Study on the oxidative stress responses of a major periodontopathogenic bacteria

口腔厌氧菌牙龈卟啉单胞菌的基因组DNA序列的检查表明，该微生物具有过氧化物敏感的转录激活因子OxyR，但没有超氧化物敏感的转录因子SoxR。使用二维凝胶电泳研究了微生物中的氧化应激响应蛋白，发现两种蛋白质，SOD和AhpC主要在氧化条件下上调。在牙龈卟啉单胞菌oxyR突变体中，这两种蛋白质在有氧条件下用过氧化氢处理不诱导。OxyR对牙龈卟啉单胞菌sod和ahpC的表达有正调控作用。发现这些启动子的推定的~ 35盒紧邻其推定的OxyR结合序列。sod和ahpC基因启动子区具有与牙龈卟啉单胞菌OxyR蛋白结合的能力。这些结果表明，牙龈卟啉单胞菌sod是OxyR的调节子之一，提示OxyR在牙龈卟啉单胞菌中起着细胞内氧化还原感受器而不是过氧化物感受器的作用。UstA的表达上调，在稳定期或暴露于大气中的氧气。UstA编码基因（ustA）位于usp基因同源物的上游。ustA基因似乎以单顺反子方式转录。ustA突变株生长速度慢于野生型亲本菌株，导致稳定期产量较低。此外，在该突变体中，SOD、Tpr和Trx的表达水平显著高于野生型。ustA突变体比野生型对二酰胺（一种巯基特异性氧化剂）更具抗性。此外，ustA突变抑制了oxyR突变体对二酰胺的超敏反应。甲硝唑和丝裂霉素C。这些结果表明，UstA可能在细菌的氧化应激反应中发挥重要作用。

Inspection of genomic DNA sequence of the oral anaerobe Porphyromonas gingivalis reveals that the microorganism possesses the peroxide-sensing transcription activator OxyR, but not the superoxide-sensing transcription factor SoxR. Oxidative stress-responsive proteins in the microorganism were investigated using two dimensional gel electrophoresis and it was found that two proteins, SOD and AhpC were predominantly upregulated in oxidative conditions. In P. gingivalis oxyR mutant these two proteins were not induced by treatment with hydrogen peroxide under aerobic conditions. P.gingivalis sod and ahpC were positively regulated by OxyR. Putative -35 boxes of these promoters were found immediately adjacent to their putative OxyR binding sequences. Moreover, the promoter regions of sod and ahpC had the ability to bind P.gingivalis OxyR protein. These results demonstrate that P.gingivalis sod is one of the OxyR regulons, suggesting that OxyR functions as an intracellular redox sensor rather than a peroxide sensor in this organism.The new protein (UstA) was also identified in this study. Expression of UstA was upregulated in stationary phase or by exposure to atmospheric oxygen. The UstA-encoding gene (ustA) was located upstream of a homologue of the usp gene. The ustA gene appeared to be transcribed in a monocistronic fashion. The ustA mutant grew slower than the wild type parent strain, resulting in a lower yield in stationary phase. Furthermore, in this mutant, the expression levels of SOD, Tpr, and Trx were markedly higher than those in the wild type. The ustA mutant was more resistant to diamide, a thiol-specific oxidant, than the wild type. In addition, the ustA mutation suppressed hypersensitivities of the oxyR mutant to diamide,. metronidazole, and mitomycin C. These results suggest that UstA may play a significant role in oxidative stress responses in the bacterium.