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1 Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, P. R. China
2 Qingdao Institute of Marine Geology, Qingdao 266071, P. R. China
3 Department of Materials and Life Science, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto City, Kumamoto 860-8555, Japan
Correspondence
Xiao-Lei Wu
xiaolei_wu{at}tsinghua.edu.cn
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9c, C16 : 0 and C12 : 0 3-OH as the predominant fatty acids. The genomic DNA G+C content was 62.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain SS011B1-4T belonged to the genus Marinobacter of the Gammaproteobacteria. The results of the phenotypic, phylogenetic and genomic analyses revealed that strain SS011B1-4T represents a novel species of the genus Marinobacter. The name Marinobacter segnicrescens sp. nov. is therefore proposed, with strain SS011B1-4T (=LMG 23928T=CGMCC 1.6489T) as the type strain.
A figure showing the growth rate of strain SS011B1-4T and two closely related Marinobacter species is available with the online version of this paper.
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with a single species Marinobacter hydrocarbonoclasticus, which was isolated from seawater in the gulf of Fos (French Mediterranean coast) near a petroleum refinery outlet (Gauthier et al., 1992). In the following years, only one other species of the genus was proposed, by Huu et al. (1999), which was later considered to be a heterotypic synonym of M. hydrocarbonoclasticus (Márquez & Ventosa, 2005). This species was isolated from an oil-producing well on an offshore platform in southern Vietnam (Huu et al., 1999). More recently, a number of novel species have been added to the genus. Aside from sites pertaining to oil (Gauthier et al., 1992; Huu et al., 1999; Gu et al., 2007), species of Marinobacter have been isolated from diverse environments, including seawater (Yoon et al., 2003, 2004; Shivaji et al., 2005), sea sand (Kim et al., 2006), marine sediment (Gorshkova et al., 2003; Romanenko et al., 2005), brine–seawater interface (Antunes et al., 2007), coastal hot spring (Shieh et al., 2003), saline soil (Martín et al., 2003), wine wastewater (Liebgott et al., 2006) and even laboratory cultures from dinoflagellates (Green et al., 2006). In this study, we isolated a novel bacterial strain from benthic sediment of the South China Sea. The results of the polyphasic analyses indicated that the strain represents a novel species of the genus Marinobacter. Strain SS011B1-4T was isolated from benthic sediment collected in 2001 from the South China Sea at a depth of 1161 m, by using the dilution-plating technique on Zobell marine agar 2216 (MA). The isolate was purified by repeated streaking on plates of MA incubated for 3–5 days at 30 °C, and was then checked for purity by microscopy and 16S rRNA gene sequencing. The pure culture was stored at –80 °C in Zobell marine liquid medium supplemented with 20 % (v/v) glycerol.
Cell morphology and flagellum type were examined by using transmission electron microscopy (Smibert & Krieg, 1994). The heat resistance of cells was determined in growth medium. After incubation for 15 and 30 h (mid-exponential and stationary phase) at 30 °C, two parallel cultures were heated at 70, 80, 90 and 100 °C for 10 min, and at 70 °C for 20 min. The heat-treated cultures were then inoculated into fresh growth medium [inoculum, 20 % (v/v)] and growth was recorded after incubation for 48 h at 30 °C. Growth was investigated at various pH values (4.0–10.0) and temperatures (4–45 °C) in marine broth 2216 (MB), and at various NaCl concentrations (0–15 %) in MB without NaCl. Oxidase activity was examined as described by Smibert & Krieg (1994) and catalase activity was determined by bubble production in 3 % (v/v) hydrogen peroxide solution. Hydrolysis of starch and Tween 80, urease activity and acid production from carbohydrates were determined according to Williams et al. (1983). Hydrolysis of gelatin was assessed as described by Smibert & Krieg (1994). Nitrate and nitrite reduction, production of indole and H2S, Voges–Proskauer reaction and methyl red tests were performed as described by Lanyi (1987). Sensitivity to antibiotics (kanamycin, tetracycline, chloramphenicol, ampicillin, streptomycin, erythromycin and gentamicin) at a concentration of 10 µg was checked by using the diffusion plate method (Cho & Giovannoni, 2003). In addition, tests for utilization of organic carbons were performed in triplicate by using MB without peptone or yeast extract. Liquid medium with organic carbon (1.0 %, w/v) was autoclaved or filter-sterilized, and then growth of the strains was tested by measuring the OD600 values of the cultures after cultivation at 30 °C for 5 days. To test the capability to degrade aliphatic hydrocarbons and crude oil, 1.0 % (w/v) n-tetradecane and toluene and 5 % (w/v) crude oil (Daqing Oilfield, China) were added to cultures of strain SS011B1-4T and four reference Marinobacter species: M. bryozoorum 50-11T, M. gudaonensis SL014B61AT, M. lipolyticus SM19T and M. excellens KMM 3809T; cultivation (in triplicate) lasted for up to 20 days.
The cellular fatty acid composition was analysed using gas chromatography following the instructions of the Microbial Identification System (MIDI). Fatty acid profiles were analysed by using the Sherlock system (Microbial ID). Respiratory lipoquinones were analysed as described by Komagata & Suzuki (1987) using reversed-phase HPLC (Shim-pack, VP-ODS, Shimadzu). Polar lipid analysis was performed following the polar lipid extraction procedure, and examined using two-dimensional thin-layer chromatography on Merck silica gel 60 F254 aluminium-backed, thin-layer plates, according to the methods of Kates (1986) and Collins et al. (1980).
Genomic DNA extraction was carried out by using the method of Marmur (1961) with the modification that the NaCl concentration was adjusted in the DNA extraction solution (Gliesche et al., 1997). DNA purity was assessed by using the A280/A260 and A230/A260 ratios (Johnson, 1994). The DNA G+C content was determined using thermal denaturation (Marmur & Doty, 1962) with DNA from Escherichia coli K-12 as a control. DNA–DNA hybridization was performed in triplicate by using the thermal denaturation and renaturation method of Huß et al. (1983), modified from that of De Ley et al. (1970). The renaturation temperature used was 78.0 °C in 2xSSC buffer (0.15 M NaCl buffered with 0.015 M trisodium citrate, pH 7.0). The 16S rRNA gene was amplified using bacterial universal primer pairs: 5'-AGAGTTTGATCCTGGCTCAG-3' (8f) and 5'-GGTTACCTTGTTACGACTT-3' (1492r). Alignment of the almost-complete 16S rRNA gene sequence (1387 bp in length) of strain SS011B1-4T against related species was performed using the CLUSTAL_X program (version 1.64b; Thompson et al., 1997). A phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987) and evaluated by bootstrap analysis based on 1000 resampling replicates by using the programs SEQBOOT, DNADIST, NEIGHBOR and CONSENSE of the PHYLIP package version 3.6 (Felsenstein, 2004).
Cells of strain SS011B1-4T were Gram-negative, ellipsoid-shaped (0.6–0.7x0.9–1.2 µm) coccobacilli, and motile by means of a single polar flagellum (Fig. 1). No spores were observed and no cells survived in the heat-resistance tests, indicating the absence of heat-resistant forms. Colonies of 1.0–2.0 mm in diameter on MA were cream-coloured or light yellow, smooth, circular, flat and slightly transparent after 3–5 days cultivation at 30 °C. The strain grew in the presence of 1–15 % (w/v) NaCl (optimum, 4–8 %). Thus the bacterium can be considered to represent a moderately halophilic species according to Kushner & Kamekura (1988). The strain grew at pH 6.0–10.0 and 15–45 °C with optimal growth at pH 7.5–8.0 and 30–37 °C. The growth rate of strain SS011B1-4T was much lower than that of other Marinobacter species (see Supplementary Fig. S1 available in IJSEM Online). Strain SS011B1-4T was oxidase- and catalase-positive. Tween 80 was hydrolysed, whereas starch, gelatin and urease were not. The strain produced indole and weakly produced H2S. In addition, the isolate was positive for nitrate-reducing activity, but negative for nitrite reduction, Voges–Proskauer and methyl red tests. Strain SS011b1-4T was sensitive to all the antibiotics tested, including kanamycin, tetracycline, ampicillin, chloramphenicol, streptomycin, erythromycin and gentamicin. In contrast to some species of the genus Marinobacter that have been reported to utilize aliphatic hydrocarbons, even crude oil (Gauthier et al., 1992; Huu et al., 1999; Shivaji et al., 2005; Antunes et al., 2007), strain SS011B1-4T could not degrade n-tetradecane, toluene or crude oil. However, we found that M. gudaonensis could utilize crude oil as a carbon source, which was not described by Gu et al. (2007). The other main characteristics of the strain are given in Table 1 and in the species description.
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. The predominant cellular fatty acids of strain SS011B1-4T were C18 : 19c (47.4 %), C16 : 0 (21.2 %), C12 : 0 3-OH (8.1 %), C19 : 010c cyclo (5.8 %), C16 : 19c (5.6 %) and C12 : 0 (4.7 %). The fatty acid profile was similar to those of other Marinobacter species, in particular that of M. bryozoorum (Spröer et al., 1998; Huu et al., 1999; Martín et al., 2003; Yoon et al., 2004; Romanenko et al., 2005). Strain SS011B1-4T contained ubiquinone-9 (Q9) as the main respiratory lipoquinone, and phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and an unknown glycolipid as the major polar lipids. The DNA G+C content of strain SS011B1-4T was 62.2 mol%, within the range of reported Marinobacter species (55.0–63.2 mol%) (Gauthier et al., 1992; Huu et al., 1999; Yoon et al., 2003, 2004; Martín et al., 2003; Gorshkova et al., 2003; Shivaji et al., 2005; Romanenko et al., 2005; Green et al., 2006; Liebgott et al., 2006; Kim et al., 2006; Gu et al., 2007; Antunes et al., 2007).
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). In addition, 16S rRNA gene sequence similarities between strain SS011B1-4T and M. lipolyticus and M. excellens were 96.8 and 95.1 %, respectively. Despite the high 16S rRNA gene sequence similarity between strain SS011B1-4T and related Marinobacter species, DNA–DNA relatedness values between strain SS011B1-4T and M. bryozoorum, M. gudaonensis, M. lipolyticus and M. excellens were 26.3 % (SD, 5.3 %), 17.3 % (SD, 6.9 %), 20.1 % (SD, 8.1 %) and 28.6 % (SD, 6.2 %), respectively, far below the threshold suggested for species delineation (Wayne et al., 1987).
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Description of Marinobacter segnicrescens sp. nov.
Marinobacter segnicrescens (seg.ni.cres'cens. L. adj. segnis slow; L. part. adj. crescens growing; N.L. part. adj. segnicrescens slowly growing, referring to the slow growth of the type strain).
Cells are Gram-negative, ellipsoid-shaped (0.6–0.7 µm in width and 0.9–1.2 µm in length) and motile coccobacilli. Colonies on MA are smooth, circular, flat, slightly transparent and cream-coloured or light yellow after 3–5 days cultivation at 30 °C. Cultivation for 3 days is required to obtain a cell concentration similar to that obtained after 1 day for other Marinobacter species. No spores are observed and cells are not heat-resistant. Growth occurs in 1–15 % (w/v) NaCl (optimum, 4–8 % NaCl), at temperatures between 15 and 45 °C (optimum, 30–37 °C) and at pH 6.0–10.0 (optimum, 7.5–8.0). Positive for catalase, oxidase, nitrate reduction, indole and H2S production, but negative for nitrite reduction, Voges–Proskauer and methyl red tests. Hydrolyses Tween 80, but not starch, gelatin or urease. Sensitive to kanamycin, tetracycline, ampicillin, chloramphenicol, streptomycin, erythromycin and gentamicin. Utilizes D-fructose, D-glucose, trehalose, D-gluconic acid, i-erythritol, myo-inositol, D-sorbitol, sucrose, acetate, citric acid, succinic acid, propionate, L-sorbose, pyruvate, D-ribose and malic acid, but not dextrin, maltose, D-mannitol, L-arabinose, cellobiose, D-galactose, 
-D-lactose, D-mannose, D-melibiose, D-raffinose, L-rhamnose, L-alanine, L-proline, formic acid, DL-lactic acid, malonic acid, L-glutamic acid, D-xylose, ethanol, L-isoleucine, L-arginine, D-melezitose, L-lysine, glycine, toluene, n-tetradecane or crude oil. The major respiratory lipoquinone is Q9 and the main cellular polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and an unknown glycolipid. The predominant cellular fatty acids are C18 : 19c, C16 : 0, C12 : 0 3-OH, C19 : 010c cyclo, C16 : 19c and C12 : 0. The DNA G+C content of the type strain is 62.2 mol%.
The type strain, SS011B1-4T (=LMG 23928T=CGMCC 1.6489T), was isolated from benthic sediment of the South China Sea.
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ACKNOWLEDGEMENTS |
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