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1 School of Biological Sciences, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea
2 DSMZ – German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
Correspondence
Jongsik Chun
jchun{at}snu.ac.kr
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ABSTRACT |
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The GenBank accession number for 16S rRNA gene sequence of strain JC1078T is AY382898.
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MAIN TEXT |
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). In addition, two genera, namely Ornithinicoccus and Ornithinimicrobium, have been proposed to harbour organisms containing ornithine in the peptidoglycan (Groth et al., 1999, 2001). The differentiation of these two genera was based on the peptidoglycan structure, polar lipid pattern, fatty acid profile and morphology as well as 16S rRNA gene analysis (Groth et al., 2001). In this study, we report the polyphasic characterization of a marine isolate containing ornithine in the peptidoglycan and propose to classify the organism in a novel genus and species, for which the name Serinicoccus marinus gen. nov., sp. nov. is proposed. Strain JC0178T was isolated from a surface sea-water-sample collected from the East Sea using the standard dilution plating technique. Isolation was achieved using Marine agar 2216 (MA; Difco) and incubation at 30 °C. The isolate was routinely cultured on MA and maintained as a glycerol suspension (20 %, w/v) at –80 °C.
The 16S rRNA gene was enzymically amplified from a single colony. Primers, PCR conditions and sequencing were described elsewhere (Chun & Goodfellow, 1995). The sequence of strain JC1078T was manually aligned with representative sequences of actinomycetes obtained from the GenBank database. Phylogenetic trees were inferred by using the Fitch–Margoliash (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Fitch, 1972) and neighbour-joining (Saitou & Nei, 1987) methods. Evolutionary distance matrices for the neighbour-joining and Fitch–Margoliash methods were generated according to the model of Jukes & Cantor (1969). The resultant neighbour-joining tree topology was evaluated by bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings. The alignment and phylogenetic analyses were carried out using the PHYDIT program (available at http://plaza.snu.ac.kr/
jchun/phydit/) and PAUP 4.0 (Swofford, 1998) as described (Chun et al., 2000).
A nearly complete 16S rRNA gene sequence of strain JC1078T was obtained (1420 bp). Preliminary sequence comparison against the 16S rRNA gene sequences deposited in the GenBank database indicated that our isolate belonged to the suborder Micrococcineae in the order Actinomycetales. On the basis of 16S rRNA gene similarity, the closest cultured relatives were Ornithinimicrobium humiphilum DSM 12362T (94·3 %), Kytococcus sedentarius DSM 20547T (94·1 %), Dermacoccus nishinomiyaensis DSM 20448T (93·9 %), Arthrobacter cumminsii DSM 10493T (93·7 %), Demetria terragena DSM 11295T (93·7 %), Arthrobacter albus CF-43T (93·5 %) and Janibacter limosus DSM 11140T (93·1 %). This relationship between our isolate and other members of the suborder Micrococcineae was also evident in the phylogenetic trees (Fig. 1). Strain JC1078T and Ornithinimicrobium humiphilum DSM 12362T formed a monophyletic clade with a relatively low bootstrap value (49 %) which was supported by all tree-making methods employed in this study. The positions of the other taxa that showed more than 93 % 16S rRNA gene sequence similarities to our marine isolate varied depending on the inferring algorithm.
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) supplemented by artificial sea water (ASW; Lyman & Fleming, 1940), nutrient agar (Difco) supplemented by ASW and basal medium (BM; Baumann et al., 1972).
Growth under anaerobic conditions was determined in an anaerobic chamber (10 % CO2, 10 % H2, 80 % N2; Sheldon Manufacturing). The growth range for temperature (between 5 and 50 °C with intervals of 5 °C), pH (between pH 4 and 12 with intervals of 1) and NaCl concentration [between 0 and 15 % (w/v) with intervals of 1 %] was determined using synthetic ZoBell medium (Zobell, 1941); growth was checked after up to 4 weeks.
Standard physiological and biochemical tests were performed as described previously (Smibert & Krieg, 1994). Acid-fastness was determined by Ziehl-Neelsen staining. Hydrolysis of alginic acid, casein, cellulose, chitin, starch and Tween 80 was tested using MA as the basal medium. DNase test agar (Difco) supplemented with 2·5 % (w/v) NaCl was used for DNase assay. Decomposition of adenine, hypoxanthine, L-tyrosine and xanthine was tested using MA according to Gordon et al. (1974). Production of H2S was detected in triple-sugar iron agar (TSI) supplemented with 2·5 % (w/v) NaCl. Arginine dihydrolase, 
-galactosidase, nitrate reduction, urease, acid production from glucose and indole production tests were performed using an API 20NE kit (bioMérieux), and other enzymic activities were determined using an API ZYM kit (bioMérieux). Strips were inoculated with a heavy bacterial suspension in half-strength ASW and AUX medium supplemented with 2·5 % (w/v) NaCl. -Galactosidase activity was additionally determined by streaking cultures onto MA agar plates amended with 0·1 mM IPTG and 20 µg X-Gal ml–1 (Gosink et al., 1998). Carbon source utilization was tested in 96-well tissue culture microplates as described by Gosink et al. (1998) using BM supplemented with 1 % (v/v) of vitamin solution (Staley, 1968) as basal medium.
The results of biochemical and physiological tests are presented in the genus and species description.
Extraction of fatty acid methyl esters and their gas chromatographic separation were performed by using the Microbial Identification System according to the instructions in the operating manual (MIDI, 1999). The peptidoglycan structure was elucidated by analysis of purified cell-wall hydrolysates according to Schleifer (1985). The amino acids and peptides were analysed by two-dimensional ascending TLC on cellulose plates using the solvent systems of Schleifer & Kandler (1972). The molar ratios of amino acids were determined by GC and GC/MS of N-heptafluorobutyryl amino acid isobutyl esters (MacKenzie, 1987; Groth et al., 1996). The amino terminal amino acid of the interpeptide bridge was determined by dinitrophenylation as described by Schleifer (1985). The glycolate content of bacterial cell walls was determined by the colorimetric method of Uchida & Aida (1984). Menaquinones were extracted according to Collins et al. (1977) and were analysed by HPLC (Groth et al., 1996).
DNA for determination of the G+C value was isolated using a French pressure cell (ThermoSpectronic) and purified on hydroxyapatite as described by Cashion et al. (1977). The G+C content was determined by reverse-phase HPLC of nucleosides according to Mesbah et al. (1989).
The peptidoglycan of strain JC1078T contained ornithine, alanine, glycine, serine and glutamic acid in an approximate molar ratio of 1 : 2 : 2 : 1 : 3. Labelling by 1-fluoro-2,4-dinitrobenzene revealed that serine represents the amino terminus of the interpeptide bridge. Dinitrophenylated glutamic acid could not be detected. The peptide L-ala–D-Glu typical of peptidoglycans of the A-type of cross-linkage was found in the partial hydrolysate (4M HCl, 100 °C, 45 min). Because the results of the peptidoglycan analysis are inconsistent with all hitherto published structures of peptidoglycans containing ornithine and serine (http://www.dsmz.de/species/murein.htm) and because of the phylogenetic proximity to Ornithinicoccus hortensis and Ornithinimicrobium humiphilum, it is likely that strain JC1078T represents a novel variation of peptidoglycan type A4. However, a detailed peptidoglycan structure can not be concluded from the available data. The acyl type of the glycan chain of peptidoglycan is acetyl. The major menaquinone of strain JC1078T was MK-8(H4). The polar lipid pattern consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and an unidentified glycolipid. The cellular fatty acids were i-14 : 0 (1·30 %), i-15 : 1 (1·88 %), i-15 : 0 (41·65 %), ai-15 : 0 (8·18 %), 15 : 0 (0·89 %), i-16 : 0 (0·79 %), i-16 : 0 (12·83 %), 16 : 0 (1·79 %), i-17 : 1
9c (12·21 %), i-17 : 0 (9·24 %), ai-17 : 0 (6·96 %), 17 : 18c (1·00 %) and 17 : 0 (1·28 %). The DNA base composition of strain JC1078T was 72 mol% G+C.
Strain JC1078T corresponds to its phylogenetic neighbour Ornithinimicrobium humiphilum and to Ornithinicoccus hortensis in displaying an A-type peptidoglycan based on ornithine, in the DNA base composition of approximately 70 mol% G+C and in the major menaquinone MK-8(H4). However, strain JC1078T can be differentiated from both genera by the occurrence of serine in the peptidoglycan and by its polar lipid pattern, fatty acid profile and physiological characteristics as shown in Table 1. All other phylogenetically related genera of the suborder Micrococcineae can be differentiated easily from strain JC1078T by phenotypic characteristics such as the peptidoglycan structure. Based on the differentiation from all validly described genera of the suborder Micrococcineae by phylogenetic analysis, chemotaxonomic investigations and physiological studies, it is proposed to classify the new isolate in a novel genus and species, Serinicoccus marinus gen. nov., sp. nov., with the type strain JC1078T.
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Gram-positive, strictly aerobic, moderately halophilic bacteria. Oxidase-negative, catalase-positive, not acid-fast. No formation of spores. Cells are non-motile cocci. The peptidoglycan type is of the A type of cross-linkage and contains ornithine, alanine, glycine, serine and glutamic acid in an approximate molar ratio of 1 : 2 : 2 : 1 : 3. The acyl type of the glycan chain of peptidoglycan is acetyl. The major menaquinone is MK-8(H4). The major cellular fatty acids are of the iso- and anteiso-methyl-branched type. The polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and one unknown glycolipid. The DNA G+C content is 72 %. Phylogenetically, this genus is affiliated to the suborder Micrococcineae. The type species is Serinicoccus marinus.
Description of Serinicoccus marinus sp. nov.
Serinicoccus marinus (ma.ri'nus. L. masc. adj. marinus of or belonging to the sea, marine).
In addition to the characteristics that define the genus, it has the characteristics described below. Growth occurs at NaCl concentrations of 0–14 % (w/v), the optimum being 2–3 %. The pH range for growth is between pH 6 and 11, the optimum being pH 8. The temperature range for growth is 10–35 °C, with the optimum being 35 °C. Extended incubation (up to 20 days) is required at 5 °C. Cells are cocci with diameters of 0·5–0·9 µm. Colonies on MA are yellow, circular, convex, entire, glistening, opaque and butyraceous. Colonies are approximately 1 mm in diameter after 3 days (on MA at 30 °C) and reach the maximum diameter of 34 mm after 5 days. Nitrate is reduced to nitrite. Does not show arginine dihydrolase activity. -Galactosidase activity is weakly present. Does not produce H2S on TSI agar, acid from glucose in API 20NE kit or indole from tryptophan. Casein, DNA, aesculin, gelatin, starch, Tween 80 and tyrosine are decomposed; adenine, alginate, cellulose, chitin, hypoxanthine, urea and xanthine are not. Tested by the API ZYM enzyme assay, strain JC1078T is positive for esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase and 
-glucosidase; variable for trypsin and -glucosidase; negative for alkaline phosphatase, lipase (C14), cystine arylamidase, -chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, -galactosidase, -galactosidase, -glucuronidase, N-acetyl--glucosaminidase, -mannosidase or -fucosidase. Utilizes acetate, D-cellobiose, D-fructose, D-galactose, D-glucose, D-mannitol, D-mannose, D-sorbitol, D-trehalose, glycerol and sucrose as a sole carbon source. Does not utilize acetamide, benzoate, citrate, D-ribose, ethanol, glycine, inulin, 2-propanol, lactose, L-arginine, L-ascorbate, L-lysine, L-rhamnose, N-acetylglucosamine, polyethylene glycol, salicylate or tartrate. The major fatty acids are i-15 : 0, i-16 : 0, i-17 : 19c, i-17 : 0, ai-15 : 0 and ai-17 : 0. The DNA G+C content is 72 mol%.
The type strain, JC1078T (=IMSNU 14026T=KCTC 9980T=DSM 15273T), was isolated from a sea-water sample from the East Sea, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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