Margaret S. Brody

A PP2C phosphatase containing a PAS domain is required to convey signals of energy stress to the sigmaB transcription factor of Bacillus subtilis.

The σB 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 σB 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 σB. According to the current understanding of σB 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 σB. 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 σB. 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 blue-light receptor YtvA acts in the environmental stress signaling pathway of Bacillus subtilis.

The general stress response of the bacterium Bacillus subtilis is regulated by a partner-switching mechanism in which serine and threonine phosphorylation controls protein interactions in the stress-signaling pathway. The environmental branch of this pathway contains a family of five paralogous proteins that function as negative regulators. Here we present genetic evidence that a sixth paralog, YtvA, acts as a positive regulator in the same environmental signaling branch. We also present biochemical evidence that YtvA and at least three of the negative regulators can be isolated from cell extracts in a large environmental signaling complex. YtvA differs from these associated negative regulators by its flavin mononucleotide (FMN)-containing light-oxygen-voltage domain. Others have shown that this domain has the photochemistry expected for a blue-light sensor, with the covalent linkage of the FMN chromophore to cysteine 62 composing a critical part of the photocycle. Consistent with the view that light intensity modifies the output of the environmental signaling pathway, we found that cysteine 62 is required for YtvA to exert its positive regulatory role in the absence of other stress. Transcriptional analysis of the ytvA structural gene indicated that it provides the entry point for at least one additional environmental input, mediated by the Spx global regulator of disulfide stress. These results support a model in which the large signaling complex serves to integrate multiple environmental signals in order to modulate the general stress response.

Catalytic function of an alpha/beta hydrolase is required for energy stress activation of the sigma(B) transcription factor in Bacillus subtilis.

The general stress response of Bacillus subtilis is controlled by the sigma(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 sigma(B), the RsbV anti-anti-sigma factor and the RsbW anti-sigma 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 sigma(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 alpha/beta 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.

Bacillus subtilis operon under the dual control of the general stress transcription factor sigma B and the sporulation transcription factor sigma H.

The σB transcription factor of Bacillus subtilis is activated in response to a variety of environmental stresses, including those imposed by entry into the stationary‐growth phase, and by heat, salt or ethanol challenge to logarithmically growing cells. Although σB is thought to control a general stress regulon, the range of cellular functions it directs remains largely unknown. Our approach to understand the physiological role of σB is to characterize genes that require this factor for all or part of their expression, i.e. the csb genes. In this study, we report that the transposon insertion csb40::Tn917lac identifies an operon with three open reading frames, the second of which resembles plant proteins induced by desiccation stress. Primer‐extension and operon‐fusion experiments showed that the csb40 operon has a σB‐dependent promoter which is strongly induced by the addition of salt to logarithmically growing cells. The csb40 operon also has a second, σH‐dependent promoter that is unaffected by salt addition. These results provide support for the hypothesis that σB controls a general stress regulon, and indicate that the σB and σH regulons partly overlap. We suggest that in addition to its acknowledged role in the sporulation process, σH is also involved in controlling a subclass of genes that are broadly involved in a general stress response.

Physical and antibiotic stresses require activation of the RsbU phosphatase to induce the general stress response in Listeria monocytogenes.

Among pathogenic strains of Listeria monocytogenes, the σB 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 σB 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, σB-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 σB. 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.

Bacillus licheniformis sigB operon encoding the general stress transcription factor σB

The general stress response of the Gram-positive soil bacterium Bacillus subtilis is controlled by the sigma B transcription factor. sigma B activity is regulated by the newly discovered partner switching mechanism of signal transduction, which integrates the two different classes of challenges which posttranslationally activate sigma B: environmental stress and energy stress. Our investigation of a possible sigma B homologue in the related soil bacterium B. licheniformis had two goals. First, this study would contribute to understanding the distribution of the sigma B general stress system among Gram-positive bacteria. Second, a phylogenetic comparison of regulatory systems can supplement genetic and biochemical analysis by revealing conserved features that are critical for function. We report here that (1) B. licheniformis cells contain a protein that closely resembles B. subtilis sigma B in size and antigenic properties; (2) the level of this potential sigma B homologue rapidly increases following environmental or energy stress; and (3) the B. licheniformis genome encodes a homologue of the sigB general stress operon, including the sigma B structural gene and seven rsb regulatory genes. Based on these results, B. licheniformis possesses a general stress system likely regulated by two coupled partner switching modules that sense and integrate the two broad classes of activating stress signals.

Bypass suppression analysis maps the signalling pathway within a multidomain protein: the RsbP energy stress phosphatase 2C from Bacillus subtilis

The network controlling the general stress response in Bacillus subtilis requires both the RsbP phosphatase and the RsbQ α/β hydrolase to convey signals of energy stress. RsbP contains three domains: an N‐terminal PAS, a central coiled‐coil and a C‐terminal PP2C phosphatase. We report here a genetic analysis that established the functional interactions of the domains and their relationship to RsbQ. Random mutagenesis of rsbP yielded 17 independent bypass suppressors that had activity in an rsbQ null strain background. The altered residues clustered in three regions of RsbP: the coiled‐coil and two predicted helices of the phosphatase domain. One helix (α0) is unique to a subfamily of bacterial PP2C phosphatases that possess N‐terminal sensing domains. The other (α1) is distinct from the active site in all solved PP2C structures. The phenotypes of the suppressors and directed deletions support a model in which the coiled‐coil negatively controls phosphatase activity, perhaps via the α0‐α1 helices, with RsbQ hydrolase activity and the PAS domain jointly comprising a positive sensing module that counters the coiled‐coil. We propose that the α0 helix characterizes an extended PP2C domain in many bacterial signalling proteins, and suggest it provides a means to communicate information from diverse input domains.

An α/β Hydrolase and Associated Per-ARNT-Sim Domain Comprise a Bipartite Sensing Module Coupled with Diverse Output Domains

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.