Pseudovibrio japonicus 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 A Gram-negative, motile, rod-shaped bacterium (WSF2 T ) was isolated from coastal seawater of the Boso Peninsula in Japan. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain WSF2 T represented a separate lineage within the genus Pseudovibrio. The DNA G+C content of strain WSF2 T was 51.7 mol%. DNA–DNA hybridization values between strain WSF2 T and the type strains of Pseudovibrio species were significantly lower than those accepted as the phylogenetic definition of a species. Furthermore, some biochemical characteristics indicated that strain WSF2 T differed from other Pseudovibrio species. Based on these characteristics, it is proposed that the isolate represents a novel species, Pseudovibrio japonicus sp. nov. The type strain is WSF2 T (5IAM 15442 T 5NCIMB 14279 T 5KCTC 12861 T ). The genus Pseudovibrio was proposed by Shieh et al. (2004) and originally contained one species, Pseudovibrio de- nitrificans, isolated from seawater in Taiwan. A second species, Pseudovibrio ascidiaceicola, isolated from a sea squirt, was described recently by Fukunaga et al. (2006). This genus contains halophilic, facultatively anaerobic bacteria that are motile by means of one to several lateral or subpolar flagella. Phylogenetic analyses based on 16S rRNA gene sequences have shown that the genus Pseudovibrio falls within the Alphaproteobacteria and is closely related to the genera Pannonibacter, Roseibium and Stappia (Shieh et al., 2004; Fukunaga et al., 2006). In this study, the taxonomic position of strain WSF2 T , which was isolated from surface seawater off the coastline of Nojimazaki in the Boso Peninsula, Chiba Prefecture, Japan, was examined. A seawater sample (0.05 ml) was spread onto a plate containing modified SP5 agar [0.56 artificial seawater (ASW; 1.5 % NaCl, 0.035 % KCl, 0.54 % MgCl 2 . 6H 2 O, 0.27 % MgSO 4 . 7H 2 O and 0.05 % CaCl 2 . 2H 2 O), 0.9 % casitone, 0.1 % extract bonito (Wako Pure Chemical Industries) and 1.5 % agar] and incubated at 25 u C for a week. Strain WSF2 T was isolated using the dilution-plating technique at 25 u C. This isolate was maintained at 25 uC on marine agar 2216 (MA; Difco). The 16S rRNA gene sequence was analysed as described by Hosoya et al. (2006). Sequences were edited and assembled using the program BIOEDIT (Hall, 1999). The most closely related sequences were found using the program BLAST of the GenBank database (Altschul et al., 1990). Multiple alignments were performed using the program CLUSTAL_X (version 1.83; Thompson et al., 1997). Nucleotide substi- tution rates (K nuc ; Kimura, 1980) were determined. Phylogenetic trees were constructed using the neighbour- joining method (Saitou & Nei, 1987) with CLUSTAL_X and the maximum-parsimony method (Fitch, 1971) with the software MEGA3 (Kumar et al., 2004). Alignment gaps and unidentified base positions were not taken into considera- tion in the calculation. The topology of the phylogenetic trees was evaluated by performing a bootstrap analysis with 1000 bootstrapped trials. The dendrogram obtained by maximum-parsimony analysis showed essentially the same topography as data determined by neighbour-joining (data not shown). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain WSF2 T falls into the genus Pseudovibrio (Fig. 1); 16S rRNA gene sequence similarity values with the type strains of P. ascidiaceicola and P. denitrificans were 98.9 and 98.3 %, respectively. For analysis of genetic relatedness, DNA–DNA hybridization was carried out at 45 u C for 4 h and measured fluorometrically using the method of Ezaki et al. (1989). Strain WSF2 T showed relatively low DNA–DNA relatedness values to P. ascidiaceicola IAM 15084 T (14.1–32.0 %) and P. denitrifi- cans JCM 12308 T (34.9–35.4 %). These values are signific- antly lower than that accepted as the phylogenetic definition of a species (Wayne et al., 1987). For determination of the G+C content, DNA was extracted by the method of Saito & Miura (1963) and G+C content was determined by the method of Mesbah et al. (1989). The G+C content of strain WSF2 T was 51.7 mol%; those of the reference strains P. ascidiaceicola IAM 15084 T and P. denitrificans JCM 12308 T were 52.1 and 51.9 mol%, respectively. Abbreviation: ASW, artificial seawater. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain WSF2 T (5IAM 15442 T 5NCIMB 14279 T 5KCTC 12861 T ) is AB246748. International Journal of Systematic and Evolutionary Microbiology (2007), 57, 1952–1955 DOI 10.1099/ijs.0.64922-0 1952 64922 G 2007 IUMS Printed in Great Britain