No protein bands other than those of 70 and

65 kDa indicated by asterisks, which might be non-specific, were detected in the hbp35 full length deletion mutant (KDP166), whereas the hbp35 insertion mutant (KDP164), which had an insertion of the ermF-ermAM DNA cassette just upstream of the F110 residue within the HBP35 protein, showed 29-and 27-kDa proteins (Figure 1). We checked independent 18 isolates of KDP164 and 5 isolates of KDP166. All of the isolates showed the same results as shown in Figure 1. The 40-kDa protein appeared as the full length gene product of hbp35, which coincided with results of previous studies [6, 7]. Figure Capmatinib mouse 1 Immunoblot analysis of cell extracts of various P. XMU-MP-1 gingivalis strains with anti-HBP35. Cell extracts (approximately 10 μg protein) of various P. gingivalis strains were analyzed by SDS-PAGE under reducing conditions

followed by immunoblotting with anti-HBP35 antibody. Lane 1, 33277 (wild type); lanes 2, 3 and 4, KDP164 (hbp35 insertion mutant); lanes 5, 6 and 7, KDP166 (hbp35 deletion mutant). Asterisks indicate protein bands with molecular masses of 70-and 65-kDa non-specifically recognized by anti-HBP35 antibody. Pigmentation and C646 in vitro gingipain activities of P. gingivalis hbp35 mutants Both full length deletion and insertion P. gingivalis hbp35 mutants formed black pigmented colonies on blood agar plates. No difference was observed in Rgp, Kgp and hemagglutinating activities between the hbp35 mutants and the wild type (data not shown). These results suggest that HBP35 does not influence expression of gingipain-encoding genes. Northern blot analysis of hbp35 To determine whether the hbp35 gene produces multiple transcripts, total RNAs were prepared from the wild type and hbp35 mutants. Northern blot analysis was then carried out with an hbp35 DNA probe that hybridized to

the hbp35 region coding for Q22-P344. The wild type showed a 1.1-kb transcript hybridizing to the hbp35 probe (Additional file 1). In the hbp35 insertion and full length Adenosine triphosphate deletion mutants, there was no 1.1-kb transcript, indicating that the 1.1-kb mRNA was produced from the hbp35 gene. The hbp35 insertion mutant produced transcripts with 1.3-2.2 kb that hybridized to the probe. The ermF probe hybridized to transcripts with similar length in the hbp35 insertion mutant (Additional file 1). Subcellular localization of HBP35 protein In an approach to understand the potential roles of HBP35 proteins with different molecular masses, we fractionated cells of the wild type and the hbp35 insertion mutant into cytoplasm/periplasm, total membrane, and inner and outer membrane fractions. These fractions were subjected to SDS-PAGE and immunoblot analysis with the anti-HBP35 antibody.