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 Hong-Hui Zhu [email protected]
State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied 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 orangepigmented, aerobic, oxidase- and catalase-positive, motile with lophotrichous flagella and rodshaped. 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 phylogenetic 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 et al. (1990) and emended subsequently by Takeuchi et al. (2001), Yabuuchi et al. (2002), Busse et al. (2003) and Chen et al. (2012). Based on analysis of phylogeny and polyamine patterns profiles, the genus Sphingomonas should be classified into five genera Sphingomonas, Novosphingobium, Sphingobium, Sphingorhabdus and Sphingopyxis (Takeuchi et al., 2001; Jogler et al., 2013). At the time of writing, approximately 68 members of the genus Sphingomonas have been described, including four species with names that have not yet been validated [‘Sphingomonas humi’ (Yi et al., 2010), ‘Sphingomonas hunanensis’ (Chen et al., 2011), ‘Sphingomonas rosea’ and ‘Sphingomonas swuensis’ (Srinivasan et al., 2011)]. They were isolated from various natural sources, i.e. rhizosphere, marine, desert sand, water and dump site. Members of the genus Sphingomonas are characterized by being yellow- or orange-pigmented, aerobic, Gram-staining-negative and non-spore-forming, and containing Q-10 as the respiratory quinone and sym-homospermidine The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 9NM-8T is JQ608326. Five supplementary figures are available with the online version of this paper.
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as the key polyamine. In this paper, we report the results from a taxonomic study using a polyphasic approach on a novel bacterium isolated from an abandoned lead-zinc ore, designated strain 9NM-8T. Based on the physiological, chemotaxonomic and phylogenetic characteristics, strain 9NM-8T represents a novel species of the genus Sphingomonas. Bacteria were isolated from a lead-zinc ore collected from a lead-zinc mine, which had been abandoned for 30 years, located in Mei county (24u 219 4499 N 116u 169 3499 E), Meizhou, Guangdong province, PR China. Isolation was performed using the standard dilution plating technique at 30 uC on R2A agar (Luqiao, China). The plates were incubated for 1 week. Strain 9NM-8T was isolated on the basis of colony morphology and purified by subculturing on R2A agar. The culture was preserved at 280 uC in R2A broth supplemented with 20 % (v/v) glycerol. The genomic DNA of strain 9NM-8T was extracted using a commercial genomic DNA isolation kit (Sangon, China). The 16S rRNA gene of strain 9NM-8T was amplified using the universal bacterial primer pair 27F (59-AGAGTTTGATCCTGGCTCAG-39) and 1492R (59-GGTTACCTTGTTACGACTT-39) (Weisburg et al., 1991) and the purified PCR product was cloned into the pCR 2.1 vector and 1697
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sequenced by Invitrogen. The 16S rRNA gene sequences of related taxa were obtained from the GenBank database and the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al. 2012). Analysis of 16S rRNA gene sequences was performed by using MEGA 5.0 software (Tamura et al., 2011). Evolutionary distances were calculated by using Kimura’s two-parameter model (Kimura, 1980). Phylogenetic trees were reconstructed by the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Fitch, 1971) and maximumlikelihood (Felsenstein, 1981) methods with bootstrap values based on 1000 replications (Felsenstein, 1985). Moreover, to verify strain 9NM-8T, PCRs were also performed using two Sphingomonas-specific primer pairs (Sph-spt 295f/Sph-spt 713r and Sph-spt 694f/Sph-spt 983r) as described by Yim et al. (2010). Biochemical and physiological tests were performed on R2A at 30 uC. The Gram reaction was tested by using a modification of the method described by Buck (1982). Catalase, cytochrome oxidase, methyl red and Voges– Proskauer tests and hydrolysis of starch, and Tweens 40 and 80 were determined according to the methods described by Smibert & Krieg (1994). Motility was assessed under a Leica DMLB phase-contrast light microscope on cells grown for 3 days at 30 uC. Strain 9NM-8T was grown on R2A agar at 30 uC for 3 days and the bacterial cells were suspended in sterile distilled water and stained with phosphotungstic acid (3 %, pH 7.0) for 2 min, air-dried and observed with Hitachi H7650 electron microscope. Anaerobic growth was determined in an anaerobic pouch (MGC) for 7 days at 30 uC on R2A agar. Ornithine decarboxylase, lysine decarboxylase, and acid production from sugars were tested by using biochemical kits (Huankai, China). Arginine dihydrolase, b-galactosidase (ONPG), urease, nitrate reduction, citrate utilization, indole production and hydrolysis of gelatin and aesculin were tested using API 20NE strips (bioMe´rieux). Growth at different pH (pH 4–10 in intervals of 1 pH unit) and temperatures (4, 10, 15, 20, 25, 30, 32, 35, 37 and 45 uC) was determined in buffered R2A broth. Tolerance to NaCl was tested in R2A broth with different NaCl concentrations (0, 0.5, 1, 1.5, 2, 2.5, 3, 5, 6, 8 and 10 %, w/v). Substrate utilization and chemical sensitive assays were evaluated by API 20NE strips and GN3 MicroPlates (Biolog) at 30 uC according to the manufacturer’s instructions. Growth of strain 9NM-8T was tested on different media: R2A agar (Luqiao), tryptic soy agar (TSA; Guangdong Huankai Microbial Science & Technology, China), PYE agar (Busse et al., 2005) and nutrient agar (NA; Huankai) at 30 uC.
extracted according to the protocol of the Sherlock Microbial Identification System (MIDI) and analysed by gas chromatography (model 7890A; Hewlett Packard) using the Microbial Identification software package with the Sherlock MIDI 6.1 system and the Sherlock Aerobic Bacterial Database (TSBA 6.1) (Sasser, 1990). The polyamines of strain 9NM-8T were determined as described by Hamana et al. (2013). Isoprenoid quinones were extracted from lyophilized cells and subsequently analysed by HPLC (UltiMate 3000; Dionex) according to the methods as described by Collins et al. (1977) and Hiraishi et al. (1996). Polar lipids were extracted and determined as described by Tindall et al. (2007). The PCR results using the Sphingomonas-specific primer pairs showed that strain 9NM-8T belonged to the genus Sphingomonas and the primer set Sph-spt 694f/Sph-spt 983r is more specific than Sph-spt 259r/Sph-spt 713r (Fig. S1, available in the online Supplementary Material). The 16S rRNA gene sequence of strain 9NM-8T determined in this study was a continuous stretch of 1437 bp. The 16S rRNA gene sequence similarities, which were calculated by the EzTaxon-e server, revealed that the closest relatives of strain 9NM-8T were Sphingomonas azotifigens KACC 14484T (96.1 %), Sphingomonas trueperi DSM 7225T (96.0 %), Sphingomonas phyllosphaerae FA2T (95.6 %), Sphingomonas endophytica YIM 65583T (95.7 %), Sphingomonas ginsenosidimutans Gsoil 1429T (95.6 %) and Sphingomonas pituitosa DSM 13101T (95.6 %). However, the neighbour-joining phylogenetic tree (Figs 1 and S2) showed that strain 9NM-8T only clustered with S. azotifigens KACC 14484T, S. trueperi DSM 7225T and S. pituitosa DSM 13101T and the topologies of the maximum-likelihood and maximum-parsimony trees were essentially the same (Figs S3 and S4). A 16S rRNA gene sequence similarity of 97.0 % was proposed by Stackebrandt & Goebel (1994) and subsequently re-evaluated to 98.7 % by Stackebrandt & Ebers (2006) as a criterion for species discrimination. Taking this into consideration, we concluded that the 16S rRNA gene sequence similarities between strain 9NM-8T and the type strains of the closest related species of the genus Sphingomonas are low enough to exclude the assignment of the novel strain to any of the recognized species of the genus Sphingomonas.
To measure the DNA G+C content of strain 9NM-8T, genomic DNA was extracted and purified using the method as described by Moore & Dowhan (1995) and the G+C content determined by HPLC as described by Mesbah et al. (1989). For fatty acid profile analysis, strain 9NM-8T, Sphingomonas azotifigens KACC 14484T, Sphingomonas trueperi DSM 7225T and Sphingomonas pituitosa DSM 13101T were grown on R2A agar for 96 h at 30 uC. Cellular fatty acids were saponified, methylated and
Cells of strain 9NM-8T were Gram-stain-negative, nonspore-forming, oxidase- and catalase-positive, aerobic, motile with lophotrichous flagella and rod-shaped (0.6– 0.8 mm wide, 1.2–1.4 mm long) (Fig. 2). Colonies grown on R2A agar were circular, convex, orange, semi-transparent and 1–2 mm in diameter after 3 days at 30 uC. Strain 9NM-8T was able to grow at 20–32 uC and pH 6–8. Growth occurred in the presence of 0–0.5 % (w/v) NaCl. The maximum growth of strain 9NM-8T was at pH 7.0 and 30 uC without NaCl on R2A agar. Cells were able to hydrolyse aesculin and Tweens 40 and 80, but not starch, gelatin or arginine. Methyl red and Voges–Proskauer tests, urease activity, indole production and citrate utilization were negative, while the nitrate reduction test was positive. Strain 9NM-8T can grow on TSA and PYE agar, but not on
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Sphingomonas guangdongensis sp. nov.
Sphingomonas yabuuchiae GTC 868T (AB071955) Sphingomonas roseiflava MK341T (D84520) Sphingomonas parapaucimobilis NBRC 15100T (D13724) Sphingomonas sanguinis IFO 13937T (D13726) 99 Sphingomonas pseudosanguinis G1-2T (AM412238) Sphingomonas paucimobilis ATCC 29837T (U37337) Sphingomonas desiccabilis CP1DT (AJ871435) Sphingomonas molluscorum An 18T (AB248285) Sphingomonas polyaromaticivorans B2-7T (EF467848) Sphingomonas ginsenosidimutans Gsoil 1429T (HM204925) Sphingomonas yunnanensis YIM 003T (AY894691)
0.005
96
90 90
86
59
Sphingomonas phyllosphaerae FA2T (AY453855) 99 Sphingomonas endophytica YIM 65583T (HM629444) Sphingomonas japonica DSM 22753T (AB428568) Sphingomonas guangdongensis 9NM-8T (JQ608326) Sphingomonas pituitosa DSM 13101T (AJ243751) 99
97
Sphingomonas azotifigens KACC 14484T (AB217471) 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 differentiate strain 9NM-8T from the reference type strains are listed in Table 1 and in the species description. The major respiratory quinone of strain 9NM-8T was ubiquinone 10 (Q-10) and the polar lipids detected were
sphingoglycolipid, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, an unidentified glycolipid and two unidentified phospholipids (Fig. S5). The polyamine patterns of strain 9NM-8T showed a predominance of sym-homospermidine. These 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), and the major fatty acids of strain 9NM-8T were C18 : 1v7c, C16 : 0, C16 : 1v7c and/or C16 : 1v6c (summed feature 3) and 11-methyl C18 : 1v7c, quite different from S. azotifigens KACC 14484T (C18 : 1v7c, C16 : 0 and 11-methyl C18 : 1v7c), S. trueperi DSM 7225T (C18 : 1v7c and C16 : 0) and S. pituitosa DSM 13101T (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).
Fig. 2. Transmission electron micrograph of a cell of strain 9NM8T. Bar, 500 nm. http://ijs.sgmjournals.org
Cells are Gram-stain-negative, non-spore-forming, aerobic, motile and rod-shaped (0.6–0.8 mm wide, 1.2–1.4 mm long) with lophotrichous flagella. Forms orange-pigmented 1699
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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 Flagellum Oxidase b-Galactosidase Nitrate reduction Hydrolysis of: Starch Gelatin Acid production from; Sucrose D-Fructose Maltose Galactose Assimilation of: D-Mannose Potassium gluconate Malic acid N-Acetylglucosamine Maltose Cellobiose D-Fructose L-Alanine D-Galactose Trehalose Melibiose DNA G+C content (mol%)*
1
2
3
4
Lophotrichous + + +
Peritrichous + + +
Peritrichous + + 2
Monotrichous 2 2 +
2 2
+ +
+ +
2 2
+ 2 2 2
2 2 + 2
2 + 2 2
2 2 2 +
2 + 2 2 2 + + + 2 2 2 69.6
+ 2 + + + + + 2 + + + 66.0–68.0a
+ 2 + + + + + + + + + 65.6b
+ 2 + + + 2 2 + + + + 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 at 30 uC for 72 h on R2A agar. Colonies on R2A agar are circular, slightly convex and semi-transparent. Growth also occurred on TSA and PYE agar, but not on NA. Growth occurs at pH 6.0–8.0 (optimum, pH 7.0), in the presence of 0–0.5 % (w/v) NaCl (optimum, 0 %) and at 20–32 uC (optimum, 30 uC). Cells are able to hydrolyse aesculin and Tweens 40 and 80, but not starch or gelatin. Catalase, oxidase, arginine decarboxylase and b-galactosidase activities and nitrate reduction are positive, while arginine dihydrolase, ornithine decarboxylase, lysine decarboxylase and urease activities, indole production and methyl red and Voges–Proskauer tests are negative. Acid is produced from sucrose, but not from sorbitol, L-arabinose, fructose, cellobiose, raffinose, D-xylose, mannitol, D-ribose, glucose, maltose, sorbose, rhamnose, lactose or galactose. Assimilates dextrin, cellobiose, gentiobiose, a-lactose, D-salicin, a-Dglucose, sucrose, D-fructose, D-fucose, glycyl L-proline, L-alanine, L-arabinose, L-aspartic acid, L-glutamic acid, Dgluconic acid, quinic acid, Tween 40, b-hydroxybutyric acid, a-ketobutyric acid, acetic acid and potassium gluconate. The
following compounds are not utilized: citrate, D-mannose, maltose, trehalose, turanose, stachyose, raffinose, melibiose, methyl b-D-glucoside, N-acetyl-D-glucosamine, N-acetyl-bD-mannosamine, N-acetyl-D-galactosamine, N-acetyl neuraminic acid, D-galactose, 3-methyl glucose, L-fucose, L-rhamnose, inosine, D-sorbitol, D-mannitol, D-arabitol, myo-inositol, glycerol, D-glucose-6-phosphate, D-fructose6-phosphate, D-aspartic acid, D-serine, gelatin, L-arginine, Lhistidine, L-pyroglutamic acid, L-serine, pectin, D-galacturonic acid, L-galactonic acid lactone, D-glucuronic acid, glucuronamide, mucic acid, D-saccharic acid, phenylacetic acid, p-hydroxyphenylacetic acid, methyl pyruvate, D-lactic acid methyl ester, L-lactic acid, citric acid, a-ketoglutaric acid, D-malic acid, L-malic acid, capric acid, adipic acid, bromosuccinic acid, c-aminobutryric acid, a-hydroxybutyric acid, acetoacetic acid, propionic acid and formic acid. Susceptible to troleandomycin, vancomycin, minocycline and aztreonam; resistant to potassium tellurite, rifamycin SV and lincomycin. The major respiratory lipoquinone is ubiquinone Q-10. The predominant polyamine is symhomospermidine. The polar lipids contain sphingoglycolipid,
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Table 2. Cellular fatty acid profiles of strain 9NM-8T and its close relatives Strains: 1, 9NM-8T; 2, S. azotifigens KACC 14484T; 3, S. trueperi DSM 7225T; 4, S. pituitosa DSM 13101T Cells were all cultured on R2A agar for 3 days at 30 uC. 2, Not detected. Fatty acid C14 : 0 C14 : 02OH Summed feature 3* C16 : 1v5c C16 : 0 C17 : 1v8c C17 : 1v6c C18 : 1v7c C18 : 1v5c C18 : 0 11-methyl C18 : 1v7c C19 : 0 cyclo v8c
1
2
3
4
0.4 1.4 6.4 1.8 14.2 2.5 1.3 66.8 0.6 0.3 4.5 2
0.3 1.1 0.9 0.5 20.1 0.6 0.7 58.6 2 0.3 15.3 1.6
2 1.1 1.2 0.7 10.4 2 0.9 84.4 0.9 0.4 2 2
0.4 1.7 2.2 1.2 13.2 0.5 1.0 70.9 2 2 7.8 1.2
Busse, H. J., Hauser, E. & Ka¨mpfer, P. (2005). Description of two
novel species, Sphingomonas abaci sp. nov. and Sphingomonas panni sp. nov. Int J Syst Evol Microbiol 55, 2565–2569. Chen, Q. H., Chen, J. H., Ruan, Y., Zhang, Y. Q., Tang, S. K., Liu, Z. X., Li, W. J. & Chen, Y. G. (2011). Sphingomonas hunanensis sp. nov.,
isolated from forest soil. Antonie van Leeuwenhoek 99, 753–760. Chen, H., Jogler, M., Rohde, M., Klenk, H. P., Busse, H. J., Tindall, B. J., Spro¨er, C. & Overmann, J. (2012). Reclassification and emended
description of Caulobacter leidyi as Sphingomonas leidyi comb. nov., and emendation of the genus Sphingomonas. Int J Syst Evol Microbiol 62, 2835–2843. Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E. (1977).
Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100, 221–230. Denner, E. B. M., Paukner, S., Ka¨mpfer, P., Moore, E. R. B., Abraham, W. R., Busse, H. J., Wanner, G. & Lubitz, W. (2001). Sphingomonas
pituitosa sp. nov., an exopolysaccharide-producing bacterium that secretes an unusual type of sphingan. Int J Syst Evol Microbiol 51, 827– 841. Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a
maximum likelihood approach. J Mol Evol 17, 368–376. 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 could not be separated by the Microbial Identification System. Summed feature 3 contained C16 : 1v7c and/or C16 : 1v6c.
Fitch, W. M. (1971). Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20, 406– 416. Hamana, K., Hayashi, H., Niitsu, M., Takeda, A. & Itoh, T. (2013).
phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, an unidentified glycolipid and two unidentified phospholipids. The major fatty acids comprise C18 : 1v7c, C16 : 0, C16 : 1v7c and/or C16 : 1v6c (summed feature 3) and 11-methyl C18 : 1v7c. The type strain, 9NM-8T (GIMCC 1.653T5CGMCC 1.12672T5DSM 27570T) was isolated from an abandoned lead-zinc ore of a mining area in Mei county, Meizhou, Guangdong province, PR China. The DNA G+C content of the type strain is 69.6±1.3 mol%.
ACKNOWLEDGEMENTS The authors are grateful to Dr Soon-Wo Kwon and the Korean Agricultural Culture Collection (KACC) for kindly providing Sphingomonas azotifigens KACC 14484T. We thank Dr Koei Hamana for his help in polyamine analysis. This work was jointly supported by the Natural Science Foundation of China, PR China (nos 31070103, 31200006) and the Key Project of Guangdong Natural Science Foundation (no. 10251007002000001).
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nov. and comb. nov., Sphingomonas parapaucimobilis sp. nov., Sphingomonas yanoikuyae sp. nov., Sphingomonas adhaesiva sp. nov., Sphingomonas capsulata comb. nov., and two genospecies of the genus Sphingomonas. Microbiol Immunol 34, 99–119. Yabuuchi, E., Kosako, Y., Fujiwara, N., Naka, T., Matsunaga, I., Ogura, H. & Kobayashi, K. (2002). Emendation of the genus
Sphingomonas Yabuuchi et al. 1990 and junior objective synonymy of the species of three genera, Sphingobium, Novosphingobium and Sphingopyxis, in conjunction with Blastomonas ursincola. Int J Syst Evol Microbiol 52, 1485–1496.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using
Yi, T. H., Han, C. K., Srinivasan, S., Lee, K. J. & Kim, M. K. (2010).
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739.
Sphingomonas humi sp. nov., isolated from soil. J Microbiol 48, 165– 169.
Tindall, B. J., Sikorski, J., Smibert, R. A. & Krieg, N. R. (2007).
Yim, M. S., Yau, Y. C. W., Matlow, A., So, J. S., Zou, J., Flemming, C. A., Schraft, H. & Leung, K. T. (2010). A novel selective growth medium-
Phenotypic characterization and the principles of comparative systematics. In Methods for General and Molecular Microbiology, pp. 365, 384–385. Edited by C. A. Reddy, T. J. Beveridge, J. A. Breznak,
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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 Hong-Hui Zhu [email protected]
State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied 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 orangepigmented, aerobic, oxidase- and catalase-positive, motile with lophotrichous flagella and rodshaped. 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 phylogenetic 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 et al. (1990) and emended subsequently by Takeuchi et al. (2001), Yabuuchi et al. (2002), Busse et al. (2003) and Chen et al. (2012). Based on analysis of phylogeny and polyamine patterns profiles, the genus Sphingomonas should be classified into five genera Sphingomonas, Novosphingobium, Sphingobium, Sphingorhabdus and Sphingopyxis (Takeuchi et al., 2001; Jogler et al., 2013). At the time of writing, approximately 68 members of the genus Sphingomonas have been described, including four species with names that have not yet been validated [‘Sphingomonas humi’ (Yi et al., 2010), ‘Sphingomonas hunanensis’ (Chen et al., 2011), ‘Sphingomonas rosea’ and ‘Sphingomonas swuensis’ (Srinivasan et al., 2011)]. They were isolated from various natural sources, i.e. rhizosphere, marine, desert sand, water and dump site. Members of the genus Sphingomonas are characterized by being yellow- or orange-pigmented, aerobic, Gram-staining-negative and non-spore-forming, and containing Q-10 as the respiratory quinone and sym-homospermidine The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 9NM-8T is JQ608326. Five supplementary figures are available with the online version of this paper.
056853 G 2014 IUMS
Printed in Great Britain
as the key polyamine. In this paper, we report the results from a taxonomic study using a polyphasic approach on a novel bacterium isolated from an abandoned lead-zinc ore, designated strain 9NM-8T. Based on the physiological, chemotaxonomic and phylogenetic characteristics, strain 9NM-8T represents a novel species of the genus Sphingomonas. Bacteria were isolated from a lead-zinc ore collected from a lead-zinc mine, which had been abandoned for 30 years, located in Mei county (24u 219 4499 N 116u 169 3499 E), Meizhou, Guangdong province, PR China. Isolation was performed using the standard dilution plating technique at 30 uC on R2A agar (Luqiao, China). The plates were incubated for 1 week. Strain 9NM-8T was isolated on the basis of colony morphology and purified by subculturing on R2A agar. The culture was preserved at 280 uC in R2A broth supplemented with 20 % (v/v) glycerol. The genomic DNA of strain 9NM-8T was extracted using a commercial genomic DNA isolation kit (Sangon, China). The 16S rRNA gene of strain 9NM-8T was amplified using the universal bacterial primer pair 27F (59-AGAGTTTGATCCTGGCTCAG-39) and 1492R (59-GGTTACCTTGTTACGACTT-39) (Weisburg et al., 1991) and the purified PCR product was cloned into the pCR 2.1 vector and 1697
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sequenced by Invitrogen. The 16S rRNA gene sequences of related taxa were obtained from the GenBank database and the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al. 2012). Analysis of 16S rRNA gene sequences was performed by using MEGA 5.0 software (Tamura et al., 2011). Evolutionary distances were calculated by using Kimura’s two-parameter model (Kimura, 1980). Phylogenetic trees were reconstructed by the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Fitch, 1971) and maximumlikelihood (Felsenstein, 1981) methods with bootstrap values based on 1000 replications (Felsenstein, 1985). Moreover, to verify strain 9NM-8T, PCRs were also performed using two Sphingomonas-specific primer pairs (Sph-spt 295f/Sph-spt 713r and Sph-spt 694f/Sph-spt 983r) as described by Yim et al. (2010). Biochemical and physiological tests were performed on R2A at 30 uC. The Gram reaction was tested by using a modification of the method described by Buck (1982). Catalase, cytochrome oxidase, methyl red and Voges– Proskauer tests and hydrolysis of starch, and Tweens 40 and 80 were determined according to the methods described by Smibert & Krieg (1994). Motility was assessed under a Leica DMLB phase-contrast light microscope on cells grown for 3 days at 30 uC. Strain 9NM-8T was grown on R2A agar at 30 uC for 3 days and the bacterial cells were suspended in sterile distilled water and stained with phosphotungstic acid (3 %, pH 7.0) for 2 min, air-dried and observed with Hitachi H7650 electron microscope. Anaerobic growth was determined in an anaerobic pouch (MGC) for 7 days at 30 uC on R2A agar. Ornithine decarboxylase, lysine decarboxylase, and acid production from sugars were tested by using biochemical kits (Huankai, China). Arginine dihydrolase, b-galactosidase (ONPG), urease, nitrate reduction, citrate utilization, indole production and hydrolysis of gelatin and aesculin were tested using API 20NE strips (bioMe´rieux). Growth at different pH (pH 4–10 in intervals of 1 pH unit) and temperatures (4, 10, 15, 20, 25, 30, 32, 35, 37 and 45 uC) was determined in buffered R2A broth. Tolerance to NaCl was tested in R2A broth with different NaCl concentrations (0, 0.5, 1, 1.5, 2, 2.5, 3, 5, 6, 8 and 10 %, w/v). Substrate utilization and chemical sensitive assays were evaluated by API 20NE strips and GN3 MicroPlates (Biolog) at 30 uC according to the manufacturer’s instructions. Growth of strain 9NM-8T was tested on different media: R2A agar (Luqiao), tryptic soy agar (TSA; Guangdong Huankai Microbial Science & Technology, China), PYE agar (Busse et al., 2005) and nutrient agar (NA; Huankai) at 30 uC.
extracted according to the protocol of the Sherlock Microbial Identification System (MIDI) and analysed by gas chromatography (model 7890A; Hewlett Packard) using the Microbial Identification software package with the Sherlock MIDI 6.1 system and the Sherlock Aerobic Bacterial Database (TSBA 6.1) (Sasser, 1990). The polyamines of strain 9NM-8T were determined as described by Hamana et al. (2013). Isoprenoid quinones were extracted from lyophilized cells and subsequently analysed by HPLC (UltiMate 3000; Dionex) according to the methods as described by Collins et al. (1977) and Hiraishi et al. (1996). Polar lipids were extracted and determined as described by Tindall et al. (2007). The PCR results using the Sphingomonas-specific primer pairs showed that strain 9NM-8T belonged to the genus Sphingomonas and the primer set Sph-spt 694f/Sph-spt 983r is more specific than Sph-spt 259r/Sph-spt 713r (Fig. S1, available in the online Supplementary Material). The 16S rRNA gene sequence of strain 9NM-8T determined in this study was a continuous stretch of 1437 bp. The 16S rRNA gene sequence similarities, which were calculated by the EzTaxon-e server, revealed that the closest relatives of strain 9NM-8T were Sphingomonas azotifigens KACC 14484T (96.1 %), Sphingomonas trueperi DSM 7225T (96.0 %), Sphingomonas phyllosphaerae FA2T (95.6 %), Sphingomonas endophytica YIM 65583T (95.7 %), Sphingomonas ginsenosidimutans Gsoil 1429T (95.6 %) and Sphingomonas pituitosa DSM 13101T (95.6 %). However, the neighbour-joining phylogenetic tree (Figs 1 and S2) showed that strain 9NM-8T only clustered with S. azotifigens KACC 14484T, S. trueperi DSM 7225T and S. pituitosa DSM 13101T and the topologies of the maximum-likelihood and maximum-parsimony trees were essentially the same (Figs S3 and S4). A 16S rRNA gene sequence similarity of 97.0 % was proposed by Stackebrandt & Goebel (1994) and subsequently re-evaluated to 98.7 % by Stackebrandt & Ebers (2006) as a criterion for species discrimination. Taking this into consideration, we concluded that the 16S rRNA gene sequence similarities between strain 9NM-8T and the type strains of the closest related species of the genus Sphingomonas are low enough to exclude the assignment of the novel strain to any of the recognized species of the genus Sphingomonas.
To measure the DNA G+C content of strain 9NM-8T, genomic DNA was extracted and purified using the method as described by Moore & Dowhan (1995) and the G+C content determined by HPLC as described by Mesbah et al. (1989). For fatty acid profile analysis, strain 9NM-8T, Sphingomonas azotifigens KACC 14484T, Sphingomonas trueperi DSM 7225T and Sphingomonas pituitosa DSM 13101T were grown on R2A agar for 96 h at 30 uC. Cellular fatty acids were saponified, methylated and
Cells of strain 9NM-8T were Gram-stain-negative, nonspore-forming, oxidase- and catalase-positive, aerobic, motile with lophotrichous flagella and rod-shaped (0.6– 0.8 mm wide, 1.2–1.4 mm long) (Fig. 2). Colonies grown on R2A agar were circular, convex, orange, semi-transparent and 1–2 mm in diameter after 3 days at 30 uC. Strain 9NM-8T was able to grow at 20–32 uC and pH 6–8. Growth occurred in the presence of 0–0.5 % (w/v) NaCl. The maximum growth of strain 9NM-8T was at pH 7.0 and 30 uC without NaCl on R2A agar. Cells were able to hydrolyse aesculin and Tweens 40 and 80, but not starch, gelatin or arginine. Methyl red and Voges–Proskauer tests, urease activity, indole production and citrate utilization were negative, while the nitrate reduction test was positive. Strain 9NM-8T can grow on TSA and PYE agar, but not on
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Sphingomonas yabuuchiae GTC 868T (AB071955) Sphingomonas roseiflava MK341T (D84520) Sphingomonas parapaucimobilis NBRC 15100T (D13724) Sphingomonas sanguinis IFO 13937T (D13726) 99 Sphingomonas pseudosanguinis G1-2T (AM412238) Sphingomonas paucimobilis ATCC 29837T (U37337) Sphingomonas desiccabilis CP1DT (AJ871435) Sphingomonas molluscorum An 18T (AB248285) Sphingomonas polyaromaticivorans B2-7T (EF467848) Sphingomonas ginsenosidimutans Gsoil 1429T (HM204925) Sphingomonas yunnanensis YIM 003T (AY894691)
0.005
96
90 90
86
59
Sphingomonas phyllosphaerae FA2T (AY453855) 99 Sphingomonas endophytica YIM 65583T (HM629444) Sphingomonas japonica DSM 22753T (AB428568) Sphingomonas guangdongensis 9NM-8T (JQ608326) Sphingomonas pituitosa DSM 13101T (AJ243751) 99
97
Sphingomonas azotifigens KACC 14484T (AB217471) 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 differentiate strain 9NM-8T from the reference type strains are listed in Table 1 and in the species description. The major respiratory quinone of strain 9NM-8T was ubiquinone 10 (Q-10) and the polar lipids detected were
sphingoglycolipid, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, an unidentified glycolipid and two unidentified phospholipids (Fig. S5). The polyamine patterns of strain 9NM-8T showed a predominance of sym-homospermidine. These 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), and the major fatty acids of strain 9NM-8T were C18 : 1v7c, C16 : 0, C16 : 1v7c and/or C16 : 1v6c (summed feature 3) and 11-methyl C18 : 1v7c, quite different from S. azotifigens KACC 14484T (C18 : 1v7c, C16 : 0 and 11-methyl C18 : 1v7c), S. trueperi DSM 7225T (C18 : 1v7c and C16 : 0) and S. pituitosa DSM 13101T (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).
Fig. 2. Transmission electron micrograph of a cell of strain 9NM8T. Bar, 500 nm. http://ijs.sgmjournals.org
Cells are Gram-stain-negative, non-spore-forming, aerobic, motile and rod-shaped (0.6–0.8 mm wide, 1.2–1.4 mm long) with lophotrichous flagella. Forms orange-pigmented 1699
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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 Flagellum Oxidase b-Galactosidase Nitrate reduction Hydrolysis of: Starch Gelatin Acid production from; Sucrose D-Fructose Maltose Galactose Assimilation of: D-Mannose Potassium gluconate Malic acid N-Acetylglucosamine Maltose Cellobiose D-Fructose L-Alanine D-Galactose Trehalose Melibiose DNA G+C content (mol%)*
1
2
3
4
Lophotrichous + + +
Peritrichous + + +
Peritrichous + + 2
Monotrichous 2 2 +
2 2
+ +
+ +
2 2
+ 2 2 2
2 2 + 2
2 + 2 2
2 2 2 +
2 + 2 2 2 + + + 2 2 2 69.6
+ 2 + + + + + 2 + + + 66.0–68.0a
+ 2 + + + + + + + + + 65.6b
+ 2 + + + 2 2 + + + + 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 at 30 uC for 72 h on R2A agar. Colonies on R2A agar are circular, slightly convex and semi-transparent. Growth also occurred on TSA and PYE agar, but not on NA. Growth occurs at pH 6.0–8.0 (optimum, pH 7.0), in the presence of 0–0.5 % (w/v) NaCl (optimum, 0 %) and at 20–32 uC (optimum, 30 uC). Cells are able to hydrolyse aesculin and Tweens 40 and 80, but not starch or gelatin. Catalase, oxidase, arginine decarboxylase and b-galactosidase activities and nitrate reduction are positive, while arginine dihydrolase, ornithine decarboxylase, lysine decarboxylase and urease activities, indole production and methyl red and Voges–Proskauer tests are negative. Acid is produced from sucrose, but not from sorbitol, L-arabinose, fructose, cellobiose, raffinose, D-xylose, mannitol, D-ribose, glucose, maltose, sorbose, rhamnose, lactose or galactose. Assimilates dextrin, cellobiose, gentiobiose, a-lactose, D-salicin, a-Dglucose, sucrose, D-fructose, D-fucose, glycyl L-proline, L-alanine, L-arabinose, L-aspartic acid, L-glutamic acid, Dgluconic acid, quinic acid, Tween 40, b-hydroxybutyric acid, a-ketobutyric acid, acetic acid and potassium gluconate. The
following compounds are not utilized: citrate, D-mannose, maltose, trehalose, turanose, stachyose, raffinose, melibiose, methyl b-D-glucoside, N-acetyl-D-glucosamine, N-acetyl-bD-mannosamine, N-acetyl-D-galactosamine, N-acetyl neuraminic acid, D-galactose, 3-methyl glucose, L-fucose, L-rhamnose, inosine, D-sorbitol, D-mannitol, D-arabitol, myo-inositol, glycerol, D-glucose-6-phosphate, D-fructose6-phosphate, D-aspartic acid, D-serine, gelatin, L-arginine, Lhistidine, L-pyroglutamic acid, L-serine, pectin, D-galacturonic acid, L-galactonic acid lactone, D-glucuronic acid, glucuronamide, mucic acid, D-saccharic acid, phenylacetic acid, p-hydroxyphenylacetic acid, methyl pyruvate, D-lactic acid methyl ester, L-lactic acid, citric acid, a-ketoglutaric acid, D-malic acid, L-malic acid, capric acid, adipic acid, bromosuccinic acid, c-aminobutryric acid, a-hydroxybutyric acid, acetoacetic acid, propionic acid and formic acid. Susceptible to troleandomycin, vancomycin, minocycline and aztreonam; resistant to potassium tellurite, rifamycin SV and lincomycin. The major respiratory lipoquinone is ubiquinone Q-10. The predominant polyamine is symhomospermidine. The polar lipids contain sphingoglycolipid,
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Table 2. Cellular fatty acid profiles of strain 9NM-8T and its close relatives Strains: 1, 9NM-8T; 2, S. azotifigens KACC 14484T; 3, S. trueperi DSM 7225T; 4, S. pituitosa DSM 13101T Cells were all cultured on R2A agar for 3 days at 30 uC. 2, Not detected. Fatty acid C14 : 0 C14 : 02OH Summed feature 3* C16 : 1v5c C16 : 0 C17 : 1v8c C17 : 1v6c C18 : 1v7c C18 : 1v5c C18 : 0 11-methyl C18 : 1v7c C19 : 0 cyclo v8c
1
2
3
4
0.4 1.4 6.4 1.8 14.2 2.5 1.3 66.8 0.6 0.3 4.5 2
0.3 1.1 0.9 0.5 20.1 0.6 0.7 58.6 2 0.3 15.3 1.6
2 1.1 1.2 0.7 10.4 2 0.9 84.4 0.9 0.4 2 2
0.4 1.7 2.2 1.2 13.2 0.5 1.0 70.9 2 2 7.8 1.2
Busse, H. J., Hauser, E. & Ka¨mpfer, P. (2005). Description of two
novel species, Sphingomonas abaci sp. nov. and Sphingomonas panni sp. nov. Int J Syst Evol Microbiol 55, 2565–2569. Chen, Q. H., Chen, J. H., Ruan, Y., Zhang, Y. Q., Tang, S. K., Liu, Z. X., Li, W. J. & Chen, Y. G. (2011). Sphingomonas hunanensis sp. nov.,
isolated from forest soil. Antonie van Leeuwenhoek 99, 753–760. Chen, H., Jogler, M., Rohde, M., Klenk, H. P., Busse, H. J., Tindall, B. J., Spro¨er, C. & Overmann, J. (2012). Reclassification and emended
description of Caulobacter leidyi as Sphingomonas leidyi comb. nov., and emendation of the genus Sphingomonas. Int J Syst Evol Microbiol 62, 2835–2843. Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E. (1977).
Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100, 221–230. Denner, E. B. M., Paukner, S., Ka¨mpfer, P., Moore, E. R. B., Abraham, W. R., Busse, H. J., Wanner, G. & Lubitz, W. (2001). Sphingomonas
pituitosa sp. nov., an exopolysaccharide-producing bacterium that secretes an unusual type of sphingan. Int J Syst Evol Microbiol 51, 827– 841. Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a
maximum likelihood approach. J Mol Evol 17, 368–376. 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 could not be separated by the Microbial Identification System. Summed feature 3 contained C16 : 1v7c and/or C16 : 1v6c.
Fitch, W. M. (1971). Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20, 406– 416. Hamana, K., Hayashi, H., Niitsu, M., Takeda, A. & Itoh, T. (2013).
phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, an unidentified glycolipid and two unidentified phospholipids. The major fatty acids comprise C18 : 1v7c, C16 : 0, C16 : 1v7c and/or C16 : 1v6c (summed feature 3) and 11-methyl C18 : 1v7c. The type strain, 9NM-8T (GIMCC 1.653T5CGMCC 1.12672T5DSM 27570T) was isolated from an abandoned lead-zinc ore of a mining area in Mei county, Meizhou, Guangdong province, PR China. The DNA G+C content of the type strain is 69.6±1.3 mol%.
ACKNOWLEDGEMENTS The authors are grateful to Dr Soon-Wo Kwon and the Korean Agricultural Culture Collection (KACC) for kindly providing Sphingomonas azotifigens KACC 14484T. We thank Dr Koei Hamana for his help in polyamine analysis. This work was jointly supported by the Natural Science Foundation of China, PR China (nos 31070103, 31200006) and the Key Project of Guangdong Natural Science Foundation (no. 10251007002000001).
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