5% bovine serum albumin in PBS; Calbiochem, Darmstadt, Germany) was added to each well. The plate was incubated at 37 °C for 30 min and washed three times with 1 × SSC. Following this, 100 μL of the substrate (4-methylumbelliferyl-β-d-galactopyranoside 100 μg mL−1) was added to each well and the fluorescence intensity was measured

using IWR-1 chemical structure a DTX 800 Multimode Detector (Beckman Coulter, Tokyo, Japan). DNA relatedness was expressed as a mean percentage of the homologous DNA-binding value. The G+C mol% content was determined by HPLC (Mesbah et al., 1989). A total of 5 μg of denatured DNA was hydrolyzed with P1 nuclease (Yamasa Syoyu, Chiba, Japan) for 1 h at 50 °C. Alkaline phosphatase (Sigma, MO) was then added, and the mixture was incubated at 37 °C for 30 min for nucleotide dephosphorylation. The nucleosides were quantified with a GC analysis standard (Yamasa Syoyu) using a model L-2400 HPLC system (Hitachi, Tokyo, Japan) and an Inertsil ODS-3 HPLC Column (GL Sciences, Tokyo, Japan). The nucleosides were eluted with a solvent containing 0.2 M NH4H2PO4 and acetonitrile (20 : 1, v/v). G+C mol% was determined using

the mean values of three experiments. Strains designated as belonging to Lancefield group M formed a precipitate with Lancefield group M antiserum and with no other Lancefield grouping sera, confirming that they were indeed Lancefield group M strains. Based on 16S rRNA gene analysis, species of the genus Streptococcus were separated Roscovitine in vivo into six major clusters (Kawamura et al., 1995). Group

M strains PAGU 653, PAGU 1331, PAGU 1332 and PAGU 1535 were located in the Epothilone B (EPO906, Patupilone) pyogenic group on the phylogenetic tree (Fig. 1) and were highly related to each other genetically (99.8–100.0% 16S rRNA gene sequence similarity). Streptococcus marimammalium strain CCUG 48494T was the closest relative to the Group M strains in this analysis. The homology values between PAGU 653 and all other streptococci were<95.6%. These data demonstrate that group M strains constitute a new species with>97% 16S rRNA gene sequence similarity between strains (Stackebrandt & Goebel, 1994). We collected additional data of the genetic relationship between group M strains and closely related species by DNA–DNA hybridization experiments including group M strains, PAGU 653, PAGU 1331 and PAGU 1332. Streptococcus marimammalium was selected for these experiments because this species was most closely related to the group M strains on the phylogenetic tree based on 16S rRNA gene, and showed similarities for some phenotypic characteristics compared with other streptococci. The DNA–DNA hybridization values obtained under optimal (30 °C) and stringent (40 °C) conditions (Table 1) indicate that group M strains possess significantly lower DNA relatedness with S. marimammalium than with each other.