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

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 Vikram2 and Rup Lal1

Correspondence 1Molecular Biology Laboratory, Department of Zoology, University of Delhi, Delhi-110007, India Rup Lal 2IMTECH-Institute of Microbial Technology, Sector-39A, Chandigarh-160036, India [email protected]

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 T T LP100 were anteiso-C17 : 0 A, iso-C15 : 0 and iso-C18 : 1 H. The polar lipid profile of strain LP100 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).

Introduction Kang et al., 2012; Dastager et al., 2010; Dwivedi et al., 2012; The genus Pontibacter, a member of the family Cytophagaceae, Singh et al., 2013). Here, we have isolated micro-organisms phylum , was first described by Nedashkovskaya from a pond primarily contaminated with HCH. We have et al. (2005). The genus Pontibacter comprises species that are previously characterized several bacterial strains from the Gram-negative, rod-shaped, aerobic and heterotrophic, and HCH dump site (Lal et al., 2010). Soil samples from a pond contain menaquinone MK-7 as the main respiratory quinone. (Lucknow, Uttar Pradesh, India: 26u 549 N81u 099 E) were At the time of writing, the genus Pontibacter comprised 11 collected, suspended in 0.9 % NaCl, serially diluted and plated species with validly published names: Pontibacter actiniarum on nystatin- and streptomycin-amended Luria Bertani (LB) (Nedashkovskaya et al., 2005), Pontibacter akesuensis (Zhou agar plates (Vanbroekhoven et al., 2004). After incubation et al., 2007), Pontibacter korlensis (Zhang et al., 2008), at 28 uC for 48 h a pigmented colony was picked and subcultured several times to get a pure culture. The colony was Pontibacter niistensis (Dastager et al., 2010), Pontibacter T roseus (Suresh et al., 2006; Wang et al., 2010), Pontibacter designated strain LP100 . A polyphasic approach (Prakash et xinjiangensis (Wang et al., 2010), ‘Pontibacter salisaro’ (Joung al., 2007) was adopted for taxonomic classification of this et al., 2011), Pontibacter populi (Xu et al., 2012), Pontibacter novel bacterium. lucknowensis (Dwivedi et al., 2012), Pontibacter saeman- 16S rRNA gene sequencing of strain LP100T was carried geumensis (Kang et al., 2012) and Pontibacter ramchanderi using the universal bacterial primer set 8F, 341F, 786F and (Singh et al., 2013). Species of the genus Pontibacter have been 1542R (Lane, 1991) using a 3100-Avant Genetic Analyzer isolated from diverse environments, including marine sequencer at the Department of Zoology, University of actinians, desert soil, muddy water, seawater, forest soil and Delhi, India. The sequence thus obtained was assembled hexachlorocyclohexane (HCH)-contaminated soil (Nedash- manually using Sequencing Analysis, version 5.1.1, and kovskaya et al., 2005; Zhou et al., 2007; Suresh et al., 2006; Clone Manager software, version 5. A continuous stretch of 1426 bp of the 16S rRNA gene of strain LP100T was Abbreviation: HCH, hexachlorocyclohexane. obtained and this sequence was subjected to similarity The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene searches using the Seqmatch tool of the Ribosomal sequence of strain LP100T is KC469980. Database Project (http://rdp.cme.msu.edu/) and BLAST Two supplementary figures and two supplementary tables are available program of the National Centre for Biotechnology with the online version of this paper. Information (http://www.ncbi.nlm.nih.gov). A non-redundant

254 055319 G 2014 IUMS Printed in Great Britain Pontibacter indicus sp. nov.

T BLASTN search of full-length sequences through GenBank fatty acids of strain LP100 were anteiso-C17 : 0 A (31.7 %), (Altschul et al.,1990),RDPII(Maidaket al., 2001) and iso-C15 : 0 (12.5 %) and iso-C18 : 1 H (11.4 %). The fatty acid EzTaxon-e Server (Kim et al., 2012) identified its closest profile of strain LP100T showed qualitative and quantita- relatives. The nearly full-length 16S rRNA gene sequences that tive differences from that of P. lucknowensis DM9T (Table were closely related to strain LP100T were retrieved from S2), further suggesting that LP100 T represents a novel GenBank for reconstruction of the phylogenetic tree. Levels of species of the genus Pontibacter. 16S rRNA gene sequence similarity between strain LP100T and Bacterial polyamines were extracted as described by Busse & the type strains of recognized species of the genus Pontibacter Auling (1988) and analysed by one-dimensional TLC. Ten ranged from 93.8 to 97.4 %. Strain LP100T showed highest microlitres of extracted sample was loaded on the TLC plate sequence similarity to P. lucknowensis DM9T (97.4 %). Levels (Silica gel 60 F254, 20620 cm; Merck) with ethyl acetate/ of similarity to the other Pontibacter species were 93.4–96.4 %. cyclohexane as the running solvent. For the detection of A phylogenetic tree was constructed using sequences of closely polyamines the TLC plate was allowed to air dry after which it related species selected from GenBank and EzTaxon Server was visualized under UV light. Polyamines were identified by version 2.1. The 16S rRNA gene sequence of Erythrobacter comparing R values by using commercially prepared standards litoralis DSM 8509T wasusedasanoutgroupandtheselected f obtained from Sigma Life Science. sym-Homospermidine was sequences were aligned using the program CLUSTAL X version detected as the major polyamine. The DNA G+Ccontentof 1.81b (Thompson et al., 1997). The alignment was checked strain LP100T was calculated according to the method manually for quality. Phylogenetic analysis was carried out described by Gonzalez & Saiz-Jimenez (2002) using an using the TREECONW software package version 1.3b (Van de Applied Biosystems 7500 Real-Time PCR system and was Peer & De Wachter, 1994). The evolutionary distance matrix found to be 58.2 mol%. was calculated using the distance model of Jukes & Cantor (1969) and an evolutionary tree was constructed using the Quinones were extracted from 200 mg dry cell mass with a neighbour-joining method of Saitou & Nei (1987). Statistical 10 % aqueous solution of 0.3 % (w/v) NaCl in methanol evaluation of the tree topology based on 1000 resamplings was and petroleum ether (boiling point 60–80 uC) at a ratio of done using the bootstrap option in the TREECONW software 1 : 1. The upper phase was collected and dried in a rotary (Fig. 1). Strain LP100T fell in the clade containing members evaporator (Bu¨chi). The residue was dissolved in 100 ml exclusively belonging to the genus Pontibacter. acetone. The extract was loaded on a TLC plate (Silica gel 60 F254, 20620 cm; Merck) using petroleum ether DNA–DNA hybridization was carried out between strain (boiling point 60–80 uC) and diethyl ether (85 : 15, v/v). LP100T and P. lucknowensis DM9T, which showed more Purified menaquinones were dissolved in diethyl ether and than 97 % 16S rRNA gene sequence similarity. Total analysed by reversed-phase TLC according to Collins et al. genomic DNA of both strain LP100T and P. lucknowensis (1977). The major respiratory quinone was MK-7, a DM9T was extracted and purified, and hybridization was characteristic feature of the genus Pontibacter. done following the protocol described by Kumar et al. (2008) and Tourova & Antonov (1988). The amount of Polar lipid analysis of strain LP100T was performed by two- bound probe DNA was calculated by using a scintillation dimensional TLC as described by Bligh & Dyer (1959). counter (1450 LSC & Luminescence counter Wallac Total polar lipids were detected by spraying with 5 % (w/v) Microbeta Trilux; PerkinElmer). All the DNA–DNA molybdatophosphoric acid dissolved in ethanol (Merck) hybridization values were below the threshold value of followed by drying at 120 uC for 15 min. Major polar lipids 70 % (Table S1, available in IJSEM Online) recommended present in strain LP100T were phosphatidylethanolamine for the delineation of bacterial species (Wayne et al., 1987), (PE), an unidentified aminophospholipid (APL1), unknown which confirms that strain LP100T represents a novel aminolipids (AL1–3) and unknown polar lipids (L1 and 2) species of the genus Pontibacter. (Fig. S1). For fatty acid analysis, cells of strain LP100T were harvested Cell morphology was examined using a light microscope from an LB agar plate after incubation at 28 uC for 2 days. (Olympus) and transmission electron microscope (TEM Analysis of fatty acid methyl esters was carried out at Royal 269D; Morgagni, Fei) (Fig. S2). Microscopic study revealed Life Sciences, Secundarabad, India. The physiological age that strain LP100T was motile. Gliding motility of the of strain LP100T was standardized by choice of the sector organism was tested on fresh LB broth culture using the from a quadrant streak on tripticase soy broth agar plates hanging drop method (Bowman et al., 2003) as well as according to the MIDI protocol. Fatty acid methyl esters motility agar, which were found to be positive. Colonies of were analysed from 2–4 loops of inocula from the third strain LP100T showed optimum growth on LB agar, quadrant of a Petri dish subjected to saponification, nutrient agar, marine agar, trypticase soya yeast agar and methylation and extraction according to the methods of brain heart infusion agar at 28 uC within 36 h of Miller (1982) and Kuykendall et al. (1988). The fatty acid incubation; only slight growth occurred on R2A agar. methyl ester mixtures were prepared and separated using Colonies were orange, circular and smooth. The Gram- the Sherlock Microbial Identification System (MIDI) and stain reaction was performed using a Gram-stain kit identification of the fatty acids was made using the Aerobe (HiMedia) which confirmed strain LP100T as a Gram- RTSBA database, version 6.0 B (Sasser,1990). The major negative bacterium. Antibiotic sensitivity tests were http://ijs.sgmjournals.org 255 A. K. Singh and others

790 Hymenobacter xinjiangensis X2-1gT (DQ888329) 545 Hymenobacter rigui WPCB131T (DQ089669) 598 Hymenobacter gelipurpurascens Txg1T (Y18836) 0.02 Hymenobacter yonginensis HMD1010T (GU808562) Hymenobacter psychrotolerans Tibet-IIU11T (DQ177475) 999 Hymenobacter actinosclerus CCUG 39621T (Y17356) 563 Hymenobacter psychrophilus BZ33rT (GQ131579) 936 Hymenobacter aerophilus DSM 13606T (EU155008) 703 1000 Hymenobacter norwichensis NS/50T (AJ549285) ‘Hymenobacter tibetensis’ XTM003 (EU382214) 999 Hymenobacter roseosalivarius AA-718T (Y18833) 1000 Hymenobacter chitinivorans Txc1T (Y18837) 594 Hymenobacter elongatus VUG-A112T (GQ454797) 983 997 Hymenobacter daecheongensis Dae14T (EU370958) 652 Hymenobacter algoricola VUG-A23aT (EU155009) Hymenobacter fastidiosus VUG-A124T (EU155015) T 1000 1000 Hymenobacter arizonensis OR 362-8 (JX294485) Hymenobacter glaciei VUG-A130T (GQ454806) 662 T 656 Hymenobacter soli PB17 (AB251884) 572 Hymenobacter antarcticus VUG-A42aaT (EU155012) 1000 Hymenobacter ocellatus Myx 2105T (Y18835) Hymenobacter deserti ZLB-3T (EU325941) 982 Adhaeribacter terreus DNG6T (EU682684) 1000 974 Adhaeribacter aquaticus MBRG1.5T (AJ626894) 1000 Adhaeribacter aerophilus 6424S-25T (GQ421850) Adhaeribacter aerolatus 6515J-31T (GQ421846) Pontibacter populi HLY7-15T (HQ223078) 1000 811 Pontibacter xinjiangenesis 311-10T (FJ004994) Pontibacter saemangeumensis GCM0142T (JN607163) 916 Pontibacter niistensis NII-0905T (FJ897494) 650 Pontibacter akesuensis AKS 1T (DQ672723) T 527 Pontibacter actiniarum KMM 6156 (AY989908) Pontibacter korlensis X14-1T (DQ888330) T 568 Pontibacter roseus SRC-1 (AM049256) ‘Pontibacter salisaro’ HMC5104 (FJ903180) Pontibacter indicus LP100T (KC469980) Pontibacter ramchanderi LP43T (JQ806111) 910 890 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 of strain LP100T and members of representative genera Pontibacter, Adhaeribacter and Hymenobacter. The tree was constructed by using the neighbour-joining (Jukes & Cantor, 1969) method of TREECONW software and the rooting was done by using Erythrobacter litoralis DSM 8509T as an outgroup. Bootstrap values .50 % based on 1000 replications are shown at branch points. The GenBank accession number for the 16S rRNA gene sequence of each strain is shown in parentheses. Bar, 0.02 substitutions per nucleotide position. performed on Muller-Hinton II medium using readymade strain LP100T was streaked on LB agar plates and incubated antibiotic sensitivity-discs (HiMedia) containing (mg per at 4, 28, 37, 45 and 55 uC. Growth at pH 3–11 (increments disc): amikacin (30), ampicillin (10), chloramphenicol of 1 pH unit) and with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 (w/v) (30), ciprofloxacin (5), gentamicin (10), kanamycin (30), NaCl was determined as described by Arden-Jones et al. nalidixic acid (30), penicillin-G (10), rifampicin (5), (1979). pH testing is important in cases where there is a tetracycline (30) and vancomycin (30). After 3 days, signs decline in pH of the media after autoclaving. This is the of growth inhibition were assumed to indicate sensitivity to case with media at pH 10 or more which undergo a drop those antibiotics. Resistance to an antimicrobial drug was by 0.5 units. Thus, to report authentic data regarding the indicated if no inhibition zone was observed. The presence growth of an organism at pH 10 or more it should always of oxidase activity was determined by oxidation of be tested so the pH can be properly maintained. Hydrolysis N,N,N9,N9- tetramethyl-p-pheneylenediamine using oxi- of Tweens 20 and 80 was tested according to Arden-Jones dase discs from HiMedia. Catalase activity was examined et al. (1979). Hydrolysis of gelatin, casein, aesculin and based on bubble production after the application of 3 % (v/ starch was determined as described by Cowan & Steel v) hydrogen peroxide solution to colonies grown on LB (1965). Urease activity was tested in accordance with agar (McCarthy & Cross, 1984). b-Galactosidase activity Christensen (1946). Indole production was tested as was tested using ONPG discs from HiMedia. Production of described by Smibert & Krieg (1994). Citrate utilization acid from carbohydrates and degradation of xanthine and was tested using Simmons citrate agar (HiMedia). The hypoxanthine were determined according to Gordon et al. nitrate reduction test was performed as described by (1974). To determine growth at different temperatures, Smibert & Krieg (1994). DNase activity was tested using

256 International Journal of Systematic and Evolutionary Microbiology 64 Pontibacter indicus sp. nov.

DNase agar (HiMedia). H2S production was tested using but oxidase- and DNase-negative. Hydrolyses gelatin, urea, triple-sugar iron agar (HiMedia). The presence of flexir- Tween 20 and Tween 80 but not aesculin or starch. Indole ubin-type pigments was examined using a 20 % (w/v) KOH and H2S production are negative. Citrate, xanthine and solution (Bernardet et al., 2002; Bowman, 2000). Degradation hypoxanthine are not utilized. b-Galactosidase activity is not of HCH isomers was carried out using the protocol described detected. Assimilates D-glucose, maltose, D-mannitol, suc- T by Kumari et al. (2002). Strain LP100 was isolated from rose, D-arabinose, sorbitol, D-fructose and xylitol but not HCH-contaminated soil sediment but it was unable to cellobiose, lactose, D-mannose, myo-inositol, rhamnose, D- degrade any of the HCH isomers. Assimilation of different ribose or D-galactose. Forms acid with the following sugars: carbohydrates was tested in basal media (Gordon et al., 1974). maltose, D-glucose, D-mannitol, sucrose, D-arabinose, D- T Differential results of biochemical tests of strain LP100 with fructose and sorbitol. Sensitive to chloroamphenicol, nali- T its closest neighbour, P. lucknowensis DM9 , are given in dixic acid, vancomycin, penicillin, polymyxcin B, oxytetra- Table 1. cycline, tetracycline, rifampicin and ciprofloxacin but Based on the combination of phenotypic, chemotaxonomic resistant to streptomycin, ampicillin, kanamycin, amikacin and phylogenetic data, strain LP100T can be differentiated and gentamicin. Although isolated from HCH-contaminated from P. lucknowensis DM9T, and it is suggested to represent soil sediment, unable to degrade HCH isomers. The major a novel species of the genus Pontibacter, for which the isoprenoid quinone is MK-7. Major fatty acids are anteiso- name Pontibacter indicus sp. nov. is proposed. C17 : 0 A, iso-C15 : 0 and iso-C18 : 1 H . The polyamine pattern showed the presence of sym-homospermidine as the major polyamine. Major polar lipids are phosphatidylethanolamine Description of Pontibacter indicus sp. nov. (PE), an unidentified aminophospholipid (APL1), unknown Pontibacter indicus (in9di.cus. L. masc.adj. indicus Indian, aminolipids (AL1–3) and unknown polar lipids (L1 and 2). referring to the isolation of the type strain from India.) The type strain, LP100T (5CCM8435T5MCC2027T), was Cells are aerobic, Gram-negative, rod-shaped, 0.5–0.9 mm isolated from HCH-contaminated soil sediment situated in diameter and 1.1–1.7 mm in length and motile. Growth around a lindane production unit in northern India. The occurs on LB agar, nutrient agar, marine agar, trypticase DNA G+C content of the type strain is 58.2 mol%. soy yeast agar, brain heart infusion agar and R2A agar. Colonies are orange, entire, smooth, translucent, circular and convex and appear after 36 h of incubation. Growth Acknowledgements occurs at 5–40 uC (optimum 28 uC). Growth occurs in 0– This research was supported by funds from the Department of 4 % NaCl and at pH 6.0–10.0 with optimal growth at pH 7.0– Biotechnology (DBT), National Bureau of Agriculturally Important 8.0. Flexirubin-type pigments are absent. Catalase-positive, 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 Table 1. Differential morphological and physiological char- and Industrial Research (CSIR), National Bureau of Agriculturally Important Micro-organisms, for providing research fellowships. We acteristics between strain LP100T and Pontibacter luck- T thank SAIF-DST (Sophesticated Analytical Instrumentation Facility, nowensis DM9 Department of Science and Technology, Department of Anatomy, All data are from this study. The two strains shared the following AIIMS) for providing the transmission electron facility. This paper was finalized during the renewed visit under an Alexander von characteristics: isolation from soil; orange-pigmented colonies; Humboldt Fellowship (at the University of Freiburg, Germany) positive for hydrolysis of casein, Tweens 20 and 80, urea and b- awarded to R. L. We thank Professor J. P. Euze´by (Ecole Nationale galactosidase, but not starch; and positive for utilization of maltose, Veterinaire, Toulouse, France) for etymological advice. a-D-glucose and D-mannitol, but not D-galactose, D-mannose or citric acid. References Characteristic LP100T P. lucknowensis DM9T Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403–410. Temperature growth range (uC) 5–40 6–45 Arden Jones, M. P., McCarthy, A. J. & Cross, T. (1979). 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258 International Journal of Systematic and Evolutionary Microbiology 64 Pontibacter indicus sp. nov.

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