Expression of the ςB-Dependent General Stress Regulon Confers Multiple Stress Resistance in Bacillus subtilis

The alternative sigma factor sigmaB of Bacillus subtilis is required for the induction of approximately 100 genes after the imposition of a whole range of stresses and energy limitation. In this study, we investigated the impact of a null mutation in sigB on the stress and starvation survival of B. subtilis. sigB mutants which failed to induce the regulon following stress displayed an at least 50- to 100-fold decrease in survival of severe heat (54 degrees C) or ethanol (9%) shock, salt (10%) stress, and acid (pH 4.3) stress, as well as freezing and desiccation, compared to the wild type. Preloading cells with sigmaB-dependent general stress proteins prior to growth-inhibiting stress conferred considerable protection against heat and salt. Exhaustion of glucose or phosphate induced the sigmaB response, but surprisingly, sigmaB did not seem to be required for starvation survival. Starved wild-type cells exhibited about 10-fold greater resistance to salt stress than exponentially growing cells. The data argue that the expression of sigmaB-dependent genes provides nonsporulated B. subtilis cells with a nonspecific multiple stress resistance that may be relevant for stress survival in the natural ecosystem.     

The influence of agr and sigmaB in growth phase dependent regulation of virulence factors in Staphylococcus aureus

The expression of many virulence determinants in Staphylococcus aureus is tightly coordinated generally by global regulatory elements such as accessory gene regulator (agr), staphylococcal accessory regulator and the alternative sigma factor sigmaB. We have compared the two-dimensional (2-D) protein pattern of extracellular protein extracts of wild-type cells with the 2-D patterns of the respective regulatory mutants in order to identify proteins whose amount is influenced by a mutation in agr or sigB. In order to quantify changes in the level of interesting proteins we used the Ettan-fluorescence difference gel electrophoresis technique (Amersham Biosciences). As in most bacteria, the amount of extracellular proteins was strongly regulated and increased mainly in the stationary phase of growth at high cell densities. By comparing the extracellular protein pattern of the RN6390 rsbU strain with that of an isogenic agr mutant RN6911 we show that the level of about 70 protein spots changed in the mutant. To analyze the role of sigmaB in virulence gene expression an RsbU+ (RN6390 RsbU+) derivative was included in this study. The protein pattern of the RsbU+ strain (RN6390 RsbU+) was very similar to that of the Deltaagr/DeltarsbU mutant strain (RN6911) indicating an opposing effect of agr and rsbU on the expression of the same genes.     

The yeast two-hybrid system detects interactions between Bacillus subtilis sigmaB regulators.

SigmaB, the general stress response sigma factor of Bacillus subtilis, is regulated by the products of seven genes (rsbR, S, T, U, V, W, and X) with which it is cotranscribed. Biochemical techniques previously revealed physical associations among RsbW, RsbV, and sigmaB but failed to detect interactions of RsbR, S, T, U, or X with each other or RsbV, RsbW, or sigmaB. Using the yeast two-hybrid system, we have now obtained evidence for such interactions. The yeast reporter system was activated when RsbS was paired with either RsbR or RsbT, RsbR was paired with RsbT, and RsbV was paired with either RsbU or RsbW. In addition, RsbW2 and RsbR2 dimer formation was detected. RsbX failed to show interactions with itself or any of the other sigB operon products.     

A phylogenomic study of the general stress response sigma factor sigmaB of Bacillus subtilis and its regulatory proteins

Regulation of expression of the general stress regulon of Bacillus subtilis is mediated by the activation of the alternative sigma factor sigmaB. Activation of sigmaB is accomplished by a complex regulatory network involving protein-protein interactions and reversible protein phosphorylation. PSI-BLAST searches were performed and phylogenetic trees for sigmaB and its regulatory proteins were constructed. Occurrence of sigmaB is restricted to a small group of gram-positive bacteria (Bacillus, Staphylococcus, Listeria). Related sigma factors also involved in stress responses are present in Mycobacterium tuberculosis, Streptomyces species and even in cyanobacteria (Synechocystis species). Putative regulatory proteins found in several other bacterial species can be broadly catagorized into three categories: Anti sigma factors, anti-anti sigma factors and phosphatases. Anti sigma factors are able to bind to sigma factors and are also kinases of anti sigma factor antagonists. Only in their nonphosphorylated state, these antagonists are able to bind to the anti sigma factor. Phosphorylated antagonists can be dephosphorylated by PP2C phosphatases. These phosphatases are of pivotal importance for activation of the sigma factor. Different phosphatases identified in this search contain a wide variety of domains found in signal transducing proteins (PAS/PAC, GAF, REC, HATase_c, HAMP). The HATPase_c domain found in several phosphatases most probably constitutes a serine/threonine kinase domain of anti sigma factors. Such proteins are most probably bifunctional anti-anti sigma factor kinases and phosphatases. The regulatory network of anti-anti sigma factors anti sigma factors and phosphatases is probably ancient and most likely evolved from a structurally similar network found in the Deinococcus radiodurans genome. In completely sequenced genomes of several bacterial species, some elements of the network are missing. The N-terminus of RsbU, a phosphatase activated in response to environmental stress exhibits similarities to a region in the beta chain of phenylalanyl-tRNA synthetases.     

Occurrence of mazEF-like antitoxin/toxin systems in bacteria

The mazEF locus of Escherichia coil located in an operon together with the upstream relA gene (encoding ATP:GTP 3'-pyrophosphotransferase; (p)ppGpp synthetase), encodes an antitoxin/toxin system which might play a role in programmed cell death under stress and starvation conditions at high cell densities. By homology searches, chromosomally encoded orthologous systems were identified in a variety of bacteria, sometimes without the MazE-like antitoxin, and several bacterial species possess multiple MazEF-like systems (paralogs). In many gram positive bacteria, the mazEF-locus is located directly upstream of the sigB (stress sigma factor sigmaB) operon in a putative operon together with the upstream dal (aIr) gene (encoding D-alanine racemase). The acidic antitoxins are less conserved than the basic toxins. The differences in genomic organization of the mazEFloci in E. coli versus those in gram positive bacteria might indicate their association with different stress response regulons in these organisms. A study on the sigmaB operon of Staphylococcus aureus showed that the mazF gene of this organism is cotranscribed with the sigmaB operon in response to heat shock, providing the first example that the expression of the mazEFlocus might be indeed associated with stress responses.     

Phosphorylation and RsbX-dependent dephosphorylation of RsbR in the RsbR-RsbS complex of Bacillus subtilis

In the pathway that controls sigmaB activity, the RsbR-RsbS complex plays an important role by trapping RsbT, a positive regulator of sigmaB of Bacillus subtilis. We have proposed that at the onset of stress, RsbR becomes phosphorylated, resulting in an enhanced activity of RsbT towards RsbS. RsbT is then free to interact with and activate RsbU, which in turn ultimately activates sigmaB. In this study with purified proteins, we used mutant RsbR proteins to analyze the role of its phosphorylatable threonine residues. The results show that the phosphorylation of either of the two RsbT-phosphorylatable threonine residues (T171 and T205) in RsbR enhanced the kinase activity of RsbT towards RsbS. However, it appeared that RsbT preferentially phosphorylates T171. We also present in vitro evidence that identifies RsbX as a potential phosphatase for RsbR T205.