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Journal of Bacteriology, September 2006, p. 6070-6080, Vol. 188, No. 17
0021-9193/06/$08.00+0     doi:10.1128/JB.00551-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Investigations into {sigma}B-Modulated Regulatory Pathways Governing Extracellular Virulence Determinant Production in Staphylococcus aureus

Lindsey N. Shaw,1* Joanne Aish,2 Jessica E. Davenport,1 Melanie C. Brown,1 James K. Lithgow,2 Kay Simmonite,2 Howard Crossley,2 James Travis,1 Jan Potempa,1,3 and Simon J. Foster2

Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602,1 Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, England S10 2TN,2 Department of Microbiology, Faculty of Biotechnology, Jagiellonian University, 7 Gronostajowa St., 30-387 Kraków, Poland3

Received 18 April 2006/ Accepted 20 June 2006

The commonly used Staphylococcus aureus laboratory strain 8325-4 bears a naturally occurring 11-bp deletion in the {sigma}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 {sigma}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 {alpha}-hemolysin compared to 8325-4. In total, 13 genes were identified displaying increased {alpha}-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 {sigma}B, these genes appeared to have a role in the regulation of {sigma}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 {sigma}B regulatory cascade.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602. Phone: (706) 542-1714. Fax: (706) 542-3719. E-mail: lesshaw{at}uga.edu.

Journal of Bacteriology, September 2006, p. 6070-6080, Vol. 188, No. 17
0021-9193/06/$08.00+0     doi:10.1128/JB.00551-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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