Development of rapid diagnostic system for periodontal disease using cell division related genes form P.gingivalis

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We have already isolated and sequenced a gene encoding the MurC protein from Porphyromonas guigivalis (PgMurC gene), an oral anaerobic rod-shaped bacterium implicated in progressive periodontal disease. The MurC protein functions in peptidoglycan synthesis and catalyzes the first step in the biosynthesis of cell wall peptidoglycan. The region including PgMurC gene appeared to be highly similar with mra region in E.coli and we found that the three ORFs had a significant similarity with FtsQ (16%), FtsA (33%), and FtsZ (54%) in F.coli respectively. The FtsZ from P.giugivalis (PgFtsZ) possessed the clear motifs for GTP binding and hydrolysis, and the purified PgFtsZ protein exhibited GTPase activity with the following properties different from other known FtsZ proteins; 1) Na^+ and K^+ ions inhibited its GTPase activity. 2) PgFtsZ exhibited its GTPase activity even without Mg^<2+> and completely retained its activity with EDTA. A series of mutants deleted from the C-teminus of PgFtsZ were … More generated, and the change of their morphology were observed. We found that the delta C-177 mutant, deleted 177 amino acid residues from C-terminus, changed to the normal cells. These results suggest that amino acid residues from T281 to E330 may be important for the functional role in PgFtsZ. Sequence comparison of the known prokaryotic FtsZs revealed that this region contained a highly conserved domain including 10 amino acids, designated A-domain, in which Ala320 and Gly322 of PgFtsZ was conserved throughout a broad variety of species. Therefore, we analyzed the role of Ala320 and GIy322 by site-directed mutagenesis. Consequently, we found that overexpression of ZA320H and ZA320R resulted in the normal phenotype, unlike the wild type. Similarly, overexpresson of ZG322P and ZG322H resulted in the normal phenotype. These results suggested that Ala320 and Gly322 are highly conserved and are crucial for cell division. The A-domain was also conserved among other periodontopathogens including A.actinomycetemcomitans, T.denticola, P.intermedia. We are currently planning to develop a novel system for differentiating these periodontopathic bacteria using the Multiplex PCR. Less