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.     

Functional and structural characterization of RsbU, a stress signaling protein phosphatase 2C

RsbU is a positive regulator of the activity of sigmaB, the general stress-response sigma factor of Gram+ microorganisms. The N-terminal domain of this protein has no significant sequence homology with proteins of known function, whereas the C-terminal domain is similar to the catalytic domains of PP2C-type phosphatases. The phosphatase activity of RsbU is stimulated greatly during the response to stress by associating with a kinase, RsbT. This association leads to the induction of sigmaB activity. Here we present data on the activation process and demonstrate in vivo that truncations in the N-terminal region of RsbU are deleterious for the activation of RsbU. This conclusion is supported by comparisons of the phosphatase activities of full-length and a truncated form of RsbU in vitro. Our determination of the crystal structure of the N-terminal domain of RsbU from Bacillus subtilis reveals structural similarities to the regulatory domains from ubiquitous protein phosphatases and a conserved domain of sigma-factors, illuminating the activation processes of phosphatases and the evolution of "partner switching." Finally, the molecular basis of kinase recruitment by the RsbU phosphatase is discussed by comparing RsbU sequences from bacteria that either possess or lack RsbT.     

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.     

Obg, an essential GTP binding protein of Bacillus subtilis, is necessary for stress activation of transcription factor sigma(B).

sigma(B), the general stress response sigma factor of Bacillus subtilis, is activated when intracellular ATP levels fall or the bacterium experiences environmental stress. Stress activates sigma(B) by means of a collection of regulatory kinases and phosphatases (the Rsb proteins), which catalyze the release of sigma(B) from an anti-sigma factor inhibitor. By using the yeast dihybrid selection system to identify B. subtilis proteins that could interact with Rsb proteins and act as mediators of stress signaling, we isolated the GTP binding protein, Obg, as an interactor with several of these regulators (RsbT, RsbW, and RsbX). B. subtilis depleted of Obg no longer activated sigma(B) in response to environmental stress, but it retained the ability to activate sigma(B) by the ATP responsive pathway. Stress pathway components activated sigma(B) in the absence of Obg if the pathway's most upstream effector (RsbT) was synthesized in excess to the inhibitor (RsbS) from which it is normally released after stress. Thus, the Rsb proteins can function in the absence of Obg but fail to be triggered by stress. The data demonstrate that Obg, or a process under its control, is necessary to induce the stress-dependent activation of sigma(B) and suggest that Obg may directly communicate with one or more sigma(B) regulators.     

Serine kinase activity of a Bacillus subtilis switch protein is required to transduce environmental stress signals but not to activate its target PP2C phosphatase

The RsbT serine kinase has two known functions in the signal transduction pathway that activates the general stress factor σ^B of Bacillus subtilis . First, RsbT can phosphorylate and inactivate its specific antagonist protein, RsbS. Second, upon phosphorylation of RsbS, RsbT is released to stimulate RsbU, a PP2C phosphatase, thereby initiating a signalling cascade that ultimately activates σ^B. Here we describe a mutation that separates these two functions of RsbT. Although the mutant RsbT protein had essentially no kinase activity, it still retained the capacity to stimulate the RsbU phosphatase in vitro and to activate σ^B when overexpressed in vivo . These results support the hypothesis that phosphatase activation is accomplished via a long-lived interaction between RsbT and RsbU. In contrast, RsbT kinase activity was found to be integral for the transmission of external stimuli to σ^B. Thus, one route by which environmental stress signals could enter the σ^B network is by modulation of the RsbT kinase activity, thereby controlling the magnitude of the partner switch between the RsbS–RsbT complex and the RsbT–RsbU complex.     

A supramolecular complex in the environmental stress signalling pathway of Bacillus subtilis

SigmaB, an alternative sigma-factor of Bacillus subtilis, mediates the response of the cell to a variety of physical insults. Within the environmental stress signalling pathway RsbU, a protein phosphatase, is stimulated by its interaction with the protein kinase RsbT. In the absence of stress RsbT is expected to be trapped by an alternative binding partner, RsbS. Here, we have demonstrated that RsbS alone cannot act as an alternative partner for RsbT, but instead requires the presence of RsbR to create a high molecular mass RsbR:RsbS complex (approximately 1 MDa) able to capture RsbT. In this complex the phosphorylation state of RsbS, and not that of RsbR, controlled the binding to RsbT, whose kinase activity towards RsbS could be counterbalanced by the activity of RsbX, the phosphatase for RsbS-P. The RsbR:RsbS complex recruited RsbT from a mixture of RsbT and RsbU. The phosphorylated form of RsbR in the complex enhanced the kinase activity of RsbT towards RsbS. This supramolecular complex thus has the functional properties of an alternative partner for RsbT. Electron micrographs of this complex are presented, and the purification of the RsbR:RsbS complex from cellular extracts provides evidence for the existence of such a complex in vivo.