Molecular Genetics of the gonococcus

描述(申请人提供)：革兰氏阴性杆菌淋病奈瑟菌(淋球菌，GC)是性传播疾病淋病的唯一病原体。这项建议的资助是为了研究GC RecA蛋白用来介导这种人类特有病原体重组和修复的机制，并确定淋球菌RecA与大肠杆菌范例的不同之处。在过去的四年中，我们已经证明了一种被低估的大肠杆菌蛋白RecX通过产生一种非活性形式的RecA-DNA细丝，与大肠杆菌RecA结合，并在SOS反应中抑制其链交换和辅酶活性。我们还表明，GC recX突变体在依赖RecA的菌毛抗原变异、DNA转化和DNA修复过程中减少，这表明在调节RecA活性方面具有积极作用。在下一个授权期，我们将通过分析GC RecX在体外对GC RecA活性的影响来确定GC RecX刺激RecA依赖过程的机制。在我们的研究过程中，我们有了新的发现，GC细胞含有多条染色体。我们将对GC细胞携带多少染色体以及它们在细胞分裂过程中如何分离进行量化。作为理解允许多条染色体的复制机制的切入点，我们将定义复制的GC起源。最后，作为一种人类特有的病原体，GC不暴露在紫外线下，也没有SOS系统。我们推测淋球菌最常见的DNA损伤是多形核细胞(PMN)的氧化猝发。我们已经使用GC微阵列开始对氧化损伤的基因表达反应进行分类。我们观察到了暴露于过氧化氢后上调的40个基因。我们将进行额外的微阵列实验，以确定哪些基因在过氧化氢暴露或非特异性DNA损伤后优先表达。我们将突变损伤反应基因，以确定在体外和在PMN存在的情况下，哪些上调的基因对于面对氧化猝发的生存是重要的。这些研究将加强我们对GC的这些基本分子过程的理解，提供对这种人类特有的病原体在重组和修复能力方面与大肠杆菌的不同之处，并为新型抗微生物药物提供潜在的靶点。

DESCRIPTION (provided by applicant): The Gram-negative bacterium Neisseria gonorrhoeae (the gonococcus, Gc) is the only causative agent of the sexually-transmitted disease gonorrhea. This proposal was funded to examine the mechanisms used by the Gc RecA protein to mediate recombination and repair in this human specific pathogen and to determine how gonococcal RecA differs from the E. coli paradigm. In the past four years, we have shown that an under appreciated E. coli protein, RecX, binds E. coli RecA and inhibits its strand exchange and co-protease activities during the SOS response by creating an inactive form of the RecA-DNA filament. We have also shown that a Gc recX mutant is reduced for the RecA-dependent processes of pilin antigenic variation, DNA transformation, and DNA repair suggesting a positive role in regulating RecA activity. During this next granting period, we will determine the mechanism by which the Gc RecX stimulates RecA-dependent processes by analyzing the effect of Gc RecX on Gc RecA activity in vitro. In the course of our investigations, we have made the novel discovery that Gc cells contain multiple chromosomes. We will quantitate how many chromosomes are carried by Gc cells and how they segregate during cell division. As an entry point into understanding the replication mechanisms that allow multiple chromosomes, we will define the Gc origin of replication. Finally, as a human-specific pathogen, Gc are not exposed to UV light and do not have an SOS system. We presume that the most common DNA damage encountered by gonococci is the oxidative burst of polymorphnuclear cells (PMNs). We have used a Gc microarray to begin to catalog the gene expression response to oxidative damage. We have observed >40 genes that are up-regulated after exposure to H2O2. We will perform additional microarray experiments to determine which genes are preferentially expressed after H2O2 exposure or nonspecific DNA damage. We will mutate damage-responsive genes to determine which upregulated genes are important for survival in the face of an oxidative burst both in vitro and in the presence of PMNs. These studies will enhance our understanding of these basic molecular processes of Gc, provide insight into how this human-specific pathogen differs in its recombination and repair capabilities from E. coli, and provide potential targets for novel antimicrobials.