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Description of a Gram-Negative Bacterium, Sphingomonas Guangdongensis Sp Nov

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Description of a Gram-Negative Bacterium, Sphingomonas Guangdongensis Sp Nov See discussions, stats, and author profiles for this publication at: http://www.researchgate.net/publication/260170275 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 CITATION DOWNLOADS VIEWS 1 56 105 7 AUTHORS, INCLUDING: Zhao Guozhen Chinese Academy of Sciences 43 PUBLICATIONS 475 CITATIONS SEE PROFILE 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, Sphingobium, Sphingorhabdus and Sphingopyxis Bacteria 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
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