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1 Pacific Institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Academy of Sciences, Pr. 100 Let Vladivostoku 159, 690022, Vladivostok, Russia
2 Department of Microbiology, School of Bioscience and Biotechnology, Chungnam National University, 220 Gung-dong, Yusong, Daejon 305-764, Republic of Korea
3 Korea Research Institute of Bioscience and Biotechnology, 52 Oun-dong, Yusong, Daejon 305-333, Republic of Korea
Correspondence
Olga I. Nedashkovskaya
olganedashkovska{at}piboc.dvo.ru
or
olganedashkovska{at}yahoo.com
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ABSTRACT |
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6c, iso-C15 : 0 3-OH and iso-C17 : 0 3-OH) and the low G+C content of the DNA (35.7 mol%), indicated that the strain should be separated from these two genera. From the results of phenotypic, genotypic, chemotaxonomic and phylogenetic analyses, the bacterium should be classified as representing a novel genus and species, for which the name Mariniflexile gromovii gen. nov., sp. nov. is proposed. The type strain of Mariniflexile gromovii is KMM 6038T (=KCTC 12570T=LMG 22578T).
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Strain KMM 6038T was isolated from the sea urchin S. intermedius inhabiting Troitsa Bay, Gulf of Peter the Great, East Sea (also known as the Sea of Japan), in September 2002. For strain isolation, 0.1 ml aliquots of homogenates of sea urchin tissues were transferred onto plates of marine agar 2216 (Difco). After primary isolation and purification, strains were cultivated at 28 °C on the same medium and stored at –80 °C in marine broth (Difco) supplemented with 20 % (v/v) glycerol.
Genomic DNA extraction, PCR and sequencing of the 16S rRNA gene followed the procedures given by Han et al. (2003)
. The sequences obtained were aligned with those of representative members of selected genera belonging to the family Flavobacteriaceae by using PHYDIT version 3.2 (http://plaza.snu.ac.kr/
jchun/phydit/). Phylogenetic trees were inferred using suitable programs of the PHYLIP package (Felsenstein, 1993). Phylogenetic distances were calculated using the Kimura two-parameter model (Kimura, 1980) and trees were constructed on the basis of the neighbour-joining (Saitou & Nei, 1987), least-squares (Fitch & Margoliash, 1967) and maximum-likelihood (Felsenstein, 1993) algorithms. Bootstrap analysis was performed with 1000 resampled datasets by using the SEQBOOT and CONSENSE programs of the PHYLIP package.
Phylogenetic analysis of the almost-complete 16S rRNA gene sequence of strain KMM 6038T (1378 nt) revealed that the strain formed a distinct lineage within the family Flavobacteriaceae (Fig. 1). The strain was clustered with Yuosuana aromativorans GW1-1T in the neighbour-joining tree shown, a relationship that was also supported by the maximum-likelihood tree. However, the level of bootstrap support was only 52 %, indicating that the topology of tree was not stable. Strain KMM 6038T showed 16S rRNA gene sequence similarities of 93.6 and 93.8 % to Y. aromativorans GW1-1T and Algibacter lectus KMM 3902T, respectively, and less than 93.1 % to other members of the family Flavobacteriaceae. A. lectus KMM 3902T shared higher sequence similarity with KMM 6038T than Y. aromativorans GW1-1T but formed a separate phylogenetic cluster, given that A. lectus KMM 3902T also shared high sequence similarities with other strains such as Formosa algae KMM 3553T (95.1 %). Y. aromativorans GW1-1T shared 93.6 % sequence similarity with Gelidibacter algens ACAM 536T. Therefore, it is evident from the phylogenetic analysis that KMM 6038T is not the closest neighbour of either A. lectus or Y. aromativorans.
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and the G+C content was determined by the thermal denaturation method (Marmur & Doty, 1962). The DNA G+C content of strain KMM 6038T was 35.7 mol%.
Analysis of fatty acid methyl esters was carried out according to the standard protocol of the Microbial Identification System (Microbial ID), except that marine agar 2216 was used to obtain cell biomass. The dominant fatty acids are detailed in the species description and in Table 1. Isoprenoid quinones were extracted from lyophilized cells and analysed as described previously (Nedashkovskaya et al., 2004); the major respiratory quinone was MK-6. These results are consistent with those obtained previously for members of the family Flavobacteriaceae (Bernardet et al., 2002).
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).
Cells of strain KMM 6038T were Gram-negative, chemo-organotrophic and motile by means of gliding. The isolate did not require Na+ ions or seawater for growth, which occurred in media that contained 0–6 % NaCl, with optimum growth at 1–2 %. The maximum growth temperature of strain KMM 6038T was 37 °C, with optimum growth occurring at 23–25 °C. The pH range for growth was 5.5–10.0, with optimum growth occurring between pH 7.5 and 8.3. Other physiological and biochemical characteristics of the strain are listed in the species description and in Table 2. Strain KMM 6038T has several characteristics that help to differentiate it from its nearest neighbours, Y. aromativorans and A. lectus (Table 2). Phenotypic features such as the ability to move by means of gliding, to ferment D-glucose, to produce oxidase and to form acids from carbohydrates, together with a lower DNA G+C content, strongly support the differentiation of strain KMM 6038T from Y. aromativorans. The sea urchin isolate is able to grow without Na+ ions or seawater and does not decompose agar or starch, in contrast to A. lectus KMM 3902T. Although KMM 6038T is related most closely to strains of A. lectus, with 16S rRNA gene sequence similarity of 93.8 %, the phylogenetic tree topology placed the strain studied more closely to Y. aromativorans GW1-1T. In addition to the above-mentioned phylogenetic and phenotypic distinctiveness between KMM 6038T and its nearest neighbours, differences in cellular fatty acid composition clearly separate them.
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Description of Mariniflexile gen. nov.
Mariniflexile (Ma.ri.ni.fle'xi.le. L. adj. marinus, -a, -um marine; L. part. adj. flexilis -e pliant, pliable, flexible; N.L. neut. n. Mariniflexile a flexible marine bacterium).
Cells are rod-shaped and motile by gliding. Gram-negative. Do not form endospores. Can ferment D-glucose. Produce non-diffusible orange pigments. Chemo-organotrophs. Cytochrome oxidase-, catalase- and alkaline phosphatase-positive. The predominant cellular fatty acids are straight-chain saturated, branched-chain saturated and unsaturated fatty acids, iso-C15 : 0, anteiso-C15 : 0, iso-C15 : 1, C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (comprising C16 : 17 and/or iso-C15 : 0 2-OH). The main lipoquinone is MK-6. As determined by 16S rRNA gene sequence analysis, the genus is a member of the family Flavobacteriaceae, within the phylum Bacteroidetes. The type species is Mariniflexile gromovii.
Description of Mariniflexile gromovii sp. nov.
Mariniflexile gromovii (gro'mo.vi.i. N.L. gen. n. gromovii of Gromov, in honour of B. V. Gromov, the Russian aquatic and marine microbiologist).
In addition to characteristics given for the genus, cells range from 0.4 to 0.5 µm in width and from 2 to 3 µm in length. Colonies are circular, 2–3 mm in diameter, convex, shiny, orange and translucent on marine agar. Does not require Na+ ions for growth. Growth occurs at 4–37 °C. Optimal temperature for growth is 23–25 °C. Growth occurs with 0–6 % NaCl. Decomposes gelatin. Does not hydrolyse agar, casein, starch, Tweens 20, 40 or 80, urea, cellulose (CM-cellulose and filter paper), chitin or DNA. Forms acid from L-fucose and DL-xylose, but not from L-arabinose, D-cellobiose, D-galactose, D-glucose, D-maltose, sucrose, D-lactose, D-melibiose, L-raffinose, glycerol, inositol or mannitol. Oxidizes L-rhamnose and N-acetylglucosamine. Utilizes D-lactose, D-mannose and sucrose, but not L-arabinose, adonitol, dulcitol, mannitol, inositol, sorbitol, malonate or citrate. Nitrate is not reduced. Indole, H2S and acetoin (Voges–Proskauer reaction) production are negative. Susceptible to ampicillin, carbenicillin, lincomycin and tetracycline. Resistant to benzylpenicillin, gentamicin, kanamycin, neomycin, oleandomycin, polymyxin B and streptomycin. Cellular fatty acids accounting for more than 1.0 % of the total are iso-C15 : 1 (16.9 %), anteiso-C15 : 1 (1.6 %), iso-C15 : 0 (15.0 %), anteiso-C15 : 0 (5.4 %), C15 : 0 (13.8 %), C15 : 16c (3.1 %), iso-C15 : 0 3-OH (4.3 %), C15 : 0 2-OH (1.4 %), C15 : 0 3-OH (1.6 %), iso-C16 : 1 (1.5 %), iso-C16 : 0 (1.0 %), C16 : 0 (1.4 %), iso-C16 : 0 3-OH (2.5 %), C16 : 0 3-OH (1.1 %), C17 : 16c (1.1 %), iso-C17 : 0 3-OH (8.9 %) and summed feature 3 (8.4 %; comprising C16 : 17 and/or iso-C15 : 0 2-OH). The G+C content of the DNA is 35.7 mol%.
The type strain, KMM 6038T (=KCTC 12570T=LMG 22578T), was isolated from the sea urchin Strongylocentrotus intermedius, collected in Troitsa Bay, East Sea (also known as the Sea of Japan).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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