| Studies of the Staphylococcus aureus LytSR two-component regulatory system have led to the identification of the cid and lrg operons that affect murein hydrolase activity, stationary phase survival, antibiotic tolerance, and biofilm formation. The cid gene products enhance murein hydrolase activity and antibiotic tolerance whereas lrg gene products inhibit these processes in a manner analogous to bacteriophage-encoded holins and antiholins, respectively. Importantly, these operons have been shown to play significant roles in biofilm development by controlling the release of eDNA. To determine the role of LytSR in biofilm development, a lytS mutant was generated in a clinical S. aureus isolate (UAMS-1) and the effects on gene expression, cell death, lysis, and biofilm formation were examined. LytSR was required for lrgAB expression and the lytS mutant formed a more adherent biofilm. The increased adherence of the mutant was attributed to the increased prevalence of matrix-associated eDNA. Transcription profiling studies indicated that the lrgAB operon is the primary target of LytSR-mediated regulation but that this regulatory system also impacts expression of many genes involved in basic metabolism. To elucidate the underlying mechanism of LytSR-mediated signal transduction, mutations in the presumed phosphorylation site of the response regulator LytR were tested for their effects on lrgAB transcription. Interestingly, complementation with lytR alone restored normal levels lrgAB transcription indicating that the sensor component LytS is dispensable under these conditions. Expression of LytR-D53A, in which the presumed phosphorylation site (D53) was changed to an alanine, did not restore lrgAB expression suggesting that this amino acid is important for LytR activity. In contrast, expression of constitutively active derivative, LytR-D53E, induced lrgAB transcription even in the absence of known inducers, confirming the role of D53 in LytR activity. EMSA studies of purified LytR protein with lrgAB promoter demonstrated that both the WT LytR and LytRD53E bound to the lrgAB promoter while LytRD53A did not. DNase protection assay revealed multiple LytR binding sites within the lrgAB promoter. Overall, the results of this study demonstrate the importance of the LytSR two-component regulatory system in S. aureus biofilm development and begin to elucidate the molecular events required for LytSR-mediated signal transduction. |
