, 2010; Shoji et al., 2011). Studies of Gram-negative bacteria have identified at least eight different protein secretion systems, including types I–VI, the two-partner

secretion system and the chaperone/usher system (Economou et al., 2006). PorSS is not related to the previously known bacterial protein secretion systems. PorK, PorL, PorM, PorN, PorW, PorT and Sov involved in the P. gingivalis PorSS share similarity in amino acid sequence with Flavobacterium johnsoniae gliding motility proteins, GldK, GldL, GldM, GldN, SprE, SprT and SprA, respectively (Braun et al., 2005; Rhodes et al., 2010; Sato et al., 2010). In F. johnsoniae, disruption of the sprT gene resulted in defects in translocation of the gliding motility protein SprB and secretion of chitinase, suggesting Fulvestrant supplier that the PorSS is linked to gliding motility of bacteria in the Bacteroidetes phylum (Sato et al., 2010). Genes homologous RO4929097 datasheet to the PorSS-related genes are also found in genomes of other members of the Bacteroidetes phylum, including important periodontal pathogens such as P. intermedia and T. forsythia (Sato et al., 2010). In the

present study, proteomic analyses of particle-free culture supernatants and vesicle fractions from porK+ and porK strains with the genetic background of rgpA rgpB kgp and outer membrane fractions from wild-type and porK strains were performed to identify P. gingivalis proteins that were secreted into the extracellular milieu by the PorSS. Bacterial strains and plasmids used in this study are listed in Table 1. Porphyromonas gingivalis cells were grown anaerobically (10% CO2, 10% H2, 80% N2) in enriched brain heart infusion medium and on enriched trypticase soy agar

(Nakayama et al., 1995). For blood agar plates, defibrinated laked sheep blood was added to enriched trypticase soy agar at 5%. For selection and GPX6 maintenance of antibiotic-resistant P. gingivalis strains, antibiotics were added to the medium at the following concentrations: erythromycin (Em), 10 μg mL−1; tetracycline (Tc), 0.7 μg mL−1. Porphyromonas gingivalis deletion mutants were constructed as follows. DNA regions upstream and downstream of a gene were PCR-amplified from the chromosomal DNA of P. gingivalis ATCC 33277T using pairs of primers (PGN gene number-U-F plus PGN gene number-U-R and PGN gene number-D-F plus PGN gene number-D-R), respectively, where ‘U’ indicates upstream, ‘F’ indicates forward, ‘D’ indicates downstream and ‘R’ indicates reverse. Primers used in this study are listed in Supporting information, Table S1. Amplified DNAs upstream and downstream of each gene were double-digested with NotI plus BamHI and KpnI plus BamHI, respectively. Both digested products were ligated together with pBluescript II SK(−) which had been digested with NotI plus KpnI, resulting in pKD945 (for rgpA mutagenesis) and pKD947 (for rgpB mutagenesis). The 1.5-kb BamHI cepA (pKD1002) and 2.