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1 Division of Biology and Ocean Sciences, Inha University, Incheon 402-751, Republic of Korea
2 Korean Collection for Type Cultures, Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Republic of Korea
Correspondence
Jang-Cheon Cho
chojc{at}inha.ac.kr
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7c (36.6 %), C16 : 17c and/or iso-C15 : 0 2-OH (26.7 %) and C16 : 0 (24.3 %). Based on the taxonomic data, only a distant relationship could be established between strain IMCC1877T and other Marinobacterium species; the strain therefore represents a novel species of the genus Marinobacterium, for which the name Marinobacterium litorale sp. nov. is proposed. The type strain is IMCC1877T (=KCTC 12756T=LMG 23872T).
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and the genus currently comprises the four species Marinobacterium georgiense (González et al., 1997), Marinobacterium stanieri (Satomi et al., 2002), Marinobacterium jannaschii (Satomi et al., 2002) and Marinobacterium halophilum (Chang et al., 2007). Marinobacterium stanieri and Marinobacterium jannaschii were transferred into the genus Marinobacterium from Pseudomonas (Baumann et al., 1983) and Oceanospirillum (Bowditch et al., 1984), respectively. In the course of studies to reveal the microbial community structure inhabiting coastal areas of the Yellow Sea, a novel Marinobacterium-like strain, designated IMCC1877T, was isolated from coastal water collected off Deokjeok island. Phenotypic characterization and phylogenetic analysis based on 16S rRNA gene sequences showed that strain IMCC1877T represents a novel species in the genus Marinobacterium. Strain IMCC1877T was isolated from a surface seawater sample collected at a coastal region near Deokjeok island, Korea, using a standard dilution-plating method on a marine agar 2216 (MA; Difco) plate. After incubating the agar plate at 20 °C for 1 month, strain IMCC1877T was initially isolated, further purified as single colonies and stored as 10 % (v/v) glycerol suspensions in liquid nitrogen. After determining the optimum growth temperature of the strain, cultures were grown and maintained routinely on MA at 30 °C.
The extraction of DNA, amplification of the 16S rRNA gene and sequencing of the PCR products were performed as described previously (Cho & Giovannoni, 2003) and resulted in the determination of an almost-complete 16S rRNA gene sequence (1508 bp) of the strain. To determine the phylogenetic relationships of strain IMCC1877T, its 16S rRNA gene sequence was aligned against approximately 52 500 aligned reference 16S rRNA gene sequences using the ARB package (Ludwig et al., 2004), and only 1248 unambiguously aligned nucleotide positions were used for phylogenetic analyses in the ARB package and PAUP* version 4.0 beta 10 (Swofford, 2002). Comparative 16S rRNA gene sequence analyses in the ARB database showed that the closest related type strain of a species with a validly published name to strain IMCC1877T was Marinobacterium stanieri ATCC 27130T (93.7 % sequence similarity), followed by Marinobacterium halophilum KCTC 12240T (93.5 %), Marinobacterium georgiense ATCC 700074T (92.9 %), Marinobacterium jannaschii IFO 15466T (92.5 %) and Neptunomonas naphthovorans (92.1 %). To clarify the phylogenetic position of the strain further, phylogenetic trees were generated by neighbour joining (Saitou & Nei, 1987) with the Kimura two-parameter model (Kimura, 1980), maximum parsimony (Fitch, 1971) and maximum likelihood (Felsenstein, 1981), and the resulting neighbour-joining and maximum-parsimony trees were subjected to bootstrap analyses based on 1000 resamplings. In all phylogenetic trees, strain IMCC1877T and the type strains of the four Marinobacterium species formed a monophyletic clade within the order Oceanospirillales. This monophyletic clade was supported by 80 and 60 % bootstrap proportions, respectively, in the neighbour-joining and maximum-parsimony trees (Fig. 1). Phylogenetic inferences and low 16S rRNA gene sequence similarity (92.5–93.7 %) between strain IMCC1877T and the type strains of the four Marinobacterium species indicated that strain IMCC1877T represents a novel species within the genus Marinobacterium. Although there is no ‘official’ classification of the Bacteria (Staley & Krieg, 1984), the 2nd edition of Bergey's Manual of Systematic Bacteriology (Bowman & McMeekin, 2005) and the NCBI taxonomy browser (http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/) classify the genus Marinobacterium as a member of the order Alteromonadales. The phylogenetic analyses in this study, however, strongly suggest the reclassification of the genus Marinobacterium into the order Oceanospirillales (Garrity et al., 2005).
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; Smibert & Krieg, 1994) using MA as the basal medium at 30 °C. Cell morphology was examined by energy-filtering transmission electron microscopy (LIBLA120; Carl Zeiss) and phase-contrast and epifluorescence microscopy (Nikon 80i). Anaerobic growth was tested on MA at 30 °C using both the MGC anaerobic system and the AnaeroPACK.Anaero (Mitsubishi Gas Chemical Company). Colony morphology, size and colour were examined from cultures grown aerobically on MA at 30 °C for 3 days. The catalase test was performed by addition of 3.0 % hydrogen peroxide to fresh colonies and oxidase activity was determined using Kovacs' solution (Kovacs, 1956). Biochemical tests and carbon-source utilization tests were carried out on API 20NE (bioMérieux) and API ZYM (bioMérieux) (biochemical tests) and Biolog GN2 microplates (carbon utilization), with artificial seawater (ASW) (l–1: 25.0 g NaCl, 1.0 g MgCl2.6H2O, 5.0 g MgSO4.7H2O, 0.7 g KCl, 0.15 g CaCl2.2H2O, 0.5 g NH4Cl, 0.1 g KBr, 0.27 g KH2PO4, 0.04 g SrCl2.6H2O, 0.025 g H3BO3) following the manufacturers' instructions. The following antibiotics were tested: ampicillin (10 µg), chloramphenicol (25 µg), erythromycin (15 µg), gentamicin (10 µg), kanamycin (30 µg), penicillin G (10 µg), rifampicin (50 µg), streptomycin (10 µg), tetracycline (30 µg) and vancomycin (30 µg). The DNA G+C content was analysed by using HPLC with a Discovery C18 column (5 µm, 15 cmx4.6 mm; Supelco)(Mesbah et al., 1989). Quinone content was analysed using reversed-phase HPLC analysis (Komagata & Suzuki, 1987). Cellular fatty acid methyl esters were prepared from a culture grown on MA at 30 °C for 3 days and analysed according to the instructions of the Microbial Identification System (MIDI).
Detailed morphological, physiological and biochemical characteristics of strain IMCC1877T are given in the species description and in Table 1. In summary, the strain was a Gram-negative, motile rod that was chemoheterotrophic, facultatively anaerobic, requiring NaCl for growth, and was catalase- and oxidase-positive. Characteristics that differentiate strain IMCC1877T from other Marinobacterium species are listed in Table 1. The DNA G+C content of strain IMCC1877T was 60.7±0.5 mol%. The major isoprenoid quinone type detected was Q-8, which is a typical respiratory quinone found in the order Oceanospirillales. The major fatty acids found in strain IMCC1877T were C18 : 17c (36.6 %), C16 : 17c and/or iso-C15 : 0 2-OH (26.7 %), C16 : 0 (24.3 %) and C10 : 0 3-OH (5.2 %), which are generally in agreement with those in Marinobacterium halophilum (Chang et al., 2007), but in different proportions.
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Description of Marinobacterium litorale sp. nov.
Marinobacterium litorale (li.to.ra'le. L. neut. adj. litorale of the seashore, a shallow-seawater dweller).
Cells are Gram-negative, motile, facultatively anaerobic, straight rods (1.2–2.0 µm long and 0.5–0.8 µm wide). Requires NaCl for growth. Colonies on MA are circular, smooth, convex, opaque, beige–milky in colour and 0.1 mm in diameter when observed after 2–3 days of incubation. Growth occurs at 8–42 °C, optimally at 30 °C, but not at 4 °C. Growth occurs at pH 5–12 and 1.0–7.5 % NaCl, optimally at pH 9.0 and 3.0–3.5 % NaCl. Oxidase and catalase are positive. Does not produce indole. Nitrate reduction, arginine dihydrolase, glucose fermentation, aesculin hydrolysis, gelatinase and 
-galactosidase are negative. Urea is hydrolysed. Alkaline phosphatase, esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin and acid phosphatase activities are present. Based on the tests contained in the Biolog GN2 microplate, the following carbon substrates are utilized: pyruvic acid methyl ester, succinic acid monomethyl ester, acetic acid, cis-aconitic acid, citric acid, formic acid, -hydroxybutyric acid, p-hydroxyphenylacetic acid, 
-ketoglutaric acid, DL-lactic acid, propionic acid, quinic acid, succinic acid, bromosuccinic acid, L-alaninamide, D-alanine, L-alanine, L-glutamic acid, L-proline, L-serine, 
-aminobutyric acid and phenyl ethylamine. Utilizes the following carbon sources weakly: glycogen, Tween 80, i-erythritol, D-galacturonic acid, -hydroxybutyric acid, -ketobutyric acid, malonic acid, D-saccharic acid, L-alanyl glycine, L-ornithine, putrescine and 2,3-butanediol. Dextrin, -cyclodextrin, Tween 40, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, adonitol, L-arabinose, D-arabitol, D-cellobiose, D-fructose, L-fucose, D-galactose, gentiobiose, -D-glucose, myo-inositol, -D-lactose, lactulose, maltose, D-mannitol, D-mannose, D-melibiose, methyl -D-glucoside, D-psicose, D-raffinose, L-rhamnose, D-sorbitol, sucrose, D-trehalose, turanose, xylitol, D-galactonic acid lactone, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, -hydroxybutyric acid, itaconic acid, -ketovaleric acid, sebacic acid, succinamic acid, glucuronamide, L-asparagine, L-aspartic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-histidine, hydroxy-L-proline, L-leucine, L-phenylalanine, L-pyroglutamic acid, D-serine, L-threonine, DL-carnitine, urocanic acid, inosine, uridine, thymidine, 2-aminoethanol, glycerol, DL--glycerol phosphate, -D-glucose 1-phosphate and D-glucose 6-phosphate are not utilized as carbon sources. Susceptible to ampicillin, chloramphenicol, erythromycin, gentamicin, kanamycin, penicillin G, rifampicin, streptomycin and tetracycline, but resistant to vancomycin. The cellular fatty acids are composed of C18 : 17c (36.6 %), C16 : 17c and/or C15 : 0 iso 2-OH (26.7 %), C16 : 0 (24.3 %), C10 : 0 3-OH (5.2 %), C12 : 0 (3.5 %), C14 : 0 (1.2 %), C18 : 0 (0.8 %) and C10 : 0 (0.7 %). The major isoprenoid quinone type is Q-8. The DNA G+C content is 60.6±0.5 mol%.
The type strain, IMCC1877T (=KCTC 12756T=LMG 23872T), was isolated from surface seawater of the coast of Deokjeok island, Yellow Sea, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bowditch, R. D., Baumann, L. & Baumann, P. (1984). Description of Oceanospirillum kriegii sp. nov. and O. jannaschii sp. nov. and assignment of two species of Alteromonas to this genus as O. commune comb. nov. and O. vagum comb. nov. Curr Microbiol 10, 221–230.[CrossRef]
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