Porphyromonas gingivalis is asaccharolytic, and utilizes short peptides as its sole energy source (Takahashi & Sato, 2001). In oral environments, P. gingivalis may generate peptide fragments from external proteins to derive sufficient energy. Such a buy GKT137831 proliferation of this bacterium would induce the destruction of human periodontal tissue, a phenomenon which is the typical pathology seen in aggressive and chronic periodontitis. This bacterium secretes various types of proteases: endopeptidases [Arg-gingipains (RgpA and RgpB) and Lys-gingipain (Kgp)]; aminopeptidases (DPPIV, DPP-7, and PTP-A); and a carboxypeptidase (CPG70) (Banbula et al., 1999, 2000, 2001; Curtis et al., 1999; Chen et al., 2002). Among the
endopeptidases and aminopeptidases, Arg- and Lys-gingipains are essential for the growth of P. gingivalis (Oda et al., 2007, 2009), indicating that gingipains are important virulence/proliferation factors for this bacterium. We searched for genes Akt inhibitor encoding proteins participating in the biosynthesis of gingipains by screening the P. gingivalis W83 genomic database for genes encoding putative novel membrane proteins. In the present report, we identify a novel outer membrane protein, PG534, which is required for the biogenesis of gingipains. The strains and plasmids are listed in Table 1. Escherichia coli ER2566 (New England Biolabs Inc.,
Ipswich, MA) was grown in Luria–Bertani broth. Porphyromonas gingivalis was cultured anaerobically (10% CO2, 10% H2, and 80% N2) at 37 °C in a brain–heart infusion (Becton Dickinson, Franklin Lakes, NJ) supplemented with hemin (7.67 μM) and menadione (2.91 μM) (BHIHM). Ampicillin (100 μg mL−1) and erythromycin (5 μg mL−1) were added to the medium as needed. PCR was performed with Vent DNA polymerase (New England
Biolabs Inc.). A 1.3-kbp 3′-terminal half region of the PG0534 gene was amplified by PCR with 5′-ATCTGCAGCTGGGGGCGGACG-3′ (italics: PstI site) and 5′-GCCGGAGCGTCCGAGCAGCG-3′. The PCR product was digested with EcoRI (in the 3′-terminus of PG0534) and PstI, and cloned into PstI–EcoRI-digested pUC119, to generate pKS39. To construct pKS42, a 0.7-kbp downstream region of PG0534 containing PG0535 was amplified by MycoClean Mycoplasma Removal Kit PCR with 5′-GGAATTCTGAGCTCTGGATCCATATACGCTGCTCGGACGCTCCG-3′ (italics: EcoRI, SacI, and BamHI sites) and 5′-AAGGCCTATAGCTTTCGTAAGGATGGACAGCCTGG-3′ (italics: StuI site), digested with EcoRI and StuI, and ligated to the EcoRI–SfoI (in pUC119) sites of pKS39. To construct pKS41, a 0.7-kbp upstream region of PG0534 containing the tRNA genes (Fig. 1a) was amplified by PCR with 5′-CCCTGCAGTCGATAGAGCATCAGCCTTCCAAGCTG-3′ (italics: PstI site) and 5′-AGAATTCTATTAACGTATTTGAGGGAGAAAATCG-3′ (italics: EcoRI site), digested with EcoRI and PstI, and ligated to the EcoRI–PstI sites of pKS42. Next, pKS39 was digested with KpnI (in the PG0534 gene), and ligated with the 2.2-kbp KpnI-digested ermF–ermAM fragment from pKS1 (Saiki & Konishi, 2007).