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）中分离并测序了编码MurC蛋白的基因，这是一种与进行性牙周病有关的口腔厌氧棒状细菌。MurC蛋白在肽聚糖合成中起作用，催化细胞壁肽聚糖生物合成的第一步。包含PgMurC基因的区域与大肠杆菌中的mra区域高度相似，我们发现三个orf分别与大肠杆菌中的FtsQ（16%）、FtsA（33%）和FtsZ（54%）具有显著的相似性。PgFtsZ具有明确的GTP结合和水解基序，纯化后的PgFtsZ蛋白具有GTP酶活性，与其他已知的FtsZ蛋白不同：1) Na^+和K^+离子抑制其GTPase活性。2) PgFtsZ在没有Mg^<2+>的情况下仍保持GTPase活性，与EDTA的作用完全保持活性。最近，从C- 1基因中删除了一系列的突变体，产生了更多的PgFtsZ负基因，并观察了它们的形态变化。我们发现δ C-177突变体从c端删除了177个氨基酸残基，改变为正常细胞。这些结果表明，从T281到E330的氨基酸残基可能在PgFtsZ中发挥重要的功能作用。对已知的原核生物fgftsz的序列比较发现，该区域包含一个高度保守的结构域，称为a结构域，其中PgFtsZ的Ala320在许多物种中都是保守的。因此，我们通过定点诱变分析了Ala320的作用。我们发现，与野生型不同，ZA320H和ZA320R的过表达导致了正常表型。这些结果表明，Ala320是高度保守的，对细胞分裂至关重要。由于a结构域在其他牙周病病原菌中也具有保守性，因此该研究提示在不久的将来可以开发针对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