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1 The Key Laboratory for Microbial Resources of Ministry of Education, Yunnan Institute of Microbiology and Laboratory for Conservation and Utilization of Bio-Resources, Yunnan University, Kunming, Yunnan, 650091, P. R. China
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
Correspondence
Wen-Jun Li
wjli{at}ynu.edu.cn or
lihxu{at}ynu.edu.cn
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ABSTRACT |
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, with meso-diaminopimelic acid as diagnostic diamino acid. The major fatty acids were ai-C15 : 0, ai-C17 : 0 and i-C16 : 0. The menaquinones were MK-7 and MK-6. The phospholipids were diphosphatidylglycerol and phosphatidylinositol. The G+C content of total DNA was 48·5 mol%. On the basis of phylogenetic and phenotypic evidence and DNA–DNA hybridization data, this isolate should be classified as a novel species of Marinococcus, for which the name Marinococcus halotolerans sp. nov. is proposed. The type strain is YIM 70157T (=DSM 16375T=KCTC 19045T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain YIM 70157T is AY817493.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Hao et al., 1984). They have meso-diaminopimelic acid in the cell wall, a DNA G+C content ranging between 43·9 and 46·6 mol%, a menaquinone system with MK-7, grow at a concentration of 20 % NaCl, and are motile cocci. A third Marinococcus species, Marinococcus hispanicus, has been transferred to the genus Salinicoccus (Ventosa et al., 1992). Therefore, at the time of writing, there are only two Marinococcus species with validly published names. M. halophilus is the predominant coccus found in most hypersaline environments (Ventosa et al., 1983; Márquez et al., 1992).
During our investigations of the extremophilic microbial flora of China (Tang et al., 2003; Li et al., 2004a, b, 2005a, b), strain YIM 70157T was recovered and characterized by a combination of genotypic and phenotypic methods. It was evident that the isolate was sufficiently distinct from the two species of the genus Marinococcus to warrant the description of a novel species, for which the name Marinococcus halotolerans sp. nov. is proposed.
Strain YIM 70157T was isolated from a hypersaline soil sample, collected from Qinghai, north-west China, by using the dilution plating method. Detailed information about the saline soils and salt lakes in Qinghai Province was given by Zhang et al. (1987). Modified medium SG (Sehgal & Gibbons, 1960) used for selective isolation contained the following (g l–1): Casamino acids (7·5), yeast extract (10·0), trisodium citrate (3·0), NaCl (2·0), KCl (2·0), MgCl2.6H2O (250), MgSO4.7H2O (1·0), FeSO4.7H2O (0·05) and MnSO4.7H2O (0·0002). MgCl2.6H2O was sterilized separately and then added to the medium. The plate was incubated at 28 °C for 2 weeks. The isolate was maintained on modified SG agar slants that contained 10 % (w/v) MgCl2.6H2O at 4 °C and as glycerol suspensions (20 %, w/v) at –20 °C. Biomass for chemical and molecular systematic studies was obtained from enrichment agar plates of modified SG agar medium incubated at 28 °C for about 4–5 days.
The morphology and motility of cells grown for 10–48 h on modified SG agar were examined by using light microscopy (model BH 2; Olympus) and transmission electron microscopy (model H-800; Hitachi). For transmission electron microscopy observation, cells were negatively stained with 1 % (w/v) phosphotungstic acid, after air drying. Observation of flagella was also performed using the Leifson flagella staining method (Leifson, 1960). Gram staining was carried out using the standard Gram reaction combined with the KOH lysis test method (Cerny, 1978). The colony colour of the isolate grown on modified SG agar medium was determined by comparing the cultures with the most suitable colour chips from the ISCC-NBS colour charts (Kelly, 1964). Growth at different temperatures, salt (NaCl, KCl and MgCl2.6H2O) concentrations and pH values was investigated as described by Tang et al. (2003), except that modified SG was used as the basic medium. Metabolic properties were determined using the API ID 32E test kits (bioMérieux) according to the manufacturer's instructions. Other physiological and biochemical tests were performed as described previously (Li et al., 2004a, b, 2005a, b).
Peptidoglycan was purified and the cell-wall amino acids and peptides in cell-wall hydrolysates were analysed by two-dimensional ascending TLC on cellulose plates using the solvent systems of Schleifer & Kandler (1972). The diaminopimelic acid isomer was identified by using the method of Rhuland et al. (1955). Analyses of polar lipids and menaquinones were performed according to published procedures (Groth et al., 1999). Analysis of the whole-cell fatty acid pattern was performed according to previously described methods (Miller, 1982) using the MIDI system (Microbial ID).
Extraction and amplification of genomic DNA for 16S rRNA gene sequence analysis were carried out as described previously (Xu et al., 2003). The DNA G+C content of strain YIM 70157T was determined by reverse-phase HPLC of nucleosides according to Mesbah et al. (1989). DNA–DNA hybridization was carried out by applying the optical renaturation method (De Ley et al., 1970; Huß et al., 1983; Jahnke, 1992) under optimal hybridization conditions.
Multiple alignments with sequences of a broad selection of related species of the order Bacillales and calculations of levels of sequence similarity were carried out using CLUSTAL X (Thompson et al., 1997). A phylogenetic tree (Fig. 1) was reconstructed using the neighbour-joining method of Saitou & Nei (1987) from Knuc values (Kimura, 1980, 1983). The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein (1985), with 1000 replicates.
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and in the species description.
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based on meso-diaminopimelic acid. The phospholipids were phosphatidylinositol and diphosphatidylglycerol. The menaquinones were MK-7 (91·6 %) and MK-6 (8·4 %). The major fatty acids were ai-C15 : 0 (37·40 %), ai-C17 : 0 (21·11 %) and i-C16 : 0 (9·07 %); the complete profile of cellular fatty acids is given in detail in the species description.
Comparison of the almost-complete 16S rRNA gene sequence (1453 nucleotide positions) of strain YIM 70157T with homologous sequences of a wide range of related type strains revealed that the closest phylogenetic relatedness is to M. halophilus DSM 20408T (99·4 % sequence similarity). The heterogeneity with respect to the second Marinococcus type strain, M. albus DSM 20748T, was significantly higher (only 89·5 % sequence similarity). A distance-matrix dendrogram is presented in Fig. 1
. The 20·8 % DNA–DNA relatedness determined between strain YIM 70157T and M. halophilus DSM 20408T was significantly lower than the 70 % value considered to be the recommended threshold value for the delineation of genomic species (Wayne et al., 1987).
The result of the 16S rRNA gene sequence comparisons clearly demonstrated that strain YIM 70157T is a member of the genus Marinococcus. Similarities in some phenotypic characteristics with respect to the only two type strains further support the inclusion of strain YIM 70157T in the genus Marinococcus. However, strain YIM 70157T differs from M. halophilus DSM 20408T by the ability to utilize mannose, dextrin and cellobiose as sole carbon sources, oxidative acid production from aesculin, and some differences in enzymic properties (Table 1). DNA–DNA hybridization confirmed the differentiation of both strains at the species level. These pieces of evidence show that the novel isolate YIM 70157T represents a hitherto unknown species of Marinococcus, for which the name Marinococcus halotolerans sp. nov. is proposed.
Description of Marinococcus halotolerans sp. nov.
Marinococcus halotolerans (ha.lo.to'le.rans. Gr. n. hals salt; L. pres. part. tolerans tolerating; N.L. part. adj. halotolerans referring to the ability of the organism to tolerate high salt concentrations).
Aerobic, Gram-positive, non-spore-forming, motile, spherical with a single polar flagellum; cell diameter is about 1·0–1·2 µm. The colony colour on most media tested is orange. Colonies are circular, opaque and approximately 1·5–1·8 mm in diameter after 24 h at 28 °C. The optimum concentration of MgCl2.6H2O for growth is 10 % (w/v) (MgCl2.6H2O can also be substituted by NaCl or KCl). The optimum growth pH and temperature are 7·0–7·5 and 28 °C, respectively. Concentration ranges of MgCl2.6H2O, NaCl and KCl for growth are 0–20, 0–25 and 0–25 %, respectively. Catalase-positive and oxidase-negative. Positive for nitrate reduction, but negative for gelatin liquefaction, ammonia production, in methyl red and Voges–Proskauer tests, and for milk peptonization and coagulation, growth on cellulose, H2S and melanin production, casein and starch hydrolysis. The following substrates are utilized: maltose, mannitol, glucose, mannose, fructose, cellobiose, salicin, acetamide, galactose, xylose and dextrin. Adonitol, arabinose, arabitol, rhamnose, inositol and sorbitol are not utilized. Acid is produced from aesculin, glucose and mannitol. The peptidoglycan type is A1 (meso-diaminopimelic acid directly cross-linked). The phospholipids contain phosphatidylinositol and diphosphatidylglycerol. The menaquinones are MK-7 (91·6 %) and MK-6 (8·4 %). The fatty acid profiles contain ai-C15 : 0 (37·4 %), ai-C17 : 0 (21·11 %), i-C16 : 0 (9·07 %), i-C17 : 0 (7·69 %), i-C15 : 0 (6·92 %), C16 : 0 (6·68 %) and small amounts of C14 : 0 (0·54 %), C15 : 0 (0·55 %), C17 : 0 (0·42 %), C18 : 0 (0·67 %), i-C14 : 0 (2·4 %), i-C18 : 0 (0·76 %), i-C19 : 0 (0·12 %), ai-C19 : 0 (0·25 %), C16 : 1
7c (0·36 %), C16 : 111c (1·16 %), C18 : 19c (0·16 %), i-C17 : 110c (0·11 %) and C16 : 17c alcohol (3·14 %). The DNA G+C content is 48·5 mol% (HPLC method).
Isolated from a saline soil sample collected from Qinghai, north-west China. The type strain is YIM 70157T (=DSM 16375T=KCTC 19045T).
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ACKNOWLEDGEMENTS |
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