International Journal of Systematic and Evolutionary Microbiology (2013), 63, 309–313 DOI 10.1099/ijs.0.040147-0

Pontibacter lucknowensis sp. nov., isolated from a hexachlorocyclohexane dump site

Vatsala Dwivedi, Neha Niharika and Rup Lal

Correspondence Molecular Biology Laboratory, Department of Zoology, University of Delhi, Delhi 110007, India Rup Lal [email protected] A Gram-negative, orange-pigmented, rod-shaped, motile and aerobic bacterial strain designated DM9T was isolated from hexachlorocyclohexane (HCH)-contaminated soil (Lucknow, India) and its taxonomic position was determined using a polyphasic approach. 16S rRNA gene sequence analysis showed that the isolate belonged to the phylum and confirmed its placement in the genus Pontibacter, with sequence similarity ranging from 93.92 to 96.21 % with other members of the genus Pontibacter. The major cellular fatty acids of the novel strain were iso-

C17 : 0 3-OH (6.00 %), iso-C15 : 0 (21.54 %) and summed feature 4 (comprising C17 : 1 iso I/ anteiso B; 32.3 %). The polar lipid profile of strain DM9T showed the presence of phosphatidylethanolamine, an unidentified aminophospholipid, two unknown aminolipids and four unknown polar lipids. Strain DM9T contained MK-7 as the predominant menaquinone and its DNA G+C content was 49.2 mol%. sym-Homospermidine was the major polyamine observed in the cell. The results obtained on the basis of phenotypic characteristics, phylogenetic analysis, biochemical and physiological tests clearly distinguished DM9T from closely related members of the genus Pontibacter. It is proposed that DM9T represents a novel , Pontibacter lucknowensis sp. nov.; the type strain is DM9T (5CCM 7955T5MTCC 11079T).

The genus Pontibacter belongs to the phylum Bacteroidetes agar plates. An orange-coloured colony, designated DM9T, and was first described by Nedashkovskaya et al. (2005). At appeared within 36 h of incubation at 28 uC which was the time of writing, there were seven species with validly picked and purified by streaking on LB agar. A polyphasic published names belonging to this genus, Pontibacter approach (Prakash et al., 2007) was adopted for taxonomic actiniarum (Nedashkovskaya et al., 2005), P. akesuensis classification of strain DM9T. (Zhou et al., 2007), P. korlensis (Zhang et al., 2008), P. 16S rRNA gene sequence determines the phylogenetic niistensis (Dastager et al., 2010), P. roseus (Suresh et al., relationship among based on its universal 2006), P. xinjiangensis (Wang et al., 2010) and P. populi distribution, highly conserved nature, the fundamental (Xu et al., 2012). In addition, ‘Pontibacter salisaro’ has also role of the ribosome in protein synthesis, no horizontal been described (Joung et al., 2011), but the name is not yet transfer and its rate of evolution, which represents an validly published. The type strains of the recognized species appropriate level of variation between organisms. The 16S of the genus are Gram-negative, rod-shaped, aerobic, rRNA gene sequence analysis of strain DM9T was carried heterotrophic and pigmented. Various species of the genus out as described by Prakash et al. (2007) using a 3100- Pontibacter have been isolated from marine actinians, Avant Genetic Analyzer sequencer at the Department of desert soil, muddy water and forest soil (Nedashkovskaya Zoology, University of Delhi, India. The sequence thus et al., 2005; Zhou et al., 2007; Suresh et al., 2006; Dastager obtained was assembled manually using Sequencing et al., 2010). In our ongoing effort (Kaur et al., 2011) to Analysis version 5.1.1 and Clone Manager software version identify the culturable and unculturable diversity at a 5. A continuous stretch of 1400 bp of the 16S rRNA gene of hexachlorocyclohexane (HCH) dump site, Lucknow, Uttar the strain DM9T was obtained and this sequence was Pradesh in North India (27u009 N and 81u099 E), we subjected to similarity search using the Seqmatch tool describe here a further species of the genus Pontibacter. For of the Ribosomal Database Project (http://rdp.cme.msu. this purpose, soil samples from the HCH dump site were edu/) and BLAST program of the National Centre for collected, serially diluted and plated on Luria–Bertani (LB) Biotechnology Information (http://www.ncbi.nlm.nih.gov/). A non-redundant BLASTN search of full sequences through Abbreviation: HCH, hexachlorocyclohexane. GenBank (Altschul et al., 1990), RDP II (Maidak et al., The GenBank accession number for 16S rRNA gene sequence of strain 2001) and EzTaxon Server version 2.1 (Chun et al., 2007) DM9T (5CCM 7955T5MTCC 11079T) is JN561788 identified its closest relatives. 16S rRNA gene sequence T Two supplementary figures and a supplementary table are available with analysis (1400 bp) revealed that strain DM9 showed T the online version of this paper. highest similarity to P. korlensis X14-1 (96.21 %). The

040147 G 2013 IUMS Printed in Great Britain 309 V. Dwivedi, N. Niharika and R. Lal nearly full-length 16S rRNA gene sequences of all members Cantor (1969) and an evolutionary tree was constructed of the genus Pontibacter were retrieved for the construction using the neighbour-joining method of Saitou & Nei (1987). of a phylogenetic tree. The 16S rRNA gene sequence Statistical evaluation of the tree topology based on 1000 similarity between strain DM9T and member of the genus resamplings was done using the bootstrap option in the T Pontibacter ranged from 93.92 to 96.21 %. Strain DM9T TREECON software (Fig. 1). Strain DM9 , falls in the clade showed highest similarity to P. korlensis X14-1T (96.21 %) containing members exclusively belonging to the genus followed by ‘P. salisaro’ HMC5104 (95.89 %), P. actiniarum Pontibacter. T T KMM 6156 (95.35 %), P. niistensis NII-0905 (95.06 %), P. Cell morphology was examined using light (Eclipse E 600; roseus SRC-1T (94.43 %), P. populi HYL7-15T (94.06 %), P. T T Nikon) and electron microscopes (transmission electron akesuensis AKS 1 (94.01 %) and P. xinjiangensis 311-10 microscope, TEM 269D; Morgagni, FEI) (see Fig. S1 (93.92 %). A phylogenetic tree was constructed using available in IJSEM Online). Microscopic analysis revealed sequences of closely related species selected from GenBank that strain DM9T is non-flagellated. Gliding motility of the and EzTaxon Server version 2.1 (Chun et al., 2007). The 16S organism was tested on a fresh LB broth culture using the rRNA gene sequence of the Reichenbachiella agariperforans hanging-drop method (Bowman et al., 2003) and was T KMM 3525 was used as an outgroup and the selected found to be positive. Colonies of strain DM9T showed sequences were aligned using the program CLUSTAL_X optimum growth on LB agar and marine agar at 28 uC version 1.81b (Thompson et al., 1997). Trimming of within 36 h of incubation. DM9T colonies were orange- terminal nucleotides that were not common to all sequences coloured, circular and smooth. The Gram staining test was was carried out manually. Phylogenetic analysis was carried performed using a Gram staining kit (HiMedia); the strain out using the TREECONW software package version 1.3b (Van was found to be Gram-negative. Antibiotic sensitivity tests de Peer & De Wachter, 1994). The evolutionary distance were performed on Muller-Hinton II medium using matrix was calculated using the distance model of Jukes & Readymade Sensi-discs (HiMedia) with varying amounts

T 0.02 757 Hymenobacter xinjiangensis X2-1g (DQ888329) 531 Hymenobacter rigui WPCB131T (DQ089669) T 571 Hymenobacter gelipurpurascens Txg1 (Y18836) Hymenobacter yonginensis HMD1010T (GU808562) Hymenobacter psychrotolerans Tibet-IIU11T (DQ177475) T 999 Hymenobacter aerophilus DSM13606 (EU155008) 552 Hymenobacter actinosclerus CCUG 39621T (Y17356) 756 Hymenobacter psychrophilus BZ33rT (GQ131579) 998 Hymenobacter tibetensis XTM003T(EU382214) 591 Hymenobacter norwichensis NS/50T (AJ549285) Hymenobacter roseosalivarius AA-718T (Y18833) 945 1000 Hymenobacter chitinivorans Txc1T (Y18837) Hymenobacter elongatus VUG-A112T (GQ454797) 981 Hymenobacter daecheongensis Dae14T (EU370958) 1000 1000 Hymenobacter soli PB17T (AB251884) Hymenobacter glaciei VUG-A130T (GQ454806) 1000 Hymenobacter ocellatus Myx 2105T (Y18835) Hymenobacter deserti ZLB-3T (EU325941) 1000 997 Adhaeribacter aerophilus 6424S-25T ( GQ421850) 965 Adhaeribacter aerolatus 6515J-31T (GQ421846) 895 Adhaeribacter aquaticus MBRG1.5T (AJ626894) T 834 Adhaeribacter terreus DNG6 (EU682684) Pontibacter populi HLY7-15T (HQ223078) 1000 Pontibacter xinjiangensis 311-10T (FJ004994) 743 ‘Pontibacter salisaro’ HMC5104 (FJ903180) 513 Pontibacter lucknowensis DM9T (JN561788) 711 T 497 Pontibacter korlensis X14-1 (DQ888330) Pontibacter actiniarum KMM 6156T (AY989908) 611 Pontibacter roseus SRC-1T (AM049256) Pontibacter akesuensis AKS 1T (DQ672723) Pontibacter niistensis NII-0905T (FJ897494) Reichenbachiella agariperforans KMM 3525T (AB058919)

Fig. 1. Phylogenetic tree based on nearly complete 16S rRNA gene sequence data showing the evolutionary relationship of strain DM9T and representative members of the 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 Reichenbachiella agariperforans KMM 3525T as the outgroup. Bar, 0.02 nt substitutions per 1000 nt positions. The GenBank accession numbers for the 16S rRNA gene sequences of each strain are shown in parentheses.

310 International Journal of Systematic and Evolutionary Microbiology 63 Pontibacter lucknowensis sp. nov. of antibiotics. Antibiotics tested were as follows (mg Smibert & Krieg (1994). Citrate utilization was tested using antibiotic per disc in parentheses) amikacin (30), ampicillin a HiMedia kit. The nitrate reduction test was performed as (10), chloramphenicol (30), ciprofloxacin (5), gentamicin described by Smibert & Krieg (1994). DNase activity was (10), kanamycin (30), nalidixic acid (30), penicillin G (10), tested using HiMedia DNase agar plates. Degradation of rifampicin (5), tetracycline (30) and vancomycin (30). HCH isomers was assessed as described by Kumari et al. Oxidase activity was tested using oxidase discs from (2002). Though isolated from the HCH dump site, strain T HiMedia. Catalase activity was tested by adding 3 % (v/v) DM9 was not found to degrade HCH. Assimilation of hydrogen peroxide solution to colonies grown on LB different carbohydrates was done in basal media (Gordon (McCarthy & Cross, 1984). Production of acid from et al., 1974). There were marked differences in several T carbohydrate and degradation of xanthine and hypox- biochemical features between strain DM9 and P. korlensis T T anthine was determined using the method of Gordon et al. X14-1 . For instance, strain DM9 was found to be positive (1974). Growth at different temperatures (4, 28, 37, 45 and for urease and assimilation of D-mannitol while P. korlensis T u X14-1 was negative for these tests. Similarly, while strain 55 C), pH (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0) and T salt concentrations [0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 % (w/v) DM9 was found to be negative for utilization and NaCl] was carried out as described by Arden-Jones et al. assimilation of fructose, D-galactose D-mannose and sucrose (1979). Hydrolysis of Tweens 20 and 80 was tested according and for hydrolysis of gelatin and starch but P. korlensis X14- 1T was positive for these tests. Differential results of to the method of Arden-Jones et al. (1979). Hydrolysis of T gelatin, casein, aesculin and starch was determined as biochemical tests of strain DM9 and its closest relatives described by Cowan & Steel (1965). Urease activity was are given in Table 1. tested in accordance with the protocol of Christensen For fatty acid analysis, cells of strain DM9T and P. korlensis (1946). Indole production was tested as described by X14-1T were harvested from LB agar plates after incubation

Table 1. Differential morphological and physiological characteristics of strain DM9T and other species of the genus Pontibacter

Strains: 1, strain DM9T (data from this study); 2, P. korlensis X14-1T (data from this study) 3, ‘P. salisaro’ HMC5104 (Joung et al., 2011); 4, P. actiniarum KMM 6156T (Nedashkovskaya et al., 2005); 5, P. niistensis NII-0905T (Dastager et al., 2010); 6, P. roseus SRC-1T (Suresh et al., 2006; Wang et al., 2010); 7, P. akesuensis AKS 1T (Zhou et al., 2007); 8, P. populi HYL7-15T (Xu et al., 2012); 9, P. xinjiangensis 311-10T (Wang et al., 2010). All species are Gram-negative and rod-shaped. Motility observed is gliding in nature. +, Positive; 2, negative; NR, not reported.

Characteristics 1 2 3 4 5 6 7 8 9

Isolation source Soil Soil Saltern Marine Soil Muddy water Soil Soil Soil Colony colour Orange Pink Red Pink Light pink Dark pink Pink Pink Pink Range for growth Temperature (uC) 6–45 7–45 20–37 6–43 15–42 4–37 4–36 4–37 4–37 Salinity (%) 0–4 0–8 0–10 0–10 0–10 0–8 0–4 0–4 0–5 Hydrolysis of: Gelatin 2 ++ + 2 ++++ Casein +++ 2 ++NR ++ Aesculin +++ ++ 2 +++ Starch 2 ++ 22++++ Tween 20 ++NR + NR 22NR NR Tween 80 ++22+ NR 222 Urea + 2 + NR 22NR 22 Carbon source utilization Cellobiose +++ NR + NR 22+ D-Fructose 2 ++ 2 +++2 + Maltose ++22+ NR + 2 + D-Glucose ++22+++2 + D-Mannitol + 22 2+ NR + 2 + D-Galactose 2 + 22++22+ Lactose +++ 2 ++22+ D-Mannose 2 + 22+ NR 2 + NR Sucrose 2 ++ 2 +++++ Citric acid 222 2NR 2222 DNA G+C content 49.2 48.2 46 48.7 51.4 59.5 51.4 44.9 47.8 (mol%)

http://ijs.sgmjournals.org 311 V. Dwivedi, N. Niharika and R. Lal at 28 uC for 2 days. Fatty acid methyl ester analysis was Cells are Gram-negative, motile by gliding, 0.6–0.7 mmin carried out at Royal Life Sciences. Fatty acid methyl esters diameter and 1.3–3.1 mm in length. Luxurious growth were analysed from two to four loops of inoculum of occurs on LB. Colonies are orange in colour, entire, culture nearly at the same phase of growth. The inoculum smooth, circular, convex and appear after 36 h of was scraped from a Petri dish and subjected to saponifica- incubation. Growth occurs at 6–45 uC; optimum growth tion, methylation and extraction using the method of is observed at 28 uC. Growth occurs in 0–4 % NaCl Miller (1982) and Kuykendall et al. (1988). The fatty acid (optimum 2 %) and at pH 6.0–9.0 (optimum pH 7.0–8.0). methyl ester mixtures were separated using the Sherlock Pigment can be extracted with organic solvents. Oxidase- Microbial Identification System (MIDI), and identification and catalase-positive. Hydrolyses aesculin, urea, Tweens 20 of the fatty acids was made using the Aerobe TSBA, 60 and 80, and casein but does not hydrolyse gelatin and version 6.0 B database. The predominant fatty acids of starch. Nitrate reduction and indole production is negative. T strain DM9 were summed feature 4 [comprising anteiso- Assimilates D-glucose, maltose, D-mannitol, lactose, cello- ] C17 : 1 B and/or iso-C17 : 1 I (32.3 %) , iso-C15 : 0 (21.54 %) biose, inositol, D-arabinose, ribose, rhamnose and sorbitol and iso-C17 : 03-OH (6.00 %). The cellular fatty acid profile but not D-fructose, D-galactose, D-mannose or sucrose. of strain DM9T was similar to those of P. korlensis X-14-1T Citrate is not utilized. Sensitive to tetracycline, gentamicin, and other closely related species. However, the fatty acid rifampicin, ciprofloxacin, streptomycin, oxytetracycline, profile of strain DM9T revealed qualitative and quantitative amikacin and kanamycin but resistant to ampicillin, differences as compared with the profile of other closely vancomycin and chloramphenicol. Although isolated from related strains (Table S1) further suggesting that DM9T an HCH-contaminated site, DM9T was unable to degrade represents a novel species of the genus Pontibacter. any of the HCH isomers. MK-7 is the major respiratory quinone. Major fatty acids are iso-C17 : 0 3-OH, C15 : 0 and Quinones were extracted from 200 mg dry cell mass with a summed feature 4 (comprising anteiso-C B and/or iso- 10 % aqueous solution of 0.3 % (w/v) NaCl in methanol 17 : 1 C17 : 1 I). sym-Homospermidine is the major polyamine. and petroleum ether (60–80 uC boiling point) at a ratio of Major polar lipids are phosphatidylethanolamine, an 1 : 1. The upper phase was collected and dried in a unidentified aminophospholipid, two unknown aminoli- m rotavapor (Buchi). The residue was dissolved in 100 l pids and four unknown polar lipids. acetone. The extract was loaded on a TLC plate (Silica gel 60 F254, 20620 cm, Merck, 1.05554.0007) using pet- The type strain is DM9T (5CCM 7955T5MTCC 11079T), roleum ether (boiling point 60–80 uC) and diethyl ether isolated from soil of an open HCH dump site situated in (85 : 15, v/v). Purified menaquinone MK-7 was dissolved in north India. The DNA G+C content of the type strain is diethyl ether and analysed by reverse-phase TLC according 49.2 mol%. to the protocol of Collins et al. (1977). For polyamine analysis, cells were cultivated in LB agar Acknowledgements medium at 28 uC. Bacterial polyamines were extracted as described by Busse & Auling (1988) and analysed by one- This research was supported by funds from Department of Biotechnology, National Bureau of Agriculturally Important Micro- dimensional TLC. A 10 ml volume of extracted sample was organisms, Indian Council of Agricultural Research, DU/DST-PURSE loaded on a TLC plate (Silica gel 60 F254, 20620 cm; Grant, Government of India. V. D. and N. N. gratefully acknowledge Merck) with ethyl acetate/cyclohexane as running solvent. the University Grants Commission and Council for Scientific and sym-Homospermidine was detected as the major poly- Industrial Research, for providing research fellowships. We thank the amine. The DNA G+C content of strain DM9T, calculated Sophisticated Analytical Instrumentation Facility, Department of by the method described by Gonzalez & Saiz-Jimenez Science and Technology, Department of Anatomy, All India Institute of Medical Sciences for providing the transmission electron (2002) using the Applied Biosystems 7500 Real-Time PCR microscopy facility. We thank Dr J. P. Euze´by (Ecole Nationale instrument, was 49.2 mol%, which is in accordance with Veterinaire, Toulouse, France) for etymological advice. We also thank that of other members of the genus Pontibacter. Deepika Kandilya for invaluable discussion. Polar lipid analysis of strain DM9T was performed by two- dimensional TLC as described by Bligh & Dyer (1959). References Total polar lipids were detected by spraying with 5 % (w/v) molybdatophosphoric acid dissolved in ethanol (Merck) Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. followed by drying at 120 uC for 15 min. Major polar lipids (1990). Basic local alignment search tool. J Mol Biol 215, 403–410. T present in strain DM9 were phosphatidylethanolamine, Arden-Jones, M. P., McCarthy, A. J. & Cross, T. (1979). Taxonomic an unidentified aminophospholipid (APL1), unknown and serologic studies on Micropolyspora faeni and Micropolyspora aminolipids (AL1, AL2) and unknown polar lipids (L1–4) strains from soil bearing the specific epithet rectivirgula. J Gen (Fig. S2). Microbiol 115, 343–354. Bligh, E. G. & Dyer, W. J. (1959). A rapid method of total lipid Description of Pontibacter lucknowensis sp. nov. extraction and purification. Can J Biochem Physiol 37, 911–917. Bowman, J. P., Nichols, C. M. & Gibson, J. A. E. (2003). Algoriphagus Pontibacter lucknowensis (luck.now.en9sis. N.L. masc. adj. ratkowskyi gen. nov., sp. nov., Brumimicrobium glaciale gen. nov., sp. lucknowensis of or belonging to Lucknow). nov., Cryomorpha ignava gen. nov., sp. nov. and Crocinitomix

312 International Journal of Systematic and Evolutionary Microbiology 63 Pontibacter lucknowensis sp. nov. catalasitica gen. nov., sp. nov., novel flavobacteria isolated from McCarthy, A. J. & Cross, T. (1984). A taxonomic study of various polar habitats. Int J Syst Evol Microbiol 53, 1343–1355. Thermomonospora and other monosporic actinomycetes. J Gen Busse, J. & Auling, G. (1988). Polyamine pattern as a chemotaxo- Microbiol 130, 5–25. nomic marker within the Proteobacteria. Syst Appl Microbiol 11, 1–8. Miller, L. T. (1982). Single derivatization method for routine analysis Christensen, W. B. (1946). Urea decomposition as a means of of bacterial whole-cell fatty acid methyl esters, including hydroxy differentiating Proteus and paracolon cultures from each other and acids. J Clin Microbiol 16, 584–586. from Salmonella and Shigella types. J Bacteriol 52, 461–466. Nedashkovskaya, O. I., Kim, S. B., Suzuki, M., Shevchenko, L. S., Chun, J., Lee, J. H., Jung, Y., Kim, M., Kim, S., Kim, B. K. & Lim, Y. W. Lee, M. S., Lee, K. H., Park, M. S., Frolova, G. M., Oh, H. W. & other authors (2005). (2007). EzTaxon: a web-based tool for the identification of Pontibacter actiniarum gen. nov., sp. nov., a novel prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst member of the phylum ‘Bacteroidetes’, and proposal of Reichen- Evol Microbiol 57, 2259–2261. bachiella gen. nov. as a replacement for the illegitimate prokaryotic generic name Nedashkovskaya et al. 2003. Int J Syst Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E. (1977). Evol Microbiol 55, 2583–2588. Distribution of menaquinones in actinomycetes and corynebacteria. Prakash, O., Kumari, K. & Lal, R. (2007). J Gen Microbiol 100, 221–230. Pseudomonas delhiensis sp. nov., isolated from fly ash dumping site of thermal power plant, Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of Delhi, India. Int J Syst Evol Microbiol 57, 527–531. Medical Bacteria. London: Cambridge University Press. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new Dastager, S. G., Raziuddin, Q. S., Deepa, C. K., Li, W. J. & Pandey, A. method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425. (2010). Pontibacter niistensis sp. nov., isolated from forest soil. Int J Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Syst Evol Microbiol 60, 2867–2870. Methods for General and Molecular Bacteriology, pp. 607–654. Edited Gonzalez, J. M. & Saiz-Jimenez, C. (2002). A fluorimetric method for by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. the estimation of G+C mol% content in microorganisms by thermal Washington, DC: American Society for Microbiology. denaturation temperature. Environ Microbiol 4, 770–773. Suresh, K., Mayilraj, S. & Chakrabarti, T. (2006). Effluviibacter roseus Gordon, R. E., Barnett, D. A., Handerhan, J. E. & Pang, C. H.-N. gen. nov., sp. nov., isolated from muddy water, belonging to the (1974). Nocardia coeliaca, Nocardia autotrophica, and the nocardin family ‘Flexibacteraceae’. Int J Syst Evol Microbiol 56, 1703–1707. strain. Int J Syst Bacteriol 24, 54–63. . Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Joung, Y., Kim, H., Ahn, T. S. & Jon, K. (2011). Pontibacter salisaro sp. Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible nov., isolated from a clay tablet solar saltern in Korea. J Microbiol 49, strategies for multiple sequence alignment aided by quality analysis 290–293. tools. Nucleic Acids Res 25, 4876–4882. Jukes, T. & Cantor, C. R. (1969). Evolution of Protein Molecules in Van de Peer, Y. & De Wachter, R. (1994). TREECON for Windows: a Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro. software package for the construction and drawing of evolutionary New York: Academic Press. trees for the Microsoft Windows environment. Comput Appl Biosci Kaur, J., Verma, M. & Lal, R. (2011). Rhizobium rosettiformans sp. nov., 10, 569–570. isolated from a hexachlorocyclohexane dump site, and reclassification Wang, Y., Zhang, K., Cai, F., Zhang, L., Tang, Y., Dai, J. & Fang, C. X. of Blastobacter aggregatus Hirsch and Muller 1986 as Rhizobium (2010). Pontibacter xinjiangensis sp. nov., in the phylum aggregatum comb. nov. Int J Syst Evol Microbiol 61, 1218–1225. ‘Bacteroidetes’, and reclassification of [Effluviibacter] roseus as Kumari, R., Subudhi, S., Suar, M., Dhingra, G., Raina, V., Dogra, C., Pontibacter roseus comb. nov. Int J Syst Evol Microbiol 60, 99–103. Lal, S., van der Meer, J. R., Holliger, C. & Lal, R. (2002). Cloning and Xu, M., Wang, Y., Dai, J., Jiang, F., Rahman, E., Peng, F. & Fang, C. X. characterization of lin genes responsible for the degradation of (2012). Pontibacter populi sp. nov., isolated from the soil of a hexachlorocyclohexane isomers by Sphingomonas paucimobilis strain Euphrates poplar (Populus euphratica) forest. Int J Syst Evol Microbiol B90. Appl Environ Microbiol 68, 6021–6028. 62, 665–670. Kuykendall, L. D., Roy, M. A., O’Neill, J. J. & Devine, T. E. (1988). Fatty Zhang, L., Zhang, Q. J., Luo, X. S., Tang, Y. L., Dai, J., Li, Y. W., Wang, acids, antibiotics resistance and deoxyribonucleic acid homology Y., Chen, G. & Fang, C. X. (2008). Pontibacter korlensis sp. nov., groups of Bradyrhizobium japonicum. Int J Syst Bacteriol 38, 358–361. isolated from the desert of Xinjiang, China. Int J Syst Evol Microbiol Maidak, B. L., Cole, J. R., Lilburn, T. G., Parker, C. T., Jr, Saxman, P. R., 58, 1210–1214. Farris, R. J., Garrity, G. M., Olsen, G. J., Schmidt, T. M. & Tiedje, J. M. Zhou, Y., Wang, X., Liu, H., Zhang, K. Y., Zhang, Y. Q., Lai, R. & Li, W. J. (2001). The RDP-II (Ribosomal Database Project). Nucleic Acids Res (2007). Pontibacter akesuensis sp. nov., isolated from a desert soil in 29, 173–174. China. Int J Syst Evol Microbiol 57, 321–325.

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