pontibacter indicus sp. nov., isolated from hexachlorocyclohexane-contaminated soil

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Pontibacter indicus sp. nov., isolated from hexachlorocyclohexane-contaminated soil Amit Kumar Singh, 1 Nidhi Garg, 1 Pushp Lata, 1 Roshan Kumar, 1 Vivek Negi, 1 Surendra Vikram 2 and Rup Lal 1 Correspondence Rup Lal [email protected] 1 Molecular Biology Laboratory, Department of Zoology, University of Delhi, Delhi-110007, India 2 IMTECH-Institute of Microbial Technology, Sector-39A, Chandigarh-160036, India An orange-pigmented bacterial strain, designated LP100 T , was isolated from hexachlorocyclohexane-contaminated soil (Lucknow, India). A neighbour-joining tree based on 16S rRNA gene sequences showed that strain LP100 T occupied a distinct phylogenetic position in the Pontibacter species cluster, showing highest similarity with Pontibacter lucknowensis DM9 T (97.4 %). Levels of similarity to strains of other Pontibacter species ranged between 94.0 and 96.8 %. Strain LP100 T contained MK-7 as the predominant menaquinone and sym- homospermidine was the major polyamine in the cell. The major cellular fatty acids of strain LP100 T were anteiso-C 17 : 0 A, iso-C 15 : 0 and iso-C 18 : 1 H. The polar lipid profile of strain LP100 T showed the presence of phosphatidylethanolamine, an unidentified aminophospholipid, three unknown aminolipids and two unknown polar lipids. The G+C content of strain LP100 T was 58.2 mol%. The results of DNA–DNA hybridization, biochemical and physiological tests clearly distinguish the novel strain from closely related species of the genus Pontibacter. Therefore, strain LP100 T represents a novel species of the genus Pontibacter for which the name Pontibacter indicus is proposed. The type strain is LP100 T (5CCM8435 T 5MCC2027 T ). Introduction The genus Pontibacter, a member of the family Cytophagaceae, phylum Bacteroidetes, was first described by Nedashkovskaya et al. (2005). The genus Pontibacter comprises species that are Gram-negative, rod-shaped, aerobic and heterotrophic, and contain menaquinone MK-7 as the main respiratory quinone. At the time of writing, the genus Pontibacter comprised 11 species with validly published names: Pontibacter actiniarum (Nedashkovskaya et al., 2005), Pontibacter akesuensis (Zhou et al., 2007), Pontibacter korlensis (Zhang et al., 2008), Pontibacter niistensis (Dastager et al., 2010), Pontibacter roseus (Suresh et al., 2006; Wang et al., 2010), Pontibacter xinjiangensis (Wang et al., 2010), ‘Pontibacter salisaro’ (Joung et al., 2011), Pontibacter populi (Xu et al., 2012), Pontibacter lucknowensis (Dwivedi et al., 2012), Pontibacter saeman- geumensis (Kang et al., 2012) and Pontibacter ramchanderi (Singh et al., 2013). Species of the genus Pontibacter have been isolated from diverse environments, including marine actinians, desert soil, muddy water, seawater, forest soil and hexachlorocyclohexane (HCH)-contaminated soil (Nedash- kovskaya et al., 2005; Zhou et al., 2007; Suresh et al., 2006; Kang et al., 2012; Dastager et al., 2010; Dwivedi et al., 2012; Singh et al., 2013). Here, we have isolated micro-organisms from a pond primarily contaminated with HCH. We have previously characterized several bacterial strains from the HCH dump site (Lal et al., 2010). Soil samples from a pond (Lucknow, Uttar Pradesh, India: 26 u 549 N 81 u 099 E) were collected, suspended in 0.9 % NaCl, serially diluted and plated on nystatin- and streptomycin-amended Luria Bertani (LB) agar plates (Vanbroekhoven et al., 2004). After incubation at 28 u C for 48 h a pigmented colony was picked and subcultured several times to get a pure culture. The colony was designated strain LP100 T . A polyphasic approach (Prakash et al., 2007) was adopted for taxonomic classification of this novel bacterium. 16S rRNA gene sequencing of strain LP100 T was carried using the universal bacterial primer set 8F, 341F, 786F and 1542R (Lane, 1991) using a 3100-Avant Genetic Analyzer sequencer at the Department of Zoology, University of Delhi, India. The sequence thus obtained was assembled manually using Sequencing Analysis, version 5.1.1, and Clone Manager software, version 5. A continuous stretch of 1426 bp of the 16S rRNA gene of strain LP100 T was obtained and this sequence was subjected to similarity searches using the Seqmatch tool of the Ribosomal Database Project (http://rdp.cme.msu.edu/) and BLAST program of the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov). A non-redundant Abbreviation: HCH, hexachlorocyclohexane. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain LP100 T is KC469980. Two supplementary figures and two supplementary tables are available with the online version of this paper. International Journal of Systematic and Evolutionary Microbiology (2014), 64, 254–259 DOI 10.1099/ijs.0.055319-0 254 055319 G 2014 IUMS Printed in Great Britain

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Page 1: Pontibacter indicus sp. nov., isolated from hexachlorocyclohexane-contaminated soil

Pontibacter indicus sp. nov., isolated fromhexachlorocyclohexane-contaminated soil

Amit Kumar Singh,1 Nidhi Garg,1 Pushp Lata,1 Roshan Kumar,1

Vivek Negi,1 Surendra Vikram2 and Rup Lal1

Correspondence

Rup Lal

[email protected]

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

2IMTECH-Institute of Microbial Technology, Sector-39A, Chandigarh-160036, India

An orange-pigmented bacterial strain, designated LP100T, was isolated from

hexachlorocyclohexane-contaminated soil (Lucknow, India). A neighbour-joining tree based on

16S rRNA gene sequences showed that strain LP100T occupied a distinct phylogenetic position

in the Pontibacter species cluster, showing highest similarity with Pontibacter lucknowensis

DM9T (97.4 %). Levels of similarity to strains of other Pontibacter species ranged between 94.0

and 96.8 %. Strain LP100T contained MK-7 as the predominant menaquinone and sym-

homospermidine was the major polyamine in the cell. The major cellular fatty acids of strain

LP100T were anteiso-C17 : 0 A, iso-C15 : 0 and iso-C18 : 1 H. The polar lipid profile of strain LP100T

showed the presence of phosphatidylethanolamine, an unidentified aminophospholipid, three

unknown aminolipids and two unknown polar lipids. The G+C content of strain LP100T was

58.2 mol%. The results of DNA–DNA hybridization, biochemical and physiological tests clearly

distinguish the novel strain from closely related species of the genus Pontibacter. Therefore, strain

LP100T represents a novel species of the genus Pontibacter for which the name Pontibacter

indicus is proposed. The type strain is LP100T (5CCM8435T5MCC2027T).

IntroductionThe genus Pontibacter, a member of the family Cytophagaceae,phylum Bacteroidetes, was first described by Nedashkovskayaet al. (2005). The genus Pontibacter comprises species that areGram-negative, rod-shaped, aerobic and heterotrophic, andcontain menaquinone MK-7 as the main respiratory quinone.At the time of writing, the genus Pontibacter comprised 11species with validly published names: Pontibacter actiniarum(Nedashkovskaya et al., 2005), Pontibacter akesuensis (Zhouet al., 2007), Pontibacter korlensis (Zhang et al., 2008),Pontibacter niistensis (Dastager et al., 2010), Pontibacterroseus (Suresh et al., 2006; Wang et al., 2010), Pontibacterxinjiangensis (Wang et al., 2010), ‘Pontibacter salisaro’ (Jounget al., 2011), Pontibacter populi (Xu et al., 2012), Pontibacterlucknowensis (Dwivedi et al., 2012), Pontibacter saeman-geumensis (Kang et al., 2012) and Pontibacter ramchanderi(Singh et al., 2013). Species of the genus Pontibacter have beenisolated from diverse environments, including marineactinians, desert soil, muddy water, seawater, forest soil andhexachlorocyclohexane (HCH)-contaminated soil (Nedash-kovskaya et al., 2005; Zhou et al., 2007; Suresh et al., 2006;

Kang et al., 2012; Dastager et al., 2010; Dwivedi et al., 2012;Singh et al., 2013). Here, we have isolated micro-organismsfrom a pond primarily contaminated with HCH. We havepreviously characterized several bacterial strains from theHCH dump site (Lal et al., 2010). Soil samples from a pond(Lucknow, Uttar Pradesh, India: 26u 549 N 81u 099 E) werecollected, suspended in 0.9 % NaCl, serially diluted and platedon nystatin- and streptomycin-amended Luria Bertani (LB)agar plates (Vanbroekhoven et al., 2004). After incubationat 28 uC for 48 h a pigmented colony was picked andsubcultured several times to get a pure culture. The colony wasdesignated strain LP100T. A polyphasic approach (Prakash etal., 2007) was adopted for taxonomic classification of thisnovel bacterium.

16S rRNA gene sequencing of strain LP100T was carriedusing the universal bacterial primer set 8F, 341F, 786F and1542R (Lane, 1991) using a 3100-Avant Genetic Analyzersequencer at the Department of Zoology, University ofDelhi, India. The sequence thus obtained was assembledmanually using Sequencing Analysis, version 5.1.1, andClone Manager software, version 5. A continuous stretch of1426 bp of the 16S rRNA gene of strain LP100T wasobtained and this sequence was subjected to similaritysearches using the Seqmatch tool of the RibosomalDatabase Project (http://rdp.cme.msu.edu/) and BLAST

program of the National Centre for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov). A non-redundant

Abbreviation: HCH, hexachlorocyclohexane.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA genesequence of strain LP100T is KC469980.

Two supplementary figures and two supplementary tables are availablewith the online version of this paper.

International Journal of Systematic and Evolutionary Microbiology (2014), 64, 254–259 DOI 10.1099/ijs.0.055319-0

254 055319 G 2014 IUMS Printed in Great Britain

Page 2: Pontibacter indicus sp. nov., isolated from hexachlorocyclohexane-contaminated soil

BLASTN search of full-length sequences through GenBank(Altschul et al., 1990), RDP II (Maidak et al., 2001) andEzTaxon-e Server (Kim et al., 2012) identified its closestrelatives. The nearly full-length 16S rRNA gene sequences thatwere closely related to strain LP100T were retrieved fromGenBank for reconstruction of the phylogenetic tree. Levels of16S rRNA gene sequence similarity between strain LP100T andthe type strains of recognized species of the genus Pontibacterranged from 93.8 to 97.4 %. Strain LP100T showed highestsequence similarity to P. lucknowensis DM9T (97.4 %). Levelsof similarity to the other Pontibacter species were 93.4–96.4 %.A phylogenetic tree was constructed using sequences of closelyrelated species selected from GenBank and EzTaxon Serverversion 2.1. The 16S rRNA gene sequence of Erythrobacterlitoralis DSM 8509T was used as an outgroup and the selectedsequences were aligned using the program CLUSTAL X version1.81b (Thompson et al., 1997). The alignment was checkedmanually for quality. Phylogenetic analysis was carried outusing the TREECONW software package version 1.3b (Van dePeer & De Wachter, 1994). The evolutionary distance matrixwas calculated using the distance model of Jukes & Cantor(1969) and an evolutionary tree was constructed using theneighbour-joining method of Saitou & Nei (1987). Statisticalevaluation of the tree topology based on 1000 resamplings wasdone using the bootstrap option in the TREECONW software(Fig. 1). Strain LP100T fell in the clade containing membersexclusively belonging to the genus Pontibacter.

DNA–DNA hybridization was carried out between strainLP100T and P. lucknowensis DM9T, which showed morethan 97 % 16S rRNA gene sequence similarity. Totalgenomic DNA of both strain LP100T and P. lucknowensisDM9T was extracted and purified, and hybridization wasdone following the protocol described by Kumar et al.(2008) and Tourova & Antonov (1988). The amount ofbound probe DNA was calculated by using a scintillationcounter (1450 LSC & Luminescence counter WallacMicrobeta Trilux; PerkinElmer). All the DNA–DNAhybridization values were below the threshold value of70 % (Table S1, available in IJSEM Online) recommendedfor the delineation of bacterial species (Wayne et al., 1987),which confirms that strain LP100T represents a novelspecies of the genus Pontibacter.

For fatty acid analysis, cells of strain LP100T were harvestedfrom an LB agar plate after incubation at 28 uC for 2 days.Analysis of fatty acid methyl esters was carried out at RoyalLife Sciences, Secundarabad, India. The physiological ageof strain LP100T was standardized by choice of the sectorfrom a quadrant streak on tripticase soy broth agar platesaccording to the MIDI protocol. Fatty acid methyl esterswere analysed from 2–4 loops of inocula from the thirdquadrant of a Petri dish subjected to saponification,methylation and extraction according to the methods ofMiller (1982) and Kuykendall et al. (1988). The fatty acidmethyl ester mixtures were prepared and separated usingthe Sherlock Microbial Identification System (MIDI) andidentification of the fatty acids was made using the AerobeRTSBA database, version 6.0 B (Sasser,1990). The major

fatty acids of strain LP100T were anteiso-C17 : 0 A (31.7 %),iso-C15 : 0 (12.5 %) and iso-C18 : 1 H (11.4 %). The fatty acidprofile of strain LP100T showed qualitative and quantita-tive differences from that of P. lucknowensis DM9T (TableS2), further suggesting that LP100 T represents a novelspecies of the genus Pontibacter.

Bacterial polyamines were extracted as described by Busse &Auling (1988) and analysed by one-dimensional TLC. Tenmicrolitres of extracted sample was loaded on the TLC plate(Silica gel 60 F254, 20620 cm; Merck) with ethyl acetate/cyclohexane as the running solvent. For the detection ofpolyamines the TLC plate was allowed to air dry after which itwas visualized under UV light. Polyamines were identified bycomparing Rf values by using commercially prepared standardsobtained from Sigma Life Science. sym-Homospermidine wasdetected as the major polyamine. The DNA G+C content ofstrain LP100T was calculated according to the methoddescribed by Gonzalez & Saiz-Jimenez (2002) using anApplied Biosystems 7500 Real-Time PCR system and wasfound to be 58.2 mol%.

Quinones were extracted from 200 mg dry cell mass with a10 % aqueous solution of 0.3 % (w/v) NaCl in methanoland petroleum ether (boiling point 60–80 uC) at a ratio of1 : 1. The upper phase was collected and dried in a rotaryevaporator (Buchi). The residue was dissolved in 100 mlacetone. The extract was loaded on a TLC plate (Silica gel60 F254, 20620 cm; Merck) using petroleum ether(boiling point 60–80 uC) and diethyl ether (85 : 15, v/v).Purified menaquinones were dissolved in diethyl ether andanalysed by reversed-phase TLC according to Collins et al.(1977). The major respiratory quinone was MK-7, acharacteristic feature of the genus Pontibacter.

Polar lipid analysis of strain LP100T was performed by two-dimensional TLC as described by Bligh & Dyer (1959).Total polar lipids were detected by spraying with 5 % (w/v)molybdatophosphoric acid dissolved in ethanol (Merck)followed by drying at 120 uC for 15 min. Major polar lipidspresent in strain LP100T were phosphatidylethanolamine(PE), an unidentified aminophospholipid (APL1), unknownaminolipids (AL1–3) and unknown polar lipids (L1 and 2)(Fig. S1).

Cell morphology was examined using a light microscope(Olympus) and transmission electron microscope (TEM269D; Morgagni, Fei) (Fig. S2). Microscopic study revealedthat strain LP100T was motile. Gliding motility of theorganism was tested on fresh LB broth culture using thehanging drop method (Bowman et al., 2003) as well asmotility agar, which were found to be positive. Colonies ofstrain LP100T showed optimum growth on LB agar,nutrient agar, marine agar, trypticase soya yeast agar andbrain heart infusion agar at 28 uC within 36 h ofincubation; only slight growth occurred on R2A agar.Colonies were orange, circular and smooth. The Gram-stain reaction was performed using a Gram-stain kit(HiMedia) which confirmed strain LP100T as a Gram-negative bacterium. Antibiotic sensitivity tests were

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performed on Muller-Hinton II medium using readymadeantibiotic sensitivity-discs (HiMedia) containing (mg perdisc): amikacin (30), ampicillin (10), chloramphenicol(30), ciprofloxacin (5), gentamicin (10), kanamycin (30),nalidixic acid (30), penicillin-G (10), rifampicin (5),tetracycline (30) and vancomycin (30). After 3 days, signsof growth inhibition were assumed to indicate sensitivity tothose antibiotics. Resistance to an antimicrobial drug wasindicated if no inhibition zone was observed. The presenceof oxidase activity was determined by oxidation ofN,N,N9,N9- tetramethyl-p-pheneylenediamine using oxi-dase discs from HiMedia. Catalase activity was examinedbased on bubble production after the application of 3 % (v/v) hydrogen peroxide solution to colonies grown on LBagar (McCarthy & Cross, 1984). b-Galactosidase activitywas tested using ONPG discs from HiMedia. Production ofacid from carbohydrates and degradation of xanthine andhypoxanthine were determined according to Gordon et al.(1974). To determine growth at different temperatures,

strain LP100T was streaked on LB agar plates and incubatedat 4, 28, 37, 45 and 55 uC. Growth at pH 3–11 (incrementsof 1 pH unit) and with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 (w/v)NaCl was determined as described by Arden-Jones et al.(1979). pH testing is important in cases where there is adecline in pH of the media after autoclaving. This is thecase with media at pH 10 or more which undergo a dropby 0.5 units. Thus, to report authentic data regarding thegrowth of an organism at pH 10 or more it should alwaysbe tested so the pH can be properly maintained. Hydrolysisof Tweens 20 and 80 was tested according to Arden-Joneset al. (1979). Hydrolysis of gelatin, casein, aesculin andstarch was determined as described by Cowan & Steel(1965). Urease activity was tested in accordance withChristensen (1946). Indole production was tested asdescribed by Smibert & Krieg (1994). Citrate utilizationwas tested using Simmons citrate agar (HiMedia). Thenitrate reduction test was performed as described bySmibert & Krieg (1994). DNase activity was tested using

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Hymenobacter xinjiangensis X2-1gT (DQ888329)Hymenobacter rigui WPCB131T (DQ089669)

Hymenobacter gelipurpurascens Txg1T (Y18836)Hymenobacter yonginensis HMD1010T (GU808562)Hymenobacter psychrotolerans Tibet-IIU11T (DQ177475)

Hymenobacter actinosclerus CCUG 39621T (Y17356)Hymenobacter psychrophilus BZ33rT (GQ131579)Hymenobacter aerophilus DSM 13606T (EU155008)

Hymenobacter norwichensis NS/50T (AJ549285)‘Hymenobacter tibetensis’ XTM003 (EU382214)

Hymenobacter roseosalivarius AA-718T (Y18833) Hymenobacter chitinivorans Txc1T (Y18837)Hymenobacter elongatus VUG-A112T (GQ454797)

Hymenobacter daecheongensis Dae14T (EU370958)Hymenobacter algoricola VUG-A23aT (EU155009)Hymenobacter fastidiosus VUG-A124T (EU155015)

Hymenobacter arizonensis OR 362-8T (JX294485)Hymenobacter glaciei VUG-A130T (GQ454806)

Hymenobacter soli PB17T (AB251884)Hymenobacter antarcticus VUG-A42aaT (EU155012)

Hymenobacter ocellatus Myx 2105T (Y18835)Hymenobacter deserti ZLB-3T (EU325941)

Adhaeribacter terreus DNG6T (EU682684)Adhaeribacter aquaticus MBRG1.5T (AJ626894)

Adhaeribacter aerophilus 6424S-25T (GQ421850)Adhaeribacter aerolatus 6515J-31T (GQ421846)

Pontibacter populi HLY7-15T (HQ223078)Pontibacter xinjiangenesis 311-10T (FJ004994)Pontibacter saemangeumensis GCM0142T (JN607163)

Pontibacter niistensis NII-0905T (FJ897494)Pontibacter akesuensis AKS 1T (DQ672723)

Pontibacter actiniarum KMM 6156T (AY989908)Pontibacter korlensis X14-1T (DQ888330)

Pontibacter roseus SRC-1T (AM049256)‘Pontibacter salisaro’ HMC5104 (FJ903180)

Pontibacter indicus LP100T (KC469980)Pontibacter ramchanderi LP43T (JQ806111)Pontibacter lucknowensis DM9T (JN561788)

Erythrobacter litoralis DSM 8509T (AB013354)

Fig. 1. Phylogenetic tree based on nearly complete 16S rRNA gene sequence data showing the evolutionary relationship ofstrain LP100T and members of representative genera Pontibacter, Adhaeribacter and Hymenobacter. The tree was constructedby using the neighbour-joining (Jukes & Cantor, 1969) method of TREECONW software and the rooting was done by usingErythrobacter litoralis DSM 8509T as an outgroup. Bootstrap values .50 % based on 1000 replications are shown at branchpoints. The GenBank accession number for the 16S rRNA gene sequence of each strain is shown in parentheses. Bar, 0.02substitutions per nucleotide position.

A. K. Singh and others

256 International Journal of Systematic and Evolutionary Microbiology 64

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DNase agar (HiMedia). H2S production was tested usingtriple-sugar iron agar (HiMedia). The presence of flexir-ubin-type pigments was examined using a 20 % (w/v) KOHsolution (Bernardet et al., 2002; Bowman, 2000). Degradationof HCH isomers was carried out using the protocol describedby Kumari et al. (2002). Strain LP100T was isolated fromHCH-contaminated soil sediment but it was unable todegrade any of the HCH isomers. Assimilation of differentcarbohydrates was tested in basal media (Gordon et al., 1974).Differential results of biochemical tests of strain LP100T withits closest neighbour, P. lucknowensis DM9T, are given inTable 1.

Based on the combination of phenotypic, chemotaxonomicand phylogenetic data, strain LP100T can be differentiatedfrom P. lucknowensis DM9T, and it is suggested to representa novel species of the genus Pontibacter, for which thename Pontibacter indicus sp. nov. is proposed.

Description of Pontibacter indicus sp. nov.

Pontibacter indicus (in9di.cus. L. masc.adj. indicus Indian,referring to the isolation of the type strain from India.)

Cells are aerobic, Gram-negative, rod-shaped, 0.5–0.9 mmin diameter and 1.1–1.7 mm in length and motile. Growthoccurs on LB agar, nutrient agar, marine agar, trypticasesoy yeast agar, brain heart infusion agar and R2A agar.Colonies are orange, entire, smooth, translucent, circularand convex and appear after 36 h of incubation. Growthoccurs at 5–40 uC (optimum 28 uC). Growth occurs in 0–4 % NaCl and at pH 6.0–10.0 with optimal growth at pH 7.0–8.0. Flexirubin-type pigments are absent. Catalase-positive,

but oxidase- and DNase-negative. Hydrolyses gelatin, urea,Tween 20 and Tween 80 but not aesculin or starch. Indoleand H2S production are negative. Citrate, xanthine andhypoxanthine are not utilized. b-Galactosidase activity is notdetected. Assimilates D-glucose, maltose, D-mannitol, suc-rose, D-arabinose, sorbitol, D-fructose and xylitol but notcellobiose, lactose, D-mannose, myo-inositol, rhamnose, D-ribose or D-galactose. Forms acid with the following sugars:maltose, D-glucose, D-mannitol, sucrose, D-arabinose, D-fructose and sorbitol. Sensitive to chloroamphenicol, nali-dixic acid, vancomycin, penicillin, polymyxcin B, oxytetra-cycline, tetracycline, rifampicin and ciprofloxacin butresistant to streptomycin, ampicillin, kanamycin, amikacinand gentamicin. Although isolated from HCH-contaminatedsoil sediment, unable to degrade HCH isomers. The majorisoprenoid quinone is MK-7. Major fatty acids are anteiso-C17 : 0 A, iso-C15 : 0 and iso-C18 : 1 H . The polyamine patternshowed the presence of sym-homospermidine as the majorpolyamine. Major polar lipids are phosphatidylethanolamine(PE), an unidentified aminophospholipid (APL1), unknownaminolipids (AL1–3) and unknown polar lipids (L1 and 2).

The type strain, LP100T (5CCM8435T5MCC2027T), wasisolated from HCH-contaminated soil sediment situatedaround a lindane production unit in northern India. TheDNA G+C content of the type strain is 58.2 mol%.

Acknowledgements

This research was supported by funds from the Department of

Biotechnology (DBT), National Bureau of Agriculturally Important

Micro-organisms (NBAIM, ICAR), DU/DST-PURSE Grant, Govern-

ment of India. A. K. S., N. G., V. N., S. V., P. L. and R. K. gratefully

acknowledge the Department of Biotechnology, Council for Scientific

and Industrial Research (CSIR), National Bureau of Agriculturally

Important Micro-organisms, for providing research fellowships. We

thank SAIF-DST (Sophesticated Analytical Instrumentation Facility,

Department of Science and Technology, Department of Anatomy,

AIIMS) for providing the transmission electron facility. This paper

was finalized during the renewed visit under an Alexander von

Humboldt Fellowship (at the University of Freiburg, Germany)

awarded to R. L. We thank Professor J. P. Euzeby (Ecole Nationale

Veterinaire, Toulouse, France) for etymological advice.

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nowensis DM9T

All data are from this study. The two strains shared the following

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Characteristic LP100T P. lucknowensis

DM9T

Temperature growth range (uC) 5–40 6–45

Hydrolysis of:

Gelatin + 2

Aesculin 2 +

Carbon source utilization

Cellobiose 2 +

D-Fructose + 2

a-Lactose 2 +

Sucrose + 2

D-Ribose 2 +

DNA G+C content (mol%) 58.2 49.2

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