The general stress response of the bacterium Bacillus subtilis is governed by a signal transduction network that regulates activity of the orb transcription factor. We show that this network comprises two partner-switching modules, RsbX-RsbS-RsbT and RsbU-RsbV-RsbW, which contribute to regulating or B. Each module consists of a phosphatase (X or U), an antagonist protein (S or V), and a switch protein/kinase (T or W). In the downstream module, the W anti-or factor is the primary regulator of orb activity. If the V antagonist is phosphorylated, the W switch protein binds and inhibits orB. If V is unphosphorylated, it complexes W, freeing orb to interact with RNA polymerase and promote transcription. The phosphorylation state of V is controlled by opposing kinase (W) and phosphatase (U) activities. The U phosphatase is regulated by the upstream module. The T switch protein directly binds U, stimulating phosphatase activity. The T-U interaction is governed by the phosphorylation state of the S antagonist, controlled by opposing kinase (T) and phosphatase (X) activities. This partner-switching mechanism provides a general regulatory strategy in which linked modules sense and integrate multiple signals by protein-protein interaction.

The alternative transcription factor sigma B of Bacillus subtilis is activated during the stationary growth phase by a regulatory network responsive to stationary-phase signals. On the basis of the results reported here, we propose that sigma B controls a general stress regulon that is induced when cells encounter a variety of growth-limiting conditions. Expression of genes controlled by sigma B, including the ctc gene and the sigB operon that codes for sigma B and its associated regulatory proteins, was dramatically induced in both the exponential and stationary phases by environmental challenges known to elicit a general stress response. After cells were subjected to salt stress, the increased expression of lacZ transcriptional fusions to the ctc and sigB genes was entirely dependent on sigma B, and primer extension experiments confirmed that the sigma B-dependent transcriptional start site was used during salt induction of sigB operon expression. Western blotting (immunoblotting) experiments measuring the levels of sigma B protein indicated that ethanol addition and heat stress also induced sigma B activity during logarithmic growth. Salt and ethanol induction during logarithmic growth required RsbV, the positive regulator of sigma B activity that is normally necessary for activity in stationary-phase cells. However, heat induction of sigma B activity was largely independent of RsbV, indicating that there are two distinct pathways by which these environmental signals are conveyed to the transcriptional apparatus.

SummaryThe s 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 s 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 s B. According to the current understanding of s B regulation, in unstressed cells the phosphorylated RsbV anti-anti-s is unable to complex the RsbW anti-s, which is then free to bind and inactivate s 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 s 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.

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,...

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-oxygenvoltage 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.

The general stress response of Bacillus subtilis is controlled by the 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 B , the RsbV anti-anti-factor and the RsbW anti-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 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 ␣/␤ 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.In Bacillus subtilis and related bacteria, a general stress response controlled by the B transcription factor confers multiple stress resistance on nongrowing cells (reviewed in references 17 and 26). The activity of B is governed by a signal transduction pathway with two distinct branches. One branch is specific for energy stresses, such as carbon, phosphorus, or oxygen limitation, and the other is specific for environmental stresses, such as acid, ethanol, heat, or salt stress (20,41,43,44). According to the model shown in Fig. 1, each branch terminates with a differentially regulated serine phosphatase: RsbU in the environmental signaling branch and RsbP in the energy signaling branch. When activated by its particular class of stress, either RsbU or RsbP engages the common regulators RsbV and RsbW. RsbV and RsbW together control B activity via a partner-switching mechanism in which alternate proteinprotein interactions are governed by the phosphorylation state of RsbV (3,8,13,41,42,44).The means by which energy and environmental stress signals enter their respective branches of the pathway are unknown, and it is clear that additional signaling components remain to be identified (1,33,41,45). Here we focus on the energy signaling branch. The amino-terminal half of the RsbP phosphatase contains a Per-ARNT-Sim (PAS) domain (41), similar to those found in a wide variety of proteins involved in sensing fluctuations in redox, light, or oxygen (38). In some proteins, such PAS domains function by binding a ligand or a chromophore and in others by controlling protein-protein interactions with other proteins. As part o...

A Bacillus thuringiensis (B.t.) cryIIIA delta-endotoxin gene was designed for optimal expression in plants. The modified cry gene has the codon usage pattern of an average dicot gene and does not contain AT-rich nucleotide sequences typical of native B.t. cry genes. We assembled the 1.8 kb cryIIIA gene in nine blocks of three oligonucleotide pairs. For two DNA blocks, the polymerase chain reaction was used to enrich for correctly ligated pairs. We compared modified cryIIIA gene with native gene expression by electroporation of dicot (carrot) and monocot (corn) protoplasts. CryIIIA-specific RNA and protein was detected in carrot and corn protoplasts only after electroporation with the rebuilt gene. Transgenic potato lines were generated containing the redesigned cryIIIA gene under the transcriptional control of a chimeric CaMV 35S/mannopine synthetase (Mac) promoter. Out of 63 transgenic potato lines, 58 controlled first-instar Colorado potato beetle (CPB) larvae in bioassays. Egg masses which produced ca. 250,000 CPB larvae were placed on replicate clones of 56 transgenic potatoes. No CPB larvae developed past the second instar on any of these plants. Plants expressing high levels of delta-endotoxin were identified by their toxicity to more resistant third-instar larvae. We show there was good correlation between insect control and the levels of delta-endotoxin RNA and protein.

Regulatory networks controlling bacterial gene expression often evolve from common origins and share homologous proteins and similar network motifs. However, when functioning in different physiological contexts, these motifs may be re-arranged with different topologies that significantly affect network performance. Here we analyze two related signaling networks in the bacterium Bacillus subtilis in order to assess the consequences of their different topologies, with the aim of formulating design principles applicable to other systems. These two networks control the activities of the general stress response factor sigma(B) and the first sporulation-specific factor sigma(F). Both networks have at their core a "partner-switching" mechanism, in which an anti-sigma factor forms alternate complexes either with the sigma factor, holding it inactive, or with an anti-anti-sigma factor, thereby freeing sigma. However, clear differences in network structure are apparent: the anti-sigma factor for sigma(F) forms a long-lived, "dead-end" complex with its anti-anti-sigma factor and ADP, whereas the genes encoding sigma(B) and its network partners lie in a sigma(B)-controlled operon, resulting in positive and negative feedback loops. We constructed mathematical models of both networks and examined which features were critical for the performance of each design. The sigma(F) model predicts that the self-enhancing formation of the dead-end complex transforms the network into a largely irreversible hysteretic switch; the simulations reported here also demonstrate that hysteresis and slow turn off kinetics are the only two system properties associated with this complex formation. By contrast, the sigma(B) model predicts that the positive and negative feedback loops produce graded, reversible behavior with high regulatory capacity and fast response time. Our models demonstrate how alterations in network design result in different system properties that correlate with regulatory demands. These design principles agree with the known or suspected roles of similar networks in diverse bacteria.