Alternative transcription factor sigmaSB of Staphylococcus aureus: characterization and role in transcription of the global regulatory locus sar

Abstract

A homolog of the multiple-stress-responsive transcription factor B of Bacillus subtilis was predicted from the DNA sequence analysis of a region of the Staphylococcus aureus chromosome. A hybrid between the coding sequence of the first 11 amino acids of the gene 10 leader peptide of phage T7 (T7.Tag) and the putative sigB gene of S. aureus was constructed and cloned into Escherichia coli BL21(DE3)pLysS for overexpression from a T7 promoter. A homogeneous preparation of the overproduced protein was obtained by affinity chromatography with a T7.Tag monoclonal antibody coupled to agarose. The amino-terminal amino acid sequence of the first 22 residues of the purified protein matched that deduced from the nucleotide sequence. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein, designated SB , indicated that it migrated as an approximately 39-kDa polypeptide. Promoter-specific transcription from the B. subtilis B -dependent P B promoter of the sigB operon was stimulated by SB in a concentration-dependent fashion when reconstituted with the S. aureus core RNA polymerase (RNAP). Specific transcript from the predicted B -dependent P B promoter of the sigB operon of S. aureus was obtained by the reconstituted RNAP in a runoff transcription reaction. The sar operon of S. aureus contains three promoter elements (P1, P2, and P3) and is known to partly control the synthesis of a number of extracellular toxins and several cell wall proteins. Our in vitro studies revealed that transcription from the P1 promoter is dependent on the primary factor SA , while that of the P3 promoter is dependent on SB . As determined by primer extension studies, the 5 end of the SB -initiated mRNA synthesized in vitro from the sar P3 promoter is in agreement with the 5 end of the cellular RNA.In prokaryotes, several factors coexist in cells (19,22,24). The transcription factors evolutionarily related to Escherichia coli 70 are subdivided into two groups (15). The primary factor is required for the expression of housekeeping genes whose products are essential for exponential cell growth. The level or the activity of the second group, comprising the alternate factors, has been shown to be modulated in response to environmental and metabolic stresses. The core RNA polymerase (RNAP) associated with a particular factor initiates transcription from a specific set of promoters with conserved sequence motifs. B , identified in sporulating Bacillus subtilis, is the first member of the alternate factors to be reported in prokaryotes (20,21). Much has been learnt recently about the regulation of expression of the sigB operon and the modulation of the function of the SigB ( B ) protein in response to changing stress (entry into stationary growth phase, nutrient depletion, and presence of uncouplers of oxidative phosphorylation) and environmental stress (heat shock and osmotic shock) in B. subtilis (2,6,8,22,36,37,41).The emergence of multiple-drug-resistant, highly pathogenic strains of Staphylococcus aureus poses a serious thr...

SarA, a Global Regulator of Virulence Determinants inStaphylococcus aureus, Binds to a Conserved Motif Essential for sar-dependent Gene Regulation

“…Because of their overlapping nature, each of these transcripts (sarA, sarC, and sarB transcripts) also includes the sarA coding region (10). With the promoters for the sarA and sarB transcripts being A-dependent (active during the exponential phase) (10,28) and that of sarC being B-dependent (active during the postexponential phase) (28,29), it is not surprising that sar transcription varies during the growth cycle. Dependent on the pattern of sar promoter activation, the SarA level may conceivably fluctuate (12,28).…”

“…Because of the multiplicity of sar promoters with diverse activation requirements (e.g. sarC activated by SigB) (28,29), the precise control of SarA protein levels as a result of differential sar promoter activation is likely to be dependent on a variety of environmental and intracellular factors. Notably, we recently identified a regulatory protein that binds to the sar promoter region to down-regulate sarC transcription (28).…”

An introduction to staphylococcus aureus, and techniques for identifying and quantifying s. aureus adhesins in relation to adhesion to biomaterials: review

“…These include sarH1 (also known as sarS; Tegmark et al, 2000;Cheung et al, 2001) and sarT . The expression of sarH1 is regulated by sarA and agr (Tegmark et al, 2000), and is transcribed, as is sarA by SigA-and SigB-dependent promoters (Deora at al, 1997;Manna et al, 1998).…”

“…This enables the bacteria to escape from the localised infection (abscess) during stationary phase and spread to new sites, where the cycle is repeated. S. aureus adhesins σ A , the primary sigma factor responsible for the expression of housekeeping genes, whose products are necessary for growth (Deora et al, 1997); and σ B , the alternative sigma factor, that regulates the expression of many genes involved in cellular functions (Deora and Misra, 1996). σ B has a role in virulence determinant production, and stress response (Horsburgh et al, 2002).…”

Adaptation beyond the Stress Response: Cell Structure Dynamics and Population Heterogeneity in Staphylococcus aureus

“…It is conserved in all bacteria and responsible for the expression of house-keeping genes (23). SigB is the alternative sigma factor for general responses to stress (24). Its levels are constant throughout the growth phase (38), but SigB can be activated under unusual pH conditions, heat shock (transient), high osmolarity, and later growth phase through the 'partner switching' mechanism, although the precise mechanism for signal transduction to the SigB regulators in S. aureus is not known (86).…”