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State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China
Correspondence
Shuang-Jiang Liu
shuangjiang{at}hotmail.com
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Sanguibacter marinus 1-19T is AJ783958.
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and currently contains only three species with validly published names, Sanguibacter inulinus (Pascual et al., 1996), Sanguibacter keddieii and Sanguibacter suarezii (Fernández-Garayzábal et al., 1995). Members of this genus were isolated from the blood of healthy cows. Data on the occurrence of some other Sanguibacter strains in marine sediments or subsurface environments, such as oil reservoirs, are available from public databases, e.g. GenBank accession no. AB126692. During an investigation into the bacterial community of coastal sediment from the Eastern China Sea, a novel strain, 1-19T, was isolated. The present study was designed to clarify the taxonomic position of this strain.
Strain 1-19T was isolated on plates of Difco marine agar 2216 (Difco) that had been seeded with a coastal sediment suspension and incubated at 30 °C for 5 days. The sediment sample was collected from the Eastern China Sea, Fujian province, China. The isolate was maintained on nutrient agar or trypticase soy agar (TSA; BBL) slants at 4 °C and as a glycerol suspension (20 %, v/v) at –20 °C. Biomass for the chemical and molecular systematic studies was prepared as previously described (Huang et al., 2002) but modified by growing the strain for 3 days.
Cultural and morphological properties were examined by routine observation and with an electron microscope (FEI Quanta) after aerobic growth on TSA for 2–5 days at 30 °C. The ability of the strain to grow anaerobically was tested using the GENbox anaerobic system (bioMérieux) by incubating the TSA plate in an anaerobic jar. Flagellum staining was performed according to Heimbrook et al. (1989). General phenotypic tests were carried out following the procedures of Gordon et al. (1974) and Schofield & Schaal (1981). Fermentation of carbohydrates was determined using the API 50 CH kit (bioMérieux) according to the manufacturer's instructions and the reactions were recorded after incubation for 2, 5, 10 and 15 days at 30 °C. Cell-wall peptidoglycan was prepared by extraction with a hot solution of 10 % trichloroacetic acid, followed by treatment with trypsin in phosphate buffer (5 mg trypsin in 10 ml 0·1 M potassium phosphate buffer, pH 7·9) according to the rapid screening procedure described by Schleifer & Kandler (1972). After two washes with distilled water, the pellet was hydrolysed with 4 M HCl at 100 °C for 60 min. Amino acids and peptides in the hydrolysates were separated by two-dimensional ascending TLC on cellulose sheets using two solvent systems. The first direction was developed with 2-propanol/acetate/H2O (75 : 10 : 15) and the second was developed using 
-picoline/25 % NH4OH/H2O (70 : 2 : 28). The developed cellulose sheets were stained by spraying with ninhydrin solution and heating at 70 °C for 5 min. Cellular menaquinones were extracted and purified as described by Collins (1985) and were analysed by HPLC (Wu et al., 1989). Mycolic acids were detected using the acid methanolysis procedure (Minnikin et al., 1975). Cellular fatty acids were extracted, methylated and analysed by GC using the Sherlock MIDI (Microbial Identification) system.
Genomic DNA of strain 1-19T was extracted according to the method of Marmur (1961). PCR amplification and sequencing of the 16S rRNA gene were performed as described previously (Zhang et al., 2003). The nucleotide sequence was obtained automatically by using a DNA sequencer (model 377; Applied Biosystems) and software provided by the manufacturer. The 16S rRNA gene sequence of strain 1-19T was aligned manually against corresponding sequences retrieved from the GenBank database using the CLUSTAL X 1.8 program (Thompson et al., 1997). Phylogenetic trees were inferred by using the least-squares (Fitch & Margoliash, 1967), maximum-parsimony (Kluge & Farris, 1969) and neighbour-joining (Saitou & Nei, 1987) algorithms from the PHYLIP package version 3.5c (Felsenstein, 1993). Evolutionary distance matrices were generated as described by Kimura (1980). The resulting unrooted tree topologies were evaluated by bootstrap analysis (Felsenstein, 1985) of the neighbour-joining method based on 1000 resamplings. The PHYLIP package (Felsenstein, 1993) was used for all phylogenetic analyses. The DNA G+C content was determined using the thermal denaturation (Tm) method (Marmur & Doty, 1962) with Escherichia coli K-12 as the standard.
Comparison of the almost complete 16S rRNA gene sequence of strain 1-19T (1486 nucleotides) with other sequences found by a BLAST search (Altschul et al., 1990) of the GenBank database showed that strain 1-19T formed a distinct phyletic line at the periphery of the evolutionary clade occupied by the genus Sanguibacter (Fig. 1). The taxonomic integrity of the strain and the three Sanguibacter species with validly published names was supported by the results obtained with all three tree-making algorithms and by a 100 % bootstrap value based on the neighbour-joining method. The 16S rRNA gene sequence similarities between strain 1-19T and its nearest neighbours were 96·9 % (S. inulinus), 96·8 % (S. keddieii) and 96·7 % (S. suarezii). These values fall into the range recorded for many coryneform bacteria at the species level (Behrendt et al., 2002; Fernández-Garayzábal et al., 1995; Osorio et al., 1999; Pascual et al., 1996; Yassin et al., 2002). Similarity values between strain 1-19T and members of other genera were below 96·0 %.
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Description of Sanguibacter marinus sp. nov.
Sanguibacter marinus (ma.ri'nus. L. masc. adj. marinus pertaining to the sea, where the isolate was found).
Cells are Gram-positive, non-endospore-forming, short, irregular, motile rods with sparse peritrichous flagella. Colonies are circular, convex with entire edges and pale yellow. Diffusible pigments are not produced. Growth occurs under aerobic and anaerobic conditions on TSA medium. Catalase-positive and oxidase-negative. Growth occurs at 15–37 °C (optimum 25–30 °C), pH 5·5–9·0 and 0–7 % (w/v) NaCl. No growth at 42 °C and 10 % NaCl. Citrate is not utilized. Aesculin, gelatin and starch are hydrolysed, but casein, cellulose and Tween 80 are not. Methyl red and indole tests are positive, but Voges–Proskauer test is negative. Acid is produced from L-arabinose, arbutin, D-cellobiose, D-fructose, D-galactose, D-glucose, glycogen, D-lactose, D-maltose, D-mannose, salicin, D-sucrose, D-trehalose and D-xylose, but not from D-adonitol, D-arabinose, DL-arabitol, dulcitol, meso-erythritol, DL-fucose, inositol, 2-ketogluconate, D-melezitose, D-raffinose, L-sorbose, D-tagatose, xylitol or L-xylose. Additional biochemical characteristics are given in Table 1
. The cell-wall peptidoglycan type is the same as that of S. keddieii. The menaquinones are MK-9(H4) and MK-9(H6) (peak area ratio, 10·4 : 1). The cellular fatty acid profile consists mainly of anteiso methyl-branched type anteiso-C15 and straight-chain saturated components. The DNA G+C content is 73·4 mol% (Tm method).
The type strain, 1-19T (=CGMCC 1.3457T=JCM 12547T), was isolated from coastal sediment collected in the Fujian province of China.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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