Bacterial murein hydrolases are essential for the normal assembly and processing of the bacterial cell wall during cell growth and division. Since these proteins also have the potential to cause autolysis, their expression must be tightly controlled. Recent studies in the applicant's laboratory have identified a putative two-component regulatory system in S. aureus (comprised of the LytS and LytR proteins) that has been shown to affect the expression of autolysis by controlling the level of murein hydrolase activity associated with the cell. Based on these preliminary studies, the present proposal concentrates on three different aspects of this system. First, the expression of genes that have been shown to be regulated by LytS and LytR will be carefully investigated using Northern blotting and primer extension studies, along with deletion mutagenesis of their operator/promoter regions. This will also include an analysis of possible environmental signals that feed into this system. Second, the components of signal transduction, mediated through LytS and LytR, will be studied by purifying the LytS and LytR proteins and then assaying the potential autophosphorylation and kinase activities common to the two-component regulatory system family of proteins. Any effects of this phosphorylation on DNA binding activity will be monitored. Third, other genes that are affected by the lyt locus will be identified and characterized. The results of the proposed studies will yield a great deal of information regarding the regulation of autolysis, providing new insight into the function of cell wall active antibiotics.
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