SGM Journals
Actinobacteria

Brevibacterium salitolerans sp. nov., an actinobacterium isolated from salt-lake sediment

Tong-Wei Guan, Ke Zhao, Jing Xiao, Ying Liu, Zhan-Feng Xia, Xiao-Ping Zhang, Li-Li Zhang

  • 1Faculty of Resource and Environmental Sciences, Sichuan Agricultural University, Yaan 625000, PR China
  • 2Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin of Xinjiang Production and Construction Corps, Tarim University, Alar, Xinjiang 843300, PR China
  • 3State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China
  • 4Key Laboratory of Marine Biological Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen, Fujian 361005, PR China
  • Correspondence
    Xiao-Ping Zhang
    zhangxiaopingphd{at}126.com
    Li-Li Zhang
    zhang63lyly{at}yahoo.com.cn
    or
    guantongwei78{at}126.com

    • International Journal of Systematic and Evolutionary Microbiology 2010; 60(12):2991–2995 · https://doi.org/10.1099/ijs.0.020214-0

    View at publisher PubMed

    Abstract

    A novel bacterium, designated TRM 415T, belonging to the genus Brevibacterium, was isolated from a sediment sample from a salt lake in Xinjiang province, China. Comparative 16S rRNA gene sequence analysis indicated that strain TRM 415T was phylogenetically most closely related to Brevibacterium album YIM 90718T (98.4 % sequence similarity) and had low similarity (<95.5 %) to other species of the genus Brevibacterium; however, DNA–DNA hybridization studies between strain TRM 415T and B. album YIM 90718T showed only 41.3 % relatedness. Strain TRM 415T possessed meso-diaminopimelic acid as the diagnostic cell-wall diamino acid, MK-8(H2) as the major menaquinone and polar lipids including phosphatidylglycerol and diphosphatidylglycerol. The major fatty acids were anteiso-C17 : 0 and anteiso-C15 : 0. The genomic DNA G+C content was 69 mol%. Based on the evidence from this polyphasic study, strain TRM 415T represents a novel species of the genus Brevibacterium, for which the name Brevibacterium salitolerans sp. nov. is proposed. The type strain is TRM 415T (=JCM 15900T =CCTCC AB 208328T =KCTC 19616T).

    • ↵†These authors contributed equally to this work.

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Brevibacterium salitolerans TRM 415T is GU117109.

    • A transmission electron micrograph and a lipid profile of cells of strain TRM 415T and a phylogenetic tree based on maximum-likelihood analysis of 16S rRNA gene sequences are available as supplementary material with the online version of this paper.

    The genus Brevibacterium was first described by Breed (1953) with Brevibacterium linens as the type species; the description was later emended by Collins et al. (1980). At the time of writing, the genus Brevibacterium contained 21 species with validly published names. In the present study, an actinobacterial isolate, designated TRM 415T, from a sediment sample collected from Lop Nur salt lake in Xinjiang province, north-west China, was investigated by means of a polyphasic taxonomic approach. The lake environment and precise sampling site location were described previously (Guan et al., 2010).

    The sample was isolated on GT2 medium using a 10-fold dilution series method with incubation at 37 °C. The GT2 medium contained (g l−1): glucose (10), yeast extract (1), tryptone (0.5), CaCO3 (1), NaCl (50), KCl (30), MgCl2 (10) and agar (16). The medium was adjusted to pH 7.0–7.5. The organism was grown and maintained on modified ISP 5 medium (Shirling & Gottlieb, 1966) with 2 % (w/v) NaCl, 1 % (w/v) KCl and 0.5 % (w/v) MgCl2. Biomass for chemical and molecular studies was determined by cultivation in flasks containing the above modified ISP 5 medium and incubation with shaking (∼180 r.p.m.) at 37 °C for 1 week.

    Cell morphology and motility were examined by optical (Olympus BX40) and transmission electron (JEM-1230) microscopy. Gram staining was carried out using the standard Gram reaction method and was confirmed by using the KOH lysis test (Cerny, 1978). The ability of strain TRM 415T to grow was tested at 4–55 °C and pH 4–11 as described by Xu et al. (2005) and tolerance to concentrations of 0–25 % (w/v) NaCl was tested using ISP 5 as the basal medium. Antimicrobial susceptibility tests were performed using antimicrobial compounds (BBL) according to Mata et al. (2002). Carbon source utilization tests were carried out using 95 substrates in GP2 MicroPlates of the Microlog system (Biolog). The physiological properties of strain TRM 415T were tested by using the API Coryne system (bioMérieux) according to the manufacturer's instructions and by using the media and methods of Gordon et al. (1974). Detailed morphological, cultural and physiological properties of strain TRM 415T are given in Table 1⇓ and in the species description.

    Table 1.

    Differential characteristics of strains TRM 415T and B. album YIM 90718T

    Strains: 1, TRM 415T; 2, B. album YIM 90718T

    The type of diamino acid in cell-wall hydrolysates of strain TRM 415T was determined following methods described previously (Lee, 2006; Staneck & Roberts, 1974). Identification of the cell-wall sugars in whole-cell hydrolysates was performed as described by Hasegawa et al. (1983). The absence of mycolic acids was demonstrated with TLC (Minnikin et al., 1980). Polar lipids were extracted, examined by two-dimensional TLC and identified using the procedures of Minnikin et al. (1984). Menaquinones were extracted using the method of Collins et al. (1977) and analysed by HPLC (Groth et al., 1997). The menaquinones detected included MK-8(H2) (64.7 %), MK-7(H2) (23.1 %), MK-6(H2) (6.8 %), MK-8 (3.4 %), MK-7 (1.5 %) and MK-6 (0.6 %). The major fatty acids were anteiso-C17 : 0 (43.8 %) and anteiso-C15 : 0 (30.3 %). Cellular fatty acid composition was determined as described by Sasser (1990) using the Microbial Identification System (MIDI). The genomic DNA G+C content of strain TRM 415T was determined by using the HPLC method (Mesbah et al., 1989). Results of the above experiments are reported in the species description and in Tables 1⇑ and 2⇓. Biomass for chemotaxonomic studies was obtained by growing strain TRM 415T in flasks containing ISP 5 liquid medium with 2 % (w/v) NaCl, 1 % (w/v) KCl and 0.5 % (w/v) MgCl2 with shaking at 37 °C for 3 days.

    Table 2.

    Cellular fatty acid compositions (%) of strains TRM 415T and B. album YIM 90718T

    Strains: 1, TRM 415T; 2, B. album YIM 90718T. Fatty acids that account for less than 1.0 % of the total fatty acids in the two strains are not shown. Data are from this study and from Tang et al. (2008). nr, Not reported.

    Genomic DNA of strain TRM 415T was extracted from cells grown on modified ISP 5 medium (as above) for 4 days at 37 °C and used as a template for subsequent PCR amplification. Reaction conditions and amplification protocols were used as described by Li et al. (2007). Multiple sequence alignments with closely related members of the genus Brevibacterium and sequence similarity calculations were carried out using the clustal_x program (Thompson et al., 1997). Phylogenetic trees were constructed by the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) methods using the mega 3 program (Kumar et al., 2004) and maximum-likelihood phylogenetic trees were constructed (Felsenstein, 1981) with the phylip 3.6 program. The topology of the resultant phylogenetic trees was evaluated by bootstrap resampling with 1000 replicates (Felsenstein, 1985). The results indicated that strain TRM 415T clustered within the genus Brevibacterium and exhibited the closest phylogenetic affinity (Fig. 1⇓) and highest sequence similarity to Brevibacterium album YIM 90718T (98.4 %) with low similarity (<95.5 %) to other species of the genus Brevibacterium. Genomic hybridization experiments between strain TRM 415T and B. album YIM 90718T were performed by using the method described by He et al. (2005). Strain TRM 415T and B. album YIM 90718T showed 41.3 % DNA–DNA relatedness to each other. Considering 70 % DNA–DNA relatedness as the cut-off point for species delineation (Wayne et al., 1987) and based on the differences in the phenotypic characteristics and the chemotaxonomic and phylogenetic data observed between the two strains, strain TRM 415T represents a novel species of the genus Brevibacterium, for which the name Brevibacterium salitolerans sp. nov. is proposed.

    Figure image not available in archive
    Fig. 1.

    Neighbour-joining tree based on 16S rRNA gene sequences, showing the phylogenetic relationships between the novel isolate TRM 415T and related taxa. Bootstrap values are percentages based on 1000 replicates; values >50 % are shown. Bar, 0.01 substitutions per nucleotide position.

    Description of Brevibacterium salitolerans sp. nov.

    Brevibacterium salitolerans (sa.li.to′.le.rans. L. n. sal, salis salt; L. part. adj. tolerans tolerating; N.L. part. adj. salitolerans salt tolerating, originating from a saline habitat).

    Cells are Gram-reaction-positive, aerobic, non-motile rods, approximately 1.3–1.8×0.6–1.0 μm. Colonies are 2.0–3.5 mm in diameter, smooth, circular and whitish-yellow when grown, in the dark or in the light, on ISP 5 medium with 2 % (w/v) NaCl, 1 % (w/v) KCl and 0.5 % (w/v) MgCl2 for 5 days. An apparent rod–coccus growth cycle is not observed after 5 days of growth. Growth occurs at 15–50 °C (optimum 28–40 °C), at pH 5–9 (optimum pH 7.0–7.5) and in concentrations of 0–18 % (w/v) NaCl [optimum 3–8 % (w/v) NaCl]. No growth occurs at 10 or 52 °C or in 20 % (w/v) NaCl. Oxidase-negative and catalase-positive. Nitrate is reduced to nitrite. Gelatin is hydrolysed but Tweens 20, 60 and 80 and starch are not. Negative for milk coagulation and peptonization, cellulose hydrolysis, production of H2S and methyl red and Voges–Proskauer tests. In the API Coryne system, tests are positive for pyrazinamidase, pyrrolidonyl arylamidase, alkaline phosphatase and β-glucosidase (aesculin hydrolysis) activities, gelatin hydrolysis and acid production from d-ribose; tests are negative for nitrate reduction, β-glucuronidase, β-galactosidase, N-acetyl-β-glucosaminidase, α-glucosidase and urease activities and acid production from d-glucose, d-xylose, d-mannitol, maltose, lactose, sucrose and glycogen. The following substrates are utilized: l-fucose, d-gluconic acid, Tween 80, d-malic acid, succinic acid, d-alanine, l-alanyl glycine, α-cyclodextrin, dextrin, glycogen, Tween 40, acetic acid, α-, β- and γ-hydroxybutyric acids, α-ketoglutaric acid, α-ketovaleric acid, lactamide, d-lactic acid methyl ester, l-lactic acid, l-malic acid, methyl pyruvate, monomethyl succinate, propionic acid, pyruvic acid, succinamic acid, N-acetyl-l-glutamic acid, l-alaninamide, l-alanine, l-asparagine, l-glutamic acid, glycyl l-glutamic acid, l-serine, 2,3-butanediol, adenosine, 2′-deoxyadenosine, inosine and thymidine. The following substrates are not utilized: β-cyclodextrin, N-acetyl-d-glucosamine, myo-inositol, lactulose, d-mannitol, methyl β-d-galactoside, methyl α-d-glucoside, palatinose, l-rhamnose, salicin, trehalose, turanose, putrescine, uridine 5′-monophosphate, fructose 6-phosphate, inulin, mannan, N-acetyl-β-d-mannosamine, amygdalin, l-arabinose, d-arabitol, arbutin, cellobiose, d-fructose, d-galactose, d-galacturonic acid, gentiobiose, α-d-glucose, lactose, maltose, d-mannose, melezitose, melibiose, methyl α-d-galactoside, 3-methyl glucose, methyl β-d-glucoside, methyl α-d-mannoside, d-psicose, raffinose, d-ribose, sedoheptulosan, d-sorbitol, stachyose, sucrose, d-tagatose, xylitol, d-xylose, p-hydroxyphenylacetic acid, l-pyroglutamic acid, glycerol, uridine, adenosine 5′-monophosphate, thymidine 5′-monophosphate, glucose 1-phosphate, glucose 6-phosphate and dl-α-glycerol phosphate. Susceptible to (μg unless stated otherwise) streptomycin (10), amoxicillin (25), chloramphenicol (30), cefoxitin (30), kanamycin (30), nalidixic acid (30), nitrofurantoin (300), ampicillin (10), sulfamide (250), cefotaxime (30), erythromycin (15), polymyxin B (300 UI), rifampicin (30) and carbenicillin (100), but resistant to tobramycin (10) and trimethoprim–sulfamethoxazole (1.25–23.75). Whole-cell hydrolysates contain meso-diaminopimelic acid. Mycolic acids are absent. Cell-wall sugars contain galactose, xylose and ribose and the phospholipids include phosphatidylglycerol and diphosphatidylglycerol. The menaquinones are MK-8(H2), MK-7(H2), MK-6(H2), MK-8, MK-7 and MK-6. The major fatty acids are anteiso-C17 : 0 and anteiso-C15 : 0. The genomic DNA G+C content of the type strain is 69 mol%.

    The type strain, TRM 415T (=JCM 15900T =CCTCC AB 208328T =KCTC 19616T), was isolated from a soil sediment sample from a hypersaline lake habitat in Xinjiang province, north-west China.

    Acknowledgments

    The authors are grateful to Professor Dr Wen-Jun Li (Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming, Yunnan, PR China) for providing the type strain B. album YIM 90718T. This research was supported by the National Natural Science Foundation of China (project numbers 30660005 and 30570062), the Key Project for Department of Science and Technology of Ministry of Education of China (project no. 209145), The Foundation of National Science Program of China (project no. 30570062), the Opening Project by State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences (project no. SKLMR-20090603), the Opening Project by Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin of Xinjiang Production & Construction Corps (project no. BR0803) and the Program of the Higher Education Institution of Xinjiang (project no. XJEDU2005G07).

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