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

我们已经分离并测序了一个编码MurC蛋白的基因，该基因来自于与进行性牙周病有关的口腔厌氧杆状细菌-Guigivalis（PgMurC基因）。MurC蛋白在肽聚糖合成中发挥作用，并催化细胞壁肽聚糖生物合成的第一步。包括PgMurC基因的区域与大肠杆菌的mra区域高度相似，与大肠杆菌的FtsQ（16%）、FtsA（33%）和FtsZ（54%）的同源性最高。猪巨噬细胞FtsZ（PgFtsZ）具有清晰的GTP结合和水解基序，纯化的PgFtsZ蛋白具有与已知FtsZ蛋白不同的性质：1）Na ^+和K ^+抑制其GTP酶活性。2)PgFtsZ即使在无Mg ^2+存在下也表现出GT3活性，而在EDTA存在下完全保持其活性。从PgFtsZ的C-末端缺失的一系列突变体是 ...更多信息 并观察其形态变化。我们发现Δ C-177突变体从C-末端缺失177个氨基酸残基，改变为正常细胞。这些结果表明，T281至E330的氨基酸残基可能对PgFtsZ的功能作用很重要。与已知的原核生物FtsZ序列比较发现，该区域包含一个高度保守的结构域，包括10个氨基酸，命名为A-结构域，其中PgFtsZ的Ala320和Gly322在广泛的物种中是保守的。因此，我们通过定点突变分析了Ala320和Gly322的作用。因此，我们发现，ZA320 H和ZA320 R的过表达导致正常表型，与野生型不同。类似地，ZG322P和ZG322H的过表达导致正常表型。这些结果表明，Ala320和Gly322是高度保守的，是细胞分裂的关键。A-结构域在其他牙周病原体包括放线菌共生放线菌、齿垢毛癣菌、中间型放线菌中也是保守的。我们目前正计划开发一种新的系统，用于区分这些牙周病细菌使用多重PCR。少

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