Pest-sequencing analysis of periodontal pathogen Porphyromonas gingivalis : expression of its proteins by oral environmental fectors.

牙龈卟啉单胞菌是一种专性厌氧细菌，是慢性牙周炎发生和发展的主要病原体。虽然已经发现细菌的几种毒力因子的表达受到环境胁迫的影响，如进入固定生长期和热量，但关于细菌应对环境胁迫的机制的信息相对较少。在这项研究中，我们研究了新的蛋白（UstA），最初是通过二维凝胶分析确定的。UstA的表达在固定期或暴露于大气氧中上调。n端测序和数据库分析表明，编码ustA的基因（ustA）位于染色体上编码通用胁迫蛋白的usp基因同源物的上游。引物延伸和Northern blot分析显示，ustA基因以单反子方式转录。为了阐明UstA在细菌中的作用，构建了携带UstA基因中断的染色体突变体。ustA突变体在富培养基中的生长速度比野生型亲本慢，导致其稳定期产量较低。此外，在该突变体中，超氧化物歧化酶、硫醇过氧化物酶和硫氧还蛋白同源物的表达水平显著高于野生型，特别是在固定期。与野生型相比，ustA突变体对二胺（一种硫醇特异性氧化剂）更具抗性。此外，ustA突变抑制了oxyR突变体对二胺、甲硝唑和丝裂霉素c的超敏反应。这些结果表明，ustA可能在细菌的氧化应激反应中起重要作用。

Porphyromonas gingivals, an obligate anaerobic bacterium, is implicated as a major pathogen in the development and progression of chronic periodontitis. Although expression of several virulence factors of the bacterium has been found to be affected by environmental stress such as entrance into the stationary growth phase and heat, there is relatively little information on the mechanisms that may operate in the bacterium in response to environmental stress. In this study, we investigated the new protein (UstA) that was initially identified following the two dimensional gel analysis. Expression of UstA was upregulated in stationary phase or by exposure to atmospheric oxygen. N-terminal sequencing and database analysis with the P.gingivalis genome sequence revealed that the UstA-encoding gene (ustA) was located upstream of a homologue of the usp gene encoding the universal stress protein on the chromosome. The ustA gene appeared to be transcribed in a monocistronic fashion as revealed by primer extension and Northern blot analysis. To elucidate the role of UstA in the bacterium, chromosomal mutants carrying a disruption of the ustA gene were constructed. The ustA mutant grew slower than the wild type parent strain in rich medium, resulting in a lower yield in stationary phase. Furthermore, in this mutant, the expression levels of the P.gingivalis homologues of superoxide dismutase, thiol peroxidase, and thioredoxin were markedly higher than those in the wild type especially in stationary phase. 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.