The σB regulon in Staphylococcus aureus and its regulation
Introduction
Many, but not all, bacterial species make use of distinct sigma factors with different promoter specificities to direct transcription in response to various stimuli. In Staphylococcus aureus there are three known sigma factors: σA, the housekeeping sigma factor, that directs the transcription of the bulk cellular RNA, and the two alternative sigma factors σB (Kullik et al., 1998; Wu et al., 1996) and σH (Morikawa et al., 2003). The S. aureus σB protein is closely related to the σB protein of Bacillus subtilis (Mittenhuber, 2002). In B. subtilis, σB is the master regulator of a large regulon which provides the cell with a multiple stress-resistant phenotype (for review (Hecker and Völker, 2004; Price, 2000)).
The regulation of σB activity in B. subtilis involves multiple protein–protein interactions, responding to a variety of stress conditions. Under non-stress conditions σB is held in an inactive complex by its antagonist RsbW, the anti-sigma factor. Following stress, RsbW is antagonized by the anti-anti-sigma factor RsbV, releasing σB free to interact with core RNA polymerase and ultimately leading to transcription initiation of about 150 σB-dependent genes (Petersohn et al., 2001; Price et al., 2001). However, only non-phosphorylated RsbV is able to compete with σB for RsbW binding. The phosphorylation state of RsbV is regulated by the kinase activity of RsbW and the action of two PP2C-type phosphatases, RsbP and RsbU. The RsbP phosphatase is required to activate σB in response to perturbations of the cellular energy level (energy stress pathway) whereas RsbU is responsible for transmitting physical and chemical stress stimuli to σB (environmental stress pathway) (Hecker et al., 1996; Price, 2000; Vijay et al., 2000; Voelker et al., 1995). To be functional, RsbP is dependent upon a second protein, RsbQ (Brody et al., 2001). However, the mechanism underlying this dependency is unclear. RsbU in turn, to achieve its activity against the substrate RsbV-P, requires the presence of free RsbT to be active (Kang et al., 1998). In vitro, RsbT was shown to be a part of a high-molecular-weight complex (∼1 MDa) which includes at least two additional proteins, RsbR and RsbS (Chen et al., 2003; Dufour et al., 1996). Under in vivo conditions, however, RsbT failed to be part of this complex (Kuo et al., 2004). Instead, RsbT was found to be present in two forms: a low- and a high-molecular-mass form. The latter one probably represents aggregated RsbT and might be in an inactive state (Kuo et al., 2004). In response to environmental stress, RsbT acts as a kinase towards RsbS and activates its target, RsbU, by binding (Chen et al., 2003; Kang et al., 1998; Voelker et al., 1996b). In the environmental stress pathway, the kinase activity of RsbT is counterbalanced by RsbX, the third PP2C phosphatase in the σB regulatory network (Smirnova et al., 1998; Voelker et al., 1997). Seven proteins from that network are organized together with σB in an eight-gene cluster with the structure rsbR–rsbS–rsbT–rsbU–rsbV–rsbW–sigB–rsbX (Kalman et al., 1990; Wise and Price, 1995).
In the case of S. aureus, the sigB gene is located in an operon similar to that of B. subtilis (Kullik et al., 1998; Wu et al., 1996). However, in S. aureus, this gene cluster lacks the upstream rsbR–rsbS–rsbT genes and the downstream rsbX gene. A blast search revealed that orthologues of rsbR, rsbS, rsbT, rsbX, and rsbP/rsbQ are absent from the available S. aureus genome sequences (Baba et al., 2002; Kuroda et al., 2001). There is experimental proof that the RsbW protein in S. aureus acts as an anti-sigma factor (Miyazaki et al., 1999) and that the activity of σB in this organism following heat shock depends on RsbU (Giachino et al., 2001). A proteomic study by Gertz et al. (2000) identified 23 proteins as regulated by σB. Recently, Bischoff et al. (2004) presented microarray data leading to a more comprehensive description of the σB regulon of S. aureus grown in LB medium.
In this study, we have identified conditions that lead to activation of σB in S. aureus and investigated the signaling pathway that controls the σB activity. In contrast to B. subtilis, energy stress is not a signal for σB activation in S. aureus. We also present DNA microarray data of S. aureus exposed to alkaline stress, a strong inducer of σB, extending the list of genes, the expression of which is regulated in a σB-dependent manner. A comparison of the function of the gene products of σB-dependent genes in S. aureus with those in B. subtilis leads us to the suggestion that the function of both regulons differs between these organisms.
Section snippets
Bacterial strains, plasmids and culture conditions
The bacterial strains and plasmids used in this study are listed in Table 1. Bacteria were routinely grown in Luria–Bertani (LB) medium at 37 °C and 130 rpm. For stress kinetic experiments, 160 ml LB medium were inoculated with exponentially growing cells of the appropriate S. aureus strain to an initial OD540 of 0.05. At an OD540 of 0.7, 20 ml of cell culture were transferred to new preheated Erlenmeyer flasks. Cells were harvested in fixed time intervals (1, 3, 6, 9, and 12 min) after imposition
A decrease of the cellular ATP pool is not a signal for σB activation in S. aureus
In B. subtilis σB-directed transcription can be induced by an uncoupler of oxidative phosphorylation (Alper et al., 1994). This induction is independent of RsbU (Voelker et al., 1995, Voelker et al., 1996a). The RsbU-independent pathway also responds to a challenge with MnCl2, which was shown to lower the cellular ATP level (Voelker et al., 1995). The PP2C phosphatase RsbP identified by Vijay et al. (2000) is required to convey signals of energy stress to σB in B. subtilis. Using a blastp
Discussion
In B. subtilis, σB provides the non-growing cell with a multiple and non-specific stress resistance. A sigB null mutant shows compromised survival if exponentially growing cells are subjected to heat, cold, osmotic, acid, and oxidative stress (Antelmann et al., 1996; Brigulla et al., 2003; Engelmann and Hecker, 1996; Völker et al., 1999). Importantly, induction of the general stress response by one stress also provides cross-protection against other stresses (Antelmann et al., 1996; Engelmann
Acknowledgments
Britta Jürgen und Stephanie Leja are acknowledged for their help in DNA microarray analyses. Furthermore, we thank Dirk Höper for helpful discussion, Renate Gloger for excellent technical assistance, and Rick Lewis for critical reading of the manuscript. This research was supported by grants of the “BMBF” (Pathogenomic-Network) and Fonds der chemischen Industrie to M. Hecker.
References (82)
- et al.
An adenosine nucleotide switch controlling the activity of a cell type-specific transcription factor in B. subtilis
Cell
(1994) - et al.
Genome and virulence determinants of high virulence community-acquired MRSA
Lancet
(2002) - et al.
Impaired oxidative stress resistance of Bacillus subtilis sigB mutants and the role of katA and katE
FEMS Microbiol. Lett.
(1996) - et al.
Optimization of a two-plasmid system for the identification of promoters recognized by RNA polymerase containing Staphylococcus aureus alternative sigma factor sigmaB
FEMS Microbiol. Lett.
(2004) - et al.
Specific contacts between the bacteriophage T3, T7, and SP6 RNA polymerases and their promoters
J. Biol. Chem.
(1991) - et al.
Whole genome sequencing of meticillin-resistant Staphylococcus aureus
Lancet
(2001) - et al.
Overexpression of sigma factor, varsigma(B), urges Staphylococcus aureus to thicken the cell wall and to resist beta-lactams
Biochem. Biophys. Res. Commun.
(2001) - et al.
General and oxidative stress responses in Bacillus subtilis: cloning, expression, and mutation of the alkyl hydroperoxide reductase operon
J. Bacteriol.
(1996) - et al.
Expression of a stress- and starvation-induced dps/pexB-homologous gene is controlled by the alternative sigma factor sigmaB in Bacillus subtilis
J. Bacteriol.
(1997) Insertional inactivation of staphylococcal methicillin resistance by Tn551
J. Bacteriol.
(1983)
Teicoplanin stress-selected mutations increasing sigma(B) activity in Staphylococcus aureus
Antimicrob. Agents Chemother.
Influence of a functional sigB operon on the global regulators sar and agr in Staphylococcus aureus
J. Bacteriol.
Microarray-based analysis of the Staphylococcus aureus sigmaB regulon
J. Bacteriol.
Chill induction of the SigB-dependent general stress response in Bacillus subtilis and its contribution to low-temperature adaptation
J. Bacteriol.
Catalytic function of an alpha/beta hydrolase is required for energy stress activation of the sigma(B) transcription factor in Bacillus subtilis
J. Bacteriol.
Regulation of transcription of compatible solute transporters by the general stress sigma factor, sigmaB, in Listeria monocytogenes
J. Bacteriol.
Comparative genomics reveal novel heat shock regulatory mechanisms in Staphylococcus aureus and other Gram-positive bacteria
Mol. Microbiol.
A supramolecular complex in the environmental stress signalling pathway of Bacillus subtilis
Mol. Microbiol.
Alternative transcription factor sigmaSB of Staphylococcus aureus: characterization and role in transcription of the global regulatory locus sar
J. Bacteriol.
CtsR, a novel regulator of stress and heat shock response, controls clp and molecular chaperone gene expression in gram-positive bacteria
Mol. Microbiol.
Identification of genes controlled by the essential YycG/YycF two-component system of Staphylococcus aureus
J. Bacteriol.
Relative levels and fractionation properties of Bacillus subtilis sigma(B) and its regulators during balanced growth and stress
J. Bacteriol.
Role of sigmaB in the expression of Staphylococcus aureus cell wall adhesins ClfA and FnbA and contribution to infectivity in a rat model of experimental endocarditis
Infect. Immun.
Bacillus subtilis functional genomics: global characterization of the stringent response by proteome and transcriptome analysis
J. Bacteriol.
Negative regulation by RpoS: a case of sigma factor competition
Mol. Microbiol.
General stress transcription factor sigmaB and sporulation transcription factor sigmaH each contribute to survival of Bacillus subtilis under extreme growth conditions
J. Bacteriol.
Regulation of sigmaB-dependent transcription of sigB and asp23 in two different Staphylococcus aureus strains
Mol. Gen. Genet.
Characterization of the sigma(B) regulon in Staphylococcus aureus
J. Bacteriol.
Sigma(B) activity depends on RsbU in Staphylococcus aureus
J. Bacteriol.
The global transcriptional response of Bacillus subtilis to manganese involves the MntR, Fur, TnrA and sigmaB regulons
Mol. Microbiol.
Towards a comprehensive understanding of Bacillus subtilis cell physiology by physiological proteomics
Proteomics
Heat-shock and general stress response in Bacillus subtilis
Mol. Microbiol.
Automated assembly of protein blocks for database searching
Nucleic Acids Res.
A putative multisubunit Na+/H+ antiporter from Staphylococcus aureus
J. Bacteriol.
Comprehensive characterization of the contribution of individual SigB-dependent general stress genes to stress resistance of Bacillus subtilis
J. Bacteriol.
sigmaB modulates virulence determinant expression and stress resistance: characterization of a functional rsbU strain derived from Staphylococcus aureus 8325-4
J. Bacteriol.
Genes controlled by the essential YycG/YycF two-component system of Bacillus subtilis revealed through a novel hybrid regulator approach
Mol. Microbiol.
Measuring genome evolution
Proc. Natl. Acad. Sci. USA
mrp, a multigene, multifunctional locus in Bacillus subtilis with roles in resistance to cholate and to Na+ and in pH homeostasis
J. Bacteriol.
Regulation of sigma factor competition by the alarmone ppGpp
Genes Dev.
Similar organization of the sigB and spoIIA operons encoding alternate sigma factors of Bacillus subtilis RNA polymerase
J. Bacteriol.
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