International Journal of Systematic and Evolutionary Microbiology (2007), 57, 1327–1330 DOI 10.1099/ijs.0.64977-0 Flammeovirga kamogawensis sp. nov., isolated from coastal seawater in Japan Shoichi Hosoya and Akira Yokota Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032, Japan Correspondence Shoichi Hosoya shouichi.hosoya@mbio.jp Two strains of gliding, agarolytic bacteria, strains YS10T 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 YS10T (=IAM 15451T=NCIMB 14281T). The genus Flammeovirga, belonging to the family ‘Flammeovirgaceae’ (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, YS10T 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 % MgCl2.6H2O (w/v), 0.54 % MgSO4.7H2O (w/v) and 0.1 % CaCl2.2H2O (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 YS10T 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 (Knuc; 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 Abbreviation: ASW, artificial seawater. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequences of strains YS10T (=IAM 15451T=NCIMB 14281T) and YML5 are AB251933 and AB251934, respectively. 64977 G 2007 IUMS Printed in Great Britain 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 YS10T 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 hybridization was carried out at 40 uC for 4 h and measured fluorometrically using the method of Ezaki et al. (1989). A high level of DNA–DNA relatedness (78–106 %) was found between strains YS10T and YML5. The novel isolates showed relatively low DNA–DNA relatedness values with F. aprica IAM 14298T (3.7–5.8 %), F. arenaria NBRC 15982T (6.5– 11.1 %) and F. yaeyamensis NBRC 100898T (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 14298T, F. arenaria NBRC 15982T and F. yaeyamensis NBRC 100898T, 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 temperatures (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 1327 S. Hosoya and A. Yokota Fig. 1. Neighbour-joining phylogenetic tree showing the position of Flammeovirga kamogawensis sp. nov. and related members of the phylum Bacteroidetes based on 16S rRNA gene sequence analysis. Bootstrap values greater than 500 are given at branchpoints. Bar, 0.02 Knuc. were determined according to previously described procedures (Hosoya et al., 2006). Degradation of starch was determined using methods described by Smibert & Krieg (1994). Degradation of Tweens (20, 40, 60 and 80) and L-tyrosine was determined by the methods described by Barrow & Feltham (1993). Acid production from carbon sources was assessed using modified O/F medium (Nogi et al., 2004). Tests with the API ZYM and API 20E commercial systems (bioMérieux) were generally performed according to the manufacturer’s instructions. The API ZYM tests were read after 4 h incubation at 37 uC and API 20E tests were read after 48 h incubation at 30 uC. Cells for inoculation onto the API test strips were suspended in halfstrength ASW. Cell movement at colony edges was verified by using phase-contrast microscopy. For analysis of cellular fatty acids, cells were grown for 48 h at 30 uC on trypticase soy broth (BBL) containing 1.2 % Gellan gum, supplemented with half-strength ASW. Cellular fatty acids were analysed by using the GC-based Microbial Identification system (MIDI). Strains YS10T and YML5 were Gram-negative, aerobic, nonsporulating, non-fruiting, gliding bacteria. The novel isolates formed reddish-orange colonies in the exponential growth phase, but turned white in the late stationary growth phase. Cells were long rods, 0.6–1.0 mm wide by 2.0–40 mm long. The phenotypic characteristics are given in the species description. Strains YS10T and YML5 can be differentiated from species of the genus Flammeovirga by several characteristics (Table 1). Phenotypic differences between the novel isolates and F. aprica IAM 14298T, F. arenaria NBRC 15982T and F. yaeyamensis NBRC 100898T were seen in the degradation of gelatin, activity of esterase (C4), chymotrypsin, a-galactosidase, b-glucuronidase and a-glucosidase and acid production from rhamnose and xylose. 1328 On the basis of DNA–DNA hybridization, phylogenetic analysis and phenotypic characteristics, the isolates are proposed as representing a novel species of the genus Flammeovirga, for which the name Flammeovirga kamogawensis sp. nov. is proposed. Description of Flammeovirga kamogawensis sp. nov. Flammeovirga kamogawensis (kamo.ga.wen9sis. N.L. fem. adj. kamogawensis pertaining to Kamogawa, Japan, from where the organisms were isolated). Table 1. Differential characteristics of strains YS10T, YML5 and species of the genus Flammeovirga Species: 1, F. kamogawensis sp. nov. (2 strains); 2, F. aprica IAM 14298T; 3, F. arenaria NBRC 15982T; 4, F. yaeyamensis NBRC 100898T. +, Positive; 2, negative. Characteristic 1 2 3 4 Degradation of gelatin Activities of: Chymotrypsin Esterase (C4) a-Galactosidase a-Glucosidase b-Glucuronidase Acid produced from: Rhamnose Xylose DNA G+C content (mol%) + 2 2 + 2 2 2 2 2 + 2 2 + 2 + + 2 + + 2 2 + 2 2 2 2 32–33 2 + 35.6 2 2 32.7 + + 35.5 International Journal of Systematic and Evolutionary Microbiology 57 Flammeovirga kamogawensis sp. nov. Table 2. Fatty acid profiles of species of the genus Flammeovirga major fatty acids are iso-15 : 0, 16 : 0 3-OH and 20 : 4v6c (Table 2). The DNA G+C content is 32–33 mol%. Species: 1, F. kamogawensis (2 strains); 2, F. aprica IAM 14298T; 3, F. arenaria NBRC 15982T; 4, F. yaeyamensis (5 strains; data from Takahashi et al., 2006). Values are percentages (w/w) of total fatty acids. 2, Not detected; tr, trace amount (<1.0 %). The type strain, YS10T (=IAM 15451T=NCIMB 14281T), was isolated from coastal seawater off Yoshiura in Kamogawa in Japan. Fatty acid iso-13 : 0 13 : 1 AT 12-13 iso-14 : 0 14 : 0 iso-15 : 0 anteiso-15 : 0 15 : 0 iso-16 : 1 H 16 : 0 N alcohol 16 : 1v5c 16 : 0 iso-15 : 0 3-OH 15 : 0 3-OH 16 : 0 3-OH iso-17 : 0 3-OH 20 : 4v6c 18 : 0 3-OH Summed feature 2* 1 2 3 4 1.9–2.5 1.6–1.8 4.3–4.8 7.8–9.0 21.2–23.0 1.5–1.9 6.4–7.1 0–3.2 1.0–1.1 4.0–4.7 2.8–4.4 4.9–5.0 2.2–2.6 12.0–14.6 2 11.4–12.8 1.6–1.9 1.1–2.7 3.1 2.5 4.4 6.7 31.0 1.3 2.2 2 2 2 2 5.5 2.1 4.1 2 24.7 2 1.4 2.5 2.9 5.0 4.9 25.0 2.0 8.1 1.1 1.8 1.8 4.2 5.4 2.8 7.7 2 14.4 3.2 2 1.2–1.5 0.8–1.5 tr 3.7–6.2 30.5–54.3 1.5–2.6 0.7–2.1 tr 0.3–1.5 2.7–4.4 3.3–4.8 3.9–5.0 tr 8.4–10.5 2.0–2.4 15.0–23.2 0–4.6 tr *Summed feature 2 contains 14 : 0 3-OH and/or 16 : 1 iso I. References Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. 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