ς B Activity Depends on RsbU in Staphylococcus aureus

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Abstract
Derivatives of the widely used laboratory strainStaphylococcus aureus NCTC8325, which are naturalrsbU mutants, were shown to be unable to produce RsbU, a positive regulator of the alternative sigma factor ςB. The lack of RsbU prevented the heat-dependent production of ςB-controlled transcripts and resulted in reduced H2O2 and UV tolerance, enhanced alpha-hemolysin activity, and the inability to produce the alkaline shock protein Asp23. After 48 h of growth, rsbU mutant strains failed to accumulate staphyloxanthin, the major stationary-phase carotenoid. Transcription of Asp23 was found to be exclusively controlled by ςB, making it an excellent target for the study of ςB activity in S. aureus. Reporter gene experiments, using the firefly luciferase gene (luc+) fused to the ςB-dependent promoter(s) ofasp23, revealed that ςB is almost inactive in 8325 derivatives. cis complementation of the 8325 derivative BB255 with the wild-type rsbU gene from strain COL produced the rsbU + derivative GP268, a strain possessing a ςB activity profile comparable to that of the rsbU + wild-type strain Newman. In GP268, the heat inducibility of ςB-dependent genes, Asp23 production, alpha-hemolysin activity, pigmentation, and susceptibility to H2O2 were restored to the levels observed in strain Newman, clearly demonstrating that RsbU is needed for activation of ςB in S. aureus.