| The LytSR two-component regulatory system of Staphylococcus aureus has been hypothesized to play a role in penicillin-induced lysis and the regulation of murein hydrolase activity. The effects of LytS and LytR have been shown to involve, in part, two LytSR-regulated genes, lrgA and lrgB, recently shown to inhibit the activity of extracellular murein hydrolases and promote penicillin tolerance. In the first part of this study, it was shown that several different environmental factors and genetic elements produce differential effects on the rates of induced lysis. Specifically, cells grown in the presence of different salts displayed differential rates of lysis and in all cases except one, the rate of lysis corresponded with lrgAB expression, consistent with the ability of these gene products to act as an inhibitor of murein hydrolase activity. Next, it was shown that mutations in the global virulence regulatory genes agr and sar affected the rates of induced lysis, increased rates of penicillininduced killing and altered murein hydrolase activities. Again, the affects of these mutations correlated with lrgAB expression and penicillin-induced killing as demonstrated by Northern and promoter fusion analysis. To identify cis-acting regulatory elements, several fragments of the lrgAB promoter region were fused to the xylE reporter gene and integrated into the chromosome. Reporter gene assays revealed the importance of DNA sequences containing an inverted repeat sequence and a region of intrinsic curvature (containing two homopolymeric AT-tracts in phase with a helical turn) in lrgAB expression. Electrophoretic mobility shift assays (EMSA) determined that purified LytR and SarA proteins specifically bind to DNA fragments containing the lrgAB promoter region. Overall conclusions of these studies are the lrgAB gene expression is subject to complex environmental and genetic regulatory mechanisms, which impact murein hydrolase activity and penicillin tolerance. |
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