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Int J Syst Evol Microbiol 56 (2006), 1635-1638; DOI  10.1099/ijs.0.64293-0
© 2006 International Union of Microbiological Societies

Mariniflexile gromovii gen. nov., sp. nov., a gliding bacterium isolated from the sea urchin Strongylocentrotus intermedius

Olga I. Nedashkovskaya1, Seung Bum Kim2, Jangryul Kwak3, Valery V. Mikhailov1 and Kyung Sook Bae3

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


   ABSTRACT
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A marine bacterium, designated strain KMM 6038T, was subjected to taxonomic analysis via a polyphasic approach. Cells of the strain were heterotrophic, orange-pigmented, Gram-negative and motile by means of gliding. 16S rRNA gene sequence analysis indicated that strain KMM 6038T was closely related to the type species of the genera Algibacter and Yeosuana, members of the family Flavobacteriaceae, with sequence similarities of 93.8 and 93.6 % to the respective type strains. However, several chemotaxonomic and phenotypic characteristics, such as the cellular fatty acid profile (iso-C15 : 0, anteiso-C15 : 0, iso-C15 : 1, C15 : 0, C15 : 1{omega}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).

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Mariniflexile gromovii KMM 6038T is DQ312294.


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In the course of a study of a microbial population of the sea urchin Strongylocentrotus intermedius, a common echinoderm from the north-western Pacific Ocean, a heterotrophic, Gram-negative, gliding and orange-pigmented marine bacterium was recovered. Based on a polyphasic study of the isolate, designated KMM 6038T, which included phylogenetic, genotypic, chemotaxonomic and phenotypic approaches, the erection of a new genus within the family Flavobacteriaceae is proposed.

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)Go. 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, 1993Go). Phylogenetic distances were calculated using the Kimura two-parameter model (Kimura, 1980Go) and trees were constructed on the basis of the neighbour-joining (Saitou & Nei, 1987Go), least-squares (Fitch & Margoliash, 1967Go) and maximum-likelihood (Felsenstein, 1993Go) 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. 1Go). 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.


Figure 1
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  		Fig. 1. Phylogenetic tree based on the 16S rRNA gene sequences of strain KMM 6038T and representative members of the family Flavobacteriaceae, constructed by the neighbour-joining method. Asterisks indicate branches that were also recovered by using the maximum-likelihood algorithm. Numbers at nodes indicate bootstrap values (%) from 1000 resamplings. Bar, 0.01 substitutions per nucleotide position.

 
DNA was isolated following the method of Marmur (1961)Go and the G+C content was determined by the thermal denaturation method (Marmur & Doty, 1962Go). 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 1Go. Isoprenoid quinones were extracted from lyophilized cells and analysed as described previously (Nedashkovskaya et al., 2004Go); the major respiratory quinone was MK-6. These results are consistent with those obtained previously for members of the family Flavobacteriaceae (Bernardet et al., 2002Go).


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  		Table 1. Fatty acid composition of Mariniflexile gromovii gen. nov., sp. nov. KMM 6038T and its close relatives

Taxa: 1, M. gromovii KMM 6038T; 2, Yeosuana aromativorans; 3, Algibacter lectus; 4, Formosa; 5,Gelidibacter; 6, Psychroserpens burtonensis; 7, Winogradskyella. Those fatty acids accounting for <1 % of the total for all taxa are not given. Data for reference taxa are taken from Bowman et al. (1997)Go, Macián et al. (2002)Go, Nedashkovskaya et al. (2004Go, 2005Go, 2006Go), Bowman & Nichols (2005)Go and Kwon et al. (2006)Go.

 
Phenotypic analysis was performed by using our previously described methods (Nedashkovskaya et al., 2004Go).

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 2Go. Strain KMM 6038T has several characteristics that help to differentiate it from its nearest neighbours, Y. aromativorans and A. lectus (Table 2Go). 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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  		Table 2. Differential characteristics of Mariniflexile gromovii gen. nov., sp. nov. KMM 6038T and its close relatives within the family Flavobacteriaceae

Data are taken from Bowman et al. (1997)Go, Macián et al. (2002)Go, Bowman & Nichols (2005)Go, Kwon et al. (2006)Go, Lau et al. (2005)Go, Nedashkovskaya et al. (2004Go, 2005Go, 2006)Go and this study. V, Variable. Strain KMM 6038T and all members of the genera shown are positive for catalase activity.

 
Thus, the polyphasic data presented herein support the conclusion that the sea urchin bacterium studied could not be affiliated to any taxa currently included in the family Flavobacteriaceae. Consequently, we propose that KMM 6038T is the type strain of a novel species in a new genus, Mariniflexile gromovii gen. nov., sp. nov.

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 : 1{omega}7 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 : 1{omega}6c (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 : 1{omega}6c (1.1 %), iso-C17 : 0 3-OH (8.9 %) and summed feature 3 (8.4 %; comprising C16 : 1{omega}7 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).


   ACKNOWLEDGEMENTS
 
This research was supported by grants from the Russian Foundation for Basic Research no. 05-04-48211, Presidium of the Far Eastern Branch of the Russian Academy of Sciences no. 06-04-96067 and Presidium of the Russian Academy of Sciences ‘Molecular and Cell Biology’. J. K. and K. S. B. acknowledge support from the KRIBB Research Initiative programme.


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Felsenstein, J. (1993). PHYLIP – Phylogeny Inference Package, version 3.5c. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, USA.

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