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1 Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung 811, Taiwan
2 Department of Marine Biotechnology, National Kaohsiung Marine University, Kaohsiung, Taiwan
3 Department of Aquaculture, National Kaohsiung Marine University, Kaohsiung, Taiwan
4 Division of Biology and Ocean Sciences, Inha University, Yonghyun Dong, Incheon 402-751, Republic of Korea
Correspondence
Wen-Ming Chen
p62365{at}ms28.hinet.net
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ABSTRACT |
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7c (49.1 %), and significant amounts of 19 : 0 cyclo (24.6 %) and 16 : 0 (14.7 %) were present. The DNA–DNA relatedness of the strain with respect to recognized species of the genus Oceanicola was less than 70 %. The isolate was also distinguishable from members of the genus Oceanicola on the basis of phenotypic and biochemical characteristics. It is evident from the genotypic, chemotaxonomic and phenotypic data, therefore, that strain AZO-CT represents a novel species of the genus Oceanicola, for which the name Oceanicola marinus sp. nov. is proposed. The type strain is AZO-CT (=LMG 23705T=BCRC 17591T).
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MAIN TEXT |
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, belongs to the order Rhodobacterales of the class Alphaproteobacteria and currently comprises three species with validly published names: Oceanicola granulosus, Oceanicola batsensis and Oceanicola nanhaiensis. O. granulosus strains HTCC2516T and HTCC2523 and O. batsensis HTCC2597T are poly-
-hydroxybutyrate-producing marine bacteria that were isolated from the Bermuda Atlantic Time-series Study (BATS) site by means of a high-throughput culturing method based on dilution to extinction in an oligotrophic seawater-based medium (Cho & Giovannoni, 2003b). O. nanhaiensis SS011B1-20T was isolated from samples of sediments from the South China Sea (Gu et al., 2007). The aim of the present study was to determine the taxonomic position of an Oceanicola-like isolate, strain AZO-CT, obtained from seawater collected off the coast of Taiwan. A seawater sample was collected (by a diver) in a sterile bottle at a depth of 4–5 m off the Eluanbi coast (Pingtung County, southern Taiwan), and was transported to the laboratory within 3–4 h. Strain AZO-CT was isolated using a standard dilution plating method on marine agar 2216 (MA; BD Difco). After incubation of the plates at 25 °C for 5 days, strain AZO-CT was purified as single colonies. The strain was preserved at –80 °C as a 20 % (v/v) glycerol suspension in marine broth 2216 (MB; BD Difco) or by lyophilization with 20 % (w/v) skimmed milk as the cryopreservation solution. Strain AZO-CT was the subject of a polyphasic taxonomic study.
The bacterial cells were observed by phase-contrast microscopy (DM 2000; Leica) in the lag, exponential and stationary phases of growth to ascertain their morphology. The motility of cells was tested by using the hanging drop method. The Spot Test flagella stain (BD Difco) was used to stain any flagella that might be present. A Gram-stain set (BD Difco) and the Ryu non-staining KOH method (Powers, 1995) were used to ascertain the Gram reaction of strain AZO-CT. Poly--hydroxybutyrate granule accumulation was observed under light microscopy after staining of the cells with Sudan black. Colony morphology was examined using a stereoscopic microscope (SMZ 800; Nikon). The pH range for growth was examined in MB with the appropriate biological buffers (pH 4–10, using increments of 0.5 pH units) (Chung et al., 1995). Tolerance of various NaCl concentrations was tested in nutrient broth prepared according to the formula of the Difco medium, except that the NaCl concentration was altered as required (0, 0.5 and 1.0–10 %, w/v, using increments of 1.0 %). Growth at various temperatures (4–45 °C) was measured in MB. Cellular growth was determined by measuring the turbidity (OD600) of cultures grown at various pH values, NaCl concentrations and temperatures. Anaerobic cultivation was performed on MA, using the Oxoid AnaeroGen system.
Extraction of genomic DNA, PCR amplification and sequencing of the 16S rRNA gene were carried out as described previously (Chen et al., 2001). The 16S rRNA gene sequences of strain AZO-CT were obtained using a DNA sequencer (ABI Prism 310; Applied Biosystems) and the DNA sequences were then assembled using the Fragment Assembly System program from the Wisconsin package version 9.1 (GCG, 1995). An almost-complete 16S rRNA gene sequence (1396 nt) of the strain was compared against 16S rRNA gene sequences available from the Ribosomal Database Project and GenBank databases. Multiple sequence alignment including strain AZO-CT and its closest relatives was performed using BioEdit software (Hall, 1999) and MEGA, version 3.1 (Kumar et al., 2004). Phylogenetic trees were inferred by using the least-squares (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Kluge & Farris, 1969) and neighbour-joining (Saitou & Nei, 1987) algorithms. An evolutionary distance matrix was generated for the neighbour-joining algorithm by using the Jukes & Cantor (1969) distance model; bootstrap analysis for the neighbour-joining tree was performed on the basis of 1000 resamplings. A comparison of the 16S rRNA gene sequence of strain AZO-CT with those of members of genera of the order Rhodobacterales in the Alphaproteobacteria showed that the strain fell within the evolutionary radiation occupied by the genus Oceanicola (Fig. 1). In the phylogenetic tree based on the neighbour-joining algorithm, strain AZO-CT formed a coherent clade with O. batsensis HTCC2597T and O. nanhaiensis SS011B1-20T, and this clade had moderate bootstrap support (>70 %). The relationship between strain AZO-CT and O. granulosus HTCC2516T, however, did not have robust bootstrap support (<50 %). Similar topology was obtained in phylogenetic trees generated with the maximum-parsimony and maximum-likelihood algorithms (data not shown). According to the pairwise sequence comparisons, strain AZO-CT was most closely related to O. batsensis HTCC2597T (95.6 % similarity), followed by O. nanhaiensis SS011B1-20T (94.5 % similarity) and O. granulosus HTCC2516T (94.0 % similarity). The levels of 16S rRNA gene sequence similarity between strain AZO-CT and other species with validly published names within the order Rhodobacterales of the Alphaproteobacteria were below 94 %.
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. O. batsensis HTCC2597T and O. granulosus HTCC2516T showed only 25.6±5.1 % and 7.2±2.8 % DNA–DNA relatedness, respectively, with respect to strain AZO-CT. These DNA–DNA relatedness values were significantly lower than the level accepted as demarcating species (Wayne et al., 1987). On the basis of these DNA–DNA relatedness data, isolate AZO-CT warrants separate species status within the genus Oceanicola.
The DNA G+C content of strain AZO-CT was estimated, in duplicate, as described by Mesbah et al. (1989). The nucleoside mixture was separated by means of HPLC. The DNA G+C content of strain AZO-CT was found to be 70.9±1.0 mol% and was within the range previously reported for Oceanicola species (64.7–71.5 mol%). Biomass of AZO-CT was obtained after growing the strain on MA at 30 °C for 3 days. Fatty acid methyl esters were prepared, separated and identified according to the instructions for the Microbial Identification System (Microbial ID; MIDI) (Sasser, 1990). The predominant fatty acid constituents of strain AZO-CT were 18 : 17c (49.1 %), 19 : 0 cyclo (24.6 %) and 16 : 0 (14.7 %) (Table 1). The fatty acid profile of the strain was in good agreement with those of members of the genus Oceanicola (Cho & Giovannoni, 2004; Gu et al., 2007).
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, b). Additional biochemical tests were performed using the Biolog GN2, API ZYM (bioMérieux) and API 20NE (bioMérieux) microtest systems according to the methods outlined by the manufacturers. The presence of flexirubin, carotenoid and bacteriochlorophyll a pigments was investigated as described by Reichenbach (1992), Richards (1994) and Schmidt et al. (1994). Sensitivity to antibiotics was examined after spreading cells (0.5 McFarland standard) on MA and applying antibiotic discs containing the following: ampicillin (10 µg), chloramphenicol (30 µg), erythromycin (15 µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid (30 µg), novobiocin (30 µg), rifampicin (5 µg), penicillin G (10 µg), streptomycin (10 µg) or tetracycline (30 µg). The effects of the antibiotics on cell growth were assessed after 3 days incubation, and susceptibility was scored on the basis of the distance from the edge of the clear zone to the disc. The cells were scored as ‘susceptible’ if the distance was greater than 3 mm, as ‘moderately susceptible’ if the distance was 1–3 mm and as ‘resistant’ if the distance was less than 1 mm. Detailed results from the phenotypic and biochemical analyses of strain AZO-CT are provided in Table 2 and in the species description. Phenotypic characteristics that serve to differentiate strain AZO-CT from the type strains of the genus Oceanicola are presented in Table 2.
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) and biochemical traits (Table 2) of the strain. It is clear from the genotypic and phenotypic data that strain AZO-CT represents a novel species within the genus Oceanicola. The name Oceanicola marinus sp. nov. is proposed for this taxon.
Description of Oceanicola marinus sp. nov.
Oceanicola marinus (ma'ri.nus. L. masc. adj. marinus of the sea, marine, referring to the isolation of the type strain from seawater).
Cells are Gram-negative (according to Gram staining and the KOH test), non-motile, non-spore-forming, short rods that are 0.5 µm wide and 0.9–1.0 µm long. Colonies on MA are cream–white in colour, circular and convex with entire edges. Colonies are approximately 0.8–2.0 mm in diameter on MA after 72 h incubation at 25 °C. Growth occurs at 4–42 °C, 2–8 % NaCl and pH 6–9. Optimal growth occurs at 28–35 °C, 3–5 % NaCl and pH 7.0. Cells are facultatively anaerobic. Neither carotenoid/flexirubin pigments nor bacteriochlorophyll a is found. Positive for the following characteristics: indole production, aesculin hydrolysis, oxidase, catalase, urease, alkaline phosphatase, esterase (C4), lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and 
-glucosidase. Negative for the following characteristics: nitrate reduction, glucose fermentation, arginine dihydrolase, gelatin hydrolysis, trypsin, -chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase. The following carbon substrates (Biolog GN2) are oxidized: dextrin, adonitol, arabinose, arabitol, cellobiose, D-fructose, D-galactose, -D-glucose, maltose, D-mannitol, D-mannose, L-rhamnose, sucrose, D-trehalose, turanose, xylitol, methyl pyruvate, monomethyl succinate, acetic acid, cis-aconitic acid, citric acid, formic acid, D-galactonic acid lactone, -hydroxybutyric acid, 
-hydroxybutyric acid, -ketoglutaric acid, DL-lactic acid, propionic acid, succinic acid, bromosuccinic acid, succinamic acid, D-alanine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-histidine, L-proline, L-pyroglutamic acid, L-serine, -aminobutyric acid, urocanic acid, inosine, uridine, thymidine and 2-aminoethanol. Cannot oxidize -cyclodextrin, glycogen, Tweens 40 and 80, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, i-erythritol, L-fucose, gentiobiose, myo-inositol, -D-lactose, lactulose, melibiose, methyl -D-glucoside, D-psicose, D-raffinose, D-sorbitol, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, -hydroxybutyric acid, p-hydroxyphenylacetic acid, itaconic acid, -ketobutyric acid, -ketovaleric acid, malonic acid, quinic acid, D-saccharic acid, sebacic acid, glucuronamide, alaninamide, glycyl L-glutamic acid, L-alanyl glycine, glycyl L-aspartic acid, hydroxy-L-proline, L-leucine, L-ornithine, L-phenylalanine, D-serine, L-threonine, DL-carnitine, phenylethylamine, putrescine, 2,3-butanediol, glycerol, DL--glycerol phosphate, glucose 1-phosphate or glucose 6-phosphate. Resistant to ampicillin, nalidixic acid and penicillin G and sensitive to chloramphenicol, erythromycin, gentamicin, kanamycin, novobiocin, rifampicin, streptomycin and tetracycline. The predominant fatty acid is 18 : 17c and there are significant amounts of 19 : 0 cyclo and 16 : 0. The DNA G+C content is 70.9 mol%.
The type strain, AZO-CT (=LMG 23705T=BCRC 17591T), was isolated from a seawater sample collected off the Eluanbi coast of Pingtung County, southern Taiwan.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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