The σ B transcription factor of the bacterium Bacillus subtilis is activated by growth‐limiting energy or environmental challenge to direct the synthesis of more than 100 general stress proteins. Although the signal transduction pathway that conveys these stress signals to σ B is becoming increasingly well understood, how environmental or energy stress signals enter this pathway remains unknown. We show here that two PP2C serine phosphatases — RsbP, which is required for response to energy stress, and RsbU, which is required for response to environmental stress — each converge on the RsbV regulator of σ B . According to the current understanding of σ B regulation, in unstressed cells the phosphorylated RsbV anti‐anti‐σ is unable to complex the RsbW anti‐σ, which is then free to bind and inactivate σ B . We can now advance the model that either PP2C phosphatase, when triggered by its particular class of stress, can remove the phosphate from RsbV and thereby activate σ B . The action of the previously described RsbU is known to be controlled by dedicated upstream signalling components that are activated by environmental stress. The action of the RsbP phosphatase described here requires an energy stress, which we suggest is sensed, at least in part, by the PAS domain in the amino‐terminal region of the RsbP phosphatase. In other bacterial signalling proteins, similar PAS domains and their associated chromophores directly sense changes in intracellular redox potential to control the activity of a linked output domain.
The RsbQ α/β hydrolase and RsbP serine phosphatase form a signaling pair required to activate the general stress factor σB of Bacillus subtilis in response to energy limitation. RsbP has a predicted N-terminal Per-ARNT-Sim (PAS) domain, a central coiled-coil, and a C-terminal protein phosphatase M (PPM) domain. Previous studies support a model in which RsbQ provides an activity needed for PAS to regulate the phosphatase domain via the coiled-coil. RsbQ and the PAS domain (RsbP-PAS) therefore appear to form a sensory module. Here we test this hypothesis using bioinformatic and genetic analysis. We found 45 RsbQ and RsbP-PAS homologues encoded by adjacent genes in diverse bacteria, with PAS and a predicted coiled-coil fused to one of three output domains: PPM phosphatase (Gram positive bacteria), histidine protein kinase (Gram negative bacteria), and diguanylate cyclase (both lineages). Multiple alignment of the RsbP-PAS homologues suggested nine residues that distinguish the class. Alanine substitutions at four of these conferred a null phenotype in B. subtilis, indicating their functional importance. The F55A null substitution lay in the Fα helix of an RsbP-PAS model. F55A inhibited interaction of RsbP with RsbQ in yeast two-hybrid and pull-down assays but did not significantly affect interaction of RsbP with itself. We propose that RsbQ directly contacts the PAS domains of an RsbP oligomer to provide the activating signal, which is propagated to the phosphatase domains via the coiled-coil. A similar mechanism would allow the RsbQ-PAS module to convey a common input signal to structurally diverse output domains, controlling a variety of physiological responses.
In Bacillus subtilis, activity of the general stress transcription factor sigma 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 sigma B activity by influencing the binding decision of the RsbW anti-sigma factor. In the absence of stress, RsbW binds to sigma B and prevents its association with RNA polymerase core enzyme. However, following stress, RsbW binds instead to the RsbV anti-anti-sigma factor, thereby releasing sigma B to direct transcription of its target genes. These two principal regulators of sigmaB activity are encoded in the eight-gene sigB operon, which has the gene order rsbR-rsbS-rsbT-rsbU-rsbV-rsbW-sig B-rsbX (where rsb stands for regulator of sigma B). Notably, the predicted rsbS product has significant amino acid identity to the RsbV anti-anti-sigma factor and the predicted rsbT product resembles the RsbW anti-sigma factor. To determine the roles of rsbS and rsbT, null or missense mutations were constructed in the chromosomal copies or each and tested for their effects on expression of a sigma 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 or" 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.
Among pathogenic strains of Listeria monocytogenes , the σ B transcription factor has a pivotal role in the outcome of food-borne infections. This factor is activated by diverse stresses to provide general protection against multiple challenges, including those encountered during gastrointestinal passage. It also acts with the PrfA regulator to control virulence genes needed for entry into intestinal lumen cells. Environmental and nutritional signals modulate σ B activity via a network that operates by the partner switching mechanism, in which protein interactions are controlled by serine phosphorylation. This network is well characterized in the related bacterium Bacillus subtilis . A key difference in Listeria is the presence of only one input phosphatase, RsbU, instead of the two found in B. subtilis . Here, we aim to determine whether this sole phosphatase is required to convey physical, antibiotic and nutritional stress signals, or if additional pathways might exist. To that end, we constructed L. monocytogenes 10403S strains bearing single-copy, σ B -dependent opuCA – lacZ reporter fusions to determine the effects of an rsbU deletion under physiological conditions. All stresses tested, including acid, antibiotic, cold, ethanol, heat, osmotic and nutritional challenge, required RsbU to activate σ B . This was of particular significance for cold stress activation, which occurs via a phosphatase-independent mechanism in B. subtilis . We also assayed the effects of the D80N substitution in the upstream RsbT regulator that activates RsbU. The mutant had a phenotype consistent with low and uninducible phosphatase activity, but nonetheless responded to nutritional stress. We infer that RsbU activity but not its induction is required for nutritional signalling, which would enter the network downstream from RsbU.
ABSTRACT The general stress response of Bacillus subtilis is controlled by the ς B transcription factor, which is activated in response to diverse energy and environmental stresses. These two classes of stress are transmitted by separate signaling pathways which converge on the direct regulators of ς B , the RsbV anti-anti-ς factor and the RsbW anti-ς factor. The energy signaling branch involves the RsbP phosphatase, which dephosphorylates RsbV in order to trigger the general stress response. The rsbP structural gene lies downstream from rsbQ in a two-gene operon. Here we identify the RsbQ protein as a required positive regulator inferred to act in concert with the RsbP phosphatase. RsbQ bound RsbP in the yeast two-hybrid system, and a large in-frame deletion in rsbQ had the same phenotype as a null allele of rsbP —an inability to activate ς B in response to energy stress. Genetic complementation studies indicated that this phenotype was not due to a polar effect of the rsbQ alteration on rsbP . The predicted rsbQ product is a hydrolase or acyltransferase of the α/β fold superfamily, members of which catalyze a wide variety of reactions. Notably, substitutions in the presumed catalytic triad of RsbQ also abolished the energy stress response but had no detectable effect on RsbQ structure, synthesis, or stability. We conclude that the catalytic activity of RsbQ is an essential constituent of the energy stress signaling pathway.
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