|Ray, Jessica - UNIV. OF ARKANSAS|
|Smeltzer, Mark - UNIV. OF ARKANSAS|
Submitted to: Journal of Bacteriology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 10, 2004
Publication Date: November 2, 2004
Citation: Koenig, R.L., Maleki, S.J., Hurlburt, B.K., Ray, J.L., Smeltzer, M.S. 2004. Staphylococcus aureus agra a binding to the rna111-agr regulatory region. Journal of Bacteriology. 186(22):7549-7555. Interpretive Summary: The bacterium, Staphylococcus aureus, causes a wide variety of diseases in humans and animals. There is a mechanism by which the bacteria senses whether there is a sufficient population necessary to trigger the production of the toxins associated with these diseases. In this paper, we isolate and examine one protein component of this mechanism, and begin to explain how this protein participates in the regulation of toxin production. By understanding this mechanism of disease in S. aureus, we may be able to more effectively combat the diseases it causes.
Technical Abstract: The control of virulence gene expression in the human pathogen, Staphylococcus aureus, is under the partial control of the two-component quorum sensing system encoded by genes of the agr locus. The product of the agrA gene has been shown by amino acid sequence similarity, to be the putative response regulator, however, binding of AgrA to promoters under its control has not yet been demonstrated. In this study, we isolated and purified soluble AgrA by expression under osmotic shock conditions and ion-exchange chromotography. Purified AgrA showed high-affinity binding to the RNAIII-agr intergenic region by electrophoretic mobility shift assays (EMSAs). Binding was localized by DNase I protection assays to a pair of direct repeats in the P2 and P3 promoter regions of the agr locus. We found that this binding was enhanced by the addition of the small phosphoryldonor, acetyl phosphate. The difference in binding affinity between these two promoters was found to result from a two base pair difference between the downstream direct repeat of the P2 and P3 sites. Mutation of these base pairs in the P3 site to match those found in the P2 site increased the affinity of AgrA for the P3 site relative to the P2 site. These results are consistent with the function of AgrA as a response regulator with recognition sites in the promoter regions of RNAIII and the agr locus.