|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 10, 2004
Citation: Koenig, R.L., Ray, J., Maleki, S.J., Smeltzer, M., Hurlburt, B.K. 2004. Staphylococcus aureus AgrA Binding to the RNAIII-agr regulatory region. Journal of Bacteriology. 7549-7555. Interpretive Summary: The bacteria Staphylococcus aureus is responsible for a wide range of infections in humans and animals. As the causative agent in bovine staphyloccocal mastitis, it was responsible for an estimated loss of $1.8 billion to the US dairy industry in 1996 alone (Current Concepts in Bovine Mastitis, 1996. National Mastitis Council). In this study we examine on of the proteins responsible for regulation of virulence in S. aureus. Previous researchers have had difficulty obtaining the protein in a pure, active, and soluble form. By changing growth conditions, we were able to overcome these difficulties. Furthermore, we were able to use the purified protein to demonstrate how the protein works to activate virulence in the bacteria. With this knowledge, it may be possible to develop new antibiotics to treat these infections.
Technical Abstract: The control of virulence 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 agrA gene has been shown by amino acid sequence similarity to be a putative member of the LytR family of response regulators, whose binding domain is thought to recognize a novel pair of direct sequence repeats (Nikolskaya and Galperin, 2002). Pairs of such repeats are found in the promoter regions of both the regulatory RNA, RNAIII, and the agr locus itself. Thus far, binding to these promoter regions by AgrA has not been demonstrated, partially due to difficulties surrounding the expression of soluble and active recombinant AgrA. In this study, we isolate and purify soluble AgrA by expression under osmotic shock conditions and ion-exchange chromotography respectively. Using the purified protein, we demonstrated the binding of AgrA to the promoter region of RNAIII in electrophoresis mobility shift assays (EMSAs), specifically to a pair of direct repeats previously identified by Morfeldt, et al., 1996b. We further found that mutation of the conserved aspartate residue at position 59 to an alanine significantly inhibited this binding. 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.