The PAS domains of the major sporulation kinase in Bacillus subtilis play a role in tetramer formation that is essential for the autokinase activity.
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ABSTRACT:
SUBMITTER: Kiehler B
PROVIDER: S-EPMC5552956 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Histidine kinases are sophisticated molecular sensors that are used by bacteria to detect and respond to a multitude of environmental signals. KinA is the major histidine kinase required for initiation of sporulation upon nutrient deprivation in Bacillus subtilis. KinA has a large N-terminal region (residues 1 to 382) that is uniquely composed of three tandem Per-ARNT-Sim (PAS) domains that have been proposed to constitute a sensor module. To further enhance our understanding of this "sensor" region, we defined the boundaries that give rise to the minimal autonomously folded PAS domains and analyzed their homo- and heteroassociation properties using analytical ultracentrifugation, nuclear magnetic resonance (NMR) spectroscopy, and multiangle laser light scattering. We show that PAS(A) self-associates very weakly, while PAS(C) is primarily a monomer. In contrast, PAS(B) forms a stable dimer (K(d) [dissociation constant] of <10 nM), and it appears to be the main N-terminal determinant of KinA dimerization. Analysis of KinA mutants deficient for one or more PAS domains revealed a critical role for PAS(B), but not PAS(A), in autophosphorylation of KinA. Our findings suggest that dimerization of PAS(B) is important for keeping the catalytic domain of KinA in a functional conformation. We use this information to propose a model for the structure of the N-terminal sensor module of KinA.
| S-EPMC3650535 | biostudies-literature
Project description:Endospore formation in the Gram-positive bacterium Bacillus subtilis initiates in response to nutrient depletion and involves a series of morphological changes that result in the creation of a dormant spore. Early in this developmental process, the cell undergoes an asymmetric cell division that produces the larger mother cell and smaller forespore, the latter destined to become the mature spore. The mother cell septal membrane then engulfs the forespore, at which time an essential channel, the so-called feeding-tube apparatus, is thought to cross both membranes to create a direct conduit between the cells. At least nine proteins are required to form this channel including SpoIIQ under forespore control and SpoIIIAA-AH under the mother cell control. Several of these proteins share similarity to components of Type-II, -III and -IV secretion systems as well as the flagellum from Gram-negative bacteria. Here we report the X-ray crystallographic structure of the cytosolic domain of SpoIIIAB to 2.3 Å resolution. This domain adopts a conserved, secretion-system related fold of a six membered anti-parallel helical bundle with a positively charged membrane-interaction face at one end and a small groove at the other end that may serve as a binding site for partner proteins in the assembled apparatus. We analyzed and identified potential interaction interfaces by structure-guided mutagenesis in vivo. Furthermore, we were able to identify a remarkable structural homology to the C-subunit of a bacterial V-ATPase. Collectively, our data provides new insight into the possible roles of SpoIIIAB protein within the secretion-like apparatus essential to bacterial sporulation.
| S-EPMC5864543 | biostudies-literature
Project description:To characterize the role of the three PAS domains in KinA, the major sporulation kinase in Bacillus subtilis, we constructed a series of systematic PAS domain deletion mutants and analyzed their activities using an IPTG (isopropyl-beta-d-thiogalactopyranoside)-inducible artificial sporulation induction system, which we have developed recently. The results showed that any one of the three PAS domains is sufficient to maintain the kinase activity and trigger sporulation, if not fully then at least partially, when the protein levels increase beyond a certain level.
| S-EPMC2832533 | biostudies-literature
Project description:While vegetative Bacillus subtilis cells and mature spores are both surrounded by a thick layer of peptidoglycan (PG, a polymer of glycan strands cross-linked by peptide bridges), it has remained unclear whether PG surrounds prespores during engulfment. To clarify this issue, we generated a slender ?ponA mutant that enabled high-resolution electron cryotomographic imaging. Three-dimensional reconstructions of whole cells in near-native states revealed a thin PG-like layer extending from the lateral cell wall around the prespore throughout engulfment. Cryotomography of purified sacculi and fluorescent labelling of PG in live cells confirmed that PG surrounds the prespore. The presence of PG throughout engulfment suggests new roles for PG in sporulation, including a new model for how PG synthesis might drive engulfment, and obviates the need to synthesize a PG layer de novo during cortex formation. In addition, it reveals that B. subtilis can synthesize thin, Gram-negative-like PG layers as well as its thick, archetypal Gram-positive cell wall. The continuous transformations from thick to thin and back to thick during sporulation suggest that both forms of PG have the same basic architecture (circumferential). Endopeptidase activity may be the main switch that governs whether a thin or a thick PG layer is assembled.
| S-EPMC3893088 | biostudies-literature
Project description:This series of experiments was designed to identify the program of gene transcription for a single differentiating cell type during sporulation in Bacillus subtilis. The mother cell is one of two cell types generated by asymmetric division of sporulating cells approximately two hours after initiation of sporulation. The program is governed by a hierarchical cascade consisting of the transcription factors: sigmaE, sigmaK, GerE, GerR (YlbO) and SpoIIID. The characterization of the sigmaE regulon was reported in Eichenberger et al. (2003), J. Mol. Biol. 327, 945-972. Here we report the data for sigmaK, GerE, GerR and SpoIIID.
2004-09-01 | GSE1620 | GEO
Project description:Starving Bacillus subtilis cells execute a gene expression program resulting in the formation of stress-resistant spores. Sporulation master regulator, Spo0A, is activated by a phosphorelay and controls the expression of a multitude of genes, including the forespore-specific sigma factor σ(F) and the mother cell-specific sigma factor σ(E). Identification of the system-level mechanism of the sporulation decision is hindered by a lack of direct control over Spo0A activity. This limitation can be overcome by using a synthetic system in which Spo0A activation is controlled by inducing expression of phosphorelay kinase KinA. This induction results in a switch-like increase in the number of sporulating cells at a threshold of KinA. Using a combination of mathematical modeling and single-cell microscopy, we investigate the origin and physiological significance of this ultrasensitive threshold. The results indicate that the phosphorelay is unable to achieve a sufficiently fast and ultrasensitive response via its positive feedback architecture, suggesting that the sporulation decision is made downstream. In contrast, activation of σ(F) in the forespore and of σ(E) in the mother cell compartments occurs via a cascade of coherent feed-forward loops, and thereby can produce fast and ultrasensitive responses as a result of KinA induction. Unlike σ(F) activation, σ(E) activation in the mother cell compartment only occurs above the KinA threshold, resulting in completion of sporulation. Thus, ultrasensitive σ(E) activation explains the KinA threshold for sporulation induction. We therefore infer that under uncertain conditions, cells initiate sporulation but postpone making the sporulation decision to average stochastic fluctuations and to achieve a robust population response.
| S-EPMC3528541 | biostudies-literature
Project description:In microbial communities, bacteria chemically and physically interact with one another. Some of these interactions are mediated by secreted specialized metabolites that act as either intraspecies or interspecies signals to alter gene expression and to change cell physiology. Bacillus subtilis is a well-characterized soil microbe that can differentiate into multiple cell types, including metabolically dormant endospores. We were interested in identifying microbial interactions that affected sporulation in B. subtilis Using a fluorescent transcriptional reporter, we observed that coculturing B. subtilis with Escherichia coli promoted sporulation gene expression via a secreted metabolite. To identify the active compound, we screened the E. coli Keio Collection and identified the sporulation-accelerating cue as the siderophore enterobactin. B. subtilis has multiple iron acquisition systems that are used to take up the B. subtilis-produced siderophore bacillibactin, as well as to pirate exogenous siderophores such as enterobactin. While B. subtilis uses a single substrate binding protein (FeuA) to take up both bacillibactin and enterobactin, we discovered that it requires two distinct genes to sporulate in response to these siderophores (the esterase gene besA for bacillibactin and a putative esterase gene, ybbA, for enterobactin). In addition, we found that siderophores from a variety of other microbial species also promote sporulation in B. subtilis Our results thus demonstrate that siderophores can act not only as bacterial iron acquisition systems but also as interspecies cues that alter cellular development and accelerate sporulation in B. subtilisIMPORTANCE While much is known about the genetic regulation of Bacillus subtilis sporulation, little is understood about how other bacteria influence this process. This work describes an interaction between Escherichia coli and B. subtilis that accelerates sporulation in B. subtilis The interaction is mediated by the E. coli siderophore enterobactin; we show that other species' siderophores also promote sporulation gene expression in B. subtilis These results suggest that siderophores not only may supply bacteria with the mineral nutrient iron but also may play a role in bacterial interspecies signaling, providing a cue for sporulation. Siderophores are produced by many bacterial species and thus potentially play important roles in altering bacterial cell physiology in diverse environments.
| S-EPMC5411514 | biostudies-literature
Project description:Proteins of the YidC/OxaI/Alb3 family play a crucial role in the insertion, folding, and/or assembly of membrane proteins in prokaryotes and eukaryotes. Bacillus subtilis has two YidC-like proteins, denoted SpoIIIJ and YqjG. SpoIIIJ and YqjG are largely exchangeable in function, but SpoIIIJ has a unique role in sporulation, while YqjG stimulates competence development. To obtain more insight into the regions important for the sporulation specificity of SpoIIIJ, a series of SpoIIIJ/YqjG chimeras was constructed. These chimeras were tested for functionality during vegetative growth and for their ability to complement the sporulation defect of a spoIIIJ deletion strain. The data suggest an important role for the domain comprising transmembrane segment 2 (TMS2) and its flanking loops in sporulation specificity, with lesser contributions to specificity by TMS1 and TMS3.
| S-EPMC3993340 | biostudies-literature
Project description:Sporulation is a highly sophisticated developmental process adopted by most Bacilli as a survival strategy to withstand extreme conditions that normally do not support microbial growth. A complicated regulatory cascade, divided into various stages and taking place in two different compartments of the cell, involves a number of primary and secondary regulator proteins that drive gene expression directed toward the formation and maturation of an endospore. Such regulator proteins are highly conserved among various spore formers. Despite this conservation, both regulatory and phenotypic differences are observed between different species of spore forming bacteria. In this study, we demonstrate that deletion of the regulatory sporulation protein SpoVT results in a severe sporulation defect in Bacillus cereus, whereas this is not observed in Bacillus subtilis. Although spores are initially formed, the process is stalled at a later stage in development, followed by lysis of the forespore and the mother cell. A transcriptomic investigation of B. cereus ΔspoVT shows upregulation of genes involved in germination, potentially leading to premature lysis of prespores formed. Additionally, extreme variation in the expression of species-specific genes of unknown function was observed. Introduction of the B. subtilis SpoVT protein could partly restore the sporulation defect in the B. cereus spoVT mutant strain. The difference in phenotype is thus more than likely explained by differences in promoter targets rather than differences in mode of action of the conserved SpoVT regulator protein. This study stresses that evolutionary variances in regulon members of sporulation regulators can have profound effects on the spore developmental process and that mere protein homology is not a foolproof predictor of similar phenotypes.
| S-EPMC5061766 | biostudies-literature
Project description:Sensor histidine kinases are widely used by bacteria to detect and respond to environmental signals. In Bacillus subtilis, KinA is a major kinase providing phosphate input to the phosphorelay that activates the sporulation pathway upon starvation via the phosphorylated Spo0A transcription factor. KinA contains three PAS domains in its amino-terminal sensor domain, which appear to be involved in the sensing of an unidentified sporulation signal(s) produced upon starvation. Prior biochemical studies have suggested that KinA forms a homodimer as a functional enzyme and that the most amino-terminal PAS domain (PAS-A) plays an important role in sensing the signal(s) to activate an ATP-dependent autophosphorylation reaction to a histidine residue. To analyze the structure and function of the kinase in vivo, we have used a strain in which the synthesis of KinA is under the control of an isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible promoter. In vivo functional studies in combination with domain-based deletion analysis show that the cytosolic KinA forms a homo-oligomer as an active form under both nutrient-rich and nutrient-depleted conditions via its amino- and carboxyl-terminal domains independently. Furthermore, we found that a mutant in which the PAS-A domain was deleted was still able to induce sporulation at a wild-type level irrespective of nutrient availability, suggesting that PAS-BC domains are sufficient to maintain the kinase activity. Based on these results, we propose that the primary role of the amino-terminal sensor domain is to form a stable complex as a functional kinase, but possibly not for the binding of an unidentified sporulation signal(s).
| S-EPMC2725609 | biostudies-literature