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Description of a Gram-negative bacterium, Sphingomonas guangdongensis sp nov.

ARTICLE in INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY · FEBRUARY 2014 Impact Factor: 2.8 · DOI: 10.1099/ijs.0.056853-0 · Source: PubMed

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Available from: Zhao Guozhen Retrieved on: 09 September 2015 International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1697–1702 DOI 10.1099/ijs.0.056853-0

Description of a Gram-negative bacterium, Sphingomonas guangdongensis sp. nov.

Guang-Da Feng, Song-Zhen Yang, Yong-Hong Wang, Xiu-Xiu Zhang, Guo-Zhen Zhao, Ming-Rong Deng and Hong-Hui Zhu

Correspondence State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Hong-Hui Zhu Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied [email protected] Microbiology, Guangdong Institute of Microbiology, Guangzhou 510070, PR China

A Gram-stain-negative bacterial strain, designated 9NM-8T, was isolated from an abandoned lead-zinc ore in Mei county, Meizhou, Guangdong province, PR China. The isolate was orange- pigmented, aerobic, oxidase- and catalase-positive, motile with lophotrichous flagella and rod- shaped. Strain 9NM-8T grew optimally at pH 7.0 and 30 6C and in the absence of NaCl on R2A agar. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 9NM-8T belongs to the genus Sphingomonas, with highest sequence similarities to Sphingomonas azotifigens KACC 14484T (96.1 %), Sphingomonas trueperi DSM 7225T (96.0 %) and Sphingomonas pituitosa DSM 13101T (95.6 %). Strain 9NM-8T contained Q-10 as the

predominant ubiquinone. The major fatty acids included C18 : 1v7c,C16 : 0,C16 : 1v7c and/or

C16 : 1v6c (summed feature 3) and 11-methyl C18 : 1v7c. The DNA G+C content was 69.6±1.3 mol%. The major component in the polyamine pattern was sym-homospermidine and the polar lipid profile contained sphingoglycolipid, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, an unidentified glycolipid and two unidentified phospholipids. Based on comparative analysis of physiological, chemotaxonomic and phylogen- etic characteristics, strain 9NM-8T should be considered to represent a novel species of the genus Sphingomonas, for which the name Sphingomonas guangdongensis sp. nov. is proposed. The type strain is 9NM-8T (5GIMCC 1.653T5CGMCC 1.12672T5DSM 27570T).

The genus Sphingomonas was first proposed by Yabuuchi as the key polyamine. In this paper, we report the results from et al. (1990) and emended subsequently by Takeuchi et al. a taxonomic study using a polyphasic approach on a novel (2001), Yabuuchi et al. (2002), Busse et al. (2003) and bacterium isolated from an abandoned lead-zinc ore, Chen et al. (2012). Based on analysis of phylogeny and designated strain 9NM-8T. Based on the physiological, polyamine patterns profiles, the genus Sphingomonas should chemotaxonomic and phylogenetic characteristics, strain be classified into five genera Sphingomonas, Novosphingo- 9NM-8T represents a novel species of the genus Sphingomonas. bium, , Sphingorhabdus and were isolated from a lead-zinc ore collected from a (Takeuchi et al., 2001; Jogler et al., 2013). At the time of lead-zinc mine, which had been abandoned for 30 years, writing, approximately 68 members of the genus Sphingo- located in Mei county (24u 219 4499 N 116u 169 3499 E), monas have been described, including four species with [ Meizhou, Guangdong province, PR China. Isolation was names that have not yet been validated ‘Sphingomonas humi’ performed using the standard dilution plating technique at (Yi et al., 2010), ‘Sphingomonas hunanensis’ (Chen et al., 30 uC on R2A agar (Luqiao, China). The plates were 2011), ‘Sphingomonas rosea’and‘Sphingomonas swuensis’ T ] incubated for 1 week. Strain 9NM-8 was isolated on the (Srinivasan et al., 2011) . They were isolated from various basis of colony morphology and purified by subculturing natural sources, i.e. rhizosphere, marine, desert sand, water on R2A agar. The culture was preserved at 280 uC in R2A and dump site. Members of the genus Sphingomonas are broth supplemented with 20 % (v/v) glycerol. characterized by being yellow- or orange-pigmented, aerobic, T Gram-staining-negative and non-spore-forming, and contain- The genomic DNA of strain 9NM-8 was extracted using a ing Q-10 as the respiratory quinone and sym-homospermidine commercial genomic DNA isolation kit (Sangon, China). The 16S rRNA gene of strain 9NM-8T was amplified using The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene the universal bacterial primer pair 27F (59-AGAGTTTG- sequence of strain 9NM-8T is JQ608326. ATCCTGGCTCAG-39) and 1492R (59-GGTTACCTTGT- Five supplementary figures are available with the online version of this TACGACTT-39) (Weisburg et al., 1991) and the purified paper. PCR product was cloned into the pCR 2.1 vector and

056853 G 2014 IUMS Printed in Great Britain 1697 G.-D. Feng and others sequenced by Invitrogen. The 16S rRNA gene sequences of extracted according to the protocol of the Sherlock related taxa were obtained from the GenBank database and Microbial Identification System (MIDI) and analysed by the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim gas chromatography (model 7890A; Hewlett Packard) et al. 2012). Analysis of 16S rRNA gene sequences was using the Microbial Identification software package with performed by using MEGA 5.0 software (Tamura et al., 2011). the Sherlock MIDI 6.1 system and the Sherlock Aerobic Evolutionary distances were calculated by using Kimura’s Bacterial Database (TSBA 6.1) (Sasser, 1990). The poly- two-parameter model (Kimura, 1980). Phylogenetic trees amines of strain 9NM-8T were determined as described by were reconstructed by the neighbour-joining (Saitou & Nei, Hamana et al. (2013). Isoprenoid quinones were extracted 1987), maximum-parsimony (Fitch, 1971) and maximum- from lyophilized cells and subsequently analysed by HPLC likelihood (Felsenstein, 1981) methods with bootstrap (UltiMate 3000; Dionex) according to the methods as values based on 1000 replications (Felsenstein, 1985). described by Collins et al. (1977) and Hiraishi et al. (1996). Moreover, to verify strain 9NM-8T, PCRs were also Polar lipids were extracted and determined as described by performed using two Sphingomonas-specific primer pairs Tindall et al. (2007). (Sph-spt 295f/Sph-spt 713r and Sph-spt 694f/Sph-spt 983r) The PCR results using the Sphingomonas-specific primer as described by Yim et al. (2010). pairs showed that strain 9NM-8T belonged to the genus Biochemical and physiological tests were performed on Sphingomonas and the primer set Sph-spt 694f/Sph-spt R2A at 30 uC. The Gram reaction was tested by using a 983r is more specific than Sph-spt 259r/Sph-spt 713r (Fig. modification of the method described by Buck (1982). S1, available in the online Supplementary Material). The Catalase, cytochrome oxidase, methyl red and Voges– 16S rRNA gene sequence of strain 9NM-8T determined in Proskauer tests and hydrolysis of starch, and Tweens 40 this study was a continuous stretch of 1437 bp. The 16S and 80 were determined according to the methods rRNA gene sequence similarities, which were calculated by described by Smibert & Krieg (1994). Motility was assessed the EzTaxon-e server, revealed that the closest relatives of under a Leica DMLB phase-contrast light microscope on strain 9NM-8T were Sphingomonas azotifigens KACC 14484T cells grown for 3 days at 30 uC. Strain 9NM-8T was grown (96.1 %), Sphingomonas trueperi DSM 7225T (96.0 %), on R2A agar at 30 uC for 3 days and the bacterial cells were Sphingomonas phyllosphaerae FA2T (95.6 %), Sphingomonas suspended in sterile distilled water and stained with endophytica YIM 65583T (95.7 %), Sphingomonas ginseno- phosphotungstic acid (3 %, pH 7.0) for 2 min, air-dried sidimutans Gsoil 1429T (95.6 %) and Sphingomonas pituitosa and observed with Hitachi H7650 electron microscope. DSM 13101T (95.6 %). However, the neighbour-joining Anaerobic growth was determined in an anaerobic pouch phylogenetic tree (Figs 1 and S2) showed that strain 9NM-8T (MGC) for 7 days at 30 uC on R2A agar. Ornithine only clustered with S. azotifigens KACC 14484T, S. trueperi decarboxylase, lysine decarboxylase, and acid production DSM 7225T and S. pituitosa DSM 13101T and the topologies from sugars were tested by using biochemical kits of the maximum-likelihood and maximum-parsimony trees (Huankai, China). Arginine dihydrolase, b-galactosidase were essentially the same (Figs S3 and S4). A 16S rRNA gene (ONPG), urease, nitrate reduction, citrate utilization, sequence similarity of 97.0 % was proposed by Stackebrandt indole production and hydrolysis of gelatin and aesculin & Goebel (1994) and subsequently re-evaluated to 98.7 % by were tested using API 20NE strips (bioMe´rieux). Growth at Stackebrandt & Ebers (2006) as a criterion for species different pH (pH 4–10 in intervals of 1 pH unit) and discrimination. Taking this into consideration, we con- temperatures (4, 10, 15, 20, 25, 30, 32, 35, 37 and 45 uC) cluded that the 16S rRNA gene sequence similarities between was determined in buffered R2A broth. Tolerance to NaCl strain 9NM-8T and the type strains of the closest related was tested in R2A broth with different NaCl concentrations species of the genus Sphingomonas are low enough to (0, 0.5, 1, 1.5, 2, 2.5, 3, 5, 6, 8 and 10 %, w/v). Substrate exclude the assignment of the novel strain to any of the utilization and chemical sensitive assays were evaluated by recognized species of the genus Sphingomonas. API 20NE strips and GN3 MicroPlates (Biolog) at 30 uC Cells of strain 9NM-8T were Gram-stain-negative, non- according to the manufacturer’s instructions. Growth of strain spore-forming, oxidase- and catalase-positive, aerobic, 9NM-8T was tested on different media: R2A agar (Luqiao), motile with lophotrichous flagella and rod-shaped (0.6– tryptic soy agar (TSA; Guangdong Huankai Microbial Science 0.8 mm wide, 1.2–1.4 mm long) (Fig. 2). Colonies grown on &Technology,China),PYEagar(Busseet al., 2005) and R2A agar were circular, convex, orange, semi-transparent nutrient agar (NA; Huankai) at 30 uC. and 1–2 mm in diameter after 3 days at 30 uC. Strain To measure the DNA G+C content of strain 9NM-8T, 9NM-8T was able to grow at 20–32 uC and pH 6–8. genomic DNA was extracted and purified using the Growth occurred in the presence of 0–0.5 % (w/v) NaCl. method as described by Moore & Dowhan (1995) and The maximum growth of strain 9NM-8T was at pH 7.0 and the G+C content determined by HPLC as described by 30 uC without NaCl on R2A agar. Cells were able to Mesbah et al. (1989). For fatty acid profile analysis, strain hydrolyse aesculin and Tweens 40 and 80, but not starch, 9NM-8T, Sphingomonas azotifigens KACC 14484T, Sphingo- gelatin or arginine. Methyl red and Voges–Proskauer tests, monas trueperi DSM 7225T and Sphingomonas pituitosa urease activity, indole production and citrate utilization DSM 13101T were grown on R2A agar for 96 h at 30 uC. were negative, while the nitrate reduction test was positive. Cellular fatty acids were saponified, methylated and Strain 9NM-8T can grow on TSA and PYE agar, but not on

1698 International Journal of Systematic and Evolutionary Microbiology 64 Sphingomonas guangdongensis sp. nov.

59 Sphingomonas yabuuchiae GTC 868T (AB071955) 0.005 Sphingomonas roseiflava MK341T (D84520) Sphingomonas parapaucimobilis NBRC 15100T (D13724) T 99 Sphingomonas sanguinis IFO 13937 (D13726) Sphingomonas pseudosanguinis G1-2T (AM412238) Sphingomonas paucimobilis ATCC 29837T (U37337) Sphingomonas desiccabilis CP1DT (AJ871435) 96 Sphingomonas molluscorum An 18T (AB248285) Sphingomonas polyaromaticivorans B2-7T (EF467848) Sphingomonas ginsenosidimutans Gsoil 1429T (HM204925) 90 Sphingomonas yunnanensis YIM 003T (AY894691) 90 Sphingomonas phyllosphaerae FA2T (AY453855) 99 Sphingomonas endophytica YIM 65583T (HM629444) Sphingomonas japonica DSM 22753T (AB428568) Sphingomonas guangdongensis 9NM-8T (JQ608326) T 86 Sphingomonas pituitosa DSM 13101 (AJ243751) 99 Sphingomonas azotifigens KACC 14484T (AB217471) 97 Sphingomonas trueperi DSM 7225T (X97776)

Fig. 1. Sub-tree captured from the neighbour-joining phylogenetic tree (Fig. S2) based on 16S rRNA gene sequence of strain 9NM-8T. Filled circles denote branches that are also present in the maximum-likelihood and maximum-parsimony trees (Figs S3 and S4). Numbers at nodes are bootstrap values expressed as a percentage of 1000 replications; only values .50 % are shown. Bar, 0.005 substitutions per nucleotide position.

NA. Details of physiological characteristics that differenti- sphingoglycolipid, phosphatidylcholine, phosphatidylgly- ate strain 9NM-8T from the reference type strains are listed cerol, diphosphatidylglycerol, phosphatidylethanolamine, in Table 1 and in the species description. an unidentified glycolipid and two unidentified phospho- lipids (Fig. S5). The polyamine patterns of strain 9NM-8T The major respiratory quinone of strain 9NM-8T was showed a predominance of sym-homospermidine. These ubiquinone 10 (Q-10) and the polar lipids detected were traits of strain 9NM-8T are all in accordance with the characteristics of Sphingomonas sensu stricto (Takeuchi et al., 2001). The DNA G+C content of strain 9NM-8T was 69.6±1.3 mol%, a value significantly higher than those of closely related species of the genus Sphingomonas (Table 1), T and the major fatty acids of strain 9NM-8 were C18 : 1v7c, C16 : 0,C16 : 1v7c and/or C16 : 1v6c (summed feature 3) and 11-methyl C18 : 1v7c, quite different from S. azotifigens T KACC 14484 (C18 : 1v7c,C16 : 0 and 11-methyl C18 : 1v7c), T S. trueperi DSM 7225 (C18 : 1v7c and C16 : 0) and S. T pituitosa DSM 13101 (C18 : 1v7c,C16 : 0 and 11-methyl C18 : 1v7c) (Table 2). Based on physiological, chemotaxonomic and phylogenetic analyses, it is clear that strain 9NM-8T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas guangdongensis sp. nov. is proposed.

Description of Sphingomonas guangdongensis sp. nov. Sphingomonas guangdongensis (guang.dong.en9sis. N.L. fem. adj. guangdongensis pertaining to Guangdong, a province of south China). Cells are Gram-stain-negative, non-spore-forming, aerobic, Fig. 2. Transmission electron micrograph of a cell of strain 9NM- motile and rod-shaped (0.6–0.8 mm wide, 1.2–1.4 mm 8T. Bar, 500 nm. long) with lophotrichous flagella. Forms orange-pigmented http://ijs.sgmjournals.org 1699 G.-D. Feng and others

Table 1. Physiological characteristics of strain 9NM-8T and type strains of closely related species of the genus Sphingomonas

Strains: 1, 9NM-8T;2,S. azotifigens KACC 14484T;3,S. trueperi DSM 7225T;4,S. pituitosa DSM 13101T. All data were from this study unless mentioned otherwise. All strains were Gram-stain-negative, rod-shaped and positive for catalase activity, hydrolysis of aesculin and Tween 80 and assimilation of lactose, D-glucose, L-arabinose and sucrose. All strains were negative for urease and arginine dihydrolase activities, indole production, assimilation of citrate, D-mannitol, capric acid, adipic acid and phenylacetic acid, and acid production from sorbose, raffinose, rhamnose, lactose, D-xylose, L-arabinose, D-glucose, sorbitol, mannitol and D-ribose. +, Positive; 2, negative.

Characteristic 1 2 3 4

Flagellum Lophotrichous Peritrichous Peritrichous Monotrichous Oxidase +++2 b-Galactosidase +++2 Nitrate reduction ++2 + Hydrolysis of: Starch 2 ++ 2 Gelatin 2 ++ 2 Acid production from; Sucrose + 22 2 D-Fructose 22+ 2 Maltose 2 + 22 Galactose 222+ Assimilation of: D-Mannose 2 ++ + Potassium gluconate + 22 2 Malic acid 2 ++ + N-Acetylglucosamine 2 ++ + Maltose 2 ++ + Cellobiose +++2 D-Fructose +++2 L-Alanine + 2 ++ D-Galactose 2 ++ + Trehalose 2 ++ + Melibiose 2 ++ + DNA G+C content (mol%)* 69.6 66.0–68.0a 65.6b 64.5c

*Data from: a, Xie & Yokota (2006); b,Ka¨mpfer et al. (1997); c, Denner et al. (2001).

colonies with a diameter of about 1–2 mm after incubation following compounds are not utilized: citrate, D-mannose, at 30 uC for 72 h on R2A agar. Colonies on R2A agar are maltose, trehalose, turanose, stachyose, raffinose, melibiose, circular, slightly convex and semi-transparent. Growth also methyl b-D-glucoside, N-acetyl-D-glucosamine, N-acetyl-b- occurred on TSA and PYE agar, but not on NA. Growth D-mannosamine, N-acetyl-D-galactosamine, N-acetyl neur- occurs at pH 6.0–8.0 (optimum, pH 7.0), in the presence aminic acid, D-galactose, 3-methyl glucose, L-fucose, of 0–0.5 % (w/v) NaCl (optimum, 0 %) and at 20–32 uC L-rhamnose, inosine, D-sorbitol, D-mannitol, D-arabitol, (optimum, 30 uC). Cells are able to hydrolyse aesculin and myo-inositol, glycerol, D-glucose-6-phosphate, D-fructose- Tweens 40 and 80, but not starch or gelatin. Catalase, 6-phosphate, D-aspartic acid, D-serine, gelatin, L-arginine, L- oxidase, arginine decarboxylase and b-galactosidase activ- histidine, L-pyroglutamic acid, L-serine, pectin, D-galacturo- ities and nitrate reduction are positive, while arginine nic acid, L-galactonic acid lactone, D-glucuronic acid, dihydrolase, ornithine decarboxylase, lysine decarboxylase glucuronamide, mucic acid, D-saccharic acid, phenylacetic and urease activities, indole production and methyl red acid, p-hydroxyphenylacetic acid, methyl pyruvate, D-lactic and Voges–Proskauer tests are negative. Acid is produced acid methyl ester, L-lactic acid, citric acid, a-ketoglutaric from sucrose, but not from sorbitol, L-arabinose, fructose, acid, D-malic acid, L-malic acid, capric acid, adipic acid, cellobiose, raffinose, D-xylose, mannitol, D-ribose, glucose, bromosuccinic acid, c-aminobutryric acid, a-hydroxybuty- maltose, sorbose, rhamnose, lactose or galactose. Assimilates ric acid, acetoacetic acid, propionic acid and formic acid. dextrin, cellobiose, gentiobiose, a-lactose, D-salicin, a-D- Susceptible to troleandomycin, vancomycin, minocycline glucose, sucrose, D-fructose, D-fucose, glycyl L-proline, and aztreonam; resistant to potassium tellurite, rifamycin L-alanine, L-arabinose, L-aspartic acid, L-glutamic acid, D- SV and lincomycin. The major respiratory lipoquinone is gluconic acid, quinic acid, Tween 40, b-hydroxybutyric acid, ubiquinone Q-10. The predominant polyamine is sym- a-ketobutyric acid, acetic acid and potassium gluconate. The homospermidine. The polar lipids contain sphingoglycolipid,

1700 International Journal of Systematic and Evolutionary Microbiology 64 Sphingomonas guangdongensis sp. nov.

Table 2. Cellular fatty acid profiles of strain 9NM-8T and its Busse, H. J., Hauser, E. & Ka¨ mpfer, P. (2005). Description of two close relatives novel species, Sphingomonas abaci sp. nov. and Sphingomonas panni sp. nov. Int J Syst Evol Microbiol 55, 2565–2569. T T Strains: 1, 9NM-8 ;2,S. azotifigens KACC 14484 ;3,S. trueperi DSM Chen, Q. H., Chen, J. H., Ruan, Y., Zhang, Y. Q., Tang, S. K., Liu, Z. X., T T 7225 ;4,S. pituitosa DSM 13101 Li, W. J. & Chen, Y. G. (2011). Sphingomonas hunanensis sp. nov., isolated from forest soil. Antonie van Leeuwenhoek 99, 753–760. Cells were all cultured on R2A agar for 3 days at 30 uC. 2, Not Chen, H., Jogler, M., Rohde, M., Klenk, H. P., Busse, H. J., Tindall, detected. B. J., Spro¨ er, C. & Overmann, J. (2012). Reclassification and emended description of Caulobacter leidyi as Sphingomonas leidyi comb. nov., Fatty acid 1 2 3 4 and emendation of the genus Sphingomonas. Int J Syst Evol Microbiol 62, 2835–2843. C14 : 0 0.4 0.3 2 0.4 C14 : 02OH 1.4 1.1 1.1 1.7 Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E. (1977). Summed feature 3* 6.4 0.9 1.2 2.2 Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100, 221–230. C16 : 1v5c 1.8 0.5 0.7 1.2 C16 : 0 14.2 20.1 10.4 13.2 Denner, E. B. M., Paukner, S., Ka¨ mpfer, P., Moore, E. R. B., Abraham, W. R., Busse, H. J., Wanner, G. & Lubitz, W. (2001). C17 : 1v8c 2.5 0.6 2 0.5 Sphingomonas pituitosa sp. nov., an exopolysaccharide-producing bacterium that C17 : 1v6c 1.3 0.7 0.9 1.0 secretes an unusual type of sphingan. Int J Syst Evol Microbiol 51, 827– C18 : 1v7c 66.8 58.6 84.4 70.9 841. C18 : 1v5c 0.6 2 0.9 2 Felsenstein, J. (1981). C18 : 0 0.3 0.3 0.4 2 Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376. 11-methyl C18 : 1v7c 4.5 15.3 2 7.8 C19 : 0 cyclo v8c 2 1.6 2 1.2 Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. *Summed features represent groups of two or three fatty acids that Fitch, W. M. (1971). Toward defining the course of evolution: could not be separated by the Microbial Identification System. minimum change for a specific tree topology. Syst Zool 20, 406–

Summed feature 3 contained C16 : 1v7c and/or C16 : 1v6c. 416. Hamana, K., Hayashi, H., Niitsu, M., Takeda, A. & Itoh, T. (2013). Cellular polyamines of alkaliphilic, halophilic and thermophilic methanogens and acidophilic and alkaliphilic extreme halophiles, phosphatidylcholine, phosphatidylglycerol, diphosphati- and acidophilic thermophiles belonging to the domain Archaea. J Jpn dylglycerol, phosphatidylethanolamine, an unidentified Soc Extremophiles 12, 15–28. glycolipid and two unidentified phospholipids. The major Hiraishi, A., Ueda, Y., Ishihara, J. & Mori, T. (1996). Comparative fatty acids comprise C18 : 1v7c,C16 : 0,C16 : 1v7c and/or lipoquinone analysis of influent sewage and activated sludge by high- C16 : 1v6c (summed feature 3) and 11-methyl C18 : 1v7c. performance liquid chromatography and photodiode array detection. The type strain, 9NM-8T (GIMCC 1.653T5CGMCC J Gen Appl Microbiol 42, 457–469. 1.12672T5DSM 27570T) was isolated from an abandoned Jogler, M., Chen, H., Simon, J., Rohde, M., Busse, H. J., Klenk, H. P., lead-zinc ore of a mining area in Mei county, Meizhou, Tindall, B. J. & Overmann, J. (2013). Description of Sphingorhabdus planktonica gen. nov., sp. nov. and reclassification of three related Guangdong province, PR China. The DNA G+C content ± members of the genus Sphingopyxis in the genus Sphingorhabdus gen. of the type strain is 69.6 1.3 mol%. nov. Int J Syst Evol Microbiol 63, 1342–1349. Ka¨mpfer, P., Denner, E. B. M., Meyer, S., Moore, E. R. B. & Busse, ACKNOWLEDGEMENTS H.-J. (1997). Classification of ‘‘Pseudomonas azotocolligans’’ Anderson 1955, 132, in the genus Sphingomonas as Sphingomonas trueperi sp. The authors are grateful to Dr Soon-Wo Kwon and the Korean nov. Int J Syst Bacteriol 47, 577–583. Agricultural Culture Collection (KACC) for kindly providing Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Sphingomonas azotifigens KACC 14484T. We thank Dr Koei Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e: Hamana for his help in polyamine analysis. This work was jointly a prokaryotic 16S rRNA gene sequence database with phylotypes that supported by the Natural Science Foundation of China, PR China represent uncultured species. Int J Syst Evol Microbiol 62, 716–721. (nos 31070103, 31200006) and the Key Project of Guangdong Natural Kimura, M. (1980). A simple method for estimating evolutionary rates Science Foundation (no. 10251007002000001). of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120. REFERENCES Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high- performance liquid chromatography. Int J Syst Bacteriol 39, 159–167. Buck, J. D. (1982). Nonstaining (KOH) method for determination of gram reactions of marine bacteria. Appl Environ Microbiol 44, 992–993. Moore, D. D. & Dowhan, D. (1995). Preparation and analysis of DNA. Busse, H. J., Denner, E. B. M., Buczolits, S., Salkinoja-Salonen, M., In Current Protocols in Molecular Biology, pp. 2–11. Edited by Bennasar, A. & Ka¨ mpfer, P. (2003). Sphingomonas aurantiaca sp. F. W. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, nov., Sphingomonas aerolata sp. nov. and sp. nov., J. A. Smith & K. Struhl. New York: Wiley. air- and dustborne and Antarctic, orange-pigmented, psychrotolerant Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new bacteria, and emended description of the genus Sphingomonas. Int J method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406– Syst Evol Microbiol 53, 1253–1260. 425. http://ijs.sgmjournals.org 1701 G.-D. Feng and others

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