Flammeovirga kamogawensis sp. nov., isolated from coastal seawater in Japan Shoichi Hosoya and Akira Yokota Correspondence Shoichi Hosoya shouichi.hosoya@mbio.jp Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032, Japan Two strains of gliding, agarolytic bacteria, strains YS10 T and YML5, were isolated from coastal seawater off Kamogawa, Japan. Phylogenetic analysis based on 16S rRNA gene sequences showed that the novel isolates represent a separate lineage within the genus Flammeovirga. DNA–DNA hybridization values between these isolates and the type strains of species of the genus Flammeovirga were significantly lower than those accepted as threshold values for the phylogenetic definition of a species. Furthermore, some of the phenotypic characteristics indicate that the isolates differ from other Flammeovirga species. Based on these differences, it is suggested that the isolates represent a novel species, for which the name Flammeovirga kamogawensis sp. nov. is proposed. The type strain is YS10 T (=IAM 15451 T =NCIMB 14281 T ). The genus Flammeovirga, belonging to the family ‘Flam- meovirgaceae’ (Garrity & Holt, 2001), was described by Nakagawa et al. (1997). At present, this genus consists of three species, Flammeovirga aprica (Nakagawa et al., 1997), Flammeovirga arenaria and Flammeovirga yaeyamensis (Takahashi et al., 2006). In this study, the taxonomic positions of two novel strains, YS10 T and YML5, isolated from seawater collected from the Yoshiura coastline (Kamogawa, Chiba Prefecture, Japan) were determined. The sample (0.05 ml) was spread onto plates of SP5 agar [half-strength artificial seawater (ASW; full-strength ASW consists of 3 % NaCl (w/v), 0.07 % KCl (w/v), 1.08 % MgCl 2 .6H 2 O (w/v), 0.54 % MgSO 4 .7H 2 O (w/v) and 0.1 % CaCl 2 .2H 2 O (w/v)), 0.9 % Casitone (w/v), 0.1 % yeast extract (w/v) and 1.5 % agar (w/v)] and marine agar 2216 (MA; Difco) and incubated at 15 uC for a week. The novel agarolytic strains YS10 T and YML5 were purified and maintained at 25 uC on marine agar. The 16S rRNA gene sequences were obtained by direct sequencing of PCR-amplified DNA, as described by Hosoya et al. (2006). The most similar sequences were obtained from the GenBank database using the BLAST program (Altschul et al., 1990). Nucleotide substitution rates (K nuc ; Kimura, 1980) were determined and a distance matrix tree was constructed using the neighbour-joining method (Saitou & Nei, 1987) with the CLUSTAL_X program (version 1.83; Thompson et al., 1997). Alignment gaps and unidentified base positions were not taken into consideration in the calculation. Bootstrap analysis was based on 1000 trials. The results of the phylogenetic analysis based on 16S rRNA gene sequences showed that strains YS10 T and YML5 fall into the genus Flammeovirga (Fig. 1). The highest 16S rRNA gene sequence similarity values were found with F. aprica (95.0 %), F. arenaria (95.7 %) and F. yaeyamensis (93.5 %). For analysis of genetic relatedness, DNA–DNA hybridiza- tion was carried out at 40 uC for 4 h and measured fluoro- metrically using the method of Ezaki et al. (1989). A high level of DNA–DNA relatedness (78–106 %) was found be- tween strains YS10 T and YML5. The novel isolates showed relatively low DNA–DNA relatedness values with F. aprica IAM 14298 T (3.7–5.8 %), F. arenaria NBRC 15982 T (6.5– 11.1 %) and F. yaeyamensis NBRC 100898 T (3.8–16.8 %). This is significantly lower than the value generally accepted as the threshold value for the phylogenetic definition of a species (Wayne et al., 1987). For determination of the G+C content, DNA was extracted using the method of Saito & Miura (1963). The DNA G+C content was determined according to the method of Mesbah et al. (1989). The DNA G+C content of the novel isolates was 32–33 mol%. The values obtained for the reference strains, F. aprica IAM 14298 T , F. arenaria NBRC 15982 T and F. yaeyamensis NBRC 100898 T , were 35.6 mol%, 32.7 mol% and 35.5 mol%, respectively. The following physiological tests were performed. The respiratory quinone was analysed by the method of Komagata & Suzuki (1987). Growth at different tempera- tures (8–37 uC), salt tolerance, growth at different pH values, oxidase and catalase activities, degradation of DNA and alginate and hydrolysis of agar and carboxymethylcellulose Abbreviation: ASW, artificial seawater. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequences of strains YS10 T (=IAM 15451 T =NCIMB 14281 T ) and YML5 are AB251933 and AB251934, respectively. 64977 G 2007 IUMS Printed in Great Britain 1327 International Journal of Systematic and Evolutionary Microbiology (2007), 57, 1327–1330 DOI 10.1099/ijs.0.64977-0