Por Secretion System-Dependent Secretion and Glycosylation of Porphyromonas gingivalis Hemin-Binding Protein 35

The anaerobic Gram-negative bacterium Porphyromonas gingivalis is a major pathogen in severe forms of periodontal disease and refractory periapical perodontitis. We have recently found that P. gingivalis has a novel secretion system named the Por secretion system (PorSS), which is responsible for secretion of major extracellular proteinases, Arg-gingipains (Rgps) and Lys-gingipain. These proteinases contain conserved C-terminal domains (CTDs) in their C-termini. Hemin-binding protein 35 (HBP35), which is one of the outer membrane proteins of P. gingivalis and contributes to its haem utilization, also contains a CTD, suggesting that HBP35 is translocated to the cell surface via the PorSS. In this study, immunoblot analysis of P. gingivalis mutants deficient in the PorSS or in the biosynthesis of anionic polysaccharide-lipopolysaccharide (A-LPS) revealed that HBP35 is translocated to the cell surface via the PorSS and is glycosylated with A-LPS. From deletion analysis with a GFP-CTD[HBP35] green fluorescent protein fusion, the C-terminal 22 amino acid residues of CTD[HBP35] were found to be required for cell surface translocation and glycosylation. The GFP-CTD fusion study also revealed that the CTDs of CPG70, peptidylarginine deiminase, P27 and RgpB play roles in PorSS-dependent translocation and glycosylation. However, CTD-region peptides were not found in samples of glycosylated HBP35 protein by peptide map fingerprinting analysis, and antibodies against CTD-regions peptides did not react with glycosylated HBP35 protein. These results suggest both that the CTD region functions as a recognition signal for the PorSS and that glycosylation of CTD proteins occurs after removal of the CTD region. Rabbits were used for making antisera against bacterial proteins in this study

An energy transduction mechanism used in bacterial flagellar type III protein export

Flagellar proteins of bacteria are exported by a specific export apparatus. FliI ATPase forms a complex with FliH and FliJ and escorts export substrates from the cytoplasm to the export gate complex, which is made up of six membrane proteins. The export gate complex utilizes proton motive force across the cytoplasmic membrane for protein translocation, but the mechanism remains unknown. Here we show that the export gate complex by itself is a proton–protein antiporter that uses the two components of proton motive force, Δψ and ΔpH, for different steps of the protein export process. However, in the presence of FliH, FliI and FliJ, a specific binding of FliJ with an export gate membrane protein, FlhA, is brought about by the FliH–FliI complex, which turns the export gate into a highly efficient, Δψ-driven protein export apparatus