Molecular Genetics of the gonococcus

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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.