, 1996). In the genus Flavobacterium, several new species have been described with a rather high 16S rRNA gene sequence similarity, for example Venetoclax the type strains of Flavobacterium weaverense and Flavobacterium segetis share 98.9% 16S rRNA gene sequence similarity, and yet, they have a DDH value of only 34% (Yi & Chun, 2006). Because protein-encoding genes are generally less conserved (Ochman & Wilson, 1987), they may be more appropriate for phylogenetic analysis of closely related species.

Several protein-encoding genes such as glnA, recA and hsp60 have been used for typing and taxonomical purposes within genera in the Bacteroidetes (Gutacker et al., 2002; Sakamoto et al., 2010). In this study, the gyrB gene, encoding for the B subunit of the DNA gyrase, was selected because it was previously used successfully to distinguish between closely related taxa affiliated with the genus Flavobacterium Sunitinib (Suzuki et al., 1999, 2001). Izumi et al. (2003) reported on the use of gyrB primers in a PCR-restriction fragment length polymorphism analysis for the genotyping of F. psychrophilum, and Suzuki et al. (1999) designed gyrB primers to study the phylogenetic relationship for the genus Marinilabilia (Bacteroidetes) and related taxa. We tested all primers reported in these studies in silico on the gyrB sequences available from related genera and from the complete genome of F. johnsoniae DSM 2064

and found considerable mismatches with all groups included in the comparison. Therefore, more general primers were designed based on the available sequence Phospholipase D1 information. As expected

for a more variable housekeeping gene, the distance between the Flavobacterium groups and the type strains is significantly higher in the gyrB gene dendrogram (Figs 2 and S2) in comparison with the 16S rRNA gene dendrogram (Figs 1, S1 and Table 3). The threshold for species definition has been suggested to be 98.7–99.0% 16S rRNA gene sequence similarity byStackebrandt & Ebers (2006), hereas for the gyrB phylogeny, this is less well documented. Suzuki et al. (2001) reported that the proposed limit for species identity, the 70% DNA reassociation value, corresponds to 88.8%gyrB sequence similarity in the subset of the Bacteroidetes they studied, whereas several other studies revealed a wide range of interspecies similarity values [60.0–89.0%gyrB gene sequence similarity within the genus Helicobacter (Epsilonproteobacteria) (Hannula & Hanninen, 2007), 75.4–95.0% within the genus Bacillus (Firmicutes) (Wang et al., 2007), 85.0–97.5% within the genus Aeromonas (Gammaproteobacteria) (Yanez et al., 2003), 77.5–97.6% within the genus Gordonia (Actinobacteria) (Kang et al., 2009), 89.5–98.2% within the genus Kribbella (Actinobacteria) (Kirby et al., 2010) and 70.1–98.7% within the genus Streptococcus (Firmicutes) (Itoh et al., 2006)].