Polaromonas Eurypsychrophila Sp. Nov., Isolated from an Ice Core

International Journal of Systematic and Evolutionary Microbiology (2016), 66, 2497–2501 DOI 10.1099/ijsem.0.001079

Polaromonas eurypsychrophila sp. nov., isolated from an ice core Tingting Xing,1 Tandong Yao,2,3 Yongqin Liu,2,3 Ninglian Wang,2,4 Bainqing Xu,2,3 Liang Shen,1 Zhengquan Gu,1 Bixi Gu,1 Hongcan Liu5 and Yuguang Zhou5

Correspondence 1Key Laboratory of Alpine Ecology and Biodiversity, Institute of Tibetan Plateau Research, Yongqin Liu Chinese Academy of Sciences, Beijing 100101, China [email protected] 2CAS Center for Excellence in Tibetan Plateau Earth Sciences, Chinese Academy of Sciences, Beijing 100101, China 3Key Laboratory of Tibetan Environment Changes and Land Surface Processes, Institute of Tibetan Plateau Research, Chinese Academy of Sciences, Beijing 100101, China 4College of Urban and Environmental Science, Northwest University, Xi’an 710000, China 5Institute of Microbiology, China General Microbiological Culture Collection Center, Chinese Academy of Sciences, Beijing 100101, China

A Gram-stain-negative, aerobic, rod-shaped, beige bacterium, strain B717-2T, was isolated from an ice core drilled from Muztagh Glacier on the Tibetan Plateau, China. According to phylogenetic analyses based on 16S rRNA gene sequences, the novel strain was related most closely to Polaromonas vacuolataand shared 97.7 % similarity with the type strain of this species. It grew optimally at pH 7, at 15 C and with 2 % (w/v) NaCl. Major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The major fatty acids

were summed feature 3 (C16 : 1!7c and/or iso-C15 : 0 2-OH), summed feature 8 (C18 : 1!7c,

C18 : 1!6c) and C16 : 0. The sole respiratory quinone was Q-8. The DNA G+C content was 63.4 mol %. In DNA–DNA hybridization tests, strain B717-2T shared 37.0±1.9, 30.0±1.7, 26.0±0.9, 23.4±0.5 and 18.4±1.9 % DNA–DNA relatedness with Polaromonas jejuensisJS12-13T, P. vacuolata 34-PT, Polaromonas aquatica CCUG 39402T, Polaromonas glacialisCr4-12T and Polaromonas cryoconitiCr4-35T, respectively. Based on the phenotypic, phylogenetic and genetic characteristics, strain B717-2T represents a novel species of the genus Polaromonas, for which the name Polaromonaseurypsychrophila sp. nov. is proposed. The type strain is B717- 2T (=CGMCC 1.15322T=JCM 31171T).

The genus Polaromonas was first proposed by Irgens et al. (Irgens et al., 1996), tap water (K€ampfer et al., 2006), soil (1996) for a psychrophilic, marine bacterium isolated from (Weon et al., 2008; Sizova & Panikov, 2007), sediment Antarctica, Polaromonas vacuolata. At the time of writing, (Jeon et al., 2004) and glacier cryoconite (Margesin et al., this genus comprised seven recognized species: Polaromonas 2012). A novel bacterial strain, B717-2T, was isolated from naphthalenivorans (Jeon et al., 2004), Polaromonas aquatica an ice core at a depth of 38 m. The whole ice core was € (Kampfer et al., 2006), Polaromonas hydrogenivorans (Sizova 120 m in length drilled from Muztagh Glacier (83.7 E & Panikov, 2007), Polaromonas jejuensis (Weon et al., 26.4 N) on the Tibetan Plateau, China. Climate on the gla- 2008), Polaromonas glacialis (Margesin et al., 2012), Polaro- cier is extremely cold. Therefore, bacteria recovered from monas cryoconiti (Margesin et al., 2012) and the type species, P. vacuolata (Irgens et al., 1996). this area may survive in an inactive state at low temperature. The taxonomic position of the novel strain was investigated Members of the genus Polaromonas have been isolated from by using a polyphasic approach. various environmental sources, such as marine water The ice core was preserved at À20 C before analysis. The ice core was cut into 5 cm long sections using a band saw The GenBank/EMBL/DDJB accession number for the 16S rRNA gene within walk-in freezers. All samples were decontaminated sequence of strain B717-2T is KP013181. by cutting away an outer annulus of about 4 mm with a

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Downloaded from www.microbiologyresearch.org by IP: 124.16.145.97 On: Sat, 08 Oct 2016 08:31:15 T. Xing and others sterilized saw tooth knife, before rinsing the remaining phylogenetic position of the novel strain was reconstructed inner cores with cold ethanol (95 %) and finally with cold by treeing using the software package MEGA 5.0.5 (Tamura autoclaved water. No particulate sterile suits were worn et al., 2011). A phylogenetic tree was reconstructed using the during the sampling process. All work was done in a sterile neighbour-joining and maximum-likelihood methods with operating room. The samples were then melted in sterile bootstrap values based on 1000 replications (Fig. 1). In the Nalgene bottles at 4 C. We subsequently incubated 200 µl neighbour-joining phylogenetic tree, strain B717-2T formed of meltwater at 4 C for 30 days on an R2A agar plate (con- a distinct cluster in a phyletic line with P. vacuolata 34-PT, taining 0.05 % yeast extract, 0.05 % peptone, 0.05 % Casa- and the maximum-likelihood tree displayed a similar topol- mino acids, 0.05 % glucose, 0.05 % starch, 0.03 % sodium ogy (Fig. 1). DNA–DNA hybridization experiments were pyruvate, 0.03 % K2HPO4, 0.005 % MgSO4, 1.5 % agar; pH carried out applying the optical renaturation method (De 7.2) (Reasoner & Geldreich, 1985), and several bacterial col- Ley et al., 1970; Huß et al., 1983; Jahnke, 1992). The geno- onies were recovered. mic DNA G+C content of strain B717-2T, which was esti- mated from the midpoint value (T ) of the thermal DNA of the novel strain was extracted according to the m denaturation profile, was 63.4 mol% (Mandel et al., 1970). method of Marmur (1961) from cells grown on R2A agar The temperature used in the optical renaturation method for 10 days at 15 C. Purity was assessed using a NanoDrop was 79 C. Mean levels of DNA–DNA relatedness between spectrophotometer (2000c; Thermo). PCR amplification strain B717-2T and P. jejuensis JS12-13T, P. vacuolata 34-PT, was via universal primers 27F (5¢-AGAGTTTGATCCTGGC P. aquatica CCUG 39402T, P. glacialis Cr4-12T and P. cryoco- TCAG-3¢) and 1492R (5¢-CGGTTACCTTGTTACGACTT- niti Cr4-35T were 37.0±1.9, 30.0±1.7, 26.0±0.9, 23.4±0.5 3¢) (Embley, 1991), and the PCR products were sequenced and 18.4±1.9 %, respectively. These data again indicate that at Sangon using an ABI PRISM 3730xl sequencer. The strain B717-2T may represent a novel species of the genus sequence was then analysed against the GenBank database Polaromonas. with the BLAST program (NCBI) and EzBioCloud (http:// www.ezbiocloud.net/eztaxon) (Kim et al., 2012) to reveal To further determine the taxonomic position of the novel the most similar sequences deposited in GenBank. In isolate, a series of phenotypic and genotypic approaches comparisons with the type strains of Polaromonas species, were used. Cell morphology was examined by transmission strain B717-2T appeared to be related most closely to P. electron microscopy (JEM-1230; JEOL) (Fig. 2), using cells vacuolata 34-PT, with a 16S rRNA gene sequence similarity that had been grown on R2A agar at 15 C. Gram staining of 97.7 %, followed by similarities of 97.4, 97.4, 97.1 and and catalase activity tests were conducted according to the 97.1 % with P. jejuensis JS12-13T, P. aquatica CCUG 39402T, methods described by Smibert & Krieg (1994). Physiologi- P. glacialis Cr4-12T and P. cryoconiti Cr4-35T, respectively. cal and biochemical characteristics and other enzyme activ- Based on the threshold value of 98.65 % (Kim et al, 2014), ities were determined by using API 20E, API 20NE and strain B717-2T may thus represent a novel species. The API ZYM systems (bioMerieux) at 15 C. All tests were

63 Variovorax boronicumulans BAM-48 T(AB 300597) 64 Variovorax paradoxus IAM 12373T(D 88006) 0.01 99 Variovorax ginsengisoli Gsoil 3165 T(AB 245358) Variovorax defluvii 2C1-b T (HQ 385753) 86 88 Variovorax soli GH 9-3T(DQ 432053) Pseudorhodoferax aquiterrae NAFc-7T(GU 721026)

T 100 Pseudorhodoferax caeni SB1 (AJ 606333) 100 Pseudorhodoferax soli TBEA3T(EU 825700)

98 B717-2T(KP 013181) Polaromonas vacuolata 34-PT(U 14585) T 100 Polaromonas jejuensis JS12-13 (EU 030285) Polaromonas cryoconiti Cr4-35 T(HM 583567) 42 Polaromonas glacialis Cr4-12T(HM 583568) 87 T 49 Polaromonas hydrogenivorans DSM 17735 (DQ 094183) 100 Polaromonas naphthalenivorans CJ2T(CP 000529) Roseateles terrae CCUG 52222T(AM 501445)

Fig. 1. Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the position of strain B717-2T. Numbers at nodes indicate bootstrap percentages (based on 1000 replications). Bar, 0.01 accumulated changes per nucleotide. Filled circles indicate that the corresponding nodes were also obtained in the maximum-likelihood tree.

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Downloaded from www.microbiologyresearch.org by IP: 124.16.145.97 On: Sat, 08 Oct 2016 08:31:15 Polaromonas eurypsychrophila sp. nov. carried out simultaneously with strain B717-2T and the seven reference strains. P. vacuolata 34-PT, P. aquatica CCUG 39402T, P. hydrogenivorans DSM 17735T and P. jejuensis JS12-13T were obtained from China General Microbiological Culture Collection Center, while P. naphthalenivorans CJ2T, P. glacialis Cr4-12T and P. cryoconiti Cr4-35T were obtained from Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures. Growth at various temperatures (0–35 C) was tested in R2A broth at increments of 5 C. The pH range (5.0–11.0) for growth was determined in R2A broth at 15 C. The pH values of <6, 6–9 and >9 were obtained by using sodium acetate/acetic acid, Tris/HCl and Na2CO3 buffers, respectively. Growth in the absence of NaCl and in the presence of 1, 2, 3, 4, 5 and 6 % (w/v) NaCl was also investigated in the same medium at 15 C. Differences in the physiological Fig. 2. Transmission electron micrograph of a cell of strain B717- characteristics between strain B717-2T and the seven refer- 2T after 10 days of growth on R2A agar. Bar, 1 µm. ence strains (Irgens et al., 1996; Jeon et al., 2004; K€ampfer et al., 2006; Sizova & Panikov, 2007; Weon et al., 2008; Margesin et al., 2012) are given in Table 1. B717-2Tgrown on R2A agar for 10 days at 15 C. Biomasses Whole-cell fatty acid methyl esters were extracted and used for fatty acid analyses were harvested from the loga- prepared using the standard protocol of the Microbial Iden- rithmic phase of growth. The fatty acid profile of strain tification System (MIDI, version 6.0) with cells of strain B717-2Tresembled those of members of the genus

Table 1. Phenotypic characteristics that differentiate strain B717-2T from closely related members of the genus Polaromonas

Strains: 1, B717-2T; 2, P. vacuolata 34-PT (Irgens et al., 1996); 3, P. aquatica CCUG 39402T (K€ampfer et al., 2006); 4, P. hydrogenivorans DSM 17735T (Sizova & Panikov, 2007); 5, P. jejuensis JS12-13T (Weon et al., 2008); 6, P. naphthalenivorans CJ2T (Jeon et al., 2004); 7, P. glacialis Cr4- 12T (Margesin et al., 2012); 8, P. cryoconiti Cr4-35T (Margesin et al., 2012). All eight strains were positive for catalase, cytochrome c oxidase and leucine arylamidase activities and the assimilation of Tweens 40 and 80. All were negative for lysine decarboxylase, ornithine decarboxylase, trypto- phan decarboxylase, H2S production, indole production, gelatin hydrolysis, inositol, D-sorbitol, L-arabinose, b-galactosidase, a-glucosidase and N-acetyl-b-glucosaminidase. +, Positive; À, negative; W, weakly positive; ND, not determined. Data are from this study except where indicated.

Characteristic 1 2 3 4 5 6 7 8

Morphology Rods Rodsa Rodsb Coccic Rodsd Coccie Rodsf Rodsf Temperature range ( C) 5–20 0–12a 25–30b 0–25c 5–30d 4–25e 1–25f 1–25f Isolation source Ice core Soila Tap waterb Soilc Soild Sedimente Glacier cryoconitef Glacier cryoconitef a c d e f f pH range 7.0–9.0 6.0–9.5 ND 6.0–7.0 5.0–9.0 6.0–9.0 6.0–7.0 7.0 Nitrate reduction + ÀÀ + À + + + Arginine dihydrolase À ND w À w ÀÀÀ Citrate utilization À + ÀÀ + ÀÀÀ Urease production À + + À w ÀÀÀ Acid production from: D-Glucose À + À + À + ÀÀ D-Mannitol w ÀÀ + ÀÀÀÀ L-Rhamnose w ÀÀ + À + À w Sucrose ÀÀÀ + À + ÀÀ Melibiose À + À + ÀÀÀ + Amygdalin ÀÀÀ w À w w À Enzyme activities: Alkaline phosphatase + + + + + À w + Lipase À + + + + + w + DNA G+C content (mol%) 63.4 52.0a ND 62.5c 63.7d 61.5e 61.3f 60.7f

Data taken from: a, Irgens et al. (1996); b,K€ampfer et al. (2006); c, Sizova & Panikov (2007); d, Weon et al. (2008); e, Jeon et al. (2004); f, Margesin et al. (2012). http://ijs.microbiologyresearch.org 2499

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Polaromonas (Irgens et al., 1996; Jeon et al., 2004; K€ampfer et al., 2006; Sizova & Panikov, 2007; Weon et al., 2008; Margesin et al., 2012). As shown in Table 2, the predomi- nant cellular fatty acids of strain B717-2T were summed feature 3 (C16 : 1!7c and/or iso-C15 : 0 2-OH; 52.7 %), summed feature 8 (C18 : 1!7c,C18 : 1!6c; 23.1 %) and C16 : 0 (21.9 %). There were no significant differences in the fatty acid com- ponents between strain B717-2T and the reference strains, but some quantitative differences were observed (Table 2). Strain B717-2T had a relatively higher proportion of summed feature 8 compared with recognized species of the genus Polaromonas. For respiratory quinone analyses, cells were harvested after 10 days of growth at 15 C. Respiratory quinones were extracted and purified according to the method of Collins (1985). They were then analysed by HPLC according to Wu et al. (1989). The only respiratory quinone of strain B717- 2T was Q-8. The reference strains of the genus Polaromonas showed a quinone system with ubiquinone Q-8 predomi- nant (Jeon et al., 2004; K€ampfer et al., 2006; Sizova & Pani- kov, 2007; Weon et al., 2008; Margesin et al., 2012). Polar Fig. 3. Polar lipid profiles of strain B717-2T after two-dimensional lipids were extracted and examined by two-dimensional TLC and staining with molybdatophosphoric acid. PE, phosphati- TLC (Altenburger et al., 1996; Tindall, 1990), using molyb- dylethanolamine; PG, phosphatidylglycerol; DPG, diphosphatidyl- datophosphoric acid, molybdenum blue, ninhydrin and a- glycerol; PL, unknown phospholipid; L1, L2, unknown polar lipids. naphthol to detect total lipids, phospholipids, aminolipids and glycolipids, respectively. The major polar lipids of strain T B717-2 were similar to those of species of the genus Polaro- T monas (Fig. 3) (Jeon et al., 2004; K€ampfer et al., 2006; Sizova 2 represents a novel species of the genus Polaromonas, for & Panikov, 2007; Weon et al., 2008; Margesin et al., 2012). which the name Polaromonas eurypsychrophila sp. nov. is The profile of polar lipids mainly consisted of phosphatidyl- proposed. ethanolamine, phosphatidylglycerol and diphosphatidylglycerol. One unknown phospholipid and two unknown polar lipids were also found in strain B717-2T (Fig. 3). The major quinone and polar lipids of strain B717-2T were consistent Description of Polaromonas with the genus Polaromonas. eurypsychrophila sp. nov. Based on the phenotypic and genotypic data presented in Polaromonas eurypsychrophila [eu.ry.psy.chro’phi.la. Gr. adj. this study, the eurypsychrotolerant bacterium strain B717- eurys wide, broad; Gr. adj. psychros cold; N.L. adj. philus -a -

Table 2. Major cellular fatty acid compositions (%) of strain B717-2T and related members of the genus Polaromonas

Strains: 1, B717-2T; 2, P. aquatica CCUG 39402T; 3, P. naphthalenivorans CJ2T; 4, P. glacialis Cr4-12T; 5, P. vacuolata 34-PT; 6, P. hydrogenivorans DSM 17735T; 7, P. jejuensis JS12-13T; 8, P. cryoconiti Cr4-35. À, <1 % or not detected. Data are from this study.

Fatty acid 1 2 3 4 5 6 7 8

C10 : 0 3-OH ÀÀ 2.5 1.6 À 2.3 ÀÀ

C12 : 0 ÀÀÀÀÀÀ 2.5 À

C14 : 0 ÀÀÀÀ 1.0 ÀÀÀ

C16 : 0 21.9 34.2 21.8 16.4 24.5 20.1 32.0 28.0

C16 : 15c ÀÀ 1.0 ÀÀ 1.5 ÀÀ

C17 : 0 cyclo À 25.8 1.8 ÀÀ 1.5 25.5 À Summed feature 3* 52.7 25.2 68.1 67.3 62.9 64.3 26.9 60.5 Summed feature 7* ÀÀÀÀ 2.1 ÀÀÀ Summed feature 8* 23.1 12.8 3.4 12.7 8.2 10.1 10.9 10.3

*Summed feature 3 included C16 : 1!7c and/or iso-C15 : 0 2-OH. Summed feature 7 included C19 : 1!6c,C17 : 0 cyclo and/or an unknown fatty acid with an equivalent chain length of 18.846. Summed feature 8 included C18 : 1!7c,C18 : 1!6c.

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Downloaded from www.microbiologyresearch.org by IP: 124.16.145.97 On: Sat, 08 Oct 2016 08:31:15 Polaromonas eurypsychrophila sp. nov. um (from Gr. adj. philos -^e -on) friend, loving; N.L. fem. adj. Huss, V. A., Festl, H. & Schleifer, K. H. (1983). Studies on the spectro- eurypsychrophila loving a broad range of low temperatures]. photometric determination of DNA hybridization from renaturation rates. Syst Appl Microbiol 4, 184–192. Cells are aerobic, Gram-stain-negative and rod-shaped Irgens, R. L., Gosink, J. J. & Staley, J. T. (1996). Polaromonas vacuolata (1.8–2.0 µm long and 0.71–0.77 µm wide). Colonies on R2A gen. nov., sp. nov., a psychrophilic, marine, gas vacuolate bacterium agar after incubation at 15 C for 10 days are beige, round, from Antarctica. Int J Syst Bacteriol 46, 822–826. smooth, convex and opaque. Growth occurs at 5–20 C Jahnke, K. D. (1992). Basic computer program for evaluation of spec- (optimally at 15 C) on R2A agar, at an initial pH of 7–9 troscopic DNA renaturation data from Gilford System 2600 spectro- (optimally at pH 7) and with 0–2 % (w/v) NaCl (optimally photometer on a PC/XT/AT type personal 219 computer. J Microbiol with 2 % NaCl). The fatty acid profile is mostly composed Methods 15, 61–73. of summed feature 3 (C16 : 1!7c and/or iso-C15 : 0 2-OH), Jeon, C. O., Park, W., Ghiorse, W. C. & Madsen, E. L. (2004). Polaromo- summed feature 8 (C18 : 1!7c,C18 : 1!6c) and C16 : 0. The nas naphthalenivorans sp. nov., a naphthalene-degrading bacterium major polar lipids are phosphatidylethanolamine, phospha- from naphthalene-contaminated sediment. Int J Syst Evol Microbiol – tidylglycerol and diphosphatidylglycerol. The sole respira- 54, 93 97. tory quinone is Q-8. In API ZYM tests, cells are positive for Kampfer,€ P., Busse, H. & Falsen, E. (2006). Polaromonas aquatica sp. – alkaline phosphatase, esterase (C4), leucine arylamidase, nov., isolated from tap water. Int J Syst Evol Microbiol 56, 605 608. acid phosphatase and N-acetyl-b-glucosaminidase. Negative Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., for esterase lipase (C8), lipase (C14), valine arylamidase, Park, S. C., Jeon, Y. S., Lee, J. H. & other authors (2012). Introduc- trysin, cystine arylamidase, a-galactosidase, b-galactosidase, ing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol b-glucosidase, a-glucosidase, N-acetyl-b-glucosaminidase, 62, 716–721. a-mannosidase and a-fucosidase. In API 20NE and API Kim, M., Oh, H. S., Park, S. C. & Chun, J. (2014). Towards a taxonomic 20E tests, does not degrade gelatin, aesculin or starch. coherence between average nucleotide identity and 16S rRNA gene Assimilates glucose, N-acetylglucosamine, capric acid and sequence similarity for species demarcation of prokaryotes. Int J Syst trisodium citrate, but not L-arabinose, D-mannose, maltose, Evol Microbiol 64, 346–351. malic acid, adipic acid or phenylacetic acid. Mandel, M., Igambi, L., Bergendahl, J., Dodson, M. L. & Scheltgen, E. The type strain, B717-2T (=CGMCC 1.15322T =JCM (1970). Correlation of melting temperature and cesium chloride 31171T), was isolated from an ice core in Muztagh Glacier, buoyant density of bacterial deoxyribonucleic acid. J Bacteriol 101, 333–338. Tibet, China. The DNA G+C content of the type strain is Margesin, R., Spröer, C., Zhang, D. C. & Busse, H. J. (2012). 63.4 mol % (T ). Polaromo- m nas glacialis sp. nov. and Polaromonas cryoconiti sp. nov., isolated from alpine glacier cryoconite. Int J Syst Evol Microbiol 62, 2662–2668. Acknowledgements Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J Mol Biol 3, 208–218. This study was financially supported by the National Natural Science Reasoner, D. & Geldreich, E. (1985). A new medium for the enumera- Foundation of China (Grant Nos. 41425004, 41190084, 41571076, tion and subculture of bacteria from potable water. Appl Environ 41125003), Science and technology basic work of science and techo- Microbiol 49, 1–7. nology (2015FY110100), and Strategic Priority Research Program (B) of the Chinese Academy of Sciences (Grant No. XDB05030100). We Sizova, M. & Panikov, N. (2007). Polaromonas hydrogenivorans sp. are grateful to Dr Bernhard Schink for his help with the species nov., a psychrotolerant hydrogen-oxidizing bacterium from Alaskan name. soil. Int J Syst Evol Microbiol 57, 616–619. Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. 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