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1 Marine Biotechnology Research Center, Korea Ocean Research and Development Institute, Ansan PO Box 29, 425-600, Republic of Korea
2 Polar BioCenter, Korea Polar Research Institute, Korea Ocean Research and Development Institute, Ansan PO Box 29, 425-600, Republic of Korea
Correspondence
Hong Kum Lee
hklee{at}kopri.re.kr
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ABSTRACT |
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10, 15 : 0 and 16 : 19. The major respiratory quinone was MK-6. The DNA G+C content was 35.8 mol%. On the basis of this polyphasic taxonomic evidence, strain KOPRI 13342T should be classified as a representative of a novel species in a new genus in the family Flavobacteriaceae; the name Costertonia aggregata gen. nov., sp. nov. is proposed. The type strain of Costertonia aggregata is KOPRI 13342T (=KCCM 42265T=JCM 13411T).
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). In oligotrophic marine environments, bacterial colonization on surfaces is regarded as a microbial survival strategy that provides micro-organisms with important advantages, including increased access to nutrients, protection against toxins and antibiotics and retention of signal molecules (Jefferson, 2004; Pasmore & Costerton, 2003). Direct observations of a wide variety of natural ecosystems have established that the vast majority of bacteria in most aquatic environments grow within matrix-enclosed biofilms (Costerton et al., 1994). Therefore, biofilms could serve as a source of diverse micro-organisms and many novel bacterial strains have been reported from marine biofilm matrices including algal surfaces (Gillan et al., 1998; Golyshin et al., 2002; Matsuo et al., 2003; Patel et al., 2003; Nedashkovskaya et al., 2004a, b, 2005; Bowman & Nichols, 2005; Lau et al., 2004). Of these, up to 30 % could be affiliated with the phylum Bacteroidetes (Webster et al., 2004). We have also isolated many bacteria belonging to the family Flavobacteriaceae from mature biofilms; taxonomic analysis of a novel strain, KOPRI 13342T, is described herein. Approximately 10 cm3 biofilm consisting of diverse algal species on a rock-bed was harvested using a razor blade and dispersed in 30 ml sterilized seawater. The dispersed biofilm was spread on marine agar 2216 (MA; Difco) after serial dilution with sterilized seawater and cultivated at 25 °C for a week. Among the distinct colonies that grew on MA, a tiny, orange-coloured colony was isolated, strain KOPRI 13342T, and preserved in 20 % glycerol solution at –80 °C. The isolate was further cultivated on MA for morphological and biochemical characterization.
Unless otherwise stated, methods used for physiological and morphological characterization were as described previously (Sohn et al., 2004b; Kwon et al., 2005). The degradation of starch and casein by strain KOPRI 13342T was tested according to Smibert & Krieg (1994). Physiological, biochemical and morphological characteristics of strain KOPRI 13342T are given in the genus and species descriptions and in Table 1.
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. However, no growth was observed in the presence of NaCl alone or in the presence of Mg2+ and Ca2+ ions, so other components found in seawater were tested. Tested elements and their concentrations were described by Parsons et al. (1984). Combinations of four major components (CaCl2.2H2O, KCl, MgCl2.6H2O and Na2SO4) and a mixture of five trace components (H3BO3, KBr, Na2CO3, NaF and SrCl2.6H2O) were supplied to the DW substituted (including 3 % NaCl) ZoBell 2216e medium. Growth was observed in the presence of Ca2+ and K+ ions in addition to NaCl.
The profile of cellular fatty acid methyl esters was determined according to Sohn et al. (2004b). The dominant fatty acids of KOPRI 13342T were i-15 : 0 (39.7 %), i-15 : 110 (22.4 %), 15 : 0 (7.8 %) and 16 : 19 (4.6 %). The strain also contained small amounts of 16 : 0 (2.7 %), C18 polyunsaturated fatty acids (2.5 %), 18 : 0 (2.2 %), 13 : 0 (2.0 %), 10-methyl 16 : 0 (1.6 %), 18 : 19 (1.5 %) and 14 : 0 (1.2 %). The isolate contained a relatively large amount of i-15 : 0 compared with that found in members of closely related genera in the family Flavobacteriaceae (Table 1
).
The major respiratory quinone was determined to be menaquinone by the reverse-phase-TLC method described by Kim et al. (2000) and confirmed to be MK-6 by HPLC analysis according to the method described by Collins (1985). The DNA G+C content was 35.8 mol%, as determined by the thermal denaturation method (Kim et al., 2000).
Genomic DNA extraction and amplification and sequencing of the 16S rRNA gene were carried out according to Sohn et al. (2004b). A phylogenetic tree including strain KOPRI 13342T and members of closely related genera was generated based on the maximum-likelihood distance model and the neighbour-joining method. 16S rRNA gene sequences of Bacteroides fragilis ATCC 25285T (GenBank accession no. NC_003228) and Sphingobacterium spiritivorum DSM 2582T (AJ459411) served as outgroups. A total of 1331 unambiguously aligned positions was compared. The closest neighbour was Maribacter dokdonensis DSW-8T (92.4 % sequence similarity), followed by Maribacter sedimenticola KMM 3903T (92.1 %) and Maribacter orientalis KMM 3947T (92.0 %). Members of the genera Zobellia and Muricauda showed a similar range of similarities (90.7–91.5 and 90.7–91.4 %, respectively) to strain KOPRI 13342T. Phylogenetic analysis of 16S rRNA gene sequences from organisms with validly published names revealed that strain KOPRI 13342T shared a phyletic line with members of the genus Muricauda. This small clade lies within a larger clade containing the genera Arenibacter, Maribacter, Pibocella and Zobellia (Fig. 1).
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) and contained a larger amount of i-15 : 0 fatty acid, enabling it to be differentiated from other members of the family Flavobacteriaceae. Hence, it is proposed that strain KOPRI 13342T should be identified as a representative of a novel species in a new genus in the family Flavobacteriaceae, Costertonia aggregata gen. nov., sp. nov.
Description of Costertonia gen. nov.
Costertonia (Cos.ter.ton'i.a. N.L. fem. n. Costertonia honouring J. W. Costerton, a famous American biofilm microbiologist).
Cells are aerobic, motile, Gram-negative rods. Gliding motility is absent. Orange-coloured colonies form on MA. Produce non-diffusible carotenoid pigments, but flexirubin-type pigments are absent. The major respiratory quinone is MK-6. The major cellular fatty acids are i-C15 : 0, i-C15 : 1 and 15 : 0. Oxidase- and catalase-positive. As determined by 16S rRNA gene sequence analysis, the genus Costertonia is a member of the family Flavobacteriaceae, phylum Bacteroidetes. The type species is Costertonia aggregata.
Description of Costertonia aggregata sp. nov.
Costertonia aggregata (ag.gre.ga'ta. L. fem. adj. aggregata joined together, referring to the formation of aggregates during cultivation in liquid medium).
Cells are 0.50–0.57 µm in length and 0.35–0.41 µm in diameter. However, rods can sometimes be longer than 4 µm. Properties are as described for the genus in addition to the following. Growth occurs at 10–35 °C, pH 6.5–9.0 and with 1.5–12.0 % sea salts. Cells form irregular aggregates during growth in liquid medium. Obligately requires NaCl, Ca2+ and K+ for growth. Optimal growth is observed at pH 7.5–8.0 and 26–32 °C and requires the presence of 3 % (w/v) sea salts. Reduces nitrate to nitrogen gas in API 20 E test strip. Positive for 
-glucosidase, -galactosidase, urease, arginine dihydrolase and protease. Degrades dextrin, glycogen, Tweens 40 and 80, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, adonitol, L-arabinose, D-arabitol, cellobiose, L-erythritol, 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-raffinose, L-rhamnose, D-sorbitol, sucrose, D-trehalose, turanose, xylitol, methyl pyruvate, acetic acid, cis-aconitic acid, citric acid, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid, D-glucuronic acid, -hydroxybutyric acid, 
-hydroxybutyric acid, p-hydroxyphenylacetic acid, -ketoglutaric acid, -ketovaleric acid, DL-lactic acid, malonic acid, propionic acid, quinic acid, D-saccharic acid, succinic acid, bromosuccinic acid, glucuronamide, alaninamide, D-alanine, L-alanine, L-alanyl–glycine, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-histidine, hydroxy-L-proline, L-leucine, L-ornithine, L-proline, L-pyroglutamic acid, D-serine, L-serine, L-threonine, -aminobutyric acid, urocanic acid, inosine, uridine, phenylethylamine, putrescine, 2-aminoethanol, glycerol, DL--glycerol phosphate, glucose 1-phosphate and glucose 6-phosphate as sole carbon sources. The dominant fatty acids are i-15 : 0 (39.7 %), i-15 : 110 (22.4 %), 15 : 0 (7.8 %) and 16 : 19 (4.6 %).
The type strain is KOPRI 13342T (=KCCM 42265T=JCM 13411T), isolated from a mature marine biofilm formed on a rock-bed in Jungdongjin, Korea. The DNA G+C content of the type strain is 35.8 mol%.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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