Gene_locus Report for: bacsu-yvfQ

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.

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

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

The RsbQ alpha/beta hydrolase and RsbP serine phosphatase form a signaling pair required to activate the general stress factor sigma(B) 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 Falpha 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.

BACKGROUND: Bacillus subtilis natto is closely related to the laboratory standard strain B. subtilis Marburg 168, and functions as a starter for the production of the traditional Japanese food "natto" made from soybeans. Although re-sequencing whole genomes of several laboratory domesticated B. subtilis 168 derivatives has already been attempted using short read sequencing data, the assembly of the whole genome sequence of a closely related strain, B. subtilis natto, from very short read data is more challenging, particularly with our aim to assemble one fully connected scaffold from short reads around 35 bp in length.
RESULTS: We applied a comparative genome assembly method, which combines de novo assembly and reference guided assembly, to one of the B. subtilis natto strains. We successfully assembled 28 scaffolds and managed to avoid substantial fragmentation. Completion of the assembly through long PCR experiments resulted in one connected scaffold for B. subtilis natto. Based on the assembled genome sequence, our orthologous gene analysis between natto BEST195 and Marburg 168 revealed that 82.4% of 4375 predicted genes in BEST195 are one-to-one orthologous to genes in 168, with two genes in-paralog, 3.2% are deleted in 168, 14.3% are inserted in BEST195, and 5.9% of genes present in 168 are deleted in BEST195. The natto genome contains the same alleles in the promoter region of degQ and the coding region of swrAA as the wild strain, RO-FF-1. These are specific for gamma-PGA production ability, which is related to natto production. Further, the B. subtilis natto strain completely lacked a polyketide synthesis operon, disrupted the plipastatin production operon, and possesses previously unidentified transposases.
CONCLUSIONS: The determination of the whole genome sequence of Bacillus subtilis natto provided detailed analyses of a set of genes related to natto production, demonstrating the number and locations of insertion sequences that B. subtilis natto harbors but B. subtilis 168 lacks. Multiple genome-level comparisons among five closely related Bacillus species were also carried out. The determined genome sequence of B. subtilis natto and gene annotations are available from the Natto genome browser http:\/\/natto-genome.org/.

The network controlling the general stress response in Bacillus subtilis requires both the RsbP phosphatase and the RsbQ alpha/beta 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 (alpha0) is unique to a subfamily of bacterial PP2C phosphatases that possess N-terminal sensing domains. The other (alpha1) 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 alpha0-alpha1 helices, with RsbQ hydrolase activity and the PAS domain jointly comprising a positive sensing module that counters the coiled-coil. We propose that the alpha0 helix characterizes an extended PP2C domain in many bacterial signalling proteins, and suggest it provides a means to communicate information from diverse input domains.

Growth-limiting stresses in bacteria induce the general stress response to protect the cells against future stresses. Energy stress caused by starvation conditions in Bacillus subtilis is transmitted to the sigma(B) transcription factor by stress-response regulators. RsbP, a positive regulator, is a phosphatase containing a PAS (Per-ARNT-Sim) domain and requires catalytic function of a putative alpha/beta hydrolase, RsbQ, to be activated. These two proteins have been found to interact with each other. We determined the crystal structures of RsbQ in native and inhibitor-bound forms to investigate why RsbP requires RsbQ. These structures confirm that RsbQ belongs to the alpha/beta hydrolase superfamily. Since the catalytic triad is buried inside the molecule due to the closed conformation, the active site is constructed as a hydrophobic cavity that is nearly isolated from the solvent. This suggests that RsbQ has specificity for a hydrophobic small compound rather than a macromolecule such as RsbP. Moreover, structural comparison with other alpha/beta hydrolases demonstrates that a unique loop region of RsbQ is a likely candidate for the interaction site with RsbP, and the interaction might be responsible for product release by operating the hydrophobic gate equipped between the cavity and the solvent. Our results support the possibility that RsbQ provides a cofactor molecule for the mature functionality of RsbP.

The kdp locus of Listeria monocytogenes encodes products with homology to structural proteins of a high-affinity potassium uptake system and to a two-component signal transduction system commonly involved in controlling gene expression. We have investigated the role of kdpE, encoding the transcriptional response regulator, as well as of the downstream gene, orfX, in adaptation of L. monocytogenes EGD to NaCl and low temperature. When grown in chemically defined medium the addition of NaCl to 2% decreased the growth rate of a mutant with an insertional inactivated kdpE, while mutants carrying in-frame deletions of either kdpE or orfX were unaffected by high osmolarity. Transcriptional analysis of kdpE and orfX revealed that their products are encoded by the same transcript. Thus, our data indicate that the absence of both KdpE and OrfX influences growth under osmotic pressure. Interestingly, OrfX contains a conserved domain of alpha/beta-hydrolases and resembles RsbQ that in Bacillus subtilis participates in the activation cascade of the general stress sigma factor SigB. When shifted to low temperature the deletion mutant lacking orfX resumed growth slightly faster than the wild-type. This phenotype was shared by a mutant carrying an in-frame deletion of sigB supporting the notion that OrfX could be a RsbQ homologue.

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.

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

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.