Molecular Genetics of the gonococcus

描述（由申请人提供）：革兰氏阴性菌淋病奈瑟菌（淋球菌，Gc）是性传播疾病淋病的唯一病原体。本研究旨在研究Gc RecA蛋白介导这种人类特异性病原体重组和修复的机制，并确定淋球菌RecA与大肠杆菌的不同之处。在过去的四年里，我们已经证明了一种被低估的大肠杆菌蛋白RecX与大肠杆菌RecA结合，并通过产生一种无活性形式的RecA- dna丝，在SOS反应中抑制其链交换和共蛋白酶活性。我们还发现Gc recX突变体在依赖RecA的匹林抗原变异、DNA转化和DNA修复过程中减少，这表明它在调节RecA活性中起积极作用。在接下来的研究期间，我们将通过分析Gc RecX对体外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，即Gc细胞含有多条染色体。我们将量化Gc细胞携带的染色体数量以及它们在细胞分裂过程中如何分离。作为理解允许多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没有暴露在紫外线下，也没有SOS系统。我们推测淋球菌最常见的DNA损伤是多态核细胞（pmn）的氧化爆发。我们已经使用Gc微阵列开始编目基因表达对氧化损伤的反应。我们观察到bbb40个基因在暴露于H2O2后表达上调。我们将进行额外的微阵列实验，以确定哪些基因在H2O2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，以确定哪些上调基因在体外和pmn存在下面对氧化爆发时对存活很重要。这些研究将增强我们对Gc的基本分子过程的理解，深入了解这种人类特异性病原体在重组和修复能力方面与大肠杆菌的不同之处，并为新型抗菌剂的开发提供潜在的靶点。

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.