222-Supplement_1-29.pdf - Oxford Academic

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MICROBIOLOGICAL ANALYSIS OF MUCOSA ANDFAECES OF WEANED PIGLETS FED WITH PROBI-OTICS LACTOBACILLUS GASSERI LF221 AND K7

B. Bogovic› Matijas›ic¤, S. Stojkovic¤, I. Rogelj

University of Ljubljana, Biotechnical Faculty, Chair ofDairy Science, Groblje 3, 1230 Domz›ale, Slovenia

In in vivo study on 24 weaned piglets (8 per group), thesurvival rate of human isolates Lactobacillus gasseri K7and LF221 was quanti¢ed by selective enumeration onMRS agar with rifampicin, and the presence of bothstrains in intestinal mucosa was examined. Faeces fromindividual animals during 2-weeks probiotic applicationperiod (5x1010 cfu of individual strains/day) and 1 weekafter the probiotic addition had been ceased, was analysedfor the number (cfu/g) of coliforms, lactic acid bacteria,clostridia and either of two probiotic strains. The samplesof duodenum, jejunum and ileum of sacri¢ed animals (5thor 21st day) were microbiologically examined as well. Agreat variability in the micro£ora of faeces and mucosawas observed even between equally treated animals. Thecounts on MRS agar were signi¢cantly higher in the K7and LF221 groups than in the control group at 9 daysonly. The survival of both strains was good (2,5x105 to3,3x105 cfu of K7/g faeces ; 4,5x105 to 5x105 cfu of LF221/g). In two animals, the LF221/K7 viable cells were foundin the faeces 6 days after ceasing of probiotic application.In both animals from the group fed with K7 that weresacri¢ed 5 days after weaning, the presence of K7 wasfound either in the mucosa of duodenum (140 cfu/10cm2) and jejunum (170 cfu/10 cm2) or in the ileum (1600cfu/10 cm2). LF221 cells were detected in the ileal mucosaof one piglet (820 cfu/10 cm2). The probiotic strains werenot detected in mucosa of the animals sacri¢ced after threeweeks.

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GI-TRACT SPECIFIC GENE EXPRESSION IN LAC-TOBACILLUS PLANTARUM

P. A. Bron, D. Molenaar, S. M. Ho¡er, W. M. de Vos, andM. Kleerebezem

Wageningen Centre for Food Sciences; NIZO food re-search, P.O. Box 20, 6710 BA Ede, The Netherlands

Lactobacillus plantarum is a lactic acid bacterium thatafter ingestion reaches the small intestine in an activeform and can persist in the GI-tract for more than 7days. Moreover, this bacterium has been shown to possesmucosal adherence properties. The genes and regulationmechanisms involved in these characteristics are highlyrelevant in view of the utilization of L. plantarum as adelivery vehicle for health promoting factors in the GI-tract of the consumer. The work presented here aims atidenti¢cation of L. plantarum promoter elements that arespeci¢cally activated in the GI-tract, which will generateinsight in in situ behavior of this bacterium and will allowthe investigation of the underlying regulatory mechanisms.The availability of the complete genome sequence of L.plantarum WCFS1 is of critical importance for e⁄cientinterpretation and evaluation of the experimental data.A genetic screen based on complementation of the essen-tial gene alr, encoding alanine racemase, was developedand led to the identi¢cation of L. plantarum promoterelements that are speci¢cally activated by bile acids invitro. Moreover, DNA micro-array analysis was used tofurther unravel the bile acid response of this organism.Resolvase-based in vivo expression technology (R-IVET)is a powerful method that has mainly been used for theidenti¢cation of up-regulated genes of pathogens duringinfection. R-IVET was implemented and employed in L.plantarum to identify GI-tract activated promoter ele-ments, using a mouse model. The promoters and genesidenti¢ed using this strategy and their functional analysiswill be presented.

0378-1097 @ 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

FEMSLE Congress 2-6-03

1st FEMS Congress / Afternoon Sessions and Seminars 29^102

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BIODIVERSITY OF THE PIGMENTED MICRO-FLORA ISOLATED FROM THE RIND OF FRENCHRED-SMEAR RIPENED SOFT CHEESES

L. Dufosse¤, P. Galaup

Universite¤ de Bretagne Occidentale, Laboratoire de Micro-biologie Applique¤e, LUMAQ EA 2651, Po“le de Cre¤ac’hGwen, F-29000 Quimper, France

Red-smear ripened soft cheeses are popular dairy productsin Europe. Among these are french cheeses such as Mar-oilles, Livarot, Epoisses..., german ones (e.g. Tilsit, Lim-burger...), italian (Taleggio) or belgium (Herve) cheeses.Most of them are protected by the European Unionthrough the PDO (Protected Designation of Origin) sys-tem which covers the term used to describe foodstu¡swhich are produced, processed and prepared in a givengeographical area using recognised know-how. The redsmear of these cheeses originates in the synthesis of pig-ments by bacteria, however research e¡orts have to beconducted in order to improve our knowledge on the pig-mented micro£ora. The work from our lab focused on: a)objective measurement of the colour of various cheeserinds, b) isolation and characterisation of the pigmentedbacteria, c) spectrocolorimetric measurements on singlestrains, d) HPLC analysis of pigmented extracts. The bac-terial genera and species active in this dairy process arenot yet really known and described, except Brevibacteriumlinens which is the only bacteria sold for color develop-ment of cheese rind. Other genera such as Arthrobacter,Corynebacterium, Micrococcus were found in our experi-ment and may have an impact. Moreover, in our study,each PDO cheese seem to have speci¢c ratios of unpig-mented / yellow / orange strains.

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MODELLING INACTIVATION AND RESISTANCEOF THE BACTERIOCIN-SENSITIVE LISTERIA IN-NOCUA LMG 13568 STRAIN WHEN GROWN IN AS-SOCIATION WITH THE BACTERIOCIN-PRODUC-ING LACTOBACILLUS SAKEI CTC 494 STRAIN

F. Leroy, K. Lievens and L. De Vuyst

Research Group of Industrial Microbiology, FermentationTechnology and Downstream Processing (IMDO), Depart-ment of Applied Biological Sciences, Vrije Universiteit Brus-sel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium

The use of bacteriocin-producing lactic acid bacteriumstrains as novel, functional starter cultures for the foodfermentation industry o¡ers an attractive way of natural

food preservation, in particular to combat Listeria mono-cytogenes. Bacteriocin (sakacin K) production by Lactoba-cillus sakei CTC 494 under sausage-making conditions waspromising and in situ experiments have con¢rmed its ef-fectiveness. However, its antibacterial e⁄ciency may becompromised since bacteriocin resistance of Listeria hasbeen observed. Therefore, the interaction of Lb. sakeiCT 494 and Listeria innocua LMG 13568 were studiedand mathematically modelled. Bacteriocin production byLb. sakei CTC 494 resulted in the rapid inactivation of L.innocua LMG 13568 from an initial population of 107

CFU ml-1 to a resistant population of 103-104 CFU ml-1,when both strains were grown in association. Even verylow activities of bacteriocin resulted in rapid inactivation,indicating that low numbers of Listeria can be easily killedwithout optimisation of bacteriocin production. It is how-ever critical that environmental conditions prevail in thefood which prevent resistant Listeria growing out. At highpH values (pH 6.5), resistant L. inncoua LMG 13568 wereable to grow to high numbers, whereas at sausage fermen-tation conditions of low pH (pH 5.2) and typical salt andnitrite levels, their growth was strongly inhibited. No dif-ference was observed between the growth rates of resistantL. innocua LMG 13568 cells and the total population. Inconclusion, antibacterial action of the sakacin producerLb. sakei CTC 494 should be su⁄cient to eliminate com-mon initial contaminations of Listeria in the early stagesof sausage fermentation, without subsequent regrowth ofresistant target cells.

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DEVELOPMENT OF POLYETHYLENE FILMS FORFOOD PACKAGING ACTIVATED WITH A FOOD-GRADE ANTILISTERIAL BACTERIOCIN

G. Mauriello, A. Casaburi and F. Villani

Dipartimento di Scienza degli alimenti, Universita' di NapoliFederico II, via Universita' 100, 80055 Portici, Napoli

Food packaging can be de¢ned ‘‘functional packaging’’when a material and/or an accessory are used to improvethe generic food protection of traditional food packaging.In the active packaging there is an interaction betweenpackage and food, implying a gas modi¢cation of head-space and/or a release of antimicrobial, antioxidant orother substances that improve food quality. Therefore,the ¢nal purposes of functional packaging are long timeprotection and safety of food as well as increase of appre-ciation and satisfaction of the consumers. Research onactive packaging is already aiming the development of¢lms capable to exercise an antimicrobial e¡ect on foodand drinks. In this study the antimicrobial e¡ect of activepolyethylene ¢lms was evaluated against Listeria monocy-togenes. The ¢lm was made active by using the anti-liste-



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rial bacteriocin 32Y by Lactobacillus curvatus with threedi¡erent procedures: soaking, coating and incorporation.While the soaking and coating treatments led to anti-lis-terial activity, no growth inhibition of the indicator strainListeria monocytogenes OH was observed when the bacter-iocin was incorporated in the polyethylene ¢lm. The anti-microbial activity of the soaked and coated ¢lms was alsotested in experiments of food packaging involving porksteak and ground beef contaminated by Listeria monocy-togenes OH at roughly103 cfu per cm2 and gram, respec-tively. The results of the challenge tests showed the highestantimicrobial activity after 24 hours at 4‡C, with a de-crease of about 1 Log of the Listeria monocytogenes pop-ulation. Finally, experiments of migration of the coatedbacteriocin in water were also performed resulting in aslow release of the antimicrobial substance.

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ADHERENCE, ERYTHROCYTE AGGLUTINATIONAND IMMUNE MODULATION PROPERTIES OFLACTOBACILLI

N. O. Yelinska, N. V. Kuryata, V. O. Ivanitsa

Odessa National University, Microbiology and Virology De-partment, Dvoryanskaya St., 2, Odessa, 65 026, Ukraine

Dairy products and probiotic preparations containing lac-tobacilli play the important role in contemporary immu-norehabilitation concept. Thus the investigation of adher-ence, erythrocyte agglutination and immune modulationproperties of lactobacilli is actual. Also the in£uence ofdi¡erent factors on the surface structures of lactobacilliis important. It has been shown that among 63 lactobacillistrains 14,3 % had high, 66,7 % ^ moderate and 19,1 % ^law adherence ability. At the same time, only 11,9 % ofthese strains had high erythrocyte agglutination ability,14,9 % had moderate, 10,5 % ^ law agglutination ability,and 62,3 % of strains did not have this ability. The studyof several vitamins and carbohydrates on the adherenceand erythrocyte agglutination ability of lactobacilli hasshown that di¡erent receptors are responsible for thesetwo processes. The in£uence of pepsin, trypsin and chemo-trypsin on the adherence ability of lactobacilli has beenstudied. It has been shown that pepsin in£uenced thisproperty most of all : it decreased the adherence abilityof 80,0 % of the investigated strains. The e¡ect of per osintake of alive lactobacilli strains on the functional activityof macrophages and delayed hypersensitivity in mice hasbeen studied. It has been found that lactobacilli make apositive e¡ect on the cell-mediated non-speci¢c immunityand do not increase speci¢c immune reaction intensity.Thus, the strains resistant to the di¡erent medium factorswere selected. Positive e¡ect of per os intake of lactobacillion the non-speci¢c and speci¢c immunity has been shown.

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CLONING AND CHARACTERIZATION OF THESCRK GENE ENCODING A FRUCTOKINASEFROM BIFIDOBACTERIUM LONGUM A10C

C. I. Caescu(1,2), O. Vidal(1), F. Krzewinski(1), E.A|«ssi(1), C. Brassart(1), V. Artenie(2) and S. Bouque-let(1)

(1) Laboratoire de Chimie Biologique UMR 8576 duCNRS, Universite¤ des Sciences et Technologies de LilleI,59655Villeneuve d’Ascq, France; (2) Universitatea ‘‘Al. I.Cuza’’, 6600 Iasi, Romania

The members of the genus Bi¢dobacterium are pleomor-phic and obligate anaerobic microorganisms in the Actino-mycetales branch of the high G+C DNA content Gram-positive bacteria. Di¡erent authors have associated thepresence of bi¢dobacteria, as an essential part of the nor-mal intestinal micro£ora, with bene¢cial health e¡ectssuch as prevention of diarrhea, decrease of lactose intol-erance or immunomodulation. Recently, Bi¢dobacteriumlongum genome has been sequenced and bioinformaticanalyses revealed genes and gene clusters involved in thecatabolism of a wide variety of sugars. In this way, theavailability of carbohydrates that escape metabolism andadsorption in the small intestine would have a major in-£uence on the micro£ora established in the colon. If cer-tain carbohydrates, such as fructooligosaccharides (FOS)are fermented only by speci¢c bacterial genera like Bi¢do-bacterium, then diets containing so-called ‘‘prebiotic’’ sub-strates could select for those probiotic species. The abilityof several Bi¢dobacterium longum strains to ferment fruc-tose and FOS was assessed in vitro. Anion exchange chro-matography data indicates that the key enzyme of fructosemetabolism would be a fructokinase. The analysis of Bi¢-dobacterium longum genome revealed the presence of onesingle gene, scrK, encoding a putative fructokinase(E.C.2.7.1.4.). Speci¢c primers were de¢ned and used toamplify and clone a 1 kb DNA fragment containing theentire coding sequence of the gene. ScrK was then ex-pressed in Escherichia coli from a pET28 vector with anattached N-terminal histidine tag. The expressed enzymewas puri¢ed by a⁄nity chromatography on Co2+-agaroseto apparent homogeneity and its biochemical pro¢le wasinvestigated. The results corroborate that a L-D-fructose-6-phosphotransferase (fructokinase) has been cloned andcharacterized.


1st FEMS Congress / Afternoon Sessions and Seminars 29^102 31

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A2^2

THE OCCURRENCE OF MALOLACTIC FERMENTA-TION IN BRANDY BASE WINE AND ITS INFLU-ENCE ON BRANDY QUALITY

H. W. du Plessis(1,3), M. G. Lambrechts(1,4), C. L. C.Steger(1,4), L. M. T. Dicks(2), I. S. Pretorius(1) and M.du Toit(1)

(1) Institute for Wine Biotechnology, Department of Viti-culture and Oenology and (2) Department of Microbiology,Stellenbosch University, Stellenbosch ZA-7600; (3) ARCInfruitec-Nietvoorbij, Wine and Fermentation Technology,Private Bag X5026, Stellenbosch, 7599, and (4) Distell,Adam Tas Road, Private Bag 184, Stellenbosch, 7599,South Africa

In this study we determined the extent to which lactic acidbacteria (LAB) occurred in brandy base wines, their abilityto catalyse the malolactic fermentation (MLF) and thee¡ect of MLF on the quality of the base wine and thebrandy distillate. Lactic acid bacteria were isolated andenumerated from grape juice, experimental and commer-cially produced brandy base wines. Spontaneous MLFoccurred in approximately 50% of the commercial basewines. In samples where MLF occurred there was a lossof fruitiness and in the intensity of aroma. Volatile com-pounds like iso-amyl acetate, ethyl acetate, ethyl caproate,2-phenethyl acetate and hexyl acetate decreased in sampleshaving undergone MLF, while ethyl lactate, acetic acidand diethyl succinate increased in the same samples. Spon-taneous malolactic fermentation does occur in commercialbrandy base wines and it has an in£uence on base wineand brandy quality. The strains were identi¢ed using nu-merical analysis of total soluble cell protein patterns, 16SrRNA sequence analyses and PCR using species-speci¢cprimers. The presence of the Lactobacillus spp. could becorrelated to the decrease in quality of the base wine anddistillate, while O oeni strains were found to have a morefavourable in£uence on the quality of base wine and dis-tillates. These results shed some light on the ecology andoenological in£uence of LAB on the quality of South Afri-can brandy. This study showed that MLF in£uences thequality of the base wine and the resulting distillate andwith this in mind commercial base wine producers shouldbe able to produce brandy of higher quality.

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A COUPLED EFFECT OF IRON AND OXYGEN ONTHE CITRIC ACID PRODUCTION OF YEAST YAR-ROWIA LIPOLYTICA

S. V. Kamzolova, I. G. Morgunov

G.K. Skryabin Institute of Biochemistry and Physiology ofMicroorganisms, Russian Academy of Sciences, pr-t Nauki5, Pushchino, Moscow region, Russia 142290

The results presented in this study on a coupled e¡ect ofiron and oxygen on citric acid production argue againstthe opinion of high oxygen requirement for citric acidsyntheses. The application of iron-enriched media makesit possible to cultivate citrate producers at relatively lowoxygen concentrations. The ¢nding is special importance,since oxygen supply is generally a limiting factor in indus-trial aerobic processes. The developed process for citricacid production by a batch culture of Y. lipolytica N1grown on ethanol at a low pO2 value (20% saturation)and at a high iron concentration showed high citric acidaccumulation of 120 g/l, the speci¢c rate of citric acidsynthesis of 120 mg citric acid/gcellsh; the mass yield co-e⁄cient of 0.87 and the energy yield of 0.31.

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ISOLATION AND CHARACTERIZATION OF BAC-TERIAL FLORA FROM YUGOSLAV FARMHOUSEFERMENTED MILK PRODUCTS

B. A. Martinovic(1), Z. Radulovic(2), A. Wind(3), D.Radin(2), D. Obradovic(2), N. Adzic(1)

(1) Biotechnical Institute- Podgorica. Department for ani-mal husbandry, Trg Kralja Nikole bb, 81 000 Podgorica,Yugoslavia; (2) Agricultural Faculty, Dep. of industrialmicrobiology, 11000 Belgrade, Yugoslavia; (3) Chr. Han-sen A/S, Dep. of Appl. Biotechnology, Hoersholm, Denmark

The natural community of lactic acid bacteria isolatedfrom farmhouse fermented milk products has been inves-tigated. In such products where no starters are added,fermentation occurs as a result of natural £ora presentin the surrounding environment. A total of 200 isolatedstrains were examined for their acidi¢cation activity andEPS formation. After further selection, strains were iden-ti¢ed by 16S rRNA sequencing in order to achieve iden-ti¢cation at species level. Out of 200 isolated strains, 15strains belong to Lactococcus lactis subsp. lactis. To di¡er-entiate between strains the pulsed-¢eld gel electrophoresis(PFGE) patterns, of 15 isolated lactococcal strains, wasgenerated using SmaI or AscI. Unrelated strains yieldeddi¡erent patterns of digestion products.The plasmid isola-



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tion of these strains has also been conducted in order tocompare these results to patterns of PFGE. Phage typingof the Lactococcus sp. strains has been conducted. AllLactococcal strains were resistant against 41 phages(Chr. Hansen phage collection) representing the majorphage groups known for Lactococcus. These results indi-cate that the strains represent a possible tool for culturesthat have not been exposed to any industrial selection.

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MOLECULAR CHARACTERISATION OF A NEWTYPE OF KILLER YEAST ISOLATED FROM TOKAJWINE REGION

K. Perka¤tai(1), J. Szamos(2), A. Mara¤z(1)

(1) Szent Istva¤n University, Department of Microbiologyand Biotechnology, 1118 Budapest, Somlo¤i u¤t 14-16; (2)Central Food Research Institute, 1022 Budapest, HermanO. u¤t 15

Killer toxins (zymocins), produced by several Saccharomy-ces sensu stricto strains, have a limited range of activityand show e¡ect only at low pH and below 23‡C. The killerproperty of these strains is mostly encoded by cytoplasmicdsRNA viral nucleic acids. The viral genome has twodsRNA segments denoted L- (large) and M- (medium).The L-dsRNA encodes the RNA-dependent RNA poly-merase and the capsid protein. The M-dsRNA is respon-sible for the production of toxin and the immunity pro-tein. Based on the characteristics of toxin produced andkiller genes, three types of killer yeasts have been di¡er-entiated till now: K1, K2 and K28. We studied the killeryeast biota of Tokaj Wine Region in Hungary and isolateda number of killer wine yeast strains. Minor di¡erenceswere found in the length of M-dsRNA molecules har-boured by the strains, which produced di¡erent or eventhe same type of toxins. Our aim was to study the back-ground of the M-dsRNA polymorphism by moleculartechniques. We determined cross-sensitivity/resistance of13 killer wine yeast isolates against the K1, K2 and K28reference strains. No strain belonged to the K1 group, 6strains proved to be K2 killer-type and 3 strains belongedto K28. The other 4 strains were sensitive to all threekiller-types but were resistant to each other’s toxins, indi-cating that they probably belonged to a new killer-type.We designed primers to selected regions of the M-dsRNAmolecules of K1-, K2- and K28-type strains and preparedcDNA by RT-PCR. Killer wine yeast isolates were furthercharacterised by molecular hybridisation with speci¢cprobes of K1, K2 and K28 cDNA. Our results indicatedthat the toxin types are in close correlation with the tar-geted sequences of the M-dsRNA molecules. We did notget, however, hybridisation signals in the case of the newtype of killer strains. Characterisation of this new type of

toxin by SDS-PAGE and isoelectric focusing revealed dif-ferences in the mobility of proteins.

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A ROLE FOR AQUAPORINS IN FREEZE TOLER-ANCE REVEALED BY GENOME-WIDE GENE EX-PRESSION ANALYSIS IN BAKER’S YEAST

A. Tanghe(1), P. Van Dijck(1), A. Teunissen(2), S. Hoh-mann(3), J. Thevelein(1)

(1) Laboratory of Molecular Cell Biology, Katholieke Uni-versiteit Leuven and Department of Molecular Microbiol-ogy, Flemish Interuniversity Institute of Biotechnology, Kas-teelpark Arenberg 31, 3001 Leuven-Heverlee, Belgium; (2)Department of Pharmacology, ErasmusMC, Box 1738,3000 DR-Rotterdam, The Netherlands; (3) Departmentof Cell and Molecular Biology, Go«teborg University, Box462, S-40530 Go«teborg, Sweden

Although much correlative evidence is available, the pre-cise mechanisms and determinants of freeze resistance inSaccharomyces cerevisiae are largely unknown. Genome-wide gene expression analyses of freeze-resistant andfreeze-sensitive strains have revealed a correlation betweenfreeze resistance and expression of the aquaporin encodinggenes AQY1 and AQY2. This relationship was con¢rmedby deletion and overexpression of those genes, reducingand enhancing yeast freeze tolerance, respectively. Similare¡ects were obtained in Schizosaccharomyces pombe andCandida albicans, as well as Nicotiana tabacum cells.Moreover, heterologous expression of the human aqua-porin encoding gene hAQP1 but not the poorly functionalhAQP1-A73M allele, enhanced yeast freeze tolerance, sup-porting a role for plasma membrane water transport ac-tivity in determination of yeast freeze tolerance. We sug-gest that rapid osmotically driven e¥ux of water duringthe freezing process reduces intracellular ice crystal forma-tion and resulting cell damage. Aquaporin overexpressionalso improved maintenance of yeast viability of industrialstrains, both in liquid cultures and in small doughs, with-out a¡ecting commercially important characteristics.These results open new perspectives for the successful de-velopment of freeze-resistant baker’s yeast strains for fro-zen dough applications. Unfortunately, the aquaporin-mediated improvement of freeze tolerance seems to be re-stricted to rapid freezing conditions. Further research isrequired to evaluate the putative physiological importanceof aquaporin-mediated freeze resistance in yeast and pos-sibly other microorganisms, as well as its usefulness in thecurrent industrial environment.



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A3^1

AN EN ISO 11290-1-COMPATIBLE PCR-BASEDMETHOD FOR THE QUALITATIVE DETECTIONOF LISTERIA MONOCYTOGENES IN RAW MILK

M. D’Agostino(1), M. Wagner(2), T. Kuchta(3), J. Hoor-far(4) and N. Cook(1)

(1) DEFRA Central Science Laboratory (CSL), SandHutton, York, YO41 1LZ, UK; (2) Institute for Milk Hy-giene, Milk Technology and Food Science, Veterinaerplatz1, Vienna 1210, Austria; (3) Food Research Institute, Bra-tislava, Slovakia; (4) Department of Bacteriology, DanishVeterinary Institute, 27 Bulowsvej, Copenhagen 1790, Den-mark

If analytical laboratories are to use PCR as a screeningmethod for pathogen-contaminated foods, any positive re-sults would have to be con¢rmed by re-analysing the sam-ple using the standard culturing method. This means thatprior to PCR, the sample will have to be enriched asstipulated in the standard. For the detection of Listeriamonocytogenes in milk, the standard method begins witha primary enrichment in half-Fraser’s broth (EN ISO11290-1: 1996). As this broth contains many inhibitorsof PCR, the enriched culture cannot be used directly inthe reaction. To remove inhibitory substances, chemicalextraction and puri¢cation of the DNA can be performed.However, many time-consuming steps are then required. Asimpler approach would to be to employ a secondary en-richment in a PCR-compatible broth, after which the cul-ture can be used directly in the PCR. A PCR-compatiblebroth approach has been used for the detection of L.monocytogenes. To demonstrate its e¡ectiveness, rawmilk taken directly after milking was used, as it was con-sidered that this would contain more inhibitory substancesand more background £ora than other forms of milk e.g.skimmed or pasteurised. Twenty ¢ve ml samples were in-oculated with approximately 2000, 200, and 20 cells ml-1.PCR signals were obtained from all inoculated samples,and from none of the uninoculated samples. This methodcan be performed in less than 48 hours, unlike the stan-dard culture-based method which requires 3 to 5 daysbefore a foodstu¡ can be declared to be L. monocyto-genes-free.

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THE DEVELOPMENT OF NOVEL rRNA BASED OLI-GONUCLEOTIDES FOR SPECIFIC PCR DETEC-TION OF SALMONELLA

D. Ferme, G. Avgus›tin and M. Trkov‰

University of Ljubljana, Biotechnical Faculty, ZootechnicalDepartment, Groblje 3, 1230 Domz›ale, Slovenia. ‰ presentaddress: Ministry of Agriculture, Forestry and Food of Re-public of Slovenia, Dunajska c. 56-58, 1000 Ljubljana, Slov-enia

The development of molecular methods for rapid detec-tion of bacterial pathogens has become an ongoing pro-cess, thriving towards ever faster and more sensitive meth-ods. We report here of the construction of novel sets ofPCR primers, based on 16S and 23S rRNA sequences,that make possible the reliable detection of Salmonella.The primers that were developed on the basis of the small-er rRNA subunit sequences allow the detection of isolatesbelonging to the species S. enterica (as de¢ned by Le Mi-nor and Popo¡, Int. J. Syst. Bacteriol., 1987, 37: 465-468)which accounts for the vast majority of all Salmonellaisolates. The primer pair developed on the basis of thelarger rRNA subunit sequences makes possible the detec-tion of all Salmonella isolates, including those belonging tothe species S. bongori. The developed primers were testedon 78 Salmonella strains, representing 31 di¡erent Salmo-nella serovars and 23 non-Salmonella strains, which wereanalysed as negative controls. These strains were fromnine di¡erent genera of the family Enterobacteriaceae, allclosely related to the genus Salmonella. The increasingincidence of salmonellosis in the industrialised countrieswhich is observed over the past decade and the excessivetime consumption of the traditional microbiological meth-ods for the detection and reliable identi¢cation of Salmo-nella, which may require up to seven days, justi¢es thee¡ort invested into the development of novel moleculartools.

A3^3

DETECTION OF PATHOGENIC FOODBORNE MI-CROORGANISMS USING REAL-TIME-PCR

T. Grewing, K. Ko«rner, K. Rottengatter, M. Hess, M.Krieger

Artus Biotech GmbH, Koenigstrasse 4A, 22767 Hamburg,Germany

The most important bacterial zoonoses in humans likesalmonellosis (caused by Salmonella sp.), listeriosis (Liste-ria monocytogenes, L. ivanovii), as well as infections with



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campylobacter species (Campylobacter jejuni, C. coli, C.pylori), EHEC (entero hemorrhagic Escherichia coli) andYersinia enterocolitica in humans are transmitted by con-taminated food of animal origin. Reliable detection (spec-i¢city) of this kind of pathogens in combination with ahigh sensitivity of the assay system is a major requirementfor clinical diagnostics and therapy of patients as well asfor food quality assurance. The principle of Real-Time-PCR not only combines a highly speci¢c and highly sensi-tive detection of pathogenic microorganism DNA, it isalso a very rapid method. With these advantages ofReal-Time-PCR we developed detection systems forsome of the most important foodborne pathogens. Aninternal control in combination with the high speci¢cityof the detection system e⁄ciently excludes false-negativeand false-positive results. The reliable and rapid detectionof salmonella, listeria, and campylobacter is now possibleusing Real-Time-PCR reagents (artus GmbH) and con-tributes to an improvement in food quality assurance.

A3^4

DETECTION, PREVALENCE AND INACTIVATIONOF FOOD- AND WATERBORNE VIRUSES

G. T. Lamothe(2), R. Meyer(1), T. Putallaz(1), H. Joos-ten(1), J. D. Marugg(1)

(1) Nestle¤ Research Center Lausanne, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland; (2)Nestle¤ Waters Technology Center, F-88805 Vittel, France

Norovirus (NV; formerly known as Norwalk-like virus)and hepatitis A (HAV) play a major role in nonbacterialoutbreaks of acute gastroenteritis and are regarded as themain foodborne viruses. Rotavirus (RV) is the majoragent of severe diarrhoea in children. Detection of entericviruses in food and water has recently become feasible forroutine laboratories by using molecular biological detec-tion methods such as RT-PCR. However, this approachsu¡ers from the drawback that it does not allow to dis-criminate between infectious and non-infectious virus par-ticles. Methods based on membrane ¢ltration and RT-PCR were optimised to investigate for the presence ofNV and RV RNA sequences in bottled and mineralwaters. Preliminary results on the prevalence and persis-tence of NV, RV and HAV in raw materials and on thee¡ect of processing and storage on viability will be pre-sented.

A3^5

APPLICATION OF MULTIPLEX PCR TO DETECTNOVEL ENTEROTOXIN GENES IN STRAINS OFSTAPHYLOCOCCUS AUREUS FROM GOATS,SHEEP, PIGS AND RABBITS

D. S. Smyth(1), P. J. Hartigan(2), W. J. Meaney(3), andC. J. Smyth(1)

(1) Department of Microbiology, Moyne Institute of Pre-ventative Medicine and (2) Department of Physiology,Trinity College, Dublin 2, Ireland; (3) Teagasc, Dairy Pro-duction Research Centre, Moorepark, Fermoy, Republic ofIreland

In the last ¢ve years rapid expansion in the discovery ofnew staphylococcal extracellular toxins has occurred, inparticular superantigens, including novel enterotoxinsand ¢fteen so-called exotoxins (SETs). Superantigenshave immuno-modulatory e¡ects on the hosts T cell pop-ulation, can cause in£ammation emesis, food poisoningand can aggravate endotoxic shock. The role of superan-tigens in mastitis remains to be elucidated. The frequencyof the novel enterotoxin genes remains unknown in strainsof Staphylococcus aureus associated with goats, sheep, pigsand rabbits. The aim of this study is to determine thenovel enterotoxin gene content of strains of S. aureus as-sociated with mastitis from goats, sheep, pigs and rabbits.S. aureus strains from Austria, Italy, Ireland, Denmarkand Norway were analysed by PCR. A number of bovineand human S. aureus isolates were included as controls. Amultiplex PCR was used (S.R.Monday and G.A. Bohach(1999). J Clin Microbiol 37, 3411-3414) to screen for en-terotoxin genes, sea-sej and the gene encoding ToxicShock Syndrome Toxin. A multiplex PCR was designedto facilitate the screening for the more recently describedgenes sek-seq. Variation in enterotoxin gene content wasobserved in the strains tested by multiplex PCR. The re-sults of this study emphasise the need to include the novelgenes in any assay screening for enterotoxin genes andhave implications for food safety.



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A3^6

THE MOLECULAR BASIS FOR THE DEVELOP-MENT OF MULTIPLE ANTIBIOTIC RESISTANCEBY SALMONELLA ENTERICA SEROTYPE VIRCH-OW

H. Solnik and S. Yaron

Department of Food Engineering and Biotechnology, Techn-ion-Israel Institute of Technology, Haifa 32000, Israel

Strains of Salmonella with resistance to antimicrobialdrugs are now spread worldwide. This study was under-taken to trace the generation of multiple antibacterial re-sistant Salmonella enterica serotype Virchow, and to de-termine the role of plasmidial and chromosomalmechanisms in conferring tolerance to antibiotics. Ourmodel is S. Virchow, which has emerged as the prevalentSalmonella serotype in Israel. Like S. Enteritidis, S. Virch-ow is normally associated with poultry products. How-ever, while antibiotic resistance in S. Enteritidis has re-mained very low, S. Virchow strains have acquiredresistance to multiple antibiotic agents. More then 40%of clinical isolates, isolated between 1997-2001, were resis-tant to four antibiotics or more. Signi¢cant increase wasobserved in number of isolates resistant to chloramphen-icol, nalidixic acid, trimethoprim-sulfamethoxazole andampicillin. All the resistant isolates were analyzed for thepresence of Class 1 integrons and plasmids. The role of theAcrAB e¥ux pump and its regulatory systems AcrR, Mar-RA and SoxRS was studied by means of a panel of re-porter plasmids, in which GFP is under the control of oneof the promoters. Less than 20% of the resistant isolatesharbored plasmids, suggesting that chromosomal elementsare more dominant in development of resistance. An in-teresting 2.3kb plasmid was isolated from a strain that wasresistant to four antibiotics, but sensitive to ampicillin.Unexpectedly, transformation of the puri¢ed plasmid toEscherichia coli strains resulted with ampicillin resistantcolonies. Results indicate that susceptible bacteria mayserve as gene reservoirs harboring resistant genes thatmay function in other strains.

A4^1

THE EFFECT OF A 5-DAY TREATMENT OF ENRO-FLOXACIN ON ENTERIC ZOONOTIC BACTERIA INTHE PIG

A. A. Delsol(1), L. Pumbwe(2), L. Piddock(2), E. Lieba-na(3), L. P. Randall(3), M. J. Woodward(3), J. Sunder-land(4), and J. M. Roe1

(1) Division of Farm Animal Science, Department of Clin-ical Veterinary Science, University of Bristol, Langford,BS40 5DU, UK; (2) Department of Infections, Universityof Birmingham, Birmingham, B15 2TT; (3) Department ofBacteriological Diseases, Veterinary Laboratories Agency(Weybridge), Woodham Lane, Addlestone, Surrey, KT133NB, UK; (4) Bristol Centre for Antimicrobial Researchand Evaluation, North Bristol, BS10 5NB, UK

There are concerns that the use of enro£oxacin in livestockproduction is contributing to the increase in £uoroquino-lone resistance in zoonotic bacteria. We report here thee¡ect of a single 5-day enro£oxacin treatment on Campy-lobacter coli and Salmonella Typhimurium DT 104 in a pigmodel. Pigs were used for this study as they represent themost heavily medicated sector in livestock production.Our results show an emergence of resistance in C. coli tonalidixic acid (MIC=128mg/l) and cipro£oxacin, the ho-mologue of enro£oxacin used in human medicine(MICs 4mg/l). Resistant C. coli were isolated up to 35days post-treatment, only in treated pigs. A mutation inthe quinolone resistance-determining region (QRDR) hasbeen identi¢ed in these resistant isolates. We have alsoshown that enro£oxacin treatment positively selects fornalidixic acid resistant S. Typhimurium DT104 (gyrAAsn87) and cyclohexane resistant S. TyphimuriumDT104 (upregulated e¥ux pumps). Mutations in the gy-rase region and upregulation of e¥ux pumps are bothmechanisms associated with reduced sensitivity to £uoro-quinolones, and could act as a ¢rst step towards develop-ing full resistance. We have also measured enro£oxacinlevels in the pig faeces by high performance liquid chro-matography (HPLC), and detected residues up to 3 dayspost treatment. The e¡ects of enro£oxacin on C. coli andS. Typhimurium DT104 outlasted the current withdrawaltime of 10 days. Therefore our study provides direct evi-dence that treated pigs are potentially entering abattoirswith higher numbers of resistant zoonotic bacteria, and inturn increasing the risk of these entering the foodchain.



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A4^2

THE EFFECT OF OZONE ON AEROSOLISED ANDSURFACE ATTACHED BACTERIA AND ITS APPLI-CATION IN THE FOOD INDUSTRY

L. M. Fielding(1), R. A. Bailey(1), A. Young(2), and C.J. Gri⁄th(1)

(1) Food Research and Consultancy Unit, School of Ap-plied Sciences, University of Wales Institute Cardi¡, Col-chester Avenue Campus, Cardi¡, CF23 9XR, UK; (2)Ozone Industries Ltd., unit 2b, Armstrong Mall, SouthwoodSummit Centre, Farnborough, Hampshire, GU14 0NR, UK

It is anticipated that the adoption of the Biocidal ProductsDirective (98/8/EC) within the European Union, and therequirements from agencies such as the Soil Associationfor organic produce, will lead to a reduction in the choiceof commercial disinfectants. Ozone has long been used inthe treatment of drinking water, is among the few disin-fectants not covered by the Directive and is acceptable foruse in the manufacture of organic produce. These facts,along with the concern about bioaerosols, have led to in-creased interest in the use of ozone. A purpose built testchamber has been constructed at UWIC and includes anozone generator, which emits variable, controlled concen-trations of ozone. The e¡ect of 0.1 and 2 ppm ozoneagainst Micrococcus luteus was investigated in aerosolisedform and attached to coupons. There was a signi¢cantdecrease in number of aerosolised bacteria at 2 ppm after20 minutes exposure and at 0.1 ppm no bacteria weredetected after 1 hour. There was also a signi¢cant reduc-tion in the number of organisms on the coupons at 2 ppmbut not at 0.1 ppm. The orientation of the coupons (ver-tical, horizontal, inverted) made little di¡erence to the e⁄-cacy of the ozone. The application of ozone in the foodindustry was assessed in a cheese-making plant. The levelsof Enterobacteriaceae and the aerobic plate count, showeda downward trend with continued use of ozone. When usewas discontinued, however, the levels of both in the envi-ronment and in food samples increased almost immedi-ately.

A4^3

SURVIVAL AND TRANSFER OF FOOD-BORNEPATHOGENIC BACTERIA IN THE ENVIRONMENTOF VEGETABLE CROPS

H. Girardin(1), C. Nguyen-The(1), C. Albagnac(1), C.Glaux(2), N. Dreux(1) and C. E. Morris(2)

(1) UMR 408 Qualite¤ et Se¤curite¤ des Produits d’OrigineVe¤ge¤tale, INRA, Site Agroparc, 84914 Avignon Cedex 9.France; (2) Unite¤ de Pathologie Ve¤ge¤tale, INRA, DomaineSt Maurice, BP 94, 84143 Montfavet Cedex, France

Some food pathogenic bacteria, as Clostridium botulinumand Listeria monocytogenes can be naturally present insoils and organic fertilisers used for vegetable cultivation.To evaluate the risk of contamination of vegetables duringtheir cultivation, we measured the survival in soil andtransfer to plants (parsley) of non pathogenic modelstrains of Clostridium and Listeria (i.e. Clostridium spor-ogenes and Listeria innocua). The two bacteria were arti-¢cially inoculated in composted sewage sludge applied tothe soil as an organic fertiliser. The spores of Clostridiasurvived at least 16 months in soil (decrease of 1.5 logCFU/g within this period) and were found on parsleyleaves during during the entire season (up to 13 weeks).Conversely, Listeria populations rapidly decreased in soil(decrease of 7 log cfu or MPN/g within 13 weeks) andwere detected on parsley leaves as long as the Listeriapopulation in soil remained high (s 3 log cfu/g). Studieson the route of transfer are under way. Survival under¢eld conditions of Listeria populations directly inoculatedon aerial plant surfaces was extremely brief (decrease s 7log cfu or MPN/g within few hours and non detectable,i.e. 6 1 cell/25 g, in less than 48 h). Because Listeria canbe isolated from raw vegetables, it is presumably able topersist better under ¢eld conditions. The persistence ofListeria may have been underestimated. Studies on surviv-al strategies of Listeria populations on aerial plant surfa-ces would contribute to a better understanding of the ecol-ogy of this bacterium in vegetable crop environments.

A4^4

INACTIVATION OF BACTERIAL SPORES BY PRES-SURE ASSISTED HEATING

V. Heinz, A. Ardia, R. Buckow and D. Knorr

Dept. Food Biotechnology & Process Engineering, BerlinUniversity of Technology, Koenigin-Luise-Str.22, D-14195Berlin, Germany

It is well known that high hydrostatic pressure processingat ambient temperatures can produce quality bene¢ts



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when applied as a food pasteurization process. The widelyused heat treatment often results in a loss of typical char-acteritics of the fresh product and the nutritional valuemay be impaired. High pressure treatment can preventthese drawbacks since even at levels in the Gigapascalrange covalent bonds are hardly a¡ected. Main target ofhydrostatic compression in biological cells are membraneassociated enzymatic systems which are crucial for meta-bolic control. In proteins the compressibility is largely re-£ected by changing spatial positions of secondary strucuredomains and the pressure induced exposure of hydropho-bic groups in the interior of the protein to the solvent.Spore resistance to pressure may be related to a numberof factors which were acquired at di¡erent stages duringsporulation. Core dehydration, impermeability of coatlayers and membranes, and the presence of SASPs areconsidered to be general components of passive protectionmechanisms. Hence, no signi¢cant spore kill is observed atambient temperatures even at 1000 MPa. In combinationwith mild heat (T " 50‡C) this situation is completelychanged. Using adiabatic heating which occurs duringcompression of aqueous media (" 4 K/100MPa) sporesare irreversibly inactivated. The combined action of tem-perature and pressure allows to keep the sterilization tem-peratures about 30 K below the conventional autoclavingregimes. Additionally, it is advantageous that the volumet-ric temperature shift (during compression and decompres-sion) produces volumetric heating and cooling indepen-dent of product geometry and dimensions. Inactivationresults of Bacillus stearothermophilus and Alicyclobacillusacidoterrestris will be presented in a pressure range up to1500 MPa.

A4^5

ENTOMOPATHOGENIC FUNGI AS BIOCONTROLAGENTS: A MODEL APPROACH TO GENERATEDATA FOR A SUCCESSFUL RISK ASSESSMENTUNDER FIELD CONDITIONS

T. Laengle(1), T. Bauer(2), J. Ra¡alt(2), B. Pernfuss(1),H. Strasser(1)

(1) Institute of Microbiology, Leopold-Franzens UniversityInnsbruck, TechnikerstraUe 25, A-6020 Innsbruck, Austria;(2) ATC-Agro Trial Center GmbH, Am Futterplatz, A-2471 Rohrau, Austria

Entomopathogenic fungi have signi¢cant potential for thedevelopment of biological control agents (BCAs) againstsubterranean insect pests. However, a responsible risk-as-sessment of fungal biological control agents is necessaryfrom an ecological perspective. This should include inves-tigations on the environmental persistence of BCAs, thestudy of the signi¢cance of secreted major metabolites,and their possible hazards to human and animal health.

These data are required for a successful registration in theEuropean Union. Beauveria brongniartii (Ascomycota,anamorph of Clavicipitales) is already used against Melo-lontha melolontha larvae (Coleoptera, Scarabaeidae) inAustria, Italy, Switzerland and France. Due to the expe-riences gained in these countries for almost 20 years Beau-veria is an ideal model organism for the evaluation ofexisting guidelines and the setting up of standard proce-dures. This will help to generate data relevant for riskassessment and registration. In the context of the EU-RTD-project RAFBCA (QLK1-2001-01391) an extensivethree year ¢eld trial to study potential phytotoxic e¡ects ofBeauveria brongniartii has been started on the facilities ofthe GLP-certi¢ed ATC-Agro Trial Center GmbH in Aus-tria. For the trial layout the guidelines of the EPPO, theU.N. FAO, the U.S. EPA, and the European Union werescreened and applied as required in the implementation ofEU-commission directive 91/414. An overview of the legalrequirements for the noti¢cation of fungal BCAs on aEuropean level will be presented. Challenges in the appli-cation of these legal requirements and results of the studywill be discussed, respectively.

A4^6

REDUCING THE POTENTIAL OF MICROBIALRISKS IN NEW ZEALAND SEAFOOD

Q. Shu, G. C. Fletcher, C. M. Osborne and G. J. Lu

The New Zealand Institute for Crop and Food ResearchLimited, Private Bag 92 169, Auckland, New Zealand

Although New Zealand seafood is very safe for consumersto eat, both the government and seafood industry are in-vesting in research to enhance the safe-to-eat reputation ofseafood products. Several pathogenic micro-organismsthat are a risk to the food safety of seafood productsfrom other countries have not been a problem for theNew Zealand seafood industry because extensive researchhas enabled the industry to put appropriate measures (in-cluding guidelines for the safe preparation of seafood inNew Zealand) in place. A new micro-well plate-based‘most probable number’ technique for quantifying poten-tial risk pathogens such as Listeria monocytogenes hasbeen developed. This technique allows processors and reg-ulatory authorities to quantify the extent of L. monocyto-genes contamination in products. Factors to determineand optimize seafood plant hygiene have been identi¢edwhile technologies for e⁄ciently improving the hygiene ofseafood processing plants have been evaluated. The opti-mum pH and concentration of chlorine to dramaticallyimprove the ability to clean bio¢lms containing L. mono-cytogenes from stainless steel benches, for example, arenow well understood. Technologies to control potentialrisk micro-organisms in seafood packing, such as modi¢ed



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atmosphere-packaging technology, have also been devel-oped. Our results demonstrated that well designed pack-aging systems retard the growth of potential pathogenicand spoilage organisms during the period of preservation.Further studies and the application of research outcomesby industry will continue to reduce the potential of micro-bial risks and enhance the safe-to-eat reputation of NewZealand seafood.

A5^1

RECOMBINATION AND SEQUENCE EVOLUTIONWITHIN THE NATURAL POPULATION OF STAPH-YLOCOCCUS AUREUS

J. E. Cooper and E. J. Feil

Department of Biology and Biochemistry, University ofBath, Claverton Down, Bath, BA2 7A7, UK

Staphylococcus aureus is an important opportunistic hu-man pathogen and represents an increasing public-healthburden owing to the spread of drug-resistant clones withinthe hospital environment. A knowledge of the populationstructure, and the impact of recombination on clonal di-versi¢cation, is essential for the development of methodsfor e¡ective disease management both in the hospital set-ting and in the community. Multi-locus sequence typing(MLST) is a powerful typing scheme which can be usedfor local and global epidemiological surveillance, andthese data also provide evidence that recombination isrelatively rare in S. aureus. We have used existing MLSTdata as a framework to sample 25 diverse staphylococcalgenotypes, recovered both from invasive disease andasymptomatic carriage, and have extended the MLSTdata to include approximately 25 extra loci. These locihave been chosen to represent both diverse functionalclasses and physical location on the chromosome, andthese data provide evidence regarding the Phylogeneticconsistency, and relative rates of recombination, aroundthe genome. More speci¢cally, these data examine the sug-gestion that the genome consists of a stable ‘‘core’’ (house-keeping genes) interspersed with less stable genes associ-ated with virulence. Outgroup sequences from all theseloci are available from the genome of S. epidemidis and,because recombination is relatively rare in S. aureus, thereconstruction of a consensus intra-speci¢c phylogenetictree is feasible. Such an analysis is also providing evidenceconcerning the evolutionary relationships between impor-tant clones and the acquisition of speci¢c virulence orantibiotic-resistance genes.

A5^2

16S-23S rDNA SPACER SEQUENCES AS TAXONOM-IC AND DIVERSITY MARKER FOR STUDYING EN-VIRONMENTAL PSEUDOMONAS SPP.

N. Fromin, S. Tarnawski, J. Hamelin, and M. Aragno

Laboratoire de Microbiologie, Universite¤ de Neucha“tel, RueE. Argand 9, CP 2, CH- 2007 Neucha“tel, Switzerland

Direct PCR ampli¢cation of phylogenetically meaningfulDNA fragments are now widely used for microbial diver-sity studies. However, their comparison with standard(cultivation) approaches was often neglected, because ofthe low proportion of culturable bacteria in natural envi-ronments. In this study, the diversity of Pseudomonas insoil and root environment of the perennial grass Moliniacoerulea was assessed by direct ampli¢cation of 16S-23SrDNA spacer (ITS1) from total DNA extract using aPseudomonas-speci¢c PCR protocol [1]. The retrievedDNA fragments were cloned and analysed for the a⁄lia-tion of corresponding organisms to the Pseudomonas ge-nus. The polymorphism of these ITS1 fragments was eval-uated by restriction analysis and representatives weresequenced. At the same time, a similar approach (PCR-RFLP of ITS1) was applied to Pseudomonas strains iso-lated on the mS1 Pseudomonas-selective culture medium.The a⁄liation of corresponding non-cultured / culturedorganisms was achieved by comparison of their ITS1 se-quence, as the taxonomic positionning based on this se-quence was shown to be relevant. The characterization ofPseudomonas diversity con¢rmed that root and soil pop-ulations were di¡erent whatever the approach used. Alarger diversity of ITS1 sequence types was obtained usingthe direct PCR-based approach. We also present someperspectives in the use of ITS1 sequences as taxonomicand diversity markers for Pseudomonas.[1] L. Locatelli et al (2002). Speci¢c PCR ampli¢cation ofthe genus Pseudomonas targeting the 3’ half of 16S rDNAand the whole 16S-23S rDNA spacer. Syst. Appl. Micro-biol. 25: 220-227.



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A5^3

ASSOCIATIONS BETWEEN REPEATS AND THESTRUCTURAL EVOLUTION OF BACTERIAL GE-NOMES

E. P. C. Rocha

Unite¤ GGB, URA 2171, Institut Pasteur & Atelier de Bio-Informatique, Universite¤ Pierre et Marie Curie, 12 rue Cuv-ier, 75005 Paris, France

The structure of bacterial genomes is challenged by recom-bination events involving homologous recombination be-tween inverted repeats. We have analyzed the presence anddistribution of such repeats and their relation to the con-servation of chromosomal structure in 63 bacterial chro-mosomes. We show a strong under-representation of in-verse repeats, relative to direct repeats, in mostchromosomes, especially among the genomes commonlyregarded as most stable. Further, repeats are located inthe chromosomes in such a way that rearrangements pro-duce smaller chromosomal asymmetries and strand com-positional biases disruption than expected. Closely relatedgenomes reported to di¡er in terms of stability are foundto di¡er inversely in the number of inverted repeats. Sincethese results highlight the challenges imposed on the ge-nome structure by the presence of inverted repeats we havetried to measure the impact of repeats in the disruption ofchromosomal synteny by further analysing 17 genomes ofQ-proteobacteria. When controlling for the phylogeny, syn-teny loss correlates very well with the square root of theevolutionary distance. Further, the deviations of the syn-teny loss to the expected values, given evolutionary dis-tances, correlates well with the number of potential syn-teny breakpoints caused by the presence of repeats in thesegenomes. Therefore, repeats should be considered as im-portant actors in the structural change of bacterial chro-mosomes. Since repeats can disrupt the chromosomalstructure but are also under positive selection to producedosage or variation e¡ects (e.g. antigenic variation), theirpresence in genomes is likely to be under stabilizing selec-tion.

A5^4

DO SPECIES EXIST?

R. Rossello¤-Mora

Grup d’Oceanogra¢a Interdisciplinar, Institut Mediterranid’Estudis AvancVats, IMEDEA (CSIC-UIB), C/MiquelMarque¤s 21, E-07190 Esporles, Mallorca, Spain

Do species exist? This is a very di⁄cult question to an-swer! Species exist at least in our minds. Classi¢cations

appear in any kind of society, and responds to the needof the human mind to understand and use order of naturefor its own purposes. The species category is created bytaxonomists to ¢t the recurrent patterns that they observein nature to construct a scienti¢c classi¢cation. The cir-cumscription of the unit is achieved by a concept, and thesuccess in circumscribing units responds to the observatio-nal methods available. Prokaryotic species concept resultsas an empirical construction of what has been thought tobe a unit. We are currently assisting to a discussion aboutif our current conception of species is adequate to theneeds of microbiologists. In this regard, there are manycriticisms to it just because it seems that what we circum-scribe as species is not comparable to what eukaryote tax-onomists do. In this regard it is important to note thatamong eukaryote taxonomies, there is no agreement aboutwhat a species is. Each taxonomy does apply a conceptthat is specially tailored for the organisms under study.Over 22 concepts of species have been conceived but notall of them can universally be applied. Although not per-fect, microbiologists have designed a concept that is ac-ceptable to circumscribe prokaryotic units. Here it will beargued that the species is an arti¢cial construction, and itis not easy to ¢nd a universally applicable concept. Thisimplies that we should be pluralistic when regarding to thebasic taxonomic category.

A5^5

THE SIGNIFICANCE OF CHEMOTAXONOMY INTHE EVALUATION OF THE SYSTEMATICS OFPROKARYOTES

B. J. Tindall

DSMZ-Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH., Mascheroder Weg 1b, D-38124Braunschweig, Germany

In the last two decades more emphasis has been placed onthe evaluation of molecular data (i.e. protein and genesequence data) in order to investigate the evolutionaryrelationships of prokaryotes. This in turn has led to anincreased use of such data in the systematics of prokary-otes. It is also generally accepted that only protein or genesequences can serve as suitable markers to investigate suchevolutionary relationships and that phenotypic data isgenerally unreliable. However, the latter assumption isbased on a misunderstanding of the original work ofZuckerkandl and Pauling, as well as the failure to appre-ciate the signi¢cance of the phenotype (structure and func-tion) in evaluating the evolutionary context of a data set.The chemical composition of the cell is a phenotypic dataset which gives us important information on the evolu-tionary groupings of prokaryotes and provides an interest-



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ing way of testing systematic hypotheses based on theresults of protein and gene sequence analysis.

A5^6

GENE ENCODING PQQ-DEPENDENT ALCOHOLDEHYDROGENASE: A PHYLOGENETIC MARKERAND TARGET FOR SPECIES IDENTIFICATION OFACETIC ACID BACTERIA

J. Trcek(1), K. Matsushita(2), O. Adachi(2) and D. Tre-bar(3)

(1) Limnos, Podlimbarskega 31, Ljubljana, Slovenia; (2)Department of Biological Chemistry, Faculty of Agricul-ture, Yamaguchi University, Yamaguchi, Japan; (3) Kolin-ska, Kolinska ul. 1, Ljubljana, Slovenia

The acetic acid bacteria (AAB) are wide spread microor-ganisms in nature and are involved in many food-produc-ing processes. Molecular identi¢cation approaches havemade possible the description of several new species andgenera of these bacteria in the last years. At present thereare six validly described genera: Acetobacter, Glucono-bacter, Gluconacetobacter, Asaia, Acidomonas and Koza-kia. The phylogenetic a⁄liation of the AAB species hasbeen established on the basis of 16S rRNA genes. How-ever, the pairwise comparison among these sequencesshows very high similarity (91.1 to 99.9 %), which maycause problems when trying to di¡erentiate them amongspecies. In searching a new phylogenetic marker that hasevolved with higher evolutionary rate than rDNA sequen-ces, we analyzed genes encoding PQQ-dependent alcoholdehydrogenase (ADH). The ADH is a membrane-boundcomplex of quinohemoprotein dehydrogenase with a cyto-chrome c subunit and pyrroloquinoline quinone (PQQ) asa prosthetic group. This type of PQQ-dependent ADH hasbeen found only in AAB. In this report, we sequenced apart of the ¢rst subunit of the ADH, originating fromdi¡erent species and genera of AAB. The sequences exhib-it lower similarity (61.4 to 95.1 %) than 16S rDNA, andare showing very similar calculated phylogenetic relation-ships among AAB to those inferred from 16S rRNAgenes. The conserved and variable regions in adh sequen-ces enable the construction of species-speci¢c oligonucleo-tides. We con¢rmed an anticipated speci¢city of con-structed PCR-primer for identi¢cation of A. aceti on 31well-de¢ned strains of AAB. All these depict a gene encod-ing PQQ-dependent ADH as an optional molecularmarker in phylogenetic studies and as a promising targetfor molecular identi¢cation of AAB.

A6^1

METABOLIC DIVERSITY OF ANAEROBIC MODER-ATELY THERMOPHILIC BACTERIA FROM TER-RESTRIAL AND DEEP-SEA VULCANIC HABITATS

E. A. Bonch-Osmolovskaya, M. L. Miroshnichenko, D. G.Zavarzina, T. G. Sokolova and A. I. Slobodkin

Institute of Microbiology, Russian Acvademy of Sciences,117312 Moscow, Russia

Diversity of anaerobic thermophilic bacteria inhabitingterrestrial hot springs of Kamchatka and deep-sea hydro-thermal vents of East Paci¢c Rise was studied by culturalmethods. Molecular hydrogen, acetate or CO were used asthe electron donors, and elemental sulfur, ferric iron, ornitrate as the electron acceptors. As a result, many newmoderately thermophilic taxa were isolated, growing opti-mally at 55-60 ‡C. Lithotrophic sulfur-reducers were rep-resented by Nautilia lithotrophica gen. nov., sp. nov., asso-ciated with polychaeta worm Alvinella pompejana,inhabiting hydrothermal chimneys. The ability of lithotro-phic growth with molecular hydrogen and nitrate wascharacteristic of several other new deep-sea isolates ^ ob-ligately anaerobic Caldithrix abissi gen. nov., sp. nov. andmicroaerophilic Oceanothermus profundus gen. nov., sp.nov. and Vulcanithermus medioatlanticus gen. nov., sp.nov. From terrestrial hot springs of Kamchatka two newthermophilic bacteria were isolated, capable of lthio-trohpic growth with molecular hydrogen as growth sub-strate and ferric iron as the electron acceptor ^ Thermove-nabulum ferriorganovorum gen. nov., sp. nov. and‘‘Thermaecola ferrireducens’’. Latter organism was alsoable to by iron reduction with acetate as the energy sub-strate. Its another capacity was the ability to grow onanaerobically on CO as a sole energy and carbon source,producing molecular hydrogen from water. This reactionwas found to be widely spread between phylogeneticallydiverse thermophilic anaerobes. Among them there is Car-boxydocella thermoautotrophica gen. nov., sp. nov. isolatedfrom Kamchatka, and ‘‘Thermosinus carboxydovorans’’from Yellowstone National Park.



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A6^2

GENUS EUROTIUM ^ HALOPHILIC FUNGAL IN-HABITANTS OF HYPERSALINE WATERS IN THESALTERNS

N. Gunde-Cimerman(1), J. C. Frisvad(2), L. Butinar(3)and P. Zalar(1)

(1) Biology Department, Biotechnical Faculty, Universityof Ljubljana, Vec›na pot 111, 1000 Ljubljana, Slovenia;(2) BioCentrum-DTU, Technical University of Denmark,2800 Kgs. Lyngby, Denmark; (3) National institute ofChemistry, Department of Biotechnology, Hajdrihova 19,1000 Ljubljana, Slovenia

In the course of the mycodiversity study of hypersalineenvironments, di¡erent species of the known food-bornexerophilic genera, such as Wallemia, Penicillium, Aspergil-lus, and its teleomorphic stage, Eurotium as well as halo-philic black yeasts were isolated from the man made salt-erns. Genus Eurotium was represented by ¢ve species: E.amstelodami, E. herbariorum, E. repens, E. rubrum and E.chevalieri. Strains of E. amstelodami were most consis-tently isolated from the Slovenian salterns and later aswell in other salterns (Spain, Israel, Dominican Republic,Namibia), while E. herbariorum, E. repens and E. rubrumwere isolated frequently. Therefore these species probablycontribute to the indigenous fungal community in hyper-saline environments. To determine their halophilic adap-tation to long-term survival in hypersaline environments,spores of E. amstelodami, E. herbariorum, E. repens and E.rubrum were in vitro exposed to prolonged suspensions inwater with di¡erent salt concentrations. They survivedfrom 0-30% NaCl for three months and more. Only E.chevalieri spores did not survive the long-term exposureto NaCl concentrations from 20-30% and were recoveredas well from the hypersaline waters just once. Therefore E.chevalieri is probably a temporal inhabitant of brine atlower salinities.

A6^3

NOVEL ARCHAEAL LINEAGES IN MICROBIAL AS-SEMBLAGES ASSOCIATED WITH ATLANTIC ANDPACIFIC HYDROTHERMAL VENTS

O. G. Nercessian(1), A.-L. Reysenbach(2), S. C. Cary(3),Y. Fouquet(4) and C. Jeanthon(1)

(1) UMR 6539, Centre National de la Recherche Scienti-¢que and Universite¤ de Bretagne Occidentale, Institut Uni-versitaire Europe¤en de la Mer, 29280 Plouzane¤, France; (2)Portland State University, Department of EnvironmentalBiology, Portland, OR 97201, USA; (3) University of Del-aware, Graduate College of Marine Studies, Lewes, Dela-ware 19958, USA; (4) Laboratoire de Ge¤ochimie et Me¤tal-loge¤nie, IFREMER, 29280 Plouzane¤, France

Microbial communities associated with chimneys and sedi-ments from hydrothermally active deep-sea vents of theEast Paci¢c Rise (EPR; 9 and 13‡N) and the Mid-AtlanticRidge (MAR; 36‡N) were studied by the combined use ofcultural and molecular techniques. The archaeal diversityassociated with microbial collectors deployed for di¡erenttimes on geographically distant vent emissions at 13‡Nwas wide. A large percentage of clones were highly similarto known archaeal isolates recovered from similar habi-tats. Among the 24 di¡erent phylotypes identi¢ed, Ther-mococcales-related sequences dominated in all the librariesbut sequences related to members of orders Methanopyr-ales, Methanococcales, Archaeoglobales, and Desulfurococ-cales were also recovered. Consistent with earlier investi-gations, the presence of these phylogenetic groups was alsocon¢rmed in enrichment cultures performed at tempera-tures ranging from 60 to 90‡C. Additional sequenceswith no known cultivated relatives consisted of Marinegroup I Crenarchaeota, Korarchaeota, and Deep-sea Hy-drothermal Vent Euryarchaeota within which a novel lin-eage was identi¢ed (DHVE8). This new lineage was alsodetected in diverse deep-sea hydrothermal vent samplesfrom 9‡N (EPR) and 36‡N (MAR). Other experimentsdemonstrated that the archaeal sequences retrieved fromsediment samples collected at 36‡N (MAR) consisted ex-clusively in new lineages. Our results give new insights onthe diversity and distribution of archaea in hydrothermalvents. They also strongly suggest that spatial and temporalvariations of microbial assemblages occur in this habitat.



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A6^4

METABOLIC AND ECOLOGICAL VERSATILITY OFHALOARCHAEA

T. J. McGenity(1), A. M. Sass(1), H. Lu«nsdorf(2), J. R.Ascott(3), S. Grant(4), H. M. Lappin-Scott(3), W. D.Grant(4), K. N. Timmis(1,2)

(1) Department of Biological Sciences, University of Essex,Wivenhoe Park, Colchester CO4 3SQ, UK; (2) GBF ^National Research Centre for Biotechnology, MascheroderWeg 1, D-38124 Braunschweig, Germany; (3) School ofBiological Sciences, University of Exeter, Hatherly Labora-tory, Prince of Wales Road, Exeter EX4 4PS, UK; (4)Department of Microbiology and Immunology, Universityof Leicester, P.O. Box 138, Leicester LE1 9HN, UK

In the 1970s, halobacteria, now more commonly referredto as haloarchaea, were considered to form a nutritionallylimited group of microorganisms that were restricted tohigh-salt environments. During the 1980s new haloarchaeawere identi¢ed, for example obligate alkaliphiles fromsoda lakes, and in the 1990s cultivation-free methodologiessuggested the existence of additional haloarchaeal taxa,many of which remain uncultivated. Throughout these de-cades it has become apparent that haloarchaea are bothphylogenetically and metabolically diverse, and that indi-vidual species are highly versatile. We will draw on recentexamples to illustrate this. Haloarchaea, together with oth-er halophiles, have been found growing on oil, some havebeen isolated directly from oil-¢eld brines. We are begin-ning to investigate the way halophilic consortia work to-gether to degrade oil. Haloarchaea have been isolatedfrom anoxic environments such as the deep hypersalinebrines found on the £oor of the Mediterranean. We areaddressing the question of whether haloarchaea, whichhave long been thought of as strict or facultative aerobes,are metabolically active under such conditions. Haloarch-aea have been isolated from salt marshes, and whereasmost species require a salt concentration that is three timesgreater than seawater, these isolates grow at or just aboveseawater salinity. We are investigating the adaptationsthat enable these haloarchaea to grow in such ‘low-salt’environments. Finally, we will consider how the ability ofhaloarchaea to degrade oil and grow anaerobically may, inpart, explain their presence in deep subsurface salt depos-its.

A6^5

SALINIBACTER, A GENUS OF RED, EXTREMELYHALOPHILIC BACTERIA: A COMPARISON WITHTHE HALOPHILIC ARCHAEA

A. Oren

Division of Microbial and Molecular Ecology, the Instituteof Life Sciences, and the Moshe Shilo Minerva Center forMarine Biogeochemistry, The Hebrew University of Jerusa-lem, 91904 Jerusalem, Israel

Salinibacter is a genus of red, rod-shaped, extremely hal-ophilic prokaryotes, phylogenetically a⁄liated with theCytophaga/Flavobacterium branch of the bacterial domain.Salinibacter ruber, the type species, was isolated from salt-ern crystallizer ponds in Spain, the environment in whichthe presence of this organism was ¢rst suggested based onsequencing of environmental 16S rDNA clones. Isolates tobe classi¢ed as a new species of Salinibacter have beenobtained from a salt crust in the Badwater area, DeathValley National Park, California. Salinibacter ruber growsoptimally between 20 and 30% salt, and is unable to growbelow 15% salt. Its red pigment (‘‘salinixanthin’’) has beenidenti¢ed as a novel acylated C40-carotenoid glycoside.Salinibacter grows aerobically on amino acids, and in ad-dition metabolizes glycerol and a small number of sugars.Glucose is probably degraded via the Entner-Doudoro¡pathway. Although belonging to the bacterial domain,Salinibacter shares many characteristic properties with ar-chaea of the order Halobacteriales, such as a high require-ment for chloride for growth, accumulation of KCl toprovide osmotic balance, the possession of a large excessof acidic amino acids in its cellular proteins, and the vir-tual absence of organic osmotic solutes. Many of the en-zymes tested depend on high salt concentrations for activ-ity. In view of the metabolic similarity betweenSalinibacter and the members of the Halobacteriales, ex-periments are now in progress to assess the advantagesthat either group may have when colonizing hypersalineenvironments approaching salt saturation.



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A6^6

BIOLOGY OF MODERATELY HALOALKALIPHILICMETHANOTROPHS AND METHYLOBACTERIA

Y. A. Trotsenko, V. N. Khmelenina, B. Ts. Eshinimaev, Ts.D. Darmaeva, N. E. Suzina, A. S. Reshetnikov, N. V. Dor-onina

G.K. Skryabin Institute of Biochemistry and Physiology ofMicroorganisms, Russian Academy of Sciences, Pushchino,Moscow region, 142290, Russia

A wide spectrum of methane- and methanol-utilizing bac-teria has been isolated from saline soda lakes of south-eastern Siberia, Mongolia, Egypt, and USA. They areaerobic moderate (halo)alkaliphiles able to grow optimallyat pH 8.5-9.5 and 0.1-3 % NaCl. Pheno- and genotypicstudies showed that the newly isolated obligate methano-trophs represent the new species of novel genus ‘Methyl-ohalonatronum’ : M. alcaliphilum, M. buryatense andMeth-ylocystis gottschalkii, whereas the methylobacteria wereclassi¢ed as Ancylobacter natronum, Methylophaga alcalicaand Methylophaga natronica. spp. nov. During growth insaline medium these bacteria accumulated the compatiblesolutes: ectoine, glutamate and sucrose. Ectoine wasshown to be a major organic osmoprotectant in the cellsgrown in C-limited saline medium while sucrose prevailedunder N-limitation. In M. alcaliphilum 20Z, ectoine is syn-thesized from aspartate by sequential action of ATP-as-partate kinase, NADPH-aspartyl semialdehyde dehydro-genase, aspartyl semialdehyde transaminase (EctB),diaminobutyrate transacetylase (EctA) and ectoine syn-thase (EctC). The ectABC genes have been characterizedand the deduced protein sequences had 43 ^ 63% identitieswith those of other halophilic bacteria. Remarkably, thecells of all haloalkaliphilic methanotrophs were covered byregularly arrayed macromolecular structures of hexagonal(p6) or linear (p2) symmetry. The cup-shaped structureswere lost during growth of M. alcaliphilum 20Z underCH4 at pH 7.2 without NaCl, thus implying their involve-ment in CH4 trapping or energy coupling in moderatelyhaloalkaliphilic methanotrophs having the enhanced car-bon and energy demands for the osmoprotectant synthesisor ionic homeostasis.

A7^1

NITRIFICATION AND NITRIFYING BACTERIA INTHE LOWER SEINE RIVER ESTUARY

A. Ce¤bron, T. Berthe and J. Garnier

UMR 7619 Sisyphe CNRS, UPMC, BP 123, Tour 26, Et-age 5, 4 place Jussieu, 75005 Paris, France

The Ache'res wastewater treatment plant (WWTP), locatedjust downstream from Paris, discharges its treated e¥uentsinto the lower Seine river. These bring a large quantity ofheterotrophic bacteria, organic matter and ammonium,and are responsible for a seeding of nitrifying bacteria.As a result, degradation of organic matter by heterotro-phic bacteria and subsequent oxygen depletion occur im-mediately downstream of the e¥uent outlet, whereas ni-trifying bacteria need to build up a signi¢cant biomass,before ammonium oxidation signi¢cantly depletes the oxy-gen downstream. During summer, the maximum nitrifyingactivity indeed occurs in the upstream estuary, about 200km downstream from the WWTP. In order to betterunderstand the nitri¢cation process, we quanti¢ed the po-tential nitrifying activity and the two steps of the nitri¢-cation process, ammonia- and nitrite-oxidation, along thatriver sector and analysed the associated bacterial popula-tions. The quantity of nitrifying bacteria (number of amoAgene copy and of Nitrobacter cells) determined by compet-itive PCR correlated with the potential nitrifying activity.The species composition of ammonia-oxidizing bacteriawas investigated at Triel station just downstream fromAche'res (Km 84) and in the Seine estuary at Duclair(Km 278). Using PCR primers targeting amoA, phyloge-netic analysis revealed that the majority of the clones atboth sites were a⁄liated to nitrosomonads: Nitrosomonasmarina-like bacteria (including N. urea and N. oligotropha)represented 81% and 60% of the population of ammonia-oxidizing bacteria at the Triel and Duclair stations, respec-tively. The other two clusters, the Nitrosomonas europaea-like bacteria and Nitrosospira were in lesser proportion butthe percentage of Nitrosospira-like clones was higher atDuclair (23%) than at Triel (6%). Some strains seemedto be adapted to both sites studied. Nevertheless, the bac-terial community appeared to be composed by speci¢cclones not found at both stations; indeed 12.5% of theTriel clones, were closely related to Nitrosomonas euro-paea-like strains isolated from freshwater environment,but not found at Duclair station where speci¢c clonesclosely related to Nitrosomonas europaea-like strains wereisolated from bio¢lm of WWTP. We conclude that a se-lection or/and an adaptation of the ammonia-oxidizingbacterial population would occur along the Seine Rivercontinuum, from Paris to the estuary.



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A7^2

RHIZOPLANE DIVERSITY OF NITRITE REDUC-TASE IN UPLAND PASTURE

T. J. Daniell, J. Squires and N. Nunan

Plant Soil Interactions, Scottish Crop Research Institute,Invergowrie, Dundee, DD2 5DA, Scotland, UK

Denitri¢cation is an anaerobic process in which nitrogencompounds are utilised as alternative electron acceptorsfor respiration. Incomplete denitri¢cation can lead to therelease of nitrogen oxide gases which are potent green-house gases. Denitri¢cation is widespread in prokaryoticsystems with a wide range of bacterial and archaeal speciescapable of the process. Nitrite reductase is a key enzyme indenitri¢cation. We have analysed the diversity of a nitritereductase (NirK) gene in the rhizoplane of a range of up-land grasses sampled at the MICRONET ¢eld site in theScottish Borders. DNA extracted directly from root frag-ments, collected from the organic horizon was utilised. Afragment of the NirK gene was ampli¢ed by PCR, clonedand around 3360 clones analysed via a high throughputsequencing programme and, following screening, over2600 true clones remained for analysis. Rarefaction anal-ysis demonstrates that with the exception of single clones(each representing circa 0.04% of the population) the levelof throughput utilised in this analysis has exhausted typespresent in this system. The diversity of types present oneach grass species was relatively uniform with the rhizo-plane communities being heavily dominated by a singlesequence type (representing approximately 75% of the to-tal community). However, despite this dominance acrossgrasses, principal co-ordinate analysis demonstrates selec-tivity within this system. There was separation of sequencetypes between the two dominant genera of grasses, Agro-stis and Festuca.

A7^3

THE ROOTS OF MOLINIA COERULEA HARBOURUNCULTURABLE BACTERIA ACTIVE IN DINITRO-GEN FIXATION UNDER NORMAL AND ELEVATEDATMOSPHERIC CO2

J. Hamelin, M. Jossi, S. Tarnawski, M. Aragno and N.Fromin

Laboratoire de Microbiologie, Universite¤ de Neucha“tel,Emile Argand 11, CH-2007 Neucha“tel, Switzerland

Molinia coerulea is a perennial hemicryptophytic grass nat-urally occurring in various oligonitrophilic places. Itpresents a root system permanently anchored in the soil.Plants were transferred from a littoral meadow at the

south shore of lake Neucha“tel to the FACE installationsat Eschikon near Zu«rich. M. coerulea plants were grownunder normal (350 ppm) and elevated (600 ppm) atmo-spheric pCO2. Assuming that associative N2 ¢xation maybe important for plant growth in N-limited conditions, thepresence and the diversity of N2-¢xers in the rhizosphereof M. coerulea was determined in the littoral meadow [1].Among the nifH (the gene encoding for the nitrogenase)sequences detected, an abundant cluster (56% of clones,named clusterA) was found either in the root or in thesurrounding soil fractions. No nifH sequence belongingto known culturable bacteria was found in this group.The present study aims to detect nifH genes and nifHmRNAs in the rhizosphere of M. coerulea under both at-mospheric conditions. Universal nifH and clusterA-speci¢cnifH PCR and RT-PCR were carried out on genomicDNA and total RNA extracts. ClusterA-nifH sequenceswere detected either in soil or roots, but clusterA-mRNAwere only recovered in the root fraction, for both ambientand elevated pCO2. We postulate that the soil environ-ment plays the role of reservoir for clusterA bacteriaand the root environment represents a favourable nichefor the expression of dinitrogen ¢xation by these uncul-tured bacteria.[1] J. Hamelin et al (2002) Environmental Microbiology 4:477-481.

A7^4

PHYLOGENETIC ANALYSIS OF MICROBIAL POP-ULATION IN A MEDIATOR-LESS MICROBIALFUEL CELL ENRICHED WITH ACETATE

J. Y. Lee, T. N. Phung, I. S. Chang, A. C. Pham and B. H.Kim

Bioelectrochemistry Laboratory, Korea Institute of Science& Technology, 39-1 Hawolgok, Sungpook, Seoul 136-791,Korea

A mediator-less microbial cell had been inoculated withactivateD sludge and fed continuously with a non-fer-mentable electron donor, acetate to enrich electrochemi-cally active microbial consortium. The microbial fuel cellhad been operated continuously for over a year. The fuelcell generated a current of around 4 mA stably. Denatur-ing gradient gel electrophoresis showed that the microbialpopulation in the enriched microbial fuel cell is di¡erentfrom that of the inoculum. The colony forming units ofthe enriched electrode were several orders of magnitudelower than that expected from chemical analyses. DNAwas extracted from the electrode and ampli¢ed by poly-merase chain reaction using primers of bacterial 16SrDNA. Out of 340 clones 55 were selected by restrictionfragment length polymorphism for sequencing. The smallribosomal RNA gene sequence analysis showed a diverse



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bacterial population including Deltaproteobacteria (25/55),Bacteroides (12/55), Gammaproteobacteria (7/55), Alphap-roteobacteria (4/55), Firmicutes (3/55), Betaproteobacteria(2/55), and one clone each of Flavobacterium and un-known eubacteria. This result is di¡erent from the elec-trode enriched using marine sediment (Bond et al., Science295:483, 2002), which showed over 70% of the populationwas Deltaproteobacteria. A strain of Aeromonas hydrophilahas been isolated, and found to be electrochemically ac-tive.

A7^5

STRUCTURAL AND FUNCTIONAL CHARACTER-IZATION OF MICROBIAL DIVERSITY IN THE RHI-ZOSPHERES OF THREE GRAIN LEGUMES

S. Sharma, M. Aneja, and M. Schloter

Institute of Soil Ecology, GSF-National Research Centerfor Environment and Health, Ingolstaedter Landstrasse 1,D-85764 Neuherberg, Germany

The bacterial diversity of rhizospheres of three economi-cally important grain legumes (Pisum sativum, Vicia fabaand Lupinus albus) was assessed by cultivation-indepen-dent methods. Nucleic acids were extracted from rhizo-sphere soil samples of the three legumes at maturity.PCR and RT-PCR were used to amplify 16S rDNA and16S rRNA respectively. The PCR products were separatedby Denaturing Gradient Gel Electrophoresis (DGGE).PCR products of representative samples were also clonedand sequenced. The most dominant population in the rhi-zosphere of all three plants belonged to ¢rmicute and pro-teobacteria groups. However, plant dependent rhizospheree¡ects were evident by absence of L-subdivision membersin peas and Q-subdivision members of proteobacteria infaba bean rhizosphere. Functional diversity of the bacte-rial population was analyzed by RNA extraction and ¢n-gerprinting using 10-mer primer. Lupinus and Pisum rhizo-spheres were more similar to each other than to Vicia.Subsequent studies based on genes playing an importantrole in the nitrogen cycle were performed. Marked plant-dependent e¡ect on the rhizosphere could be observedwith an absence of bacterial neutral metallopeptidase tran-scripts from Lupinus rhizosphere.

A7^6

NEW VIBRIOID BACTERIA FORMING WHITISH-TRANSLUCENT VEILS ON SULFIDIC MARINESEDIMENT

R. Thar and M. Ku«hl

Marine Biological Laboratory, University of Copenhagen,Strandpromenaden 5, 3000 HelsingWr, Denmark

We describe a hitherto unknown obligate microaerophilicbacterial species forming whitish-translucent veils on topof marine sul¢dic sediment. The new bacteria were en-riched on complex sul¢dic medium within a benthic gra-dient chamber in oxygen-sul¢de countergradients, but thebacteria have so far not been isolated in pure culture. TheGram-negative colorless vibrioid bacteria with typical ex-tensions of 2 by 6Wm show a unique swimming pattern bymeans of bipolar £agellar bundles, as they rotate aroundas well as translate along their short axis. The cells senseoxygen gradients across their lateral extensions and aggre-gate at 2 WM oxygen concentration by true chemotaxis,where they are able to attach by a mucous stalk formingcohesive whitish veils at the oxic-anoxic interface. At-tached bacteria keep rotating around their short axis in-ducing a homogeneous water-£ow across the veil, e¡ec-tively increasing their oxygen uptake up to 6 times. Theveils show a pronounced succession pattern. New veils aregenerated de novo within 24 h and have a homogeneouswhitish translucent appearance. Bacterial competitors oreukaryotic predators are apparently kept away by thelow oxygen concentration prevailing at the veil surface.Frequently, within 2 days the veil develop a honeycombpattern of regularly spaced holes. After 4 days, most veilsare colonized by grazing ciliates, leading to the fast dis-appearance of the new bacteria. Several-week-old veils ¢-nally develop into microbial mats consisting of green, pur-ple, and colorless sulfur bacteria.

A8^1

DIVERSITY OF FUNGI IN SOME BALKAN FRESH-WATER ECOSYSTEMS

Lj. CX omic¤

Faculty of Science, 34000 Kragujevac, Serbia

Mycological investigations have to date encompassed thefollowing freshwater ecosystems in the Balkans: OhridLake, Skadar Lake, Dojran Lake, Vlasina Reservoir,Grosnica Reservoir, Gruza Reservoir, CŁ elije Reservoir,Neretva river, Stavnja river and Lepenica River. In theten investigated aquatic ecosystems the autochthonouscommunity is made up of 31 species of fungi identi¢ed



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to date. Most of the identi¢ed species belong to the orderSaprolegniales and the dominant genera are Achlia(29,03%) and Saprolegnia (22,58%). Saprolegnia feraxand S. hypogina are constant and eurytopic species. Thenext widespread species are Achlia americana and Pythiumultimum. Dissemination of the remaining species varieddepending on ecological factors, above all in relation tothe quantity and nature of organic matter. The allochth-onous community of fungi is composed of 164 speciesbelonging to 63 genera. It should be observed that mostof them are found rarely or only once and that only rep-resentatives of some genera had a relatively high degree ofmass presence. These fungi primarily belong to the generaAspergillus (15 determined species) and Penicillium (31species). Aspergillus £avus, A. niger, Penicillium brevi-com-pactum and P. verrucosum are the most frequently foundspecies They are dominate in all reservoirs. Besides, thefollowing genera were also frequently founded: Cladospo-rium (12 species), Fusarium, Phoma, Rhizopus, Mucor andVerticilium.

A8^2

ISOLATION AND CHARACTERIZATION OF TYPI-CAL FRESHWATER BACTERIA

M. W. Hahn

Institute for Limnology, Mondseestrasse 9, A-5310 Mon-dsee, Austria

Studies with culture-independent methods demonstratedthat the majority of freshwater bacterioplankton taxahave never been cultured and isolated. Most of the bac-teria detected by these culture-independent methods areindigenous to freshwater habitats and form phylogeneticclusters of typical freshwater organisms. Several in situstudies showed that bacteria belonging to the Actinobac-teria and Betaproteobacteria frequently dominate fresh-water bacterioplankton. At least seven Actinobacteria clus-ters indigenous to freshwater habitats do exist, but none ofthese clusters contained cultivated organisms. Six typicalfreshwater clusters of Betaproteobacteria have been found.Only two of these clusters contain cultivated strains. Wedeveloped a novel method for cultivation and isolation offreshwater bacteria and isolated more than 50 strains af-¢liated to four di¡erent clusters of typical freshwater bac-teria. We obtained the ¢rst isolates a⁄liated with fresh-water Actinobacteria clusters, as well as the ¢rst isolatesa⁄liated to the Polynucleobacter necessaries cluster. Se-quences a⁄liated with the latter cluster have been detectedfrequently, if not most frequently in 16S rRNA gene clon-ing studies. All isolated freshwater Actinobacteria and P.necessarius. cluster strains are obligate ultramicrobacteria.This means that they always posses cell sizes smaller than0.1Wm3, even when cultured in rich media.

A8^3

CULTIVATING THE UNCULTURED

M. Keller(1), K. Zengler(1), G. Toledo(1), M. Rappe¤(2),J. Elkins(1), E. J. Mathur(1) and J. M. Short(1)

(1) Diversa Corporation, San Diego, California 92121,USA; (2) Department of Microbiology, Oregon State Uni-versity, Corvallis, Oregon 97331, USA

The recent application of molecular phylogeny to environ-mental samples has resulted in the discovery of an abun-dance of unique and previously unrecognized microorgan-isms. The vast majority of this microbial diversity hasproved refractory to cultivation. Here we describe a uni-versal and novel method that provides access to this im-mense reservoir of untapped microbial diversity. This tech-nique combines encapsulation of cells in gel microdropletsfor massively parallel microbial cultivation under low nu-trient £ux conditions, followed by £ow cytometry to detectmicrodroplets containing microcolonies. The ability togrow and study previously uncultured organisms in pureculture will enhance our understanding of microbial phys-iology and metabolic adaptation, and will provide newsources of novel microbial metabolites. We show thatthis technology can be applied to samples from severaldi¡erent environments, including seawater and soil.Zengler et al. (2002). PNAS 99, 15681-15686

A8^4

THE LUMINESCENCE AS CRITERIUM FOR DE-TECTION OF ECOLOGY HABITATION OF MARINELUMINOUS BACTERIA

D. A. Kotov, S. E. Medvedeva, L. Yu. Popova

Institute of Biophysics SB RAS, 660036, Krasnoyarsk, Rus-sian Federation

During prolonged evolution marine luminous bacteriaproduced the strong system of regulation of lux-genes ex-pression that can cause the variations of luminescence lev-el in wide diapasons. It was carried out serial experimentsto receive information concerning the e¡ect of cell meta-bolic activity of marine luminous bacteria Photobacteriumleiognathi, P. phosphoreum, Vibrio harveyi on lux-gene ex-pression under their cultivation in model medium imagingthe habitation conditions. On the basis of growth andluminescence parameters the studied strains can be sepa-rated in several types with characteristic features. 1) Free-living bacteria emitted light better under nutrition de¢-ciency (sea water) ; latent period of luminescence inductionwas less 3 hours; maximum quanta yield was equally ormore than 3.00.109 quanta/sec. 2) Migrants were capable



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to grow and emit light under nutrition de¢ciency as well asnutrition abundance (gastric-internal tract) ; latent periodof luminescence induction was more than 3 hours; max-imum quanta yield was 1.00.109 ^ 3.00.109 quanta/sec. 3)Symbiotic were capable to grow and emit light well onlyunder nutrition abundance; latent period of luminescenceinduction was more than 3 hours; maximum quanta yieldwas less than 109 quanta/sec. Analysis of growth and lu-minescence curves allowed detecting the true habitation ofluminous bacteria with great validity and estimating thepossibilities of every natural luminous bacteria species tokeep lux-genes expression in di¡erent habitation condi-tions. Such information is needed to maintain the collec-tion strains and describe the bacteria features in databasebiolumbase.This work was supported by RFBR (grants 00-07-9011;02- 07-06093).

A8^5

MICROBIAL COMMUNITIES IN ANTARCTICFRESHWATER LAKES HAVE BEEN DESCRIBEDAS SIMPLE ECOSYSTEMS. TO WHAT EXTENTCAN THESE SYSTEMS BE CONSIDERED REPRE-SENTATIVE OF LAKE SYSTEMS ELSEWHERE?

D. A. Pearce

British Antarctic Survey, High Cross, Madingley Road,Cambridge, CB3 OET, UK

Over the past ¢ve years, a combination of cultural andmolecular identi¢cation methods has been applied to arange of Antarctic aquatic environments in order to studythe biodiversity and stability of microbial communitystructures present. This research has combined the directculture of di¡erent groups of microorganisms with fattyacid methyl ester analysis, £uorescence in situ hybridisa-tion, direct sequencing of 16S rDNA fragments, denatur-ing gradient gel electrophoresis and comprehensive clonelibrary construction. This work has shown that di¡erentAntarctic environments, in close proximity, and sharingsimilar physical and chemical characterisitics, can oftenhave very di¡erent microbial community structures. Usingthis information, we hope to demonstrate that microbialcommunities in Antarctic freshwater lakes can indeed beconsidered simple ecosystems with low trophic complexityand limited diversity, and to determine the extent to whichthese systems can be considered representative of lake sys-tems elsewhere.

A8^6

DIVERSITY OF MICROORGANISMS CULTIVATEDFROM DEEP-SEA HYPERSALINE ANOXIC BASINS

A. M. Sass, B. McKew, K. N. Timmis and T. J. McGenity

University of Essex, Department of Biological Sciences,Colchester CO4 3SQ, U.K.

Several basins ¢lled with highly saline waters, ‘‘brinelakes’’, have been discovered on the £oor of the EasternMediterranean Sea. Because of their high salinity thebrines do not mix with seawater and so the basins becomeanoxic. The brine lakes are extreme environments in termsof high pressure, high salt concentrations, steep chemicalgradients at the interface, and anoxic conditions in thebrine body. Brines, interfaces and sediments from fourbasins containing brines of di¡erent salt compositionswere sampled in the summer of 2001 and 2002 with arosette sampler and a multicorer. A total of 208 aerobicand 43 anaerobic strains were cultivated and tentativelyidenti¢ed by their 16S rRNA gene sequence. Extremelyhalophilic anaerobic strains belonging to the Haloanaero-biales were cultivated from each basin, extremely halo-philic Archaea, methanogens and a haloarchaeaon, werecultivated from one of the basins. Aerobic and anaerobicspore-formers predominated in sediments, whereas at theinterfaces aerobic halotolerant and extremely halotolerantorganisms were most abundant. Many of the cultivatedorganisms potentially represent new species or genera,such as anaerobic Cytophaga-related bacteria from oneof the interfaces. Cultivation-independent ¢ngerprintingof 16S rRNA genes using t-RFLP showed that the brinesof each basin were inhabited by a unique microbial com-munity, although the chemical composition of the brinesand microbial community pro¢le were clearly related. In-vestigation of the 1.5 m thick interface of the Bannockbasin with high spatial resolution revealed that it wasalso inhabited by a distinct microbial community di¡erentfrom seawater or brines.



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A9^1

NEW SPIROCHETES FROM BOVINE RUMEN: GE-NETIC AND PHENOTYPIC CHARACTERISATION,ABUNDANCE AND POPULATION DYNAMICS

T. Accetto(1), L. Fanedl(1), S. ZX itnik(2), M. Trkov(1‰),G. Gorenc(1), R. Kostanjs›ek(3), J. Ambroz›ic›(3) and G.Avgus›tin(1)

(1) University of Ljubljana, Biotechnical Faculty, Zootech-nical department, Groblje 3, 1230 Domz›ale, Slovenia; (2)SPE Veterina, Lek d.d., Lipovci 251a, 9231 Beltinci, Slov-enia; (3) University of Ljubljana, Biotechnical Faculty, De-partment of biology, Vec›na pot 111, 1000 Ljubljana, Slov-enia. ‰ present address: Ministry of agriculture, forestryand food of Republic of Slovenia, Dunajska c. 56-58, 1000Ljubljana, Slovenia

Several bacterial isolates from the phylogenetic group Spi-rochaeta were isolated recently from bovine rumen. Thegenetic and phenotypic characterisation showed, thatthey all belong to the genus Treponema, but cannot beassigned to the established species of this genus due tothe genotypic or phenotypic variations. One group of iso-lates relates to Treponema bryantii on genotypic level, butdi¡ers substantially in regard to certain phenotypic char-acteristics e.g. polysaccharide degradation, cell size andnumber of axial ¢laments. The other group of isolatesrelates, according to the 16S rRNA phylogenetic analysis,to the strain Treponema sp. CA, which represents a uniquephylogenetic lineage among the members of the genusTreponema, only distantly related to T. succinifaciens, ananaerobic spirochete from the swine intestine. The strainCA was never characterised in full taxonomic sense as arepresentative of a novel bacterial species, however. Thedata are going to be presented, supporting the possibleproposal of two novel Treponema species inhabiting thebovine rumen, and allowing the estimation of the geneticdiversity of ruminal spirochaetes. The ecological role ofruminal spirochaetes is going to be discussed too, as de-duced from the results of the molecular monitoring ofruminal spirochetes and the discovery of active polysac-charide degrading enzymatic systems. The molecular mon-itoring was performed by the Treponema speci¢c compet-itive PCR analysis of rumen £uid samples obtained duringanimal feeding trials.

A9^2

THE GENETIC DIVERSITY OF CFB BACTERIA(THE BACTEROIDETES) INHABITING THE BOVINEHINDGUT

G. Avgus›tin and K. Teps›ic›

University of Ljubljana, Biotechnical Faculty, Zootechnicaldepartment, Groblje 3, 1230 Domz›ale, Slovenia

Certain members of the phylogenetic group Cytophaga-Flexibacter-Bacteroides or the Bacteroidetes constitute im-portant and in certain cases dominant parts of the micro-bial communitiy inhabiting the gastrointestinal tract ofanimal and man. Members of the genus Prevotella arecommon inhabitants of human oral cavity and representthe major Gram-negative bacterial population in the oth-erwise Gram-positive bacteria dominated rumen microbialecosystem. Members of the genus Bacteroides dominatethe hindgut of non-ruminants, including carni and omni-vores as well as large herbivores. In contradiction to thewell investigated ruminal microbial community, very littleis known about the microorganisms inhabiting the hindgutof ruminant herbivores, despite of the fact that between 10and 30% of host energy demands may be covered by hind-gut microbiota activities. Bacterial 16S rRNA genes werePCR ampli¢ed from black-and-white Holstein cattle fecalDNA, cloned and hybridised to three digoxygenin labelledoligonucleotide probes covering the majority of the CFBbacteria. 6,7 % of clones hybridised to the oligonucletideprobe BacPre speci¢c for genera Bacteroides, Prevotellaand Porphyromonas. Comparative sequence analysis ofcloned 16S rDNA showed, that most of the sequencesbelong to bacteria distantly related to B. fragils phyloge-netic subgroup, whereas only one sequence showed slightresemblance to bacteria from the genus Prevotella. Themicrobiota of the hindgut appears thus to contain signi¢-cantly di¡erent bacterial populations in relation to the ru-men microbiota and the question appears, how this mayin£uence the gene £ow throughout the gastrointestinaltract, speci¢cally with consideration to antibiotic resis-tance dissemination.



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A9^3

A HITHERTO UNKNOWN BACTERIUM SHOWINGA NEW SOCIAL BEHAVIOR

U. Bo«ckelmann(1), A. Jahnke(1), T. R. Neu(2), J.Wecke(3), U. Szewzyk(1) and J. R. Lawrence(4)

(1) Department of Microbial Ecology, Technical Univer-sity, Franklinstrasse 29, 10587 Berlin, Germany; (2) De-partment of Inland Water Research Magdeburg, UFZCentre for Environmental Research Leipzig-Halle, Brueck-strasse 3A, 39114 Magdeburg, Germany; (3) Robert KochInstitute, Nordufer 20, 13353 Berlin, Germany; (4) Nation-al Water Research Institute, 11 Innovation Boulevard, Sas-katoon, Saskatchewan, Canada, S7N 3H5

Thirty eight di¡erent bacterial strains were recently iso-lated from microbial aggregates of the South Saskatche-wan River, Canada, using an oligotrophic medium. One ofthem produced a unique extracellular compound. This ma-terial was involved in the development of a complex phys-ical network along which bacteria moved to form aggre-gates. The behavioral pattern consisted of ¢ve steps: 1)typical rod shaped bacterial cells produced a visible mate-rial around them; 2) from the initial material ¢ne ¢bersdeveloped resulting in the formation of ¢laments of vari-ous lengths; 3) the ¢laments extended and became inter-connected forming a network with attached cells ; 4) cellsmoved along the network aggregating into small and sub-sequently larger cell clusters; 5) the process ended withlarge aggregates of cells and a cell free ¢lament network.The ¢laments were initially visualized through fortuitousstaining with the £uorescent nucleic acid speci¢c stainSYTO9. In contrast, no binding was observed using othernucleic acid stains or a £uorescent protein stain (Sypro).MALDI-TOF analysis of the ¢laments resulted in a spec-trum which was consistent with the material being a re-peating unit of a cyclic polymer. It was found that strainF8 was a producer of N-(3-oxo-octanoyl)-L-homoserinelactone only on low nutrient media which also supported¢lament production and aggregation behavior. Phyloge-netic analysis revealed isolate F8 as a deep branching gam-ma-Proteobacterium with 96% sequence similarity to itsnext relative Rheinheimera baltica a blue pigment produc-ing bacterial strain. In conclusion, this behavior may playan important role in £oc formation in aquatic environ-ments.

A9^4

LARGE DIVERSITY OF BACTERIAL PREDATORS:EVOLUTIONARY AND FUNCTIONAL IMPLICA-TIONS

Y. Davidov and E. Jurkevitch

Department of Plant Pathology and Microbiology, Facultyof Agricultural, Food and Environmental Quality Sciences,76100, Rehovot, Israel

The largest part of living biomass on earth is constitutedof microbial cells. This tremendous amount of material£ows through various trophic levels. Bacterial predators,which are at the bottom of the predation ladder, havebeen largely overlooked. More than 70 bacterial predatorsbelonging to the Bdellovibrio and like organisms (BLOs)from various habitats were isolated. Ampli¢ed 16S ribo-somal DNA restriction analysis provided a classi¢cationtool for BLOs at the species level for habitat analysis.Sequencing of the 16S rRNA gene of representative strainsshowed that BLOs mainly belong to two largely heteroge-neous, separate monophyletic groups, both in the deltaProteobacteria. Using various analytical tools, models,and signature sequences, the exact position of these twoclusters within the delta class could not be resolved, indi-cating that these are deep branching or fast evolvinggroups. The Bdellovibrio cluster exhibited more than 10%heterogeneity, with nine stable clusters forming a similarnumber of putative species. Bacteriovorax exhibited morethan 14% heterogeneity with nine clusters forming fourclades (corresponding to similar numbers of putative spe-cies and genera, respectively), one clade being mainly com-posed of marine and another of freshwater strains. Anumber of BLOs not belonging to the delta Proteobacteriawere also isolated. Nevertheless, ¢rst phylogenetic analysisusing 16S rDNA and other genes suggests a common linkbetween the three groups, indicating that bacterial preda-tors belong to an ancient, lineage. The wide distributionand heterogeneity of bacterial predators also suggest thatpredation is not a fortuitous but common event in theenvironment.



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A9^5

UNRAVELLING OF THE BACTERIAL PARTNER-SHIP OF THE ARCHAEZOAN MIXOTRICHA PARA-DOXA INVOLVED IN MOVEMENT SYMBIOSIS

H. Ko«nig(1), M. Wenzel(1), R. Radek(2) and G. Bruger-olle(3)

(1) Institute of Microbiology and Wine-Research, JohannesGutenberg University, 55099 Mainz, Germany; (2) Insti-tute of Zoology, FU Berlin, 14195 Berlin, Germany; (3)Laboratoire Biologie des Protistes, Universite¤ Blaise Pascalde Clermont-Fd., 63177 Aubiere, France

Mixotricha paradoxa, a trichomonad from the hindgut ofthe Australian termite Mastotermes darwiniensis Froggatt,is a rare example of a movement symbiosis between eu-karyotic and prokaryotic microorganisms. The surface ofMixotricha paradoxa is covered with spirochaetes and arod-shaped bacterium. The four £agella at the anteriorend seem only to alter the direction of movement, whilethe ectosymbiotic spirochaetes propel the £agellate cells.Based on a 16S rDNA sequence analysis after a semi-spe-ci¢c PCR and subsequent £uorescence in situ hybridiza-tion applying helper oligonucleotides and a denaturingstep of the 16S rRNA, three di¡erent spirochaete clonescould be clearly identi¢ed on the surface of the protozoalcells. They belonged to the Treponema cluster. The rodshaped bacterium showed highest 16S rDNA sequencesimilarity to Bacteroides-related species. Due to its lowphylogenetic relationship to its nearest relatives in the da-tabase it should represent a so far undescribed species.

A9^6

INTRACELLULAR BACTERIA FROM HEPATO-PANCREATIC CELLS OF PORCELLIO SCABER

R. Kostanjs›ek(1), D. Drobne(1), J. SX trus(1), G. Avgus›-tin(2)

(1) University of Ljubljana, Biotechnical Faculty, Biologi-cal department, Vec›na pot 111, 1000 Ljubljana, Slovenia;(2) University of Ljubljana, Biotechnical Faculty, Zooteh-nical department, Groblje 3, 1234 Domz›ale, Slovenia

The tissues of isopod crustaceans are often infected withintracellular bacteria with complex developmental cycle.Beside infections with Rickettsiella grylli two other intra-cellular bacteria with di¡erent morphological and patho-logical properties as R. grylli were described in digestiveglands of terrestrial isopod Porcellio scaber. In spite ofobvious morphological resemblance and common host,both subsequently described bacteria were a⁄liated to dif-ferent bacterial taxonomic groups. In order to clarify the

phylogenetic position of intracellular bacteria from diges-tive glands of P. scaber, 16S rRNA sequence analysis andmicroscopic observations were performed. Phylogeneticanalysis revealed that intracellular bacteria represent anindependent lineage within order Chlamydiales, clearlyseparated from known chlamydial families. The lineageis most closely related to lineage ‘ECL VI’ which clustersthe rRNA sequences retrieved from activated sludge andmore distantly to the family Simkaniaceae. The presenceof speci¢c oligonucleotide sequences in intracellular bac-teria was con¢rmed by in situ hybridisation and £uores-cent microscopy. Although the complex developmentalcycle of intracellular bacteria from P. scaber con¢rms theira⁄liation to chlamydia, their elementary bodies exhibitdistinctive morphological features, not observed in otherchlamydia. According to the results of the phylogeneticanalysis, unique morphology of the elementary bodiesand the speci¢c host, we propose that the intracellularbacteria from digestive glands of P. scaber represent anew chlamydial genus and species.

A10^1

METABOLIC ROBUSTNESS: IN SILICO AMMO-NIUM ASSIMILATION IN ESCHERICHIA COLI

F. C. Boogerd, F. J. Bruggeman and H. V. Westerho¡

Department of Molecular Cell Physiology, Faculty of Earthand Life Sciences, Free University, De Boelelaan 1085, NL-1081 HV Amsterdam, The Netherlands

Ammonium assimilation in Escherichia coli can take placevia two di¡erent pathways: the GS-GOGAT system (glu-tamine synthetase and glutamine-oxoglutarate amino-transferase) and the GDH system (glutamate dehydroge-nase). Multiple regulatory processes are involved in main-taining an optimal £ux in response to a continually chang-ing environment. The metabolic network is responsive tothe nitrogen status (ammonium, glutamine), the carbonstatus (2-oxoglutarate), and the free energy status (ATP).A kinetic model is presented of the regulatory processes onthe metabolic time scale of ammonium assimilation. Thein silico model is entirely based upon literature data of thekinetic parameters of all the enzymes engaged in ammo-nium assimilation: GS, GOGAT, and GDH, including theproteins involved in modi¢cation of GS via adenylylation:ATase, UTase/UR, and PII. We analysed the regulation ofammonium assimilation at varying levels of 2-oxoglutaratefor cells equipped with di¡erent amounts of GS, GOGAT,and GDH to mimic a range of growth conditions. Tran-sient and steady-state analyses of the model indicate thatthe ammonium assimilation £ux is robust against changesin the availability of ammonium at the metabolic timescale. The model illustrates that the interplay betweenthe carbon and nitrogen status, as re£ected in the concen-



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trations of ammonium, glutamine and 2-oxoglutarate, hasa complex e¡ect on the regulation of the ammonium as-similation £ux and strongly depends on the physiologicalhistory of Escherichia coli.

A10^2

METABOLIC AND ENGINEERING INTEGRATIONAPPROACH FOR THE OPTIMISATION OF RE-COMBINANT MICROBIAL FERMENTATION PRO-CESSES

M. Cserjan-Puschmann, H. Reischer, G. Striedner, K.Du«rrschmid, F. Clementschitsch , F. Po«tschacher, J. Kernand K. Bayer

Institute of Applied Microbiology, University of NaturalResources and Applied Life Sciences, Nussdorfer La«nde11, 1190 Vienna, Austria

The e⁄cient production of recombinant protein relieslargely on the maximal exploitation of the cell factory.To cope with the often limited productivity of biopro-cesses an integrated systems approach aiming at optimalcontrol of the £ux-ratios between biosynthesis of (1) hostcell proteins and (2) recombinant proteins will substan-tially contribute to the optimisation of process develop-ment. To achieve these goals methods for (1) monitoringof the metabolic load derived from overexpression of re-combinant protein and (2) regimes for expression rate con-trol in relation to the metabolic potential have been estab-lished. For comprehensive determination of the metabolicload imposed by recombinant protein synthesis a varietyof analytical procedures was set up. Due to its widespreadregulatory role in the stringent response network the signalmolecule guanosinetetraphosphate (ppGpp) was selectedas the key analyte for monitoring metabolic load. To cir-cumvent extensive sample preparation and its complex de-termination by ionpair-HPLC, a stress promoter depen-dent GFP reporter gene-fusion is being constructed foron-line signal acquisition. Whole genome E. coli micro-arrays were used to identify signi¢cant stress promoters,which are activated immediately after induction and dur-ing the whole period of recombinant gene expression. Inaddition, 2-D gel electrophoresis is applied to assesschanges on proteome level to derive stress speci¢c markerproteins. The speci¢c metabolic load data are used to tunerecombinant gene expression by feeding limited amountsof inducer in a constant ratio to biomass (repressor titra-tion). The integration of metabolic and engineering re-gimes largely contributes to the systematic optimisationof recombinant fermentation processes.

A10^3

ENGINEERING MANNITOL PRODUCTION IN LAC-TOCOCCUS LACTIS

P. Gaspar(1), A. R. Neves(1), A. Ramos(1), M. J. Gas-son(2), C. A. Shearman(2) and H. Santos(1)

(1) Instituto de Tecnologia Qu|¤mica e Biolo¤gica, Universi-dade Nova de Lisboa, Rua da Quinta Grande, 6, Apt 127,2780-156 Oeiras, Portugal; (2) Institute of Food Research,Norwich Research Park, Colney, Norwich, NR4 7UA, UK

The increasing demand for food products enriched in com-pounds with bene¢cial properties for human healthprompted the construction of strains with suitable traits.Interestingly, disruption of lactate dehydrogenase in Lac-tococcus lactis FI9630 resulted in a transient accumulationof mannitol, a polyol claimed to have health-promotingproperties since it is a low-calorie edulcorant and an anti-oxidant. Aiming at increased mannitol production andaccumulation by L. lactis, two genes of the mannitolPTS-system (mtlA and mtlF) were deleted by doublecross-over recombination in the strain FI9630, leading tothe two mutant strains v(ldh-mtlA) and v(ldh-mtlF). Themetabolism of [1-13C] glucose and [6-13C]glucose in restingcells suspensions of the mutant strains was characterizedby in vivo 13C-NMR. Mannitol was the major end prod-uct; in addition, minor amounts of lactate, ethanol, 2,3-butanediol, acetoin, and acetate were produced regardlessof the strain used. Disruption of the PEP:PTSMtl com-pletely blocked mannitol catabolism in L. lactis. In con-trast to the parental strain FI9630, mannitol was not usedby the mutants after glucose depletion. A pronounced shiftto a mixed-acid fermentation (formate, ethanol, acetate)was observed during growth under controlled conditionsof pH, mannitol being no longer the major end product.The requirement of energy for the anabolic processes ismost likely the reason for this alteration in the patternof end-products formed during growth as compared toresting cells. New metabolic engineering strategies will beattempted with the goal of increasing mannitol productionduring growth.



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A10^4

OPTIMISING THE DIRECTED EVOLUTION PRO-CESS TO PRODUCE NOVEL PROTEIN MOLE-CULES

H. D. Gri⁄ths(1), L. Pritchard(1), C. Evans(1), M. A.Kaderbhai(1), D. B. Kell(2), M. K. Winson(1)

(1) Institute of Biological Sciences, University of WalesAberystwyth, Aberystwyth UK; (2) Department of Chem-istry, UMIST, Manchester, UK

Directed evolution (DE) is a powerful method throughwhich proteins of completely novel or greatly enhancedfunction can be produced without the need for a detailedunderstanding of structure-function relationships. Thiscan, for example, be used to engineer changes in enzymebinding e⁄ciency, substrate speci¢city or stereoselectivityand the technique thus has the potential for signi¢cantbene¢t in metabolic engineering of microbes. The methodnormally involves performing multiple rounds of mutationof a target gene with selection for the phenotypic charac-teristic of interest after each round. Mutations are intro-duced using a PCR reaction in which the conditions in-duce the polymerase to make errors in the copying of thetemplate (error-prone (epPCR) or ‘sloppy’ PCR). Typi-cally recombinant DNA from the best mutants after oneround of mutation is used to seed the next PCR reaction.In some cases e⁄cient evolution of the relevant proteinactivity may require a number of mutations to be intro-duced and thus achieving an optimal mutation rate in theepPCR process is of importance for obtaining the desiredproduct. In order to predict the optimal conditions we areconducting epPCR experiments in which mutation ratesare varied. Output from these experiments is being usedin conjunction with new computer models of PCR to op-timise parameters for searching sequence-space and en-hance protein function more e⁄ciently. Using informationgained from this systematic analysis we are currentlyevolving novel £uorescent protein tags for use in bindingand interaction studies and altering the substrate speci¢c-ities of bacterial enzymes with proteolytic activity.

A10^5

MODELLING NATURAL CARBON CYCLE WITHMICROBES ^ CAN WE GET FUEL GASES FROM IT?

A. Netrusov(1), E. Slepova(1), L. Gassanova(2) and V.Teplyakov(2)

(1) Microbiology Department, Moscow State University;Moscow 119992; (2) A.V.Topchiev Institute for Petro-chemical Synthesis RAS; Moscow 119991, Russia

Complete carbon cycle was modelled in the laboratory,based on light-dependent growth and decomposition ofalgae biomass in sequenced aerobic photobioreactor, an-aerobic digester and anaerobic photobioreactor, all oper-ating in continuous mode. Biomass from producing reac-tor was fed into anaerobic methane tank, where biogasproduced by methanogenic consortium. The e¥uentfrom anaerobic digester after ultra¢ltration was fed in an-aerobic photobioreactor with purple phototrophic bacteriaproducing hydrogen. Gas phases of all the reactors wereconnected to the membrane contactors for the gas mix-tures separations on pure methane and CO2 or on purehydrogen and CO2, respectively. Resulting CO2 from tworeactors was fed into the ¢rst, biomass-producing photo-bioreactor with algae, therefore completing natural carboncycle of production/digestion, based on sunlight, withmethane and hydrogen obtaining as valuable fuels.

A10^6

MULTIVITAMIN PRODUCTION IN LACTOCOCCUSLACTIS BY USING METABOLIC ENGINEERING:DEVELOPMENT OF FUNCTIONAL FOODS FORNUTRIGENOMICS

W. Sybesma(1), C. Burgess(2), D. van Sinderen(2) and J.Hugenholtz(1)

(1) Wageningen Centre for Food Sciences; NIZO FoodResearch, P.O. Box 20, 6710 AB Ede, The Netherlands;(2) Departments of Microbiology, Food Science, FoodTechnology and Nutrition, University College Cork, Cork,Ireland

The dairy starter bacterium Lactococcus lactis is able tosynthesise folate, vitamin B11, and ribo£avin, vitamin B2.Both vitamins are essential nutrients in the human dietand are important for the prevention of diseases such asmegaloblastic anaemia and hyperhomocysteinemia (riskfactor for cardiovascular disease), birth defects, pregnancycomplications and Alzheimer disease. Hyperhomocysteine-mia occurs in 10-15% of the human, white, populationcarrying a mutation in the gene coding for methylenete-trahydrofolate reductase (MTHFR). The diet of this mu-



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tant population requires both extra folate and ribo£avin,to increase the stability of MTHFR. In the present workwe describe an example of successful metabolic engineer-ing of the complicated pathways of folate and ribo£avinbiosynthesis : The nisin controlled expression system in L.lactis was used to induce overexpression of folKE and ribAencoding GTP cyclohydrolase I and GTP cyclohydrolaseII respectively, the ¢rst enzymes involved in folate andribo£avin biosynthesis. Both vitamins could be detectedsimultaneously by using HPLC. The engineered strainshows almost three-fold increased folate production andmore than hundred-fold increased ribo£avin production.Further control of expression of genes involved in folatebiosynthesis, such as folC, encoding polyglutamyl folatesynthetase, has shown to change the folate polyglutamyltail length, which could in£uence folate bioavailability,because only monoglutamyl folate can directly be ab-sorbed in the human gut. Conclusions: Our work providesa basis for development of functional foods with increasedlevels of vitamins that could be bene¢cial for health of thegeneral population, especially since B-vitamin de¢ciency iscommon even in well-developed countries.

A11^1

DEVELOPMENT AND VALIDATION OF A REAL-TIME PCR ASSAY FOR THE DETECTION OF MY-COBACTERIUM AVIUM SUBSPECIES PARATUBER-CULOSIS (MAP) IN FECAL SAMPLES AND ITSCORRELATION TO CULTURE

K. Boegli-Stuber(1), B. Glanemann(2), G. Seitert(1), C.Kohler(1), M. Wittwer(1), M. M. Wittenbrink(2) and B.Bissig-Choisat(1)

(1) Swiss Federal Veterinary O⁄ce, Schwarzenburgstrasse161, CH-3003 Bern, Switzerland; (2) Institute of Veteri-nary Bacteriology, University of Zurich, Winterthurerstr.270, CH-8057 Zurich, Switzerland.

Molecular based detection strategies like real-time PCRfor Mycobacterium avium subsp. paratuberculosis (MAP)o¡er the great bene¢t of being time consuming over thetraditional culturing methods. But using PCR for the anal-ysis of fecal samples the sensitivity is often very low. Sev-eral points may be responsible for the failure: di⁄cultiesin the lysis of the MAP cell wall and the presence ofinhibitors. By using a commercial DNA extraction kit(High Pure PCR Template Preparation Kit, Roche) com-bined with mechanical methods of lysis and the optimiza-tion of the real-time PCR conditions (primer concentra-tion and higher cycle numbers) by using SYBR0 Green weobtained good results. To assess the sensitivity of our real-time PCR we tested fecal samples spiked with knownamounts of bacteria. By using our extraction methodand real-time PCR protocol we could detect down to

125 CFU/g feces in 100%, whereas 62 and 32 CFU/g feceswere detected in 71%. In conclusion we have provided anoptimized extraction method combined with a sensitivereal-time PCR assay for the detection of MAP. We furtherwill compare the PCR and culture results of our fecalsamples. Results will be discussed at the congress.

A11^2

Withdrawn.



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A11^3

HUMORAL IMMUNOMODULATION BY PASTEUR-ELLA MULTOCIDA : A POTENTIALLY IMPOR-TANT VIRULENCE FACTOR?

R. W. Jordan and J. M. Roe

Department of Clinical Veterinary Science, University ofBristol, Langford House, Langford, Bristol BS40 5DU, UK

Pasteurella multocida is an important veterinary pathogen,causing a variety of diseases in many animals. We havedeveloped a mouse model to examine the e¡ects of thisorganism upon host humoral immune responses. In thisstudy we have demonstrated that colonisation of the mu-rine upper respiratory tract with toxigenic strains of P.multocida can suppress humoral immune responses to aparenterally administered novel antigen. Non-toxigenicP. multocida strains do not cause an equivalent e¡ect.We examined the e¡ects of soluble products of these dif-ferent strains on the growth and di¡erentiation of murinedendritic cells in vitro. Cells exposed to lysates of toxigenicP. multocida exhibited changes in morphology and theexpression of cell-surface markers that may a¡ect dendriticcell functionality. We hypothesise that Pasteurella multo-cida toxin, a novel protein toxin and potent mitogen, is themediator of these e¡ects. The observed immunomodula-tion may be the true function of this multipotent toxin.Dendritic cells may be one of possibly several importanttargets for the toxin to exert this e¡ect. This immunomo-dulation may constitute a hitherto unknown virulence de-terminant of P. multocida, and may have important impli-cations in aiding survival of the organism within apopulation of host animals.

A11^4

MORPHOLOGICAL AND IMMUNOHISTOCHEMI-CAL STUDIES OF MARBURG VIRUS INFECTIONIN GUINEA PIGS

E. Ryabchikova, E. Malkova, O. Taranov, A. Shestopalov

State Research Center of Virology and Biotechnology ‘‘Vec-tor’’, Koltsovo, Novosibirsk region, Russia 630559

Marburg virus (MV) belonging to Filovoridae family causelethal disease in green monkeys, while the infection inguinea pigs becomes lethal after the virus adaptation bysequential passages [1]. Morphological characteristics ofMV infection in guinea pigs have been not fully examined.A goal of our work was to study visceral organs of guineapigs fatally infected with MV (Popp strain) by immuno-histochemistry, light and electron microscopy. Guinea pigsinfected with 100 LD50 of MV developed acute disease and

died on days 7-8 post-infection. Light microscopy revealeddi¡use lesions of liver, kidneys, lung and spleen not asso-ciated with in£ammation. In electron microscope replica-tion of the MV was observed in macrophages, hepato-cytes, adrenal cortical cells, ¢broblasts and endothelialcells. The infected cells di¡ered by the shapes of viral in-clusions, amount of nucleocapsids and progeny virions.Characteristic feature of MV infection was pronounceddamage of the lymphatic tissue. Decrease of lymphocytenumber, necroses of macrophages, stroma and dendriticcells were seen in spleen, lymphatic nodes and lymphatictissue of lungs and intestines. No evident signs of thedevelopment of humoral and cellular immune responseswere found. Commercial antibodies (Novocastra Lab.)were used to examine the changes of the lymphatic tissueof MV infected guinea pigs. Presence, number and distri-bution of B-, T-, follicular dendritic and macrophage cellshave been studied, using CD21, CD56 and macrophagemarker NCL-LN5 antibodies. Proliferating and apoptoticcells were examined using PCNA and NCL-DFFp anti-bodies.[1] R. Siegert (1972) R. Marburg virus. Virology Mono-graphs, 11, 98-153

A11^5

IMMUNOLGICAL COMPARISON OF MAJOR PRO-TEINS IN MYCOBACTERIUM AVIUM SPP. PARA-TUBERCULOSIS

S. J. Shin(1), Y. F. Chang(2) and H. S. Yoo(1)

(1) Department of Infectious Disease, College of Veteri-nary Medicine and School of Agricultural Biotechnology,Seoul National University, San 56-1, Shinlim-dong, Kwa-nak-gu, Seoul, 151-742, Korea; (2) Department of Popula-tion Medicine and Diagnostic Sciences, College of Veteri-nary Medicine, Cornell University, Ithaca, NY 14853, USA

Mycobacterium avium ssp. paratuberculosis (M. paratuber-culosis), the causative agent of Johne’s disease, is an im-portant animal pathogen that has also been implicated inhuman disease. Several mycobacterial antigens are knownto play a key role in the pathogenesis and immune stim-ulations. Among these antigens, antigen 85 complex ( 85A,85B and 85C), 35-kDa protein and superoxide dismutase(SOD) have known as the most powerful T-cell antigen inother mycobacteial infection. These proteins expressedwith PET-22b(+) vector in E. coli were puri¢ed for thisstudy. The aim of this study was to compare various my-cobacterial proteins for their capacity to stimulate T-cellsfrom each of high infected, middle infected, low infectedand healthy cattle using lymphoproliferation assay, £owcytometric analysis and IFN-Q assay. We showed that85B and 35-kDa proteins were most powerful antigensthat could elicit cellular immune response in highly in-



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fected cows and middle infected cows. Also, 85C and SODwere antigens that could stimulate T and B-cells simulta-neously. These results suggested that 85B, 85C, 35-kDaand SOD could be a useful candidate for combined vac-cine against M. paratuberculosis infection.

A11^6

MOLECULAR CHARACTERISATION OF MYCO-BACTERIUM AVIUM SUBSP. AVIUM ISOLATEDFROM RED DEER WITH TUBERCULOSIS

J. Spergser(1), W. Glawischnig(2), T. Steineck(3) and R.Rosengarten(1)

(1) Institute of Bacteriology, Mycology and Hygiene, Uni-versity of Veterinary Medicine, Vienna, Austria; (2) Fed-eral Veterinary Diagnostic Laboratory, Innsbruck, Austria;(3) Research Institute of Wildlife Ecology, University ofVeterinary Medicine, Vienna, Austria

Since 2001, tuberculosis in free-ranging red deer caused byMycobacterium avium subsp. avium emerged in di¡erentgeographic regions in Austria. Common pathologicaland clinical ¢ndings involved the digestive tract and asso-ciated lymph nodes with persistent diarrhoea and severeweight loss. Strains were identi¢ed by cultural character-istics and were additionally characterised by a variety ofPCR-based molecular techniques. Typing of strains wasperformed by PCR detection of IS901 and the virulence-associated mig sequence, inverted repeat (IR) typing(IS1245/IS1311) and RAPD analysis. While all 10 M.avium strains recovered from a¡ected lymph nodes con-tained the mig gene, IS901 was detected in only nine iso-lates. IS901 containing strains were shown to be genotypi-cally closely related, as they exhibit identical patterns inIR typing and in RAPD analysis. Interestingly, geograph-ically related M. avium isolates from bird species withtuberculosis shared a high degree of similarity with thecervide isolates. IR typing and RAPD analysis representsimple, rapid and reproducible molecular techniques pro-viding a relevant characterisation of M. avium strains. It isintended to apply these PCR-based techniques on numer-ous isolates in order to have a validated test system forlarge epidemiological studies designed to identify the sour-ces of the observed wildlife infection. The reasons for anincreasing number of M. avium infections in red deer inAustria and the possible predisposing conditions are notknown so far. The epidemiology of M. avium infections ispoorly understood, but contamination of feeding places byinfected birds as source of M. avium infections should beconsidered.

A12^1

EXPERIMENTAL LEGIONELLA LONGBEACHAELUNG INFECTION IN INTRATRACHEALLY INOCU-LATED MICE

I. Gobin(1), M. SXus›a(2) and M. Doric¤(1)

(1) Department of Microbiology, Medical Faculty, Univer-sity of Rijeka, B. Branchetta 20, 51000 Rijeka, Croatia;(2) Robert Bosch Krankenhaus, Universita«t Tu«bingen,Anerbach Strasse 110, D-70376 Stuttgart, Germany

Legionella longbeachae was described as a new species ofLegionellaceae in 1981, when it was isolated from a patientwith pneumonia. Since then many infections due to L.longbeachae have been reported in North America, Europeand Australia. Most of the studies undertaken to under-stand the pathogenesis of Legionella infections have fo-cused on L. pneumophila and L. micdadei. Wishing to con-tribute to the understanding of the pathogenicmechanisms, we established an experimental model of rep-licative L. longbeachae (D4968 strain) infection in A/Jmice. The animals were infected by intratracheal inocula-tion using various doses of L. longbeachae (from 104 to109). In comparison to the results of our earlier studiesusing Legionella pneumophila we showed that the letaldose of L. longbeachae was more than 10-fold lowerthan that of L. pneumophila. Furthermore, the histologicalappearance of the lungs taken from infected mice wasconsistent with severe acute pneumonia, characterised byengorgment of the alveolar spaces with £uid and a largenumber of neutrophils and monocytes. Besides analysinglung pathohistology and bacterial counts in the lungs wealso followed the dissemination of L. longbeachae intoother organs. The bacteria could be also isolated fromthe blood, liver, spleen, kidney and brain tissues, already24-hours after intratracheal inoculation. The obtained re-sults underline the importance of this species as a causa-tive agent of acute lung infection and further studiesshould elucidate the factors included in the pathogenicprocess.

A12^2

SALMONELLA GENE EXPRESSION DURING IN-FECTION OF MAMMALIAN CELLS

J. Hinton, S. Eriksson, S. Lucchini, A. Thompson and M.Rhen

Institute of Food Research, Norwich Research Park, Nor-wich, NR4 7UA, UK

For intracellular pathogens such as Salmonellae, Myco-bacteriae and Brucellae, infection requires adaptation to



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the intracellular environment of the phagocytic cell. Thetransition from extracellular to intravacuolar environmenthas been expected to involve a global modulation of bac-terial gene expression, but the precise events have beendi⁄cult to pin down. We have determined the completetranscriptional pro¢le of intracellular Salmonella entericasv. Typhimurium following macrophage infection. Duringreplication in murine macrophage-like J774-A.1 cells, 919of 4451 S. Typhimurium genes showed signi¢cant changesin transcription. The expression pro¢le identi¢ed altera-tions in numerous virulence and SOS response genes,and revealed unexpected ¢ndings concerning the biologyof the Salmonella/macrophage interaction. Our data shedlight on the levels of phosphate, iron, magnesium in theintravacuolar environment and suggest that intracellularSalmonella are not starving. S. Typhimurium appears tobe using the Entner-Doudero¡ pathway to utilise carbonsources within macrophages. Almost half of the in vivo-regulated genes were of unknown function, suggesting thatintracellular growth involves novel bacterial properties.

A12^3

STUDIES ON THE POTENTIAL VIRULENCE FAC-TORS OF CLINICAL TRICHODERMA ISOLATES

Z. Antal(1), L. Kredics(1), I. Do¤czi(2), L. Man-czinger(3), F. Kevei(3), and E. Nagy(1,2)

(1) Hungarian Academy of Sciences and University ofSzeged, Microbiological Research Group, P.O. Box 533,H-6701 Szeged, Hungary; (2) Department of Clinical Mi-crobiology, Faculty of Medicine, University of Szeged, So-mogyi Be¤la te¤r 1, H-6725 Szeged, Hungary; (3) Depart-ment of Microbiology, Faculty of Sciences, University ofSzeged, P.O. Box 533, H-6701 Szeged, Hungary

The genus Trichoderma is already on the growing list ofpotential fungal pathogens in immunocompromised hosts.Most of the Trichoderma infections are reported from pa-tients undergoing peritoneal dialysis and from transplantrecipients. We studied the antifungal susceptibilities andthe potential virulence factors of ten clinical Trichodermaisolates. Most isolates proved to be resistant to £uconazoleand 5-£uorocytosine, and susceptible or intermediate toamphotericin B, itraconazole, and ketoconazole. The lowsusceptibility level of some clinical Trichoderma isolates toantifungal drugs may cause di⁄culties in the therapy ofinfected patients. The ecophysiological and enzymologicalinvestigation of clinical Trichoderma isolates may result inbetter understanding of their virulence factors. The exam-ined strains were able to grow at temperatures rangingfrom 10 to 40 ‡C and at pH-values ranging from 2.0 to9.0. The utilization of 80 compounds as carbon sources,and 34 compounds as nitrogen sources were also investi-gated. A series of amino acids were utilized by all of the

examined clinical Trichoderma isolates both as carbon andnitrogen source. The production of trypsin-like protease,chymotrypsin-like protease and Leu-aminopeptidase activ-ities were found to be common among the examinedstrains. The isoenzyme pro¢les of proteases were foundto be dependent on the culturing conditions in many cases.Growth at elevated temperatures, the ability to tolerateneutral pH and to utilize amino acids as carbon and nitro-gen sources, as well as the production of extracellular pro-teolytic enzymes are among the potential virulence factorsof Trichoderma strains involved in opportunistic infec-tions.

A12^4

MOLECULAR CHARACTERISATION OF HELICO-BACTER SPECIES IN NORMAL-, ATROPHIC- ANDTUMOUR-TISSUE OF PATIENTS WITH PANCREASCANCER

H.-O. Nilsson(1), H. Stafihlbrandt(1), W. Abu Al-Soud(1),A[ . Andre¤n-Sandberg(3), A[ . Ljungh(1), I. Ihse(2), U. Sten-ram(3), T. Wadstro«m(1)

(1) Med. Microbiology, Dermatology and Infection, (2)Dept. of Surgery and (3) Dept. of Pathology, Lund Uni-versity, Lund, Sweden

Non-gastric Helicobacter species are increasingly detectedin liver- and gallbladder tissue of patients with chronicliver diseases and liver cancer. Helicobacter sp. are fastid-ious and molecular assays are important to identify thesepathogens. Embedded pancreatic tissues (n=106), withnormal- (n=35), atrophic- (n=15) or tumour histology(n=51), diagnosed as ductal carcinoma (DC, n=43), neuro-endocrine cancer (NE, n=8), or multiple endocrine neo-plasia type 1 (MEN 1, n=4), were de-embedded, extractedusing the Qiagen Tissue protocol and ampli¢ed using aHelicobacter-speci¢c PCR-assay. Positive samples were an-alysed by denaturing gradient gel electrophoresis (DGGE)and compared with the migration distance of PCR-prod-ucts of reference Helicobacter strains. PCR-products weresequenced, aligned and subjected to BLAST and phyloge-netic analysis. In the DC patient group, 37.5%, 71%, and47.5% of normal-, atrophic-, and tumour tissues, respec-tively, were positive in the Helicobacter PCR-assay. Of theNE patients, 14% normal tissues and 71% of tumour tis-sues were Helicobacter positive. None of 4 normal- and 3/4tumour tissues of MEN 1 patients were positive. ByDGGE analysis, samples migrated similarly to either ofthree reference DNAs; H. pylori, H. rappini, and H. cinae-di. DNA-sequence analyses showed close similarity to H.sp liver, H. rappini, and H. cinaedi, and were in accor-dance with the DGGE. H. sp liver was uniformly distrib-uted among all tissue types, whereas H. rappini and H.cinaedi were detected only in tumour tissues. This PCR-



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protocol showed the same sensitivity as a method previ-ously applied on fresh pancreas tissue (Nilsson et al. JNatl Cancer Inst. 2002, 94).

A12^5

THE TYPE V SECRETION SYSTEM OF GRAM (-)BACTERIAL PATHOGENS: SECRETION ANDFUNCTIONAL ANALYSIS OF THE Tsh AUTO-TRANSPORTER OF A PATHOGENIC ESCHERI-CHIA COLI (APEC) 078:K80 STRAIN

C. Stathopoulos, M. Kostakioti, C. Newman and Y. Li

Department of Biology and Biochemistry, University ofHouston, Houston, TX 77204, USA

Avian pathogenic E. coli (APEC) is capable of colonizingthe respiratory track of chickens and turkeys, and causinga disease known as airsacculitis, which can develop into afatal systemic infection with pericarditis, perihepatiti, andcolisepticemia. We have isolated a temperature-regulatedvirulence factor, termed Tsh, from an APEC 078:K80strain. Our results show that Tsh is a 139 kDa extracellu-lar protein that is secreted across the bacterial cell envo-lope via the autotransporter pathway and is processed bya mechanism that it does not appear to be autoproteolytic.The precursor of Tsh consists of three functional domains:(i) an atypical amino-terminal signal sequence which is 52aa long and mediates secretion across the cytoplasmicmembrane, (ii) an 106 kDa Amino-terminal domain, and(iii) a 33-kDa carboxyl-terminal domain. After secretionand processing, the 106 kDa Amino-terminal domain ofTsh is released from the bacterial cell, whereas the 33-kDacarboxyl-terminal domain remains attached to the outermembrane. The 106-kDa Tsh domain displays the follow-ing functional properties : (i) it functions a hemagglutininthat agglutinates chicken erythrocytes, (ii) it functions as ahemoglobin-binding protein, and (iii) it is a serine-pro-tease-motif autotransporter protein. Our analysis providesinsight into the secretion, function, and structure of theTsh protein, which belongs to a growing family of bacte-rial extracellular virulence factors, named autotransport-ers, which utilize a unique, self mediated mechanism toachieve delivery by the host pathogen.

A12^6

TRANSDUCTION OF SHIGA TOXIN ENCODINGPHAGES OF ENTEROHAEMORRHAGIC ESCHERI-CHIA COLI UNDER DIFFERENT CONDITIONS

I. To¤th(1), H. Schmidt(2), M. Dow(1), E. Oswald(3), B.Nagy(1)

(1) Veterinary Medical Research Institute of the HungarianAcademy of Sciences, Budapest, Hungary; (2) TechnicalUniversity of Dresden, Carl Gustav Carus Medical Faculty,Department of Microbiology and Hygiene, Dresden, Ger-many; (3) UMR960 INRA de Microbiologie Moleculaire,Ecole Nationale Veterinaire de Toulouse, France

Dissemination of Shiga toxin (Stx) ^ encoding phages isthe most likely mechanism for the emergence of newSTEC serotypes and for the intergeneric spread of stxgenes. We proposed that Stx-negative enteropathogenicE. coli (EPEC) can be lysogenized by Stx-encoding phagesin vivo leading to novel E. coli pathogens. To model ourhypothesis we have investigated the ability of detoxi¢edStx2-encoding bacteriophages x3538(vstx2 : :cat) (Schmidtet al., Appl Environ Microbiol 65: 3855-3861, 1999) andStx1-encoding H-19B: :Tn10d-bla (Acheson et al., InfectImmun 66: 4496-4498, 1998) phages to lysogenize entero-pathogenic Escherichia coli (EPEC) strains in vitro and invivo. We were able to transduce porcine EPEC O45 strain1390 with x3538(vstx2 : :cat) in porcine ligated ileal loopsbut not EPEC prototype strain E2348/69. In the same invivo experiments neither strain 1390 nor strain E2348/69were lysogenised when E. coli K-12 containing H-19B: :Tn10d-bla was used as phage donor. Furthermore,none of the phages lysogenized none of the above strainsin vitro. The repeated success in the in vivo transductionexperiments of stx2-encoding phage to a porcine EPECstrain in pig loops, in contrast to failures in the in vitrotrials with these and other EPEC strains, let us suggestthat in vivo conditions are more e¡ective for phage trans-duction (and for this type of new pathogen evolution) thanin vitro conditions.



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A13^1

HIGH THROUGHPUT RAPID ARRAY HYBRIDISA-TIONS FOR MICROBIAL DIAGNOSTICS; DEVEL-OPMENT OF FLOW THROUGH MICROARRAYASSAYS FOR MYCOBACTERIAL CHARACTERISA-TION

R. M. Anthony(1), A. R. J. Schuitema(1), R. Hilhorst(2),P. R. Klatser(1), L. Oskam(1)

(1) Biomedical Research, KIT, Meibergdreef 39, 1105 AZ,Amsterdam, The Netherlands; (2) PamGene BV, PO Box1345 ‘s-Hertogenbosch, 5200 BJ, The Netherlands

Highly parallel assays such as microarrays have great po-tential for diagnostic microbiology as they provide thepossibility of testing for a wide range of bacterial geno-types using a single test. At present the utility of this typeof multiplex assay is limited by the cost, speed, and thecomplexity of results produced by high density arraybased assays. We have explored the possibility of using atargeted £ow through array system (the PamChip) thatallows real time hybridisation monitoring of up to 200elements for bacterial detection and characterisation. Ini-tial arrays have been produced to determine the sensitivityand speci¢city of the system. Results demonstrate that asingle nucleotide polymorphism can be detected in up to18 positions within a 20 base probe. Arrays have also beenproduced to a range of mycobacterial 16S rRNAs andshown, after 10 minutes hybridisation time, to accuratelyclassify rDNA PCR products from mycobacterial isolates.This proof of principle work is now being extended andarrays are under development that will allow the identi¢-cation of drug resistance in M. tuberculosis by the detec-tion of point mutations associated with resistant pheno-types. As PamChip arrays are also suitable for themeasurement of mRNA levels, we are additionally explor-ing the possibility of using targeted arrays of gene speci¢c60 mer probes to predict the response of cultured bacteriato antimicrobial agents.

A13^2

GENO(SERO)TYPING OF YERSINIA PSEUDOTU-BERCULOSIS

T. M. Bogdanovich(1), E. Carniel(2), H. Fukushima(3)and M. Skurnik(1,4)

(1) Department of Medical Biochemistry, University ofTurku, Kiinamyllynkatu 10, Turku 20520, Finland; (2) In-stitute Pasteur, Paris, France; (3) The Shimane PrefecturalInstitute of Public Health and Environmental Science, Ja-pan; (4) University of Helsinki, The Haartman Institute

Yersinia pseudotuberculosis strains are divided into se-rogroups (O:1-O:15) based mainly on antigenic di¡erencesin the lipopolysaccharide O-antigen (O-ag). The O-ag geneclusters of 21 reference strains of Y. pseudotuberculosisrepresenting all known serotypes fall into distinct groupsre£ecting the di¡erences in the chemical structure of theO-ags. Based on this information we developed a multi-plex PCR assay for genetic serotyping of Y. pseudotuber-culosis as an alternative to the conventional serotyping.Reference strains of Y. pseudotuberculosis were used toset up conditions for the assay. A combination of O-aggene cluster speci¢c primers was selected to produce aunique PCR product pattern for each reference strain.PCR conditions were optimized such that each PCR prod-uct ampli¢ed speci¢cally without interference from theother ampli¢cations running in the same tube. The result-ing multiplex PCR consisted of 9 pairs of primers andallowed identi¢cation of 14 serotypes and 2 serogroups,the O:4a-O:8 and the O:12-O:13 groups. The remaining3 serotypes (O:7, O:9, O:10) were negative in the multi-plex PCR and required an additional double-PCR analy-sis. Y. pseudotuberculosis strains of known serotypes,rough and non-typable strains from Japan, France andFinland were used to test the multiplex assay. Our resultsproved that conventional serotyping is vulnerable to errorsand that rough strains can be genetically (sero)typed. Wedetected heterogeneity within some serotypes and genetic(sero)typing can be useful for classi¢cation of such strains.Though the presented genetic typing is not perfect, it rep-resents a useful tool for rapid and reliable characterizationof Y. pseudotuberculosis.



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A13^3

A NOVEL IS ELEMENT, ISMpa1, IN MYCOBACTE-RIUM AVIUM SUBSP. PARATUBERCULOSIS

I. Olsen, T. B. Johansen, H. Bilman-Jacobe, S. F. Nilsenand B. Djonne

Department of Animal Health, National Veterinary Insti-tute, Oslo, Norway

Di¡erent insertion sequences have been identi¢ed in di¡er-ent strains of the M. avium complex. IS900 is found in M.avium subsp. paratuberculosis, IS901 in some isolates of M.avium subsp. avium, IS902 in M. avium subsp. silvaticum,IS1311 in all MAC strains and IS1245 in M. avium subsp.avium. A novel insertion element has been identi¢ed in M.avium subsp. paratuberculosis. The IS element, ISMpa1, is1500 bp and has one ORF encoding a putative transpo-sase. Three copies were identi¢ed in the M. avium subsp.paratuberculosis genome. The element had inserted intothe mycobacterial genes prrB and a homologue ofRv1593c, and between a putative cytochrome p450 oxy-genase and a putative hydrolase. The IS element waspresent in all (n = 11) M. avium subsp. paratuberculosisstrains and not detected in other mycobacterial speciesincluding 10 M. avium subsp. avium isolates of human,avian and porcine origin. However a porcine isolate ofM. avium subsp. avium and the reference strain IWGMT49did harbour ISMpa1. These strains belong to a previouslydescribed subgroup of M. avium subsp. avium based onIS1245 restriction fragment length polymorphism(RFLP) pattern and serovars. All the M. avium subsp.paratuberculosis strains examined had identical RFLP pat-tern when probed with sequences corresponding to the 5’end of ISMpa1 while a di¡erent pattern was seen in thepositive M. avium subsp. avium strains. This novel IS ele-ment might be a useful tool in strain classi¢cation andidenti¢cation of M. avium subsp. paratuberculosis whenused in combination with IS900.

A13^4

CROSS-PLATFORM COMPARISON OF FLUORES-CENT-AFLP FOR EPIDEMIOLOGICAL TYPING OFLEGIONELLA PNEUMOPHILA SEROGROUP 1

N. K. Fry(1), B. Afshar(1), P. Visca(2), D. Jonas(4), A.Underwood(3), T. G. Harrison(1)

(1) Respiratory & Systemic Infection Laboratory; (2) Uni-versity of Roma Tre, Rome, Italy; (3) Bioinformatics Unit,PHLS Central Public Health Laboratory, London UK; (4)Institut fu«r Umweltmedizin und Krankenhaushygiene, Frei-burg, Germany

A standardised non-£uorescent single-enzyme AFLP pro-tocol has been used widely by members of the EuropeanWorking Group on Legionella Infections for the epide-miological typing of L. pneumophila (http//www.ew-gli.org). Fluorescent-AFLP (f-AFLP) has also been re-ported to be a reproducible and highly discriminatorymethod suitable for typing, but there are few data on itsuse for L. pneumophila. This study compared a double-enzyme f-AFLP performed on three di¡erent platforms(one gel-based and two capillary) in di¡erent laboratories,using a well-characterised set of 50 strains. These were (i)an ALF Express (ii) a Beckman CEQ 8000 DNA AnalysisSystem, and (iii) an ABI Prism 3100 Genetic Analyzer.Data were analysed using either the Pearson correlationor categorical coe⁄cient in GelCompar or BioNumerics(Applied Maths) and compared to those obtained usingthe standard method. Preliminary data indicates that f-AFLP yields comparative results to AFLP. Using f-AFLP, the number of types ranged from 7-12, reproduc-ibility 0.70-.89 and epidemiological concordance from0.70-1.00, compared to 18, 1.00 and 1.00 respectively forAFLP. However, further optimisation of the f-AFLP pro-tocols is required.

A13^5

PYROSEQUENCING1, A NEW RAPID AND RELI-ABLE DNA-SEQUENCING TECHNOLOGY FOR MI-CROBIAL TYPING

B. Gharizadeh and P. Nyre¤n

Department of Biotechnology, Stockholm Center for Phys-ics, Astronomy and Biotechnology, Royal Institute of Tech-nology, Roslagstullbacken 21, SE-106 91 Stockholm, Swe-den

The Pyrosequencing1 technology has been utilized foridenti¢cation of di¡erent clinically relevant microorgan-isms. The Pyrosequencing method is a novel non-electro-phoretic DNA sequencing method based on sequencing-



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by-synthesis. The method employs a four-enzyme mixtureto sequentially determine the sequence of a target DNA inreal-time. For microbial typing, analyses were performedon amplicons derived from the 16S rRNA gene for bacte-rial detection, the 18S rRNA gene for fungal detection andthe L1 ‘‘consensus region’’ for genotyping of human pap-illomaviruses using PCR universal primers for ampli¢ca-tion. Multiple infections and unspeci¢c ampli¢cationproducts have been a problem for DNA sequencing tech-nologies when using general PCR primers for ampli¢ca-tion, as all available species are ampli¢ed. We have there-fore developed a multiple sequencing primer assay forspecimens harboring multiple infections/species and unspe-ci¢c ampli¢cation from genomic DNA when using generalprimer sets. The new assay is £exible and could be easilytailored according to the distribution and prevalence ofvarying microbial species by geographic region or otherdemographic factors for speci¢c typing of relevant species.Our results show the Pyrosequencing method is a fast ande⁄cient tool for microbial typing. The method is robustand well suited for large-scale programs as 96 or 384 sam-ples could be sequenced in parallel. Microbial typing bythe Pyrosequencing technology is also cost e¡ective, asevery genotyping will cost 1-2 USD.

A13^6

DETECTION OF ORGANOTIN COMPOUNDS WITHA SENSITIVE MICROBIAL BIOASSAY AND BIO-SENSOR

G. Thouand(1), M. J. Durand(1), H. Horry(1), P. Pic-art(2), Ph. Daniel(3), L. Bendriaa(2) and M. S. Dubow

(1) Universite¤ de Nantes, IUT de la Roche-sur-Yon, de¤-partement Ge¤nie Biologique, Laboratoire de Capteur Bac-te¤rien pour l’Analyse et le Contro“le, 18 Bd G. De¡erre,85035 La Roche-sur-Yon, France; (2) EŁ cole NationaleSupe¤rieure d’Inge¤nieurs du Mans, rue Aristote, 72085 LeMans Cedex 9, France, Laboratoire d’Acoustique de l’Uni-versite¤ du Maine, UMR CNRS 6613, Avenue Olivier Mes-siaen, 72085 Le Mans Cedex 9, France; (3) Laboratoire dePhysique de l’EŁ tat Condense¤, UMR CNRS 6087, AvenueOlivier Messiaen, 72085 Le Mans Cedex 9, France

A new diagnostic method was developed for the detectionof the highly toxic organotin compounds. The strain Es-cherichia coli TBT3 (Ec: :luxAB TBT3), isolated after arandom insertion of the luxAB (promoter-less) genes ofVibrio harveyi into the E. coli DH1 chromosome, speci¢-cally detects tributyltin (TBT) and dibutyltin (DBT). Thestrain Ec: :luxAB TBT3 produced light mainly at highgrowth rates either in batch or continuous culture. Thespectral emission of the recombinant E. coli was similarto that of the wild type Vibrio harveyi. Two peaks wereclearly identi¢ed, one at 491-500 nm and a second at 585-

595 nm with a highly sensitive spectrometer initially de-voted to Raman scattering. Growth conditions and culturemedium were not found to modify the spectrum of lightemission. The bioassay was conducted either after over-night culture of the bacterial strain on a dedicated glucosemedium or with lyophilized cells. Induction was recordedas signi¢cant after 60 min of contact time with organotins.The detection limits were 0.08 WM for TBT and 0.0001WM for DBT with a linear range of one logarithm. Inaddition, the repeatability and the reproducibility were 8% and 14% respectively. In the second step, a biosensorwas developed. Real time monitoring of bioluminescenceafter TBT induction occurred with continuous addition ofdecanal up to 300 WM that was not toxic throughout a 7day experiment. The design of our biosensor and the opti-misation of the main parameters that in£uence microbialactivity led to the capacity for on line detection of TBT.

A14^1

THE POSTANTIBIOTIC EFFECT OF 18 ANTIBIOT-ICS ON B. ANTHRACIS

A. Athamna(1), M. Massalha(1), M. Athamna(1,2), A.Nura(1), B. Medlej(1), I. Ofek(2) and E. Rubinstein(2,3)

(1) The Triangle Research And Development Center, Kfar-Qaraa, Israel; (2) The Department of Human Microbiol-ogy and (3) The Infectious Diseases Unit, Sheba MedicalCenter, Tel-Aviv University School Of Medicine, Tel-Aviv,Israel

The MIC’s and Postantibiotic e¡ect (PAE) of 18 antibac-terial agents on 2 strains of B anthracis (Sterne and theRussian Vaccine Strain ST-1) was tested. The PAE wasde¢ned as the time required for the viable counts of anti-biotics exposed bacteria to increase by one log10 abovethe counts observed after antibiotic removal. Methods:The MIC’s were determined by the microdilution methodThe PAE was determined in the recovery period of B.an-thracis strains following antibiotic exposure for 2h at 370Cat a conc. of 10X MICs. Antibiotics were removed byrepeated washings. Samples were obtained at -2h, at 0h(before and after washing) and then hourly for 7 h. Bac-terial count was determined by OD.The £uoroquinolones:cipro£oxacin, o£oxacin, levo£oxacin and moxi£oxacin.Penicillin G and amoxicillin, clarithromycin, telithromy-cin, clindamycin, rifampin and quinupristin-dalfopristinhad a MIC’s in the range of 0.03- 0.25Wg/ml. Cipro£oxacinand penicillin G being the most active with a MIC of 0.03Wg/ml. Erythromycin, vancomycin and linezolid were lessactive (MIC 0.5-2.5Wg/ml). Ceftriaxone was the least activehaving a MIC of 9.8Wg/ml. Chloramphenicol was inactiveMICs 250Wg/ml. The PAEs of the £uoroquinolones was2-5h. The macrolide’s PAE was 1-4h. The PAEs for thebeta-lactams, vancomycin, linezolid and chloramphenicol



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were 1-2h. The PAE of rifampin was 4-5h. Q/D had thelongest PAE duration 7-8h. Our results indicate that thePAE is unrelated to the MIC but may be associated withthe rapidity of bacterial kill. These observations may bearimportance on treatment regimens of human anthrax.

A14^2

FIRST APPEARANCE OF A BLAIMP GENE IN ANACINETOBACTER GENOSPECIES 10 CLINICALSTRAIN ASSOCIATED WITH REDUCED SUSCEPTI-BILITY TO IMIPENEM

G. J. Da Silva(1), M. Correia(2), C. Vital(3), G. Ri-beiro(3), L. Peixe(4) and A. Duarte(2)

(1) Fac. of Pharmacy of Coimbra Univiversity and Centerof Pharmaceutical Studies, Coimbra; (2) Fac. of Pharmacyof Lisboa University; (3) University Hospitals of Coimbra;(4) Fac. of Pharmacy of Porto University, Portugal

Two Acinetobacter genospecies 10 isolates were recoveredfrom hemocultures (24 and 48h) of an inpatient of Coim-bra University Hospital, Portugal. MIC of imipenem was8 mg/L, a value rather high for the majority of Acineto-bacter isolates. The main purpose of this study was tocharacterize the imipenem resistance mechanism in theseisolates. Methods. MICs of L-lactams were determined bythe E-test method. Carbapenemase activity was quanti¢edin extracts by spectrophotometry using imipenem as sub-strate in the absence and presence of zinc or EDTA. Genedetection was performed by PCR using blaIMP and class 1integron speci¢c primers. Amplicons were cloned ontopPCR-Script Cam SK(+) vector, transformed on Epicur-ian Coli XL10-Gold Kan competent cells. Results. Theisolates were resistant to cipro£oxacin, gentamicin, tobra-mycin and netilmicin. MICs of amoxicillin, cefoxitin andaztreonam were s 256 mg/L, 32 mg/L for ceftazidime and8 mg/L for imipenem. Extracts hydrolyzed imipenem (spe-ci¢c activity of 2,6 and 1,7 mU/mg protein). Analysis ofnucleotide sequence revealed the presence of a blaIMP genecassette inserted between two cassettes of aminoglycosideacetyltransferases genes (aacA7 and aac(3’)-I’). The threecassettes were inserted in a class 1 integron, designatedIn64. Conclusions. To our knowledge, this is the ¢rst de-scription of a blaIMP gene in an Acinetobacter genospecies10, a species not usually associated with nosocomial infec-tion. The results show that other species than A. bauman-nii may carry imipenem resistance genes. These genes in-serted in integrons can emerge in hospital environment byselective pressure due to the overuse of imipenem as wellas other antibiotics, like aminoglycosides.

A14^3

XENOBIOTIC EXPORT BY THE RND-TRANSPORT-ERS RECOGNIZING AND EXPELLING THE SUB-STRATES AT THE PERIPLASMIC SPACE

T. Nakae and S. Eda

Department of Molecular Life Science, Tokai UniversitySchool of Medicine, Isehara 259-1113, Japan

Most membrane transporters select and transport the sub-strate at the transmembrane domains. We show, here, thatresistance-nodulation-division (RND) family xenobiotictransporter in Pseudomonas aeruginosa selects substratesin the periplasmic domains. Transmembrane domain ismay not be involved in the substrate selectivity. The xeno-biotic transporters MexB and MexY export L-lactams andaminoglycosides, respectively. Taking an advantage ofhigh amino acid identity between these two, we carriedout domain swapping experiments. When two large peri-plasmic loop-domains of MexY were substituted with thatof MexB without touching the transmembrane domains,this MexY-MexB hybrid protein transported L-lactamsbut not aminoglycoside implying that periplasmic loopsselected the substrates. Substitution of either one of largeperiplasmic domain of MexB with that of MexY producednon-functional hybrid proteins. Next, we replaced thetransmembrane domains of MexB by that of MexY one-by-one. All hybrid proteins showed MexB-type (L-lactams)selectivity without exception suggesting that transmem-brane domains were less likely recognizingthe substrate.These results let us to formulate a model how the RND-family xenobiotic transporters export xenobiotics. TheRND-transporters of P. aeruginosa select xenobiotics bytwo large periplasmic domains and expel directly fromperiplasm. These results explain the following unansweredquestions. (i) How the transporters protect cells frommembrane deteriorating surfactants? (ii) How the antibi-otics with low membrane permeability such as L-lactamsand aminoglycosides could be e⁄ciently transported? (iii)How these low substrate-selective transporters preventleakage of intracellular materials? This is a novel and el-egant means of safeguard.



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A14^4

6-DESMETHYL ERYTHROMYCINS BY REPLACINGAN ACYLTRANSFERASE DOMAIN OF THE ERYTH-ROMYCIN POLYKETIDE SYNTHASE

H. Petkovic(1), R. E. Lill(1), R. M. Sheridan(1), B. Wil-kinson(1), E. L. McCormick(3), E. Hafner(3), H. A. I.McArthur(3), J. Staunton(1,2), P. F. Leadlay(1,2) S. G.Kendrew(1)

(1) Biotica Technology Limited, Cambridge, UK; (2) Uni-versity of Cambridge, UK; (3) P¢zer Inc, USA

The structural diversity found among polyketides arises inpart from the selection of starter and extender units by theacyltransferase domain (AT) of the polyketide synthase(PKS). It has been demonstrated that PKS engineeringto replace natural AT domains by Ats specifying alterna-tive units results in the production of novel polyketideanalogues. However, it has been claimed that some ATdomain swaps do not produce the desired compounds orproduce at low yield. For example, while AT replacementsproduce the expected products in modules 0, 1, 2, 3, 5 and6 of the erythromycin PKS (DEBS), replacement of mod-ule 4 AT with an AT speci¢c for malonyl-CoA has beenrepeatedly reported to fail. In contrast to these reports,and demonstrating the versatility of PKS engineering, wedescribe a strain containing a hybrid DEBS PKS in whichAT4 is replaced by AT2 from the rapamycin PKS and itsuse to produce 6-desmethyl erythromycins.

A14^5

BIOSYNTHESIS OF LINCOSAMIDE ANTIBIOTICSAND ITS GENETIC CONTROL

J. Janata, M. Albrechtova, D. Byrtusova, K. Hola, M. Je-linkova, J. Kopecky, L. Najmanova, J. Novotna, J. Spizek

Institute of Microbiology, Academy of Sciences of theCzech Republic, Videnska 1083, Prague 4, Czech Republic

In lincomycin biosynthesis, two basic precursors are ¢rstsynthesized separately, the aglycone propyl-L-proline fromL-tyrosine, and the sugar moiety methylthiolincosamide(MTL), from D-glucose. These two parts are condensedto N-demethyllincomycin (NDL) in a reaction catalyzedby NDL synthetase, an enzyme complex of readily disso-ciable non-identical subunits. Finally, NDL is convertedto lincomycin A by S-adenosyl methionine (SAM) depen-dent NDL methyltranferase. The lincomycin gene clusterof Streptomyces licolnensis was sequenced and 26 biosyn-thetic (lmb) and 3 resistance (lmr) genes were demon-strated. Functions of several genes of the cluster and oftheir protein products were clari¢ed. Gene lmbJ codes for

a subunit of the homooctameric SAM-dependent methyl-transferase. A larger open reading frame lmbIH coding forthe LmbIH protein is located downstream of the lmbJgene and is expressed in a coordinated manner withlmbJ. Genes lmbB1 and lmbB2 encode enzymes catalyzingthe conversion of L-tyrosine and L-dihydroxyphenylala-nine, intermediates in the amino acid part of the lincomy-cin biosynthetic pathway. Genes specifying subunits ofNDL synthetase, the key enzyme of the pathway, werelooked for and interactions among speci¢c proteins invivo were investigated by means of the yeast two-hybridsystem. Protein products of the genes lmbC, lmbD, lmbEand lmbF seem to be the likely candidates. Proteins LmbC,LmbD, LmbE and lmbF were puri¢ed and their activity tocatalyze NDL synthesis and mutually interact in vitro isnow tested.

A14^6

ANTIFUNGAL ACTIVITY OF V-253

V. V. Tetz(1), J. Vazquez(2), D. Boikov(2), N. K. Arte-menko(1) and G. V. Tetz(1)

(1) Microbiology Department, St. Petersburg Pavlov StateMedical University, 6/8, L. Tolstoy str., St.Petersburg,197089, Russia; (2) School of Medicine, Wayne State Uni-versity, Detroit, MI, USA

Treatment of fungal infections especially immunocompro-mised patients is actual problem of modern medicine. Us-ing of existing drugs is limited by their preferentially fun-gistatic action and spreading of resistant forms. Memberof new class of synthetic chemical compounds (derivativeof arylidenimino-1,3-pyrimidines) possessing a wide spec-trum of biological activities was evaluated against 42strains of Candida, (5) Saccharomyces cerevisiae, (5) Geo-trichum candidum, (5) Aspergillus spp., (5) Mucor spp. and(4) Trichophyton spp. These strains include known azoleresistant variants. Evaluation was performed in-vitro andcompared to £uconazole and amphotericin B using micro-broth dilution test. Fresh 24-hr old cultures of the variousspecies to be evaluated were subcultured in YEPD broth.In-vitro susceptibility studies (MICs & MLCs) were deter-mined. Acute toxicity was evaluated on the mice afterintrabdominal and oral administration. The minimal in-hibitory concentration (MIC50/MLC) of V-253 for di¡er-ent fungi varied from 1.0Wg/ml to 64 Wg/ml. The resultsindicate that V-253 demonstrated the best activity with thelowest MICs against C. parapsilosis, C. stellatoidea, S. ce-revisiae, Trichophyton spp. and Mucor spp., less activityagainst C. lusitaniae, G. candidum, C. krusei, and C. dub-liniensis, and the least activity against C. glabrata, C. tro-picalis, C. albicans and Aspergillus spp. In addition, it wasdetermined that V-253 has fungicidal activity as demon-strated by MLC for most of the isolates evaluated except



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for C. parapsilosis. Overall, V-253 demonstrated a broadspectrum of activity with good e¡ectiveness against nu-merous Candida spp. and Mucor spp. No toxic e¡ectswere registered during two weeks after administration of1000 mg/kg orally and 500 mg/kg intrabdominally.

A15^1

A SPONTANEOUS DELETION IN THE GLYCINEREPEAT REGION OF THE CTSR REGULATOR OFLISTERIA MONOCYTOGENES AFFECTS PIEZO-TOLERANCE, STRESS RESISTANCE AND VIRU-LENCE

K. A. G. Karatzas(1,2), J. A. Wouters(1), C. G. M. Ga-han(3), C. Hill(3), T. Abee(1) and M. H. J. Ben-nik(1,2,4)

(1) Wageningen Centre of Food Sciences (WCFS), Wage-ningen, The Netherlands; (2) ATO B.V., Wageningen, TheNetherlands; (3) University College Cork, Cork, Ireland;(4) current address: Institute of Food Research, Norwich,UK

A spontaneous piezotolerant mutant of Listeria monocyto-genes ScottA, named AK01, was previously isolated. Thismutant was immotile and showed cross-resistance to heat,acid, and H2O2 compared with the wildtype (wt) (KaratzasKA, Bennik MHJ. Appl Environ Microbiol. 68: 3183-319,2002). We now found that the observed phenotypic char-acteristics of the mutant are due to a single codon deletionin a key regulator, ctsR (class three stress gene repressor),in the region encoding the highly conserved glycine repeat.CtsR negatively regulates the expression of Class III heatshock genes (clpP, clpE, and the clpC operon which en-compasses ctsR itself). In strain AK01 and in allelic re-placement mutants we observed increased levels of ClpPprotein and clpP mRNA and ctsR mRNA, which indicatesthe involvement of Class III heat shock proteins in in-creased piezotolerance and other stresses. To our knowl-edge, this is the ¢rst report describing a mechanism under-lying piezotolerance of a Gram-positive bacterium.Importantly, strains expressing the mutant CtsR show sig-ni¢cantly attenuated virulence compared with the wtstrain. Ongoing studies indicate that a codon deletion inthis repeat region occurs relatively frequently in wt pop-ulations of L. monocytogenes, suggesting a role in adapta-tion to environmental stresses.

A15^3

ROLE OF NITRIC OXIDE IN THE RESPONSE OFSACCHAROMYCES CEREVISIAE CELLS TO HEATSHOCK AND HIGH HYDROSTATIC PRESSURE

T. Domitrovic(1), F. L. Palhano(1), C. Barja-Fidalgo(3),M. de Freitas(3), M. T. D. Orlando(2) and P. M. B.Fernandes(1)

(1) Dept. Cie“ncias Fisiolo¤gicas and (2) Dept. F|¤sica, Uni-versidade Federal do Esp|¤rito Santo, Vito¤ria, ES, 29040-090; (3) Dept. Farmacologia, Universidade do Estado doRio de Janeiro, RJ 20.551-030, Brazil

Nitric oxide (NO) is a simple and unique molecule thathas diverse functions in organisms, including intracellularand intercellular messenger. NO is produced by a nitricoxide synthase (NOS). Mammalian NOS occurs in bothconstitutive (NOS1 and NOS3) and inducible (NOS2) iso-forms. Recently, it has been shown that NO is produced incells from organisms as diverse as invertebrates, plants andmicroorganisms. Nevertheless, little is known about itsaction on cellular metabolism. The in£uence of NO onSaccharomyces cerevisiae cell growth and as a signal mol-ecule in stress response was evaluated. Aerobically grow-ing cells were more sensitive to an increase in NO intra-cellular concentration than the fermentative ones.However, low levels of NO revealed a cytoprotective prop-erty for both heat-shock and high hydrostatic pressurestresses. NOS expression analysis was performed in cellsat di¡erent growth phases. No signal could be detected onthe stationary phase cells. However, during ¢rst and sec-ond exponential growing phase, a single band could bevisualized for each isoform tested. The expression ofNOS1 and NOS3 seems to be constitutive throughout ex-ponential growth. Conversely, the expression of NOS2 wasclearly induced by fermentation. On the other hand, theWestern blot experiment showed that, di¡erently fromheat treatment, NOS2 expression was not decreased incells submitted to barotreatment. Those results reinforcethe hypothesis that an increase in NO intracellular con-centration leads to a stress protection.Financial Support: CNPq



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A15^4

ROLE OF CHARGE PROPERTIES OF BACTERIALENVELOPE IN BACTERICIDAL ACTION OF HU-MAN GROUP IIA PHOSPHOLIPASE A2 AGAINSTSTAPHYLOCOCCUS AUREUS

T. Koprivnjak, J. P. Weiss

Department of Microbiology, The University of Iowa, 3-403BSB, Iowa City, 52246 IA, USA

Mammalian Group IIA phospholipases A2 (PLA2) po-tently kill Staphylococcus aureus. Highly cationic proper-ties of these PLA2 are important for Ca

2+-independentbinding and cell wall penetration, pre-requisites forCa2+-dependent degradation of membrane phospholipidsand bacterial killing. To further delineate charge proper-ties of the bacterial envelope important in Group IIAPLA2 action against S. aureus, we examined the e¡ectsof mutations that prevent speci¢c modi¢cations of cellwall (dltA) and cell membrane (mprF) polyanions. In com-parison to the parent strain, isogenic dltA- bacteria areV30-100x more sensitive to the PLA2, whereas mprF

- bac-teria are 6 3 fold more sensitive. Di¡erences in PLA2sensitivity of intact bacteria re£ect di¡erences in cellwall, not cell membrane, properties since protoplastsfrom all three strains are equally sensitive to the PLA2.Diminished positive charge in PLA2 reduces PLA2 bindingand antibacterial activity. In contrast, diminished cell wallnegative charge by substitution of (lipo)teichoic acids withD-alanine reduces antibacterial activity of bound PLA2,but not initial PLA2 binding. Therefore, the potent anti-staphylococcal activity of Group IIA PLA2 depends oncationic properties of the enzyme that promote bindingto the cell wall, and polyanionic properties of cell wall(lipo)teichoic acids that promote attack of membranephospholipids by bound PLA2.

A15^5

REGULATION OF THE STRESS RESPONSE IN RHI-ZOBIA : AT LEAST THREE DISTINCT MECHA-NISMS REGULATE CHAPERONIN GENE EXPRES-SION IN RHIZOBIUM LEGUMINOSARUM

P. Gould, M. Maguire, P. A. Lund

School of Biosciences, University of Birmingham, Birming-ham, B15 2TT, UK

The GroE proteins GroES and GroEL (also known asCpn60 and Cpn10) are encoded in most bacteria by asingle heat shock inducible groE operon. Rhizobium. legu-minosarum has three groE operons, and analysis of mRNAand protein levels shows that the three operons are di¡er-

entially expressed. One operon (cpn-1) is highly expressed;the GroEL homologue encoded by this operon (Cpn60-1)constitutes ca. 90% of the overall chaperonin pool, and isessential for growth. The second operon (cpn-2) is ex-pressed more weakly, and the third operon (cpn-3) is ex-pressed more weakly still and only under microaerobicconditions. Expression of this operon is NifA regulated.All three operons are heat shock inducible. Sequence anal-ysis of the promoters for the three operons suggests regu-lation by both positive (alternative sigma factor) and neg-ative (HrcA repressor) mechanisms. Mutagenesis of thepromoters of the cpn-1 and cpn-2 operons suggested thatthe former is HrcA regulated but the latter is not. This wascon¢rmed by the cloning and knockout mutagenesis ofhrcA. An rpoH homologue has also been identi¢ed,cloned, and mutagenised. Unlike the situation with E.coli, mutation of the rpoH gene does not produce a severephenotype, consistent with this protein not having a rolein the regulation of the essential cpn-1 operon. The doublehrcA rpoH mutant grows better than does the rpoH singleknockout, suggesting that over-expression of the cpn-1operon can partially compensate for the decreased expres-sion of the rpoH-dependent operons.

A15^6

MITOGEN-ACTIVATED PROTEIN KINASEHWHOG1P FROM HALOPHILIC BLACK YEASTHORTAEA WERNECKII

M. Turk(1), N. Gunde-Cimerman(1), A. Plemenitas›(2)

(1) Department of Biology, Biotechnical Faculty, Univer-sity of Ljubljana, Vec›na pot 111, SI-1000 Ljubljana, Slov-enia; (2) Institute of Biochemistry, Faculty of Medicine,University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana,Slovenia

Halophilic black yeast Hortaea werneckii, previosly knownas a causative agent of a human super¢cial mycotic infec-tion tinea nigra, was found to occur naturally in hypersa-line waters of marine salterns. It could grow at salinitiesranging from 0% to saturated solution of NaCl (35% NaCl(w/v)). Besides the accumulation of glycerol as a compat-ible solute, di¡erent responses to increased salinity wereobserved in halophilic H. werneckii at the level of mem-brane composition and £uidity as well as in metabolicresponses. In adapting to hyperosmotic stress high-osmo-larity glycerol response signaling pathway (HOG pathway)is essential for the induction of a set of osmoadaptiveresponses required for cell survival. In Saccharomyces ce-revisiae exposure of yeast to hyperosmotic stress activatesa key mitogen-activated protein kinase (MAPK), Hog1p.A homologue of HOG1 gene was isolated from H. wer-neckii encoding a putative 359 amino acid protein,HwHog1p. Its activation by increased salinity is regulated



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at the post-transcriptional level. Phylogenetic analysistogether with functional classi¢cation revealed that it be-long to the subgroup of MAP kinases, the stress-activatedprotein kinases (SAPKs). Similarly to other SAPKsHwHog1p contains a TGY motif within a protein kinasecatalytic domain. On the basis of the comparison of pre-dicted HwHog1p amino acid sequence to a number ofpreviously identi¢ed Hog1p homologues from di¡erent or-ganisms a phylogenetic tree was constructed.

A15^2

PLASTIDS OF UNICELLULAR EUKARYOTES ASDRUG TARGETS

J. Krajc›ovic›, L. Ebringer

Institute of Cell Biology, Comenius University, 811 07 Bra-tislava, Slovakia

Plastids of the photosynthetic £agellate Euglena gracilisare extremely sensitive to various chemical and physicalagents. Plastids were acquired through establishment ofan endosymbiotic relationship between a eukaryotic hostand a photosynthetic prokaryote ^ a cyanobacterium.Evolution of an endosymbiont into a plastid has beenassociated with a loss of host cell function resulting incell growth and viability being dependent upon a func-tional plastid. Apart from the plastid’s own biosynthesisand photosynthesis, plastids perform a wide range of met-abolic functions, from heme, fatty acid and aminoacidbiosynthesis, nitrate assimilation, etc. to storage function.A reduced but functional plastid genome is retained innonphotosynthetic residual plastids of holoparasitic £ow-ering plants, some heterotrophic algae, and in protozoanapicomplexan parasites. The plastid’s own machineries,being cyanobacterial in origin, o¡ers conventional targetsfor antibacterial drugs inhibiting plastid functions. Wehave demonstrated that practically all inhibitors of bacte-rial protein- and DNA-synthesis a¡ected chloroplasts inthe £agellate E. gracilis inducing transformation of greencells to white ones without any loss of cell viability ^bleaching phenomenon. Identi¢cation of bleaching agentshas taken on additional signi¢cance with the discovery ofa residual plastid genome contained within the apicoplastof protozoan parasites of the phylum Apicomplexa. Or-ganisms within this phylum such as Plasmodium, Toxo-plasma and Eimeria are major disease causing agents. Bac-terial metabolic pathways in the relict plastid ofapicomplexan parasites make this organelle a promisingnew parasite-speci¢c target for therapeutic agents andfor drug development. Bleaching in Euglena could providea rapid screening method for these agents.

A16^1

IDENTIFICATION OF OSMOTIC STRESS-INDUCEDGENES IN ENTEROBACTER SAKAZAKII

P. Breeuwer, J. E. Germond, I. Jankovic, M. Peterz, and H.Joosten

Nestle¤ Research Center, Nestec Ltd, Vers-chez-les-Blanc,1000 Lausanne 26, Switzerland

In the last decades Enterobacter sakazakii has been impli-cated in several incidents as the cause of meningitis andenterocolitis in premature infants. The mode of transmis-sion is not always clear, but in some cases there wereindications that powdered milk formulas might havebeen the source of E. sakazakii. Why this bacterium waspresent and could survive in this type of products is stillunclear. However, since dried infant formulas have a lowwater activity (approx. 0.2), survival in such a dry environ-ment will largely depend on the osmotic and/or dry stressresistance of the bacteria. The objective of this study wasto identify the genes in E. sakazakii, which are inducedupon osmotic stress. Inducible promoters were selected bycloning partially SAU3A digested DNA fragments up-stream the promoterless red £uorescent protein reportergene. The resulting clones, after transformation of theplasmids into E. sakazakii 1387, were arrayed in 96-wellplates, and screened for red £uorescence upon exposure toosmotic stress. The inserted DNA regions of the positiveclones were sequenced and analysed. Using this proceduremore than 30 genes potentially involved in osmotic stresswere identi¢ed. The genes could be subdivided in a num-ber of categories including those related to heat stress, ironuptake, oxidative stress, stringent response, and ATP pro-duction. Of particular interest was a gene, designatedEs181, of which we could not ¢nd any homology, butshowing a large induction after osmotic stress. Knockoutmutants should give further insight in the function of thisgene.



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A16^2

GENOMICS ASSISTED IDENTIFICATION OF ANTI-FUNGAL STRATEGIES

S. Brul(1,2), H. de Nobel(3), I. Bom(2), A. Zakrzew-ska(1), A. Boorsma(1),C. Resende(2), P. Coote(4), H.van der Spek(1) and F. Klis(1)

(1) Swammerdam Institute for Life Sciences, University ofAmsterdam, Nieuweachtergracht 166, 1018 WV Amster-dam; (2) Unilever R&D, Vlaardingen; (3) Genencor R &D Leiden, The Netherlands; (4) University of St. Andrews,St. Andrews, Five, United Kingdom

Fungi cause major food spoilage and health problems. Weprovide an example of how genomics can help identifynew antifungal targets for food preservation. As a modelsystem Saccharomyces cerevisiae was used. Sorbic acid, alipophilic weak-organic acid food preservative, was addedto growing yeasts at 0.9 mM. Following a clearly en-hanced lag-time, cells gained resistance to the weak-acid.At the point were the cells just resumed growth, sampleswere analysed using both proteomics and genome-widearray technologies. The results showed that cells stressedwith sorbic acid activated genes encoding the multidrugresistance pump Pdr12p, HSP26 encoding a general stressprotein involved in correct protein folding, genes encodingenergy generating proteins, and mating factors. A large setof genes encoding proteins involved in the cell integrity(protein kinase C) stress response pathway were induced,corroborating the membrane perturbing e¡ect sorbic acidis presumed to have, and including genes encoding glyco-sylphosphatidylinositol (GPI) anchored proteins. Finally,many genes involved in the functioning of transposonswere induced, suggesting that cells might ultimately at-tempt to ‘mutate’ their way out of the stress imposed.All these stress responses represent possible leads fornew antifungal strategies. As an accessible target, we fur-ther analysed biogenesis of the extracellular GPI linkedcell wall proteins. We used heat resistant beta-1,6 glucanfragments to compete with the wall incorporation of GPI-anchored proteins. A fusion protein of Cwp2p-alphagalac-tosidase was mistargeted to the extracellular medium inthe presence of beta-1,6 glucan oligomers. In the presenceof these compounds, cells were indeed sensitized to mem-brane active compounds. Currently, we are extending ourexperiments to include next to sorbic acid also sorbic al-cohol, the presumably membrane active antimicrobialagent chitosan and membrane perturbing antifungal pep-tides. Initial results with these compounds will be dis-cussed.

A16^3

GENE EXPRESSION PROFILES OF ACID TOLER-ANT SALMONELLA TYPHIMURIUM DT104 ISO-LATES

A. P. H. M. Hermans(1,2), T. Abee(2), H. J. M. Aarts(1)

(1) RIKILT Institute of Food Safety, P.O. box 230, 6700AE Wageningen, The Netherlands; (2) Food Hygiene andMicrobiology Group, Department of Agrotechnology andFood Sciences Wageningen University, Wageningen, TheNetherlands

During the last decades, the incidence of foodborne dis-eases has increased in many parts of the world and newfoodborne pathogens, like Salmonella TyphimuriumDT104, have been identi¢ed. These pathogens have thepotential to adapt to a wide variety of food preservationrelated stress conditions, i.e. starvation, temperature ex-tremes and (weak) acids, which can result in an increasedsurvival in the GI-tract. The objective of our research is toanalyze gene expression pro¢les of acid tolerant Salmonel-la Typhimurium DT104 isolates to gain detailed insight inthe molecular pathways involved in low pH survival. Acidtolerant and acid sensitive variances were selected from anumber of food Salmonella Typhimurium DT104 isolates,by exposure to pH=2.5 after adaptation at pH=5. A the-matic microarray was constructed containing oligo’s ho-mologous to Salmonella Typhimurium LT2 stress responseand virulence related genes. Cells of both isolates wereharvested in the exponential, transitionary and stationarygrowth phase at pH=7 and pH=5 from the high and lowresponder Salmonella Typhimurium DT104 isolates andthe obtained RNA samples were hybridized to the micro-array. The microarray data analysis resulted in many dif-ferential expressed genes between growth phases of bothpHs. Furthermore a comparison of the acid tolerant andacid sensitive isolates resulted in a limited number of dif-ferential expressed genes. Nevertheless some interestingacid survival related genes were up regulated in the acidtolerant isolate. The thematic microarray provides indeeda tool to gain detailed insight in the molecular pathwaysinvolved in low pH survival.



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A16^4

STRESS RESPONSES OF ESCHERICHIA COLI TOTHE NEUTROPHIL BACTERICIDAL / PERMEABIL-ITY-INCREASING PROTEIN

P. Prohinar and J. P. Weiss

Department of Microbiology, The University of Iowa, 3-403BSB, Iowa City, 52246 IA, USA

The bactericidal/permeability-increasing protein (BPI) ofneutrophils is a cytotoxic and endotoxin-neutralizing pro-tein with high potency and selectivity for Gram-negativebacteria. E. coli can tolerate initial sublethal attack by lowdoses of BPI in part by use of the global transcriptionalregulator, OmpR. The fate of E.coli exposed to BPI de-pends on both OmpR-independent mechanisms engaged inouter membrane repair and OmpR-dependent processesthat modulate porin synthesis and retard progression ofinjury from the outer to the inner membrane. The sensorkinase EnvZ that normally works in concert with OmpR isnot needed for BPI-induced changes in OmpF/C synthesissuggesting involvement of alternative phospho-donors tomodify OmpR in response to BPI. We demonstrate herethat both acetyl-phosphate and outer membrane phospho-lipase (pldA) are needed for BPI-induced porin alterations.Our ¢ndings suggest that the conserved outer membranephospholipase plays a sensory role, activated by outermembrane perturbations induced by BPI to release freefatty acids and thereby increase levels of acetyl-phosphatewhich in turn phosphorylates OmpR to a¡ect gene expres-sion. Initial micro-array analyses have identi¢ed a subsetof genes di¡erentially regulated in bacteria exposed to BPIincluding genes encoding products engaged in TCA cycle-dependent oxidative metabolism (down-regulated) andthose mediating protective e¥ux systems (e.g. marA ; up-regulated). Given the prominence of BPI in innate immunedefenses against Gram-negative bacteria, identi¢cation ofprotective stress responses may reveal mechanisms of bac-terial tolerance not only to isolated BPI but also to intactneutrophils.

A16^5

POST GENOMIC ANALYSIS OF LISTERIA MONO-CYTOGENES USING TARGETED PLASMID INTE-GRATION

R. Rea, C. Hill, and C. G. M. Gahan

Department of Microbiology, National University of Ire-land, Cork, Ireland

The Gram positive foodborne pathogen Listeria monocy-togenes must adapt to adverse environmental conditions

encountered in the food processing environment and dur-ing host infection. We have analysed the contribution ofputative regulatory loci to growth under sub-optimal en-vironmental conditions and during murine infection. Dis-ruption of two in vivo inducible loci encoding a type Itopoisomerase and a putative DNA methylase revealed asigni¢cant role for the DNA methylase in growth at alka-line pH and in the presence of ethanol (5%). A MarRfamily regulator was required for growth at alkaline pHand in the presence of ethanol. Neither the topoisomerase,methylase, nor marR were involved in virulence in themurine model of infection. Disruption of the alternativesigma factor Sigma H resulted in a mutant which wasmildly a¡ected in virulence. Murine studies indicated aminor role for this sigma factor in the infectious process.Strikingly, disruption of both perR and fur genes resultedin mutants that are signi¢cantly a¡ected in virulence, withthe fur mutant demonstrating the greatest reduction invirulence potential. Both perR and fur mutants demon-strated signi¢cantly increased sensitivity to ethanol andalkaline conditions, and exhibited increased resistance tohydrogen peroxide. The fur mutant was sensitive to lowiron conditions in vitro. The virulence defect of both furand perR mutants was rescued by iron-overload followingesculetin treatment of mice suggesting that the in vivo roleof these loci is to procure iron for bacterial growth. Futurework will concentrate on de¢ning the roles of these genesand their associated regulons in the pathogenesis of L.monocytogenes.

A16^6

OXIDATIVE STRESS RESPONSE IN FRANKIASTRAIN R43: A PROTEOMIC APPROACH

J. Vieira(1), P. Moradas-Ferreira(1,2), A. Sellstedt(3)and F. Tavares(1,4)

(1) Instituto de Biologia Molecular e Celular, Microbiolo-gia Celular e Aplicada, Universidade do Porto, Rua doCampo Alegre, 823, 4150-180 Porto, Portugal; (2) Institu-to de Cie“ncias Biome¤dicas Abel Salazar, Universidade doPorto, Portugal; (3) Umeafi Plant Science Centre, UmeafiUniversity, S-901 87 Umeafi, Sweden; (4) Faculdade deCie“ncias, Departamento de Bota“nica, Universidade do Por-to, Portugal

Frankia is a group of actinomycetes, which are able toinduce symbiotic nitrogen-¢xing root nodules in a widerange of plant species. Although the cellular aspects ofactinorhizal infections have been extensively studied, theearly interactions between the microsymbiont and the hostplants are still poorly understood. The conceivable releaseof reactive oxygen radicals by the host plants during in-fection, points towards the existence of an e⁄cient oxida-tive defence mechanism in Frankia during infection. This



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work aims to reveal the mechanisms involved in the oxi-dative stress response in symbiotic infectious bacteria. Re-cently we have characterised two hydroperoxidases forFrankia strain R43: a monofunctional catalase and a bi-functional catalase-peroxidase. Activity staining of non-denaturing PAGE together with Western-blot analysesshowed that both enzymes were constitutive and upregu-lated during the stationary growth-phase and by treatmentwith H2O2 and paraquat in free-living Frankia strain R43.A proteomic survey using 2-D electrophoresis and MAL-DI-TOF analyses was carried out to identify di¡erentiallyexpressed proteins in Frankia cells stressed with H2O2 orparaquat. Three proteins were shown to be upregulated:one by treatment with H2O2; another by paraquat; and athird by both H2O2 and paraquat. Partial sequence data ofthe two peptides whose expression increased with paraquatdid not show a high homology with known protein se-quences. At present we are aiming at obtaining DNAprobes using the data generated from the partial sequencesof these proteins to clone and to characterise the genes.This work was supported by FCT, Portugal (grant ^ POC-TI/35283/BCI/2000)

A17^1

REGULATION AND PHYSIOLOGICAL ROLE OFCYANIDE-RESISTANT OXIDASE IN FUNGI

A. Yu. Arinbasarova, A. G. Medentsev, V. K Akimenko

G.K. Skryabin Institute of Biochemistry and Physiology ofMicroorganisms, Russian Academy of Sciences, 142290Pushchino, Moscow region, Russia

Cyanide-insensitive respiration is widespread among high-er plants, fungi, yeasts and protozoa. The alternative ox-idase is located in the inner mitochondrial membrane andinsensitive to cyanide, azide, antimycine A, myxothiazoleand CO, but speci¢cally inhibited by benzohydroxamicacid and propyl gallate. The respiration is due to the func-tioning of cyanide-insensitive oxidase, which transfers theelectrons from reduced ubiquinone (coenzyme Q) to oxy-gen independently of the main cytochrome respiratorychain. Data on the induction and regulation of cyanide-resistant oxidase in eukaryotic microorganisms are pre-sented. A decrease in the cell energetic parameters maybe one of the signals inducing the expression of the alrter-native oxidase gene. Some evidence exists suggesting thatcAMP and Ca2+ act as intracellular signals in expressionof the gene encoding alternative oxidase in fungi. Undercertain conditions alternative oxidase is synthesized bycell, but it is in the unactive state. Activation of the alter-native pathway of cell respiration is usually observed whenelectron transport via cytochrome pathway is inhibited.The question about activity of the alternative pathway invivo and the mechanism of electron partitioning between

the main and alternative pathways is very important. Weshowed that alternative oxidase is unable to compete withthe cytochrome respiratory chain for electrons and onlytransfers the electrons that are super£uous for the mainpathway. Possible physiological role of alternative oxidaseis discussed (compensation of respiration when the cyto-chrome pathway activity is impaired, thermogenic func-tion, protection against reactive oxygen species , partici-pation in protection against phytopathogens).

A17^2

CELL DEATH PROCESSES IN STREPTOMYCETES

C. Hardisson, E. M. Migue¤lez and M. B. Manzanal

Facultad de Medicina, Universidad de Oviedo, Oviedo,Spain

A major feature of streptomycetes is their ability to carryout a complex developmental life cycle that, phylogeneti-cally, can be considered as one of the probably severalevolutionary attempts at multicellularity. Recent observa-tions, however, have revealed that during the life cycle ofthe streptomycetes the hyphae die by following two quitedistinct sequences of degenerative events : they either au-tolyse or undergo physiological cell death. Autolytic hy-phal death, which a¡ects a minority of the hypha through-out the colony, is characterized by the early degradationof the cell wall as a consequence of an uncontrolled, lyticaction of murein hydrolases. In physiological cell death,however, the hyphae undergo an orderly process of inter-nal cell dismantling that takes a long time: progressivedisorganization of the nucleoid, and degradation ofDNA and cytoplasmic constituents followed by shrinkageand distortion of the hyphal shape.Whereas cell deathphenomena in streptomycetes have some traits of theirown, they also display striking similarities with those oc-curring in the higher eukaryotic developmental systems.Thus, whereas physiological hyphal death mimics manyaspects of apoptosis, autolysis shows similarities to ne-crotic cell death. Regardless of the exact mechanism whichcauses hyphal death, the present observations reveal thatmulticellular behavior of streptomycetes resembles that ofhigher organisms much more than has been assumed pre-viously.



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A17^3

PENETRATION OF CHROMIUM COMPOUNDSTHROUGH YEAST CELL ENVELOPE

M. Pas›(1), R. Milac›ic›(2), K. Dras›lar(1), N. Pollak(1)and P. Raspor(1)

(1) Biotechnical Faculty, University of Ljubljana, Jamni-karjeva 101, 1000 Ljubljana, Slovenia; (2) ‘‘Joz›ef Stefan’’Institute, Department of Environmental Sciences, Jamova39, 1000 Ljubljana, Slovenia

Chromium bioaccumulation by yeast cells has been exten-sively studied in the last years, mainly from the point ofview of two possible applications, i.e. detoxi¢cation ofenvironment polluted by hexavalent chromium and prep-aration of yeast biomass enriched with organically boundforms of chromium that possess biological activity. Suchbiomass has a good potential to be used as a dietary sup-plement in animal or human nutrition. The oxidation stateof chromium plays an important role in transport of chro-mium ions through yeast cell envelope. Our study inves-tigated the in£uence of di¡erent Cr(III) and Cr(VI) com-pounds in the cultivation medium on the uptake andlocalization of chromium in the cell structure of the yeastCandida intermedia. The morphology of the yeast cell sur-face was observed by the scanning electron microscopy.Results demonstrated that the growth inhibitory concen-tration of Cr(III) in the cultivation medium inducedchanges in the yeast cell shape and a¡ected the buddingpattern, while inhibitory concentration of Cr(VI) did notcause any visible e¡ects on morphological properties ofthe yeast cells. The amount of total accumulated chromi-um in yeast cells and the distribution of chromium be-tween the yeast cell walls and spheroplasts were deter-mined by atomic absorption spectroscopy. No signi¢cantdi¡erences were found neither in total chromium accumu-lation nor in the distribution of chromium in yeast cellwalls and spheroplasts between the two of Cr(VI) com-pounds. Conversely, substantial di¡erences betweenCr(III) compounds were demonstrated in the total uptakeas well as the localization of chromium in yeast cells.Thestudy was supported by Ministry of Science and Technol-ogy, Republic of Slovenia (Project No. J4-7454).

A17^4

TRANSCRIPTION OF THE NOS GENE CLUSTERENCODING NITROUS OXIDE REDUCTASE OFPARACOCCUS DENITRIFICANS

R. J. M. van Spanning, N. Saunders, S. Ferguson, R. Doo-deman, J. Hornberg and H. V. Westerho¡

Department of Molecular Cell Physiology, Free Universityof Amsterdam, De Boelelaan 1087, 1081 HV Amsterdam,The Netherlands

The nosCRZDFYLX gene cluster encoding nitrous oxidereductase in Paracoccus denitri¢cans has been cloned andsequenced. The nosZ gene encodes the subunits of nitrousoxide reductase and the nosDFYLX genes encode proteinsrequired for copper uptake and assemblage in the enzyme.The nosC gene is so far unique to P. denitri¢cans. Up-stream of but divergently transcribed from the nos genes,the paz gene, encoding pseudoazurin, was found. The 200bp intergenic region between paz and the nos genes con-tains two FNR-boxes. Studies with promoter lacZ genefusions revealed that this region encompasses the pazand nosC promoters. The latter was positively regulatedby NNR and, to a lesser extent, by FnrP. A third pro-moter is present in front of the nosZ gene. NosR is atransmembrane protein which contains six transmembranehelices, a large periplasmic loop and two [4Fe-4S] clustersat the C-terminal cytoplasmic end. The protein is homol-ogous to NirI, which controls transcription of the nitritereductase gene cluster of P. denitri¢cans, and to NosRsequences from other bacteria. The putative periplasmicand cytoplasmic loops were fused to alkaline phosphataseand ^-galactosidase, respectively, and analyses of their ac-tivities con¢rmed the predicted topology of NosR. ThenosR mutation resulted in a Nos- phenotype as a resultof impaired transcription activation of the nosZ promoter.

A17^5

THE ENTEROBACTIN-DIHYDROXYBENZOYLSER-INE COMPLEX IS INVOLVED IN VIRULENCE OFSALMONELLA ENTERICA

P. H. Williams(1), W. Rabsch(2), U. Methner(3), W.Voigt(2), H. Tscha«pe(2), R. Reissbrodt(2)

(1) Department of Microbiology and Immunology, Univer-sity of Leicester, Leicester, UK; (2) Robert Koch-Institut,Wernigerode, Germany; (3) Bundesforschungsanstalt fu«rViruskrankheiten der Tiere, Jena, Germany

The role of enterobactin, the principal siderophore of Sal-monella enterica, and its cognate receptors, the iron regu-lated outer membrane proteins (IROMPs) FepA and



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IroN, in virulence is controversial. To clarify the matter,we constructed single, double and triple mutants of S.enterica serovar Typhimurium and S. enterica serovar En-teritidis defective in the expression of the IROMPs pro-posed to be catecholate receptors, FepA, IroN and Cir.The mutants were characterized by assessing the uptake ofa number of catecholate type siderophores (naturally-oc-curring and chemically synthesised compounds) in growthpromotion tests and by MIC tests with a siderophore-cephalosporin conjugate. Unique patterns of uptake wereidenti¢ed for each IROMP. Speci¢cally, FepA and IroNwere con¢rmed to be required for transport of enterobac-tin and Cir was shown to function as a receptor for theenterobactin breakdown products, trimeric, dimeric andmonomeric 2,3-dihydroxybenzoyl-serine. In vitro assaysfor adherence to and invasion of the rat small intestinalepithelial cell line IEC6 gave similar results for the parentS. enterica serovar Enteritidis strain and its fepA and fepAiroN derivatives. However, adherence and in particularinvasion were markedly reduced in the triple mutant miss-ing FepA, IroN and Cir proteins. Similar results were seenin vivo in experimental infections of day-old chicks. Theresults point to a contribution of the enterobactin-dihy-droxybenzoylserine complex in the virulence of S. entericaserovar Enteritidis, particularly for the process of host cellinvasion.

A17^6

PSEUDOMONAS CHLORITIDIMSUTANS : A NON-DENITRIFYING, CHLORATE-REDUCING BACTE-RIUM

A. F. W. M. Wolterink, S. W. M. Kengen and A. J. M.Stams

Laboratory of Microbiology, Wageningen University, H.van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands

A Gram-negative, facultatively anaerobic, dissimilatorychlorate-reducing bacterium, Pseudomonas chloritidismu-tans, was isolated from biomass of an anaerobic chlo-rate-reducing bioreactor. Chlorate is reduced to chloriteby a chlorate reductase. Chlorite is converted into chlorideand oxygen by a chlorite dismutase. Phylogenetic analysisof the 16S rDNA sequence showed 100% sequence simi-larity to Pseudomonas stutzeri DSM 50227 and 98.6% se-quence similarity to the type strain of P. stutzeri(DSM5190T). However, DNA-DNA hybridization indi-cated that P. chloritidismutans and P. stutzeri DSM50227were not related at the species level. P. chloritidismutanswas di¡erentiated from P. stutzeri with respect to the ca-pacity for dissimilatory chlorate reduction, and its inabil-ity of denitri¢cation. Puri¢cation and characterization ofthe heterotrimeric chlorate reductase of P. chloritidismu-tans showed resemblance to membrane bound nitrate re-

ductase in number of subunits (3), cytoplasmic localiza-tion, molybdenum content (EPR spectrum) and N-terminal sequence of the L-subunit. The major di¡erenceis the substrate speci¢city; only chlorate and bromate re-ductase activity was demonstrated. Although P. stutzerialso showed chlorate reductase activity, it cannot growon chlorate because chlorite dismutase was absent. Chlo-rite dismutase was puri¢ed from the periplasmic fractionof P. chloritidismutans and was found to be a homotetra-mer composed of 31 kDa subunits. The pyridine-NaOH-dithionite-reduced dismutase showed absorption peaks in-dicative for a protoheme IX. The catalytic e⁄ciency (kcat/Km) resembles the values of other puri¢ed chlorite dismu-tases of strain GR-1 and Ideonella dechloratans.

A18^1

PSEUDOMONAS BACTERIA DEGRADING POLYCY-CLIC AROMATIC HYDROCARBONS

A. M. Boronin

Institute of Biochemistry and Physiology of Microorgan-isms, Russian Academy of Sciences, Pushchino, MoscowRegion, 142290, Russia

The diversity of Pseudomonas bacteria capable of degrad-ing polycyclic aromatic hydrocarbons (PAH) and the prev-alence of plasmids controlling these processes were re-vealed. IncP-9 plasmids for naphthalene degradationdi¡er in the structure of their backbone segments fromthose for caprolactam degradation and R-plasmids ofthe same incompatibility group and comprise two sub-groups due to their replicon structure. There is a high levelof DNA homology among genes for naphthalene catabo-lism of di¡erent plasmids. However, catabolic operons andregulatory genes occur on di¡erent replicons: a plasmid, achromosome, a plasmid and a chromosome, two di¡erentplasmids. Varying host-bacteria and PAH-plasmids com-binations, one can profoundly change growth character-istics and competitiveness of Pseudomonas strains and in-crease PAH degradation. Strains of plant growthpromotion rhizosphere Pseudomonas (PGPRP) able to de-grade PAH and resistant to heavy metals were con-structed. The role of plasmids and horizontal gene transferin the evolution of the degradative potential of bacterialpopulations and development of bioremediation strategiesbased on the PGPRP-plants concurrent application arediscussed.This work was supported by the U.S. CRDF grant RB2-2029 and a grant of the Russian Federal Scienti¢c andTechnical Program, # 43.073.1.1.2502 ‘‘



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A18^2

DIOXIN-DECHLORINATORS IN ANAEROBIC CUL-TURES FROM CONTAMINATED SITES: IDENTIFI-CATION, ENRICHMENT STRATEGIES AND PROP-ERTIES OF ISOLATED BACTERIA

M. Bunge, A. Wagner and U. Lechner

Institute for Microbiology, Martin-Luther-Universita«tHalle, Kurt-Mothes-Str. 3, 06114 Halle, Germany

Anaerobic mixed cultures were established with samplesfrom river sediments of the Bitterfeld area (Germany) his-torically contaminated with polychlorinated dioxins andfurans. These cultures were able to dehalogenate spiked1,2,3,4-tetrachlorodibenzo-p-dioxin, 1,2,3- and 1,2,4-tri-chlorodibenzo-p-dioxin (TrCDD) to 1,3- and 2,3-dichlor-odibenzo-p-dioxin and 2-monochlorodibenzo-p-dioxin.Several transfers in synthetic medium with a mixture oforganic acids and 50 WM 1,2,4-TrCDD resulted in sedi-ment-free mixed cultures. Most-probable-number (MPN)analyses showed that only 104 dioxin dechlorinators perml were present in the microbial communities compared toa total cell number of about 108 cells. Therefore, otherchlorinated aromatic compounds with higher water solu-bility were tested as potential electron acceptors. 1,2,3-tri-chlorobenzene (TrCB) was transformed to 1,3-dichloro-benzene. A two-phase enrichment system with 200 mM1,2,3-TrCB dissolved in hexadecane was established andthe reductive dehalogenation was monitored by measuringthe chloride release. Within 170 days, up to 42% of thesubstrate was dechlorinated to 1,3-dichlorobenzene. Thenumber of dioxin-dechlorinating bacteria (as monitoredby MPN) increased by a factor of up to 4.4 x 103 duringchlorobenzene incubation resulting in a maximum numberof 1.1 x 107 cells ml-1. The changes in microbial commu-nity structure were studied by restriction fragment lengthpolymorphism and by cloning and sequencing of the PCRampli¢ed 16S rDNA. A comparison of clone libraries be-fore and after the two-phase enrichment indicated an in-creased abundance of some bacteria, partly with a knowndechlorination potential, and identi¢ed other organismsamong them syntrophic bacteria. From agar-shake dilu-tions of the trichlorobenzene-grown cultures 125 singlecolonies were picked under anaerobic conditions andtransferred into liquid medium with 1,2,4-TrCDD. Threecolonies showed dioxin-dehalogenating activity and werestudied in detail.

A18^3

METAL BIOREMOVAL BY TWO EXOPOLYSAC-CHARIDE-PRODUCING, FILAMENTOUS CYANO-BACTERIA

R. De Philippis, R. Paperi and M. Vincenzini

Dipartimento di Biotecnologie Agrarie, Universita’ degliStudi, Piazzale delle Cascine 24, I 50144 Firenze, Italy

The use of exopolysaccharide(EPS)-producing microor-ganisms for the bioremoval of toxic metal cations frompolluted waters seems to be very promising, owing tothe anionic charge that usually characterizes these poly-mers. Thus, two capsulated, EPS-producing cyanobacte-ria, Cyanospira capsulata and Nostoc PCC7936, has beentested with regard to their metal removal capability byusing the whole cyanobacterial cultures con¢ned into dya-lisis tubings dipped into metal solutions. The maximumcopper removal capability (qmax) of the cyanobacterial cul-tures amounted to 109 ` 2 and 86 ` 2 mg of Cu(II) per gof protein, for C. capsulata and Nostoc PCC793, respec-tively. Pure solutions of the two cyanobacterial EPSs werealso tested, showing qmax values higher than those of mostof the microbial polysaccharides so far tested. When testedfor the removal capability towards Zn(II) and Ni(II), Ccapsulata showed qmax values of 41.5 and 55.4 and NostocPCC7936 of 18.8 and 21.5 mg of metal removed per g ofprotein, for Zn and Ni respectively. The simultaneouspresence of the three metals strongly a¡ected the qmaxvalues observed with the single metals, the new valuesdecreasing to 62 and 78% for Cu, 48 and 50% for Ni,and to 30 and 50% for Zn, with C. capsulata and NostocPCC7936, respectively. In conclusion, the good metal re-moval showed by C. capsulata and Nostoc PCC7936, com-parable with the best performances obtained with othermicrobial biomasses, suggests further investigations onthe use of these two cyanobacteria for the bioremoval ofheavy metals from polluted water bodies.

A18^4

IMMOBILISATION OF METALS AND METAL-LOIDS BY SULPHATE-REDUCING BACTERIAL BI-OFILMS

S. L. Hockin and G. M. Gadd

University of Dundee, Division of Environmental and Ap-plied Biology, School of Life Sciences, Dundee, DD1 4HN,Scotland

Sulphate-reducing bacteria (SRB) are key participants inthe biological cycling of many metals. While much re-search interest has focused on precipitation of insoluble



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transition metal sulphides, SRB can also reduce metal sol-ubility by altering pH and Eh and in addition posses en-zymes capable of directly reducing metalloid oxyanions.There is considerable interest in the use of SRB in biore-mediation and a number of commercial applications al-ready exist. In nature SRB commonly occur in bio¢lmsand the bio¢lm growth-mode o¡ers potential advantagesfor use in bioreactors. Attached cells display altered phe-notype and metabolism, while the presence of an extensiveextracellular matrix can directly in£uence metal immobili-sation through ligand binding, entrapment, and establish-ment of di¡usion gradients. We investigated the immobi-lisation and fate of manganese (II), selenium (IV) andselenium (VI) in laboratory-grown bio¢lms of Desulfomi-crobium norvegicans. Metabolic experiments were used tocharacterise underlying processes, while X-ray analysisand novel cryo-electron microscopy techniques revealedthe nature and location of precipitates in situ withinfully-hydrated bio¢lms. Manganese (II) sulphide formedin the medium and was entrapped at the bio¢lm surface.Selenium (IV) was precipitated by a redox reaction withSRB-generated sulphide. Precipitates formed within thebio¢lm matrix, and colloidal selenium particles were fur-ther entrapped by the bio¢lm matrix. Selenium (VI) wasenzymatically reduced to cell-associated selenium and tovolatile selenide. SRB bio¢lms were shown to be highlye¡ective in the sequestration of manganese and selenium.However, oxidation state of the target metal/metalloid andthe concentration of other ions in solution a¡ected boththe e⁄ciency and location of precipitation.This work was supported by a BBSRC postgraduate re-search grant. Additional funding from British NuclearFuels Ltd is gratefully acknowledged.

A18^5

PYRENE BIODEGRADATION BY MYCOBACTE-RIUM SPECIES: IDENTIFICATION OF THE CATA-BOLIC ENZYMES INVOLVED

Y. Jouanneau, S. Krivobok, S. Kuony, C. Meyer, J. C.Willison

DRDC/BBSI, CNRS UMR 5092, CEA-Grenoble, 38054Grenoble, France

A bacterial strain able to use pyrene as sole carbon sourcefor growth was isolated from a PAH-contaminated soiland identi¢ed as a Mycobacterium gilvum species. Thisstrain, called 6PY1, which also degraded phenanthrene,showed no pyrene mineralization activity when grown onacetate. As a means to identify speci¢c catabolic enzymes,pyrene-induced proteins were identi¢ed by two-dimension-al gel electrophoresis and peptide sequence analysis. Halfof them resemble enzymes known to be involved in phe-nanthrene degradation, with closest similarity to the en-

zymes from Nocardioides sp. KP7. The genes encoding theterminal components of two distinct ring-hydroxylatingdioxygenases have been cloned. Sequence analysis revealedthat the two enzymes, designated Pdo1 and Pdo2, belongto a subfamily of dioxygenases exclusively found in Gram-positive bacteria. When overproduced in E. coli, Pdo1 norPdo2 showed low dioxygenase activity, suggesting thatboth enzymes require speci¢c electron carrier proteins.Pdo1 converted both pyrene and phenanthrene to corre-sponding dihydrodiols whereas Pdo2 preferentially oxi-dized phenanthrene. Immunoblot analysis of cell extractsfrom strain 6PY1 revealed that Pdo1 was present in cellsgrown on benzoate, phenanthrene or pyrene and absent inacetate-grown cells. In contrast, Pdo2 could only be de-tected in PAH-grown cells. These results indicated that thetwo enzymes were di¡erentially regulated depending onthe carbon source used for growth.

A18^6

GFP-MARKED BACTERIA: WHAT CAN WE LEARNABOUT LOCAL BIOAVAILABILITY OF CARBONSUBSTRATES?

J. R. van der Meer, A. Baehler, S. Grimberg

Swiss Fed Institute for Env Science and Technol (EA-WAG), Ueberlandstrasse 133, Postfach 611, CH 8600 Due-bendorf, Switzerland

The gene for Green Fluorescent Protein (GFP) has beenextensively used for tagging bacteria, looking at their pres-ence in the environment and in some cases studying theiractivity under in-situ conditions. When regulatory path-ways of interest are su⁄ciently understood, more speci¢cgene fusions between pathway promoters and the gfp genecan be made, thus allowing the study of pathway expres-sion under natural conditions in individual cells. Sincecells in the natural environment usually experience gra-dients and £uxes of substrates rather than constant con-centrations, individually marked reactive cells could beused to study local bioavailability of carbon substrates.Bioavailability of carbon substrates is an issue in microbialdegradation of environmental pollutants. Environmentalpollutants are often poorly water soluble compoundsand their availability may be limiting the microbial degra-dation rates. Here we describe the use of a gfp fusion tothe promoter of the phn phenanthrene degradation genesin Burkholderia sp. RP007 to study the response of indi-vidual cells to di¡erent sources of phenanthrene. Hereto,phenanthrene crystals, dissolved phenanthrene, the e¡ectof surfactants and the presence of oils were tested. It wasfound that at very low bioavailability of phenanthrene, theproportion of cells inducing the phenanthrene pathwaydecreases, rather than a homogeneous decrease of phenan-threne pathway gene expression in all cells. On the other



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hand, the £ux of phenanthrene to the cells could not bearti¢cially increased to levels high enough to obtain high-est expression of the phn genes, showing that the cells aremostly limited for phenanthrene.

A19^1

SELECTION OF SOME PUTATIVE VIRULENCEGENES OF NON-O1/NON-O139 VIBRIO CHOLERAEIN SEWAGE TREATMENT WORKS (ESTUARY OFRANCE, BRITTANY, FRANCE)

S. Baron, S. Chevalier and J. Lesne

Ecole Nationale de la Sante¤ Publique Laboratoire d’Etudeset de Recherche en Environnement et Sante¤, C.S. 74312Rennes cedex, France

Vibrio cholerae is an inhabitant of natural aquatic ecosys-tems and wastewaters. Some strains belonging to otherserotypes than O1/O139, the causative agents of cholera,have been associated with gastroenteritis, and/or extrain-testinal infections, but their virulence factors are still un-known. It can be speculated that the chromosomic genesimplicated in the virulence cascade of cholera are putativevirulence genes of Vibrio cholerae non-O1/non-O139. Theaim of this study was to know (i) if water-borne sanitationsystems may contribute to the selection of these genes, (ii)if these genes could improve the ¢tness of the bacteria forthe natural aquatic environment.108 strains of V. choleraenon-O1/non-O139, isolated from raw and treated waste-waters (48 and 60 respectively), and 44 strains fromcockles growing downstream the treated wastewater dis-charge were examined using PCR multiplex for the pres-ence of the mobile genetic elements ctxA, zot and tcpA, forthe presence of the putative virulence genes toxR, andompU and for the hlyA gene encoding for an hemolysin-cytolysin. The mobile genetic elements were absent in the152 strains. Frequencies of toxR and ompU genes werehigher in the treated wastewater isolates than in the rawwastewater ones (respectively 44.0 vs 26.6%, 26.6 vs 6.4%).Moreover, the proportion of strains where toxR ompUand hlyA genes were present together was 4 times higherin the treated wastewater than in the raw sewage (23.9 vs5.5 %). These observations are consistent with the hypoth-esis of the selection of some chromosomic genes associatedto virulence by the sanitation system. In cockles, frequen-cies of toxR and ompU genes were higher than in treatedwastewater (90.9 and 70.5 % respectively), whereas thefrequencies of hlyA gene stay stable (68.2 % vs 69.6 %).That could mean an involvement of toxR and ompU genesbut not of hlyA gene, in the ¢tness for the aquatic ecosys-tem.

A19^2

USE OF QUANTITATIVE RT-PCR OF HOUSEKEEP-ING AND VIRULENCE GENES TO MONITOR SUR-VIVAL AND ACTIVITY OF ENTEROPATHOGENICBACTERIA IN WATER SAMPLES

A. Fey, S. Eichler, M. G. Ho«£e and C. A. Guzma'n

GBF ^ German Research Centre for Biotechnology, Divisionof Microbiology, Mascheroder Weg 1, D-38124 Braunsch-weig, Germany

The presence of pathogenic bacteria in drinking and bath-ing waters is a major health concern. Water quality wastraditionally monitored by staining and culturing tech-niques, which provide poor tools for the detection of via-ble but non-culturable bacteria. Furthermore, very little isknown concerning survival and activity of pathogenic bac-teria in water samples, which are critical issues for theassessment of human health riks. A novel approach toquantify the number, activity and pathogenicity of bacte-ria present in water samples was developed, which is basedon quantitative reverse transcription PCR (Q-RT-PCR).Speci¢c primers were designed for 16S rRNA and tufmRNA, as taxonomic and metabolic activity markers,and main virulence genes of Salmonella enterica and enter-ohaemorrhagic Escherichia coli, as model pathogens caus-ing food- and waterborne infections. Special RNA stan-dards were developed for each target gene To overcomequanti¢cation problems associated with the lack of stablyexpressed bacterial housekeeping reference genes. For val-idation, bacterial survival and activity was evaluated inwater samples spiked with de¢ned numbers of pathogenicmicroorganisms. Kinetics studies were performed to deter-mine cell numbers (total and live), as well as the expres-sion of 16S rRNA, tuf and virulence genes mRNAs bystaining techniques and/or Q-RT-PCR. The obtained re-sults demonstrated that the developed Q-RT-PCR-basedmethod constitutes a powerful tool, which enables an ac-curate determination of bacterial number and activity inwater samples. This allows to perform a more e⁄cient riskassessment of the infectious potential of water samples.

A19^3

THE ROLE OF THE ANTIFUNGAL COMPOUNDPRODUCED BY THE MARINE BACTERIUM PSEU-DOALTEROMONAS TUNICATA

A. Franks(1,2), S. Egan(1,2), C. Holmstro«m(1,2), H.Lappin-Scott(3) and S. Kjelleberg(1,2)

(1) School of Biotechnology and Biomolecular Sciences,Microbiology and Immunology, University of New SouthWales, Sydney, New South Wales, Australia; (2) Centre



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for Marine Biofouling and Bioinnovaition, University ofNew South Wales, Sydney ,New South Wales, Australia;(3) School of Biological Sciences, University of Exeter,Exeter, Devon, United Kingdom.

Members of the marine genus Pseudoalteromonas com-monly inhabit the surface of sessile marine organismsand express a variety of bioactive compounds. These com-pounds appear to provide protection against biofouling ofthe host surface. Strains belonging to the species P. tuni-cata have been isolated from the surface of eukaryoteswhich lack recognised biofouling defences. The dark greenpigmented bacterium P. tunicata has been shown to pro-duce a broad range of highly inhibitory antifouling com-pounds, speci¢cally targeting bacteria, diatoms, barnaclelarvae, algal spores and fungi. This study identi¢es theyellow pigment, a hydrophobic low molecular weight com-pound containing an aromatic ring and a fatty acid chain,as the antifungal compound expressed by P. tunicata.Transposon mutagenesis was used to generate a mutantof P. tunicata unable to inhibit fungal growth. Geneticanalysis of this mutant revealed the involvement of agene with a high similarity to fadD, encoding a long chainfatty acid CoA ligase in E. coli, and suggested to regulateantimicrobial synthesis in Streptomyces coelicolor. Theantifungal mutant and the wild type strains where em-ployed in competition with a marine fungal isolate duringsurface colonisation and establishment of an arti¢cial ma-rine bio¢lm. It was found that established bio¢lms of P.tunicata inhibit fungal attachment. Moreover wild type P.tunicata was found to attach to an established fungal bio-¢lms, remove up to 65% of the bio¢lm, and inhibit furthersurface colonisation by the fungus. These results stronglysupport the proposal that bacterial species such as P. tu-nicata are important in the development and compositionof microbial fouling communities on surfaces in the ma-rine environment.

A19^4

AsaP1, A MAJOR EXOTOXIN OF SOME AEROMO-NAS SALMONICIDA STRAINS IS AN ASPZINCINMETALLOENDOPEPTIDASE

B. K. Gudmundsdottir, I. Hvanndal, S. Gudmundsdottir, B.Magnadottir and V. Andresdottir

Institute for Experimental Pathology, University of Iceland,Reykjav|¤k, Iceland

Infections by various strains of the bacterium Aeromonassalmonicida cause furunculosis and related diseases of feraland cultivated ¢sh stocks in freshwater and marine envi-ronment. A major exotoxin of a group of A. salmonicidastrains has been described and identi¢ed as a metalloen-dopeptidase, AsaP1. According to gel ¢ltration chroma-

tography the toxin is a single polypeptide with a MW closeto 20 kDa. The 24 h LD50 of AsaP1 in salmon is 0.03 Wgg-1 and in mice 0.18 Wg g-1. Animals receiving lethal dosesof the toxin display symptoms comparable to toxic shocksyndrome. Sublethal doses injected into salmon or miceinduce all the pathology seen by injection of the extracel-lular products. Passive immunization of Atlantic salmonwith anti-AsaP1 sera confers signi¢cant protection. Thetoxin was found to be mitogenic in leukocyte culturesfrom the head-kidney of Atlantic salmon and mousespleen cell cultures and to induce the production of theacute phage cytokines TNF-a and IL-6 in mouse. Se-quencing of the complete AsaP1 gene revealed an openreading frame of 1029 bp (343 aa) with 87% aa sequenceidentity to the EprA1 peptidase of A. hydrophila. It sharesthe conserved HexxH and GTxDxxYG sequences of theaspzincin family of metalloendopeptidases. A mature ac-tive protein with a predicted molecular mass of V19kDawas identi¢ed using N-terminal aa sequencing data. Thecomplete asaP1 gene was expressed in E. coli as aV22kDa caseinase, recognized by anti-AsaP1 antibodies.The recombinant protein induced tissue damages in sal-mon comparable to those induced by native AsaP1.

A19^5

MARINE SPONGES AS MICROBIOLOGICAL FER-MENTERS

U. Hentschel

Institute of Molecular Infectious Disease, Ro«ntgenring 11,D-97070 Wu«rzburg, Germany

Sponges (Porifera) represent the oldest, still extant animalphylum with a fossil record dating back more than 580million years in time. Sponges may contain large amountsof bacteria that are embedded within the animal matrixand that can amount to 40% of the biovolume. This pop-ulation consists mostly of extracellular bacteria that areenclosed within the sponge extracellular matrix and thatare physically separated from the seawater by contiguoushost membranes. Because sponge-bacteria interactions arepresumably evolutionarily ancient, widely distributed, andin some cases speci¢c to their host, it is generally believedthat symbiotic interactions exist between sponges and mi-croorganisms. In order to provide insights into the speciesrichness of the microbial community of sponges, a diver-sity survey was performed based on 190 16S rDNA se-quences. As a result, a uniform microbial communitywas discovered that is distinctly di¡erent from that of ma-rine plankton or marine sediments. Altogether 14 mono-phyletic, sponge-speci¢c sequence clusters were identi¢edthat belong to at least seven di¡erent bacterial divisions.These monophyletic clusters comprise 70% of all publiclyavailable, sponge-derived sequences re£ecting the general-



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ity of the phenomenon. None of the sequence clusterscontain cultured representatives. A picture emerges wheresponges can be viewed as highly concentrated microbialfermenters of so far unculturable, elusive and possiblyevolutionarily ancient marine microorganisms.

A19^6

THE CHIRONOMID ^ VIBRIO CHOLERAE CON-NECTION

Y. Kashi(1), M. Halpern(2), H. Gancz(1) and M. Bro-za(2)

(1) Department of Food Engineering and Biotechnology,Technion-Israel Institute of Technology, Haifa 32000, Isra-el; (2) Department of Biology, Faculty of Science and Sci-ence Education, University of Haifa, Oranim, Tivon 36006,Israel

Vibrio cholerae is an environmental Gram (-) bacteriumthat speci¢c serogroups of cause the cholera disease.Lately it was proposed that the non-biting midge Chiro-nomus sp. (Diptera) egg-mass harbor V. cholerae. Here weshow that the midge egg-mass is a proteoglycan biopoly-mer that serves as a nutrient source for V. cholerae. Thefactor responsible for the degradation of the egg-mass isthe secreted Hemagglutinin/Protease (HA/P), which disin-tegrates the egg-mass gelatin-like matrix. Homologuesproteases are found in the genome of other members ofthe Vibrionacae family that were also isolated from theegg-masses. To date, more than 20 di¡erent serogroupsof V. cholerae were found on the egg-masses collectedfrom di¡erent freshwater environments. The isolation ofmore than 10 di¡erent V. cholerae serogroups from adult,£ying midges suggests the possible aerial transfer of V.cholerae by the adult, £ying midge. V. cholerae taggedwith Green Fluorescent Protein was allocated to the inter-segmental membrane of the adult midge, a location proneto microbial attachment. In the laboratory, the attachedV. cholerae were able to enter air-separated water bodies.The relationship between large invertebrate invasion towater bodies and V. cholerae presence in that water wasdemonstrated in a preleminary ¢eld observation.

A20^1

PARTIAL PURIFICATION OF EXTRACELLULARPROTEOLYTIC ENZYMES OF BACTERIAL AGGRE-GATES FROM ACTIVATED SLUDGE AND IDENTI-FICATION OF POTENTIAL PRODUCTIVE BACTE-RIA

A. Cadoret, A. Visvikis and J.-C. Block

LCPME-Po“le de l’Eau, UMR Universite¤ Henri Poincare¤-CNRS 7564, 15 avenue du Charmois, F-54500 Vand]uvre,France

Bacterial communities in activated sludge mobilise com-plex organic matter by sharing various extracellular hy-drolytic enzymes which remained mainly associated tothe extracellular polymeric substances (EPS) involved inthe bacterial aggregates cohesiveness (Cadoret et al.,2002, Enzyme and Microbial Technology, 31, 179-186).Temperature, bacterial starvation, and aggregation in£u-ence the level of production of extracellular enzymes. Asthe extracellular enzymes associated to EPS consist of ahighly complex mixture, this work ¢rst focused on extra-cellular proteolytic enzymes.Then the purpose of the studywas to purify few proteolytic extracellular enzymes asso-ciated to the EPS extracted from activated sludge by com-bining ultrasounds with cation exchange resin. In parallel¢ve bacteria responsible of proteolytic activity were iso-lated from activated sludge on R2A-agar medium contain-ing 1 % casein as substrate and were used to provide ahigher amount of target enzymes to study their environ-mental regulation factors. Brie£y, the extracellular en-zymes associated to the EPS recovered from activatedsludge were submitted to the puri¢cation procedure in-cluding ammonium sulfate concentration, ionic exchangechromatography and gel ¢ltration. The chromatographyrevealed ¢ve peaks, among which one exhibited Ala-ami-nopeptidase activity and two exhibited Leu-aminopepti-dase activity with a 110-fold and 65-fold puri¢cation re-spectively. The molecular weights of these partiallypuri¢ed enzymes were compared with those provided bydata-bank and with those of the extracellular proteolyticenzymes puri¢ed from the isolated strains. In conclusionthe release of extracellular enzymes appears as a prevalentstrategy in bacterial aggregates for organic matter retriev-al. The ecological signi¢cance of the highly diverse extra-cellular enzymes in activated sludge will be discussed.



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A20^2

PROFILING DENITRIFYING BACTERIA IN ACTI-VATED SLUDGE BY METABOLIC PROPERTIESAND DGGE ANALYSIS OF PARTIAL nirK GENES

S. Hallin(1), J. Dicksved(1), I. Throba«ck(1), P.-E Lindg-ren(2) and M. Pell(1)

(1) Department of Microbiology, Swedish University ofAgricultural Sciences, P.O. Box 7025, S-750 07 Uppsala,Sweden; (2) IFM/Department of Biology, Linko«ping Uni-versity, S-581 83 Linko«ping, Sweden

An external carbon source can enhance denitri¢cationrates in activated sludge and improve nitrogen removal.Nevertheless, it may in£uence the functional propertiesand select for certain denitrifying bacteria. The structureand function of denitrifying communities in pre-denitri¢-cation was determined. Functional diversity was assessedwith non-growth substrate-use pro¢ling of sludge samplesfrom activated sludge processes supplemented with eithermethanol or ethanol. Potential denitri¢cation activity(PDA) with ten separate electron donors was determinedin 1 h incubations to produce substrate-use pro¢les for 17sludge samples. The data was subjected to principal com-ponent analysis to elucidate the pro¢les’ patterns. Thepopulation structure in 8 of the 17 samples was describedas denaturing gradient gel electrophoresis (DGGE) ¢nger-prints of partial nirK genes followed by sequencing of thedominating bands. The substrate-use pro¢les divided thesamples into three clusters; control, methanol, and ethanolsludge. The discriminating variables were the alcohols andthe fatty acids. Methanol-fed plants had an increased ac-tivity with methanol and ethanol and a decreased activitywith fatty acids, while the ethanol adapted sludge had anoverall higher capacity to use even-numbered fatty acidsand alcohols. The DGGE analysis showed that the deni-trifying communities in the samples had a high degree ofdiversity with at least 20 bands. Yet, the patterns wereidentical for all samples, which suggest that the same de-nitri¢ers were present in all the treatments despite the ex-ternal carbon source. The adaptation to di¡erent carbonsources could therefore be explained by physiological al-terations rather than by community shifts.

A20^3

PHENOTYPING OF BACTERIAL POPULATIONS: AUSEFUL METHOD FOR MICROBIAL SOURCETRACKING

I. Ku«hn(1), A. Iversen(1), X. Bonjoch(2), J. Wallis(3), J.Ebdon(3), H. Taylor(3), S. Skraber(4), N. Pissarides(5),T. G. Papageorgiou(4), A. R. Blanch(2)

(1) MTC, Karolinska Institute, SE 171 77 Stockholm,Sweden; (2) Departament de Microbiologia, Universitatde Barcelona, 08028 Barcelona, Spain; (3) School of theEnvironment, University of Brighton, BN2 4GJ Brighton,UK; (4) Faculte¤ de Pharmacie, Universite¤ de Nancy I,5400 Nancy, France; (5) State General Laboratory, Micro-biological Section, 1451 Nicosia, Cyprus

Microbial source tracking may be used to identify theorigin of faecal pollution in the environment. Variousmethods, including geno- and phenotyping, can be usedto compare microorganisms in a polluted environmentwith those found in a suspected faecal source. In thepresent study we have used a biochemical ¢ngerprintingmethod (the PhenePlateTM system) to type more than 8000coliform and enterococcal isolates from sewage and ani-mal manure in ¢ve di¡erent European regions. The aimwas to ¢nd out if certain parameters (diversity, populationstructure, species distribution) were associated with certainkinds of sewage, and also to determine if there were re-gional di¡erences in the compositions of the bacterial pop-ulations related to humans and various animal species. Itwas found that high diversities were associated with urbansewage, lower diversities with samples from slaughter-houses, and the lowest diversities were associated withsamples from animal manure. Among the enterococci, E.faecalis and E. faecium dominated in samples of humanand poultry sources. E. hirae was most common in cattle,also common in pigs and humans, but almost absent inpoultry. The samples of urban sewage also showed a lowerincidence of E.coli among the coliforms than samples ofanimal origin. These patterns were similar in samples fromall countries studied (Spain, Sweden, UK, Cyprus, andFrance). Thus, The PhenePlateTM system might be a use-ful method for microbial source tracking, but a combina-tion with other methods is needed to verify the results



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A20^4

IDENTIFICATION OF TRAITS IMPLICATED INTHE RHIZOSPHERE COMPETENCE OF FLUORES-CENT PSEUDOMONADS: DESCRIPTION OF ASTRATEGY BASED ON POPULATION AND MODELSTRAIN STUDIES

Ph. Lemanceau, S. Delorme, X. Latour and P. Mirleau

UMR INRA/Universite¤ de Bourgogne ‘Microbiologie etGe¤ochimie du Sol’, INRA-CMSE, BP 86510 21065 DijonCedex, France

The lack of consistency of the bene¢cial e¡ects of inocu-lated £uorescent pseudomonads has often been related totheir bad survival in the rhizosphere. In this review, wedescribe the strategy followed over the last decade to studytraits involved in the rhizosphere competence of these bac-teria. The diversity of indigenous populations associatedwith plant roots was ¢rst compared to that of populationsassociated with uncultivated soils in order to identify traitsthat discriminate these populations. The involvement ofthese bacterial traits in the rhizosphere competence wasthen assessed by comparing the competitiveness of awild-type strain to that of mutants a¡ected in the corre-sponding phenotypes. Finally, traits shared by populationsadapted to the rhizosphere were identi¢ed by comparingboth the competitiveness in the rhizosphere and the me-tabolism of a collection of bacterial strains. The datayielded indicated that rhizosphere competent pseudomo-nads show a speci¢c metabolism especially characterizedby the e⁄ciency of the pyoverdine-mediated iron uptakeand by the ability to reduce nitrogen oxides.

A20^5

DETECTION AND DIVERSITY OF THE FLORFENI-COL RESISTANCE GENE £oR IN SOIL BACTERIA

C. Lerondelle, V. Srinivasan, P. Mavinguy, T. M. Vogel, P.Simonet and X. Nesme

Microbial Ecology, UMR CNRS 5557, Lyon University,Bat G. Mendel, 43 bd du 11 Novembre 1918, 69622 Villeur-banne Cedex, France

The development of multi-drug resistance among patho-genic bacteria has emerged as a major public health con-cern. The relatively short time periods between the intro-duction of an antimicrobial agent into clinical use and theoccurrence of resistant bacteria con¢rms that bacteria areable to rapidly and e⁄ciently adapt to altered environmen-tal conditions. Florfenicol is a £uorinated derivative ofchloramphenicol, which has been used since 1995 in ani-mal therapy for treatment of bovine respiratory pathogens

such as Pasteurella spp. While clinical isolates (mainlySalmonella typhimurium) have been investigated for pres-ence and diversity of £orfenicol among other antibioticresistance genes, little has been done with bacterial popu-lations in soil and water. Traditional microbiological tech-niques include isolation of resistant bacteria on nutrientmedia containing the targeted antibiotic or by screeningbacterial isolates for their antibiotic resistance patterns.On the other hand, molecular techniques that include ex-traction and puri¢cation of DNA from the environment(metagenome) on which DNA probes or PCR-based de-tection systems can be used increase the range of testedbacteria to many of those which are not accessible bytraditional cultivation. In this study, we focused our studyon the £orfenicol resistance gene (£oR) in soil bacteriacollected from farms where pigs were treated with £orfe-nicol. These results were compared to that of control soils.Techniques used included traditional cultivation methodsand metagenome-based molecular techniques. Results con-cerning the diversity of the £oR gene in the various soilstested, the 16S rDNA-based identi¢cation of bacteriabearing the gene, the possible gene location on plasmidsand integrons will be presented and discussed.

A20^6

IN SITU VISUALIZATION OF PLASMID TRANSFERFROM PSEUDOMONAS PUTIDA TO THE INDIGE-NOUS BACTERIAL POPULATION OF ALFALFASPROUTS

L. MWlbak(1,2), T. R. Licht(2), T. Kvist(2), N. Kroer(1)and S. R. Andersen(2)

(1) National Environmental Research Institute, Departmentof Marine Ecology and Microbiology, Roskilde, Denmark;(2) Danish Veterinary and Food Administration, SWborg,Denmark.

Transfer of the plasmids pJKJ5 and TOL from Pseudomo-nas putida to the indigenous bacterial population wasstudied in situ on alfalfa sprouts grown in a home sprout-ing kit. Tagging with £uorescent protein markers alloweddirect visualization of the introduced donor bacteria aswell as of indigenous bacteria that had received the plas-mids. Transfer of both plasmids occurred during a periodof nine days, in which alfalfa seeds, inoculated with donorbacteria, were developing to edible and subsequently de-caying sprouts. Scanning confocal laser microscopy re-vealed that most transconjugants were located aroundthe cotyledon and root areas. Randomly selected membersof the indigenous bacterial population from inoculated aswell as un-inoculated sprouts, and a representative part ofthis population, which had received the plasmids, werecharacterized by PCR ampli¢cation of rDNA spacer poly-morphisms, followed by partial 16S rDNA sequencing.



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This showed that the initially dominating genera Erwinia,Paenibacillus were gradually replaced by Pseudomonas onthe fully developed sprouts. Consistently, transconjugantsthat for both of the investigated plasmids belonged mainlyto the genera Pseudomonas and Erwinia, did not reachhigh numbers until six days after onset of germination.In conclusion, alfalfa sprouts constitute a bacterial envi-ronment that allows considerable frequencies of plasmidtransfer, and might be a reservoir for genes conferringantibiotic resistance.

A21^1

COMMUNICATION AND SIGNALLING IN PLANT-BACTERIAL SYMBIOSES

L. P. Antonyuk, V. E. Smirnova, A. V. Tugarova

Institute of Biochemistry and Physiology of Plants and Mi-croorganisms, Saratov, Russia

The question how bacteria and plants communicate witheach other for establishing e⁄cient symbioses in mostcases remains a mystery. A single well-known example ofplant-bacterial communication is the functioning of £avo-noids produced by legume roots as signal molecules toinduce nod genes for the symbiotic rhizobium partner.We studied interactions of the partners in the Azospiril-lum-wheat endophytic symbiosis and found that wheatlectin (wheat germ agglutinin, WGA) can act as a molec-ular signal for A. brasilense Sp245, a native wheat sym-biont [1]. The A. brasilense cell response to WGA hasappeared to be pleiotropic, in line with the action of an-other proteinaceous signal, the C factor known as a signalfor Myxococcus xanthus [2]. The wheat lectin in nanomo-lar concentrations stimulated certain processes in azospir-illum which underlie the e⁄ciency of the Azospirillum-wheat endosymbiosis, viz. N2-¢xation, ammonia excretion,phytohormone indole-3-acetic acid production. WGA alsocaused some other changes; e.g. it induced protein biosyn-thesis in azospirillum and in£uenced the cell surface prop-erties of the bacterium. There is evidence that proteins canact as molecular signals in other types of symbioses.[1] L.P. Antonyuk and V.V. Ignatov. Russ. J. Plant Phys-iol., 2001, 48: 427-433. [2] P.J. Bonner and L.J. Shimkets.Trends in Microbiology, 2001, 9: 419-462.Supported by the Russian Foundation for Basic Research,Project 01-04-48755, the Russian Academy of Sciences’Commission, 6th Competition-Expertise, Grant 205, andNATO, grant LST.CLG.977664.

A21^2

PATHOGEN SELF DEFENSE: FUSARIUM SIGNAL-ING BLOCKS BIOCONTROL GENE EXPRESSIONIN ANTAGONISTIC PSEUDOMONAS AND TRICHO-DERMA ON PLANTS

B. Du¡y(1), M. Lutz(2), R. Notz(2) and G. De¤fago(2)

(1) Swiss Federal Institute of Technology (ETH), CH-8092 Zu«rich; (2) Swiss Federal Research Center for FruitProduction, Viticulture and Horticulture (FAW), CH-8820Wa«denswil, Switzerland

Fusarium species are among the most economically impor-tant and di⁄cult to control groups of plant pathogens. Wefound that Fusarium produce toxins that act as negativesignals blocking biosynthesis of antimicrobial metabolitesin natural antagonists and thereby interfere with biologicalcontrol of plant disease. We investigated the e⁄cacy of thebacterium Pseudomonas £uorescens and fungus Trichoder-ma atroviridae for biocontrol of root-pathogenic Fusariumon tomato and mycotoxigenic Fusarium on maize. InPseudomonas, production of the antibiotic 2,4-diacetyl-phloroglucinol (DAPG) was the primary biocontrol mech-anism on tomato as determined with mutational analysis.However, DAPG biosynthesis was blocked (and biocon-trol e⁄cacy greatly diminished) in certain Pseudomonasgenotypes in the presence of the pathogen. Fusaric acid,a phyto-/mycotoxin produced by Fusarium oxysporum inthe rhizosphere, was identi¢ed using a lacZ reporter fusionas the signal speci¢cally repressing the DAPG promoterphlA. In Trichoderma, biocontrol is largely attributed toproduction of cell-wall-degrading chitinases. We foundthat the mycotoxin deoxynivalenol (DON) produced byF. graminearum and F. culmorum on maize blocked expres-sion of the nag1 chitinase gene, but had no e¡ect on theech42 endochitinase gene in Trichoderma (determined us-ing goxA reporter fusions). However, since these chitinasesfunction synergistically, repression of one by the pathogenmay be su⁄cient to thwart antagonism. No signaling wasobserved for Fusarium insertion mutants with a non-func-tional tri5 trichothecene biosynthetic gene (DON-nega-tive). These results suggest that fungal toxins are negativesignals that serve as a pathogen self defense strategy toovercome microbial competition in plant habitats with anadverse impact of biocontrol.



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A21^3

IDENTIFICATION OF SYNTROPHICALLY PROPIO-NATE-OXIDIZING MICROBIAL POPULATIONS INANOXIC SOIL BY STABLE ISOTOPE PROBING OFrRNA AND rDNA

M. W. Friedrich and T. Lueders

Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch Str., 35043 Marburg, Germany

In anoxic environments such as £ooded soils, propionateis a prominent intermediate of the anaerobic degradationof organic matter. In the absence of alternative electronacceptors such as nitrate, ferric iron, or sulphate, propio-nate is oxidized syntrophically, i.e. by the cooperation ofpropionate-oxidizing, proton-reducing bacteria and H2- orformate-consuming methanogens. So far, only few delta-Proteobacteria (Syntrophobacter spp., Smithella propioni-ca) and thermophilic Desulfotomaculum spp. are knownto oxidize propionate syntrophically. We have studied pro-pionate-metabolising microbial populations in anoxic ricesoil slurries by stable isotope probing (SIP) analyses. SIPallows to directly link structure and in-situ function ofmicrobial populations in natural habitats. 13C-labelledpropionate was added to rice ¢eld soil slurries after alter-native electron acceptors had been reduced. DNA andrRNA were separated by density gradient centrifugations.Gradient fractions were analysed for rDNA and rRNA byreal time PCR over time (11, 18, 25 days) to detect theactivation of community members. After 70 days of incu-bation at low propionate concentrations (V5 mM), clonelibraries of both, 13C-labelled rDNA and rRNA, revealedthe presence of Syntrophobacter spp. In addition, Geo-bacter spp. as well as gram-positive Syntrophomonas, De-sulfotomaculum, Clostridium, and Bacillus spp. were de-tected, which have not been associated with syntrophicpropionate oxidation before. Moreover, we detectedmostly 13C-labelled rDNA of the yet-uncultured methano-gens of ‘‘Rice Cluster I’’, whereas acetoclasticMethanosar-cina spp. were less abundant. We conclude that more spe-cies appear to be involved in syntrophic propionateoxidation in anoxic rice ¢eld soil than were previouslyanticipated.

A21^4

MICROBIAL MOVEMENT WITHIN TERRESTRIALBIOFILMS

P. M. Gaylarde, C. C. Gaylarde

Federal University of Rio Grande do Sul, Porto Alegre,Brazil

Bio¢lms on the external surfaces of buildings are complexecosystems containing bacteria, phototrophs, fungi, proto-zoa, nematodes and higher life forms. Various types ofmovement occur in these bio¢lms, resulting in constantchanges in community structure. Bacterial movement isalmost entirely con¢ned to boundary regions, exceptwhere they are transported by eukaryotic organisms suchas protozoa. Movement is constrained at interfacial re-gions by physical forces arising from interaction betweencell movement and the electrical double layer. A voltagegradient is produced along the axis of movement (Dorne¡ect), which is dependent on cell velocity, ionic strengthand composition and pH. This induced voltage a¡ects theorientation of cells on any surface carrying an electricaldouble layer. This phenomenon will be shown in a shortvideo of cells in a bio¢lm. When cells move rapidly nearsuch surfaces, the hydrated shell of counter-ions is lost,resulting in an apparent reduction in viscosity (a modi¢edWein e¡ect) ; when cells are in channels barely wider thantheir own diameter, movement can be extremely rapid. Wehave estimated a rate of 3000Wm/sec for bacteria movingin a channel produced by a motile Leptolyngbya (approx.1.5Wm diameter). These observations show that motilebacteria are entrained into the boundary layers of surfacesby physico-chemical forces. This controls the indigenouscommunities of a natural bio¢lm. The principles can beapplied to various systems. For instance, they are certainlya major factor shaping the special populations of the rhi-zoplane community, which is still incompletely explained.

A21^5

BACTERIAL PREY EXHIBIT RESISTANCE TO PRE-DATION BY BDELLOVIBRIO AND LIKE ORGAN-ISMS

Y. Shemesh and E. Jurkevitch


At every level in the tree of life, organisms are confrontedwith parasites and predators but are able to maintain ex-tant populations. This dilemma is integrated in ecologicaltheories and considered a property of the ecosystem for



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large organisms. Predation by Bdellovibrio and like organ-isms (BLOs) obligate, semi-generalist bacterial predatorsof gram negative cells has been used as a model for pred-ator-prey interactions because it is a convenient and accu-rate system. The BLOs are ubiquitously found in water, insoil, associated with bio¢lms and with marine and landanimals. Under laboratory conditions, the interaction ofa BLO predator with a bacterial prey results in the lysis ofthe prey culture and the proliferation of the predator.However, prey cells are never eradicated from the suspen-sion, and a small residual population always remains. Us-ing red- and green-£uorescent protein labeled prey popu-lations, confocal, video and electron microscopy, we showthat the surviving prey cell population is able to sustainitself and grow, as it exhibits increased resistance to pre-dation. This phenomenon, which seems not to originatefrom a stable mutational event, is reversible and is accom-panied by a phenotypic change in the prey cells. Increasedresistance may be a phenotypic plastic response to preda-tion stress. Increased resistance may lead to dumping ofpredator-prey oscillations, thereby creating conditions inwhich both predator and prey can survive.

A21^6

LACCASE AND ABC-TRANSPORTERS CONFER RE-SISTANCE IN BOTRYTIS CINEREA TO ANTIBIOT-ICS PRODUCED BY ANTAGONISTIC PSEUDOMO-NAS

A. Schouten, G. van den Berg, Y. C. Arenas, H. J. Schoon-beek, M. A. de Waard, and J. M. Raaijmakers

Laboratory of Phytopathology, Wageningen University,P.O. Box 8025, 6709 PG Wageningen, The Netherlands

Antagonistic Pseudomonas species produce a wide varietyof secondary metabolites that adversely a¡ect the growthor metabolic activity of plant pathogenic fungi. Antibioticsare produced by introduced and indigenous Pseudomonasspecies in plant-associated environments and play an im-portant role in in situ interactions with plant pathogenicfungi. Consequently, antibiosis is recognized as an impor-tant mechanism by which introduced Pseudomonas strainscan control multiple fungal diseases, including ‘‘greymould’’ caused by Botrytis cinerea. Its enormous hostrange and saprophytic and parasitic life style indicatethat B. cinerea has the potential to cope with a varietyof toxic compounds. In this study, we examined mecha-nisms of resistance in B. cinerea against 2,4-diacetylphlor-oglucinol (2,4-DAPG) and phenazines, two antifungal me-tabolites produced by antagonistic Pseudomonas species.Northern analyses showed that phenazine antibiotics and2,4-DAPG induced expression of the ABC transportergene BcatrB. Furthermore, BcatrB replacement mutantsof B. cinerea were more sensitive to phenazine antibiotics

and 2,4-DAPG than their parental strain. Uptake experi-ments further indicated that phenazine antibiotics act as asubstrate of BcatrB. In addition to membrane-bound ef-£ux, degradation plays a role in resistance of B. cinerea to2,4-DAPG. Studies with speci¢c gene replacement mutantsand induction assays indicated that a laccase contributesto 2,4-DAPG-resistance in B. cinerea. In conclusion, thisstudy shows that the plant pathogenic fungus B. cinereaharbours multiple mechanisms to cope with antibiotic me-tabolites produced by antagonistic Pseudomonas.

A22^1

A PROTEOMIC STUDY OF BDELLOVIBRIO BAC-TERIOVORUS

G. Barel and E. Jurkevitch


The Bdellovibrio and Bacteriovorax (or Bdellovibrio andlike organisms-BLO) are predatory bacteria, ubiquitouslyfound in water, in soil, associated with bio¢lms and withmarine and land animals. Most BLOs exhibit a dimorphiclife pattern as rapidly swimming, £agellated, small attackphase cells and as intracellular growing ¢laments. Uponattachment onto a gram negative cell, attack cells perfo-rate the outer prey cell membrane, sheds their £agellumwhile penetrating the periplasmic space of the prey to en-gage in cellular growth and replication at the expense ofthe prey’s cellular content. Progeny attack cells lyse thehost ghost through the action of lytic enzymes to start anew cycle. A comparative proteomic study of the attackand elongation phases of B. bacteriovorus 109J has beeninitiated. De¢ned axenic host-independent mutants (Bareland Jurkevitch, 2001) are used along with the wild typestrain to avoid contamination with prey protein and nosuch contamination is apparent in our system. The impactof di¡erent prey on the proteome of attack cells and thedi¡erences between the various mutants are also exam-ined. Protein spots appearing in the attack phase cellsoriginating from a lysate with Escherichia coli as a preyhave been identi¢ed by MALDI-QTOF and appear tocorrespond to a number of porins of prey origin. Thesespots were not observed using Pseudomonas syringae asprey but other spots appeared instead. Master gel mapsof the various phases and their di¡erences will be pre-sented as well as selected identi¢ed di¡erential spots.



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A22^2

NEF AND VPR/VPX ACCESSORY PROTEINS OFPRIMATE LENTIVIRUSES ACT ALSO ON NON-IN-FECTED CELLS TO INDUCE THE IMMUNODEFI-CIENCY

B. Bouzar, S. Villet, F. Guiguen, K. Gallay, C. Garnier, G.Verdier, and Y. Chebloune

Laboratoire de Biologie Mole¤culaire, Cellulaire et MaladiesEmergentes, UMR 754 INRA/ENVL/UCBL, Re¤trovirus etPathologie Compare¤e, Universite¤ Claude Bernard, Bat B. 50Avenue Tony Garnier, 69366 Lyon cedex 07, France

Human and Simian Immunode¢ciency Viruses (HIV, SIV)are the etiologic agents of AIDS in infected patients andmonkeys, and they belong to of the lentivirus genus of theRetrovirus family. SIV and HIV genomes are character-ized by the presence of a number of accessory nef, vpu, vpr,vpx. Caprine Arthritis Encephalitis Virus (CAEV) is apathogen lentivirus closely related to HIV and SIV butdoes not induce immunode¢ciency in infected goats. Thisproperty correlates with both a lack of productive infec-tion of CD4+ T-lymphocytes and a simpler genome orga-nization lacking nef, vpu, vpx and vpr accessory genes. Theaim of this study was to develop a model for both in vitroand in vivo studies of primate lentivirus accessory genefunctions. We therefore constructed CAEV recombinantgenomes in which SIV nef or vpr/vpx genes were inserted.The resulting recombinant viruses CAEVnef andCAEVvpxvpr were shown to be infectious and replicationcompetent. We found that Vpr/Vpx induce G2 arrest ininfected goat cells. Interestingly, infection of goat PBMCswith CAEVvpxvpr resulted in lymphocyte apoptosis de-spite the lack of their infection. We demonstrated thatSIV Nef protein induces CD4 and MHC-I down-regula-tion of expression on the surface of non infected goatlymphocytes. CAEVnef-infected goat PBMCs were foundto be activated with an over-expression of IL-2R associ-ated with an increase of their proliferation. On the otherhand, we showed that Nef exert a dual role on lymphocyteapoptosis regulation. Since CAEV is not a lymphocytetropic virus, our results raise the question about the im-plication of other pathways like an extra-cellular signalingin Nef and Vpr/Vpx inducing pathogenesis. This CAEVrecombinant virus system is a unique lentivirus model thathelp to study in vitro and in vivo unknown functions ofprimate lentivirus accessory genes, and in particular theirimplication in the alteration of functions of non-infectedcells.

A22^3

A GENOMIC ANALYSIS OF THE DYNAMIC ADAP-TATION OF YEAST TO HIGH ETHANOL PRODUC-TION

M. Cot(1), L. Benbadis(1), S. Alfenore(1), X. Camel-eyre(1), V. le Berre(1,2), S. Sokol(2), S. Jasson(3), C.Molina-Jouve(1), J.-L. Uribelarrea(1), S. Guillouet(1), G.Goma(1) and J. FrancVois(1,2)

(1) Centre de Bioingenierie Gilbert Durand, UMRCNRS5504, UR INRA 792; (2) Transcriptome ^ Biopchipsplateform of Genopole Toulouse, 135 Avenue de Rangeuil,F-31077 Toulouse, France; (3) INRA, Chemin BordeRouge, F-31326, Castanet-Tolosan

S. cerevisiae is well known for its high ethanol productionperformance. However, an Energy recommendation fromthe EU requires that we raise the bulk production of etha-nol by a factor of 10 within the next 5 years. Throughoptimisation of nutrient feeding, we were able to produce150 g l-1 ethanol (i.e. 18.9‡GL) in 45 h (Alfenore et al.(2002). Appl. Microbiol Biotech 60, 67 -72). The yeastcells adapt to this increased ethanol concentration by amechanism that is probably di¡erent of the stress responsetriggered by sudden ethanol addition to exponentiallygrowing cells. In order to investigate the dynamic adapta-tion of yeast to high ethanol production, macrokineticsand genomic analyses were carried out during fed-batchprocess. Data were collected during a fermentation whereglucose feeding kept the residual glucose concentration ataround 40 g l-1. A ¢nal ethanol titer of 120 g l-1 wasreached in 52 h. Growth kinetics showed a short exponen-tial growth phase at Wmax 0.5 h

-1 followed by a decreasedue to ethanol inhibition. At an ethanol concentration of90 g l-1, an uncoupling phase occurred when cells keptproducing ethanol while not dividing and with an in-creased loss of viability. We used DNA chip technologyto investigate on a genome scale the expression changesthat occur during this process. We found increased expres-sion of stress genes at the uncoupling phase, and a pro-nounced drop in MCT1 gene encoding malonyl COA ace-tyl transferase and an increase in expression of YAT1encoding mitochondrial/peroxisomal carnitine acetyltransferase, suggesting a reorganisation in the use of lipids.Clustering analysis identifed two major groups: one thatcharacterizes the declining growth phase and whose genesare mostly down regulated; while the second group gath-ers up-regulated genes that mainly appear shortly aftergrowth arrest. Thus we have uncovered remodelling ofgene expression involved in lipid, phospholipid and sterolmetabolism as well as in redox balance, unlinked to glyc-erol production. Further analysis of our genomic data willprovide a comprehensive overview of the e¡ects induced



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by high ethanol production, and identify the primary tar-gets for ethanol tolerance.

A22^4

ASPERGILLUS NIGER STRAIN AND PROCESS IM-PROVEMENT USING TRANSCRIPTOMICS

R. Meulenberg, S. Breestraat, H. H. Menke, N. N. M. E.van Peij, H. J. Pel and H. Stam

DSM Food Specialties, Research & Development, P.O. Box1 (bag 624-0295), 2600 MA Delft, The Netherlands

The ¢lamentous fungus Aspergillus niger is one of themost important microorganisms used for production ofenzymes in the food and feed industry. Strains of A. nigerare able to secrete large amounts of various enzymes and,therefore, this organism is a suitable host for homologousand heterologous gene expression. Classical strain im-provement programs and de¢ned genetic modi¢cationshave been successfully used to improve enzyme productiv-ity. In order to rationalize and speed up the ongoing strainand process improvement programs, as well as to identifypotentially new homologous business products, DSM se-quenced and annotated the genome of A. niger. Over14,000 open reading frames (ORF’s) were identi¢ed within35.9 Mbp (s 98%, rDNA excluded), of which over 50%have an unknown function. Recently, A¡ymetrix A. nigerGeneChips were designed and manufactured, enabling thesimultaneous analysis of the genome-wide transcriptome.A strain and process improvement program using tran-scriptomics has now started. For a good interpretationof microarray results, a high reproducibility of experimen-tal procedures is essential. Validation of GeneChip analy-sis and RNA sample treatment has been extensively testedand will be discussed. On top of this, improved strains andprocesses, tested and selected on labscale, ultimately haveto be implemented in our production facilities. For thisreason, labscale fermentations were exactly downscaledfrom full scale and were strictly controlled during all ourstudies. Reproducibility of our labscale fermentation pro-cess and results of the sophisticated control protocol willbe discussed on the basis of the biological variation asdeduced from transcriptome analysis.

A22^5

CHARACTERIZATION AND FUNCTIONAL ANALY-SIS OF PLASMIDS OF AN F18+ PORCINE ENTER-OTOXIC ESCHERICHIA COLI STRAIN

F. Olasz(1), P. Zs. Fekete(2), Zs. Boldogkoi(1), G. Blum-Oehler(3), and B. Nagy(2)

(1) Agricultural Biotechnology Center, Go«do«llo, Hungary;(2) Veterinary Medical Research Institute of the HungarianAcademy of Sciences, Budapest, Hungary); (3) Institut fu«rMoleculare Microbiologie, Universita«t Wu«rzburg, Wu«rz-burg, Germany

An enterotoxigenic Escherichia coli (ETEC) strain (namedEc2173), isolated in Hungary from a weaned pig that diedas a result of post-weaning diarrhoea, was characterized.The strain harboured three large plasmids (individual sizes48-85 kb) and three relative smaller ones (13-16 kb). The85 kb plasmid carried a haemolysin gene and an F18acoperon (as proven by phenotyping and DNA hybridisa-tion). Further hybridisation experiments with heat stabiletoxin (STa and STb) genes showed that both genes werelocalized on a 75 kb plasmid. This large plasmid alsocarried the tetracycline resistance gene, therefore calledpTc. In order to asses the pathonegic potential of theheat labile toxin gene stb, it was to be replaced by aninsertional mutant gene (carrying kanamycin resistancegene) using homologous recombination. However, inspiteof intensive selection pressure through s 200 generationsno replacements occurred. The loss of pTc was ¢nallyachieved by plasmid incompatibility and segregation.The pTc cured strains lost their sta and stb genes andand their toxic phenotypes, together with tetracycline re-sistance. These traits all appeared in a K12 strain trans-formed with pTc. Subsequent experiments using K12strains with and without chromosomal transfer regionhave proven that pTc is a self conjugative plasmid. Epi-demiologic studies on porcine ETEC strains from Austria,Hungary and the USA indicated that similar strains withpTc-like plasmids are quite common. It is also recognizedthat pTc cured F18+ derivatives of the Ec2173 strain havea potential as a live oral vaccine against post-weaningdiarrhoea of pigs.



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A22^6

TEMPERATURE AND GROWTH PHASE REGULATETHE TRANSCRIPTION OF THE O-ANTIGEN GENECLUSTER OF YERSINIA ENTEROCOLITICA O:3

M. Skurnik(1,2), M. Biedzka(1), A. Brzezinska(2), R.Venho(2) and P. Lahtinen(2)

(1) The Haartman Institute, University of Helsinki, Helsin-ki; (2) Department of Medical Biochemistry and MolecularBiology, University of Turku, Turku, Finland

Temperature regulates very precisely the expression of Y.enterocolitica virulence factors in vitro including that ofYadA, Ail, invasin, urease, lipopolysaccharide (LPS)such that some factors are optimally expressed at 37‡Cand others, at 25‡C. In addition to change in temperaturesome other environmental factors such as pH and ionicstrength are modulating the gene expression in Yersinia.This modulation must also be very important in vivo andtherefore it is important to know the molecular details ofthe regulation mechanisms to be able to study their role invirulence. Here we report the construction of two reporterstrains where the ¢re£y luciferase gene lucFF was intro-duced into the genome of Yersinia enterocolitica serotypeO:3 downstream of both promoter regions of the O-anti-gen gene cluster to monitor the transcriptional activities ofthe promoters. We found that both temperature andgrowth phase have a strong in£uence on the transcription-al activity of both promoters. The in£uence of temperaturewas most pronounced in stationary phase bacteria. In log-arithmically growing bacteria the e¡ect of temperature wasvery little. The role of di¡erent temperature-regulated fac-tors on serum resistance of Y. enterocolitica was studied.

A23^1

TRANSCRIPTIONAL REGULATION OF LACCASEGENES IN PLEUROTUS SAJOR-CAJU

C. M. Tang, W. Ge and J. A. Buswell

Department of Biology, and the Centre for InternationalServices to Mushroom Biotechnology, The Chinese Univer-sity of Hong Kong, Shatin, New Territories, Hong KongSAR, China

The edible mushroom, Pleurotus sajor-caju, produces mul-tiple forms of extracellular laccase (p-diphenol oxidase,EC1.10.3.2) when grown in liquid culture and on lignocel-lulosic materials of the type used for industrial-scale mush-room cultivation. In order to assign physiological roles toeach laccase isoenzyme, we have set out to clone and se-quence the cDNA sequences encoding for di¡erent laccaseisoforms, and to determine gene expression levels in both

liquid culture and in solid-state systems throughout themushroom developmental cycle. Using a PCR-based strat-egy, we have cloned ¢ve full-length P. sajor-caju laccasecDNAs (designated lcc1, lcc2, lcc3, lcc4 and lcc5) fromfungal mycelium that was grown on potato dextrose brothsupplemented with vanillin as an enzyme inducer. ThecDNAs of lcc1, lcc2, lcc3, lcc4 and lcc 5 contained pre-dicted ORFs of 1602, 1605, 1572, 1605 and 1584 bp, en-coding for 534, 535, 524, 535 and 528 amino acids, respec-tively. Transcripts of all ¢ve laccase genes were detectedthroughout the di¡erent stages of the developmental cyclein fungal mycelium grown on straw. Both lcc2 and lcc5 arestrongly expressed throughout the substrate colonisationphase and during fruit body development up to the elon-gation stage, whereas lcc1 transcription is highest just pri-or to the onset of fruiting and throughout the pinhead andbutton stages. Expression of lcc2, lcc3 and lcc5 is alsostrongly correlated with extracellular manganese peroxi-dase activity, and transcription of lcc1, lcc3, lcc4 andlcc5 increased when the fungus was grown in media sup-plemented with various phenols.

A23^2

ENZYMATIC AND WOOD DEGRADING CAPABIL-ITIES OF A NOVEL FUNGUS SUITABLE FOR BIO-PULPING OF WOOD CHIPS

A. Hatakka, T. K. Hakala, V. Salo, T. Lundell and P.Maijala

Department of Applied Chemistry and Microbiology, P.O.Box 56 (Biocenter 1, Viikinkaari 9), 00014 University ofHelsinki, Helsinki, Finland

Successful pretreatment of wood chips by white-rot fungiis usually connected to selective lignin removal from woodcell walls, which is considered to be essential for energysaving in re¢ner pulping. We have isolated white-rot fungifrom old forests and other sources. More than 300 isolateswere screened on agar plate tests, and the ability of about90 fungi to degrade spruce wood blocks in 10 weeks wasfurther assessed. The most interesting fungus for biopulp-ing was Physisporinus rivulosus T241i. Spruce wood chipstreated by P. rivulosus and the well-known fungus Ceripor-iopsis subvermispora that was used as a reference, weremechanically re¢ned and the handsheet properties of thepulps were studied. A two-week fungal treatment de-creased 30% energy needed for re¢ning compared to un-treated chips. Fungal growth in spruce wood was studiedby light and confocal laser scanning microscopy. Growthhabit and rate on malt agar, in and on wood blocks re-vealed di¡erences among di¡erent Physisporinus strainsobtained from culture collections. The strain T241ishowed the most e⁄cient capacity to degrade sprucewood. The main lignin-degrading enzyme produced in



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spruce wood chips by the strain T241i was manganeseperoxidase (MnP), which was produced as several iso-forms with pIs between 3.8-4.3. Molecular weights variedbetween 47-54 kDa. The N-terminal amino acid sequenceof one of the isoforms revealed close resemblance of P.rivulosus MnP to MnP1 from the Japanese isolate IZU-154 and MnPs from C. subvermispora, Phanerochaetechrysosporium, and Dichomitus squalens.

A23^3

PHLEBIA RADIATA MnP AND LiP ENZYMES: PHY-LOGENETIC DIVERSITY AND A NEW CLUSTERWITHIN THE FUNGAL LIGNINOLYTIC PEROXI-DASES

T. Lundell(1), K. Hilde¤n(1), A. Hatakka(1) and A. T.Martinez(2)

(1) Department of Applied Chemistry and Microbiology,P.O.Box 56, Biocenter 1, Viikinkaari 9, University of Hel-sinki, FIN-00014 Helsinki, Finland; (2) Department ofMolecular Microbiology, Centro de InvestigacViones Biolo¤g-icas, CSIC, Vela¤zquez 144, E-28006 Madrid, Spain

Phlebia radiata strain 79 is e⁄cient lignin-degrading, whiterot in wood causing and thereby, biotechnologically prom-ising basidiomycetous fungus belonging to the family ofCorticiaceae. Previously, we isolated two manganese per-oxidases (MnPs), three lignin peroxidases (LiPs) and oneglyoxal oxidase from nitrogen-limited and manganese-sup-plied cultures of this fungus. We have recently cloned andfully sequenced all the ¢ve genes coding for the multipleMnP and LiP isozymes using RT-, RACE- and genomewalking PCR approaches. The three LiP encoding genes(Pr-lip1, Pr-lip2, Pr-lip3) and the gene Pr-mnp3 coding forMnP3 isozyme are all disrupted by 11 short introns. Theiropen reading frames are translated to 339-342 amino-acidlong mature proteins preceded by a secretion leader pep-tide of pre-pro structure. Genetically and at protein se-quence level with 60-73 % similarity, these four peroxi-dases form a new, distinct cluster within the recentlycreated dendrogram of fungal ligninolytic peroxidases(Martinez, A. 2002. Enz. Microb. Technol. 30:425-444).However, the second manganese peroxidase, encoded bythe gene Pr-mnp2, is phylogenetically divergent from thePhlebia LiP/MnP3 cluster and shows a high degree ofsimilarity at amino acid level (over 70 %), length of matureprotein (367 amino acids) and in conserved intron-exonstructure (7 introns) with the group of typical MnPsfrom the fungi Dichomitus squalens, Phanerochaete chrys-osporium, P. sordida and Ceriporiopsis subvermispora. The3-D modelled folded structures of Pr-MnP2 and Pr-MnP3further con¢rm the divergency detected at primary se-quence level. The expression and potential use of thesetwo enzymes is discussed.

A23^4

ACCUMULATION OF HEAVY METALS IN MUSH-ROOMS GROWING IN TISZA RIVER BASIN AFTERECOLOGICAL ACCIDENT

M. P. Niksic(1) S. Blagojevic(2), B. Zarkovic(2) and A.Klaus(1)

(1) Faculty of Agriculture, Institute for Food Technologyand Biochemistry, Department of Microbiology, Nemanjina6, 11080 Zemun-Beograd, Yugoslavia; (2) Department ofAgricultural Chemistry

In spring 2000 large quantities of heavy metals togetherwith cyanide-tainted water over£owed from a gold miningcenter in Baia Mare caused ecological disaster in the TiszaRiver in Romania, Hungary and Yugoslavia, causingwhat’s been, termed the worst environmental disaster sinceChernobyl. Certain species of mushrooms have capabilityto accumulate heavy metals in fruit body and can act as‘‘bio accumulators’’ of metals. This study contains theresults of six surveys of metals in fungi collected afterthe accident and every 6 months in last 3 years. A numberof common fungi were analysed for their contents of Pb,Cu, Fe, Cr, Ni, Co, Zn, and Mn from di¡erent placesalong the river in order to estimate the pollution of envi-ronment. The investigated heavy metals were determinedby £ame atomic absorption spectrophotometry AAS (Var-ian SpectrAA-10). After the accident accumulation of Pb,Fe and Zn, in fungi from 4 di¡erent locations along theriver, were much higher than it is regulated. The contentof Pb was from 2-5 times higher (12-25 mg/kg of drymatter) in Panus tigrinus and Coprinus micaceus. The re-sults also indicated that the content of Cu, Mn, Cr, and Nilevel in analysed species can be higher several times than itis allowed in water. After the 3 years the contents of met-als in fungi were still high but in same species (Panustigrinus and Laetiporus sulphureus) at the same 3 locations,the content of some metals in fruit bodies decreased reg-ularly. The average content of Zn decreased in both fungi.Also content of Mn and Cr were decreasing for both fun-gi, indicating that these 3 metals could be good indicatorfor measuring decreasing of accumulation.



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A23^5

FAST ANALYSIS AND ISOLATION OF LIGNINO-LYTIC ENZYMES

H. Podgornik(1), A. Podgornik(2), A. Perdih(1)

(1) Faculty of Chemistry and Chemical Technology, As›ker-c›eva 5, 1000 Ljubljana, Slovenia; (2) BIA Separationsd.o.o., Teslova 30, 1000 Ljubljana, Slovenia

Ligninolytic enzymes represent a big potential on di¡erentareas like paper and textile industry, bioremediation etc.due to their ability to degrade lignin as well as di¡erentxenobiotics. Three groups of ligninolytic enzymes, ligninperoxidases (LiP), manganese peroxidases (MnP) and lac-case (Lac) are excreted by white rot fungi. While severalmethods for determination of LiP, MnP, and Lac activityare known, isolation of particular ligninolytic enzyme isstill time consuming since LiP as well as MnP are isoen-zyme families. Di¡erent isoforms can only be determinedusing electrophoresis or chromatography. Both methodsrequire close to an hour or even more, before the resultis obtained. In the chromatography, the main reason forlong analysis time can be attributed to the slow masstransfer between mobile and stationary phase occurringinside the chromatography column. In last decay, this bot-tleneck was overcome introducing a new group of station-ary phase called monoliths. They enable extremely fastseparation of macromolecules, eg. separation of proteinsor DNA within few seconds. In this work we introducechromatographic methods using CIM0 monolithic col-umns for LiP, MnP and Lac analysis and their isolationfrom the growth medium of two di¡erent white rot fungi,Phanerochaete chrysosporium and Ceriporiopsis subvermis-pora. We were able to determine ligninolytic enzymes com-position in few minutes, time comparable to determinationof enzyme activity. Di¡erent cultivation conditions wereapplied to vary LiP isoenzyme composition in P. chryso-sporium growth medium. MnP analysis revealed that dif-ferent isoforms are produced by tested fungi. Additionally,a good separation of MnP isoenzymes and Lac wasachieved in C. subvermispora growth medium.

A23^6

IS OSTREOLYSIN, A CYTOLYTIC PROTEIN FROMTHE OYSTER MUSHROOM (PLEUROTUS OSTREA-TUS), INVOLVED IN FUNGAL FRUITING?

S. Berne(1), T. Turk(1), P. Mac›ek(1), F. Pohleven(2), K.Sepc›ic¤(1)

(1) Department of Biology, Biotechnical Faculty, Univer-sity of Ljubljana, Vec›na pot 111, 1000 Ljubljana, Slovenia;(2) Department of Wood Science and Technology, Biotech-nical Faculty, University of Ljubljana, Roz›na dolina, CestaVIII/34, 1000 Ljubljana, Slovenia

Ostreolysin, a 16 kDa cytolytic protein, was recently pu-ri¢ed from the fruiting bodies of the edible oyster mush-room (Pleurotus ostreatus). Its N-terminal amino acid se-quence (50 residues) is highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 proteinfrom the mushroom Agrocybe aegerita, Asp-hemolysinfrom Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found thatostreolysin expression confers with development of pri-mordia and fruiting bodies of P. ostreatus, which is inaccord with previous ¢nding that the Aa-Pri1 gene is spe-ci¢cally expressed during fruiting initiation. Indeed, whennutrient media plates had been supplemented with puri¢edostreolysin before inoculation with P. ostreatus mycelium,a considerably faster formation of primordia was ob-served, compared to the control plates without ostreolysin.It is suggestive that the isolated protein plays an importantrole in the initial phase of fungal fruiting.

A24^1

BIOACTIVE POLYSACCHARIDES ISOLATEDFROM SOME MUSHROOMS AND THEIR INFLU-ENCE ON THE HUMAN T AND B CELLS

A. S. Klaus(1), M. P. Niksic(1) and L. J. L. D. VanGriensven(2)

(1) Faculty of Agriculture, Institute for Food Technologyand Biochemistry, Department of Microbiology, Belgrade,Yugoslavia; (2) Plant Research International, Departmentof Cell Cybernetics, Wageningen

The aim of these experiments was to examine bioactivepolysaccharide’s and protein’s in£uences on the humanT and B lymphocytes. Bioactive polysaccharides and pro-teins were fractionated from di¡erent industrial and med-ical basidiomycetous fungi ^ Ganoderma lucidum, Coprinuscomatus, Agaricus bisporus, Agaricus blazei, Agaricus sub-rufescens, Grifola frondosa, Lentinus edodes. Di¡erentmethods and techniques were used for fractionating, puri-



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¢cation and researching activities of bioactive compo-nents. The in£uences of bioactive components were testedon JY and Jurkat cell lines. JY cells are human B lym-phocytes that are transformed by Epstein-Bar virus; theyare not producing EB virus, but they are producing im-munoglobulin. Jurkat cells are human T lymphocytes. Ex-periments started with a dilution of 100.000 cells per ml inRPMI 1640 + 10 % fetal calf serum, seeded in a 24 wellplate with six rows. First row was control without anymushroom extract, second contained 1Wg mushroom ex-tract per well, and rows 3, 4, 5 and 6 were serially dilutedwith 100 micro liters from row 2 into row 3 respectivelyfrom 3 into 4, etc. Counting of cells was performed in ahemocytometer after 72 h incubation at 37‡C. Di¡erentGanoderma lucidum extracts showed the best results be-cause elementary number of cells decreased almost morethan 50% in some cases. The results showed that bioactivepolysaccharides and proteins extracted from certain mush-rooms had bigger or smaller in£uence on human T and Blymphocytes. It is very interesting, because these bioactivecomponents from mushrooms can be potentially used insome tumor treatment.

A24^2

MOLECULAR CHARACTERIZATION OF THE HAE-MOGLOBIN AND HAEMIN IRON UTILIZATIONPATHWAY IN CANDIDA ALBICANS

Z. Weissman and D. Kornitzer

Dept. of Microbiology, Faculty of Medicine, Technion ^Israel Institute of Technology, Haifa 31096, Israel

The ability to acquire iron from host tissues is a majorvirulence factor of pathogenic microorganisms. Candidaalbicans is an important fungal pathogen, responsible foran increasing proportion of bloodstream infections. Likeother pathogens, C. albicans is able to utilize haemin andhaemoglobin as iron sources. However, the molecular ba-sis of this pathway in fungal pathogens is unknown. Tostart delineate the haemoglobin / haemin iron utilizationpathway in C. albicans, we performed a screen for genesable to confer the ability to utilize haemoglobin iron toSaccharomyces cerevisiae. The single gene isolated in thisscreen, RBT51, shows very high homology to the formerlyidenti¢ed RBT5 gene (Repressed By Tup1). Both RBT5and RBT51 are induced by iron deprivation, but RBT51is much more weakly expressed in C. albicans, even underinductive conditions. Accordingly, the C. albicans rbt5 de-letion, as well as the double deletion rbt5 rbt51, but notthe rbt51 deletion by itself, display a signi¢cant defect inhaemin and haemoglobin iron utilization. RBT5 andRBT51 encode predicted extracellular GPI-anchored pro-teins, which contain a repeated cysteine motif found in anumber of additional fungal proteins. By immuno£uores-

cence, Rbt5 and Rbt51 and are found to localize to theexternal face of the cell membrane. We suggest that theseproteins serve as ligands for haemin, haemoglobin, andmay facilitate their uptake by increasing their concentra-tion at the cell membrane.

A24^3

UPDATE ON FUSARIUM INFECTIONS IN ADULTSAND CHILDREN

V. Krcmery and A. Doczeova

School of Public Health, University of Trnava, Dept. OfOncology, Trnava, Slovakia

Fusarium infections are the number 3 among invasive fun-gal infection in adults after Candida and Aspergillus IFI.Within last 20 years, we see some change in this trend:1. Before, Fusarium was infecting only immunocompro-mised host; currently, several cases inimmunocompetent individuals have been seen.2. Children were infected exceptionally; nowadays, there isan increasing trend in children as well.3. Respiratory tract and skin were considered to be theportals of entry; currently, water and food were discov-ered to be the source in most of cases.4. In vitro tests showed nowadays increasing resistance toamphotericin B in vitro and even lipid formulations ofAMB were of little help.5. Fusarium infections were considered to be deadly beforedisease (80-100% mortality). However, new classes of anti-fungals (Voriconazol, Caspofungin) showed some im-provement in survival and mortality recently dropped to50-70%.

A24^4

CONTROL OF VIRULENCE GENE EXPRESSION INCANDIDA ALBICANS

J. Morschha«user

Institut fu«r Molekulare Infektionsbiologie, Universita«tWu«rzburg, Ro«ntgenring 11, 97070 Wu«rzburg, Germany

Many di¡erent traits of the opportunistic fungal pathogenCandida albicans contribute to its capacity to cause infec-tions in immunocompromised patients, including the abil-ity to switch between yeast and hyphal growth modes andthe secretion of hydrolytic enzymes. C. albicans possessesa large gene family encoding secreted aspartic proteinases(Saps). The individual SAP genes are di¡erentially inducedby host signals at various stages of an infection, suggestingthat they have speci¢c roles in the fungus-host interaction.Expression of a subset of these genes, SAP-SAP6, is asso-



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ciated with the switch from yeast to hyphal growth due tothe use of common signal transduction pathways, a MAPkinase cascade and a cAMP-dependent pathway. Disrup-tion of both signaling pathways inhibits hyphae formationand SAP4-SAP6 expression in vitro and in vivo. However,the expression of hyphae-associated virulence genes doesnot depend on morphogenesis. Mutants with a defect inpolarized growth are unable to form hyphae and grow asunusually large yeast cells under normally hyphae-induc-ing conditions. Nevertheless, they still express known hy-phae-associated genes in response to appropriate stimuli.Therefore, C. albicans uses speci¢c signal transductionpathways to coordinate the expression of various virulencetraits in response to host signals. This allows an optimaladaptation to host niches encountered at di¡erent stagesof an infection.

A24^5

THERAPEUTIC ACTIVITY OF KILLER ANTIIDIO-TYPIC ANTIBODIES AND MIMOTOPES

L. Polonelli

Department of Pathology and Laboratory Medicine, Sectionof Microbiology, University of Parma, Parma, Italy

Antiidiotypic antibodies with fungicidal activity (KTan-tiId) have been produced by systemic or mucosal idiotypicvaccination with a monoclonal antibody (mAbKT4) neu-tralizing in vitro the activity of a Pichia anomala killertoxin (KT) characterized by a wide spectrum of microbi-cidal activity against pathogenic fungi presenting speci¢csuper¢cial receptors (KTR). Signi¢cantly, anti-KTR can-didacidal antibodies have been detected in the serum andsecretions of animals and individuals experimentally ornaturally infected with KT-sensitive Candida albicansstrains. KTantiId produced in the polyclonal, monoclonal(KTmAb) or single chain recombinant format (KTscFv)have shown to kill in vitro KT-sensitive, Candida albicans,£uconazole-resistant C. glabrata and C. krusei, Cryptococ-cus neoformans, Aspergillus fumigatus and Pneumocystiscarinii strains and to confer active and passive immuno-protection in animal models of experimental aspergillosis,candidiasis, cryptococcosis and pneumocystosis. KTscFvexpressed at the surface or secreted by recombinant strainsof a human commensal bacterium, Streptococcus gordonii,an e⁄cient colonizer of mucosal membranes, have showna potent therapeutic e¡ect in an experimental model of ratvaginal candidiasis. Decapeptides with antibiotic activityhave been selected and synthesized on the basis of theaminoacidic sequence of the variable region of KTscFv.Such synthetic killer mimotopes (KM) have shown to ex-ert a potent candidacidal activity in vitro and a markedtherapeutic e¡ect in vivo in murine experimental models ofvaginal and systemic candidiasis (manuscript in prepara-

tion). Killer antibodies or killer mimotopes administeredas antibiotics could represent innovative and exclusivemodels for prophylactic and immunotherapeutic purposesagainst further relevant microorganisms potentially sus-ceptible to KT.

A24^6

G-CSF AND POLYENES IN TREATMENT OF EX-PERIMENTAL ASPERGILLOSIS

E. Segal and E. Sionov

Department of Human Microbiology, Sackler School ofMedicine, Tel-Aviv University, Tel-Aviv, Israel

Aspergillosis is a life-threatening invasive mold infectionthat a¡ects severely immunosuppressed patients. The inci-dence of aspergillosis continues to increase and remains amain cause of morbidity and mortality in this population.Treatment of aspergillosis is problematic and still unsatis-factory. We found in previous studies that polyene-intra-lipid admixtures (amphotericin B-intralipid (AMB-IL) andnystatin-intralipid (Ny-IL)) were e¡ective and less toxicthan the standard formulations of these polyenes. In thisstudy we investigated the e¡ect of granulocyte colony-stimulating factor (G-CSF) in combination with AMB,AMB-IL or Ny-IL admixtures in an experimental murineaspergillosis model. An experimental animal model of sys-temic aspergillosis was obtained by intravenous (iv) inoc-ulation of immunocompromised ICR mice with spores ofA. fumigatus (ATCC 64026). Immunosuppression wasachieved by intraperitoneal (ip) administration of 200mg/kg of cyclophosphamide, 3 days prior to infectionwith the fungus. Treatment began 2 hrs after inoculationof A. fumigatus. Treatment consisted of 5 consecutive dai-ly administrations of a combination of G-CSF (ip at adose of 150 Wg/kg/day) and either AMB, AMB-IL (iv, 1mg/kg/day) or Ny-IL (iv, 4 mg/kg/day). The mean survivalrate (MSR) and mean survival time (MST) were evaluatedduring an observation period of 30 days. The experimentsrevealed that the combination therapy, particularly G-CSFwith AMB or AMB-IL, led to about 90% survival of thetreated mice, in comparison to 0% of the untreated con-trols and 39.7-48% of the animals treated by the polyeneswithout the cytokine. In addition, the combination ther-apy prolonged the MST of the mice up to 29.2 days, incomparison to MST of 6.13 days in untreated controls and12.4-17.3 days in animals treated with polyenes alone.These results suggest that a similar approach could possi-bly be promising for management of aspergillosis in hu-mans.



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A25^1

CHARACTERISATION OF PIGEON PARAMYXOVI-RUS TYPE-1 (PPMV-1) ISOLATES BY PHYLOGE-NETIC ANALYSIS OF FUSION AND MATRIX PRO-TEIN GENES

D. Barlic›-Maganja, U. Krapez›, I. Toplak, S. Mankoc›, J.Grom, O. Zorman Rojs

University of Ljubljana, Veterinary Faculty, Gerbic›eva 60,1115 Ljubljana, Slovenia

Paramyxoviruses of type-1 (PMV-1) obtained from pi-geons were isolated in the allantoic £uid of SPF eggsand identi¢ed as PMV-1 through a haemagglutination-in-hibition test using speci¢c PMV-1 polyclonal antisera. Thedetermined ICPI values of all PMV-1 isolates were about1.0. Single step RT-PCR method was used to amplify nu-cleotide sequences from the fusion and matrix protein generegions. For genotyping of PPMV-1 isolates direct nucle-otide sequencing of ampli¢ed products was performed.Among four PPMV-1 isolates included in our study threeof them had the fusion protein cleavage sequence109SGGGRQKR/FIG119 and one of them109SGGRRQKR/FIG119. For comparative purposes, nu-cleotide analysis of the conserved matrix protein codinggene was also performed. The phylogenetic analysis ofboth genome regions grouped PMV-1 isolates of all path-otypes in two major groups. Inside one group PPMV-1from di¡erent parts of the world formed separate lineageshowing close relationship among pigeon isolates. Theseviruses may presumably share a common progenitor andevolutionary pathway.

A25^2

PRECLINICAL EVALUATION IN RH. MACAQUE OFSIV THERAPEUTIC VACCINATION BASED ONDNA/FOWLPOX PRIME-BOOST REGIMEN

C. De Giuli Morghen, C. Zanotto, V. Elli, V. Basavecchia,M. Paganini, M. Neri and A. Radaelli

Laboratory of Molecular Virology, Dept. of Medical Phar-macology, University of Milan, Via Vanvitelli 32, 20129Milan, Italy

Two groups of primates were immunized with SIVgag/pol-expressing vectors to assess their ability to restore the im-mune response in SIV-infected ART-treated Rhesus mac-aques. If present, this activity would be very useful, incombination with the structured therapy interruption(STI), to abolish viremia in long-term non-progressors.In SIVmac-infected macaques, the expansion of helperCD4+ T-cells may be of even greater importance since a

signi¢cant loss of CD4+ T-cells occurs in several compart-ments early in SIVmac251 infection. The broadening ofthe CD8+ T-cell response would also be necessary inboth therapeutic and preventive vaccine inasmuch as itmay reduce the risk of the immune escape that occursduring viral rebound. We therefore designed immunizationregimens to directly compare, in SIVmac251-infectedMamu-A*01-positive Rhesus macaques, the relative im-munogenicity of a DNA/poxvirus to a poxvirus/poxvirusimmunization. Macaques were divided into two groups offour macaques each. Macaques of group A were ¢rst im-munized with plasmid DNA (DNA-SIV-gp) encoding theGag and Pol SIV proteins and, eight weeks later, with 2 x108 pfu of recombinant FP-SIV-gp encoding the same pro-teins, whereas macaques of group B received two immu-nizations with FP-SIV-gp. While a negligible increase inthis CD8+ T-cell population was found following inocu-lation with DNA-SIV-gp in the animals of group A, asigni¢cant increase in the frequency of tetramer-stainingCD8+ T-cells was observed in macaques from group Bwithin a few weeks from immunization with the FP-SIV-gp. Vaccination with DNA-SIV-gp and FP-SIV-gp combi-nation induced higher frequency of functional Gag181-189CM9-speci¢c CD8+ T-cells than the FP-SIV-gp vaccina-tion alone. These results suggest that a therapeutic immu-nization can be a useful approach to restore the cell-medi-ated immunity in SIV-infected animals.

A25^3

DETECTION OF ENTEROVIRUSES FROM DIAG-NOSTIC SAMPLES USING 5’- NTR SPECIFIC PRIM-ERS BETWEEN 1st JULY 2000 AND 31st DECEMBER2002 IN HUNGARY

B. Kapusinszky, M. Vollain, E. Toth, I. Mezey, Gy. Berenc-si

Division of Virology ‘Be¤la Johan’ National Center for Epi-demiology, H-1097 Budapest, Gya¤li str. 2-6, Hungary

The recent eradication of wild polioviruses in the Euro-pean Region has drawn our attention to the non-polioenteroviruses circulating in the country. Because of thelow e⁄ciency of the classical virological techniques thestrategy of laboratory diagnostic had to be modi¢ed. Alldiagnostic samples have been tested ¢rst using RT-PCR,and only samples found to be positive with nested pairs ofprimers speci¢c to the 5’-NTR were processed using tissueculture techniques. Several hundred virus positive sampleswere found in stool, throat swab, cerebrospinal £uid orserum samples every year. Less than 25 % of the virusescould be grown on RD, L20B or VERO cell lines. New-born mice supported only the replication of Coxsackievi-rus A16 from Hand-Foot-Mouth diseases. The circulationof echovirus types 4, 6, 7, 13, 20 and 30 could be veri¢ed



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using mixtures of type speci¢c immune sera. The etiolog-ical role, however, could not be supported by the detectionof the enteroviruses even in those cases when serum neu-tralisation test were possible, since the submitted sampleshad been taken sometimes weeks after hospitalisation ofthe patients. In these cases the passanger role of the de-tected or isolated viruses is very probable. It has to bementioned that in a few patients long term presence ofenteroviruses could be detected in serum samples. Un-fortunately the antibodies prevented the virus isolationfrom the vast majority of serum samples. In the case ofa few serotypes (echovirus 4) the epidemic spread of thevirus could be followed within the country.

A25^4

PHYLOGENETIC CHARACTERIZATION OF RE-CENT AFRICAN HEPATITIS B VIRUS SEQUENCESREVEALS PREDOMINANCE OF GENOTYPE E INWEST AFRICA

I. Ma|«ga(1), P. Edorh(1), M. N. Mulders(2), C. P. Mul-ler(2), J. P. Bronowicki(3), V. Venard(1) and A. LeFaou(1)

(1) UMR 7565 UHP-CNRS, Laboratoire de Bacte¤riologie-Virologie, Faculte¤ de Me¤decine, Vandoeuvre-le's-Nancy; (2)De¤partement d’Immunologie, Laboratoire National deSante¤, Luxembourg, Luxembourg; (3) Service d’He¤pato-gastro-ente¤rologie, Ho“pital d’Adultes, Vandoeuvre-le's-Nancy

We have obtained human sera from young adults living in3 di¡erent African countries : Benin (120), Togo (148) andMali (152). Sera were collected from healthy volunteers.The prevalence of infectivity (HBsAg and/or DNA posi-tivity) is 22% in the Togolese sera, 14% in the Beninesesera and 67% in the Malinese sera. The sequence of thePreS/S gene was established and compared to representa-tive sequences of each of the 8 di¡erent human Hepatitis Bvirus genotypes. Previously obtained sera from Nigeria,Burkina Faso, Cameroun, already described are includedin this study for comparison. Within usually a genotypethe genetic variability lies between 6.4% and 10.4%. Allwest African HBV strains belonged to genotype E excepta few strains of Cameroun which are of genotype A. With-in this genotype E, a 1.5% genetic variability was ob-served. Throughout the rest of Africa multiple genotypescocirculate, whereas in West Africa so far only genotype Ehas been found. Genotype E seems to more on more con-served sequence than the other genotypes. The occurrenceof 4 to 7 amino acid codons deletion in the PreS2 region isa common occurrence for viruses belonging to genotype E.

A25^5

CIRCULATION OF THE NOVEL G9 ROTAVIRUSGENOTYPE IN SLOVENIA IN 2001/2002

M. Poljs›ak-Prijatelj(1), A. Steyer(1), T. Bufon(2), D.Barlic›-Maganja(3), J. Marin(1)

(1) Institute of Microbiology and Immunology, MedicalFaculty, University of Ljubljana; (2) Medical Centre,Ljubljana; (3) Veterinary Faculty, University of Ljubljana,Slovenia

Group A rotaviruses are the most important cause of se-vere dehydrating, diarrhoeal illness in young children andare associated with approximately one million deaths peryear in this age group. These viruses belong to the familyReoviridae and contain a genome of 11 segments of dou-ble-stranded RNA. Two major proteins VP7 and VP4present the basis for classi¢cation of group A rotavirusesinto G (VP7) and P (VP4) genotypes. Stool samples wereobtained from children admitted for treatment of severeacute diarrhoea and dehydration to hospital and routinelytested for the presence of rotavirus by the enzyme immu-noassay and electron microscopy. Double-stranded RNAextracted from stool specimens with TRIzol reagent wasused as the template for reverse transcription, followed byampli¢cation in a single tube, two-enzyme system. Theselected primer pairs permitted the ampli¢cation of theentire selected genome segment. Genotypes were identi¢edusing the type-speci¢c primers, derived from distinct re-gions of the gene 9 and 4. For the G-typing, six geno-type-speci¢c primers were used (determined genotypesG1, G2, G3, G4, G8, and G9), which were complementaryto the RNA strand of gene 9 variable regions. For the P-typing, six genotype-speci¢c primers selected from gene 4variable regions were used and genotypes P4, P6, P8, P9,P10 and P14 were determined. The predominant genotypewas G1P[8], followed by G9P[8] and G4 P[8]. In somespecimens the coexistence of G1 and G4 were determined.

A25^6

QUICK PROTOCOL FOR DNA EXTRACTION INCERVICAL SWABS USED FOR HUMAN PAPILLO-MAVIRUS (HPV) DIAGNOSIS

A. Santos, H. Sousa, A. Vasconcelos, R. Medeiros

Unidade de Oncologia Molecular, Instituto Portugue“s deOncologia Francisco Gentil ^ Porto, 4200 Porto, Portugal

Many epidemiological and molecular biology studies havedemonstrated that human papillomavirus (HPV) is asso-ciated with the development of cervical carcinoma. Di¡er-ent HPV types relative prevalence have been reported in



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distinct populations. The aim of the present study was totest the quality of DNA using a fast protocol to extractDNA in 59 samples of cervical swabs, generally used forcytological studies due to the di⁄culty in DNA extraction,with the purpose of detecting the presence of HPV DNA.We evaluated the InViSorb1 Spin DNA Micro Kit III onwhich it was suggested that cervical swabs could be di-rectly submerged into extraction tubes. Taking into con-sideration that samples were collected a long time ago andthat could present low cell quantity, good results wereaccomplished in the DNA extraction process (93%) despitehaving used cervical swabs samples stored at low temper-atures for more than 3 months. In addition to this, theInViSorb1 Spin DNA Micro Kit III’s protocol revealedto be a good method for DNA extraction from swabs in ashort period of time considering price and simplicity. Inconclusion, besides the fact that these preliminary studiesdemonstrate to be fast and e⁄cient to extract DNA fromcervical swabs it also allows to con¢rm diagnosis of HPVfrom the same extractions.Authors 1 and 2 contributed equally to this work (Stu-dent’s of Microbiology in Escola Superior de Biotecnolo-gia ^ Universidade Cato¤ lica Portuguesa)

A26^1

XYLANASE PRODUCTION BY ASPERGILLUSSTRAINS ON SULPHITE WASTE LIQUOR AS CAR-BON FEEDSTOCK

J. C. du Preez(1), Z. A. Chipeta(1), L. Christov(1,2) andG. Szakacs(3)

(1) Department of Microbial, Biochemical & Food Biotech-nology ,University of the Free State, P.O. Box 339, 9300Bloemfontein, South Africa; (2) Sappi Management Serv-ices, P.O. Box 3252, 1560 Springs, South Africa; (3) De-partment of Agricultural Chemical Technology, TechnicalUniversity of Budapest, Gellert ter 4, 1111 Budapest, Hun-gary

Pulp mill sulphite waste liquor (SWL) constitutes a cheapcarbon feedstock (mainly D-xylose) for microbial xylanaseproduction. SWL induced xylanase production at varyinglevels by certain ¢lamentous fungi, obviating the need forexpensive inducers such as xylan. In shake £ask cultures,strains of Aspergillus oryzae and A. phoenicis yielded rela-tively high xylanase activities of up to 164 U/ml in a me-dium based on concentrated SWL. These values werehigher than those obtained on xylan (up to 77 U/ml) orD-xylose (27 U/ml) with these strains. Higher xylanaseactivities (up to 200 U/ml) were obtained with A. oryzaein SWL at pH 7.5 in bioreactor cultivations. Crude xylan-ase preparations from these strains of A. oryzae and A.phoenicis exhibited optimal activities at assay pH values of6.5 and 5.0 and at assay temperatures of 65 and 55‡C,

respectively. The xylanases produced by these strainswith SWL as carbon substrate had potential applicationin biobleaching to enhance pulp brightness and/or de-crease the usage of bleaching chemicals. Pre-treatment ofpulp with the crude xylanases from these fungi, grown onthe SWL concentrate, enhanced pulp brightness by up to1.5 brightness points over that of the control. The appli-cation of crude xylanases from SWL to unbleached pulpreduced the subsequent consumption of chlorine dioxideup to 30% without sacri¢cing brightness. These ¢ndingsindicated that on-site enzyme production for biobleachingmay be feasible, thereby signi¢cantly reducing the negativeenvironmental impact of aqueous e¥uents from the pulpand paper industry.

A26^2

THE CHANGES OF CARBON FLUX DURING PHOS-PHATE STARVATION OF CORYNEBACTERIUMGLUTAMICUM

R. Ionina(1), M. Ruklisha(1), N. D. Lindley(2)

(1) Institute of Microbiology & Biotechnology, Universityof Latvia, 4 Kronvalda boulevard, LV-1586 Riga, Latvia;(2) Institut National des Sciences Appliquees, 135 Avenuede Rangueil, 31077 Toulouse, France

Corynebacterium glutamicum is of special interest for theindustrial production of amino acids and other metabo-lites. The restriction of bacterial grow is one of the widelyused strategies to increase the amino acids biosynthesis.The modi¢cations of carbon and the associated energetic£uxes through central metabolism redirect anabolic path-ways linked to biomass synthesis towards metabolite over-production. But it is important to ¢nd a way to reach therestricted bacterial growth without complete halt of thegrowth, and subsequently, restriction of protein synthesisde novo. One of the methods could be the cultivation ofbacteria under phosphate limiting conditions. Phospho-rous is one of the most important elements of cellularcontent. Thus phosphate depletion is likely to have a di-rect e¡ect on the global expression pro¢le of a large num-ber of genes and thereby modify carbon £ux distribution.We have examined the e¡ect of phosphate starvation onthe growth, intracellular metabolite concentrations, pro-duction of amino and organic acids in the wild-type C.glutamicum in the fermentation processes. The speci¢cgrowth rate decreases under phosphate-limiting condi-tions, but bacteria keep the ability to grow for one gen-eration of cells without duplication of RNA content. Theconcentrations of most of intracellular glycolytic inter-mediates upstream of pyruvate decrease. At the sametime the increasing of NADH/NAD+ ratio is observed.The synthesis of alanine and valine, as well as pyruvate,increases drastically after phosphorus depletion. These re-



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sults allow us to assume that conditions of phosphatestarvation of C. gluatmicum could be useful for an improv-ing of some amino acids biosynthesis, especially alanineand valine.

A26^3

THE ROLE OF INTERCELLULAR SIGNALLING ININDUSTRIAL WASTEWATER TREATMENT

M. Mane¢eld, A. Valle, A. Whiteley, M. Bailey

CEH Oxford, Mans¢eld Rd, Oxford, OX13SR, UK

Much is understood about the molecular biology and bio-chemistry underpinning bacterial intercellular signallingmechanisms. The N-acylated-L-homoserine lactone(AHL) dependent gene expression mechanism commonto a number of Proteobacterial genera is a particularlystriking example of where our understanding of the regu-latory mechanism far outweighs our appreciation of therole of this mechanism in mediating interactions betweenbacteria and their biotic and abiotic environment. Wehave screened isolates from a phenol degrading bioreactorfor AHL production and discovered an unexpectedly richpopulation of novel AHL producing bacterial species. Be-cause this rich community of AHL producing isolates isnot associated with higher organisms and a microbialcommunity function (phenol degradation) is clearly de-¢ned, the bioreactor constitutes a simple model ecosystemin which to explore the question ‘‘What is the role ofbacterial intercellular signalling in microbial ecology?’’through AHL and AHL antagonist amendments. AHLproducing isolates have been identi¢ed through 16SrDNA sequencing and the AHLs produced under labora-tory conditions characterised in a preliminary manner bythin layer chromatography. The e¡ects of amending thebioreactor community with AHLs and halogenated fura-nones known to antagonise AHL activity, on communitycomposition as determined by denaturing gradient gelelectrophoresis, community structure as determined by£uorescence in situ hybridisation targeting numerically orfunctionally dominant groups and community function bymeasuring phenol degradation rates, will be presented.

A26^4

GENETIC APPROACH TO STRAIN IMPROVEMENTFOR THE OVERPRODUCTION OF ANTITUMORANTIBIOTIC LANDOMYCIN E

B. P. Matselyukh, V. Ya. Lavrenchuk, L. V. Polishchukand V. V. Lutchenko

Institute of Microbiology and Virology of the NationalAcademy of Sciences of Ukraine, 03143, Kyiv-143, Zabo-lotny Str., 154, Ukraine

The Gram-positive soil bacterium Streptomyces globispo-rus 1912 produces a new polyketide antibiotic landomycinE which belong to angucycline family and possesses anantitumor activity especially against leukemia cells. It con-sists of tetracyclic chromophoric landomycinon A glycosi-lated with a trisaccharide D-olivose ^ D-olivose ^ L-rho-dinose. Series of di¡erent lanE mutants defective inlandomycin E production were obtained by means of pro-toplast mutagenesis.Some of lanE mutants have long dele-tion in chromosomal DNA (lanE genes cluster) and neverreverted to Lan+ phenotype. Another part of lanE mutantswere de¢cient in daidzein production and can restore anti-biotic biosynthesis after addition to the medium this exo-genic iso£avon. Third part of lanE mutants accumulates inthe medium di¡erent yellow, red and blue metabolites asthe precursors of landomycin E biosynthesis. Very inter-esting group of lanE mutants represents yellow and orangestrains producing lycopene and unidenti¢ed carotenoid.The last compounds have no relation to biosynthesis oflandomycin E and appear after activation of cryptic ctrgenes in consequence of chromosome rearrangement.These ctr mutants are not stable and produced with highfrequency the colorless clones. Among lanE mutants highpercentage of ultraviolet sensitive strains were identi¢edhaving de¢ciency in reparation system of DNA damages.A gene cloning system for S. globisporus 1912 has beendeveloped. It is presented by vector pSG1912-3 con-structed on the basis of endogenes plasmid and tsr gene,and protoplasts of di¡erent lanE strains. Self-cloning ex-periments resulted in high active strain overproducinglandomycin E.



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A26^5

OIL UTILISATION DURING OXYTETRACYCLINEFERMENTATIONS BY STREPTOMYCES RIMOSUS

P. A. Papapanagiotou, A. W. Nienow and C. J. Hewwit

Centre for Bioprocess Engineering, University of Birming-ham, Edgbaston, Birmingham B15 2TT, West Midlands,UK

Project industrially sponsored by Cognis Corporation andP¢zer Ltd (UK). Previous project on the utilisation oflipids during oxytetracycline fermentation by S. rimosusshowed that when the oil feed is supplied in the form ofa microemulsion, leads to lower residual oil amounts atthe end of the process. In addition, the antibiotic (oxyte-tracycline) yield increases towards the end of fermentationwhen compared to the control process. The current projectconcentrates on the utilisation of several oil feeds (oil andemulsion nature) with varying oil droplet sizes, therefore,investigates in more detail the project of the past. At themoment work has been carried on an older strain as wellas the production plant strain, used at P¢zer (results sub-ject to secrecy agreement). Analytical techniques such asHPLC, GC (and solvent extraction), image analysis arecurrently being used for determination of the antibioticyield, residual oil, and microbial dimensions, respectively.Image analysis has provided additional information on themorphology of the microorganism when utilizing oil ofdi¡erent droplet size. The results from the analytical tech-niques in combination with the ones from image analysishave been used to con¢rm the theory suggesting that uti-lisation of oil is highly dependent on the oil droplet size.The ‘‘Rigid Sphere Hypothesis’’ has been proposed as apossible explanation to the phenomenon. Repeated experi-ments at P¢zer (Sandwich, UK) have also concentrated onthe utilisation of the surfactants during 300-hour fermen-tations using the production plant culture.

A26^6

LACTIC ACID PRODUCTION DIRECTLY FROMSTARCH AND INULINE CONTAINING SOURCESUSING STREPTOCOCCUS BOVIS BACTERIA

M. Shamtsyan

St. Petersburg State Technological Institute (TechnicalUniversity), Dept. of Technology of Microbiological Syn-theses, Moskovsky Prospekt, 26, St. Petersburg, 198013,Russia

Over the past years new applications of lactic acid, espe-cially the possibilities of its utilization as a source of afamily of new bio- and photodegradable polymers were

been pointed, with high hopes for the market expansion.The main factor, limiting wide usage of lactic acid to syn-thesize other products is it’s relatively high cost. To realizee⁄cient production of lactic acid and its wide utilization infood industry, as well as a source of biodegradable andphotodegradable polymers, we isolated rumen bacteriaStreptococcus bovis, which is characterized by high amylo-lytic and inulinase activities. This bacteria isolated fromthe calf rumen has the ability to digest raw starch andinnulin and is characterized as a homofermentative pro-ducer of L(+) lactic acid. Since S. bovis was not studied asproducer of lactic acid we investigated all the technolog-ical stages, including preparation of inoculate, optimiza-tion of the fermentation process and isolation of the endproduct. Fermentations using di¡erent starch containingsubstrates, such as rice bran, low- grade wheat £our, sor-ghum grains and potato tubers, as well as, topinambourtubers containing inuline and fructose were carried out.The usage of S. bovis strains allows for direct and e⁄cientlactic acid fermentation from starch and inuline containingsources.These studies were supported by INTAS FOOD-00-876grant.

A27^1

USE OF FLOW CYTOMETRY FOR ASSESSMENTOF PHYSIOLOGICAL STATE OF YEAST DURINGSTORAGE

A. C. Gabier(1), J. Reitz(2), P. Gourdon(2), J. Y. Lev-eau(1), M. Bouix(1)

(1) ENSIA ^ Laboratoire de Microbiology Industrielle, 1Avenue des Olympiades, 91744 Massy cedex; (2) SORE-DAB, Chemin de la Tremblaye, 78125 La -Boissiere-Ecole

Flow cytometric methods using carboxy£uorescein diace-tate (CFDA) and DiBAC 4(3) bis (1-3 dibutylbarbituricacid) trimethine oxonol (DIBAC) have been described asrapid and relevant to estimate viable and depolarised cells,respectively. We used both labelling to asses the physio-logical status of cells upon storage at +4‡C for referenceand non conventional yeast. Physiology of yeasts wascharacterised during storage through culturability, treha-lose content, £uorescence intensity and number of cellsloaded with CFDA and DIBAC. Use of trehalose reservewas concomitant with the decrease of CFDA £uorescenceintensity and the increase of the cells stained with DIBAC.Culturability proved to decrease sharply when all the cellswere loaded with DIBAC and £uorescence intensity wasmaximal, cells were depolarised but still viable as indicatedby the CFDA labelling. Then viability decreased simulta-neously with the reduction of the DIBAC £uorescenceintensity which might indicate membrane damages. Ourstudy demonstrates that the characterisation of the di¡er-



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ent physiological states of yeast throughout cold storagerequires both £ow cytometric methods.

A27^2

STRESS IN RECOMBINANT PROTEIN PRODUCINGPICHIA PASTORIS : EFFECTS OF GENETIC ANDENVIRONMENTAL VARIABLES ON EXPRESSIONOF RECOMBINANT HUMAN TRYPSINOGEN

H. Hohenblum, M. Maurer, B. Gasser and D. Mattanovich

Institute of Applied Microbiology, BOKU ^ University ofNatural resources and Applied Life Sciences, Vienna

Eukaryotic expression systems such as Pichia pastoris playan important role in research and pharmaceutical industryproducing a wide variety of foreign proteins. Yeasts com-bine the ease of microbial growth and gene manipulationwith the ability to perform many eukaryotic posttransla-tional modi¢cations. Pichia pastoris is known as a highlye⁄cient expression system, but not all proteins could beproduced with the desired yield. Di¡erent strategies existfor optimising the genetic construct (inducible or constitu-tive promoters, secretion leaders, codon usage, hoststrains, gene copy number) and the cultivation conditions,which otherwise can lead to an overload of the expressionmachinery and the host cell. Especially the secretion ofrecombinant proteins can lead to severe stress reactionsof the host cell, as it was shown mainly for S. cerevisiae.We are investigating Pichia pastoris as an expression sys-tem for recombinant human trypsinogen. By comparingdi¡erent host strains, gene copy numbers, leader sequen-ces, promoters and culture conditions, we could identifyseveral stress reactions. The retention of unfolded productin the cell obviously exerts stress on the cell, and an in-crease of the gene copy number leads to a severe reductionof productivity and viability. Di¡erences in the cultureconditions can also lead to severe stress reactions. Theresults will be discussed with the aim to identify bottle-necks for recombinant protein production and strategiesto overcome them.

A27^3

STUDY OF STRESS RESPONSE IN THE YEASTCANDIDA INTERMEDIA EXPOSED TO CR(VI)

P. Jamnik and P. Raspor

Food Science and Technology Department, Chair of Bio-technology, Biotechnical faculty, Jamnikarjeva 101, 1000Ljubljana, Slovenia

Changes in the chemical or physical conditions of the cellthat impose a negative e¡ect on growth demand rapid

cellular response, which is essential for survival. The mo-lecular mechanisms induced upon exposure of cells to suchadverse conditions are commonly designated as stress re-sponse. In our study stress response of the yeast Candidaintermedia exposed to Cr(VI) was investigated. Cr(VI)compounds have widespread industrial uses and thereforeare contaminants in the environment. Yeast cells weretreated with Cr(VI) in concentration of 50, 100, 300 and500 WM in the mid-exponential growth phase. Monitoringof some bioprocess parameters during yeast growth, spe-ci¢cally pO2 showed that Cr(VI) addition, particularly inconcentration of 100 and partially 50 Wmol/l increasedmetabolism intensity, which is connected to induced stressresponse. Cr(VI) belongs to redox active metals, whichplay an important role in the generation of reactive oxy-gen species in the cell. Therefore, intracellular oxidant lev-el after Cr(VI) addition was estimated using 2’,7’-dichlor-o£ourescin and it was elevated, speci¢cally at 100 WMCr(VI). Yeast cells posses various primary and secondaryantioxidant defence systems against reactive oxygen spe-cies. Enzymatic, speci¢cally superoxide dismutase and cat-alase as well as non-enzymatic (glutathione level in thereduced form) primary antioxidant defence systems wereinvestigated. We found that catalase and superoxide dis-mutase do not participate in the yeast cell defence in con-trast to glutathione, which increased signi¢cantly in thecells exposed to 100 Wmol Cr(VI)/l. Therefore, we demon-strated that glutathione plays an important role in theyeast stress response to Cr(VI).We thank the Ministry of Education and Sport of theRepublic of Slovenia for grant (no.: 35/8) and the Minis-try of Science and Technology of the Republic of Slovenia(Project no.: J4-7454-490) for ¢nancial support.

A27^4

ANHYDROBIOSIS OF YEASTS: CELL ADAPTA-TION TO STRESS CONDITIONS

A. Rapoport(1), G. Khroustalyova(1), I. Guzhova(2)

(1) Laboratory of Cell Biology, Institute of Microbiologyand Biotechnology, University of Latvia, Kronvald Blvd.,4,LV-1586 Riga, Latvia; (2) Laboratory of Cell ProtectionMechanisms, Institute of Cytology, Russian Academy ofScience, Tihoretsky Pr., 4, 194064 Saint-Petersburg, Russia

Dehydration is one of the most usual natural stresses fordi¡erent microorganisms including yeasts which are peri-odically subjected to the extreme conditions of very strongdrought. Rather signi¢cant amount of organisms is able tomaintain their viability in these conditions by the way oftemporary change of their status ^ from active life to an-hydrobiosis in which their metabolism is reversibly sus-pended or delayed. Di¡erent adaptive and protective reac-tions which take place at all structural levels of live



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organisms were worked out during their evolution. Struc-tural-and-functional changes are characteristic practicallyfor any intracellular component and organella (cell wall,plasma membrane, ribosomes, mitochondria, vacuoles, ly-sosomes, endoplasmic reticulum, peroxisomes, nucleus).Speci¢c protective substances can be synthesized duringcell transition into the state of anhydrobiosis. It wasshown by us that besides wellknown e¡ects of trehalosesome other compounds, such as polyols, can be of greatimportance for the maintenance of cell viability. In thecase of absense of trehalose polyols (inositol, sorbitol, xy-litol and probably some others too) can substitute it forthe protection of molecular organization of intracellularmembranes. In the case of the presense of adequateamounts of trehalose polyols most probably protect intra-cellular macromolecules ^ proteins and nucleic acids. An-other potent protective system can involve molecularchaperons or heat shock proteins found in all living or-ganisms. Anaerobically grown yeast cells do not possessany resistansce to conditions of strong dehydration.Therefore it is necessary to work out special new ap-proaches for their arti¢cial transfer into the state of anhy-drobiosis.

A27^5

GLUTATHIONE-DEPENDENT DEFENCE OF METH-YLOTROPHIC YEAST HANSENULA POLYMOR-PHA AGAINST STRESSES CAUSED BY PEROX-IDES, ELECTROPHILES AND HEAVY METALS

V. M. Ubiyvovk(1), O. V. Blazhenko(1), T. Y. Naza-rko(1), O. V. Stasyk(1), J. Maszewski(2), G. Bartosz(2),A. A. Sibirny(1,3)

(1) Institute of Cell Biology, National Academy of Sciencesof Ukraine, Drahomanov Street, 14/16, Lviv 79005 Uk-raine; (2) Department of Molecular Biophysics, Universityof Jo¤dz¤, Banacha 12/16, 90-237 Jo¤dz¤, Poland; (3) Instituteof Biotechnology, Rzeszow University, Rejtana 16C, 35-310Rzeszo¤w, Poland

Glutathione (GSH) participates in oxidative stress re-sponse and defends cells from toxic action of heavy metalsand xenobiotics. The utilization of methanol in methylo-trophic yeasts starts with its conversion to formaldehydeand hydrogen peroxide, potential inducers of electrophilicand peroxidative stresses. Methylotrophic yeast H. poly-morpha responds to methanol metabolism by elevation ofGSH level (cofactor of formaldehyde dehydrogenase andglutathione peroxidase), as well as by increasing activitiesof membrane-bound and soluble forms of GSH peroxi-dase. Methylotrophic yeast H. polymorpha CBS 4732leu2 detoxi¢es some electrophilic xenobiotics by theirGSH-dependent accumulation in vacuoles as well as byGSH-dependent and GSH-independent extracellular ex-

port of xenobiotic derivatives as follows from £uorescencemicroscopy and HPLC studies. Conjugates of GSH andN-acetylcysteine with tested electrophiles, monobromobi-mane and N-[1-pyrene]maleimide, were observed amongextruded HPLC fractions. Isolated GSH de¢cient mutantsof H. polymorpha NCYC 495 leu1-1 gsh1 and leu1-1 gsh2,failed to ful¢l such detoxi¢cation of electrophilic xenobi-otics and acquired elevated susceptibility to cadmium ionsand methanol. The corresponding genes GSH1 and GSH2were cloned from H. polymorpha gene library by comple-mentation of the corresponding mutant defects and se-quenced. It was shown that GSH2 gene of H. polymorphais homologous to GSH1 gene of Saccharomyces cerevisiaeencoding Q-glutamylcysteine synthetase, whereas H. poly-morpha GSH1 gene has homology to S. cerevisiae MET1gene encoding S-adenosyl-L-methionine uroporphyrinogenIII methyltransferase. Current studies with mutants de-leted in genes of GSH metabolism (GSH1, GSH2, and Q-glutamyltransferase gene) will additionally elucidate theirrole in GSH-dependent defense of methylotrophic yeast H.polymorpha against stresses caused by peroxides, electro-philes and heavy metals.

A27^6

THE GLUTATHIONE-SYNTHETASE OF SCHIZO-SACCHAROMYCES POMBE : AN ENZYME WITH APECULIAR SUBUNIT STRUCTURE

N. Phlippen(1), K. Ho¡mann(2), K. Wolf(1) and M. Zim-mermann(1)

(1) Institute for Microbiology and Genetics and (2) Insti-tute for Molecular Biotechnology, Aachen University, D-52056 Aachen, Germany

The tripeptide glutathione is synthesised enzymatically intwo steps. The second step is the addition of glycine toQglutamyl-cysteine. This ATP-dependent reaction is cata-lysed by glutathione-synthetase. In almost all eukaryotes,this enzyme is a homodimer. In the ¢ssion yeast S. pombethe enzyme has been puri¢ed and described as a hetero-tetramer consisting of two identical small and two identi-cal large subunits. We show that the enzyme is functionalin vivo and in vitro as a homodimer as well as a heterotet-ramer. We demonstrate that the small and the large sub-units are encoded by the same reading frame and arisefrom a proteolytic cleavage of a precursor protein. Site-directed mutagensis has led to the formation of stable,functional homodimers. On the other hand the smalland large subunits could be synthesised separately, yield-ing active proteins.



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A28^1

CLINICAL CHARACTERISTICS OF LYME BORRE-LIOSIS

F. Strle

Department of Infectious Diseases, University MedicalCentre, Ljubljana, Slovenia

Lyme borreliosis (LB) is the most common tick-transmit-ted human disease on the Northern hemisphere. It a¡ectsboth sexes and all ages. The most common clinical man-ifestation of LB is erythema migrans. This enlarging eryth-ematous skin lesion develops at the site of a tick-bite,usually 10 days (a few days to more than one month) afterthe bite, though bites are often not noticed by patients.Initially homogeneous patch later-on often becomes ring-like as a result of central clearing. About 50% of Europeanpatients report local symptoms while some patients expe-rience systemic symptoms (more often in the USA than inEurope). Erythema migrans is regularly the ¢rst and often^ especially when recognized and treated in due time ^ theonly clinical manifestation of LB. When the disease pre-sentation is complete (that is an exception) a tick bite isfollowed by erythema migrans, which is followed by heartand nervous involvement and later on by arthritis. Eye,late nervous system, joint and skin involvement are alsoknown. Clinical signs and symptoms should obligatoryform a basis for the diagnosis of LB. The only sign thatenables a reliable clinical diagnosis of LB is erythema mi-grans. Highly supportive for the diagnosis of LB are alsoear lobe lymphocythoma, meningoradiculitis (Garin-Buja-doux-Bannwarth’s syndrom) and acrodermatitis chronicaatrophicans. The large majority of numerous other symp-toms and signs have only minimal or even symbolic diag-nostic value. Laboratory con¢rmation of borrelial infec-tion is needed for all manifestations of LB with theexception of typical early skin lesions.

A28^2

COMPARISON OF FINDINGS IN PATIENTS WITHLYME BORRELIOSIS CAUSED BY DIFFERENTBORRELIA STRAINS

F. Strle(1), E. Ruzic-Sabljic(2), M. Logar(1), S. Lotric-Furlan(1), J. Cimperman(1), T. Jurca(1), V. Maras-pin(1)

(1) University Medical Centre, Department of InfectiousDiseases, Ljubljana, Slovenia; (2) Institute for Microbiol-ogy and Immunology, University of Ljubljana, Ljubljana,Slovenia

Lyme borreliosis is a widely distributed multi-system dis-order caused by the tick-borne spirochete Borrelia burg-dorferi sensu lato. Human disease is associated with atleast three Borrelia species. There are indications that in-fection with di¡erent genospecies of B. burgdorferi sensulato results in distinct clinical manifestations of Lyme bor-reliosis : in Europe skin manifestations of the disease aremost often associated with B. afzelii, neuroborreliosis withB. garinii, whereas B. burgdorferi sensu stricto, the onlyagent causing human disease in the USA, has a propensityfor joint involvement. While data indicating the associa-tion of di¡erent B. burgdorferi sensu lato genospecies withdistinct clinical manifestations of Lyme borreliosis are rel-atively abundant and obvious, comparisons of character-istics of individual manifestation caused by di¡erent Bor-relia genospecies are quite scarce. In fact, publishedinformation is limited to only one report on patientswith erythema migrans: comparison of culture con¢rmederythema migrans caused by B. afzelii in Slovenia anderythema migrans caused by B. burgdorferi sensu strictoin New York showed di¡erences in epidemiological, clin-ical, and laboratory ¢ndings, suggesting two distinct syn-dromes that are caused by di¡erent agents. Recent ¢ndingsfrom Slovenia further support the idea that the course ofindividual Lyme borreliosis manifestation depends on thespecies of B. burgdorferi sensu lato: adult patients withculture con¢rmed erythema migrans caused by B. afzeliirevealed several distinctions in comparison to B. gariniigroup. Pronounced di¡erences were found also evaluatingpatients with neuroborreliosis caused by B. garinii and B.afzelii.



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A28^3

BORRELIA IN RESERVOIR ANIMALS

G. Khanakah(1), E. Ruzic-Sabljic(2), A. Zore(2), F.Strle(3), E. Kocianova(4), V. Vyrostekova(5), G. Sta-nek(1)

(1) Institute of Hygiene and Medical Microbiology, Uni-versity of Vienna, Austria; (2) Institute for Microbiologyand Immunology, University of Ljubljana, Ljubljana, Slov-enia; (3) University Medical Centre, Department of Infec-tious Diseases, Ljubljana, Slovenia; (4) Institute of Virol-ogy, Slovak Academy of Sciences, Bratislava, Slovakia; (5)Institute of Epidemiology, Comenius University Bratislava,Slovakia

Middle Europe is a highly endemic area of Lyme borre-liosis. The causative agents are transmitted to humans al-most exclusively by Ixodes ricinus ticks. Mice were caughtin the northeastern part of Austria, most frequently Apo-demus £avicollis (44%), followed by Clethrionomys glareo-lus (34%), Microtus arvalis (9%), A. sylvaticus (7%) andMus musculus (6%). Signi¢cant di¡erences were seen inthe total number of catch per area (Hohenau, Ernstbrunn,Vienna at 10:9:2) and in the distribution of rodent speciesin the catchment areas. Borrelia were more frequently cul-tured from bladder wall than from heart muscle and onlyonce from brain. Heart specimens of 226 animals wereborrelia PCR positive (24%) and most frequently of therodent species A. £avicollis (43%) and C. glareolus (38%).B. afzelii was most frequently identi¢ed, followed by B.burgdorferi sensu stricto and by mixed infection of B. af-zelii and B. burgdorferi sensu stricto. B. garinii was onlyonce detected in a sample of M. arvalis. In about 20% ofborrelia PCR positive samples the identi¢cation of one ofthe three genomic species could not be ascertained withthe test panel used. In Slovenia, Ixodes ricinus ticks wereremoved from di¡erent bird species including the blackbird (Turdus merulae), song thrush (Turdus philomelos),robin (Erithacus rebecula) and hedge accentor (Prunellamodularis). The black bird was the species most heavilyinfested with ticks. Borrelia were cultured from ticks andidenti¢ed by sequence analysis as B. garinii, B. valaisianaand B. afzelii. Mixed infection was observed with B. gar-inii and B. valaisiana. The rodent species A. £avicollis, M.arvalis and C. glareolus proved especial reservoirs for B.afzelii and B. burgdorferi sensu stricto. Noteworthy is theabsence of B. garinii which has no apparent reservoir inrodents of the areas investigated. But the reservoir com-petent animals of B. garinii are seemingly birds, and Tur-dus merulae is most probably the natural reservoir of B.valaisiana.

A28^4

MICROBIOLOGICAL DIAGNOSIS

G. Stanek(1), E. Ruzic-Sabljic(2), F. Strle(3)

(1) Institute of Hygiene and Medical Microbiology Univer-sity Vienna, Austria; (2) Institute for Microbiology andImmunology, University of Ljubljana, Ljubljana, Slovenia;(3) University Medical Centre, Department of InfectiousDiseases, Ljubljana, Slovenia

The microbiological con¢rmation of infection is neededfor all manifestations of LB with the exception of typicalearly skin lesions. Ideally, detection of the causative agentby culture or by ampli¢cation of speci¢c nucleic acid se-quences from lesional skin, other tissue, cerebrospinal £u-id, synovial £uid or blood would prove the aetiology.However, direct detection is most successful in early skinmanifestations which are usually identi¢ed clinically. Cul-tivation of cerebrospinal £uid in neuroborreliosis is posi-tive shortly after the onset of clinical signs and symptomsbut is as a rule successful in less than 10% of patients.Only single isolates are available so far from joint £uidor tissue but PCR was found positive in up to 80% in aretrospective study. However, culture and nucleic acid am-pli¢cation assays (NAA) can only be performed satisfac-torily in specialised laboratories. Indications for microbio-logical testing (culture, NAA) related to di¡erent clinicalsigns and suspected clinical manifestations are always giv-en. Positive results will aetiologically con¢rm the clinicaldiagnosis, however, negative results with direct detectionmethods do not exclude a suspected aetiology. Recom-mended specimens for culture and NAA are usually le-sional skin and synovial £uid or synovial tissue. Cerebro-spinal £uid and biopsies other than synovial may be usefulfor culture and NAA under special circumstances.

A28^5

LYME DISEASE SEROLOGY: STANDARD PROCE-DURES AND RESULTS OF EXTERNAL QUALITYCONTROL

K.-P. Hunfeld and V. Brade

Central Laboratory of the German Pro¢ciency Testing Pro-gram for Bacteriological Infection Serology, Institute ofMedical Microbiology, University Hospital of Frankfurt,Germany

Despite substantial advances in our knowledge of theproperties of B. burgdorferi and many new diagnostic ap-proaches, serological detection of speci¢c antibody re-sponse remains the method of choice for many routinemicrobiological laboratories in diagnosing Lyme disease



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(LD). Speci¢c antibodies usually can be detected 3 to 6weeks after the infection. Today, rational stepwise diag-nostic testing consisting of a screening test (ELISA) incombination with a con¢rmatory assay (immunoblot) isthe diagnostic procedure of choice. This test standard isin most cases su⁄cient to assess the immune status ofpatients suspected to have LD. Test combinations otherthan the classical two tier protocol increases the risk offalse positive and false negative test results. Moreover,external quality control surveys are an important tool inregulating the quality of infection serology in general andof borreliosis serology in particular. We re£ect on poten-tial and limitations of current serological standard proce-dures and report on the results of a LD pro¢ciency testingprogram established in 1999 which is regularly organisedtwo times a year by our institution in close cooperationwith the Institute of Standardisation in the Medical Lab-oratory (INSTAND). From 1999 to 2002 between 229 and350 microbiological laboratories from Germany and from13 other European countries participated in each of theseven surveys that were held so far. Test results werefound to be in part highly variable and clearly correlatedto the manufacturers and the applied test methodology.Obviously, IgM tests were more di⁄cult to handle thanwere IgG-tests. ELISA-testing was more reproducible andproved to be more sensitive and speci¢c than IFA andIHA testing. Quanti¢cation of test results and reportingof speci¢c immunoblot bands showed high variability. Inaddition, a high number of false positive and false nega-tive test results were reported for some assays by the par-ticipants. In the view of our results further standardisationof LD disease serology is not just desirable but is urgentlyneeded to increase the medical quality of laboratory diag-nosis in LD patients. Moreover, stronger criteria must beapplied to approve the available test kits.

A28^6

TYPING METHODS FOR BORRELIA BURGDOR-FERI SENSU LATO STRAIN IDENTIFICATION

E. Ruzic-Sabljic(1), F. Strle(2), G. Khanakah(3) and G.Stanek(3)

(1) Institute of Microbiology and Immunology, Universityof Ljubljana Medical Faculty; (2) Department of InfectiousDiseases, University Medical Centre, Ljubljana, Slovenia;(3) Institute of Hygiene and Medical Microbiology, ViennaUniversity, Austria

The etiological agent of Lyme borreliosis is a spirochete ofthe genus Borrelia collectively named B. burgdorferi sensulato. Molecular techniques used for identi¢cation and typ-ing of microorganisms indicate that Borreliae are pheno-typically and genetically divergent. At least three of 11 B.burgdorferi sensu lato species are associated with human

diseases : B. afzelii, B. garinii, and B. burgdorferi sensustricto. Methods for their identi¢cation and di¡erentiationcan be categorized as either phenotypic or genetypic. Theprotein pro¢le and serotyping represent the most com-monly used method for B. burgdorferi sensu lato pheno-typic analysis ; OspA and OspC are the most commonlyused proteins for typing. They vary in molecular mass (31-34 and 20-25 kD) and reactivity with monoclonal antibod-ies. At least 8 OspA and 13 OspC serotyps have beendescribed. A number of di¡erent methods for genotypinghave been used to assess B. burgdorferi sensu lato charac-teristics. Among them PFGE, PCR and PCR -based re-striction are frequently used for identi¢cation on the spe-cies level, and plasmid pro¢le analysis for discriminationbetween strains within individual species. Molecular typ-ing provides valuable data for geographic, animal reser-voir, vector and patient distribution of individual Borreliaspecies as well as an e⁄cient tool for the analysis of thepotential association of particular Borrelia species with thedistinct clinical manifestations of Lyme borreliosis.

A28^7

DEVELOPMENT OF BORRELIA IN TICKS ANDTRANSMISSION TO HOSTS

L. Gern and M. Crippa

Institut de Zoologie, University of Neucha“tel, Neucha“tel,Switzerland

Four main tick vectors transmit Borrelia burgdorferi sensulato (sl) to humans: Ixodes ricinus in Europe, I. scapularisand I. paci¢cus in North America and I. persulcatus inRussia and Asia. Strains that can cause Lyme borreliosisare grouped into a species complex which comprises 11species and some unnamed groups. Of these species, B.garinii, B. afzelii, B. tanuki, B. turdi, B. japonica, B. va-laisiana and B. sinica have been isolated in Asia. B. burg-dorferi sensu stricto (ss), B. andersoni and B. bissettii occurin North America whereas in Europe B. burgdorferi ss, B.garinii, B. afzelii, B. valaisiana and B. lusitaniae have beencultured from I. ricinus ticks. Infection rates and distribu-tion of the various Borrelia species in I. ricinus ticks inendemic areas may greatly vary according to year, asshown in a focus in Neucha“tel. This implies that humanrisk to be infected by one Borrelia species or another aftera tick bite is variable. Most of the studies on the interac-tions of B. burgdorferi sl with its vectors have concentratedon the events that occur when infected nymphal and adultticks transmit the spirochetes to the vertebrate hosts. Ithas been shown that in unfed free living ticks, B. burgdor-feri sl is present in the lumen of the gut and when ticksfeed the spirochetes cross the gut epithelium and migrateinto the hemocoel, reach the salivary glands from wherethey are transmitted to the host via saliva. From North



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American studies it is known that transmission of B. burg-dorferi ss by I. scapularis does not occur immediately afterticks attach to hosts and that high level of transmission isreached only after at least 48 h of tick attachment. In I.ricinus, an early transmission of B. burgdorferi sl with highe⁄ciency was described by Kahl et al. (1998): after 16.7hof tick attachment, 50% of animals were infected. In arecent study, we investigated the interactions of di¡erentBorrelia species with I. ricinus and their transmission tohost. The e⁄ciency of I. ricinus ticks to transmit B. afzeliiand B. burgdorferi ss and their infectivity for mice wereexamined in relation to the duration of the blood meal.The success of transmission of Borrelia from I. ricinusnymphs to mouse increased with duration of tick attach-ment. However, B. afzelii infected ticks started to transmitinfection earlier (9 48h) than B. burgdorferi ss infectedticks. We also observed that B. burgdorferi ss and B. afzeliispirochetes are infectious in the tick before natural trans-mission can occur. I. ricinus appears to be a more e⁄cientvector of B. afzelii than of B. burgdorferi ss.

A29^1

ADVANTAGES AND LIMITATIONS OF POSTHARV-EST BIOLOGICAL CONTROL: TOWARD THE NEXTGENERATION PRODUCTS

W. J. Janisiewicz

USDA, ARS, Appalachian Fruit Research Station, 45 Wilt-shire Rd. Kearneysville, West Virginia 25430, USA

Most of the postharvest decay on pome fruits results frominfection through wounds made during harvest and post-harvest handling. Synthetic fungicides have been, by far,the most widely used remedy against this decay. However,their use has been increasingly curtailed by the perceivedhazard to humans and the environment. Recently, biolog-ical control of postharvest diseases (BCPD) with antago-nistic bacteria and yeasts, that naturally occur on fruits,has emerged as the most e¡ective alternative to fungicides.The ¢rst commercial products have been registered for usein the United States, and the fruit industry has acceptedthis control method. The major advantages of BCPD are:controlled and stable environmental conditions in storagerooms, which favors antagonist survival, ability to applyantagonists directly to targeted area (fruit), ease in manip-ulation of the postharvest system, and exemption fromtolerance of the registered antagonists. The major limita-tions are: limited spectrum of activity and e⁄cacy undersome environmental conditions, speci¢city against variousdiseases or fruits, and lack of eradicative activity. Thoselimitations are addressed by developing antagonist mix-tures with superior biocontrol potential to individual an-tagonists, improving an antagonist’s biocontrol potentialthrough physiological and genetic manipulation, combin-

ing antagonists with non-fungicidal methods such as heattreatment, calcium in¢ltration, cultural methods, and anti-microbial substances that are generally regarded as safe.Knowing the mechanism of biocontrol would be veryhelpful in enhancing biocontrol, but so far this approachhas not been realized. Expansion of research and the re-lated successes creates an optimistic picture for the futureof BCPD of fruits.

A29^2

IMPROVEMENT OF ENVIRONMENTAL STRESSRESISTANCE OF BIOCONTROL AGENTS

N. Teixido¤(1), M. Abadias(1), J. Usall(1), N. Magan(2)and I. Vi•as(1)

(1) Pathology Laboratory, Postharvest Unit, CeRTA,Centre UdL-IRTA, 191 Rovira Roure, 25198-Lleida, Cata-lonia, Spain; (2) Applied Mycology Group, BiotechnologyCentre, Cran¢eld University, Silsoe, Bedford MK45 4DT,U.K.

Biological control is usually limited by £uctuating environ-ment and the narrow range of conditions over which mi-croorganisms are able to survive, establish and e¡ectivelycontrol pests and diseases. It is thus important to developan improved understanding of the mechanisms of howbiocontrol agents may survive environmental stresses (os-motic, thermal, pH, oxidative stresses and starvation) andenable an improvement in ecological competence andquality, by ecophysiological manipulation. The noveltyand innovative components of our approach is to takeadvantage of the capacity shown by microorganisms todevelop natural protection mechanisms under stress con-ditions, in order to improve and optimise their behaviour,and to translate this into improved production, formula-tion and subsequent application as biocontrol agents. Ourstudies are focussed on Candida sake CPA-1 and Pantoeaagglomerans CPA-2, both known antagonists against post-harvest fungal diseases on fruits. Studies on osmotic, pHand thermal stresses have been carried out. Physiologicalmethods were used to improve stress tolerance of bothbiocontrol agents. The intracellular content of major poly-ols and sugars was signi¢cantly modi¢ed by reducingwater activity (aw) of the growth medium using glucoseor glycerol in the case of C. sake. This change improvedviability of the antagonist over a wider range of relativehumidity maintaining and even enhancing biocontrol e⁄-cacy. The physiologically manipulated inocula of C. sakesurvived better than unmodi¢ed yeast cells when they weresprayed on apples in orchard conditions. P. agglomeransgrown in a reduced aw medium with NaCl demonstratedmore desiccation tolerance than the cells grown in an un-modi¢ed medium.



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A29^3

FRUIT QUALITY PRESERVATION BY MEANS OFBIOLOGICAL AND INTEGRATED CONTROL

G. Arras

CNR ^ (ISPA) ^ Istituto di Scienze delle Produzioni Ali-mentari ^ Sez. Sassari, Via dei Mille, 48 07100 Sassari,Italy

Three commercial tests were conducted in two commercialpacking-houses (Muravera and Villacidro) located in Sar-dinia, Italy, to evaluate the e⁄cacy of biological, chemicaland integrated treatments against Penicillium digitatumand P. italicum on naturally inoculated orange fruit. Dam-age caused by the packing-house processing line was alsoassessed. Treating orange fruits with the yeast Pichia guil-liermondii (strain 5A) in the processing line generally led toa signi¢cant reduction of post-harvest decay compared tothe processed control, while the commercial product As-pire0, based on Candida oleophila, was ine¡ective in inhib-iting the pathogen when applied alone. The integratedapplication of thiabendazole or imazalil with the biocon-trol agents signi¢cantly improved the control of fruit de-cay. Using thiabendazole at concentrations of 0.1 and 1.2g l-1, led to similar results in inhibiting fruit decay in twotrials. Both yeasts were equally able to actively colonisethe fruit during storage. Passing fruits through the packingline caused a signi¢cant increase in fruit decay. Pichiaguilliermondii cells were viable and numerous on the fruitexocarp at both the beginning and end of the inoculationtrial, whereas C. oleophila cells were initially lower in num-ber but rapidly increased at the end of the trial. It isimportant to restore an e¡ective antagonist presence onthe surface of the fruit after the oranges have passed alongthe processing line and lost most of the micro£ora.

A29^4

ANTIFUNGAL LACTIC ACID BACTERIA AS POST-HARVEST BIOCONTROL AGENTS

J. Schnu«rer

Department of Microbiology, Swedish University of Agri-cultural Science, Box 7025, SE-750 07, Uppsala, Sweden

Lactic acid bacteria (LAB) have been used since ancienttimes to preserve food and animal feed and to improve thearoma and texture of various fermented foods. Bacteria,yeasts and moulds all contribute to spoilage problems andmay also constitute health hazards. The ability of LAB toinhibit growth of spoilage and pathogenic bacteria hasbeen the focus of a multitude of studies, with bacteriocinsrecieving particular attention. The antifungal activities of

LAB have only been investigated to a limited extent. Wehave isolated a number of antifungal LAB from plantmaterial stored under anaerobic conditions and character-ised antifungal compounds from Lactobacillus corynifor-mis subsp. coryniformis and Lactobacillus plantarum. Theantifungal compounds include a broad-spectrum 3 kDaprotein, 3-phenyllactic acid and the cyclic dipeptides cy-clo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro).The latter three molecules inhibit fungi at concentrationssimilar to that of the wellknown preservative sodium ben-soate, i e mg/ml. We are presently characterising otherfungal inhibitory compounds active at 10 Wg/ml concen-trations. The emerging picture suggests that LAB are ableto inhibit fungal growth through the production of a mul-titude of metabolites, possibly acting in synergi with thefermentation products lactic acid, acetate and ethanol.This presentation will provide an overview of our currentknowledge of the antifungal abilities of LAB, as well as anevaluation of their potential as biocontrol agents in thepostharvest environment.

A30^1

MOLECULAR BIOLOGY OF MYCOPLASMAS

S. Razin

Department of Membrane and Ultrastructure Research, TheHebrew University Hadassah Medical School, Jerusalem,Israel 91120

Due to their minute genome and cell simplicity mycoplas-mas were among the ¢rst organisms subjected to completegenome sequencing. The genomic projects have contrib-uted most conspicuously to our understanding of the mo-lecular biology and evolution of mycoplasmas. Accord-ingly, mycoplasmas evolved as a branch of gram-positivebacteria by reductive evolution, losing considerable por-tions of their ancestors’ chromosomes in this process. Thesigni¢cant genome compaction was made possible byadopting a parasitic lifestyle. Supply of nutrients fromtheir host facilitated the loss of genes for many assimila-tive, metabolic and regulatory processes. To keep to theparasitic lifestyle, mycoplasmas developed rather sophisti-cated mechanisms to colonize their host and resist thehost’s immune system. This required a signi¢cant numberof genes devoted to adhesion and generation of antigenicvariation systems, consisting mostly of membrane lipopro-teins. The mycoplasmal genomic projects have brought usmuch closer to achieving the goal of complete deciphering,in molecular terms, of the machinery of a self-replicatingcell. M. genitalium is, thus far, the organism closest to thetheoretical minimal cell capable of self-replication. Globaltransposon mutagenesis was applied to test the e¡ects ofspeci¢c gene disruptions on growth. Of the 480 protein-coding genes in M. genitalium 265 to 350 appear essential



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under laboratory growth conditions. In the case of DNAreplication, transcription, and translation, it seems that theminimal set of genes has already been established in M.genitalium and M. pneumoniae. There is still very much todo to identify the unclassi¢ed open-reading frames (ORFs)that have no database match, prove experimentally theDNA-based predictions, and assign functions to proposedORFs with hitherto unknown functions. Cell protein anal-ysis, applying the ‘Proteome’ and ‘Transcriptome’ ap-proaches, are amongst the major directions currently tak-en to de¢ne structurally and functionally the entirecomplement of mycoplasmal cell proteins.

A30^2

EPIDEMIOLOGY OF MYCOPLASMAL INFEC-TIONS BASED ON ANALYSES OF GENE POLY-MORPHISMS

S. H. Kleven and M. Garc|¤a

Department of Avian Medicine, University of Georgia,Athens, GA 30605, USA

Knowledge of relatedness among isolates of pathogenicavian mycoplasmas is important in studying the epidemi-ology of such infections. Random Ampli¢ed PolymorphicDNA (RAPD) analysis has been used to study relation-ships among strains when pure cultures are available.RAPD results are useful when strains being comparedgenerate clearly di¡erent gel patterns; however, whenRAPD patterns are similar, further analysis is needed.DNA sequence analysis of variable regions of major im-munogenic surface protein genes of M. gallisepticum,gapA, mgc2, pvpA, and an uncharacterized lipoproteingene (LP), are useful in such instances. Unfortunately,strains which appear to be identical when examining anyone of these genes, may be completely di¡erent if othergene sequences are examined. We have found sequenceanalysis of a combination of the above genes to be moreprecise in determining genetic relatedness. Because purecultures of pathogenic mycoplasmas are often di⁄cult toobtain, a major goal has been to determine relatednessamong strains by analysis of a PCR product obtainedfrom clinical specimens. PCR primers for a variable regionof the mgc2 gene of M. gallisepticum which may be usefulfor such purposes. There is size polymorphism in this PCRproduct from various strains, which is of value in someinstances. In cases where the product size does not di¡er,DNA sequence analysis may determine whether strains areunrelated. If the mgc2 PCR product from two strainsbeing compared is identical, DNA sequences of othergenes may be necessary to determine relatedness betweentwo strains.

A30^3

IMPACT OF GENOMICS ON MYCOPLASMA RE-SEARCH

A. Blanchard, C. Lartigue, E. Rocha and P. Sirand Pugnet

INRA-Universite¤ de Bordeaux 2, Institut de Biologie Ve¤ge¤-tale Mole¤culaire, UMR 1090 GDPP, BP 81, 33883 Ville-nave D’Ornon, France

The bacterial class of Mollicutes includes species that havelost a signi¢cant portion of their genomes during evolu-tion. The genomes of ¢ve mollicute species have now beensequenced and several others are underway. In some func-tional categories, these genomes share what seems to be aminimal set of genes. Due to this limited amount of in-formation, the mycoplasmas have been considered as bio-logical models by investigators willing to evaluate the lim-its of innovative global approaches. Among these, werenew strategies for genome sequencing, proteomic analysisand in silico analyses such as predictions of secondarystructure. In addition, there was also a strong bene¢t formycoplasmology. Indeed, until recently, the use of molec-ular genetics for these bacteria was hampered because thelack of suitable genetic vectors. Comparative analysis ofthe putative chromosomal origins of replication allowed topredict functional oriC which was con¢rmed by construct-ing replicative oriC-based plasmids, initially for Mycoplas-ma pulmonis and subsequently for other species belongingto the mycoides cluster. Downsizing the cloned oriC al-lowed de¢ning a minimal region supporting plasmid rep-lication and resulted in vectors with increased stability.These vectors are now being used in functional genomicstudies. In another study, repeats were identi¢ed in thesequenced mycoplasma genomes by a systematic in silicoanalysis. Striking di¡erences were found in the density andthe distribution of di¡erent types of repeats among thestudied genomes. These results suggest that these bacteriahave adopted di¡erent strategies for generating sequencediversity and this ¢nding can also be related to the evolu-tion of Mollicutes.

A31^1

LIFESTYLE OF ENDOCELLULAR BACTERIA ANDTHEIR FUNGAL HOST IN ARBUSCULAR MYCOR-RHIZAS

A. Genre, V. Bianciotto, E. Lumini, P. Bonfante

Dipartimento di Biologia Vegetale dell’Universita' di Torinoand IPP-CNR, Viale Mattioli 25, 10125 Torino, Italy

Many bacteria complete their life cycle within eukaryoticcells of Animal and Plant Kingdom. By contrast, Fungi



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o¡er a limited number of interactions with endobacteria.Arbuscular mycorrhizal (AM) fungi are unique for suchsymbioses, since they represent a specialised niche for un-culturable rod-shaped bacteria. These have been consis-tently found in many Gigasporaceae along all the stepsof the fungal cycle: spores, germinating and symbiotichyphae. On the basis of their ribosomal sequences- theendobacteria have been identi¢ed as a new bacterial taxon,Candidatus Glomeribacter gigasporarum. To date there isno evidence on the transmission mechanism operating inthe endobacteria which live in AM fungi, since the obli-gate biotrophic status of both Gigaspora margarita andCandidatus Glomeribacter gigasporarum has hamperedany experimental investigations. The set up of a systembased on transformed carrot roots colonised with singlespores of G. margarita allowed us to demonstrate that theendobacteria are transmitted vertically. The £ow of bac-teria was tracked by using speci¢c primers designed onboth 16S and 23S ribosomal DNA sequences. A verticallytransmission mechanism does not exclude -however- thepotential acquisition of bacteria from the soil, and opensnew questions on the evolutionary meaning of the bacte-rial population and on its interactions with the fungalhost. Since endobacteria prosper inside fungal sporespropagated for more than 10 years in pot-cultures, weconclude that Candidatus G. gigasporarum represents aresident genome, raising the complexity of the moleculardialogue among mycorrhizal partners and supporting thecurrent view of mycorrhizal symbioses as often tripartiteassociations.The research has been funded by the eu genomyca proj-ect, QLK5-CT-2000.

A31^2

MYCORRHIZAL SIDEROPHORES

K. Haselwandter

Department of Microbiology, University of Innsbruck,Technikerstr. 25, A-6020 Innsbruck, Austria

The phenomenon of mycorrhizal symbiosis is widespreadin the plant kingdom. Mycorrhizal fungi a¡ect the mineralnutrition of plants, including micronutrient uptake. Foralmost all forms of life iron is an essential element. How-ever, due to the formation of rather insoluble polymers inan aerobic environment, iron is not available for organ-isms in su⁄cient quantities unless they produce a solubi-lization system. Under iron-limiting conditions, most fungiexcrete siderophores as chelating agents which form solu-ble complexes with Fe3+ with very high stability constants,thus solubilizing ferric iron. Ericoid mycorrhizal fungiproduce as main siderophores ferricrocin and fusigen, re-spectively. Ferricrocin was also demonstrated to representthe main siderophore of the ectomycorrhizal fungus Cen-

ococcum, some E-strain fungi forming ectendomycorrhi-zae, and Phialocephala fortinii as typical dark septateroot endophyte. Orchidaceous mycorrhizal fungi synthe-size hydroxamate siderophores. An arbuscular mycorrhi-zal grass species, which showed greater iron uptake thannon-mycorrhizal controls, tested positively when bioas-sayed for siderophores. At present we try to elucidatethe precise chemical structure of the main siderophoresreleased by arbuscular mycorrhizal fungi.

A31^3

IMPACT OF ARBUSCULAR MYCORRHIZAL FUNGIIN PLANT GROWTH AND HEALTH

P. Je¡ries

Department of Biosciences, The University of Kent, Canter-bury, Kent, CT2 7NJ, U.K.

Arbuscular mycorrhizas are the most important microbialsymbioses for the majority of plants and, under conditionsof P-limitation, in£uence plant community development,nutrient uptake, water relations, and aboveground pro-ductivity. They are primary determinants of plant healthand soil fertility. They also act as bioprotectants againstpathogens and toxic stresses. This presentation will reviewthe mechanisms by which these bene¢ts are conferredthrough abiotic and biotic interactions in the rhizosphere.Consideration will be given to the conservation of biodi-versity in arbuscular mycorrhizal fungi (AMF). Exampleswill be provided in which the ecology of AMF has beentaken into account and has had an impact in landscaperegeneration, horticulture, alleviation of deserti¢cationand in the bioremediation of contaminated soils.



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