Homologous pairs of regulatory proteins control activity of Bacillus subtilis transcription factor sigma(b) in response to environmental stress

Abstract

In Bacillus subtilis, activity of the general stress transcription factor B is controlled posttranslationally by a regulatory network that transmits signals of environmental and metabolic stress. These signals include heat, ethanol, or osmotic challenge, or a sharp decrease in cellular energy levels, and all ultimately control B activity by influencing the binding decision of the RsbW anti-factor. In the absence of stress, RsbW binds to B and prevents its association with RNA polymerase core enzyme. However, following stress, RsbW binds instead to the RsbV anti-anti-factor, thereby releasing B to direct transcription of its target genes. These two principal regulators of B activity are encoded in the eight-gene sigB operon, which has the gene order rsbR-rsbS-rsbT-rsbU-rsbV-rsbW-sigB-rsbX (where rsb stands for regulator of sigma B). Notably, the predicted rsbS product has significant amino acid identity to the RsbV anti-anti-factor and the predicted rsbT product resembles the RsbW anti-factor. To determine the roles of rsbS and rsbT, null or missense mutations were constructed in the chromosomal copies of each and tested for their effects on expression of a B -dependent reporter fusion. On the basis of this genetic analysis, our principal conclusions are that (i) the rsbS product is a negative regulator of B activity, (ii) the rsbT product is a positive regulator, (iii) RsbS requires RsbT for function, and (iv) the RsbS-RsbT and RsbV-RsbW pairs act hierarchically by a common mechanism in which key protein-protein interactions are controlled by phosphorylation events.In response to stress and starvation signals, the gram-positive bacterium Bacillus subtilis expresses a large set of genes termed the general stress regulon, which is primarily under control of the alternative transcription factor B (3,8,16,19,20,24,36). The activity of B is itself controlled posttranslationally by a multicomponent signal transduction pathway which conveys signals of environmental stress, such as heat shock, osmotic stress, or ethanol challenge, and signals of metabolic stress, such as the drop in cellular energy levels that occurs upon challenge with uncouplers of oxidative phosphorylation or upon entry into the stationary growth phase (2,4,6,7,9,34,35).All of the known regulators in the B signal transduction pathway are encoded by genes in the sigB operon, which also contains the B structural gene (20, 37). As shown in Fig. 1, these genes are termed rsb, for regulator of sigma B. Previous genetic and biochemical data indicate that the RsbW antifactor is the primary regulator, binding directly to B and maintaining it in a transcriptionally inactive complex (4, 5, 9).B is released from this complex by the action of the RsbV anti-anti-factor, which apparently sequesters RsbW by direct protein-protein interaction (4, 9, 13). Additional in vitro and in vivo experiments have demonstrated that the RsbW antifactor can associate with either B or RsbV and that the binding decision of RsbW is controlled by the phosphorylation state of RsbV (1,...

Modulator protein RsbR regulates environmental signalling in the general stress pathway of Bacillus subtilis

“…From these results, we conclude that: (i) RsbR is a positive regulator which probably belongs to the upstream, environmental-sensing module; (ii) RsbR augments response to salt and heat stress but is not absolutely required for sensing or transmitting these signals; and (iii) RsbR is unique among the known rsb gene products in that it is differentially involved in transmitting some but not all environmental stress signals. Significantly, genetic and biochemical analysis has established that the phosphorylation state of the RsbS, RsbV, and SpoIIAA antagonist proteins is important for their function, and that a conserved serine residue is the probable site of the phosphorylation event (Alper et al, 1994;Diederich et al, 1994;Dufour and Haldenwang, 1994;Najafi et al, 1995;Duncan et al, 1996;Kang et al, 1996;Yang et al, 1996). As shown in Fig.…”

“…Because many kinases that modify serine residues also modify threonine, we hypothesized that threonine residue 205 could be phosphorylated, and that this modification was important for RsbR function in vivo. If this were the case, we would predict that changing threonine 205 either to alanine, which cannot be phosphorylated, or to aspartate, which is thought to approximate the threonine in its phosphorylated state, would have opposite regulatory effects in vivo, much like the opposite effects caused by equivalent changes in the homologous RsbS and SpoIIAA antagonist proteins (Diederich et al, 1994;Duncan et al, 1996;Kang et al 1996;Yang et al, 1996).…”

“…As the rsbR null mutant responded differentially to environmental stress, we sought to more fully determine the role of the other positive regulator in the upstream module, the RsbT phosphatase activator. Previously, Kang et al (1996) found that RsbT function was required to transmit signals of salt stress but was not needed for signals of energy stress. As shown in Fig.…”

“…In contrast, the upstream module appears to be required chiefly for response to environmental stress signals (Voelker et al, 1995b;Kang et al, 1996). The output of the upstream module is borne by the RsbT switch protein, which directly binds RsbU in order to elicit its phosphatase activity .…”

“…Model of B regulation by coupled partner-switching modules Yang et al, 1996). As described in the text, the upstream module is required for response to environmental stress and the downstream module for response to energy stress (Alper et al, 1994;Voelker et al, 1995b;Kang et al, 1996). Each module consists of three components: a serine phosphatase (U or X), an antagonist protein (S or V), and a switch protein (T or W) which is also a serine protein kinase .…”