The accessory sigma factor B controls a general stress response that is thought to be important for Staphylococcus aureus survival and may contribute to virulence. The strain of choice for genetic studies, 8325-4, carries a small deletion in rsbU, which encodes a positive regulator of B activity. Consequently, to enable the role of B in virulence to be addressed, we constructed an rsbU ؉ derivative, SH1000, using a method that does not leave behind an antibiotic resistance marker. The phenotypic properties of SH1000 (8325-4 rsbU ؉ ) were characterized and compared to those of 8325-4, the rsbU mutant, parent strain. A recognition site for B was located in the promoter region of katA, the gene encoding the sole catalase of S. aureus, by primer extension analysis. However, catalase expression and activity were similar in SH1000 (8325-4 rsbU ؉ ), suggesting that this promoter may have a minor role in catalase expression under normal conditions. Restoration of B activity in SH1000 (8325-4 rsbU ؉ ) resulted in a marked decrease in the levels of the exoproteins SspA and Hla, and this is likely to be mediated by reduced expression of agr in this strain. By using Western blotting and a sarA-lacZ reporter assay, the levels of SarA were found to be similar in strains 8325-4 and SH1000 (8325-4 rsbU ؉ ) and sigB mutant derivatives of these strains. This finding contrasts with previous reports that suggested that SarA expression levels are altered when they are measured transcriptionally. Inactivation of sarA in each of these strains resulted in an expected decrease in agr expression; however, the relative level of agr in SH1000 (8325-4 rsbU ؉ ) remained less than the relative levels in 8325-4 and the sigB mutant derivatives. We suggest that SarA is not likely to be the effector in the overall B -mediated effect on agr expression.

We report the cloning and sequencing of the gene containing cytochrome c′ (cycP) from the photosynthetic purple bacterium Rhodobacter capsulatus and the regions flanking that gene. Mutant strains unable to synthesize cytochromec′ had increased sensitivity to nitrosothiols and to nitric oxide (which binds to the heme moiety of cytochrome c′).

The commonly used Staphylococcus aureus laboratory strain 8325-4 bears a naturally occurring 11-bp deletion in the B -regulating phosphatase rsbU. We have previously published a report (M. J. Horsburgh, J. L. Aish, I. J. White, L. Shaw, J. K. Lithgow, and S. J. Foster, J. Bacteriol. 184:5457-5467, 2002) on restoring the rsbU deletion, producing a B -functional 8325-4 derivative, SH1000. SH1000 is pleiotropically altered in phenotype from 8325-4, displaying enhanced pigmentation, increased growth yields, and a marked decrease in secreted exoproteins. This reduction in exoprotein secretion appears to result from a sixfold reduction in agr expression. In this study we have undertaken transposon mutagenesis of SH1000 to identify components involved in the modulation of extracellular proteases and ␣-hemolysin compared to 8325-4. In total, 13 genes were identified displaying increased ␣-hemolysin transcription and extracellular proteolysis. Phenotypic analysis revealed that each mutant also had decreased pigmentation and a general increase in protein secretion. Interestingly this phenotype was not identical in each case but was variable from mutant to mutant. None of the genes identified encoded classic regulatory proteins but were predominantly metabolic enzymes involved in amino acid biosynthesis and transport. Further analysis revealed that all of these mutations were clustered in a 35-kb region of the chromosome. By complementation and genetic manipulation we were able to demonstrate the validity of these mutations. Interestingly transcriptional analysis revealed that rather than being regulated by B , these genes appeared to have a role in the regulation of B activity. Thus, we propose that the loss of individual genes in this chromosomal hot spot region results in a destabilization of cellular harmony and disruption of the B regulatory cascade.