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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 Institute of Marine Biology of the Far-Eastern Branch of the Russian Academy of Sciences, Pal'chevskogo St. 17, 690032, Vladivostok, Russia
Correspondence
Olga I. Nedashkovskaya
olganedashkovska{at}piboc.dvo.ru
olganedashkovska{at}yahoo.com
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ABSTRACT |
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-galactosidase- and alkaline phosphatase-positive marine bacterium, designated strain KMM 6220T, was isolated from seawater and studied by using a polyphasic taxonomic approach. The DNA G+C content of strain KMM 6220T was 59.9 mol%. The predominant fatty acids were iso-C15 : 1, iso-C15 : 0, iso-C15 : 0 3-OH, iso-C17 : 0 3-OH and C16 : 1
7/iso-C15 : 0 2-OH. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that strain KMM 6220T formed a cluster with the misclassified strains [Flexibacter] aggregans NBRC 15974 and [Flexibacter] tractuosus NBRC 16035 and with the type strains of Reichenbachiella agariperforans and Roseivirga ehrenbergii with levels of similarity of 95.9, 94.4, 92.0 and 91.8 %, respectively. On the basis of its phenotypic, chemotaxonomic, genotypic and phylogenetic characteristics, strain KMM 6220T is considered to represent a novel species of a new genus in the phylum Bacteroidetes, for which the name Fulvivirga kasyanovii gen. nov., sp. nov. is proposed. The type strain of the type species is KMM 6220T (=CCTCC AB 206119T=KCTC 12832T).
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MAIN TEXT |
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conducted the complete 16S rRNA gene sequence-based phylogenetic analysis of NBRC (National Institute of Technology and Evaluation Biological Resource Center, Tokyo, Japan) strains previously placed in the genera Flexibacter, Microscilla and Flexithrix on the basis of their phenotypic properties. They proposed restricting the genera Flexibacter and Microscilla, known by their extreme heterogeneity, to the type species Flexibacter flexilis and Microscilla marina, respectively. Nakagawa et al. (2002) suggested that other species of these genera be reclassified as new taxa based on their genomic and phenotypic characteristics. Indeed, Kämpfer et al. (2006) proposed the transfer of several species of the genus Flexibacter, [Flexibacter] sancti, [Flexibacter] filiformis and [Flexibacter] japonensis, to the genus Chitinophaga based on phylogenetic, genomic and phenotypic evidence. In addition, the species ‘Microscilla arenaria’ was recently placed in the genus Flammeovirga as Flammeovirga arenaria (Takahashi et al., 2006). Moreover, members of the genus ‘Taxeobacter’ erected by Reichenbach (1992) (name not validly published) were reclassified as species of the genus Hymenobacter, the closest phylogenetic relative of ‘Taxeobacter’ (Buczolits et al., 2006). Here, we report the isolation of a novel bacterium of the phylum Bacteroidetes isolated from seawater and its identification, based on a polyphasic approach, as a representative of a new genus.
Strain KMM 6220T was isolated from a seawater sample collected from a mussel farm located in a lagoon of Nha Trang Bay, South China Sea, Viet Nam, in January 2005. After primary isolation by direct plating on medium containing 0.5 % (w/v) Bacto peptone (Difco), 0.2 % (w/v) casein hydrolysate (Merck), 0.2 % (w/v) Bacto yeast extract (Difco), 0.1 % (w/v) glucose, 0.02 % (w/v) KH2PO4, 0.005 % (w/v) MgSO4 and 1.5 % (w/v) Bacto agar (Difco) in 50 % (v/v) natural seawater and 50 % (v/v) distilled water, the strain was cultivated on marine agar (Difco) at 25 °C for 48 h and stored at –80 °C in marine broth (Difco) supplemented with 20 % (v/v) glycerol.
Genomic DNA extraction, PCR and 16S rRNA gene sequencing were carried out as described by Kim et al. (1998). Sequence data obtained were aligned with those of representative members of the phylum Bacteroidetes using PHYDIT version 3.2 (http://plaza.snu.ac.kr/
jchun/phydit/). Phylogenetic trees were inferred by using suitable programs from the PHYLIP package (Felsenstein, 1993). Phylogenetic distances were calculated with the Kimura two-parameter model (Kimura, 1980) and trees were constructed on the basis of the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Kluge & Farris, 1969) 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 sequences revealed that strain KMM 6220T formed a distinct branch within the phylum Bacteroidetes (Fig. 1). Its nearest neighbours were [Flexibacter] aggregans IFO 15974, [Flexibacter] tractuosus IFO 16035, Reichenbachiella agariperforans KMM 3525T and Roseivirga ehrenbergii KMM 6017T with 16S rRNA gene sequence similarities of 95.9, 94.4, 92.0 and 91.8 %, respectively. Other close relatives were the misclassified species [Flexibacter] tractuosus ATCC 23168T, ‘Microscilla sericea’ ATCC 23182 and ‘Microscilla furvescens’ ATCC 23129 with levels of similarity of 89–89.3 %.
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and its G+C content was determined by using the thermal denaturation method (Marmur & Doty, 1962). The DNA G+C content of strain KMM 6220T was 59.9 mol%.
Analysis of fatty acid methyl esters was carried out according to the standard protocol of the Sherlock Microbial Identification System (MIDI Inc.). The predominant cellular fatty acids of strain KMM 6220T were iso-C15 : 0 (31.2 %), iso-C17 : 0 3-OH (23.7 %), iso-C15 : 0 3-OH (7.0 %), iso-C15 : 1 G (5.1 %) and summed feature 3 (24.7 %; comprising C16 : 17c and/or iso-C15 : 0 2-OH). Isoprenoid quinones were extracted from lyophilized cells and analysed as described by Nedashkovskaya et al. (2004a). Menaquinones were identified by comparison with known quinones from the reference strain Reichenbachiella agariperforans KMM 3525T. The main isoprenoid quinone of the novel isolate was MK-7.
Physiological and biochemical properties of strain KMM 6220T were examined as described by Nedashkovskaya et al. (2004a, b, 2005c).
Cells of strain KMM 6220T were Gram-negative, chemo-organotrophic, motile by gliding and formed irregular, smooth and yellow–brownish colonies on marine agar. The main physiological and biochemical characteristics are given in Table 1 and in the genus and species descriptions below. Strain KMM 6220T differed from its closest recognized relatives, which include species of the genera Reichenbachiella and Roseivirga, by the production of caseinase and a higher DNA G+C content (Table 1). Strain KMM 6220T could also be distinguished from Reichenbachiella agariperforans by its inability to form acid from carbohydrates. Phenotypic characteristics such as production of flexirubin-type pigments, NaCl requirement for growth and hydrolysis of agar also discriminated the novel isolate from Reichenbachiella agariperforans KMM 3525T (Table 1). The presence of amylase and production of hydrogen sulfide differentiated strain KMM 6220T from recognized species of the genus Roseivirga.
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Description of Fulvivirga gen. nov.
Fulvivirga (Ful.vi.vir'ga. L. adj. fulvus yellow–brownish; L. fem. n. virga rod; N.L. fem. n. fulvivirga a yellow–brownish-coloured rod).
Cells are strictly aerobic, Gram-negative rods. Do not form endospores. Produce non-diffusible and/or diffusible pigments. Chemo-organotrophic. Cytochrome oxidase-, catalase- and alkaline phosphatase-positive. The predominant cellular fatty acids are branched-chain saturated and unsaturated and straight-chain unsaturated, iso-C15 : 0, iso-C15 : 1, iso-C15 : 0 3-OH, iso-C17 : 0 3-OH and summed feature 3 (comprising C16 : 17 and/or iso-C15 : 0 2-OH fatty acids). The main respiratory quinone is MK-7. 16S rRNA gene sequence analysis indicates that the genus Fulvivirga is a member of the phylum Bacteroidetes. The type species is Fulvivirga kasyanovii.
Description of Fulvivirga kasyanovii sp. nov.
Fulvivirga kasyanovii [ka.sy.a'no.vi.i. N.L. gen. n. kasyanovii of Kasyanov, in honour of Vladimir L. Kasyanov (1940–2005), the famous Russian marine biologist, for his contributions to the development of marine biology and microbiology in the Far East].
The main characteristics are the same as those given for the genus. In addition, cells range from 0.2 to 0.3 µm in width and from 2.3 to 2.5 µm in length and are motile by means of gliding. On marine agar, colonies are 2–3 mm in diameter, irregular, shiny and yellow–brownish-coloured after cultivation for 1–2 days. Weakly diffusible black pigment is also produced after prolonged incubation (4–7 days) on marine agar. Growth is observed at 14–44 °C, with an optimal temperature for growth of 35–37 °C. Growth occurs with 0–10 % (w/v) NaCl, with optimum growth with 2–3 % (w/v) NaCl. Flexirubin-type pigments are not produced. Decomposes casein, gelatin, starch, elastin, DNA and Tweens 40 and 80. Does not degrade agar, Tween 20, cellulose (carboxymethylcellulose and filter paper) or chitin. Does not form acid from L-arabinose, D-cellobiose, L-fucose, D-galactose, D-glucose, D-lactose, D-maltose, D-melibiose, L-raffinose, L-rhamnose, L-sorbose, D-sucrose, DL-xylose, N-acetylglucosamine, citrate, adonitol, dulcitol, glycerol, inositol or mannitol. Nitrate is not reduced. Hydrogen sulfide is produced, but indole is not. Susceptible to carbenicillin, doxycycline and tetracycline. Resistant to ampicillin, benzylpenicillin, chloramphenicol, erythromycin, gentamicin, kanamycin, lincomycin, neomycin, oleandomycin, polymixin B and streptomycin. Fatty acids accounting for more than 1 % of the total acid content are iso-C15 : 0 (31.2 %), summed feature 3 (24.7 %; comprising C16 : 17c and/or iso-C15 : 0 2-OH), iso-C17 : 0 3-OH (23.7 %), iso-C15 : 0 3-OH (7.0 %), iso-C15 : 1 G (5.1 %), C15 : 0 (2.9 %), C16 : 15c (2.5 %) and C16 : 0 (1.2 %). The DNA G+C content of the type strain is 59.9 mol%.
The type strain, KMM 6220T (=CCTCC AB 206119T=KCTC 12832T), was isolated from a seawater sample collected from a mussel farm located in a lagoon of Nha Trang Bay, South China Sea, Viet Nam.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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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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Nedashkovskaya, O. I., Kim, S. B., Han, S. K., Rhee, M. S., Lysenko, A. M., Falsen, E., Frolova, G. M., Mikhailov, V. V. & Bae, K. S. (2004a). Ulvibacter litoralis gen. nov., sp. nov., a novel member of the family Flavobacteriaceae isolated from the green alga Ulva fenestrata. Int J Syst Evol Microbiol 54, 119–123.
Nedashkovskaya, O. I., Vancanneyt, M., Van Trappen, S., Vandemeulebroecke, K., Lysenko, A. M., Rohde, M., Falsen, E., Frolova, G. M., Mikhailov, V. V. & Swings, J. (2004b). Description of Algoriphagus aquimarinus sp. nov., Algoriphagus chordae sp. nov. and Algoriphagus winogradskyi sp. nov., from sea water and algae, transfer of Hongiella halophila Yi and Chun 2004 to the genus Algoriphagus as Algoriphagus halophilus comb. nov. and emended descriptions of the genera Algoriphagus Bowman et al. 2003 and Hongiella Yi and Chun 2004. Int J Syst Evol Microbiol 54, 1757–1764.
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