Hyperproduction of Alpha-Hemolysin in a sigB Mutant Is Associated with Elevated SarA Expression in Staphylococcus aureus

Staphylococcus aureus is a major cause of human infections, such as superficial abscesses, pneumonia, endocarditis, and sepsis (6). The control of a multitude of extracellular and cell wall virulence determinants in S. aureus is growth phase dependent. In particular, cell wall proteins are normally synthesized in the logarithmic phase, while exoproteins are generally produced postexponentially. The growth phase dependence of these virulence factors is mediated in part by global regulatory loci, such as sar (12) and agr (22). These modulators may either interact with the target gene directly (e.g., RNAIII with hla [alpha-hemolysin gene] mRNA) or control another regulatory molecule (e.g., sar regulation of the agr gene product) which, in turn, alters the transcription of the target gene. The sar locus is composed of three overlapping transcripts, designated sarA (0.56 kb), sarC (0.8 kb), and sarB (1.2 kb), initiated from the P1, P3, and P2 promoters, respectively. Because of this multiplicity of promoters, the activation of sar leading to the expression of SarA, the major sar regulatory molecule, is complex and may be growth phase dependent. Whereas the sarB transcript and the more abundant sarA transcripts are maximally expressed during the exponential phase, the transcription of sarC from the P3 promoter is most active during the postexponential phase (3). Additional transcriptional analysis indicated that the P3 promoter is ςB dependent (17, 20, 25). In contrast to the primary sigma factor (ςA), which is required for the expression of housekeeping genes, SigB (ςB) is an alternate transcription factor that has been shown to respond to environmental stresses (e.g., stationary phase of growth) in gram-positive bacteria (20). The core RNA polymerase associated with a particular sigma factor recognizes a specific set of promoters with conserved sequence motifs to initiate the transcription of genes programmed to respond to certain environments (20, 22). For Bacillus subtilis, ςB activity is regulated posttranslationally by complex networks of protein-protein interactions governed by a variety of environmental stresses (1, 20). Because one of the promoters (P3) within the sar locus is ςB dependent, it is conceivable that the SigB protein influences sar expression. As the sar locus activates the synthesis of alpha-hemolysin at the transcriptional level, presumably in part through the interaction of SarA with the agr locus (15), we speculate that sigB may modulate sarA expression and the ensuing hla transcription. In this study, we report the construction and characterization of a sigB mutant of RN6390, a prototypic S. aureus strain. The specificity of the mutation was confirmed by the absence of the SigB protein on an immunoblot, but the protein was restored in the mutant by a shuttle plasmid carrying the sigB gene. Phenotypic analysis revealed that the sigB mutant strain secreted more alpha-hemolysin than the parental strain, as determined by immunoblotting and Northern analysis. Complementation of the mutant with the sigB gene in trans reestablished alpha-hemolysin expression to near parental levels. Interestingly, the hyperproduction of alpha-hemolysin coincided with elevated SarA expression in the sigB mutant. Using the rabbit endocarditis model, we found that the sigB mutation was stable in vivo. We hypothesize that the hyperproduction of alpha-hemolysin in S. aureus as a result of the sigB mutation is mediated by an increase in the SarA level which, in turn, enhances the transcription of hla via a direct pathway (i.e., agr independent).