Sphingobium baderi sp. nov., isolated from ahexachlorocyclohexane dump site

Jasvinder Kaur,13 Hana Moskalikova,23 Neha Niharika,13Miroslava Sedlackova,3 Ales Hampl,2,3 Jiri Damborsky,2,4 Zbynek Prokop4

and Rup Lal1

Correspondence

Zbynek Prokop

zbynek@chemi.muni.cz

Rup Lal

ruplal@gmail.com

1Molecular Biology Laboratory, Department of Zoology, University of Delhi, Delhi-110007, India

2International Clinical Research Centre, St Anne’s University Hospital Brno, Pekarska 53, 656 91Brno, Czech Republic

3Department of Histology and Embryology, Faculty of Medicine, Masaryk University, 625 00 Brno,Czech Republic

4Loschmidt Laboratories and Centre for Toxic Compounds in the Environment, Faculty of Science,Masaryk University, 628 00 Brno, Czech Republic

A Gram-stain-negative; , rod-shaped and white-coloured bacterial strain, designated LL03T, was

isolated from hexachlorocyclohexane-contaminated soil at Spolana, Neratovice, Czech Republic,

where lindane was formerly produced. Strain LL03T was found to be a degrader of a-, c- and d-

isomers of hexachlorocyclohexane, although no significant degradation activity was observed for

the b-isomer. A neighbour-joining tree based on 16S rRNA gene sequences showed that strain

LL03T occupied a distinct phylogenetic position in the Sphingobium cluster, showing the highest

similarity with Sphingobium wenxiniae CC-FH12-1T< (99.2%). The DNA G+C content of strain

LL03T was 67.0 mol%. DNA–DNA relatedness values of strain LL03T with its close phylogenetic

neighbours were below the threshold level of 70%, supporting its identification as a

representative of a novel species of the genus Sphingobium. The predominant respiratory quinone

was ubiquinone Q-10. The polar lipid profile of strain LL03T also corresponded to those reported

for other Sphingobium species (phosphatidylethanolamine, diphosphatidylglycerol, phosphati-

dylcholine, phosphatidylglycerol, phosphatidylmonomethylethanolamine and sphingoglycolipid),

supporting its identification as a member of the genus Sphingobium. Spermidine was identified as

the major polyamine. The predominant fatty acids were 16 : 0, summed feature 3 (16 : 1v7c and/

or 16 : 1v6c), summed feature 8 (18 : 1v7c and/or 18 : 1v6c) and 14 : 0 2-OH. The polar lipid

pattern, the presence of spermidine and ubiquinone Q-10, the predominance of the cellular fatty

acids C18 : 1v7c, C16 : 0 and C14 : 0 2-OH and the G+C content of the genomic DNA supported

the affiliation of the strain to the genus Sphingobium. The results obtained after DNA–DNA

hybridization, biochemical and physiological tests clearly distinguished it from closely related

species of the genus Sphingobium. Therefore, strain LL03T represents a novel species of the

genus Sphingobium for which the name Sphingobium baderi LL03T sp. nov. is proposed; the type

strain is LL03T (5CCM 7981T5DSM 25433T).

Hexachlorocyclohexane (HCH) is a saturated chlorinated hydrocarbon and consists of eight variously stable isomers.Out of these isomers, c-HCH or lindane, was widely usedfrom the 1940s to the 1990s as a pesticide. Since lindaneproduction is a highly inefficient process wherein 90% ofthe production mix is waste, HCH emerged as a majorenvironmental pollutant. The toxicity assessment of HCHisomers ranked them as probable human carcinogens,endocrine disruptors with proven teratogenic, genotoxic,and mutagenic effects (ATSDR, 2005). In an attempt toselectively isolate strains capable of degrading HCH, soilsamples were taken from the ground below the former

%paper no. ije039834 charlesworth ref: ije039834&New Taxa - Proteobacteria

Abbreviations: HCH, hexachlorocyclohexane; t-HCH, technical hexa-chlorocyclohexane.

3These authors contributed equally to this work.

The GenBank accession number for 16S rRNA gene sequence of strainLL03T (5CCM 7981T5DSM 25433T) is JN695620.

Two supplementary figures and a supplementary table are available withthe online version of this paper.

Dedication: The authors dedicate this article to Dr Alfred Bader.

International Journal of Systematic and Evolutionary Microbiology (2013), 63, 000–000 DOI 10.1099/ijs.0.039834-0

039834 G 2013 IUMS Printed in Great Britain 1

production facility of Spolana, Neratovice, Czech Republic.Strain LL03T was isolated by the enrichment method of Itoand co-workers (Ito et al., 2007). Strain LL03T was foundto degrade a-, c- and d- isomers, while no significantdegradation activity was observed for the b-isomer ofHCH. It is interesting to note that the strain is novel in therespect that it lacks the linB gene which encodes theenzyme involved in the degradation of b-HCH. None ofthe other= strains could degrade HCH except forSphingobium quisquiliarum P25T, which could also degradea- and c-HCH completely but degraded d-HCH onlyslightly following 24 h incubation. Preliminary studies withthe strain showed it to be a member of the genusSphingobium. The genus Sphingobium, grouped in thefamily Sphingomonadaceae, belongs to theAlphaproteobacteria. The genus was established togetherwith Novosphingobium and Sphingopyxis by Takeuchi et al.(2001). It forms cluster II in the phylogenetic tree ofSphingomonas species (Takeuchi et al., 2001). Members ofthe family are widely distributed in nature and are knownto degrade a variety of compounds (Lal et al., 2008). At thetime of writing, the genus Sphingobium consisted of 25species. Here, we analysed the taxonomic status of strainLL03T by using a polyphasic approach (Prakash et al.,2007a).

A soil sample (1 g) was mixed with 2 ml 1/10 W medium(l21: KH2PO4, 170 mg; Na2HPO4, 980 mg; (NH4)2SO4,100 mg; MgSO4, 48.7 mg; FeSO4, 0.52 mg; MgO,10.75 mg; CaCO3, 2.0 mg; ZnSO4, 0.81 mg; CuSO4,0.16 mg; CoSO4, 0.15 mg; and H3BO3, 0.06 mg) andvortexed. The mixture was centrifuged (10006g, 30 min)and 1 ml supernatant was then inoculated into 100 ml 1/10 W medium saturated with technical HCH (t-HCH).After static incubation at 25 uC for 10 days, 1 ml of theprimary enrichment culture was transferred into 100 ml offresh 1/10 W medium, and the resultant secondaryenrichment culture was incubated under the samecondition for 4 days. The procedure of transfer andincubation was once repeated. Serial dilutions of thetertiary enriched culture were spread on 1/10 W agar platescontaining 1.8 mM t-HCH. After incubation at 25 uC for15 days, a number of colonies with a cleared zone ofdegraded t-HCH were picked up. Single-colony isolationwas repeated several times on t-HCH minimal medium toobtain pure cultures. Strain LL03T was found to be amember of the genus Sphingobium for which the nameSphingobium baderi LL03T is proposed for it.

16S rRNA gene sequence analysis was carried out asdescribed by Prakash et al. (2007b). A 1462 bp stretch of16S rRNA gene sequence was obtained, which was checkedmanually to ensure quality of sequences. Searches forclosely related species were carried out using the BLAST

program (Altschul et al., 1997) from the NCBI (http://www.ncbi.nlm.nih.gov). The nearly full-length 16S rRNAgene sequences of species closely related to LL03T wereretrieved from GenBank for the construction of phylogen-etic trees. The 16S rRNA gene sequence of Zymomonas

mobilis ATCC 10988T was used as an outgroup. All theselected sequences were aligned using the CLUSTAL_X

program, version 1.81b (Thompson et al., 1997). Thealignment was checked manually for quality. The evolu-tionary distance matrix was calculated using the distancemodel of Jukes & Cantor (1969) within the TREECON W

software package, version 1.3b (Van de Peer & De Wachter,1994). A phylogenetic tree was constructed using theneighbour-joining method of Saitou & Nei (1987) and theresultant tree topology was evaluated by bootstrap analysisbased on 1000 resamplings. The tree topology was found tobe similar with maximum-parsimony, neighbour-joiningand maximum-likelihood methods. Evaluation of thetopology revealed that strain LL03T clustered withmembers of the genus Sphingobium. The signaturenucleotides found in the 16S rRNA gene sequencecharacteristic for the genus Sphingobium sensu stricto(Takeuchi et al., 2001) were found in the 16S rRNA genesequence of strain LL03T. These nucleotides were U : A atposition 52 : 359, G at position 134, A : U at position987 : 1218 and U : G at position 990 : 1215, based onEscherichia coli numbering (Brosius et al., 1978). Thisobservation further supported the placement of strainLL03T within the genus Sphingobium.

Strain LL03T showed the highest 16S rRNA gene sequencesimilarity (99.2%) with Sphingobium wenxiniae JZ-1T

(Wang et al., 2011; Fig. 1). DNA–DNA hybridization wascarried out between strain LL03T and closely related strainsshowing more than 97% sequence similarity of 16S rRNAgene sequence with LL03T. Total genomic DNA of all theclosely related strains was extracted, purified and hybrid-ization was done by following the protocol as described byKumar et al. (2008) and Tourova & Antonov (1988). DNA(10 mg) of each strain was transferred onto a positivelycharged nylon membrane (Hybond-N; Amersham) using adot-blot apparatus (Bio-Rad). The membrane was air-dried, cross-linked and the DNA probe for each strain waslabelled with [a-P32]-ATP (BRIT) using a nick-translationkit (Amersham Pharmacia). Hybridization was performedovernight at 65 uC. After hybridization, the filter waswashed with SSC and SDS to remove unbound probe. Theamount of probe bound to the DNA was estimated using ab-scintillation counter (Perkin Elmer) and hybridizationvalues obtained were expressed as percentage of the probebound relative to the homologous reaction. All the DNA–DNA hybridization values were below the threshold valueof 70% (Table S1, available in IJSEM Online), as isrecommended for the delineation of bacterial species(Wayne et al., 1987), thus confirming that strain LL03T

represents a novel species of the genus Sphingobium. Cellmorphology was examined using electron microscopy (Fig.S1). Samples were fixed in 300 mmol glutaraldehyde l21 in100 mmol cacodylate buffer l21 and post-fixed in 40 mmolosmium tetroxide l21 in 100 mmol cacodylate buffer l21.For negative staining, the samples were spread on EM grids(coated with Formvar carbon support film), incubated for15 s in 2% solution of ammonium molybdate; then the

%paper no. ije039834 charlesworth ref: ije039834&

J. Kaur and others

2 International Journal of Systematic and Evolutionary Microbiology 63

excess solution was removed, samples were air-dried atroom temperature and examined in an FEI Morgagni 268Delectron microscope. For conventional processing, the fixedsamples were dehydrated in ethanol and embedded inaraldite resin (Durcupan ACM; Fluka). Ultrathin sections(60 nm thick) were cut using Leica EM UC6 ultramicro-tome and stained with uranyl acetate and lead citrate.Sections were examined under an FEI Morgagni 268Delectron microscope.

Fatty acid methyl ester (FAME) analysis of all eight strainswas carried out at Royal Life Sciences, Secundarabad, India,as described by Miller (1982) and Kuykendall et al. (1988).The mixture of FAMEs was separated by GC (Agilent 6890)equipped with flame-ionization detector (FID).Identification of FAMEs was done using the Aerobe(RTSBA, version 60) database of the Sherlock MicrobialIdentification System (MIDI). The predominant fatty acidswere 16 : 0 (14.3%), summed feature 3 (16 : 1v7c and/or16 : 1v6c; 21.8%), summed feature 8 (18 : 1v7c and/or18 : 1v6c; 12.1%) and 14 : 0 2-OH (8.4%). 14 : 0 2-OH wasthe major 2-hydoxy fatty acid and no 3-OH fatty acidswere found. These are characteristic fatty acids for thegenus Sphingobium (Takeuchi et al., 2001). However, thefatty acid profile of strain LL03T revealed qualitative andquantitative differences as compared to the profile of otherclosely related strains (Table 1) further suggesting thatLL03T is a novel species of the genus Sphingobium. In the

next step, quinones were extracted according to theprotocol described by Kaur et al. (2012). Purifiedubiquinones were dissolved in 2-propanol and analysedby reverse-phase TLC according to Collins & Jones (1980).Polar lipid analysis of strain LL03T was carried out by two-dimensional TLC as described by Bligh & Dyer (1959).Polar lipid analysis of strain LL03T exhibited the presenceof phosphatidylethanolamine, phosphatidylmonomethy-lethanolamine, phosphatidylglycerol, diphosphatidylgly-cerol, phosphatidylcholine, sphingoglycolipid, unknownphospholipids and glycolipids (Fig. S2). Polyamines wereextracted as described by Madhubala (1997) and analysedby one-dimensional TLC. Ten microlitres of the extractedsample was applied on a TLC plate (silica gel 60 F254,20620 cm; Merck, 1.05554.0007) and running solventused was cyclohexane/ethylacetate (3 : 2). Spermidine wasidentified as the major polyamine in strain LL03T, which ischaracteristic of the genus Sphingobium (Busse et al., 1999;Takeuchi et al., 2001).

The DNA G+C content of strain LL03T calculated by themethod described by Gonzalez & Saiz-Jimenez (2002)using Applied Biosystems 7500 Real-Time PCR system, wasfound to be 67.0 mol%. Motility of the organism was alsochecked on motility agar, the strain was found to be non-motile. The colony morphology of strain LL03T wasstudied on Luria–Bertani agar (LB), nutrient agar (NA)and tryptic soy agar (TSA) and R2A. Strain LL03T grew

%paper no. ije039834 charlesworth ref: ije039834&

Sphingobium chinhatense IP26 T (EF190507)Sphingobium indicum B90AT (AY519129)Sphingobium francense Sp+ (AY519130)

Sphingobium japonicum UT26T (AP010804)Sphingobium chungbukense DJ77T (AF159257)

Sphingobium chlorophenolicum ATCC 33790T (X87161)Sphingobium herbicidovorans MBIC 3166T (AB022428)

Sphingobium vermicompostii VC-230T (AM998824)Sphingobium fuliginis DSM 14926T (DQ092757)

Sphingobium quisquiliarium P25T (EU781657)Sphingobium ummariense RL-3T (EF207155)

Sphingobium cloacae S-3T (AB040739)Sphingobium faniae JZ-2T (FJ373058)

Sphingobium wenxiniae JZ-1T (FJ686047)Sphingobiumbaderi LL03T (JN695620)

Sphingobium vulgare HU1-GD12T (FJ177535)Sphingobium qiguonii X23T (EU095328)

Sphingobium amiense YT T (AB047364)Sphingobiumy anoikuyae GIFU 9882T (D16145)

Sphingobium scionense WP01T (EU009209)Sphingobium rhizovicinum CC-FH12-1T (EF465534)

Sphingobiumolei IMMIB HF-1T (AM489507)

Sphingobium abikonense NBRC 16140T (AB021416)Sphingobium lactosutens DS20 T (EU675846)

Sphingobium xenophagum BN6T (X94098)Sphingobium aromaticiconvertens DSM 12677T (AM181012)

Zymomonas mobilis ATCC 10988T (AF281031)

0.02

819999

821

865

635

527

913

535888836

649

5231000

871983

1000

**

*

*

*

*

*

*

*

*

Fig. 1. Phylogenetic tree based on nearly complete 16S rRNA gene sequence data, showing the evolutionary relationship ofstrain LL03T and members of genus Sphingobium. The tree was constructed by using neighbour-joining method of TREECON W

software and the rooting was done using Z. mobilis ATCC 10988T as an outgroup. Bar, 0.02 nt substitution per @1000 ntposition. The GenBank accession number for the 16S rRNA gene sequence of each strain is shown in parentheses. Asterisksindicate the branches tha At were also found using the maximum-parsimony and neighbour-joining methods of PHYLIP 3.69.

Sphingobium baderi sp. nov.

http://ijs.sgmjournals.org 3

well under aerobic conditions on LB agar, NA and TSA at28 uC within 24–48 h of incubation. LL03T colonies werewhite, circular and smooth, with a diameter of 0.5–1.2 mm. Gram staining was performed using a Gram-staining kit (HiMedia). Antibiotics tested were as follows(mg antibiotic per disc in parentheses): ampicillin (10),chloramphenicol (30), gentamicin (10), kanamycin (30),oxytetracycline (30), rifampicin (5), tetracycline (30),vancomycin (30), penicillin G> (10), ciprofloxacin (5),amikacin (30), ampicillin (10), streptomycin (10) andnalidixic acid (30). Catalase activity was confirmed byadding 3% (v/v) hydrogen peroxide solution to thecolonies grown on LB agar. Oxidase activity was testedusing bacteriological differentiation discs (HiMedia).Degradation of HCH isomers was assessed as describedby Kumari et al. (2002). Strain LL03T was found to degradea-, c- and d-isomers of HCH. All biochemical tests werecarried out at 28 uC. Degradation of xanthine andhypoxanthine was determined as described by Gordon etal. (1974). Growth at different pH (5.0, 6.0, 7.0, 8.0 and9.0) and salt concentrations (NaCl; 0, 1, 2, 3 and 4%, w/v)was carried out as described by Arden-Jones et al. (1979).Hydrolysis of Tween 20 and Tween 80 was tested accordingto Arden-Jones et al. (1979) and urease activity was testedaccording to Christensen (1946). Hydrolysis of casein,starch and aesculin was tested as described by Cowan &

Steel (1965). Production of indole and nitrate reductaseactivity (Smibert & Kreig, 1994) was tested. DNase activitywas tested using a HiMedia DNase agar plate. Severalmorphological, chemotaxonomic and biochemical differ-ences were found between strain LL03T and its nearestneighbour S. wenxiniae JZ-1T (Table 2). Some of theseinclude: the strain is oxidase and catalase-positive and canreduce nitrate to nitrite; hydrolyzes aesculin and utilizes D-mannose and maltose as carbon source, whereas the resultswith JZ-1T are negative for all these tests. Further, the straincannot utilize citrate; hydrolyse Tween 20, Tween 80 orcasein (sodium salt), whereas the results with JZ-1T werepositive for all these tests. Based on the results obtainedfrom morphological, physiological and biochemical ana-lysis, it is proposed that strain LL03T represents a novelspecies of the genus Sphingobium, for which the nameSphingobium baderi sp. nov. is proposed.

Description of Sphingobium baderi sp. nov.

Sphingobium baderi (ba9de.ri. N.L. gen. masc. n. baderi ofBader, named for Alfred Bader, a Czech-born chemist anda co-founder of Aldrich Chemical Company ?).

Cells are Gram-stain-negative and rod-shaped, and pro-duce white-coloured colonies. Growth occurs on LB, NA,R2A and TSA. The optimum temperature, pH andconcentration of NaCl for growth are 28 uC, 6–8 and 2–3%, respectively. Catalase and oxidase tests are positive.Nitrate is reduced to nitrite. Cannot hydrolyse Tween 20,starch or xanthine. Indole production is negative. Resistantto penicillin, streptomycin, nalidixic acid and ampicillin,but is sensitive to tetracycline, gentamicin, rifampicin,ciprofloxacin, vancomycin, chloramphenicol, kanamycin,polymyxin B and oxytetracycline. Degrades a-, c- and d-HCH isomers in liquid culture. Predominant respiratoryquinone is ubiquinone Q-10. Major polar lipids aresphingoglycolipid, phosphatidylethanolamine, phosphati-dymonomethylethanolamine, phosphatidylcholine, dipho-sphatidylglycerol and phosphatidylglycerol. Majorpolyamine is spermidine. The predominant fatty acids are16 : 0, summed feature 3 (16 : 1v7c and/or 16 : 1v6c),summed feature 8 (18 : 1v7c and/or 18 : 1v6c) and 14 : 02-OH.

The type strain is LL03T (5CCM 7981T5DSM 25433T),isolated from HCH-contaminated soil at Spolana,Neratovice, Czech Republic. The DNA G+C content ofthe type strain is 67.0 mol%.

Acknowledgements

This research was supported by funds from Department of

Biotechnology (DBT), National Bureau of Agriculturally Important

Microorganisms (NBAIM, ICAR), DU/DST-PURSE Grant,

Government of India, the Grant Agency of the Czech Republic

(203/08/0114 and P202/10/1435), the Grant Agency of the Czech

Academy of Sciences (IAA401630901) and the European Regional

Development Fund (CZ.1.05/2.1.00/01.0001 and CZ.1.05/1.1.00/

02.0123). J. K. and N.N. gratefully acknowledge Council for

%paper no. ije039834 charlesworth ref: ije039834&

Table 1. Cellular fatty acids of strain LL03T and closely relatedmembers of the genus Sphingobium

Data were obtained in this study (under similar conditions) unless

indicated. Strains: 1, strain LL03T; 2, Sphingobium wenxiniae JZ-1T; 3,

Sphingobium faniae JZ-2T; 4, Sphingobium cloacae S-3T; 5,

Sphingobium amiense YTT; 6, Sphingobium vermicomposti VC-230T;

7, Sphingobium quisquiliarum P25T; and 8, Sphingobium

chlorophenolicum ATCC 33790T. Values ,1% have been designated

TR (trace).

Fatty acid 1 2 3 4 5 6 7 8

14 : 0 1.0 TR TR TR TR TR TR TR

16 : 0 14.3 24.3 3.5 4.6 4.6 6.5 7.8 8.4

18 : 0 3.3 TR 2.4 2 2 2 2 1.3

16 : 1v5c TR TR TR 1.0 1.7 3.8 3.8 2.9

17 : 1v6c TR 2 2 1.8 14.6 6.0 3.5 3.8

17 : 1v8c TR TR 2 2 1.7 1.0 TR TR

18 : 1v5c TR 2 2 6.3 3.7 5.0 11.0 5.8

18 : 1v7c 11-

methyl

TR TR 1.5 2.6 2.1 2.6 4.1 1.8

14 : 0 2-OH 8.4 7.1 11.0 31.6 7.2 9.9 2.9 14.6

12 : 0 2-OH TR 5.3 2 2 2 2 TR TR

Summed fea-

ture:*

3 19.0 34.4 12.6 2.7 8.0 13.8 5.3 8.5

8 22.2 14.3 14.3 41.2 49.0 34.7 53.3 46.8

*Summed feature 3 consists of 16 : 1v7c and/or 16 : 1v6c and

summed feature 8 consists of 18 : 1v7c and/or18 : 1v6c.

J. Kaur and others

4 International Journal of Systematic and Evolutionary Microbiology 63

Scientific and Industrial Research (CSIR) for providing fellowships.

We would also like to thank J. P. Euzeby (Ecole Nationale Veterinaire,

Toulouse, France) for etymological advice and Dr Jian He (College of

life science of Nanjing Agricultural University, China) for providing

Sphingobium wenxiniae JZ-1T and Sphingobium faniae JZ-2T.

References

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Table 2. Differential physiological characteristics of strain LL03T and closely related members of the genus Sphingobium

Data were obtained in this study (under similar conditions) unless indicated. Strains: 1, LL03T; 2, S. wenxiniae JZ-1T; 3, S. faniae JZ-2T; 4, S. cloacae

S-3T; 5, S. amiense YTT; 6, S. vermicomposti VC-230T; 7, S. quisquiliarum P25T; and 8, S. chlorophenolicum ATCC 33790T.

Characteristic 1 2 3 4 5 6 7 8

Colony colour White Yellow Pale yellow Cream Yellow Yellow Yellow Yellow

HCH degradation + 2 2 2 2 2 + 2

Catalase + 2 + + + + + 2

Oxidase + 2 + + 2 + + +

Urease 2 2 + 2 2 2 2 +

Nitrate reduction + 2 2 2 2 2 + 2

Hydrolysis of:

Aesculin + 2 2 2 2 + + +

Citrate 2 + 2 2 2 2 2 2

Tween 80 2 + 2 2 2 2 2 2

Tween 20 2 + + 2 2 + + 2

Casein* 2 + 2 2 2 2 2 2

Indole 2 2 2 2 2 2 + 2

Utilization of:

D-Glucose + + + 2 + + + +

N-Acetylglucosamine 2 2 2 2 2 2 + +

Maltose + 2 2 2 2 2 + +

D-Mannose + 2 + 2 2 2 2 +

*Sodium salt.

Sphingobium baderi sp. nov.

http://ijs.sgmjournals.org 5

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%paper no. ije039834 charlesworth ref: ije039834&

J. Kaur and others

6 International Journal of Systematic and Evolutionary Microbiology 63

Dear Authors,

Please find enclosed a proof of your article for checking.

When reading through your proof, please check carefully authors’ names, scientific data, data in tables, any mathematics andthe accuracy of references. Please do not make any unnecessary changes at this stage. All necessary corrections should bemarked on the proof at the place where the correction is to be made; please write the correction clearly in the margin (if in thetext they may be overlooked).

Any queries that have arisen during preparation of your paper for publication are listed below and indicated on the proof.Please provide your answers when returning your proof.

Please return your proof by email (ijsem.proofs@sgm.ac.uk) or Fax [+44 (0)118 988 1834] within 2 days of receipt.

Query no. Query

1 I have changed Gram-negative to Gram-stain-negative to match details given in the text. OK?

2 In the abstract, CC-FH12-1T is not the type strain of S. wenxiniae. Please can you clarify thespecies and strain number.

3 In the sentence beginning ‘None of the other strains’, please could you clarify which ‘other’ strainsthese were.

4 Author: changed penicillin-9 to penicillin G. OK?

5 Author: is this addition correct?

6 Is the bar definition correct in the legend to Fig. 1? (not 0.02 substitutions pre nucleotideposition?)

7 No information is given regarding the numbers on the nodes in the phylogenetic tree. Please canyou provide this. (i.e. bootstrap values?)

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