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1 Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, China
2 Daqing Oilfield Company Ltd, Daqing 163712, China
Correspondence
Xiao-Lei Wu
xiaolei_wu{at}tsinghua.edu.cn
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ABSTRACT |
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7c (28.61 %), C19 : 1 cyclo 7c (27.97 %), C16 : 0 (19.66 %) and C12 : 0 3-OH (8.87 %) as the predominant fatty acids and Q9 as the major ubiquinone, with the G+C content of genomic DNA being 64.0 mol%. Phylogenetic analyses based on 16S rRNA gene sequences indicated that the two strains belonged to genus of Halomonas in the Gammaproteobacteria, with the highest 16S rRNA gene sequence similarities of 96.4 % with Halomonas campisalis ATCC 700597T and 96.0 % with Halomonas desiderata FB2T. DNA–DNA relatedness of strain SL014B-69T with strain SL014B-62A2, H. campisalis ATCC 700597T and H. desiderata DSM 9502T was 97.4, 42.9 and 36.8 %, respectively. On the basis of these data, a novel species of the genus Halomonas, Halomonas gudaonensis sp. nov., is proposed for strain SL014B-69T and SL014B-62A2. The type strain is SL014B-69T (=LMG 23610T=CGMCC 1.6133T).
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), the species of the genus Halomonas were described as rod-shaped, Gram-negative, halotolerant bacteria with flagella that grow aerobically on a wide range of salt concentrations of 0.1–32.5 % (w/v) NaCl (Franzmann et al., 1987). By the end of 2005, the genus Halomonas contained almost 40 species, isolated from various environments, including saline and hypersaline lakes (Franzmann et al., 1987; Mormile et al., 1999), a saltern (Hebert & Vreeland, 1987; Lim et al., 2004), saline wetland (Martínez-Cánovas et al., 2004), saline soil (García et al., 2004), a mineral pool (Romano et al., 2005), seawater (Yoon et al., 2001), seafood (Yoon et al., 2002), a deep-sea hydrothermal vent (Kaye et al., 2004) and gill tissue of an ascidian (Romanenko et al., 2002), as well as alkaline waste (Berendes et al., 1996) and a mural painting (Heyrman et al., 2002). In this study, we report the characterization of two novel strains, SL014B-69T and SL014B-62A2, that were isolated from an oil-polluted saline soil in a coastal oilfield in eastern China. The results indicate that the two isolates represent a novel species of the genus Halomonas.
Strains SL014B-69T and SL014B-62A2 were isolated from a saline soil from Gudao, in the Shengli oilfield of eastern China, that was contaminated with crude oil; they were isolated by 10-fold dilution plating on agar plates containing only oil-produced water, and incubated for 7 days at 30 °C. The isolates were purified with restreaking on plates of artificial seawater (ASW) agar (l–1: peptone, 5 g; yeast extract, 1 g; Na2SO4, 4 g; KCl, 0.68 g; KBr, 0.1 g; H3BO3, 0.025 g; MgCl2.H2O, 5.4 g; CaCl2.2H2O, 1.5 g; SrCl2.6H2O, 0.024 g; NaHCO3, 0.2 g; Na2HPO4, 0.04 g; NH4Cl, 0.5 g; NaF, 0.002 g; pH 8.0) with 2.4 % NaCl (Eguchi et al., 1996) for 2 days at 30 °C. Growth was tested at different temperatures (5–50 °C) and at pH 2.0–12.0 in ASW medium containing 5 % (w/v) NaCl. Requirement for and tolerance of different concentrations of NaCl were determined in ASW medium with NaCl concentrations of 0–20 % (w/v).
After the strains were grown to late exponential phase on ASW agar, cell morphology and flagellum type were examined using transmission and scanning electron microscopy. Oxidase activity was tested as described by Smibert & Krieg (1994). Catalase activity was determined with 3 % (v/v) hydrogen peroxide solution. Denitrification was assessed as described by Zumft (1991). Hydrolysis of starch, gelatin and Tween 80, urease activity and growth on sole carbon sources and sole nitrogen sources were examined according to Williams et al. (1983) on ASW medium after 5–7 days incubation at 30 °C. After cells of SL014B-69T, Halomonas campisalis ATCC 700597T and Halomonas desiderata DSM 9502T were grown on ASW agar at 30 °C for 2 days, cellular fatty acid methyl esters were prepared and analysed using gas chromatography according to the instructions of the Microbial Identification System (MIDI). Fatty acid profiles were analysed with the Sherlock system (Microbial ID Inc.). Isoprenoid quinones were analysed as described by Komagata & Suzuki (1987), using HPLC fitted with a reversed-phase column (Shim-pack, VP-ODS; Shimazu). Polar lipids were extracted and tested by one- and two-dimensional TLC 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 was extracted and purified by the method of Marmur (1961) from cells grown in ASW medium for 2 days at 30 °C. The purity was assessed from the A280/A260 and A230/A260 ratios (Johnson, 1994). The G+C content of genomic DNA was determined by thermal denaturation (Marmur & Doty, 1962) using DNA from Escherichia coli K-12 as a control. The 16S rRNA gene was amplified as described by Embley (1991) with universal bacterial primers 8F (5'-AGAGTTTGATCCTGGCTCAG) and 1492R (5'-GGTTACCTTGTTACGACTT). DNA–DNA hybridization was performed in triplicate following the methods of De Ley et al. (1970) and Huß et al. (1983). The 16S rRNA gene sequence of strain SL014B-69T was aligned with those of related Halomonas species by using the MEGA software (Kumar et al., 2004). The phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987) available in the MEGA software package, version 5.0 (Kumar et al., 2004), and re-evaluated with the interior branch test of phylogeny.
Cells of strains SL014B-69T and SL014B-62A2 were Gram-negative rods, 0.3–0.4x0.75–1.65 µm, with a lateral flagellum. Spores were not present (Fig. 1). The two isolates formed creamy, smooth, irregular circular colonies on ASW agar. The ranges of NaCl concentration, pH and temperature for growth were 1–20 % NaCl (w/v) (optimum 10–15 % NaCl), pH 8.0–9.0 (optimum pH 8.0) and 10–42 °C (optimum 30 °C). The isolates were positive for oxidase, catalase, urease and denitrifying activities, but negative for hydrolysis of gelatin, starch and Tween 80. The other main characteristics that differentiate the strains from other type strains of Halomonas are listed in Table 1 and in the species description.
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). The 16S rRNA gene sequence of strain SL014B-69T showed 100, 96.4 and 96.0 % sequence similarity with those of strain SL014B-62A2, H. campisalis ATCC 700597T and H. desiderata FB2T, respectively. Whole-genome DNA–DNA hybridization was carried out between strains SL014B-69T and SL014B-62A2, H. campisalis ATCC 700597T and H. desiderata DSM 9502T. The DNA–DNA relatedness values for strain SL014B-69T with SL014B-62A2, H. campisalis ATCC 700597T and H. desiderata DSM 9502T were respectively 96.4 % (SD 4.4 %), 42.9 % (SD 2.7 %) and 36.8 % (SD 5.2 %).
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. The predominant cellular fatty acids of strain SL014B-69T were C18 : 17c (28.61 %), C19 : 1 cyclo 7c (27.97 %), C16 : 0 (19.66 %), C12 : 0 3-OH (8.87 %), which are characteristic of Halomonas species. In contrast with other closely related Halomonas species, strain SL014B-69T contained a distinct unknown fatty acid ECL 11.799 (2.98 %) and did not have C14 : 0 (Table 2). The major polar lipids of strain SL014B-69T included phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and, distinctively, an unknown glycolipid that was not detected in H. campisalis ATCC 700597T or H. desiderata DSM 9502T (Fig. 3). The predominant isoprenoid quinone in strain SL014B-69T was ubiquinone with nine isoprene units (Q9). The DNA G+C content of strain SL014B-69T was 64.0 mol%, which is in the range reported previously for Halomonas (52–68 mol%; Franzmann et al., 1987).
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Description of Halomonas gudaonensis sp. nov.
Halomonas gudaonensis (gu.dao.nen'sis. N.L. fem. adj. gudaonensis pertaining to Gudao, in the Shengli oilfield, PR China, where the type strain was isolated).
Cells are Gram-negative, aerobic rods (0.3–0.4x0.75–1.65 µm) with lateral flagella. Colonies on ASW medium are creamy, circular, smooth, opaque and approximately 1.5–2 mm in diameter after incubation at 30 °C for 2 days. Growth occurs at temperatures of 10–42 °C (optimum 30 °C), at NaCl concentrations of 1.0–20.0 % (optimum 10.0–15.0 %) and at pH 8.0–9.0. Positive for catalase, oxidase, urease and denitrifying activities. Negative for hydrolysis of Tween 80, starch and gelatin. The following substrates are utilized as sole carbon sources for growth: D-glucose, galactose, mannose, sucrose, maltose, trehalose, lactose, dextrin, ethanol, glycerol, mannitol, lactate, acetate and gluconate. Fructose, cellobiose, L-arabinose, D-xylose and ribose are not utilized. The following substrates are utilized as sole nitrogen sources for growth: L-alanine, glutamine, isoleucine, L-proline and arginine. Lysine and glycine are not utilized. The predominant isoprenoid quinine is Q9 and the cellular polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and an unknown glycolipid. The major cellular fatty acids are C18 : 17c (28.61 %), C19 : 1 cyclo 7c (27.97 %), C16 : 0 (19.66 %) and C12 : 0 3-OH (8.87 %). The DNA G+C content is 64.0 mol%.
The type strain, SL014B-69T (=LMG 23610T=CGMCC 1.6133T), was isolated from a saline soil contaminated by crude oil, collected from Gudao oil-product from the coastal Shengli oilfield in China.
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ACKNOWLEDGEMENTS |
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