Development of antisense vector for bacterial cell-division related genes and antibacterial agents for the periodontopathogens

我们已经从牙龈卟啉单胞菌（PgMurC基因）中分离并测序了MurC蛋白编码基因，牙龈卟啉单胞菌是一种与进行性牙周病有关的口腔厌氧杆状细菌。MurC蛋白在肽聚糖合成中起作用，并催化细胞壁肽聚糖生物合成的第一步。包括PgMurC基因在内的区域与大肠杆菌中的mra区域高度相似。coli中的FtsQ（16%）、FtsA（33%）和FtsZ（54%）的同源性较高。coli中表达。牙龈卟啉单胞菌FtsZ（PgFtsZ）具有清晰的GTP结合和水解基序，纯化的PgFtsZ蛋白具有与其他已知FtsZ蛋白不同的性质：1）Na^+和K^+抑制其GTP酶活性。2)PgFtsZ在无Mg^2+存在下仍显示出GT3活性，而在EDTA存在下完全保持其活性。最近，一系列从C- ...更多信息 产生PgFtsZ末端，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，从T281到E330的氨基酸残基可能是重要的功能作用，在PgFtsZ。已知的原核FtsZ的序列比较显示，该区域包含一个高度保守的结构域，指定的A-结构域，其中的Ala 320的PgFtsZ是保守的在整个广泛的物种。因此，我们通过定点突变分析了Ala 320的作用。我们发现，ZA 320 H和ZA 320 R的过表达导致正常表型，与野生型不同。这些结果表明，Ala 320是高度保守的，是至关重要的细胞分裂。由于A结构域在其他牙周病原体中也是保守的，因此这项工作具有启示意义，可以在不久的将来开发针对A结构域的牙周病抗菌剂。少

We have already isolated and sequenced a gene encoding the MurC protein from Porphyromonas gingivalis (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 E. coli, respectively. The FtsZ from P. gingivalis (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. Very recently, a series of mutants deleted from the C- … More teminus of PgFtsZ were 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, designated A-domain, in which Ala320 of PgFtsZ was conserved throughout a broad variety of species. Therefore, we analyzed the role of Ala320 by site-directed mutagenesis. We found that overexpression of ZA320H and ZA320R resulted in the normal phenotype, unlike the wild type. These results suggested that Ala320 is highly conserved and is crucial for cell division. Since the A-domain was also conserved among other periodontopathogens, this work has implications that antibacterial agents for periodontal diseases targeted the A-domain could be developed in the near future. Less