The σB regulon in Staphylococcus aureus and its regulation

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Abstract

The Staphylococcus aureus genome codes for a sigma factor that shows close sequence similarity to the alternative sigma factor σB of Bacillus subtilis. However, of the proteins controlling the activity of σB in B. subtilis only RsbU, RsbV, and RsbW are encoded in the staphylococcal genome. Therefore, the regulation of the σB activity must differ between these two bacterial species. The present study was designed (i) to describe the σB regulon and (ii) to identify stimuli leading to an activation of σB-dependent transcription. All conditions under which σB was activated in S. aureus (heat shock, addition of MnCl2 or NaCl, alkaline shock) required the presence of RsbU, a positive regulator of σB. In contrast to B. subtilis, a drop in the cellular ATP level caused by the addition of carbonyl cyanide m-chlorophenylhydrazone did not lead to an activation of σB in S. aureus. Moreover, ethanol, a strong inductor of σB activity in B. subtilis, also failed to induce σB in S. aureus. Expression of sigB and σB-dependent genes was enhanced following entry into stationary phase of cells grown in complex medium (LB medium). Our DNA microarray data indicated that 122 genes are positively regulated by σB under alkaline stress conditions. Interestingly, only 12% of these genes have an orthologue in the B. subtilis σB regulon, suggesting that the function of the σB regulon in S. aureus is different from that in B. subtilis. We could show that σB of S. aureus, in contrast to B. subtilis, may have a function in more basic cellular processes such as cell envelope composition, membrane transport processes and intermediary metabolism. σB-dependent genes identified by the DNA microarray approach were subjected to detailed transcriptional analyses using primer extension and Northern blot techniques. These analyses confirmed our DNA microarray data and furthermore revealed different regulatory groups of σB-dependent genes.

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 LuriaBertani (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.

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