indeed, the

newly identified OxdK has a new primary structureand the results encourage us to use this method to iden-

tify new types of Oxds from various microorganisms.

We could not amplify Oxd genes from the strains having

Nit and NHase(Co) probably because they might con-

tain Oxd having low similarities with the known ones.

Also, we [21] and the other group [22] recently reported

primitive studies on elucidating reaction mechanisms of

OxdB and OxdA, respectively, but the details of themechanism have not yet been understood. Further stud-

ies on purification and gene cloning of Oxds from a vari-

ety of microorganisms would accumulate genetic and

enzymatic information of the new types of Oxds and

comparison of the characters of the Oxds may help to

explain the mechanisms.

As reported by Duran et al. [19] and very recently by

Novo et al. [23], parts of NHases were amplified by PCRwith primers designed based on homologies of NHase to

show the existence of NHase(Fe) and NHase(Co) in

some bacterial strains. By comparing the results shown

in Figs. 2 and 3, it is possible to say that the primary

structure of Oxds is much similar each other (80.6%

identity) than those of NHase(Fe) (63.7% identity)

found among NHase(Fe)-containing strains. Thus, we

claim that the genetic analysis of oxd gene becomes amuch better tool to identify not only Oxd, but also

NHase(Fe).

The direct cloning of genes from environmental DNA

– the metagenome [24] has recently been paid much

attentions to obtain enzymes having novel primary

structures. Since enzymes comprising ‘‘aldoxime-nitrile

pathway’’, i.e., Oxd, NHase, Nit, and Ami, have become

potential catalysts in chemical industries [1,2], it isimportant to rapidly clone and identify genes of the

pathway in nature including metagenomes. It would be

advantageous to use Oxd as a key enzyme in the path-

way, because Oxd locates at an upstream of the

‘‘branched’’ nitrile-degradation which is catalyzed by di-

verse enzymes, Nit, NHase(Fe), and NHase(Co). In

practice, we have been focusing on Oxd and have shown

that all the microorganisms having Oxd also had nitrile-degrading enzymes and Oxd and the nitrile-degrading

enzymes are linked genetically as well as enzymatically

[1,7–11]. In a separate experiment from this study, we se-

quenced flanking regions of oxd gene in the genome of

Pseudomonas sp. K-9 and found that the oxd gene was

clustered with genes coding NHase(Fe), Ami, and their


activators and regulatory proteins, as seen in NHa-

se(Fe)-containing microorganisms, such as P. chlorora-

phis B23, R. erythropolis N-771, and R. globerulus A-4

(data not shown). Based on the results, we can conclude

here that the PCR-based analysis of oxd gene is a useful

tool to detect and analyze the ‘‘aldoxime-nitrile path-way’’ in nature because Oxd is important as a key en-

zyme. The structures of the ‘‘aldoxime-nitrile

pathway‘‘ gene cluster in Pseudomonas sp. K-9 and the

enzymatic properties of OxdK will be reported

elsewhere.

References

[1] Asano, Y. (2002) Overview of screening for new microbial

catalysts and their uses in organic synthesis – selection and

optimization of biocatalysts. J. Biotechnol. 94, 65–72.

[2] Banerjee, A., Sharma, R. and Banerjee, U.C. (2002) The nitrile-

degrading enzymes: current status and future prospects. Appl.

Microbiol. Biotechnol. 60, 33–44.

[3] Cowan, D.A., Cameron, R.A. and Tsekoa, T.L. (2003) Compar-

ative biology of mesophilic and thermophilic nitrile hydratases.

Adv. Appl. Microbiol. 52, 123–158.

[4] Asano, Y., Tani, Y. and Yamada, H. (1980) A new enzyme nitrile

hydratase which degrades acetonitrile in combination with

amidase. Agric. Biol. Chem. 44, 2251–2252.

[5] Asano, Y., Fujishiro, K., Tani, Y. and Yamada, H. (1982)

Aliphatic nitrile hydratase from Arthrobacter sp. J-1. Purification

and characterization. Agric. Biol. Chem. 46, 1165–1174.

[6] Asano, Y., Yasuda, T., Tani, Y. and Yamada, H. (1982) A new

enzymatic method of acrylamide production. Agric. Biol. Chem.

46, 1183–1189.

[7] Kato, Y., Ooi, R. and Asano, Y. (2000) Distribution of aldoxime

dehydratase in microorganisms. Appl. Environ. Microbiol. 66,

2290–2296.

[8] Kato, Y., Nakamura, K., Sakiyama, H., Mayhew, S.G. and

Asano, Y. (2000) A novel heme-containing lyase, phen-

ylacetaldoxime dehydratase from Bacillus sp. strain OxB-1:

purification, characterization, and molecular cloning of the gene.

Biochemistry 39, 800–809.

[9] Kato, Y. and Asano, Y. (2003) High-level expression of a novel

FMN-dependent heme-containing lyase, phenylacetaldoxime

dehydratase of Bacillus sp. strain OxB-1, in heterologous hosts.

Protein Exp. Purif. 28, 131–139.

[10] Xie, S.-X., Kato, Y., Komeda, H., Yoshida, S. and Asano, Y.

(2003) A novel gene cluster responsible for alkylaldoxime metab-

olism coexisting with nitrile hydratase and amidase in Rhodococ-

cus globerulus A-4. Biochemistry 42, 12056–12066.

[11] Kato, Y., Yoshida, S., Xie, S.-X. and Asano, Y. (2004) Aldoxime

dehydratase co-existing with nitrile hydratase and amidase in

iron-type nitrile hydratase producer Rhodococcus sp. N-771. J.

Biosci. Bioeng. 97, 250–259.

[12] Xie, S.X., Kato, Y. and Asano, Y. (2001) High yield synthesis of

nitriles by a new enzyme, phenylacetaldoxime dehydratase, from

Bacillus sp. strain OxB-1. Biosci. Biotechnol. Biochem. 65, 2666–

2672.

[13] Yamada, H., Asano, Y. and Tani, Y. (1980) Microbial utilization

of glutaronitrile. J. Ferment. Technol. 58, 495–500.


Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor


[15] Pearson, W.R. and Lipman, D.J. (1988) Improved tools for

biological sequence comparison. Proc. Natl. Acad. Sci. USA 85,

2444–2448.

[16] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman,

D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215,

403–410.

[17] Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUS-

TAL W: improving the sensitivity of progressive multiple

sequence alignment through sequence weighting, position-specific

gap penalties and weight matrix choice. Nucleic Acid. Res. 22,

4673–4680.

[18] Saito, H. and Miura, K. (1963) Preparation of transforming

deoxyribonucleic acid by phenol treatment. Biochim. Biophys.

Acta 72, 619–629.

[19] Precigou, S., Goulas, P. and Duran, R. (2001) Rapid and specific

identification of nitrile hydratase (NHase)-encoding genes in soil

samples by polymerase chain reaction. FEMS Microbiol Lett.

204, 155–161.

[20] Oinuma, K., Hashimoto, Y., Konishi, K., Goda, M., Noguchi, T.,

Higashibata, H. and Kobayashi, M. (2003) Novel aldoxime

dehydratase involved in carbon-nitrogen triple bond synthesis of

Pseudomonas chlororaphis B23. Sequencing, gene expression,

purification, and characterization. J. Biol. Chem. 278, 29600–

29608.

[21] Lourenco, P.M., Almeida, T., Mendonca, D., Simoes, F. and

Novo, C. (2004) Searching for nitrile hydratase using the

Consensus-Degenerate Hybrid Oligonucleotide Primers strategy.

J. Basic Microbiol. 44, 203–214.

[22] Kobayashi, K., Yoshioka, S., Kato, Y., Asano, Y. and Aono, S.

(2005) Regulation of aldoxime dehydratase activity by redox-

dependent change in the coordination structure of the aldoxime-

heme complex. J. Biol. Chem. 280, 5486–5490.

[23] Konishi, K., Ishida, K., Oinuma, K., Ohta, T., Hashimoto, Y.,

Higashibata, H., Kitagawa, T. and Kobayashi, M. (2004)

Identification of crucial histidines involved in carbon-nitrogen

triple bond synthesis by aldoxime dehydratase. J. Biol. Chem.

279, 47619–47625.

[24] Streit, W.R. and Schmitz, R.A. (2004) Metagenomics – the key to

the uncultured microbes. Curr. Opin. Microbiol. 7, 492–498.



The gene encoding xylulose-5-phosphate/fructose-6-phosphatephosphoketolase (xfp) is conserved among Bifidobacterium

species within a more variable region of the genome and bothare useful for strain identification

Xianhua Yin, James R. Chambers, Kathleen Barlow, Aaron S. Park, Roger Wheatcroft *

Agriculture and Agri-Food Canada, Food Research Program, 93 Stone Road West, Guelph, Ont., Canada N1G 5C9

Received 31 December 2004; received in revised form 23 March 2005; accepted 12 April 2005


Edited by R.Y.C. Lo

Abstract

The nucleotide sequence of the xfp-gene region in six known and two unknown species of Bifidobacterium was determined and

compared with the published sequences of B. animalis subsp. lactis DSM10140 and B. longum biovar longum NCC2705. The xfp

coding sequences were 73% identical and coded for 825 amino acids in all 10 sequences. Partial sequences of an adjacent gene, guaA,

were 61% identical in six sequences for which data were available. The region between xfp and guaA was variable in both length and

sequence. Oligonucleotide sequences from the conserved and variable xfp regions were used as PCR primers, in combinations of

appropriate specificity, for the detection and identification of Bifidobacterium isolates.


Keywords: Bifidobacterium; Phosphoketolase; xfp; Strain identification; Detection

1. Introduction

Bacteria of the genus Bifidobacterium are anaerobic,

Gram-positive, non-spore-forming, non-motile bacilli

[1]. They are found in sewage and in the internal tracts

of animals, including insects and humans [2,3]; about

30 species are currently recognized [4]. Some species

are used in industry for the preparation of fermentedfoods and dietary supplements [2,5]. In the human gut,

bifidobacteria are generally regarded as safe and consis-

tent with good health [6–8]. They possess a wide range of

catabolic pathways which break down undigested food


doi:10.1016/j.femsle.2005.04.013

* Corresponding author. Tel: +1 519 780 8025; fax: +1 519 829 2600.

E-mail address: [email protected] (R. Wheatcroft).

and secretions of the host [9,10]. They are at an advan-

tage as scavengers in the large intestine where readily

fermentable carbohydrates are in short supply [11,12].

A characteristic pathway is the �bifid shunt�, by which

bifidobacteria convert hexoses to acetic acid and lactic

acid, as chief products, in a theoretical molar ratio of

3:2 [13]. Secretion of acid into the gut is likely to affect

the growth and composition of the local microflora[14,15]. When acetic acid is undissociated, for example,

it acts synergistically with lactic acid to inhibit growth

of many enteric bacteria [16]. There is evidence to sug-

gest that bifidobacteria provide protection against some

pathogens, by this mechanism, in both humans and live-

stock [17–19]: a possibility that is of considerable inter-

est for public health and food safety. Clearly, there is a

need to establish whether bifidobacteria can be reliably



Fig. 1. Southern hybridization of genomic DNA digested with EcoRI

probed with chemiluminescent xfp probe, P1. m: Size marker; lane 1:

B. animalis subsp. animalis ATCC27674; lane 2: B. gallinarum

ATCC33777, lane 3: B. longum biovar infantis ATCC15697, lane 4:

B. longum biovar longum ATCC15707, lane 5: B. pseudolongum subsp.

pseudolongum ATCC25526, lane 6: B. pullorum ATCC49618, lane 7: B.

thermophilum ATCC25525, lane 8: B. sp. BcRW10.

Table 1

Strains of bifidobacteria

Species Strain Source

B. adolescentis CFAR335 Human infant

B. animalis subsp. animalis ATCC27536 Chicken faeces

B. animalis subsp. animalis ATCC27674 Rabbit faeces

B. bifidum CFAR115 Wheat germ

B. boum ATCC27917 Bovine rumen

B. breve CFAR118 Morinaga Institute

252 X. Yin et al. / FEMS Microbiology Letters 246 (2005) 251–257

used as an adjunct or effective alternative to antibiotics

[20]; and, if so, whether natural populations can be stim-

ulated [21], supplemented [22] or genetically modified

for this purpose [23].

An early step in the �bifid shunt� is the phosphoketo-

lase reaction [EC: 4.1.2.22] by which D-fructose-6-phos-phate (F6P) is converted to erythrose-4-phosphate and

acetyl-1-phosphate [13]. This reaction is used to test

for Bifidobacterium species [24] though it is not exclusive

to them [25]. There is evidence for the existence of two

distinct F6P-phosphoketolase enzymes in bifidobacteria

[26,27]. One is specific solely for F6P; the other is less

stringent and is able to utilize D-xylulose-5-phosphate

(X5P) as an alternative substrate [EC: 4.1.2.9]. The latterreaction, which yields glyceraldehyde-3-phosphate and

acetyl-1-phosphate, is a later step in the �bifid shunt�[13]. Thus, it would seem that the same enzyme is able

to play an important double role in this pathway.

The dual-specificity X5P/F6P-phosphoketolase is en-

coded by the gene xfp, first described in B. animalis

subsp. lactis [25]. In the human isolate B. longum biovar

longum NCC2705, the genome has been completelysequenced [10] and a single copy of xfp is identified at lo-

cus BLO959. In another important contribution, a 503-

bp amplicon of xfp has been sequenced in most, if not

all, Bifidobacterium species; see GenBank Accessions:

AY574091 and AY377393 to AY377424, inclusive [28].

In the present study, we have sequenced xfp and its

neighbouring region in a selection of Bifidobacterium

species to explain the restriction-fragment-length poly-morphism (RFLP) observed (Fig. 1). We have selected

and tested primer and target sequences for the detection

of these species, and for the identification of isolates, by

PCR. This approach is but one of many molecular

methods now available for the detection and identifica-

tion of bifidobacteria [29–33].
(Japan)
B. choerinum ATCC27686 Pig faeces

B. cuniculi ATCC27916 Rabbit faeces

B. dentium ATCC27534 Human dental caries

B. gallinarum ATCC33777 Chicken caecum

B. gallinarum ATCC33778 Chicken caecum

B. longum biovar infantis ATCC15697 Human infant intestine

B. longum biovar longum ATCC15707 Human adult intestine

B. longum biovar suis ATCC27533 Pig faeces

B. magnum ATCC27540 Rabbit faeces

B. merycicum CFAR339 Bovine rumen

B. minimum ATCC27538 Sewage

B. pseudolongum

subsp. globosum

ATCC25865 Bovine rumen

B. pseudolongum

subsp. pseudolongum

ATCC25526 Pig faeces

B. pullorum ATCC27685 Chicken faeces

B. pullorum ATCC49618 Chicken faeces

B. thermophilum ATCC25525 Pig faeces

B. sp. CFAR172 Calf-Guard (Pfizer)

B. sp. BcRW10 Pig faeces

ATCC, American Type Culture Collection, Rockville, MD, USA.

CFAR, Centre for Food and Animal Research (Agriculture and Agri-

Food Canada).


2.1. Bacterial strains, plasmids and primers

Strains of bifidobacteria used in this study are listed

in Table 1. Cultures were grown anaerobically at 37

�C, in MRS medium (Difco) supplemented with 0.05%

cysteine hydrochloride, 0.02% Na2CO3 and 0.01%CaCl2. Tests were also made on the bacteria listed in

Table 2, which were grown according to ATCC recom-

mendations. Plasmids, listed in Table 3, were propa-

gated in Escherichia coli TOP10 (Invitrogen) or

GM2163 dam cells (NEB) at 37 �C, in LB broth [34] sup-

plemented with ampicillin (50 lg ml�1). T3 and T7 prim-

ers were used to sequence pRWBl10 and pRWBp10;

16S-rRNA primers (P0 and 338F), which typically pro-duce a PCR amplicon of 332 bp from genomic DNA,

were used as positive controls [35]; other oligonucleo-

Table 2

Other bacterial strains tested

Species Strain or source PCR product

obtained with

primers U1R/U2L

Actinomyces bovis ATCC13683 +

Actinomyces israelii ATCC12102 +

Arthrobacter ureafaciens ATCC7562 +

Bacillus cereus ATCC14579 �Bacillus subtilis ATCC6051 �Citrobacter freundii ATCC8090 �Clostridium lituseburense DSM797 �Clostridium perfringens D. Barnuma �Enterobacter aerogenes ATCC13048 �Enterococcus faecalis ATCC19433 �Escherichia coli ATCC11775 �Gardnerella vaginalis ATCC14018 +

Gluconacetobacter hansenii ATCC23769 �Gluconacetobacter xylinus ATCC23767 +

Klebsiella pneumoniae ATCC13883 �Lactobacillus amylovorus DSM20531 �Lactobacillus salivarius DSM20555 �Leuconostoc mesenteroides ATCC8293 +

Listeria innocua B. Blaisb �Listeria monocytogenes B. Blaisb �Propionibacterium acnes ATCC6919 �Propionibacterium

freudenreichii

ATCC8262 �

Proteus hauseri ATCC13315 �Pseudomonas aeruginosa ATCC10145 �Rhodococcus fascians ATCC12974 +

Ruminococcus torques ATCC27756 �Salmonella choleraesuis

subsp. choleraesuis

Montevideo

ATCC8387 �

Salmonella choleraesuis

subsp. choleraesuis

Typhimurium

ATCC14028 �

Serratia marcescens ATCC13880 �Shigella sonnei ATCC29930 �Staphylococcus aureus ATCC12600 �Staphylococcus epidermidis ATCC12228 �Yersinia enterocolitica ATCC9610 �ATCC, American Type Culture Collection, Rockville, MD, USA.

CFAR, Centre for Food and Animal Research (Agriculture and Agri-

Food Canada). DSM, German Collection of Microorganisms and Cell

Cultures, Braunschweig, Germany.a University of Guelph, Ont., Canada.b Canadian Food Inspection Agency, Ottawa, Canada.

X. Yin et al. / FEMS Microbiology Letters 246 (2005) 251–257 253

tides used in this study are listed in Table 3 and mapped

in Fig. 2.

2.2. DNA manipulation and analysis

Genomic DNA was isolated from 5-ml bacterial cul-

tures as previously described [36]. A commercial kit

(Qiagen) was used to extract plasmids from cells andto purify DNA from agarose gels. DNA restriction,

cloning, and PCR were carried out by routine methods

[34]. The PCR programme consisted of an initial step

at 94 �C for 4 min, followed by 35 cycles at 94 �C for

30 s, 60 �C for 30 s, and 72 �C for 1 min, followed by

a final step at 72 �C for 10 min. DNA probes, P1 and

P2, were digoxigenin labelled by PCR, as previously

described [37], using B. animalis ATCC27536 DNA as

substrate with primers A1R/A1L and A2R/A2L, respec-

tively. Southern blot and colony hybridizations werecarried out on HyBond filters (Amersham–Pharmacia

Biotech). Hybridization was detected using a commer-

cial chemiluminescence kit (Roche Diagnostic) and

XAR-2 film (Kodak). Nucleotide sequences of DNA

were determined using a Prism 377 automated sequencer

(ABI); alignments and analysis were carried out using

DNAman software (Lynnon BioSoft). GenBank acces-

sion numbers are included in Fig. 2.

3. Results

3.1. RFLP of the xfp-gene region

Southern hybridization profiles of Bifidobacterium

genomic DNA digested with EcoRI and probed withP1 are shown in Fig. 1. A single band indicates the pres-

ence of one copy of xfp in the genome and the absence

of any EcoRI-cleavage sites in the region covered by

the probe. The relative position of bands shows that

the location of EcoRI sites in the xfp region and the size

of hybridizing fragments are variable among genomes

(RFLP). A set of single- or multi-banded xfp-hybridiza-

tion profiles were obtained for several endonucleases(data not shown), whose cleavage sites were subse-

quently confirmed by DNA sequencing (Fig. 2).

3.2. DNA-sequence analysis

Blots were probed successively, with P1 and P2, to

identify the shortest restriction fragments that contained

both ends of xfp and, therefore, might be expected tocontain its full length. Thus, the 4.96-kb-HindIII/EcoRI

fragment of B. longum biovar longum ATCC15707

(pRWBl10) and the 4.43-kb-HindIII/XbaI fragment of

B. pullorum ATCC49618 (pRWBp10) were identified,

cloned and sequenced. The sequences were compared

with the published sequences of B. animalis subsp. lactis

DSM10140 and B. longum biovar longum NCC2705

(Fig. 2). Suitable oligonucleotides were synthesized (Ta-ble 3) to sequence the whole of xfp and its 5 0-neighbour-

ing region in B. pullorum ATCC49618 and in six other

Bifidobacterium isolates, including two unknown species

(Fig. 2). The 10 xfp coding sequences were found to be

73% identical at the nucleotide level. Their relatedness

is shown in Fig. 3. Each was found to encode 825 amino

acids of which 77% are conserved. By comparison, a

395-nucleotide partial sequence of guaA, a neighbouringgene encoding GMP synthase, was shown to be 61%

conserved at the nucleotide level, in six sequences for

Table 3

Plasmids and oligonucleotides

Source Designation Description or sequence (5 0–30)

Stratagene BlueScriptII SK+ Cloning vehicle for restriction fragments; Apr

Invitrogen pCR4-TOPO Cloning vehicle for PCR amplicons; Apr

B. longum biovar longum ATCC15707 pRWBl10 4.96-kb HindIII–EcoRI fragment containing xfp gene in BlueScriptII SK+; Apr

B. pullorum ATCC49618 pRWBp10 4.43-kb HindIII–XbaI fragment containing truncated xfp gene in

BlueScriptII SK+; Apr

B. animalis subsp. lactis DSM10140 [25] A1R catggcagaagctggatcgt

A2R gtcaccaagaagcagtgggac

A3R gctcaagcactgcaatcacaag

A1L ctccggcttgtaggattcca

A2L ggcctttcatcggctaagc

B. longum biovar longum NCC2705 [10] L1R gccactgcacaccatagagcttg

L2R tggctcatccacgtggtctgctc

L3R gatcacgtgcaggagtacagg

L4R atcctgcacctcaacggctac

L5R aagggctggacctgcccgaag

L1L agccctcggtcttcttgccgtc

L2L taggactcgagccagttcttgag

L3L tcactcgttgtcgccagcgg

B. pullorum ATCC49618 [this work] P1R gtctattgtggcggttcaagg

U1R acctgcccgaagtacatcgac

U1L tgtactcctgtactcctgcac

U2L gagctccagatgccgtgacg

B. sp. CFAR172 [this work] B1R cgactcagtactgattgatacc

B. sp. BcRW10 [this work] B1L tgcagcttcaggaggtcaacg

Apr, ampicillin resistant.

Fig. 2. Restriction maps to show variation (RFLP) in the xfp-gene region of Bifidobacterium species (including two cloned segments, pRWBp10 and

pRWBl10). Thick lines indicate coding sequences of xfp and part of guaA. Small arrowheads indicate the 5 0-ends of oligonucleotides (Table 3) used as

primers for PCR and sequencing (underlined). Primers, A1R/A1L and A2R/A2L, were used to make probes, P1 and P2, respectively. U1R/U2L

produced a 593-bp amplicon with all Bifidobacterium and some other species tested (Table 2). A3R, B1R and P1R with U1L produced amplification

products only with those species indicated to contain them. Dotted lines indicate the 5 0-ends of xfp and guaA delimiting the variable region between

them. Restriction sites: B, BamHI; C, BclI; G, BglII; H, HindIII; S, SphI; X, XbaI; Y, XmnI. GenBank accession numbers for the nucleotide

sequences determined in this work are given at right.


Fig. 3. Homology tree of xfp-gene coding sequences of Bifidobacterium species. DNA homology is expressed as the number of identical nucleotide

residues between sequences as a percentage of the length of the aligned sequences (2475 nucleotides). The 10 sequences studied were 73% identical

overall.


which data were available. In these species, the region

between xfp and guaA was found to be variable both

in sequence and in length (Fig. 2).

3.3. Specific detection and identification of bifidobacteria

by PCR

Primer sequences were selected from the xfp region

to use in PCR for diagnostic purposes (Table 3; Fig.

2). The combination, U1R/U2L, from the coding se-

quence of B. pullorum, generated a 593-bp amplicon

from genomic DNA of all Bifidobacterium strainstested (Table 1). The DNA of 33 strains of non-Bifido-

bacterium species was also tested. Seven species, which

were reported in the literature to possess a F6P-phos-

phoketolase, tested positive with U1R/U2L (Table 2).

The remaining strains were negative with U1R/U2L

but all tested positive with 16S-rRNA primers P0/

338F, used in controls of the PCR and template

DNA (data not shown).The Bifidobacterium strains were further resolved by

using combinations of primers chosen from both con-

served and variable sequences. For example, the combi-

nation P1R/U1L generated a 410-bp amplicon with both

B. gallinarum and B. pullorum, which are closely related

species (Fig. 3). Primers B1R/U1L generated a 565-bp

amplicon only with isolate B. sp. CFAR172, whereas

A3R/U1L gave a 362-bp amplicon only with B. animalis

strains. None of these primer combinations gave posi-

tive PCR results with any other bacterial species tested(Tables 1 and 2). These primers are now used routinely

to test directly for their respective species in samples

(Fig. 4).

4. Discussion

The gene xfp encodes one of two F6P-phosphoketo-lases reported to occur in bifidobacteria [26,27]. There

is evidence to suggest that one, if not both of these en-

zymes, is polymorphic, differing in human and animal

Bifidobacterium species [27,38]. In this work, we have

investigated the xfp region in several examples but have

been unable to detect any sequence differences that

would simply explain the enzyme polymorphism

reported. We conclude, therefore, that a species-dependent modification of the xfp-gene product takes

place or, alternatively, that the polymorphism reported

does not apply to the phosphoketolase encoded by

xfp.

We showed that the length (2475 nucleotides) and

73% of the xfp coding sequence are absolutely conserved

Fig. 4. Detection of diagnostic PCR products in Bifidobacterium species. m: size marker; and for each set, lane 1: B. pullorum ATCC49618; lane 2: B.

gallinarum ATCC33778; lane 3: B. longum biovar longum ATCC15707; lane 4: B. animalis subsp. animalis ATCC27536. Set A: 593-bp amplicon

obtained with all Bifidobacterium and some other species (Table 2), using primers U1R/U2L; set B: 410-bp amplicon only obtained with B. pullorum

and B. gallinarum, using primers P1R/U1L; set C: 362-bp amplicon obtained with all B. animalis strains tested, using primers A3R/U1L.


in the 10 examples studied; whereas an adjacent house-

keeping gene, guaA, appeared to be less conserved. This

suggests that the function of xfp, if not the whole �bifidshunt� pathway, confers a significant advantage inBifidobacterium.

The xfp gene is characteristic of Bifidobacterium, yet

some non-Bifidobacterium species also gave positive

PCR results with primers designed to amplify its core,

for example U1R and U2L. The usefulness of these

primers for the detection of bifidobacteria must there-

fore be circumscribed; they are likely to be most useful

in the study of exclusive habitats like the gut, for exam-ple. By comparing amplicon sequences generated by

U1R/U2L from purified isolates, it is possible to differ-

entiate between species and assign them to homology

groups consistent with current taxonomy (Fig. 3). It is

also possible, and most convenient, to make a positive

species identification by matching the nucleotide se-

quence of an xfp amplicon with that of an authenticated

species [28]. We have, for example, used the currentGenBank database to identify isolates B. sp. BcRW10

and B. sp. CFAR172 precisely, as B. choerinum and B.

thermophilum, respectively.

Since 5 0-sequences adjacent to xfp are variable in

Bifidobacterium, they present species-specific targets

for diagnostic PCR. The primer combination A3R/

U1L, for example, is useful for the detection of B. ani-

malis strains. In our current work in progress, we areusing the combination P1R/U1L to detect strains of

B. pullorum [39], including mutant derivatives, in a ge-

netic study of their purported probiotic effects in

chickens.

Acknowledgements

We are grateful to our AAFC colleagues for help, ad-

vice and comments on the manuscript.

References

[1] Scardovi, V. (1986) Genus Bifidobacterium Orla-Jensen, 1924 In:

Bergey�s Manual of Systematic Bacteriology (Sneath, P.H.A.,

Mair, N.S., Sharpe, M.E. and Holt, J.G., Eds.), Vol. 2, pp. 1418–

1434. Williams and Wilkins, Baltimore, MD.

[2] Rasic, J. and Kurmann, J. (1983). Bifidobacteria and Their Role.

Experientia, Vol. 39. Birkhauser Verlag, Basel, Supplementum

278 pp..

[3] Biavati, B., Vescovo, M., Torriani, S. and Bottazzi, V. (2000)

Bifidobacteria: history, ecology, physiology and applications.

Ann. Microbiol. 50, 117–131.

[4] Ventura, M., van Sinderen, D., Fitzgerald, G.F. and Zink, R.

(2004) Insights into the taxonomy, genetics and physiology of

bifidobacteria. Anton. Leeuw. 86, 205–223.

[5] Kurmann, J.A. and Rasic, J.L. (1991) The health potential of

products containing bifidobacteria In: Therapeutic Properties of

Fermented Milks (Robinson, R.K., Ed.). Elsevier, London.

[6] Tannock, G.W. (1995) Normal Microflora: An Introduction to

Microbes Inhabiting the Human Body. Chapman & Hall,

London, 115 pp..

[7] Mitsuoka, T. (1990) Bifidobacteria and their role in human

health. J. Ind. Microbiol. 6, 263–268.

[8] Ishibashi, N., Yaeshima, T. and Hayasawa, H. (1997) Bifidobac-

teria: their significance in human intestinal health. Mal. J. Nutr. 3,

149–159.

[9] Crociani, F., Alessandrini, A., Mucci, M.M. and Biavati, B.

(1994) Degradation of complex carbohydrates by Bifidobacterium

spp.. Int. J. Food Microbiol. 24, 199–210.

[10] Schell, M.A., Karmirantzou, M., Snel, B., Vilanova, D., Berger,

B., Pessi, G., Zwahlen, M.C., Desiere, F., Bork, P., Delley, M.,

Pridmore, R.D. and Arigoni, F. (2002) The genome sequence of

Bifidobacterium longum reflects its adaptation to the human

gastrointestinal tract. Proc. Natl. Acad. Sci. USA 99, 14422–

14427.

[11] Macfarlane, G.T. and Macfarlane, S. (1997) Human colonic

microbiota: ecology physiology and metabolic potential of

intestinal bacteria. Scand. J. Gastroenterol. 32 (Suppl. 222), 3–9.

[12] Hopkins, M.J., Cummings, J.H. and Macfarlane, G.T. (1998)

Inter-species differences in maximum specific growth rates and cell

yields of bifidobacteria cultured on oligosaccharides and other

simple carbohydrate sources. J. Appl. Microbiol. 85, 381–386.

[13] Scardovi, V. and Trovatelli, L.D. (1965) The fructose-6-phos-

phate shunt as peculiar pattern of hexose degradation in the genus

Bifidobacterium. Ann. Microbiol. 15, 19–29.


[14] Benno, Y. and Mitsuoka, T. (1992) Impact of Bifidobacterium

longum on human fecal microflora. Microbiol. Immunol. 36, 683–

694.

[15] Gibson, G.R. and Wang, X. (1994) Regulatory effects of

bifidobacteria on the growth of other colonic bacteria. J. Appl.

Bacteriol. 77, 412–420.

[16] Adams, M.R. and Hall, C.J. (1988) Growth inhibition of food-

borne pathogens by lactic and acetic acids and their mixtures. Int.

J. Food Sci. Technol. 23, 287–292.

[17] Savage, D.C. (1987) Factors influencing biocontrol of bacterial

pathogens in the intestine. Food Technol. 41, 82–87.

[18] Ibrahim, S.A. and Bezkorovainy, A. (1993) Inhibition of Esch-

erichia coli by bifidobacteria. J. Food Prot. 56, 713–715.

[19] Araya-Kojima, T., Yaeshima, T., Ishibashi, N., Shimamura, S.

and Hayasawa, H. (1995) Inhibitory effects of Bifidobacterium

longum BB536 on harmful intestinal bacteria. Bifidobacteria

Microflora 14, 59–66.

[20] Gomes, A.M.P. and Malcata, F.X. (1999) Bifidobacterium spp.

and Lactobacillus acidophilus: biological, biochemical, technolog-

ical and therapeutical properties relevant for use as probiotics.

Trends Food Sci. Technol. 10, 139–157.

[21] Gibson, G.R. and Wang, X. (1994) Enrichment of bifidobacteria

from human gut contents by oligofructose using continuous

culture. FEMS Microbiol. Lett. 118, 121–128.

[22] Ballongue, J., Grill, J.P. and Baratte-Euloge, P. (1993) Action sur

la flore intestinale de laits fermentes au Bifidobacterium. Lait 73,

249–256.

[23] Kullen, M.J. and Klaenhammer, T.R. (2000) Genetic modifica-

tion of intestinal lactobacilli and bifidobacteria. Curr. Issues Mol.

Biol. 2, 41–50.

[24] Orban, J.I. and Patterson, J.A. (2000) Modification of the

phosphoketolase assay for rapid identification of bifidobacteria.

J. Microbiol. Methods 40, 221–224.

[25] Meile, L., Rohr, L.M., Geissmann, T.A., Herensperger, M. and

Teuber, M. (2001) Characterization of the D-xylulose 5-phos-

phate/D-fructose 6-phosphate phosphoketolase gene (xfp) from

Bifidobacterium lactis. J. Bacteriol. 183, 2929–2936.

[26] Sgorbati, B., Lenaz, G. and Casalicchio, F. (1976) Purification

and properties of two fructose-6-phosphate phosphoketolases in

Bifidobacterium. Anton. Leeuw. 42, 49–57.

[27] Grill, J.P., Crociani, J. and Ballongue, J. (1995) Characterization

of fructose 6 phosphate phosphoketolases purified from Bifido-

bacterium species. Curr. Microbiol. 31, 49–54.

[28] Berthoud, H., Chavagnat, F., Haueter, M. and Casey, M.G.

(2005) Comparison of partial gene sequences encoding a phos-

phoketolase for the identification of bifidobacteria. Lebensm.

Wiss. Technol. 38, 101–105.

[29] Zavaglia, A.G., de Urraza, P. and De Antoni, G. (2000)

Characterization of Bifidobacterium strains using box primers.

Anaerobe 6, 169–177.

[30] Satokari, R.M., Vaughan, E.E., Smidt, H., Saarela, M., Matto, J.

and de Vos, W.M. (2003) Molecular approaches for the detection

and identification of bifidobacteria and lactobacilli in the human

gastrointestinal tract. Syst. Appl. Microbiol. 26, 572–584.

[31] Mullie, C., Odou, M.F., Singer, E., Romond, N.B. and Izard, D.

(2003) Multiplex PCR using 16S rRNA gene-targeted primers for

the identification of bifidobacteria from human origin. FEMS

Microbiol. Lett. 222, 129–136.

[32] Zhu, L., Li, W. and Dong, X. (2003) Species identification of

genus Bifidobacterium based upon partial HSP60 gene

sequences and proposal of Bifidobacterium thermacidophilum

subsp. porcinum subsp. nov. Int. J. Syst. Evol. Microbiol. 53,

1619–1623.

[33] Delcenserie, V., Bechoux, N., Leonard, T., China, B. and Daube,

G. (2004) Discrimination between Bifidobacterium species from

human and animal origin by PCR-restriction fragment length

polymorphism. J. Food Protection 67, 1284–1288.


Cloning: A Laboratory Manual, second ed. Cold Spring Harbor


[35] Ventura, M., Reniero, R. and Zink, R. (2001) Specific identifi-

cation and targeted characterization of Bifidobacterium lactis

from different environmental isolates by a combined multiplex-

PCR approach. Appl. Environ. Microbiol. 67, 2760–2765.

[36] Wheatcroft, R. and Watson, R.J. (1988) A positive strain

identification method for Rhizobium meliloti. Appl. Environ.

Microbiol. 54, 574–576.

[37] Anon (1995) The DIG System User�s Guide for Filter Hybrid-

ization. Boehringer Mannheim GmbH, Mannheim, 100 pp..

[38] Scardovi, V., Sgorbati, B. and Zani, G. (1971) Starch gel

electrophoresis of fructose-6-phosphate phosphoketolase in the

genus Bifidobacterium. J. Bacteriol. 106, 1036–1039.

[39] Trovatelli, L.D., Crociani, F., Pedinotti, M. and Scardovi, V.

(1974) Bifidobacterium pullorum sp. nov.: a new species isolated

from chicken feces and a related group of bifidobacteria isolated

from rabbit feces. Arch. Microbiol. 98, 187–198.



The stabilization of housekeeping transcripts in Trypanosoma cruziepimastigotes evidences a global regulation of RNA decay

during stationary phase

Ana Marıa Cevallos, Mariana Perez-Escobar, Norma Espinosa, Juliana Herrera,Imelda Lopez-Villasenor, Roberto Hernandez *

Departamento de Biologıa Molecular y Biotecnologıa, Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico,

Apartado Postal 70-228, 04510 Mexico D.F., Mexico



Edited by D.P. Wakelin

Abstract

The relative steady state concentration of mRNAs of four housekeeping single-copy type Trypanosoma cruzi genes (actin, triose-

phosphate isomerase, trypanothion reductase and the ribosomal protein S4) was analyzed throughout the growth curve. A distin-

guishable pattern was observed with maximal levels occurring at the logarithmic phase of growth and minimum levels occurring at

the stationary phase. The half-lives of all analyzed messenger RNAs, and also of three molecular species of immature ribosomal

RNAs were increased in cells isolated from stationary phase. These results suggest the occurrence of a novel global regulation mech-

anism that might protect transcripts from degradation in stationary epimastigotes, probably as a strategy to perpetuate through this

quiescent stage.


Keywords: Protozoa; Kinetoplastid; RNA stability; Gene expression

1. Introduction

Trypanosoma cruzi is a parasitic protozoa causative

agent of Chagas disease or American trypanosomiasis.

During its life cycle this parasite can alternate throughvertebrate and invertebrate hosts. In both hosts, T. cruzi

goes through a cycle that includes infective and non-

infective forms and these forms are morphologically

identifiable (see [1] for review). Differential gene expres-

sion occurs during development of this organism, espe-


doi:10.1016/j.femsle.2005.04.017

* Corresponding author. Tel.: +52 55 5622 3872; fax: +52 55 5550

0048.

E-mail address: [email protected] (R. Hernandez).

cially through post-transcriptional regulation of

mRNAs [2]. The extracellular epimastigote forms (pres-

ent in the digestive tract of the reduvid vector) can be

readily cultured in axenic media, and are hence amena-

ble to experimentation. The growth curve of epimastig-otes represents a useful tool to analyze differential gene

expression throughout a cellular population that

changes its morphology, from being rounded or oval

cells with a short flagellum during the logarithmic phase

of growth to elongated cells with an extended flagellum

(over 30 lm) during the stationary phase [1]. Cells in the

latter phase can perpetuate for long periods under cul-

ture conditions. The stationary phase has been consid-ered an environmental condition where differentiation



260 A.M. Cevallos et al. / FEMS Microbiology Letters 246 (2005) 259–264

towards non-dividing metacyclic trypomastigotes is trig-

gered [3]. The mechanisms that allow epimastigotes to

survive in the stationary phase are unknown. The search

for different steady state concentrations of specific

mRNAs during the growth curve showed that a and btubulin [4] and actin mRNAs [5] decrease prior to andduring the stationary phase. In order to gain insight in

the biology of epimastigotes at the stationary phase,

and to further analyze a potential modulation of addi-

tional RNAs during non proliferative conditions, we

investigated: (1) the steady state concentrations of four

mRNAs from single-copy type housekeeping genes

along the growth curve, and (2) the relative stability of

these mRNAs and of three ribosomal RNA precursormolecules from both logarithmic and stationary cells.


2.1. Parasites and culture conditions

T. cruzi epimastigotes from the CL Brener strain weregrown at 28 �C in liver infusion triptone medium supple-

mented with 10% heat inactivated fetal bovine serum [3].

In order to obtain reproducible results, the cellular pop-

ulation was homogenized as follows: epimastigotes were

maintained in logarithmic growth during at least 3 cy-

cles from 1 · 106 to 30 · 106 cells per ml, and then di-

luted to 1 · 106 cells per ml to start the actual time

course analysis. The number of cells was registered at24–48 h intervals; the cells were harvested at selected

days of the culture during the logarithmic and stationary

phases. Mid-logarithmic phase was defined as the time

when epimastigotes reached a concentration of approx-

imately 8–12 · 106 cells per ml (days 3–4 post-inocula-

tion). Stationary phase was defined as the time when

parasites stopped their growth for 72 h, (90–100 · 106

cells per ml; days 12–14 post-inoculation). Mid-logarith-mic cellular populations were devoid of metacyclic

forms while stationary phase cultures had about 5% of

metacylic trypomastigotes as estimated from fixed

stained preparations.

2.2. Gene probes

To prepare gene probes, DNA fragments were gelpurified from the following T. cruzi plasmid clones:

pD-4, actin genomic clone [5]; pBTR, plasmid bearing

a PCR derived coding sequences from the tripanothion

reductase (TR) gene [6]; recombinant plasmid composed

the pCR II vector carrying a genomic derived PCR

amplification product from the triosephosphate isomer-

ase (TIM) gene [7]; pS4-2, cDNA clone from the ribo-

somal protein S4 (S4) locus [8]. In northern blotanalysis of total RNA obtained from epimastigotes in

culture, each of these probes recognizes a single mRNA

band at every stage during growth (data not shown).

Three ribosomal genomic clones pRTC20, pRTC42

and pRTC32 were also used [9]. All DNA probes were

labeled with [a-32P] dCTP using a random prime label-

ing system (Redi prime II, Amersham Pharmacia

Biotechnology).

2.3. Northern blot analyses

Total RNA preparations and northern blot hybrid-

izations were carried out as earlier described [5]. The

amount of bound radioactivity on the membranes was

detected with the Molecular Imager FX system (Bio-

Rad), and quantitation of the samples was done usingthe Quantity One software (BioRad). The levels of spe-

cific mRNAs in each lane were normalized as a ratio to

an independent rRNA probe to correct for potential dif-

ferences in loading (mRNA/rRNA ratio). Values are

thereby expressed as a percentage of the maximal ratio

obtained for each probe. All RNA size determinations

were estimated with the 0.24–9.5 kb RNA ladder (Gibco

BRL).

2.4. Analysis of RNA precursors’ half-life

The half-life of mRNAs was quantitated in northern

blots as above with RNA isolated at several time points

from cells incubated in the presence of the transcription

inhibitor actinomycin D (10 lg/ml) [10]. Due to their cel-

lular abundance the amounts of rRNA precursors weredetected directly from ethidium bromide stained gels

with the Molecular Imager FX system. Their quantifica-

tion was done using the Quantity One software

(BioRad).


3.1. Steady state concentrations of mRNAs along

epimastigotes growth curve

In the early stages of this work, we were interested to

find out whether in culture derived epimastigotes

mRNAs other than actin transcripts showed the increase

and decrease pattern observed in our previous work [5].

The mRNAs for the TIM, TR and S4 genes were thenanalyzed in this context. Reproducible results were ob-

tained only when cultures were passaged while in loga-

rithmic growth prior to the start of the experiment.

Actin, TIM, TR and S4 mRNAs were analyzed by

northern hybridizations of total RNA isolated from

the cultures at different time points. Fig. 1 is representa-

tive of three independent experiments and shows an

increment in the concentration of mRNAs during earlystages of the curve followed by a decrease in the station-

ary phase. In order to analyze the viability of cells in

Fig. 1. mRNAs levels during growth of Trypanosoma cruzi epimastig-

otes. Total RNA was isolated from cultured epimastigotes at the

indicated days in culture and analyzed with gene specific probes in

northern hybridizations (�10 lg/lane, panel A). The observed levels of

mRNAs (actin, closed triangles, straight line; S4, closed squares,

straight line; TIM, crosses, dotted line; and TR, empty diamonds,

dotted line) are depicted in panel B as the ratio of radioactivity in the

specific mRNA band/radioactivity of rRNA in the corresponding lane

(right, Y-axis). Values were normalized to 100% with the higher ratio

for each mRNA series. Solid circles represent the density of

epimastigotes per ml (left, Y-axis).

Fig. 2. Differential stability of mRNAs in Trypanosoma cruzi epim-

astigotes from the mid logarithmic and stationary phase. Panel A:

northern blots of total RNA were carried out at different time points

after the addition of actinomycin D. Gene specific probes are the same

as those depicted in Fig. 1. Panel B, graphs corresponding to half-life

of actin (closed triangles, straight line), S4 (closed squares, straight

line) TIM (crosses, dotted line) and TR open diamonds, dotted line)

mRNAs at both mid-logarithmic and stationary phases of growth.

A.M. Cevallos et al. / FEMS Microbiology Letters 246 (2005) 259–264 261

well established stationary phase, the inoculum used to

start one of the experimental growth curves were cells

from aged cultures, that is, cells that remained in sta-

tionary phase for 14 days after growth had stopped.

This experiment reproduced a similar pattern albeitthere was a delay in the accumulation of steady state

concentration of mRNAs in correlation with the ob-

served extended lag phase of the growth curve (data

not shown).

3.2. Stability of RNAs during growth and stationary

phases

Non-dividing epimastigotes from stationary phase

can survive for few weeks when transcription of Pol I

and potentially all Pol II transcripts are down regulated

[11]. To investigate mechanisms that would support a

minimal level of transcripts needed for cellular survival

during stationary phase, the stability of the four

mRNAs here studied was analyzed from cells at both

the mid-logarithmic phase of growth and the stationaryphase of growth. A kinetics profile of RNA decay was

carried out in cells treated with actinomycin D. The pat-

terns depicted in Fig. 2B are consistent with a heteroge-

neous RNA population. In any case, it is interesting that

all four mRNAs analyzed showed an expanded half-life

(at least threefold) in cells from the stationary phase as

compared with cells from logarithmic phases of growth

(Fig. 2 and Table 1). This result agrees with the reportthat the half-lives of histone H2A mRNAs are about

twice as long in T. cruzi epimastigotes from the station-

ary phase when compared to cells at the logarithmic

phase of growth [12]. As a not understood phenomenon,

the presence of actinomycin D correlated with an initial

short and temporal increase in the concentration of

some RNA species. An analogous unexplained effect in

mRNAs from Trypanosoma brucei has been observed[13].

Table 1

Half lives of mRNAs and immature rRNAs at both mid-logarithmic

(ML) and stationary phases (S) of growtha

ML S S/ML

mRNAs

Actin 5.7 40.6 7.1

S4 4.2 18.3 4.3

TIM 6.9 22.1 3.2

TR 3.8 16.6 4.3

Immature rRNAs

7.6 kb 1.7 10.8 6.2

6.7 kb 6.8 17.9 2.6

5.3 kb 2.0 11.6 5.7

a Results were derived from the lineal regression calculations

obtained from the lineal segment of the decay curve and are expressed

in hours. The relative stabilization of transcripts was determined by the

stationary to mid-logarithmic ratio.


Immature and mature rRNAs species are so abun-

dant that can be visualized from total RNA electropher-

ograms stained with ethidium bromide. This is

demonstrated in Fig. 3 where major bands are shown

to hybridize with rRNA gene probes. The relative stabil-

Fig. 3. (Top) Diagram of the rRNA cistron. The coding regions are

depicted as solid boxes interconnected by a thin line that represents

transcribed spacer regions. The transcription start point is indicated by

an arrow [19]. The bars marked as pRTC show three genomic

fragments of this region previously cloned [9]. (Bottom) Ethidium

bromide profile of total RNA of growing epimastigotes. Total RNA

from parasites in the mid-logarithmic phase of growth was extracted

and loaded into a non-denaturing TBE, 1%, agarose gel in three

identical lanes. The whole gel was stained with ethidium bromide and

the negative version of the image was registered using a phosphor

imager (Molecular Imager FX, BioRad), only one lane is shown. For

the hybridization analysis, the stained gel was equilibrated with the

standard MOPS/formaldehyde solution and transferred to a nylon

membrane. Each lane was cut separately and hybridized to probes

pRTC20, pRTC42, and pRTC32 as indicated (lanes 20, 42 and 32).

ity analysis of the pre RNAs was therefore carried out

from ethidium bromide stained gels (Fig. 4). Similarly

to data from mRNAs, the immature rRNAs were found

to be more stable in cells from stationary phase (Fig. 4,

Table 1). Whether the observed stabilization of pre

rRNAs is due to a slower decay and/or a decrease inthe rate of processing cannot be distinguished with this

experimental approach.

All together, the stabilization of specific mRNAs and

immature rRNAs during the stationary phase suggests

the occurrence of a post-transcriptional control point

associated to the growth phase of T. cruzi epimastigotes

(at least under culture conditions). Differential stability

of mRNAs has been well documented to occur as animportant mechanism to regulate differential gene

expression during development in kinetoplastids, mainly

involving sequences present in their 3 0 untranslated re-

gions (3 0 UTR) [14]. In the case of T. cruzi, at least

two RNA sequence elements present in the 3 0 UTR of

the small type mucin mRNAs either destabilize (AU rich

sequences, ARE), or stabilize (G rich elements, GREs)

with a cis effect on their coding flanking sequences.

Fig. 4. Half-life of rRNA precursors. (A) Negative image of ethidium

bromide stained gels of total RNA (�1 lg/lane) from epimastigotes in

mid-logarithmic and stationary phases of growth, at different time

intervals after the addition of actinomycin D (10 lg/ml). (B) Kinetics

of the rRNA precursors decay after the addition of actinomycin D in

mid-logarithmic and stationary phase epimastigotes. Values represent

the mean of four independent assays; standard errors are depicted as

vertical lines. Open circles correspond to data from the 7.6 kb

precursor, closed squares to the 6.7 kb precursor and closed triangles

to the 5.3 kb precursor.

A.M. Cevallos et al. / FEMS Microbiology Letters 246 (2005) 259–264 263

Interestingly these two elements seem to function at dif-

ferent developmental stages, ARE functions in trypom-

astigotes while GREs function in epimastigotes [15,16].

We therefore explored the 3 0 UTR sequences for the

presence of common motifs either in their primary se-

quence or in the secondary structure using the RNAanalyzer software described by Bengert and Dandekar

[17]. The analyzed 3 0 UTRs varied in length and in the

percentage of A + U content. No regions with signifi-

cant homology were identified and in no case were the

ARE or GRE motifs found. We also studied the 5 0

UTRs as they could also be involved in gene expression.

The 5 0 UTR also varied in length an in percentage of

A + U content and no regions of homology were identi-fied. Therefore, no correlation between UTR�s and dif-

ferential mRNA stability could be found. It is to point

out that this phenomenon includes at least five types

of mRNAs (including histone H2A [12]) and three

immature rRNA molecular species, therefore it is

improbable that a single RNA motif could be involved

as the main recognition element. A more likely mecha-

nism may be a general down regulation of the RNA de-cay (or processing) at stationary phase. It has been

demonstrated that transcription by both Pol I and Pol

II polymerases is down regulated in non-dividing

T. cruzi stages and in non-infective cells from stationary

phase cultures [11]. Data from our laboratory is in

accordance with this observation: the incorporation rate

of labeled uridine is about sixfold higher in growing

epimastigotes than in cells from the stationary phase,whose activity is registered well above background (data

not shown). In T. brucei species the RNA synthesis is

down regulated in the stationary phase that occurs dur-

ing differentiation of bloodstream to procyclic forms

[18]. In this situation, the stabilization of housekeeping

transcripts observed in the present work may be part

of a global regulation strategy to compensate for a

reduction in transcription.It is widely accepted that during the stationary

phase of culture a small proportion of epimastigotes

spontaneously transform into metacyclic trypomastig-

otes [3]. Therefore the entrance of epimastigotes into

the stationary phase can be considered as an onset

for differentiation. Metacyclic trypomastigotes are

non-dividing infective forms of the T. cruzi parasite,

that in natural conditions reside in the cloacal regionof the alimentary tract of the Triatome vector. Metacy-

clic trypomastigotes remain there until the insect finds

an appropriate host to feed. At the time of feeding

the parasites are excreted and upon entrance into a sus-

ceptible host they differentiate into dividing amastig-

otes. A general stabilization of transcripts may

therefore be a selected mechanism in these non-dividing

forms of T. cruzi to maintain a minimal level of expres-sion of housekeeping genes that would allow the para-

site to resume growth if environmental conditions turn

favorable for proliferation. The mechanism for this sta-

bilization of RNAs during the stationary phase re-

mains to be determined.

Acknowledgements

The authors thank Dr. Jorge Tovar and Dr. Ruy

Perez-Montfort for their kind donations of recombinant

plasmids used in this study, and Dr. Joaquın Sanchez

for the critical reading of the manuscript. We acknowl-

edge Lorena Lopez-Griego for technical help. This work

was supported by grant IN209302 from PAPIIT and

Grants 28036M, 37620M and 45037Q from CONACyT,Mexico. Mariana Perez-Escobar was supported by a

scholarship from CONACyT during her Ph.D. Thesis

program.

References

[1] Tyler, K.M. and Engman, D.M (2001) The life cycle of Trypan-

osoma cruzi revisited. Int. J. Parasitol. 31, 472–481.

[2] Teixeira, S.M. (1998) Control of gene expression in Trypanoso-

matidae. Braz. J. Med. Biol. Res. 31, 1503–1516.

[3] Camargo, E.P. (1964) Growth and differentiation of Trypanosoma

cruzi, origin of metacyclic trypanosomes in liquid medium. Rev.

Inst. Med. Trop. Sao Paulo 12, 93–100.

[4] Gonzalez-Pino, M.J., Rangel-Aldao, R. and Slezynger, T.C.

(1999) Expression of alpha- and beta-tubulin genes during growth

of Trypanosoma cruzi epimastigotes. DNA Cell Biol. 18, 449–455.

[5] Cevallos, A.M., Lopez-Villasenor, I., Espinosa, N., Herrera, J.

and Hernandez, R. (2003) Trypanosoma cruzi: allelic comparisons

of the actin genes and analysis of their transcripts. Exp. Parasitol.

103, 27–34.

[6] Borges, A., Cunningham, M.L., Tovar, J. and Fairlamb, A.H.

(1995) Site-directed mutagenesis of the redox-active cysteines of

Trypanosoma cruzi trypanothione reductase. Eur. J. Biochem.

228, 745–752.

[7] Ostoa-Saloma, P., Garza-Ramos, G., Ramirez, J., Becker, I.,

Berzunza, M., Landa, A., Gomez-Puyou, A., Tuena de Gomez-

Puyou, M. and Perez-Montfort, R. (1997) Cloning, expression,

purification and characterization of triosephosphate isomerase

from Trypanosoma cruzi. Eur. J. Biochem. 244, 700–705.

[8] Hernandez, R., Palacios, S., Herrera, J., Martinez-Calvillo, S. and

Lopez, I. (1998) The deduced primary structure of a ribosomal

protein S4 from Trypanosoma cruzi. Biochim. Biophys. Acta 1395,

321–325.

[9] Hernandez, R., Diaz-de Leon, F. and Castaneda, M. (1988)

Molecular cloning and partial characterization of ribosomal RNA

genes from Trypanosoma cruzi. Mol. Biochem. Parasitol. 27, 275–

279.

[10] Charest, H., Zhang, W.W. and Matlashewski, G. (1996) The

developmental expression of Leishmania donovani A2 amastigote-

specific genes is post-transcriptionally mediated and involves

elements located in the 30-untranslated region. J. Biol. Chem. 271,

17081–17090.

[11] Elias, M.C., Marques-Porto, R., Freymuller, E. and Schenkman,

S. (2001) Transcription rate modulation through the Trypano-

soma cruzi life cycle occurs in parallel with changes in nuclear

organisation. Mol. Biochem. Parasitol. 112, 79–90.

[12] Maranon, C., Thomas, M.C., Puerta, C., Alonso, C. and Lopez,

M.C. (2000) The stability and maturation of the H2A histone


mRNAs from Trypanosoma cruzi are implicated in their post-

transcriptional regulation. Biochim. Biophys. Acta 1490, 1–10.

[13] Irmer, H. and Clayton, C. (2001) Degradation of the unstable

EP1 mRNA in Trypanosoma brucei involves initial destruction of

the 3 0-untranslated region. Nucleic Acids Res. 29, 4707–4715.

[14] Clayton, C.E. (2002) Life without transcriptional control? From

fly to man and back again. EMBO J. 21, 1881–1888.

[15] Di Noia, J.M., D�Orso, I., Sanchez, D.O. and Frasch, A.C. (2000)

AU-rich elements in the 30-untranslated region of a new mucin-

type gene family of Trypanosoma cruzi confers mRNA instability

and modulates translation efficiency. J. Biol. Chem. 275, 10218–

10227.

[16] D�Orso, I. and Frasch, A.C. (2001) Functionally different AU-

and G-rich cis-elements confer developmentally regulated mRNA

stability in Trypanosoma cruzi by interaction with specific RNA-

binding proteins. J. Biol. Chem. 276, 15783–15793.

[17] Bengert, P. and Dandekar, T. (2003) A software tool-box for

analysis of regulatory RNA elements. Nucleic Acids Res. 31,

3441–3445.

[18] Pays, E., Hanocq-Quertier, J., Hanocq, F., Van Assel, S., Nolan,

D. and Rolin, S. (1993) Abrupt RNA changes precede the first cell

division during the differentiation of Trypanosoma brucei blood-

stream forms into procyclic forms in vitro. Mol. Biochem.

Parasitol. 61, 107–114.

[19] Figueroa-Angulo, E., Martınez Calvillo, S., Lopez-Villasenor, I.

and Hernandez, R. (2003) Evidence supporting a major promoter

in the Trypanosoma cruzi rRNA gene. FEMS Microbiol. Lett.

225, 221–225.



Genotypic and phenotypic characterization of a biofilm-formingSerratia plymuthica isolate from a raw vegetable processing line

Rob Van Houdt *, Pieter Moons, An Jansen, Kristof Vanoirbeek, Chris W. Michiels

Laboratory of Food Microbiology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, B-3001 Leuven, Belgium

Received 11 March 2005; received in revised form 8 April 2005; accepted 13 April 2005


Edited by J.A. Cole

Abstract

Recently, we isolated from a raw vegetable processing line a Serratia strain with strong biofilm-forming capacity and which pro-

duced N-acyl-L-homoserine lactones (AHLs). Within the Enterobacteriaceae, strains of the genus Serratia are a frequent cause of

human nosocomial infections; in addition, biofilm formation is often associated with persistent infections. In the current report,

we describe the detailed characterization of the isolate using a variety of genotypic and phenotypic criteria. Although the strain

was identified as Serratia plymuthica on the basis of its small subunit ribosomal RNA (16S rRNA) gene sequence, it differed from

the S. plymuthica type strain in production of pigment and antibacterial compounds, and in AHL production profile. Nevertheless,

the identification as S. plymuthica could be confirmed by gyrB phylogeny and DNA:DNA hybridization.


Keywords: Serratia; Identification natural isolate; gyrB; Phylogeny; Quorum sensing; N-Acyl-L-homoserine lactone

1. Introduction

The genus Serratia, named after the Italian physicistSerafino Serrati, belongs to the family Enterobacteria-

ceae and consists of the recognized species: Serratia mar-

cescens, S. liquefaciens, S. ficaria, S. rubidaea, S.

fonticola, S. odorifera, S. plymuthica, S. grimesii, S. pro-

teamaculans, S. quinivorans, and S. entomophila [1]. All

species except S. entomophila have been frequently iso-

lated from clinical samples, and S. marcescens in partic-

ular is recognized as an important nosocomial pathogencapable of causing pneumonia, intravenous catheter-

associated infections, urinary tract infections, osteomy-

elitis and endocarditis [2]. However, the recently

described virulence-associated properties in Serratia


doi:10.1016/j.femsle.2005.04.016

* Correspondent author. Tel.: +32 16 321752; fax: +32 16 321960.

E-mail address: [email protected] (R. Van Houdt).

strains other than S. marcescens, and the increasing

number of documented infections caused by such

strains, together with the difficult identification of thesebacteria by commercial systems urges for a more de-

tailed investigation of the physiology, virulence and tax-

onomy of this genus [3]. As ubiquitous inhabitants of

soil, air and water, Serratia species are commonly asso-

ciated with food raw materials and are implicated in the

spoilage of various foods of plant and animal origin. In

addition, as opportunistic pathogens, they may pose a

foodborne health hazard. We have recently conductedan investigation on the biofilm-forming capacity and

the production of quorum-sensing signalling molecules

in Gram-negative bacteria isolated from a raw vegetable

processing line [4]. Five out of 68 isolates produced N-

acyl-L-homoserine lactones (AHLs), and two of these,

one with strong and one with weak biofilm-forming

capacity, were tentatively identified as S. plymuthica



266 R. Van Houdt et al. / FEMS Microbiology Letters 246 (2005) 265–272

using Biolog carbon utilization patterns. S. plymuthica

has been described as a non-motile, prodigiosin pig-

ment-producing Serratia and is regarded as a significant

pathogen [5] to which a variety of infections including

peritonitis, pneumonia, sepsis and wound infections

have been attributed [6–10]. The capacity to formbiofilms often contributes to pathogen virulence because

it provides protection against host defense and antibiotic

therapy, it allows cells to survive in hostile environments

and from there to disperse and colonize new niches, and

may facilitate the spread of antibiotic resistance by hor-

izontal gene transfer [reviewed in 11,12]. In the current

report, we describe the detailed identification and char-

acterization of the tentative S. plymuthica isolate withstrong biofilm-forming capacity, using a variety of geno-

typic and phenotypic criteria.


2.1. Bacterial strains, plasmids, and media

Strains used in this study are listed in Table 1. All

Serratia species type strains were obtained from the

Deutsche Sammlung von Mikroorganismen und Zellk-

ulturen (DSM, Braunschweig, Germany), except S. liq-

uefaciens DSM 4487 (=LMG 7884), which was

obtained from the Belgian Co-ordinated Collections of

Micro-organisms (BCCMe/LMG). Escherichia coli

ESS [13] and Chromobacterium violaceum CV026 [14]were obtained from Dr. Susan E. Jensen (University of

Alberta) and Dr. Rene De Mot (Katholieke Universiteit

Leuven), respectively. All strains were routinely grown

in Luria–Bertani (LB) medium at 30 �C.

Table 1

Strains used in this study

Species Straina

Chromobacterium violaceum CV026 (cviI::mini-Tn5 derivative of A

Escherichia coli ESS

Plesiomonas shigelloides DSM 8224T = ATCC 14029T

Serratia entomophila DSM 12358T = ATCC 43705T

Serratia ficaria DSM 4569T = ATCC 33105T

Serratia fonticola DSM 4576T = ATCC 29844T

Serratia grimesii DSM 30063T = ATCC 14460T

Serratia liquefaciens DSM 4487T = ATCC 27592T

Serratia marcescens DSM 30121T = ATCC 13880T

Serratia odorifera DSM 4582T = ATCC 33077T

Serratia plymuthica DSM 4540T = ATCC 183T

Serratia proteamaculans DSM 4543T = ATCC 19323T

Serratia quinivoransb DSM 4597T = ATCC 33765T

Serratia rubidaea DSM 4480T = ATCC 27593T

Serratia sp. RVH1

TType strain.a DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen,

Manassas, VA, USA.b Originally classified as S. proteamaculans subsp. quinovora the transfer

proteamaculans to Serratia proteamaculans.

2.2. Phenotypic analysis

2.2.1. Swimming and swarming motility

Motility was tested by stab inoculating the strain to

be tested in both LB and minimal AB [15] medium solid-

ified with either 0.3% agar to examine swimmingthrough the water-filled channels in the agar, or 0.7%

agar to examine swarming over the agar surface [16].

2.2.2. Proteolytic activity

Production of extracellular proteolytic enzymes was

evaluated by observation of clearing zones around stab

inoculated bacteria on LB agar supplemented with 10%

skimmed milk after 24 h of incubation at 30 �C.

2.2.3. Production of antibacterial factors

Bacteria were checked for the production of antibac-

terial compounds active against various target strains by

scoring inhibition or lysis zones. Briefly, 100 ll of an

overnight LB broth culture of the target strain was

mixed with liquid 0.7% LB agar at 50 �C and poured

into a petri dish. After solidification, potential antibacte-rial producer strains were stab inoculated onto this

lawn, and plates were scored after overnight incubation

at 30 �C for the presence of inhibition or lysis zones.

2.2.4. Analysis of the N-acyl-L-homoserine lactone

production pattern

Analysis of the AHL production pattern was per-

formed by thin-layer chromatography (TLC) on C18 re-versed-phase plates (VWR International, Leuven,

Belgium) using a methanol/water (60:40 v/v) solvent sys-

tem essentially as described by Shaw et al. [17]. Briefly,

cell-free culture supernatants from 21 h LB broth

GenBank Accession No.

16S rDNA gyrB

TCC 31532, Kmr, AHL�)

M59159 AJ300545

AJ233427 AJ300543

AJ233428 AJ300541

AJ233429 AJ300539

AJ233430 AJ300538

AJ306725 AJ300537

AJ233431 AJ300536

AJ233432 AJ300533

AJ233433 AJ300532

AJ233434 AJ300531

AJ233435

AJ233436 AJ300530

AY394724 AY787168

Braunschweig, Germany; ATCC, American Type Culture Collection,

to Serratia quinivorans [30] reduces Serratia proteamaculans subsp.

R. Van Houdt et al. / FEMS Microbiology Letters 246 (2005) 265–272 267

stationary-phase cultures (500 ml) of the Serratia spp.

were extracted twice with the same volume of ethyl ace-

tate, dried over anhydrous MgSO4, evaporated to dry-

ness, and the residue was dissolved in a small volume

of ethyl acetate and loaded onto the TLC-plates. After

chromatographic separation, the presence of AHLswas detected by overlaying the dried TLC-plates with

a thin film of AHL sensor strain C. violaceum CV026

in 1.4% LB agar, and looking for the appearance of pur-

ple spots indicative of induction of violacein production

after incubation at 30 �C for 24 h.

2.3. 16S rDNA analysis

Analysis of 16S rDNA of S. plymuthica RVH1 was

performed by BCCMe/LMG (Gent, Belgium). Briefly,

genomic DNA was extracted following the protocol of

Pitcher et al. [18] and the part of the 16S rRNA gene

corresponding to positions 28–1521 of the E. coli 16S

rRNA gene was PCR amplified with the primers

16F27 (5 0-AGAGTTTGATCCTGGCTCAG-3 0) and

16R1522 (5 0-AAGGAGGTGATCCAGCCGCA-3 0).The PCR product was purified using the QIAquick

PCR Purification Kit (Qiagen GmbH, Hilden, Ger-

many) and sequenced using five forward primers and

three reverse primers annealing to universally conserved

regions, with the ABI PRISM TM BigDye TM Termi-

nator Cycle Sequencing Ready Reaction Kit (Perkin–El-

mer, Applied Biosystems Div., Foster City, CA, USA)

and an Applied Biosystems 377 DNA Sequencer (Ap-plied Biosystems, Foster City, CA, USA). The sequence

assembly was performed using the program AutoAssem-

bler (Perkin–Elmer).

2.4. DNA:DNA hybridizations

DNA:DNA hybridizations were performed by

BCCMe/LMG (Gent, Belgium). Briefly, DNA was pre-pared according to a slightly modified procedure of Wil-

son [19] and hybridizations were performed at 46 �Cusing the method described by Ezaki et al. [20] with

some modifications.

2.5. gyrB gene amplification and sequencing

The gyrB gene of S. plymuthica RVH1 was PCRamplified as described by Dauga [21]. Briefly, 50 pmol

of each primer gyr-320 (5 0-TAARTTYGAYGAYAA-

CTCYTAYAAAGT-3 0) and rgyr-1260 (5 0-CMCCYTC-

CACCARGTAMAGTTC-3 0) were used in a reaction

mixture (100 ll) containing 10 mM Tris–HCl (pH 8.3),

50 mM KCl, 2.5 mM MgCl2. PCR amplification was

carried out as follows: 94 �C for 4 min, followed by 35

cycles of 94 �C for 1 min, 55 �C for 1 min and 72 �Cfor 2 min, with a final incubation at 72 �C for 10 min.

The amplification product was purified using the High

Pure PCR Purification Kit (Roche Diagnostics, Vilvo-

orde, Belgium) and sequenced in both directions using

the same primers as used for amplification at a commer-

cial sequencing facility (MWG-Biotech AG, Ebersberg,

Germany).

2.6. Phylogenetic data analysis

Multiple-sequence alignments were performed using

the CLUSTAL W algorithm from the European Bio-

informatics Institute (EBI) toolbox (http://www.ebi.

ac.uk/clustalw/) and were further refined by eye, intro-

ducing gaps to improve overall alignment. Sequence

distance matrices were established in pairwise compar-isons by use of the Kimura algorithm [22]. Phyloge-

netic trees were constructed by the neighbour-joining

method [23] using the PHYLIP version 3.5 software

package [24]. Statistical significance was evaluated by

bootstrap analysis [25] with 100 repeats of bootstrap

samplings.


3.1. Phenotyping of strain RVH1

In a screening of 68 biofilm-forming Gram-negative

bacteria from a raw vegetable processing line, one of

the strongest biofilm-forming isolates that also produced

different AHLs and AI-2 as quorum signalling com-pounds, designated RVH1, was a catalase negative and

oxidase positive rod-shaped organism (1.0 lm width;

1.2–1.5 lm length), and was tentatively identified as S.

plymuthica based on phenotypic analysis with the Biolog

GN2 Microplate System [4]. The results of additional

phenotypic analysis of this strain, in comparison to type

strains of the nine Serratia species most closely related

to S. plymuthica, are described below and summarizedin Table 2 and Fig. 1.

3.1.1. Proteolytic activity and swimming and swarming

motility

All bacteria, including RVH1, showed proteolytic

activity on skimmed milk plates except S. proteamacu-

lans and S. grimesii. Since all strains except S. fonticola

score positive in gelatin hydrolysis assays [26], it is pos-sible that the proteases produced by S. proteamaculans

and S. grimesii are not able to hydrolyse caseins.

All strains showed swimming motility, as expected

for the genus Serratia, but only S. ficaria showed

swarming motility on both LB and AB medium with

0.7% agar. The S. liquefaciens type strain showed no

swarming motility; contrary to S. liquefaciens strain

MG1, which we used as an internal control for ourswarming motility assay because it is a model organism

in many studies of swarming motility [16].

http://www.ebi.ac.uk/clustalw/

http://www.ebi.ac.uk/clustalw/

Table 2

Summary of phenotypic tests

Strain Pigment production Swimming motility Swarming motility Proteolytic activity ESSa RVH1b

S. entomophila � + � + � +

S. ficaria � + + + � +

S. fonticola � + � + � �S. grimesii � + � � � +

S. liquefaciens � + � + � �S. odorifera � + � + � +

S. plymuthica Red + � + � +

S. proteamaculans � + � � � +

S. quinivorans � + � + � �RVH1 � + � + + �a ESS: Production of antibacterial factor scored on E. coli ESS overlay plates.b RVH1: Production of antibacterial factor by RVH1 scored on overlay plates of listed strains.

Fig. 1. N-Acyl-L-homoserine production profile as indicated by biosensor strain Chromobacterium violaceum CV026. (a) S. ficaria DSM 4569; (b) S.

liquefaciens DSM 4487; (c) S. quinivorans DSM 4597; (d) strain RHV1; (e) S. plymuthica DSM 4540; (f) S. entomophila DSM 12358; (g) S. odorifera

DSM 4582; (h) S. proteamaculans DSM 4543; (i) S. grimesii DSM 30063; and (j) S. fonticola DSM 4576.


3.1.2. Production of antibacterial factor

Serratia strains have been reported to produce certaincompounds with antibacterial activity, such as the sim-

ple carbapenem, 1-carbapen-2-em-3-carboxylic acid,

identified in Serratia sp. strain ATCC 39006 [27]; Serra-

cin P, a phage-tail-like bacteriocin, produced by S. ply-

muthica J7 [28]; and bacteriocin 28b produced by most

S. marcescens biotypes [29]. Therefore, the production

and activity spectrum of possible antibacterial factors

produced by RVH1 and the type strains was analyzedby stab inoculating each strain onto a series of plates

each containing a lawn of one of the other Serratia

strains or of E. coli ESS, a b-lactam supersensitive strain

used in carbapenem production analysis. None of the

Serratia spp. type strains produced a halo in this test ex-

cept for S. proteamaculans, which caused a weak inhibi-

tion of S. grimesii. However, strain RVH1 caused

complete inhibition (clear halo) of S. entomophila, S. fi-caria, S. grimesii, S. odorifera, S. plymuthica, S. protea-

maculans, and E. coli ESS, but not of S. fonticola, S.

liquefaciens, and S. quinivorans. The activity spectrum

of the antibacterial factor produced by RVH1 differs

from Serracin P, which shows no activity towards the

E. coli strains tested [28]. Preliminary tests suggest that

the antibacterial activity can be ascribed to a protein,

but the presence of other compounds cannot be ex-cluded at this stage (data not shown). The antibacterial

spectra of the type strains in this study differ from thosereported by Ashelford et al. [30], possibly due to differ-

ences in growth temperature and other experimental

parameters.

3.1.3. N-Acyl-L-homoserine lactones

N-Acyl-L-homoserine lactone mediated quorum-sens-

ing is a widespread communication system in Gram-neg-

ative bacteria, in which small diffusible AHL signallingmolecules, synthesized by a LuxI homologue, interact

with a LuxR homologue and activate or repress the tar-

get genes when their concentration reaches a certain

threshold, related to population density [31]. Since quo-

rum sensing regulates a range of important biological

functions, such as antibiotic production, plasmid trans-

fer, motility, virulence and biofilm formation [reviewed

in 31], it is considered as a possible target for antibacte-rial treatment, and several studies have demonstrated

the feasibility of interfering with quorum sensing by

the use of specific antagonists of the signalling mole-

cules, an approach known as �quorum quenching�[32,33].

Different AHL production profiles and target genes

have been described in a number of Serratia spp.,

showing the specificity and diversity of quorum sensingsignal molecules and regulation in this genus. For

example, Serratia proteamaculans strain B5a produces


3-oxo-N-hexanoyl-L-homoserine lactone (3-oxo-C6-

HSL) and N-hexanoyl-L-homoserine lactone (C6-HSL)

[34], S. liquefaciens strain MG1 produces primarily N-

butanoyl-L-homoserine lactone (C4-HSL) and also C6-

HSL [35], while S. marcescens strain SS-1 produces at

least four AHLs, namely 3-oxo-C6-HSL, C6-HSL, N-heptanoyl-L-homoserine lactone (C7-HSL) and N-octa-

noyl-L-homoserine lactone (C8-HSL) [36]. Recently,

three AHLs produced by S. plymuthica IC1270 were

tentatively identified as 3-hydroxy-N-hexanoyl-L-homo-

serine lactone (3-hydroxy-C6-HSL), 3-hydroxy-N-octa-

noyl-L-homoserine lactone (3-hydroxy-C8-HSL) and

an unidentified compound by comigration with syn-

thetic compounds in thin layer chromatography [37].These AHL molecules are produced by the AHL syn-

thase from the substrates S-adenosyl-L-methionine

(SAM) and acylated acyl carrier protein (acyl-ACP)

[38] and can vary in acyl chain length (from C4 to

C14), oxidation at the C3 position and saturation

[39,40] due to the enzyme acyl chain specificity and the

available cellular pool of acyl-ACPs [39,41].

As additional phenotype, we examined the AHL pro-duction profile of strain RVH1 and the Serratia sp. type

strains by TLC analysis in combination with C. viola-

ceum CV026 biosensor overlay, and compared the

AHL profiles to those already described in Serratia

spp. In C. violaceum CV026, the proper production of

AHL molecules has been blocked by mutation of the

AHL synthase but the gene encoding the production

of the purple pigment violacein remains AHL-respon-sive. In the presence of specific AHLs with acyl chain

lengths shorter than C10, this strain will therefore pro-

duce purple pigment due to violacein production [14].

The results of the AHL profile analysis are shown in

Fig. 1, and reveal at least four different AHLs with a dif-

ferent TLC migration. The two farthest migrating spots

(spots 3 and 4 in Fig. 1) are the most common AHLs,

being present in S. ficaria, S. quinovorans, S. entomo-

phila, S. odorifera, S. proteamaculans, and RVH1. Since

another strain of S. proteamaculans (B5a) was previ-

ously reported to produce 3-oxo-C6-HSL and C6-HSL

[34], these two spots most likely correspond to these

two AHL molecules, although the existence of other

AHLs with the same migration cannot be excluded at

this stage. Strain RVH1 shows a third spot (spot 1 in

Fig. 1) which did not migrate from the point of applica-tion and which was not seen in any of the other Serratia

species. The slow migration could indicate the presence

of an AHL with a long-chain hydrophobic fatty acid res-

idue that binds strongly to the C-18 solid phase, but

such an AHL should not be able to elicit violacein pro-

duction. Even so, when a lot of material is loaded onto a

TLC-plate, molecules with shorter acyl chains some-

times get blocked, but on the other hand preliminarymass spectrometry analysis suggests indeed the presence

of 3-oxo-C12-HSL in the ethyl acetate extracts of RVH1

(data not shown). Finally, one TLC spot (spot 2 in Fig.

1) was observed only in S. ficaria and S. odorifera, but

its nature remains unknown. No AHLs capable to in-

duce violacein production were found for the S. grimesii,

S. plymuthica, and S. liquefaciens type strains, although

AHL production has been described for S. plymuthica

IC1270 [37] and S. liquefaciensMG1 [35], indicating that

the AHL synthases in S. plymuthica and S. liquefaciens

type strains are absent or mutated, resulting in the loss

of AHL production.

In spite of the tentative biochemical identification of

strain RVH1 as S. plymuthica, the difficulties in precise

phylogenetic positioning of Serratia strains combined

with the phenotypic differences between strain RVH1and the S. plymuthica type strain (see Table 2) motivated

us to perform a detailed phylogenetic study based on

16S rDNA and gyrB sequence comparison, and on

DNA:DNA hybridization.

3.2. 16S rRNA-based phylogeny

Part of the 16S rDNA gene sequence of strain RVH1was amplified and analysed (GenBank Accession No.

AY394724). Fig. 2(a) shows a neighbour-joining phylo-

genetic tree based on the alignment of the nearly com-

plete 16S rDNA gene sequence of strain RVH1 with

16S rDNA sequences of the 11 described Serratia type

strains available in GenBank and EMBL databases (see

Table 1 for corresponding accession numbers), and

rooted by using Plesiomonas shigelloides, which is themost closely related species to the Enterobacteriaceae

family [42]. The 16S rDNA sequence similarity between

strain RVH1 and the 11 described Serratia species ranged

between 99.3% and 96.3%, with the highest similarity to

S. plymuthica (99.3%) and S. ficaria (99.2%) and the low-

est to S. rubidaea (96.4%) and S. marcescens (96.3%).

Two separate clusters were obtained as described by

Sproer et al. [43]. One cluster comprised S. rubidaea, S.marcescens, S. odorifera, S. entemophila, and S. ficaria

and the second cluster comprised S. plymuthica, S. fonti-

cola, S. liquefaciens, S. quinivorans, S. grimesii, and S.

proteamaculans. The phylogenetic information obtained

from these sequences is poor due to the low rate of vari-

ation of 16S rDNA sequences. Therefore, we also deter-

mined a phylogeny based on the gyrB sequence.

3.3. gyrB-based phylogeny

Dauga [21] described the use of the gyrB sequence for

determining relationships among Serratia species. In

general, phylogenetic trees based on gyrB sequences ap-

pear to be more reliable for closely related bacterial spe-

cies than trees based on 16S rDNA. The gyrB nucleotide

sequence from strain RVH1 was determined from thePCR-amplified gyrB gene, revealing a 910 bp open read-

ing frame (GenBank Accession No. AY787168). The

Fig. 2. Neighbour-joining phylogenetic tree obtained from (a) 16S rRNA gene sequences, with the scale bar representing an estimated five base

substitutions per 1000 nt positions and (b) gyrB sequences, with the scale bar representing an estimated 25 substitutions per 1000 nt positions.

Numbers refer to significant bootstrap values of 100 calculated trees.


translated amino acid sequence had a lysine (K) at co-

don 206 (E. coli amino acid numbering system, Acces-

sion No. X04341) in a b-sheet-shaped region of the

ATP binding site, which is a Serratia signature sequence

[21]. The sequence similarity of strain RVH1 to the 10

Serratia species examined (the gyrB sequence of S. qui-

novorans was not available in a database) ranged be-

tween 98.9% and 86.5%, with the highest similarity toS. plymuthica (98.9%) and S. liquefaciens (94.2%) and

the lowest to S. rubidaea (87.8%) and S. fonticola

(86.5%). Fig. 2(b) represents a phylogenetic tree based

on the alignment of the gyrB gene sequence of strain

RVH1 and the Serratia sp. type strain gyrB sequences

available in GenBank and EMBL databases (see Table

1 for corresponding accession numbers) and rooted by

using P. shigelloides. Two phylogenetic clusters with sig-nificant bootstrap values were again found. The first

cluster (bootstrap value 93%) contained S. rubidaea, S.

marcescens, S. entomophila, and S. ficaria. The second

cluster (bootstrap value 99%) contained strain RVH1,

S. grimesii, S. proteamaculans, S. liquefaciens, and S.

plymuthica. Within this cluster strain RVH1 and S. ply-

muthica formed a coherent group validated by a signifi-

cant bootstrap value of 100%.

3.4. DNA:DNA hybridization

Bacterial strains are generally considered to belong tothe same species if they share a 16S rDNA sequence

identity of >97% and/or 70% or greater DNA–DNA

relatedness with 5 �C or less difference of melting tem-

perature (DTm), with the latter criterion being decisive

[44]. Therefore, to conclusively confirm the identity of

RVH1, we performed DNA:DNA hybridization be-

tween RVH1 and the two most closely related type

strains based on 16S rDNA sequence identity, i.e., S.plymuthica and S. ficaria. We found 100% DNA–DNA

hybridization with the S. plymuthica type strain and

46% with the S. ficaria type strain, confirming the iden-

tity of RVH1 as S. plymuthica.


4. Conclusions

We have performed a comparative characterization

of Serratia sp. RVH1, which was previously isolated

as a biofilm-forming strain from a raw vegetable pro-

cessing line [4], with the type strains of the 9 or 10 mostclosely related Serratia species. Phenotypically, the iso-

late could not be clearly assigned to any of the described

Serratia species, but 16S rRNA and gyrB sequence com-

parison and DNA:DNA hybridization unequivocally

identified the strain as S. plymuthica. Furthermore, these

observations add new phenotypic and genotypic infor-

mation to the Serratia genus, thus contributing to a

more precise phylogenetic positioning of Serratia

strains.

Acknowledgement

Rob Van Houdt is a research assistant of the Fund

for Scientific Research-Flanders (F.W.O.-Vlaanderen).

References

[1] Euzeby, J.P. (2004) List of bacterial names with standing in

nomenclature Available from: <http://www.bacterio.cict.fr> .

[2] Eisenstein, B.I. (1990) Enterobacteriaceae In: Principles and

Practice of Infectious Disease (Mandell, G.L., Douglas, R.G.

and Bennett, J.E., Eds.), 3rd edn, pp. 1658–1673. Churchill

Livingstone, New York.

[3] Stock, I., Burak, S., Sherwood, K.J., Gruger, T. and Wiedemann,

B. (2003) Natural antimicrobial susceptibilities of strains of

�unusual� Serratia species: S. ficaria, S. fonticola, S. odorifera, S.

plymuthica and S. rubidaea. J. Antimicrob. Chemother. 51, 865–

885.

[4] Van Houdt, R., Aertsen, A., Jansen, A., Quintana, A.L. and

Michiels, C.W. (2004) Biofilm formation and cell-to-cell signalling

in Gram-negative bacteria isolated from a food processing

environment. J. Appl. Microbiol. 96, 177–184.

[5] Berg, G. (2000) Diversity of antifungal and plant-associated

Serratia plymuthica strains. J. Appl. Microbiol. 88, 952–960.

[6] Carrero, P., Garrote, J.A., Pacheco, S., Garcia, A.I., Gil, R. and

Carbajosa, S.G. (1995) Report of six cases of human infection by

Serratia plymuthica. J. Clin. Microbiol. 35, 2531–2536.

[7] Clark, R.B. and Janda, J.M. (1985) Isolation of Serratia

plymuthica from human burn site. J. Clin. Microbiol. 21, 656–657.

[8] Domingo, D., Limia, A., Alarcon, T., Sanz, J.C., Del Rey, M.C.

and Lopez-Brea, M. (1994) Nosocomial septicemia caused by

Serratia plymuthica. J. Clin. Microbiol. 32, 575–577.

[9] Nough, F. and Bhandari, S. (2000) CAPD peritonitis due to

Serratia plymuthica. Periton. Dialysis Int. 20, 349.

[10] Reina, J., Borrell, N. and Llompart, I. (1992) Community-

acquired bacteremia caused by Serratia plymuthica: case report

and review of the literature. Diagn. Microbiol. Infect. Dis. 15,

449–452.

[11] Fux, C.A., Costerton, J.W., Stewart, P.S. and Stoodley, P. (2005)

Survival strategies of infectious biofilms. Trends Microbiol. 13,

34–40.

[12] Hall-Stoodley, L., Costerton, J.W. and Stoodley, P. (2004)

Bacterial biofilms: from the natural environment to infectious

diseases. Nat. Rev. Microbiol. 2, 95–108.

[13] Jensen, S.E., Elder, K.J., Aidoo, K.A. and Paradkar, A.S. (2000)

Enzymes catalyzing the early steps of clavulanic acid biosynthesis

are encoded by two sets of paralogous genes in Streptomyces

clavuligerus. Antimicrob. Agents Chemother. 44, 720–726.

[14] McClean, K.H., Winson, M.K., Fish, L., Taylor, A., Chhabra,

S.R., Camara, M., Daykin, M., Lamb, J.H., Swift, S., Bycroft,

B.W., Stewart, G.S.A.B. and Williams, P. (1997) Quorum sensing

and Chromobacterium violaceum: exploitation of violacein pro-

duction and inhibition for the detection of N-acylhomoserine

lactones. Microbiology 143, 3703–3711.

[15] Clark, J.D. and Maaloe, O. (1967) DNA replication and the

division cycle in Escherichia coli. J. Mol. Biol. 23, 99–112.

[16] Eberl, L., Molin, S. and Givskov, M. (1999) Surface motility of

Serratia liquefaciens MG1. J. Bacteriol. 181, 1703–1712.

[17] Shaw, P.D., Ping, G., Laly, S.L., Cha, C., Cronan, J.E., Rinehart,

K.L. and Farrand, S.K. (1997) Detecting and characterizing N-

acyl-homoserine lactone signal molecules by thin-layer chroma-

tography. Proc. Natl. Acad. Sci. USA 94, 6036–6041.

[18] Pitcher, D.G., Saunders, N.A. and Owen, R.J. (1989) Rapid

extraction of bacterial genomic DNA with guanidium thiocya-

nate. Lett. Appl. Microbiol. 8, 151–156.

[19] Wilson, K. (1987) Preparation of genomic DNA from bacteria In:

Current Protocols in Molecular Biology (Ausubel, F.M., Brent,

R.E., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A.

and Struhl, K., Eds.), pp. 2.4.1–2.4.5. Greene Publishing and

Wiley–Interscience, New York.

[20] Ezaki, T., Hashimoto, Y. and Yabuuchi, E. (1989) Fluorometric

deoxyribonucleic acid-deoxyribonucleic acid hybridization in

microdilution wells as an alternative to membrane filter hybrid-

ization in which radioisotopes are used to determine genetic

relatedness among bacterial strains. Int. J. Syst. Bacteriol. 39,

224–229.

[21] Dauga, C. (2002) Evolution of the gyrB gene and the molecular

phylogeny of Enterobacteriaceae: a model molecule for molecular

systematic studies. Int. J. Syst. Evol. Microbiol. 52, 531–547.

[22] Kimura, M. (1980) A simple method for estimating evolutionary

rates of base substitutions through comparative studies of

nucleotide sequences. J. Mol. Evol. 16, 111–120.

[23] Saitou, N. and Nei, M. (1987) The neighbor-joining method: a

new method for reconstructing phylogenetic trees. Mol. Biol.

Evol. 4, 406–425.

[24] Felsenstein, J. (1993). Phylogenies Inference Package (PHYLIP),

Version 3.53c. Department of Genetics, University of Washing-

ton, Seattle.

[25] Felsenstein, J. (1985) Confidence limits on phylogenies: an

approach using the bootstrap. Evolution 39, 783–791.

[26] Holt, J.G., Krieg, N.R., Sneath, P.H.A., Staley, J.T. and

Williams, S.T. (1994) Genus Serratia In: Bergey�s Manual of

Determinative Bacteriology (Holt, J.G., Krieg, N.R., Sneath,

J.T., Staley, J.T. and Williams, S.T., Eds.), 9th edn, p. 187.

Williams & Wilkins, Baltimore.

[27] Bycroft, B.W., Maslen, C., Box, S.J., Brown, A. and Tyler, J.W.

(1987) The isolation and characterisation of (3R,5R)- and

(3S,5R)-carbapenem-3-carboxylic acid from Serratia and Erwinia

species and their putative biosynthetic role. J. Chem. Soc., Chem.

Commun. 21, 1623–1625.

[28] Jabrane, A., Sabri, A., Compere, P., Jacques, P., Vandenberghe,

I., Van Beeumen, J. and Thonart, P. (2002) Characterization of

serracin P, a phage-tail-like bacteriocin, and its activity against

Erwinia amylovora, the fire blight pathogen. Appl. Environ.

Microbiol. 68, 5704–5710.

[29] Guasch, J.F., Enfedaque, J., Ferrer, S., Gargallo, D. and Regue,

M. (1995) Bacteriocin 28b, a chromosomally encoded bacteriocin

produced by most Serratia marcescens biotypes. Res. Microbiol.

146, 477–483.

[30] Ashelford, K.E., Fry, J.C., Bailey, M.J. and Day, M.J. (2002)

Characterization of Serratia isolates from soil, ecological impli-

http://www.bacterio.cict.fr


cations and transfer of Serratia proteamaculans subsp. quinovora

Grimont et al. 1983 to Serratia quinivorans corrig., sp. nov. Int. J.

Syst. Evol. Microbiol. 52, 2281–2289.

[31] Whitehead, N.A., Barnard, A.M., Slater, H., Simpson, N.J. and

Salmond, G.P. (2001) Quorum-sensing in Gram-negative bacte-

ria. FEMS Microbiol. Rev. 25, 365–404.

[32] Williams, P. (2002) Quorum sensing: an emerging target for

antibacterial chemotherapy. Expert Opin. Ther. Targets 6, 1–18.

[33] Zhang, L.H. and Dong, Y.H. (2004) Quorum sensing and signal

interference: diverse implications. Mol. Microbiol. 53, 1563–1571.

[34] Christensen, A.B., Riedel, K., Eberl, L., Flodgaard, L.R., Molin,

S., Gram, L. and Givskov, M. (2003) Quorum-sensing-directed

protein expression in Serratia proteamaculans B5a. Microbiology

149, 471–483.

[35] Eberl, L., Winson, M.K., Sternberg, C., Stewart, G.S.A.B.,

Christiansen, G., Chhabra, S.R., Bycroft, B., Williams, P., Molin,

S. and Givskov, M. (1996) Involvement of N-acyl-L-homoserine

lactone autoinducers in controlling the multicellular behaviour of

Serratia liquefaciens. Mol. Microbiol. 20, 127–136.

[36] Horng, Y.T., Deng, S.C., Daykin, M., Soo, P.C., Wei, J.R., Luh,

K.T., Ho, S.W., Swift, S., Lai, H.C. and Williams, P. (2002) The

LuxR family protein SpnR functions as a negative regulator of N-

acylhomoserine lactone-dependent quorum sensing in Serratia

marcescens. Mol. Microbiol. 45, 1655–1671.

[37] Ovadis, M., Liu, X., Gavriel, S., Ismailov, Z., Chet, I. and

Chernin, L. (2004) The global regulator genes from biocontrol

strain Serratia plymuthica IC1270: cloning, sequencing, and

functional studies. J. Bacteriol. 186, 4986–4993.

[38] Parsek, M.R., Val, D.L., Hanzelka, B.L., Cronan, J.E.J. and

Greenberg, E.P. (1999) Acyl homoserine-lactone quorum-sensing

signal generation. Proc. Natl. Acad. Sci. USA 96, 4360–4365.

[39] Fuqua, C. and Eberhard, A. (1999) Signal generation in autoin-

duction systems: synthesis of acylated homoserine lactones by

LuxI-type proteins In: Cell–cell Communication in Bacteria

(Dunny, G. and Winans, S.C., Eds.), pp. 211–230. ASM Press,

Washington.

[40] Kuo, A., Blough, N.V. and Dunlap, P.V. (1994) Multiple N-acyl-

L-homoserine lactone autoinducers of luminescence in the marine

symbiotic bacterium Vibrio fischeri. J. Bacteriol. 176, 7558–7565.

[41] Fray, R.G., Throup, J.P., Daykin, M., Wallace, A., Williams, P.,

Stewart, G.S. and Grierson, D. (1999) Plants genetically modified

to produce N-acylhomoserine lactones communicate with bacte-

ria. Nat. Biotechnol. 171, 1017–1020.

[42] Brenner, D.J. (1984) Enterobacteriaceae, 2nd edn In: Bergey�sManual of Systematic Bacteriology (Krieg, N.R. and Holt, J.G.,

Eds.), Vol. 1, pp. 408–420. Williams & Wilkins, Baltimore.

[43] Sproer, C., Mendrock, U., Swiderski, J., Lang, E. and Stacke-

brandt, E. (1999) The phylogenetic position of Serratia, Butti-

auxella and other genera of the family Enterobacteriaceae. Int. J.

Syst. Bacteriol. 49, 1433–1438.

[44] Wayne, L.G., Brenner, D.J., Colwell, R.R., Grimont, P.A.D.,

Kandler, O., Krichevsky, M.I., Moore, L.H., Moore, W.E.C.,

Murray, R.G.E., Stackebrandt, E., Strarr, M. and Truper, H.G.

(1987) Report of the ad hoc committee on reconciliation of

approaches to bacterial systematics. Int. J. Syst. Bacteriol. 37,

463–464.



Chemotypes significance of lichenized fungi bystructural characterization of heteropolysaccharides from the genera

Parmotrema and Rimelia

Elaine Rosechrer Carbonero a, Caroline Grassi Mellinger a, Sionara Eliasaro b,Philip Albert James Gorin a, Marcello Iacomini a,*

a Departamento de Bioquımica e Biologia Molecular, Universidade Federal do Parana, C.P. 19046, CEP 81531-990 Curitiba, PR, Brazilb Departamento de Botanica, Universidade Federal do Parana, C.P. 19031, CEP 81531-990 Curitiba, PR, Brazil

Received 2 September 2004; accepted 14 April 2005


Edited by G.M. Gadd

Abstract

Galactoglucomannans were isolated from the lichenized fungi of the genus Parmotrema (Parmotrema austrosinense, Parmotrema

delicatulum, Parmotrema mantiqueirense, Parmotrema schindlerii, and Parmotrema tinctorum and that of Rimelia (Rimelia cetrata

and Rimelia reticulata) via successive hot alkaline extraction and precipitation with Fehling solution. The structure of each polysac-

charide was investigated using 13C NMR and HSQC-DEPT spectroscopy, methylation analysis, and HPSEC-MALLS. The galac-

toglucomannans had a (1 ! 6)-linked main chain of a-Manp units, substituted preferentially at O-2 and O-4 by a-Galp and b-Galp

nonreducing end-units, respectively. The C-1 region of the 13C NMR spectra of these heteropolysaccharides is typical of the lichen

species, and is an additional tool in lichenized fungi classification.


Keywords: Lichenized fungi; Parmeliaceae; Galactoglucomannan; Chemical structure; 13C NMR

1. Introduction

The identification and classification of lichenized fun-

gi was originally carried out on the basis of morphology.

Since the 1860s, species differentiation was aided by the

specific color reactions of their components [1,2], pres-

ent at concentrations of 0.15% to 10%, or carotenoids

[3,4]. The chemical analysis of compounds for taxo-

nomic purposes was carried out by microcrystallization,chromatography, fluorescence and mass spectroscopy

analysis [5]. Recently, advances in DNA technology


doi:10.1016/j.femsle.2005.04.019


E-mail address: [email protected] (M. Iacomini).

and fine chemical characterization of macromoleculesserved as a useful tools in the classification of lichens.

The use of structurally different mannose-containing

polysaccharides for the classification and identification

of yeasts [6] led to the investigation of related polysac-

charides isolated from ascomycetous lichens via Fehling

precipitation. Their structure, as evidenced by chemical

and 13C NMR studies, proved to be typical of the parent

lichen and could thus be utilized in chemotyping studies[7–11].

In terms of macromolecules, the study of mannose-

containing polysaccharides as a taxonomic tool involves

the structural diversity of the galactomannans from

several lichenized fungi, and depends on their side-chain



274 E.R. Carbonero et al. / FEMS Microbiology Letters 246 (2005) 273–278

substituents on (1 ! 6)-linked a-D-mannopyranosyl

main-chains [12]. These generally include monosubstitu-

ents at O-2 of a-D-Manp or a-D-Galp, at O-4 by b-Galp

and sometimes with disubstitution occurring at O-2 and

O-4 by a-D-Galp and b-D-Galp, or a-D-Manp and b-D-Galp, respectively, although some of the main-chainunits are frequently not substituted.

Studies involving the taxonomy of lichenized fungi

from Cladonia and Cladina species were carried out

since classical taxonomy considered Cladina to be a sub-

genus of Cladonia, but thereafter lichenologists decided

to be a distinct genus. Woranovicz-Barreira et al. [11]

showed that galactoglucomannans are chemotypes

which could be significant in aiding the taxonomy ofCladonia spp. and those of related genera. Ahti and

Depriest [13] proposed, based on molecular phyloge-

netic results, that Cladina becomes a synonym of Clado-

nia. Subsequently, Carbonero et al. [14] studied the

structures of polysaccharides of Cladina spp. and, based

on their chemical characterization and when compared

to those of Cladonia species, agreed with results ob-

tained with DNA studies.Another suitable example of conflicting taxonomic

data concerns the segregation of the genus Rimelia from

the earlier Parmotrema, which was proposed by Hale

and Fletcher [15]. Studies on the chemical elucidation

of polysaccharides of species from these two genera

are now reported as a taxonomic aid.

Aq. 2% KOH at 100ºC for 3 h (x3)

LICHENIZED FUNGUS Cleaned, dried and powdered

CHCl3: MeOH (2:1; v/v) at 60ºC, 3 h (x3)

MeOH: H2O (4:1; v/v) at 80ºC for 3 h (x3)

Lipid extractLichen residue I

Extract of low molecular mass

Alcaline extractLichen residue III

Lichen residue II

Treatment with Fehling solutionCentrifugation

Felhing supernatant Fehling precipitate

Freeze-Thawing Centrifugation

PrecipitateSupernatant

GALACTOGLUCOMANNAN

Fig. 1. Scheme of extraction and purification of the galactogluco-

mannans obtained from Parmotrema spp. and Rimelia spp.


2.1. Lichenized fungi (family, Parmeliaceae)

Parmotrema austrosinense (Zahlbr.) Hale, Parmo-

trema delicatulum (Vain.) Hale, Parmotrema schindlerii

Hale, Parmotrema mantiqueirense Hale, Parmotrema

tinctorum (Nyl.) Hale, Rimelia cetrata (Ach.) Hale andFletcher and Rimelia reticulata (Taylor) Hale and

Fletcher were examined. Parmotrema spp. were col-

lected in 1996, in Lapa, State of Parana, Brazil, while

Rimelia spp. are from Curitiba, State of Parana, and

have their vouchers (no. 33886, 33354, 33890, 33355,

28838, 38057, 38118, respectively) deposited in the

UPCB (Herbarium name follows Holmgren et al. [16]).

2.2. Isolation and purification of polysaccharides

Lichenized fungus samples (P. austrosinense, 41 g;

P. delicatulum, 32 g; P. schindlerii, 35 g; P. mantiquei-

rense, 43 g; P. tinctorum, 60 g; R. cetrata, 31 g; and

R. reticulata, 26 g) were successively refluxed in

CHCl3-MeOH (2:1 v/v; 300 ml) and 80% aqueous

MeOH (300 ml), in order to extract low molecularcomponents. The residual material was then extracted

three times with 2% aq. KOH containing traces of

NaBH4 at 100 �C for 3 h. The alkaline extract was

neutralized with HOAc, dialyzed against tap water,

and after 48 h was freeze dried. The crude fraction

obtained from alkaline extraction was submitted to a

freeze-thawing process, which furnished insoluble and

soluble material, which were separated by centrifuga-tion (15 min, 9000 rpm, 25 �C). The soluble fraction

was submitted to a second purification process using

Fehling solution [17], resulting in a precipitate

(Cu2+-ppt) and a soluble fraction (Cu2+-sup) which

were separated by centrifugation under the above con-

ditions. Each fraction was neutralized with HOAc,

dialyzed against tap water and deionized with mixed

ion exchange resins.

2.3. Monosaccharide composition

Hydrolysis of the fractions were carried out with 1

M TFA at 100 �C for 8 h and the hydrolyzates then

evaporated to dryness, followed by successive reduc-

tion with NaBH4 and acetylation with Ac2O–pyridine

(1:1 v/v; 2 ml) at room temperature for 12 h [18,19].The resulting alditol acetates were analyzed by GC-

MS using a Varian model 3300 gas chromatograph

linked to a Finnigan Ion-Trap, model 810 R-12 mass

spectrometer, using a DB-225 capillary column (30

m · 0.25 mm i.d.), with helium as carrier gas. The

analysis was carried out from 50–220 �C at 40 �C/min maintaining the temperature constant to the end

of analysis (18 min). The products were identified bytheir typical retention times and electron impact

profiles.

Table 1

Yield of Fehling precipitates obtained from Parmotrema spp. and

Rimelia spp. and their monosaccharide composition

Lichenized fungus Yield (%)a Monosaccharide

composition (%)b

Man Gal Glc

Parmotrema austrosinense 6.7 50 44 5

P. delicatulum 3.8 49 44 6

P. mantiqueirense 5.6 50 43 6

P. schindlerii 3.2 50 43 7

P. tinctorum 5.4 51 42 6

Rimelia cetrata 3.4 53 40 7

R. reticulata 5.2 52 40 8

a Yields based on dry material.b Alditol acetates obtained on successive hydrolysis, NaBH4 reduc-

tion, and acetylation, analyzed by GC-MS (DB-225 column).

E.R. Carbonero et al. / FEMS Microbiology Letters 246 (2005) 273–278 275

2.4. Methylation analysis

Each sample (5 mg) was per-O-methylated according

to the method of Ciucanu and Kerek [20], using pow-

dered NaOH in Me2SO–MeI. The per-O-methylated

derivatives were hydrolyzed with 50% v/v sulfuric acid

(1 h, 0 �C), followed by dilution to 5.5% v/v (5 h, 100

�C), neutralization (BaCO3) and filtration [21]. Theresulting mixture of O-methylaldoses was reduced with

NaBH4 or NaBD4 and acetylated as cited above to give

Table 2

Partially O-methylated alditol acetates obtained from methylated galactoglu

O-Me-alditol acetatesa Molar % b,c

Pa Pd Pm

2,3,4,6-Me4Man 1.1 0.9 0.9

2,3,4,6-Me4Glc 1.6 1.4 3.1

2,3,5,6-Me4Gal – 0.6 0.5

2,3,4,6-Me4Gal 40.0 40.3 39.6

2,4,6-Me3Glc 3.3 3.6 2.5

2,4,6-Me3Man 0.4 – 0.4

2,4,6-Me3Gal 0.4 0.3 –

2,3,6-Me3Man 0.2 0.1 –

3,4,6-Me3Gal – – 1.7

2,3,4-Me3Man 22.0 21.4 15.7

2,3,4-Me3Gal 1.1 0.5 0.7

2,6-Me2Man 0.2 0.2 0.3

4,6-Me2Gal 0.6 0.2 0.3

3,6-Me2Gal 0.5 0.5 0.9

2,3-Me2Man 1.9 4.4 3.8

3,4-Me2Man 7.2 7.8 6.1

2,4-Me2Man 0.2 0.2 0.1

2,3-Me2Gal 0.4 0.1 0.2

2-MeMan 0.4 0.1 0.8

3-MeMan 18.1 16.4 19.3

Man 0.4 0.2 0.7

a O-Me-alditol acetates obtained by methylation analysis, followed by su

(column DB-225).b % of peak area relative to total peak area.c The symbols are: Pa, P. austrosinense; Pd, P. delicatulum; Pm, P. mant

reticulata).

a mixture of partially O-methylated alditol acetates,

which was analyzed by GC-MS. The analysis was car-

ried out from 50–215 �C at 40 �C/min maintaining the

temperature constant to the end analysis (31 min), and

the resulting partially O-methylated alditol acetates

identified by their typical electron impact breakdownprofiles and retention times [22,23].

2.5. Determination of homogeneity and molar mass

The elution profiles of fractions were determined by

high performance size-exclusion chromatography

(HPSEC), using a WATERS 510 HPLC pump at 0.6

ml/min with four gel permeation columns in series withexclusion sizes of 7 · 106, 4 · 105, 8 · 104, and 5 · 103

Da, using a refraction index (RI) detector. The eluent

was 0.1 mol/l aq. NaNO3 containing 200 ppm aq.

NaN3. Samples, previously filtered through a membrane

(0.22 lm; Millipore), were injected (250 ll loop) at 2 mg/

ml.

The specific refractive index increment (dn/dc) was

determined, with the samples being dissolved in 50mM NaNO3 and five increasing concentrations, rang-

ing from 0.2 to 1.0 mg/ml, were used to determine the

slope of the increment. Results were processed in

software provided by the manufacturer (Wyatt

Technologies).

comannans

Ps Pt Rc Rr

1.3 1.3 1.0 0.9

2.4 1.7 3.8 4.1

0.7 0.2 0.3 0.7

39.7 39.2 38.4 38.1

3.8 3.2 4.1 3.9

0.3 – – –

0.3 0.5 0.2 0.5

0.4 0.9 – –

– – 1.3 1.7

19.7 20.9 17.9 14.1

0.8 1.2 0.6 1.1

0.5 0.3 0.3 0.2

0.4 0.4 0.1 0.3

0.4 0.7 0.3 1.0

3.6 2.4 6.2 6.5

6.6 7.4 7.6 8.7

– 0.4 – –

– 0.4 0.3 0.4

0.3 0.3 0.2 0.3

18.4 18.3 17.1 16.8

0.4 0.3 0.3 0.7

ccessive hydrolysis, reduction and acetylation, analyzed by GC-MS

iqueirense; Ps, P. schindlerii; Pt, P. tinctorum; Rc, R. cetrata; Rr, R.


2.6. Nuclear magnetic resonance spectroscopy

NMR spectra were obtained using a 400 MHz Bru-

ker model DRX Avance spectrometer with a 5 mm

inverse probe. 13C NMR (100.6 MHz) and HSQC-

1DEPT analyses were performed at 50 or 30 �C, withsamples being dissolved in D2O, the OH groups being

exchanged with D2O followed by freeze-drying. Chem-

ical shifts of samples are expressed in ppm (d) relativeto acetone at d 30.20 and 2.22 for 13C and 1H signals,

respectively.

Fig. 2. 13C NMR spectra of heteropolysaccharide from Parmotrema austrosi

tinctorum (e), Rimelia cetratum (f), and R. reticulata (g).


Samples of seven species of lichenized fungi from

Parmotrema and Rimelia genus were submitted to puri-

fication procedures, according to Fig. 1, and after the

treatment with Fehling solution supernatant and precip-itated fractions were obtained. Table 1 shows all Fehling

precipitated fractions to contain mannose, galactose and

glucose as monosaccharide components. The average

obtained from all the fractions was 51% Man, 42%

Gal and 7% of Glc, and it is important to observe that

nense (a), P. delicatulum (b), P. mantiqueirense (c), P. schindlerii (d), P.

Fig. 3. HSQC-DEPT of heteropolysaccharide from Parmotrema

austrosinense in D2O at 30 �C (chemical shifts are expressed as d, ppm).

E.R. Carbonero et al. / FEMS Microbiology Letters 246 (2005) 273–278 277

no species showed a significant variation of monosac-

charide composition when compared to the average

data.

All Fehling precipitate fractions showed homoge-

neous elution profiles when analyzed by HPSEC-

MALLS, and as all the elution profiles were similar,the averaged specific refractive index increment was

dn/dc = 0.148. The samples had Mw of 53.7 kDa for P.

austrosinense, 68.2 kDa for P. delicatulum, 62.7 kDa

for P. schindlerii, 64.5 kDa for P. mantiqueirense, 39.4

kDa for P. tinctorum, 38.5 kDa for R. cetrata and 59.7

kDa for R. reticulata.

Methylation analysis of Fehling precipitate fractions

(Table 2) showed highly branched structures based onresulting partially O-methylated alditol acetates (GC-

MS) with high proportion of non-reducing ends of Galp,

besides small percentages of Manp, Galf and Glcp. They

also showed 2,3,4-Me3Man, 2,3-Me2Man, 3,4-Me2Man,

and 3-MeMan, corresponding to the main chains

formed by a-Manp-(1 ! 6) units, which were non-

substituted, substituted at O-2, O-4, and disubstituted

at O-2,4. Small amounts of Manp fully substituted unitswere also observed. Substitutions at O-2, O-3, and O-6;

disubstitutions at O-2,3; O-2,4; and O-4,6 were observed

for Galp units. Glcp was 3-O-substituted, besides its

nonreducing end-units.

The 13C NMR spectra of the galactoglucomannan

(Fig. 2) contained major signals in common, but there

were minor differences typical of the species. In gen-

eral, we have found that such 13C NMR spectra cor-respond to the lichen species [10,11,14,24], to the

extent that have been used for classification and iden-

tification [9–11].

The 13C NMR spectra of all species (Fig. 2), con-

tained C-1 signals that indicated predominant branched

structures with nonreducing end-units of b-D-Galp-

(1 ! 4)-a-D-Manp (d 104.6), a-D-Galp-(1 ! 2)-a-D-Manp (d 102.8) [12,25], along with 6-O-(d 101.6) and2,6-di-O-and 2,4,6-tri-O-substituted (d 99.8) units of a-D-Manp from the polysaccharide core [12,26]. The signal

at d 80.8 arose from 2-O-substituted a-D-Manp units

[27].

The HSQC spectrum of the P. austrosinense galacto-

glucomannan (Fig. 3) defined its a-and b-glycosidic con-figurations: the nonreducing end-units of Galp that had

a b-configuration by virtue of a high-field H-1 signal at4.42 (C-1 d 104.6), and an a-configuration due to a low

field H-1 signal at d 5.17 (102.8). The low-field H-1 sig-

nals in d 4.98 (101.6) and 5.23 (99.8) indicated that the

units of Manp had the a-configuration.Its HSQC-DEPT spectrum showed inverted signals in

d 67.9 and d 67.3 suggesting a substituted CH2 group,

probably from C-6 of a-Manp units, data in agreement

with the methylation analysis that gave mainly 3-Meand 2,3,4-Me3Man derivatives. The non substituted C-

6�s appeared at d 62.4 (3.92), d 62.7 (3.78) and d 62.9

(3.74) from the non reducing ends of Galp, Glcp, andManp units.

According to the present data, we can conclude that

the galactoglucomannans showed much similarity be-

tween the two distinct genera Parmotrema and Rimelia,

but with minor differences, typical of the species. The

galactoglucomannans have main chains of (1! 6)-

linked a-D-mannopyranosyl residues, that are mainly

unsubstituted and disubstituted at O-2 and O-4 witha-Galp and b-Galp side-chains, respectively. These re-

sults agree with previous data on other species of these

genera [9,27], and show the chemical method based on

polysaccharides to be useful as an additional tool in lich-

enized fungi classification.

Acknowledgements

The authors thank the Brazilian agencies, Coor-

denacao de Aperfeicoamento de Pessoal de Nıvel Supe-

rior (CAPES), Conselho Nacional de Desenvolvimento

Cientıfico e Tecnologico (CNPq), and Fundacao

Araucaria for financial assistance, and Dr. G. Torri,

from the Istituto di Ricerche Chimiche e Biochimiche

‘‘G. Ronzoni’’, Milan, Italy, for preparation of theHSQC-DEPT spectrum.

References

[1] Purvis, W. (2000) Lichens 122 p. Craft Print, Singapore.

[2] Aghoramurth, K., Sarma, K.G. and Seshadri, T.R. (1961)

Chemical investigation of Indian lichens. J. Sci. Ind. Res. 20,

166–168.


[3] Czezuga, B. and Skult, H. (1988) Carotenoids in lichens of

Southern Finland. Ann. Bot. Fennici 25, 229–232.

[4] Czezuga, B. and Xavier Filho, L. (1987) Investigations on

carotenoids in lichens. VII. Some lichens from Brazil. Rev. Bras.

Biol. 47, 243–246.

[5] Honda, N.K. and Vilegas, W. (1988) A quımica dos liquens.

Quım. Nova 21 (6), 110–125.

[6] Gorin, P.A.J. and Spencer, J.F.T. (1970) Proton magnetic

resonance spectroscopy: an aid in identification and chemotax-

onomy of yeasts. Adv. Appl. Microbiol. 13, 25–89.

[7] Gorin, P.A.J., Baron, M. and Iacomini, M. (1988) Storage

products of lichens In: CRC Handbook of Lichenology

(Galun, M., Ed.), vol. III, pp. 9–23. CRC Press, Boca Raton,

FL.

[8] Gorin, P.A.J., Baron, M., da Silva, M.L.C., Teixeira, A.Z.A. and

Iacomini, M. (1993) Lichen carbohydrates. Ciencia e Cultura

(Braz.) 45, 27–36.

[9] Teixeira, A.Z.A., Iacomini, M. and Gorin, P.A.J. (1995) Chem-

otypes of mannose-containing polysaccharides of lichens myco-

bionts: a possible aid in classification and identification.

Carbohydr. Res. 266, 309–314.

[10] Woranovicz, S.M., Pinto, B.M., Gorin, P.A.J. and Iacomini, M.

(1999) Novel structures in galactoglucomannans of the lichens

Cladonia substellata and Cladonia ibitipocae: significance as

chemotypes. Phytochemistry 51, 395–402.

[11] Woranovicz-Barreira, S.M., Gorin, P.A.J., Sassaki, P.L., Mar-

celli, M.P. and Iacomini, M. (1999) Galactomannoglucans of

lichenized fungi of Cladonia spp.: significance as chemotypes.


[12] Gorin, P.A.J. and Iacomini, M. (1985) Structural diversity

of D-galacto–D-mannan components isolated from lichens

having ascomycetous mycosymbionts. Carbohydr. Res. 142,

253–267.

[13] Ahti, T. and Depriest, P.T. (2001) New combination of Cladina

epithets in Cladonia (Ascomycotina, Cladoniaceae). Mycotaxon

78, 499–502.

[14] Carbonero, E.R., Montai, A.V., Woranovicz-Barreira, S.M.,

Gorin, P.A.J. and Iacomini, M. (2002). Phytochemistry 61, 681–

686.

[15] Hale, M.E. and Fletcher, A. (1990) Rimelia Hale & Fletcher, a

new lichen genus (Ascomycotina: Parmeliaceae). Bryologist 93,

23–29.

[16] Holmgren, P.K., Holmgren, N.H. and Barnett, L.C. (1990) Index

Herbariorum. 8 th ed. Part I: The herbaria of the world. Regnum

Veg. 120, 1–693.

[17] Jones, J.K.N. and Stoodley, R.J. (1965) Fractionation using

copper complexes. Meth. Carbohydr. Chem. 5, 36–38.

[18] Wolfrom, M.L. and Thompson, A. (1963) Reduction with sodium

borohydride. Meth. Carbohydr. Chem. 2, 65–67.

[19] Wolfrom, M.L. and Thompson, A. (1963) Acetylation. Meth.

Carbohydr. Chem. 2, 211–215.

[20] Ciucanu, I. and Kerek, F. (1984) A simple and rapid method for

the permethylation of carbohydrates. Carbohydr. Res. 131, 209–

217.

[21] Saeman, J.F., Moore, W.E., Mitchell, R.L. and Millet, M.A.

(1954) Techniques for the determination of pulp constituents by

quantitative paper chromatography. Tech. Assoc. Pulp Pap. Ind.

37, 336–343.

[22] Jansson, P., Kennec, L., Liedgren, H., Lindberg, B. and Lonn-

gren, J. (1976) A pratical guide to the methylation analysis of

carbohydrates. Chem. Commun. (Univ. of Stockholm) 48, 1–70.

[23] Carpita, N.C. and Shea, E. (1989) Linkage structure of carbohy-

drates by gas chromatography-mass spectrometry (GC-MS) of

partially methylated alditol acetates In: Analysis of Carbohy-

drates by GLC and MS (Biermann, C.J. and McGinnis, G.D.,

Eds.), pp. 157–216. CRC Press, Boca Raton, FL.

[24] Woranovicz, S.M., Gorin, P.A.J., Marcelli, M., Torri, G. and

Iacomini, M. (1997) Structural studies on the galactomannans of

lichens of the genus Cladonia. Lichenologist 29, 471–481.

[25] Gorin, P.A.J. and Iacomini, M. (1984) Polysaccharides of the

lichens Cetraria islandica and Ramalina usnea. Carbohydr. Res.

128, 129–132.

[26] Gorin, P.A.J. (1973) Rationalization of carbon-13 magnetic

resonance spectra of yeast mannans and structurally related

oligosaccharides. Can. J. Chem. 51, 2375–2383.

[27] Corradi da Silva, M.L., Gorin, P.A.J. and Iacomini, M. (1993)

Unusual carbohydrates from the lichen, Parmotrema cetratum.

Phytochemistry 34 (3), 715–717.



Isolation of genes differentially expressed during the fruitbody development of Pleurotus ostreatus by differential display

of RAPD

Masahide Sunagawa *, Yumi Magae

Department of Applied Microbiology, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan

Received 8 December 2004; received in revised form 12 April 2005; accepted 14 April 2005


Edited by G.M. Gadd

Abstract

To analyze genes involved in fruit body development of Pleurotus ostreatus, mRNAs from three different developmental stages:

i.e., vegetative mycelium, primordium, and mature fruit body, were isolated and reverse-transcribed to cDNAs. One hundred and

twenty random PCR amplifications were performed with the cDNAs, which generated 382, 394, 393 cDNA fragments from each

developmental stage. From these fragments, four cDNA clones specifically expressed in primordium or mature fruit body were

detected. Sequence analysis and database searches revealed significant similarity with triacylglycerol lipase, cytochrome P450 sterol

14 a-demethylase and developmentally regulated genes of other fungi. Northern blot analyses confirmed that all of the four cDNAs

were unexpressed in mycelium, thus stage-specific genes for fruit body formation of P. ostreatus were successfully isolated.


Keywords: Differential display; Pleurotus ostreatus; RAPD

1. Introduction

Fruit body morphogenesis is an important subject in

both basic and applied fields of mycological research.

The shift from vegetative growth to fruit body develop-

ment is a very interesting biological phenomenon for ba-

sic research; moreover, understanding the mechanism offruit body development will contribute to the advance-

ment of commercial mushroom production. Several

genes related to fruit body development have been iden-

tified in Coprinus cinereus [1,2], Schizophyllum commune

[3,4], Tuber borchii [5,6], Agaricus bisporus [7], Agrocybe


doi:10.1016/j.femsle.2005.04.018


E-mail address: [email protected] (M. Sunagawa).

aegerita [8], Lentinula edodes [9–13], and Flammulina

velutipes [14]. In Pleurotus ostreatus, Lee et al. [15] ana-

lyzed expressed sequence tags (ESTs) of cDNAs library

derived from liquid-culture mycelia and fruit bodies of

P. ostreatus.

The method of differential mRNA display (DD) [16]

has mostly been used to study differential gene expres-sions in plants [17–19]. In fungi, Leung et al. [11], have

used this method to identify differentially expressed

genes in RNA populations of four developmental stages

of L. edodes: vegetative mycelium, primordium, young

fruit body and mature fruit body. Also, DD has been

used to identify putative genes involved in the develop-

ment of fruit bodies of T. borchii [6]. In the present

study, genes expressed during fruit-body developmentof P. ostreatus were examined by DD.



280 M. Sunagawa, Y. Magae / FEMS Microbiology Letters 246 (2005) 279–284


2.1. Strain and culture conditions

A dikaryotic strain, P. ostreatus ASI2029, was pro-

vided by Dr. Beom-Gi Kim (National Institute of Agri-culture Science and Technology, Korea). ASI2029 was

cultivated in a sawdust-medium containing beech saw-

dust and rice bran 3:1 (v/v). The sawdust-medium was

adjusted to a hydrous rate of 65% with tap water and

packed into a culture bottle (850 ml). After autoclaving,

5 ml of liquid inoculum was inoculated. The cultures

were grown at 20 �C for 30 days, and then the tempera-

ture was lowered to 15 �C to induce fruit body develop-ment. During the cultivation of ASI2029, samples from

three stages of development, i.e., mycelium, primordium

(3–7 mm in diameter), and mature fruit body (Fig. 1)

were collected. These samples were immediately frozen

in liquid nitrogen and stored at �80 �C until use.

2.2. RNA preparation

Total RNAs were isolated using a Qiagen RNA Prep-

aration Kit (Qiagen K.K., Tokyo, Japan). Poly(A)+–

RNA was prepared by Oligotex-dT30 super (Takara

Bio Co., Shiga, Japan). Both procedures were carried

out according to the manufacturer�s instructions.

2.3. Differential display of mRNA

Poly(A)+–RNA (0.5 lg) was heated at 65 �C for 10

min and immediately chilled on ice. First-strand cDNA

synthesis was performed in a reaction mixture contain-

ing 50 mM Tris–HCl (pH 8.5), 40 mM KCl, 5 mM

MgCl2, 2 mM DTT, 850 lM each dNTP, 95 units of

RNAase Inhibitor (Takara Bio Co.), 0.2 mM random

primer (Takara Bio Co.), and 40 units of Superscript

II Reverse Transcriptase (Invitrogen, Tokyo, Japan).The reaction was carried out for 1 h at 42 �C. After

heat-denatureation of the enzyme at 95 �C for 5 min,

the random primers were removed by ultrafiltration with

Super-02 (Takara Bio Co.). 10-mer RAPD primers (Op-

eron Technologies, Inc., Alameda, CA) were used to

Fig. 1. Samples used for the RNA preparation. A: myc

PCR amplify the second-strand cDNA. PCR was car-

ried out as 45 cycles of the following thermal cycle: 30

s at 95 �C, 1 min at 50 �C, and 2 min at 72 �C [20].

2.4. Cloning and sequence of specific cDNA fragments

Specific cDNA fragments found in RAPD were cut

out from the agarose gel and purified with Gel purifica-

tion column (Nippon Bio-Rad Lab., Tokyo, Japan).

The purified DNA was cloned into vector pCR 2.1 with

the TA cloning System Kit (Invitrogen). DNA sequenc-

ing of the clones were performed by Dynamic ET Ter-

minater Sequencing Kit (Amersham Biosciences K.K.,

Tokyo, Japan) and Mega Base 1000 Sequencer (Amer-sham Biosciences K.K.). Homology search was done

using BlastX program [21] for the translated protein

and EST sequences in the NCBI data bank.

2.5. Northern blotting analyses

Total RNA (20 lg) of each developmental stage used

for the isolation of mRNA was fractionated in a 1.0%agarose gel containing formaldehyde, and transferred

to Hybond-N (Amersham Bioscience K.K.). Northern

blots were hybridized with DIG (Roche Diagnostics

K.K., Tokyo, Japan) labeled cDNA at 42 �C according

to the manufacture�s instructions. As a control, North-

ern blots were probed with 18S rDNA fragment of the

P. ostreatus (ASI2029). The 18S rDNA was isolated as

described by White et al. [22].

2.6. Nucleotide sequence accession numbers

All the clones have been deposited with the DDBJ

data banks under the Accession No. AB19629,

AB196292, AB196293 and AB196294.


In the present study, four genes specifically expressed

during the fruit body development of P. ostreatus were

isolated by means of differential displays of randomly

elium (My), B: primordium (P), C: fruit body (F).

M. Sunagawa, Y. Magae / FEMS Microbiology Letters 246 (2005) 279–284 281

amplified cDNA. To detect changes in transcripts dur-

ing fruit body development of P. ostreatus, mRNAs

were isolated from three stages of development: myce-

lium, primordium, and mature fruit body (Fig. 1). Then,

reverse-transcribed cDNAs were used as templates for

the following PCR. A total of 120 PCR amplificationswere performed with 10-mer RAPD primers. Each

PCR product separated by the agarose gel was resolved

into 1–9 distinct DNA bands in agarose gel. A total of

382, 394, and 393 cDNA fragments were identified in

the mycelium, primordium, and mature fruit body,

respectively. The electrophoresis patterns of the PCR-

amplified cDNA were confirmed as reproducible in three

independent experiments.Of the 120 random primers tested, three primers gen-

erated cDNA fragments analyzed in the present study

(Fig. 2). Ninety-five PCR amplifications (79%) gave

identical cDNA patterns between the three developmen-

tal stages. In 16 PCR, the same cDNA patterns were ob-

tained with primordium and fruit body. In one case,

specific cDNA was detected only in the mycelium stage.

All of the differentially expressed cDNA fragments wererecovered from the agarose gel and cloned into the vec-

tor pCR2.1. As a result, two fruit body-specific and the

Fig. 2. RAPD patterns of cDNA generated from mRNA of each

developmental stage. cDNA indicated by arrowheads were isolated,

cloned and analyzed in this study. M: molecular standard k/HindIII,

My: mycelium, P: primordium, F: fruit body.

Table 1

Characterization of differentially expressed cDNA clones of P. ostreatus

Levels Accession No. Homology

Clone Size

A8-U 777 AB196291 Cytochrome P450 sterol 14 a-demethyla

A8-D 574 AB196294 cDNA clone expressed during carbon st

T19-4 506 AB196294 Triacylglycerol lipase (E.C.3.1.1.3) [Cand

W3-7 415 AB196293 Early developmental cDNA clone GM57

two primordium-specific cDNA fragments were success-

fully cloned. Two specifically expressed cDNA were

detected in mature fruit body using primer A8

(5 0-GTGACGTAGG-3 0) (Fig. 2). The sizes of the frag-

ments were 777 and 574 bps, and were designated as

A8-U and A8-D, respectively. In the cases of primerT19 (5 0-GTCCGTATGG-3 0) and W3 (5 0-GTCC-

GGAGTG-3 0), two differentially amplified cDNA

fragments, 506 and 415 bps, were identified in the pri-

mordium (Fig. 2). They were designated as T19-4 and

W3-7, respectively. The cDNA clones were subjected

to sequence analysis.

Homology of the deduced amino acid sequences of

A8-U, A8-D, T19-4, and W3-7 with the database wassearched using the BlastX program. When no significant

homology was found with the protein databases, the

homology search was performed with EST databases

with the tBlastX program. The results are summarized

in Table 1. Predicted protein of T19-4 (+2 frame)

showed significant homology with triacylglycerol lipase

and contained a conserved domain of esterase-lipase.

The highest homology was found with triacylglycerol

E-value mRNA

My P F

se [Aspergillus fumigatus, AAF32372] 0.001 � � +

arvation [Trichoderma reesei, CF869295] 2e � 05 � � +

ida rugosa, 1LPP] 9e � 26 � + +

8 [Glomus mosseae, AJ315727] 0.008 � + +

Fig. 3. Northern analysis of four cDNA that are differentially

expressed during fruit body development of P. ostreatus. My:

mycelium, P: primordium, F: fruit body. Total RNA isolated from

each sample was hybridized with DIG-labeled cDNA clone and as a

control by DIG-labeled 18S rDNA. EtBr-stained ribosomal RNA

bands are loaded as quantitative control.

Fig. 4. Alignment of lipase sequences. The deduced amino acid sequence of T19-4 and three fungal lipase sequences were compared using Clustal W.

Residues, which are identical to P. ostreatus T19-4 are marked by asterisks. Underline shows the conserved domain of esterase and lipase. ILPP:

Triacyl glycerol Lipase (E.C.3.1.1.3) of Candida rugosa (GI:1064964), 1THG: Lipase of Galactomyces geotrichum (GI:443280), NCHP: Hypothetical

protein of Neurospora crassa (XM_322879).


lipase of Candida rugosa (9e � 26). The multiple align-

ment of the deduced T19-4 polypeptide with putative

amino acid sequences of lipase from other fungi is

shown in Fig. 4. Triacyl glycerol (TAG) is the predom-

inant acyl lipid in cultures undergoing sexual develop-

ment of Neurospora crassa [23]. In addition, as

described withMagnaporthe grisea, triacylglycerol lipase

activity increased during appressorium maturation [24].Mass transfer of storage lipid reserve to the aspersorium

occurred under the control of the MAP kinase and tur-

gor generation proceeded under the control of protein

kinase A [24]. Since TAG is known as an energy dense

substance [25], it is plausible that TAG is used as an en-

ergy source for the rapid development of P. ostreatus

fruit body while triacylglycerol lipase plays a role in lipid

degradation.As with A8-U, similarity (51%) was found with the

cytochrome P450 CYP51 (sterol 14 a-demethylase) of

Aspergillus fumigatus. Gene of cytochrome P450 has

been identified as involved in fruit body development

of A. bisporus [7], C. cinereus [26] and L. edodes [9],

but this is the first case of P450 CYP51, which is an

essential enzyme required in sterol biosynthesis and pri-

mary target of azole antimycotic drugs, isolated as agene related to fruit body development of mushroom.

The multiple alignment of the deduced A8-U polypep-

tide with putative amino acid sequences of CYP51 from

A. fumigatus and Penicillium italicum is shown in Fig. 5.

Fig. 5. The deduced amino acid sequence of A8-U was aligned with cytochro

and Penicillium italicum (GI:836642). Residues, which are identical to P. ost

No highly significant homology was found between

the function-known genes and the deduced polypep-

tide of A8-D and W3-7. But when they were com-

pared with genes deposited in the EST database,

high similarity was found with developmentally regu-

lated cDNA of other fungi. W3-7 may encode a pro-

tein with 84% similarity to the early developmental

gene of arbuscular mycorrhizal fungus Glomus mos-

seae (Table 1). On the other hand, the deduced poly-

peptide of A8-D showed significant homology with the

predicted protein of Trichoderma reesei cDNA clone

(2e � 05), expressed during carbon starvation. Interest-

ingly, A8-D codes a common gene involved in the

development of T. reesei and P. ostreatus although

its function is yet unknown.

Northern analysis showed that T19-4 and W3-7that were detected in primordium by the RAPD anal-

ysis, hybridized to total RNA in both primordium and

mature fruit body. The fact that T19-4 and W3-7

hybridized to the RNA of both primordium and ma-

ture fruit body shows that these mRNAs encode pro-

teins that play specific roles during both stages of fruit

body development. One possible reason for why T19-4

and W3-7 were undetected in the RAPD of fruit bodystage is that most of the RAPD primers were con-

sumed for amplification of more highly expressed

cDNA. In contrast, A8-U and A8-D hybridized only

to the total RNA of mature fruit body indicating that

me P450 sterol 14 a-demethylase of Aspergillus fumigatus (GI:6942241)

reatus are marked by asterisks.

M. Sunagawa, Y. Magae / FEMS Microbiology Letters 246 (2005) 279–284 283

they are specific genes in the latter stage of the devel-

opment. None of the four cDNA isolated in the pres-

ent study, hybridized to the RNA derived from

mycelium. The control probe, 18S rRNA gene of the

P. ostreatus, hybridized to RNA of all the stages

(Fig. 3).Liang and Pardee [16] reported about differential dis-

plays of mRNA using 3 0-anchored oligo-dT for the first-

strand cDNA synthesis and 10-mer arbitrary primers for

the second-strand synthesis by PCR. In their study,

numerous amplified fragments were visible after autora-

diography of labeled PCR products and the subsequent

isolation of the specific DNA band was rather difficult.

Leung et al. [11] and Zeppa et al. [6] also used 3 0-an-chored oligo-dT and various random primers for the

isolation of differentially expressed gene fragments.

The difference between the previous DD studies and

ours is that we did not use 3 0-anchored oligo-dT. The

number of cDNA fragments obtained in one PCR reac-

tion was not too large and each cDNA could be resolved

as a separate band in agarose gel.

With the basidiomycetes, not many genes related tofruit body development have been isolated but they of-

ten share common genes. For instance, Hydrophobin

is one of the most abundant genes in fruit bodies of

basidiomycetes, as reported by Penas et al. [27] and

Asgeirsdottir et al. [28] and has been isolated as fruiting

related gene with A. bisporus [7], F. velutipes [14] and L.

edodes [13]. But we did not detect hydrophobin as a spe-

cific gene for fruit body development of P. ostreatus inthe present study. ATPase has been detected in L. edodes

[11] and T. borchii [6]. PriA was detected in L. edodes

[10], Agrocybe aegerita [8] and EST of P. ostreatus

[15]. In addition, Septin has been isolated as a gene re-

lated to fruit body development [15,29,6]. However,

none of these genes were detected in this study. Presum-

ably because the method used in the previous studies

screen the elevated expression of genes, abundant genesinstead of unique genes tended to be isolated. But by

comparing RAPD patterns of ca. 1200 cDNA frag-

ments, abundant but not stage specific gene was easily

eliminated. All four cDNAs isolated in this study were

novel fruiting genes for basidiomycetes. The Northern

analysis confirmed that they were not expressed during

the mycelium stage, but expressed after the primordium

development. In conclusion, the DD technique was asimple and efficient method to detect fruit body stage-

specific cDNA of P. ostreatus.

Acknowledgements

We are grateful to Dr. Beom-Gi Kim of the National

Institute of Agriculture Science and Technology for pro-

viding the Pleurotus ostreatus strain (ASI2029).

References

[1] Boulianne, R.P., Liu, Y., Aebi, M., Lu, B.C. and Kues, U. (2000)

Fruiting body development in Coprinus cinereus: regulated

expression of two galectins secreted by a non-classical pathway.


[2] Kamada, T. (2002) Molecular genetics of sexual development in

the mushroom Coprinus cinereus. Bioessays 24, 449–459.

[3] Horton, J.S., Palmer, G.E. and Smith, W.J. (1999) Regulation of

dikaryon-expressed genes by FRT1 in the basidiomycete Schizo-

phyllum commune. Fungal Genet. Biol. 26, 33–47.

[4] Schuren, F.H., Asgeirsdottir, S.A., Kothe, E.M., Scheer, J.M.

and Wessels, J.G. (1993) The Sc7/Sc14 gene family of Schizo-

phyllum commune codes for extracellular proteins specifically

expressed during fruit-body formation. J. Gen. Microbiol. 139,

2083–2090.

[5] Lacourt, I., Duplessis, S., Abba, S., Bonfante, P. and Martin, F.

(2002) Isolation and characterization of differentially expressed

genes in the mycelium and fruit body of Tuber borchii. Appl.


[6] Zeppa, S., Guidi, C., Zambonelli, A., Potenza, L., Vallorani, L.,

Pierleoni, R., Sacconi, C. and Stocchi, V. (2002) Identification of

putative genes involved in the development of Tuber borchii fruit

body by mRNA differential display in agarose gel. Curr. Genet.

42, 161–168.

[7] De Groot, P.W., Schaap, P.J., Van Griensven, L.J. and Visser, J.

(1997) Isolation of developmentally regulated genes from the

edible mushroom Agaricus bisporus. Microbiology 143, 1993–

2001.

[8] Sirand-Pugnet, P. and Labarere, J. (2002) Molecular character-

ization of the Pri3 gene encoding a cysteine-rich protein,

specifically expressed during fruiting initiation within the Agro-

cybe aegerita complex. Curr. Genet. 41, 31–42.

[9] Hirano, T., Sato, T. and Enei, H. (2004) Isolation of genes

specifically expressed in the fruit body of the edible basidio-

mycete Lentinula edodes. Biosci. Biotechnol. Biochem. 68, 468–

472.

[10] Kajiwara, S., Yamaoka, K., Hori, K., Miyazawa, H., Saito, T.,

Kanno, T. and Shishido, K. (1992) Isolation and sequence of a

developmentally regulated putative novel gene, priA, from the

basidiomycete Lentinus edodes. Gene 114, 173–178.

[11] Leung, G.S., Zhang, M., Xie, W.J. and Kwan, H.S. (2000)

Identification by RNA fingerprinting of genes differentially

expressed during the development of the basidiomycete Lentinula

edodes. Mol. Gen. Genet. 262, 977–990.

[12] Nishizawa, H., Miyazaki, Y., Kaneko, S. and Shishido, K. (2002)

Distribution of hydrophobin 1 gene transcript in developing

fruiting bodies of Lentinula edodes. Biosci. Biotechnol. Biochem.

66, 1951–1954.

[13] Ng, W.L., Ng, T.P. and Kwan, H.S. (2000) Cloning and

characterization of two hydrophobin genes differentially

expressed during fruit body development in Lentinula edodes.


[14] Ando, A., Harada, A., Miura, K. and Tamai, Y. (2001) A gene

encoding a hydrophobin, fvh1, is specifically expressed after the

induction of fruit in the edible mushroom Flammulina velutipes.

Curr. Genet. 39, 190–197.

[15] Lee, S.H., Kim, B.G., Kim, K.J., Lee, J.S., Yun, D.W.,

Hahn, J.H., Kim, G.H., Lee, K.H., Suh, D.S., Kwon, S.T.,

Lee, C.S. and Yoo, Y.B. (2002) Comparative analysis of

sequences expressed during the liquid-cultured mycelia and

fruit body stages of Pleurotus ostreatus. Fungal Genet. Biol.

35, 115–134.

[16] Liang, P. and Pardee, A.B. (1992) Differential display of eukary-

otic messenger RNA by means of the polymerase chain reaction.

Science 257, 967–971.


[17] Stein, S., Cho, Y.J., Jackson, R.S. and Liang, P. (2003) Identi-

fication of p53 target genes by fluorescent differential display.

Methods Mol. Biol. 234, 51–63.

[18] Ueda, A., Shi, W., Nakamura, T. and Takabe, T. (2002) Analysis

of salt-inducible genes in barley roots by differential display. J.

Plant Res. 115, 119–130.

[19] Wu, L.M., Ni, Z.F., Meng, F.R., Lin, Z. and Sun, Q.X. (2003)

Cloning and characterization of leaf cDNAs that are differentially

expressed between wheat hybrids and their parents. Mol. Genet.

Genomics. 270, 281–286.

[20] Sunagawa, M., Tamai, Y., Neda, H., Miyazaki, K. and Miura, K.

(1995) Application of random amplified polymorphic DNA

(RAPD) markers (I): analyses of fussants in edible mushrooms.

Nihon Mokuzai Gakkaishi 41, 945–948.

[21] Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang,

Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSI-

BLAST: a new generation of protein database search programs.

Nucleic Acids Res. 25, 3389–3402.

[22] White, T.J., Bruns, T., Lee, S. and Taylor, J. (1990) Amplification

and direct sequencing of fungal ribosomal RNA genes for

phylogenetics In: PCR Protocols (Innis, M.A., Gelfand, D.H.,

Sninsky, J.J. and White, T.J., Eds.), pp. 315–322. Academic Press,

San Diego, CA.

[23] Goodrich-Tanrikulu, M., Howe, K., Stafford, A. and Nelson,

M.A. (1998) Changes in fatty acid composition of Neurospora

crassa accompany sexual development and ascospore germina-

tion. Microbiology 144, 1713–1720.

[24] Thines, E., Weber, R.W. and Talbot, N.J. (2000) MAP kinase and

protein kinase A-dependent mobilization of triacylglycerol and

glycogen during appressorium turgor generation by Magnaporthe

grisea. Plant Cell 12, 1703–1718.

[25] Gibbons, G.F., Islam, K. and Pease, R.J. (2000) Mobilisation of

triacylglycerol stores. Biochim. Biophys. Acta 1483, 37–57.

[26] Muraguchi, H. and Kamada, T. (2000) A mutation in the

elen2 gene encoding a chtochrome P450 of Coprinus cinereus

affects mushroom morphogenesis. Fungal Genet. Biol. 29, 49–

59.

[27] Penas, M.M., Asgeirsdottir, S.A., Lasa, I., Culianez-Macia, F.A.,

Pisabarro, A.G., Wessels, J.G. and Ramirez, L. (1998) Identifi-

cation, characterization, and In situ detection of a fruit-body-

specific hydrophobin of Pleurotus ostreatus. Appl. Environ.

Microbiol. 64, 4028–4034.

[28] Asgeirsdottir, S.A., de Vries, O.M. and Wessels, J.G. (1998)

Identification of three differentially expressed hydrophobins in

Pleurotus ostreatus (oyster mushroom). Microbiology 144, 2961–

2969.

[29] Stoop, J.M.H. and Mooibroek, H. (1999) Advances in

genetic analysis and biotechnology of the cultivated button

mushroom, Agaricus bisporus. Appl. Microbiol. Biotechnol.

52, 474–483.

FEMS Microbiology Letters

Author Index Volume 246

Alves, A., see Tacao, M. (246) 11

Ando, S., see Goto, M. (246) 33

Anne, J., see Barbe, S. (246) 67

Arciola, C.R., Campoccia, D., Gamberini, S., Baldassarri, L. and

Montanaro, L. Prevalence of cna, fnbA and fnbB adhesin genes

among Staphylococcus aureus isolates from orthopedic infections

associated to different types of implant (246) 81

Asano, Y., see Kato, Y. (246) 243

Athie-Morales, V., see O�Brien, J.B. (246) 199Azcarate-Peril, M.A., see Bruno-Barcena, J.M. (246) 91

Bala, K., see Paul, B. (246) 207

Baldassarri, L., see Arciola, C.R. (246) 81

Barbe, S., Van Mellaert, L., Theys, J., Geukens, N., Lammertyn, E.,

Lambin, P. and Anne, J. Secretory production of biologically active

rat interleukin-2 by Clostridium acetobutylicum DSM792 as a tool

for anti-tumor treatment (246) 67

Barlow, K., see Yin, X. (246) 251

Behr, T., see Lehner, A. (246) 133

Belarbi, A., see Paul, B. (246) 207

Bera, R., Nayak, A., Sen, A.K., Chowdhury, B.P. and Bhadra, R.

Isolation and characterisation of the lipopolysaccharide from

Acidiphilium strain GS18h/ATCC55963, a soil isolate of Indian

copper mine (246) 183

Bevivino, A., see Dalmastri, C. (246) 39

Bhadra, R., see Bera, R. (246) 183

Blanch, A.R., see Garcıa-Aljaro, C. (246) 55

Blanco, J., see Garcıa-Aljaro, C. (246) 55

Blanco, J.E., see Garcıa-Aljaro, C. (246) 55

Blanco, M., see Garcıa-Aljaro, C. (246) 55

Bordons, A., see Reguant, C. (246) 111

Bruno-Barcena, J.M., Azcarate-Peril, M.A., Klaenhammer, T.R. and

Hassan, H.M. Marker-free chromosomal integration of the

manganese superoxide dismutase gene (sodA) from Streptococcus

thermophilus into Lactobacillus gasseri (246) 91

Campoccia, D., see Arciola, C.R. (246) 81

Carbonero, E.R., Mellinger, C.G., Eliasaro, S., Gorin, P.A.J. and

Iacomini, M. Chemotypes significance of lichenized fungi by

structural characterization of heteropolysaccharides from the

genera Parmotrema and Rimelia (246) 273

Carrascosa, A.V., see Cebollero, E. (246) 1

Carrete, R., see Reguant, C. (246) 111

Cebollero, E., Martinez-Rodriguez, A., Carrascosa, A.V. and

Gonzalez, R. Overexpression of csc1-1. A plausible strategy to

obtain wine yeast strains undergoing accelerated autolysis (246) 1

Cevallos, A.M., Perez-Escobar, M., Espinosa, N., Herrera, J.,

Lopez-Villasenor, I. and Hernandez, R. The stabilization of

housekeeping transcripts in Trypanosoma cruzi epimastigotes

evidences a global regulation of RNA decay during stationary

phase (246) 259

Chambers, J.R., see Yin, X. (246) 251

Chiarini, L., see Dalmastri, C. (246) 39

Chowdhury, B.P., see Bera, R. (246) 183

Clarke, P., see Viguier, C. (246) 235

Constantı, M., see Reguant, C. (246) 111

Cornelis, P., see Ghysels, B. (246) 167

Correia, A., see Tacao, M. (246) 11

Dalmastri, C., Pirone, L., Tabacchioni, S., Bevivino, A. and

Chiarini, L. Efficacy of species-specific recA PCR tests in the

identification of Burkholderia cepacia complex environmental

isolates (246) 39

Dunn, M.F., see Guillen-Navarro, K. (246) 159

Eliasaro, S., see Carbonero, E.R. (246) 273

Encarnacion, S., see Guillen-Navarro, K. (246) 159

Espejo, R.T., see Gonzalez-Escalona, N. (246) 213

Espinosa, N., see Cevallos, A.M. (246) 259

Fan, K.-Q., see Wu, X.-B. (246) 103

Forster-Fromme, K. and Jendrossek, D. Malate:quinone

oxidoreductase (MqoB) is required for growth on acetate and

linear terpenes in Pseudomonas citronellolis (246) 25

Fu, X., see Qu, Y. (246) 143

Gaenge, H., see Lehner, A. (246) 133

Gamberini, S., see Arciola, C.R. (246) 81

Garcıa-Aljaro, C., Muniesa, M., Blanco, J.E., Blanco, M., Blanco, J.,

Jofre, J. and Blanch, A.R. Characterization of Shiga toxin-

producing Escherichia coli isolated from aquatic environments

(246) 55

Geukens, N., see Barbe, S. (246) 67

Ghysels, B., Ochsner, U., Mollman, U., Heinisch, L., Vasil, M.,

Cornelis, P. and Matthijs, S. The Pseudomonas aeruginosa pirA

gene encodes a second receptor for ferrienterobactin and synthetic

catecholate analogues (246) 167

Gognies, S., see Paul, B. (246) 207

Gonzalez, R., see Cebollero, E. (246) 1

Gonzalez-Escalona, N., Romero, J. and Espejo, R.T. Polymorphism

and gene conversion of the 16S rRNA genes in the multiple rRNA

operons of Vibrio parahaemolyticus (246) 213

Gorin, P.A.J., see Carbonero, E.R. (246) 273

Goto, M., Ando, S., Hachisuka, Y. and Yoneyama, T. Contamination

of diverse nifH and nifH-like DNA into commercial PCR primers

(246) 33

doi:10.1016/S0378-1097(05)00268-5



Guillen-Navarro, K., Encarnacion, S. and Dunn, M.F. Biotin

biosynthesis, transport and utilization in rhizobia (246) 159

Guo, J., see Lai, X. (246) 87

Hachisuka, Y., see Goto, M. (246) 33

Hassan, H.M., see Bruno-Barcena, J.M. (246) 91

Heinisch, L., see Ghysels, B. (246) 167

Henriques, I., see Tacao, M. (246) 11

Hernandez, R., see Cevallos, A.M. (246) 259

Herrera, J., see Cevallos, A.M. (246) 259

Hirai, H., Sugiura, M., Kawai, S. and Nishida, T. Characteristics of

novel lignin peroxidases produced by white-rot fungus

Phanerochaete sordida YK-624 (246) 19

Hu, D.-L., see Omoe, K. (246) 191

Hubalek, M., see Lenco, J. (246) 47

Iacomini, M., see Carbonero, E.R. (246) 273

Irani, V.R. and Maslow, J.N. Induction of murine macrophage TNF-asynthesis by Mycobacterium avium is modulated through

complement-dependent interaction via complement receptors 3

and 4 in relation to M. avium glycopeptidolipid (246) 221

Jansen, A., see Van Houdt, R. (246) 265

Jendrossek, D., see Forster-Fromme, K. (246) 25

Jofre, J., see Garcıa-Aljaro, C. (246) 55

Jovcic, B., see Kojic, M. (246) 175

Kato, Y., Yoshida, S. and Asano, Y. Polymerase chain reaction for

identification of aldoxime dehydratase in aldoxime- or nitrile-

degrading microorganisms (246) 243

Kawai, S., see Hirai, H. (246) 19

Kelleher, D.P., see O�Brien, J.B. (246) 199Kimura, T., see Morimoto, K. (246) 229

Klaenhammer, T.R., see Bruno-Barcena, J.M. (246) 91

Kojic, M., Jovcic, B., Vindigni, A., Odremanp, F. and Venturi, V.

Novel target genes of PsrA transcriptional regulator of

Pseudomonas aeruginosa (246) 175

Kuhl, M., see Thar, R. (246) 75

Lai, X., Guo, J., Zhang, X. and Wang, H. Identification of a novel

domain – DIM, which defines a new family composed mainly of

bacterial membrane proteins (246) 87

Lambin, P., see Barbe, S. (246) 67

Lammertyn, E., see Barbe, S. (246) 67

Lehner, A., Loy, A., Behr, T., Gaenge, H., Ludwig, W., Wagner, M.

and Schleifer, K.-H. Oligonucleotide microarray for identification

of Enterococcus species (246) 133

Lenco, J., Pavkova, I., Hubalek, M. and Stulik, J. Insights into the

oxidative stress response in Francisella tularensis LVS and its

mutant DiglC1+2 by proteomics analysis (246) 47

Lopez-Villasenor, I., see Cevallos, A.M. (246) 259

Loy, A., see Lehner, A. (246) 133

Ludwig, W., see Lehner, A. (246) 133

Magae, Y., see Sunagawa, M. (246) 279

Martinez-Rodriguez, A., see Cebollero, E. (246) 1

Maslow, J.N., see Irani, V.R. (246) 221

Matthijs, S., see Ghysels, B. (246) 167

McCabe, M.S., see O�Brien, J.B. (246) 199McDonald, G.S.A., see O�Brien, J.B. (246) 199Melin, P., see Strom, K. (246) 119

Mellinger, C.G., see Carbonero, E.R. (246) 273

Michiels, C.W., see Van Houdt, R. (246) 265

Mollman, U., see Ghysels, B. (246) 167

Montanaro, L., see Arciola, C.R. (246) 81

Moons, P., see Van Houdt, R. (246) 265

Morimoto, K., Kimura, T., Sakka, K. and Ohmiya, K. Overexpression

of a hydrogenase gene in Clostridium paraputrificum to enhance

hydrogen gas production (246) 229

Moura, A., see Tacao, M. (246) 11

Muniesa, M., see Garcıa-Aljaro, C. (246) 55

Nakane, A., see Omoe, K. (246) 191

Nayak, A., see Bera, R. (246) 183

Nı Eidhin, D.B., see O�Brien, J.B. (246) 199Nishida, T., see Hirai, H. (246) 19

O�Brien, J.B., McCabe, M.S., Athie-Morales, V., McDonald, G.S.A.,

Nı Eidhin, D.B. and Kelleher, D.P. Passive immunisation of

hamsters against Clostridium difficile infection using antibodies to

surface layer proteins (246) 199

O Cuıv, P., see Viguier, C. (246) 235

Ochsner, U., see Ghysels, B. (246) 167

O�Connell, M., see Viguier, C. (246) 235

Odremanp, F., see Kojic, M. (246) 175

Ohmiya, K., see Morimoto, K. (246) 229

Omoe, K., Hu, D.-L., Takahashi-Omoe, H., Nakane, A. and

Shinagawa, K. Comprehensive analysis of classical and newly

described staphylococcal superantigenic toxin genes in

Staphylococcus aureus isolates (246) 191

Ona, O., Van Impe, J., Prinsen, E. and Vanderleyden, J. Growth and

indole-3-acetic acid biosynthesis of Azospirillum brasilense Sp245 is

environmentally controlled (246) 125

Park, A.S., see Yin, X. (246) 251

Paul, B., Bala, K., Gognies, S. and Belarbi, A. Morphological and

molecular taxonomy of Pythium longisporangium sp. nov. isolated

from the Burgundian region of France (246) 207

Pavkova, I., see Lenco, J. (246) 47

Perez-Escobar, M., see Cevallos, A.M. (246) 259

Pirone, L., see Dalmastri, C. (246) 39

Prinsen, E., see Ona, O. (246) 125

Qu, Y., Zhou, J., Wang, J., Fu, X. and Xing, L. Microbial community

dynamics in bioaugmented sequencing batch reactors for

bromoamine acid removal (246) 143

Reguant, C., Carrete, R., Constantı, M. and Bordons, A. Population

dynamics of Oenococcus oeni strains in a new winery and the effect

of SO2 and yeast strain (246) 111

Romero, J., see Gonzalez-Escalona, N. (246) 213

Saavedra, M.J., see Tacao, M. (246) 11

Sakka, K., see Morimoto, K. (246) 229

Schleifer, K.-H., see Lehner, A. (246) 133

Schnurer, J., see Strom, K. (246) 119

Sen, A.K., see Bera, R. (246) 183

Shinagawa, K., see Omoe, K. (246) 191

Strom, K., Schnurer, J. and Melin, P. Co-cultivation of antifungal

Lactobacillus plantarum MiLAB 393 and Aspergillus nidulans,

evaluation of effects on fungal growth and protein expression

(246) 119

Stulik, J., see Lenco, J. (246) 47

Sugiura, M., see Hirai, H. (246) 19

Sunagawa, M. and Magae, Y. Isolation of genes differentially

expressed during the fruit body development of Pleurotus

ostreatus by differential display of RAPD (246) 279

Tabacchioni, S., see Dalmastri, C. (246) 39

Tacao, M., Moura, A., Alves, A., Henriques, I., Saavedra, M.J. and

Correia, A. Evaluation of 16S rDNA- and gyrB-DGGE for typing

members of the genus Aeromonas (246) 11

Takahashi-Omoe, H., see Omoe, K. (246) 191

286 Author Index Volume 246

Thar, R. and Kuhl, M. Complex pattern formation of marine gradient

bacteria explained by a simple computer model (246) 75

Theys, J., see Barbe, S. (246) 67

Van Houdt, R., Moons, P., Jansen, A., Vanoirbeek, K. and Michiels,

C.W. Genotypic and phenotypic characterization of a biofilm-

forming Serratia plymuthica isolate from a raw vegetable

processing line (246) 265

Van Impe, J., see Ona, O. (246) 125

Van Mellaert, L., see Barbe, S. (246) 67

Vanderleyden, J., see Ona, O. (246) 125

Vanoirbeek, K., see Van Houdt, R. (246) 265

Vasil, M., see Ghysels, B. (246) 167

Venturi, V., see Kojic, M. (246) 175

Viguier, C., O Cuıv, P., Clarke, P. and O�Connell, M. RirA is the iron

response regulator of the rhizobactin 1021 biosynthesis and

transport genes in Sinorhizobium meliloti 2011 (246) 235

Vindigni, A., see Kojic, M. (246) 175

Wagner, M., see Lehner, A. (246) 133

Wang, H., see Lai, X. (246) 87

Wang, J., see Qu, Y. (246) 143

Wang, Q.-H., see Wu, X.-B. (246) 103

Wheatcroft, R., see Yin, X. (246) 251

Wu, X.-B., Fan, K.-Q., Wang, Q.-H. and Yang, K.-Q. C-terminus

mutations of Acremonium chrysogenum deacetoxy/deacetyl-

cephalosporin C synthase with improved activity toward

penicillin analogs (246) 103

Xing, L., see Qu, Y. (246) 143

Yang, K.-Q., see Wu, X.-B. (246) 103

Yin, X., Chambers, J.R., Barlow, K., Park, A.S. and Wheatcroft, R.

The gene encoding xylulose-5-phosphate/fructose-6-phosphate

phosphoketolase (xfp) is conserved among Bifidobacterium species

within a more variable region of the genome and both are useful for

strain identification (246) 251

Yoneyama, T., see Goto, M. (246) 33

Yoshida, S., see Kato, Y. (246) 243

Zhang, X., see Lai, X. (246) 87

Zhou, J., see Qu, Y. (246) 143

Zwirglmaier, K. Fluorescence in situ hybridisation (FISH) – the next

generation (246) 151

Author Index Volume 246 287

FEMS Microbiology Letters

Subject Index Volume 246

Acidiphilium ATCC55963

Lipopolysaccharide; Lipid A; Lethal toxicity (Bera, R. (246) 183)

Aeromonas

DGGE; gyrB; 16S rRNA (Tacao, M. (246) 11)

Aldoxime dehydratase

Nitrile hydratase; PCR; Aldoxime-nitrile pathway; Screening (Kato,

Y. (246) 243)

Aldoxime-nitrile pathway

Aldoxime dehydratase; Nitrile hydratase; PCR; Screening (Kato, Y.

(246) 243)

Antheridia

Pythium longisporangium; Sporangia; Oogonia; Oospores; ITS region;

rRNA (Paul, B. (246) 207)

Anti-bacterial drugs

DIM; Protein domain; Bacteria; Transmembrane region (Lai, X. (246)

87)

Anti-cancer treatment

Clostridium acetobutylicum; Protein secretion; Interleukin-2 (Barbe, S.

(246) 67)

Antifungal activity

Two dimensional gel electrophoresis; 3-Phenyl lactic acid; Cyclic

dipeptide (Strom, K. (246) 119)

Autolysis

Sparkling wine; Genetic engineering; Autophagy; Saccharomyces

cerevisiae (Cebollero, E. (246) 1)

Autophagy

Sparkling wine; Autolysis; Genetic engineering; Saccharomyces

cerevisiae (Cebollero, E. (246) 1)

Bacteria

DIM; Protein domain; Transmembrane region; Anti-bacterial drugs

(Lai, X. (246) 87)

Bacterial adhesion

Collagen adhesion gene (cna); Fibronectin-binding protein;

Biomaterial-associated infections; Staphylococcus aureus (Arciola,

C.R. (246) 81)

Bifidobacterium

Phosphoketolase; xfp; Strain identification; Detection (Yin, X. (246)

251)

Bioaugmentation

Bromoamine acid; Community dynamics; Ribosomal intergenic

spacer; Sphingomonas xenophaga (Qu, Y. (246) 143)

Biomaterial-associated infections

Collagen adhesion gene (cna); Fibronectin-binding protein; Bacterial

adhesion; Staphylococcus aureus (Arciola, C.R. (246) 81)

Biotin biosynthesis

Rhizobia; Rhizobia–legume symbiosis (Guillen-Navarro, K. (246) 159)

Branched-chain carbon metabolism

Citronellol pathway; Malate:quinone oxidoreductase; Geranyl-

coenzymeA carboxylase; Pseudomonas citronellolis (Forster-Fromme,

K. (246) 25)

Bromoamine acid

Bioaugmentation; Community dynamics; Ribosomal intergenic spacer;

Sphingomonas xenophaga (Qu, Y. (246) 143)

Burkholderia cepacia complex identification

recA specific PCR; Cystic fibrosis; Rhizosphere environment

(Dalmastri, C. (246) 39)

Catecholate siderophores

Pseudomonas aeruginosa; Enterobactin; pfeA; pirA; TonB-dependent

receptors (Ghysels, B. (246) 167)

Chemical structure

Lichenized fungi; Parmeliaceae; Galactoglucomannan; 13C NMR

(Carbonero, E.R. (246) 273)

Chemotaxis

Self-organisation; Thiovulum; Microbial pattern formation; Quorum

sensing; Computer model (Thar, R. (246) 75)

Citronellol pathway

Branched-chain carbon metabolism; Malate:quinone oxidoreductase;

GeranylcoenzymeA carboxylase; Pseudomonas citronellolis (Forster-

Fromme K. (246) 25)

Clostridium acetobutylicum

Protein secretion; Interleukin-2; Anti-cancer treatment (Barbe, S. (246)

67)

doi:10.1016/S0378-1097(05)00269-7



Clostridium difficile

Diarrhoea; Surface layer proteins; Hamster model (O�Brien, J.B. (246)199)

Clostridium paraputrificum

Hydrogenase; Hydrogen gas production (Morimoto, K. (246) 229)

13C NMR

Lichenized fungi; Parmeliaceae; Galactoglucomannan; Chemical

structure (Carbonero, E.R. (246) 273)

Collagen adhesion gene (cna)

Fibronectin-binding protein; Bacterial adhesion; Biomaterial-

associated infections; Staphylococcus aureus (Arciola, C.R. (246) 81)

Community dynamics

Bioaugmentation; Bromoamine acid; Ribosomal intergenic spacer;


Complement receptor

Mycobacterium avium; GPL; TNF-a; Macrophage; Serum proteins

(Irani and J.N. Maslow V.R. (246) 221)

Computer model

Self-organisation; Chemotaxis; Thiovulum; Microbial pattern

formation; Quorum sensing (Thar, R. (246) 75)

Contamination

nifH; PCR; Primer (Goto, M. (246) 33)

C-terminus

DAOC; DAC; Deacetoxy/deacetylcephalosporin C synthase;

Mutagenesis; Acremonium chrysogenum; Kinetics (Wu, X.-B. (246)

103)

Cyclic dipeptide

Antifungal activity; Two dimensional gel electrophoresis; 3-Phenyl

lactic acid (Strom, K. (246) 119)

Cystic fibrosis

Burkholderia cepacia complex identification; recA specific PCR;

Rhizosphere environment (Dalmastri, C. (246) 39)

DAC

DAOC; Deacetoxy/deacetylcephalosporin C synthase; C-terminus;


103)

DAOC

DAC; Deacetoxy/deacetylcephalosporin C synthase; C-terminus;


103)

Deacetoxy/deacetylcephalosporin C synthase

DAOC; DAC; C-terminus; Mutagenesis; Acremonium chrysogenum;

Kinetics (Wu, X.-B. (246) 103)

Detection

Bifidobacterium; Phosphoketolase; xfp; Strain identification (Yin, X.

(246) 251)

DGGE

Aeromonas; 16S rRNA; gyrB (Tacao, M. (246) 11)

Diarrhoea

Clostridium difficile; Surface layer proteins; Hamster model (O�Brien,J.B. (246) 199)

Differential display

Pleurotus ostreatus; RAPD (Sunagawa, M. (246) 279)

DIM

Protein domain; Bacteria; Transmembrane region; Anti-bacterial

drugs (Lai, X. (246) 87)

Enterobactin

Pseudomonas aeruginosa; Catecholate siderophores; pfeA; pirA; TonB-

dependent receptors (Ghysels, B. (246) 167)

Enterococcus

rRNA gene; Oligonucleotide; Microarray; Probe; Microbial

diagnostics (Lehner, A. (246) 133)

Enterotoxin

Staphylococcus aureus; Multiplex PCR; Genotyping; Mobile genetic

elements (Omoe, K. (246) 191)

Escherichia coli

STEC; stx2; VT2 verotoxin; VTECWater (Garcıa-Aljaro, C. (246) 55)

Fermentor

Azospirillum brasilense; Indole-3-acetic acid (Ona, O. (246) 125)

Fibronectin-binding protein

Collagen adhesion gene (cna); Bacterial adhesion; Biomaterial-

associated infections; Staphylococcus aureus (Arciola, C.R. (246) 81)

Fluorescence in situ hybridisation

Tyramide signal amplification; Polynucleotide probes (Zwirglmaier K.

(246) 151)

Francisella tularensis

Oxidative stress; Quantitative proteomics; Two-dimensional

electrophoresis (Lenco, J. (246) 47)

Functional gene replacement

Manganese superoxide dismutase; Oxidative stress; Lactobacillus

gasseri; Lactic acid bacteria; Probiotics (Bruno-Barcena, J.M. (246) 91)

Fur

Siderophore; Iron response; RirA (Viguier, C. (246) 235)

Galactoglucomannan

Lichenized fungi; Parmeliaceae; Chemical structure; 13C NMR


Gene conversion

Polymorphism; rrn operons; Vibrio parahaemolyticus; 16S rRNA

(Gonzalez-Escalona, N. (246) 213)

Gene expression

Protozoa; Kinetoplastid; RNA stability (Cevallos, A.M. (246) 259)

Genetic engineering

Sparkling wine; Autolysis; Autophagy; Saccharomyces cerevisiae

(Cebollero, E. (246) 1)

Genotyping

Staphylococcus aureus; Enterotoxin; Multiplex PCR; Mobile genetic


GeranylcoenzymeA carboxylase

Citronellol pathway; Branched-chain carbon metabolism;

Malate:quinone oxidoreductase; Pseudomonas citronellolis (Forster-

Fromme, K. (246) 25)

290 Subject Index Volume 246

GPL

Mycobacterium avium; TNF-a; Macrophage; Complement receptor;

Serum proteins (Irani, V.R. (246) 221)

gyrB

Aeromonas; DGGE; 16S rRNA (Tacao, M. (246) 11)

Serratia; Identification natural isolate; Phylogeny; Quorum sensing; N-

Acyl-L-homoserine lactone (Van Houdt, R. (246) 265)

Hamster model

Clostridium difficile; Diarrhoea; Surface layer proteins (O�Brien, J.B.(246) 199)

Hydrogen gas production

Clostridium paraputrificum; Hydrogenase (Morimoto, K. (246) 229)

Hydrogen peroxide

Phanerochaete sordida YK-624; Lignin peroxidase; Ordered bi-bi ping-

pong mechanism; Lignin substructure model compounds (Hirai, H.

(246) 19)

Hydrogenase

Clostridium paraputrificum; Hydrogen gas production (Morimoto, K.

(246) 229)

Identification natural isolate

Serratia; gyrB; Phylogeny; Quorum sensing; N-Acyl-L-homoserine

lactone (Van Houdt, R. (246) 265)

Indole-3-acetic acid

Azospirillum brasilense; Fermentor (Ona, O. (246) 125)

Interleukin-2

Clostridium acetobutylicum; Protein secretion; Anti-cancer treatment

(Barbe, S. (246) 67)

Iron response

Siderophore; RirA; Fur (Viguier, C. (246) 235)

ITS region

Pythium longisporangium; Sporangia; Oogonia; Antheridia; Oospores;

rRNA (Paul, B. (246) 207)

Kinetics

DAOC; DAC; Deacetoxy/deacetylcephalosporin C synthase; C-

terminus; Mutagenesis; Acremonium chrysogenum (Wu, X.-B. (246)

103)

Kinetoplastid

Protozoa; RNA stability; Gene expression (Cevallos, A.M. (246) 259)

Lactic acid bacteria

Functional gene replacement; Manganese superoxide dismutase;

Oxidative stress; Lactobacillus gasseri; Probiotics (Bruno-Barcena,

J.M. (246) 91)

Lactobacillus gasseri


Oxidative stress; Lactic acid bacteria; Probiotics (Bruno-Barcena,

J.M. (246) 91)

Lethal toxicity

Acidiphilium ATCC55963; Lipopolysaccharide; Lipid A (Bera, R.

(246) 183)

Lichenized fungi

Parmeliaceae; Galactoglucomannan; Chemical structure; 13C NMR


Lignin peroxidase

Phanerochaete sordida YK-624; Ordered bi-bi ping-pong mechanism;

Lignin substructure model compounds; Hydrogen peroxide (Hirai, H.

(246) 19)

Lignin substructure model compounds

Phanerochaete sordida YK-624; Lignin peroxidase; Ordered bi-bi ping-

pong mechanism; Hydrogen peroxide (Hirai, H. (246) 19)

Lipid A

Acidiphilium ATCC55963; Lipopolysaccharide; Lethal toxicity (Bera,

R. (246) 183)

Lipopolysaccharide

Acidiphilium ATCC55963; Lipid A; Lethal toxicity (Bera, R. (246) 183)

Macrophage

Mycobacterium avium; GPL; TNF-a; Complement receptor; Serum

proteins (Irani, V.R. (246) 221)

Malate:quinone oxidoreductase


GeranylcoenzymeA carboxylase; Pseudomonas citronellolis (Forster-

Fromme, K. (246) 25)

Malolactic fermentation

Oenococcus oeni; RAPD multiplex; Strain typification; Sulphur

dioxide; Wine (Reguant, C. (246) 111)

Manganese superoxide dismutase

Functional gene replacement; Oxidative stress; Lactobacillus gasseri;

Lactic acid bacteria; Probiotics (Bruno-Barcena, J.M. (246) 91)

Microarray

Enterococcus; rRNA gene; Oligonucleotide; Probe; Microbial


Microbial diagnostics

Enterococcus; rRNA gene; Oligonucleotide; Microarray; Probe

(Lehner, A. (246) 133)

Microbial pattern formation

Self-organisation; Chemotaxis; Thiovulum; Quorum sensing;

Computer model (Thar, R. (246) 75)

Mobile genetic elements

Staphylococcus aureus; Enterotoxin; Multiplex PCR; Genotyping

(Omoe, K. (246) 191)

Multiplex PCR

Staphylococcus aureus; Enterotoxin; Genotyping; Mobile genetic


Mutagenesis

DAOC; DAC; Deacetoxy/deacetylcephalosporin C synthase; C-

terminus; Acremonium chrysogenum; Kinetics (Wu, X.-B. (246) 103)

Mycobacterium avium

GPL; TNF-a; Macrophage; Complement receptor; Serum proteins

(Irani and J.N. Maslow V.R. (246) 221)

Subject Index Volume 246 291

N-Acyl-L-homoserine lactone

Serratia; Identification natural isolate; gyrB; Phylogeny; Quorum

sensing (Van Houdt, R. (246) 265)

nifH

Contamination; PCR; Primer (Goto, M. (246) 33)

Nitrile hydratase

Aldoxime dehydratase; PCR; Aldoxime-nitrile pathway; Screening

(Kato, Y. (246) 243)

Oenococcus oeni

Malolactic fermentation; RAPD multiplex; Strain typification;

Sulphur dioxide; Wine (Reguant, C. (246) 111)

Oligonucleotide

Enterococcus; rRNA gene; Microarray; Probe; Microbial diagnostics

(Lehner, A. (246) 133)

Oogonia

Pythium longisporangium; Sporangia; Antheridia; Oospores; ITS

region; rRNA (Paul, B. (246) 207)

Oospores

Pythium longisporangium; Sporangia; Oogonia; Antheridia; ITS

region; rRNA (Paul, B. (246) 207)

Ordered bi-bi ping-pong mechanism

Phanerochaete sordida YK-624; Lignin peroxidase; Lignin substructure

model compounds; Hydrogen peroxide (Hirai, H. (246) 19)

Oxidative stress

Francisella tularensis; Quantitative proteomics; Two-dimensional

electrophoresis (Lenco, J. (246) 47)


Lactobacillus gasseri; Lactic acid bacteria; Probiotics (Bruno-

Barcena, J.M. (246) 91)

Parmeliaceae

Lichenized fungi; Galactoglucomannan; Chemical structure; 13C

NMR (Carbonero, E.R. (246) 273)

PCR

Contamination; nifH; Primer (Goto, M. (246) 33)

Aldoxime dehydratase; Nitrile hydratase; Aldoxime-nitrile pathway;

Screening (Kato, Y. (246) 243)

pfeA

Pseudomonas aeruginosa; Enterobactin; Catecholate siderophores;

pirA; TonB-dependent receptors (Ghysels, B. (246) 167)

Phanerochaete sordida YK-624

Lignin peroxidase; Ordered bi-bi ping-pong mechanism; Lignin

substructure model compounds; Hydrogen peroxide (Hirai, H. (246)

19)

3-Phenyl lactic acid

Antifungal activity; Two dimensional gel electrophoresis; Cyclic

dipeptide (Strom, K. (246) 119)

Phosphoketolase

Bifidobacterium; xfp; Strain identification; Detection (Yin, X. (246)

251)

Phylogeny

Serratia; Identification natural isolate; gyrB; Quorum sensing; N-Acyl-

L-homoserine lactone (Van Houdt, R. (246) 265)

pirA


pfeA; TonB-dependent receptors (Ghysels, B. (246) 167)

Pleurotus ostreatus

Differential display; RAPD (Sunagawa, M. (246) 279)

Polymorphism

Gene conversion; rrn operons; Vibrio parahaemolyticus; 16S rRNA


Polynucleotide probes

Fluorescence in situ hybridisation; Tyramide signal amplification

(Zwirglmaier K. (246) 151)

Primer

Contamination; nifH; PCR (Goto, M. (246) 33)

Probe

Enterococcus; rRNA gene; Oligonucleotide; Microarray; Microbial


Probiotics


Oxidative stress; Lactobacillus gasseri; Lactic acid bacteria (Bruno-

Barcena, J.M. (246) 91)

Protein domain

DIM; Bacteria; Transmembrane region; Anti-bacterial drugs (Lai, X.

(246) 87)

Protein secretion

Clostridium acetobutylicum; Interleukin-2; Anti-cancer treatment

(Barbe, S. (246) 67)

Protozoa

Kinetoplastid; RNA stability; Gene expression (Cevallos, A.M. (246)

259)

Pseudomonas aeruginosa

Enterobactin; Catecholate siderophores; pfeA; pirA; TonB-dependent

receptors (Ghysels, B. (246) 167)

Pseudomonas citronellolis


Malate:quinone oxidoreductase; GeranylcoenzymeA carboxylase

(Forster-Fromme, K. (246) 25)

PsrA regulon

Stationary phase (Kojic, M. (246) 175)

Pythium longisporangium

Sporangia; Oogonia; Antheridia; Oospores; ITS region; rRNA (Paul,

B. (246) 207)

Quantitative proteomics

Francisella tularensis; Oxidative stress; Two-dimensionalelectro-

phoresis (Lenco, J. (246) 47)


Quorum sensing

Self-organisation; Chemotaxis; Thiovulum; Microbial pattern

formation; Computer model (Thar, R. (246) 75)

Serratia; Identification natural isolate; gyrB; Phylogeny; N-Acyl-L-

homoserine lactone (Van Houdt, R. (246) 265)

RAPD

Differential display; Pleurotus ostreatus (Sunagawa, M. (246) 279)

RAPD multiplex

Malolactic fermentation; Oenococcus oeni; Strain typification; Sulphur


recA specific PCR

Burkholderia cepacia complex identification; Cystic fibrosis;

Rhizosphere environment (Dalmastri, C. (246) 39)

Rhizobia

Biotin biosynthesis; Rhizobia–legume symbiosis (Guillen-Navarro, K.

(246) 159)

Rhizobia–legume symbiosis

Biotin biosynthesis; Rhizobia (Guillen-Navarro, K. (246) 159)

Rhizosphere environment

Burkholderia cepacia complex identification; recA specific PCR; Cystic

fibrosis (Dalmastri, C. (246) 39)

Ribosomal intergenic spacer

Bioaugmentation; Bromoamine acid; Community dynamics;


RirA

Siderophore; Iron response; Fur (Viguier, C. (246) 235)

RNA stability

Protozoa; Kinetoplastid; Gene expression (Cevallos, A.M. (246) 259)

rrn operons

Gene conversion; Polymorphism; Vibrio parahaemolyticus; 16S rRNA


rRNA

Pythium longisporangium; Sporangia; Oogonia; Antheridia; Oospores;

ITS region (Paul, B. (246) 207)

rRNA gene

Enterococcus; Oligonucleotide; Microarray; Probe; Microbial


Saccharomyces cerevisiae

Sparkling wine; Autolysis; Genetic engineering; Autophagy


Screening

Aldoxime dehydratase; Nitrile hydratase; PCR; Aldoxime-nitrile

pathway (Kato, Y. (246) 243)

Self-organisation

Chemotaxis; Thiovulum; Microbial pattern formation; Quorum


Serratia

Identification natural isolate; gyrB; Phylogeny; Quorum sensing; N-

Acyl-L-homoserine lactone (Van Houdt, R. (246) 265)

Serum proteins

Mycobacterium avium; GPL; TNF-a; Macrophage; Complement

receptor (Irani, V.R. (246) 221)

Siderophore

Iron response; RirA; Fur (Viguier, C. (246) 235)

Sparkling wine

Autolysis; Genetic engineering; Autophagy; Saccharomyces cerevisiae


Sphingomonas xenophaga

Bioaugmentation; Bromoamine acid; Community dynamics;

Ribosomal intergenic spacer (Qu, Y. (246) 143)

Sporangia

Pythium longisporangium; Oogonia; Antheridia; Oospores; ITS region;

rRNA (Paul, B. (246) 207)

16S rRNA

Aeromonas; DGGE; gyrB (Tacao, M. (246) 11)

Gene conversion; Polymorphism; rrn operons; Vibrio parahaemolyticus


Staphylococcus aureus

Collagen adhesion gene (cna); Fibronectin-binding protein;

Bacterial adhesion; Biomaterial-associated infections (Arciola,

C.R. (246) 81)

Enterotoxin; Multiplex PCR; Genotyping; Mobile genetic elements

(Omoe, K. (246) 191)

Stationary phase

PsrA regulon (Kojic, M. (246) 175)

STEC

Escherichia coli; stx2; VT2 verotoxin; VTECWater (Garcıa-Aljaro, C.

(246) 55)

Strain identification

Bifidobacterium; Phosphoketolase; xfp; Detection (Yin, X. (246) 251)

Strain typification

Malolactic fermentation; Oenococcus oeni; RAPD multiplex; Sulphur


stx2Escherichia coli; STEC; VT2 verotoxin; VTECWater (Garcıa-Aljaro,

C. (246) 55)

Sulphur dioxide

Malolactic fermentation; Oenococcus oeni; RAPD multiplex; Strain

typification; Wine (Reguant, C. (246) 111)

Surface layer proteins

Clostridium difficile; Diarrhoea; Hamster model (O�Brien, J.B. (246)199)

Subject Index Volume 246 293

Thiovulum

Self-organisation; Chemotaxis; Microbial pattern formation; Quorum


TNF-aMycobacterium avium; GPL; Macrophage; Complement receptor;

Serum proteins (Irani, V.R. (246) 221)

TonB-dependent receptors


pfeA; pirA (Ghysels, B. (246) 167)

Transmembrane region

DIM; Protein domain; Bacteria; Anti-bacterial drugs (Lai, X. (246) 87)

Two dimensional gel electrophoresis

Antifungal activity; 3-Phenyl lactic acid; Cyclic dipeptide (Strom, K.

(246) 119)

Two-dimensional electrophoresis

Francisella tularensis; Oxidative stress; Quantitative proteomics

(Lenco, J. (246) 47)

Tyramide signal amplification

Fluorescence in situ hybridisation; Polynucleotide probes (Zwirglmaier

K. (246) 151)

Vibrio parahaemolyticus

Gene conversion; Polymorphism; rrn operons; 16S rRNA (Gonzalez-

Escalona, N. (246) 213)

VTECWater

Escherichia coli; STEC; stx2; VT2 verotoxin (Garcıa-Aljaro, C. (246)

55)

VT2 verotoxin

Escherichia coli; STEC; stx2; VTECWater (Garcıa-Aljaro, C. (246) 55)

Wine

Malolactic fermentation; Oenococcus oeni; RAPD multiplex; Strain

typification; Sulphur dioxide (Reguant, C. (246) 111)

xfp

Bifidobacterium; Phosphoketolase; Strain identification; Detection

(Yin, X. (246) 251)


Novel target genes of PsrA transcriptional regulator of Pseudomonas aeruginosa

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