As shown in Fig. 4, a 92-kDa protein band (between two nonspecific protein bands that are represented by * in Fig. 4) was detected in the outer membrane fraction of W83 (lane 5) but not of mutant 83K25 (lane 6) or other fractions (lanes 1–4, 7 and 8) (the expected molecular weight of PG534 click here is calculated as 92 000). This result suggests that PG534 is an outer membrane protein. A recent study revealed 13 proteins involved in gingipain secretion (PorK-N, PorP, PorQ, PorT, PorU,

PorW-Y, Sov, and PG27 (Sato et al., 2005, 2010; Saiki & Konishi, 2007; Ishiguro et al., 2009). Homologues of the Por proteins, Sov, and PG27 are found in Cytophaga–Flavobacterium–Bacteroidetes phylum members. Importantly, bioinformatics analyses also identified PG534 homologues in Cytophaga–Flavobacterium–Bacteroidetes phylum members Bacteroides spp., Parabacteroides spp., Prevotella spp., Flavobacterium spp., and Cytophaga spp (data not shown). In this study, we found that PG534 is required for normal gingipain activity (Fig. 1c). 83K3 (Δsov) and 83K10

(ΔPG0027) secrete few gingipains into the extracellular milieu. However, appreciable amounts of abnormal learn more forms of Arg-gingipains were detected in the HSP fraction from 83K25 (Fig. 2a, lane 8). Lysis of 83K25 cells was unlikely because 60- and 62-kDa forms of Lys-gingipain (Fig. 2b, lane 4) were not well-detected in the HSS or the HSP fraction from 83K25 (lanes 8 and 12). This suggests Casein kinase 1 that 83K25 still contains gingipain secretory activity, unlike 83K3 or 83K10. The observed phenotypes of 83K25 resemble those of the vimA, vimE, or vimF mutants that are defective in carbohydrate biogenesis of gingipains (Abaibou et

al., 2001; Vanterpool et al., 2004, 2005a, b). The mechanism of action is unclear, but the vimA, vimE, and vimF defective mutants all share the production of truncated forms of lipopolysaccharide. In contrast, 83K25 produced normal lipopolysaccharide, suggesting that PG534 affects the biogenesis of gingipains, but not lipopolysaccharide. Therefore, the function of PG534 is likely different from that of proteins shown to be involved in gingipain biogenesis: Por proteins, Sov, PG27, or Vim proteins. In Fig. 3, there appears to be a difference in the signal intensity of lipopolysaccharide bands; 83K3, 83K10, and 83K25 likely exhibited denser lipopolysaccharide bands than those of W83. The generation and/or the glycosylation of lipopolysaccharide might be facilitated by a defect of glycosylation in gingipains (Fig. 2a, lanes 2–4). In this study, we showed that PG534 is an outer membrane protein (Fig. 4).