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Journal of Bacteriology, December 2005, p. 8006-8019, Vol. 187, No. 23
0021-9193/05/$08.00+0     doi:10.1128/JB.187.23.8006-8019.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Molecular Analysis and Organization of the ^B Operon in Staphylococcus aureus

Department of Medical Microbiology, University of Zürich, 8006 Zürich, Switzerland,¹ Institute of Molecular Biology, Centre of Excellence for Molecular Medicine, Slovak Academy of Sciences, Dubravska cesta 21, 845 51 Bratislava, Slovak Republic,² Division of Infectious Diseases, University Hospitals Basel, Basel, Switzerland,³ Institute of Plant Sciences, Swiss Federal Institute of Technology, 8092 Zürich, Switzerland⁴

Received 25 July 2005/ Accepted 15 September 2005

The alternative sigma factor ^B of Staphylococcus aureus controls the expression of a variety of genes, including virulence determinants and global regulators. Genetic manipulations and transcriptional start point (TSP) analyses showed that the sigB operon is transcribed from at least two differentially controlled promoters: a putative ^A-dependent promoter, termed sigB_p1, giving rise to a 3.6-kb transcript covering sa2059-sa2058-rsbU-rsbV-rsbW-sigB, and a ^B-dependent promoter, sigB_p3, initiating a 1.6-kb transcript covering rsbV-rsbW-sigB. TSP and promoter-reporter gene fusion experiments indicated that a third promoter, tentatively termed sigB_p2 and proposed to lead to a 2.5-kb transcript, including rsbU-rsbV-rsbW-sigB, might govern the expression of the sigB operon. Environmental stresses, such as heat shock and salt stress, induced a rapid response within minutes from promoters sigB_p1 and sigB_p3. In vitro, the sigB_p1 promoter was active in the early growth stages, while the sigB_p2 and sigB_p3 promoters produced transcripts throughout the growth cycle, with sigB_p3 peaking around the transition state between exponential growth and stationary phase. The amount of sigB transcripts, however, did not reflect the concentration of ^B measured in cell extracts, which remained constant over the entire growth cycle. In a guinea pig cage model of infection, sigB transcripts were as abundant 2 and 8 days postinoculation as values found in vitro, demonstrating that sigB is indeed transcribed during the course of infection. Physical interactions between staphylococcal RsbU-RsbV, RsbV-RsbW, and RsbW-^B were inferred from a yeast (Saccharomyces cerevisiae) two-hybrid approach, indicating the presence of a partner-switching mechanism in the ^B activation cascade similar to that of Bacillus subtilis. The finding that overexpression of RsbU was sufficient to trigger an immediate and strong activation of ^B, however, signals a relevant difference in the regulation of ^B activation between B. subtilis and S. aureus in the cascade upstream of RsbU.

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