Background: Dipeptidyl-peptidases (DPPs) are key factors for amino acid metabolism and bacterial growth of asaccharolytic Porphyromonas gingivalis. Results: DPP5, which is specific for Ala and hydrophobic residues, is expressed in the periplasmic space of P. gingivalis. Conclusion: DPP5 was discovered in prokaryotes for the first time. Significance: The discovery of DPP5 expands understanding of amino acid and energy metabolism in prokaryotes.

The Gram-negative anaerobe Porphyromonas gingivalis is recognized as a major pathogen for aggressive and chronic periodontitis, infection and inflammation of the ligaments and bones that support the teeth (4,18). P. gingivalis is asaccharolytic, therefore, its growth is strictly dependent on external proteins as the carbon and nitrogen source. P. gingivalis secretes proteases to digest external proteins into small peptides which are then imported into the cells of the bacterium and metabolized. Among these proteases, gingipains are known to be the very important virulence factors of P. gingivalis owing to their strong proteolytic activities and their ability to destruct human periodontal tissue where P. gingivalis colonizes (5). Gingipains include three proteases. Arg-gingipains (RgpA and RgpB) are specific for the Arg-X peptide bond, whereas Lys-gingipain (Kgp) is specific for the Lys-X peptide bond (5, 10). Gingipains are translated into huge proteins consisting of four domains (signal sequence domain, propeptide domain, catalytic domain, and adhesion domain), and may cross an inner membrane by the Sec system followed by processing of their signal sequence. The resulting precursor forms of gingipains are suggested to be secreted and matured on an outer membrane (14, 15); however, the secretion of gingipains is poorly understood. The secretion of proteins in pathogenic Gram-negative bacteria is known to be mediated via highly conserved pathways throughout bacterial species. At present, these pathways have generally been classified into the six systems (type I to VI) (8, 13). Type I, III, and VI secretion systems are Sec-independent pathways; however, none of these systems is substantiated by genome analysis. Therefore, gingipains are not likely secreted by any of these secretion systems. Type II, (part of) IV, and V secretion systems are Sec-dependent pathways; however, gingipains show no domain homology with the β-barrel structure found in the type V secretion system, and the genome database of W83 shows no protein homology with the apparatus for the type II and type VI secretion systems, suggesting Abstract: Gingipains are extracellular proteases important for the virulence of Porphyromonas gingivalis; however, the mechanism for the secretion of gingipains is poorly understood. In this report, we found that insertion mutants for PG0809 (83K1 and 83K2) were defective in black pigmentation and hemolysis. We cloned and sequenced PG0809 and found that PG0809 contains two additional nucleotides that are not deposited in the W83 genome database. The revised sequence reveals an in-frame fusion of PG0810 and PG0809 and is designated the sov gene. We constructed a sov deletion mutant (83K3) and showed that 83K3 was defective in the activities of black pigmentation, hemolysis, and hemagglutination. Furthermore, in 83K3, the activities of gingipains were severely reduced whereas those of other secreted proteases DPPIV, DPP-7, and PtpA were not affected. Immunoblot analysis using anti-RgpB antiserum showed that Arg-gingi...

Porphyromonas gingivalis secretes endopeptidase gingipains, which are important virulence factors of this bacterium. Gingipains are transported across the inner membrane via the Sec system, followed by transport across the outer membrane via an unidentified pathway. The latter transport step is suggested to be mediated via a novel protein secretion pathway. In the present study, we report a novel candidate as an essential factor for the latter transport step. The PG0027 gene of P. gingivalis W83 encodes novel protein PG27. In a PG0027 deletion mutant (83K10), the activities of Arg-gingipain and Lys-gingipain were severely reduced, while the activities of secreted exopeptidases DPPIV, DPP-7, and PTP-A were unaffected. Protein localization was investigated by cell-surface biotinylation, subcellular fractionation, and immunoblot analysis. In the wild-type W83, Arg-gingipains in membrane fraction were detected as cell surface proteins. In contrast, in 83K10, Arg-gingipains were trapped in the periplasm and hardly secreted into an extracellular milieu. PG27 was suggested to be exposed to the cell surface by a cell surface biotinylation experiment; however, PG27 was detected in both inner and outer membrane fractions by subcellular fractionation experiments. Taken together, we suggest that PG27 is a unique membrane protein essential for a novel secretion pathway.

Haem O and/or haem A are specifically synthesized for the haem-copper respiratory oxidases. A 17-carbon hydroxyethylfarnesyl chain at the pyrrole ring A of the haems seems essential for catalytic functions at the oxygen-reduction site. The discovery of haem O in the cytochrome bo complex from Escherichia coli was a breakthrough in the studies on haem A biosynthesis. Molecular biological and biochemical studies in the past three years demonstrated that the cyoE/ctaB/COX10 genes are indispensable for functional expression of the terminal oxidases and encode a novel enzyme haem O synthase (protohaem IX farnesyltransferase). It has recently been suggested that the ctaA gene adjacent to the ctaB-ctaCDEF gene cluster in Bacillus subtilis encodes haem A synthase (haem O monooxygenase). In this article, we review current knowledge of the genes for haem O and haem A biosyntheses, the location and regulation of haem O synthase, the possible enzymatic mechanism of farnesyl transfer to haem B and the possible roles of the farnesylated haems.

Cytolethal distending toxin (CDT) secreted by Actinobacillus actinomycetemcomitans induces cell cycle arrest of cultured cells in the G2 phase. The crystal structure of the natural form of A. actinomycetemcomitans DCT (aCDT) has been determined at 2.4 Å resolution. aCDT is a heterotrimer consisting of the three subunits, aCdtA, aCdtB, and aCdtC. Two crystallographically independent aCDTs form a dimer through interactions of the aCdtB subunits. The primary structure of aCDT has 94.3% identity with that of Haemophilus ducreyi CDT (hCDT), and the structure of aCDT is quite similar to that of hCDT reconstituted from the three subunits determined recently. However, the molecular packings in the crystal structures of aCDT and hCDT are quite different. A careful analysis of molecular packing suggests that variation of the amino acid residues in a nonconserved loop ( 181 TSSPSSPERRGY192 of aCdtB and 181 NSSSSPPERRVY 192 of hCdtB) is responsible for the different oligomerization of very similar CDTs. The loop of aCdtB has a conformation to form a dimer, while the loop conformation of hCdtB prevents hCDT from forming a dimer. Although dimerization of aCDT does not affect toxic activity, it changes the stability of protein. aCDT rapidly aggregates and loses toxic activity in the absence of sucrose in a buffered solution, while hCDT is stable and retains toxic activity. Another analysis of crystal structures of aCDT and hCDT suggests that the receptor contact area is in the deep groove between CdtA and CdtC, and the characteristic "aromatic patch" on CdtA.Keywords: Cytolethal distending toxin; CDT; Actinobacillus actinomycetemcomitans; crystal structure; oligomerization; stability and toxic activity Abbreviations: A. actinomycetemcomitans, Actinobacillus actinomycetemcomitans; CDT, cytolethal distending toxin; aCDT, A. actinomycetemcomitans CDT; hCDT, Haemophilus ducreyi CDT; aCdtA, subunit A of aCDT; aCdtB, subunit B of aCDT; aCdtC, subunit C of aCDT; hCdtA, subunit A of hCDT; hCdtB, subunit B of hCDT; hCdtC, subunit C of hCDT; C A RMSD, root-mean-square-deviation of C A positions between aCDT and hCDT.Article and publication are at