Genetic and biochemical analyses of sensor kinase A in Bacillus subtilis sporulation (original) (raw)
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Role of the PAS Sensor Domains in the Bacillus subtilis Sporulation Kinase KinA
2013
Histidine kinases are sophisticated molecular sensors that are used by bacteria to detect and respond to a multitude of environ- mental 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 het- eroassociation properties using analytical ultracentrifugation, nuclear magnetic resonance (NMR) spectroscopy, and multiangle laser light scattering. We show that PASA self-associates very weakly, while PASC is primarily a monomer. In contrast, PASB forms a stable dimer (Kd [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 PASB, but not PASA, in autophosphorylation of KinA. Our findings suggest that dimerization of PASB 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 mod- ule of KinA.
Journal of Bacteriology, 2010
Sporulation in Bacillus subtilis is controlled by a complex gene regulatory circuit that is activated upon nutrient deprivation. The initial process is directed by the phosphorelay, involving the major sporulation histidine kinase (KinA) and two additional phosphotransferases (Spo0F and Spo0B), that activates the master transcription factor Spo0A. Little is known about the initial event and mechanisms that trigger sporulation. Using a strain in which the synthesis of KinA is under the control of an IPTG (isopropyl--D-thiogalactopyranoside)-inducible promoter, here we demonstrate that inducing the synthesis of the KinA beyond a certain level leads to the entry of the irreversible process of sporulation irrespective of nutrient availability. Moreover, the engineered cells expressing KinA under a H -dependent promoter that is similar to but stronger than the endogenous kinA promoter induce sporulation during growth. These cells, which we designated COS (constitutive sporulation) cells, exhibit the morphology and properties of sporulating cells and express sporulation marker genes under nutrient-rich conditions. Thus, we created an engineered strain displaying two cell cycles (growth and sporulation) integrated into one cycle irrespective of culture conditions, while in the wild type, the appropriate cell fate decision is made depending on nutrient availability. These results suggest that the threshold level of the major sporulation kinase acts as a molecular switch to determine cell fate and may rule out the possibility that the activity of KinA is regulated in response to the unknown signal(s).
Molecular Microbiology, 2013
Sporulation initiation in Bacillus subtilis is controlled by the phosphorylated form of the master regulator Spo0A which controls transcription of a multitude of sporulation genes. In this study, we investigated the importance of temporal dynamics of phosphorylated Spo0A (Spo0A∼P) accumulation by rewiring the network controlling its phosphorylation. We showed that simultaneous induction of KinC, a kinase that can directly phosphorylate Spo0A, and Spo0A itself from separately controlled inducible promoters can efficiently trigger sporulation even under nutrient rich conditions. However, the sporulation efficiency in this artificial two-component system was significantly impaired when KinC and/or Spo0A induction was too high. Using mathematical modelling, we showed that gradual accumulation of Spo0A∼P is essential for the proper temporal order of the Spo0A regulon expression, and that reduction in sporulation efficiency results from the reversal of that order. These insights led us to identify premature repression of DivIVA as one possible explanation for the adverse effects of accelerated accumulation of Spo0A∼P on sporulation. Moreover, we found that positive feedback resulting from autoregulation of the native spo0A promoter leads to robust control of Spo0A∼P accumulation kinetics. Thus we propose that a major function of the conserved architecture of the sporulation network is controlling Spo0A activation dynamics.
BMC Systems Biology, 2014
Background: Bacterial spores are important contaminants in food, and the spore forming bacteria are often implicated in food safety and food quality considerations. Spore formation is a complex developmental process involving the expression of more than 500 genes over the course of 6 to 8 hrs. The process culminates in the formation of resting cells capable of resisting environmental extremes and remaining dormant for long periods of time, germinating when conditions promote further vegetative growth. Experimental observations of sporulation and germination are problematic and time consuming so that reliable models are an invaluable asset in terms of prediction and risk assessment. In this report we develop a model which assists in the interpretation of sporulation dynamics.
Novel Modulators Controlling Entry into Sporulation in Bacillus subtilis
Journal of Bacteriology, 2013
Upon nutrient deprivation, Bacillus subtilis initiates the developmental process of sporulation by integrating environmental and extracellular signals. These signals are channeled into a phosphorelay ultimately activating the key transcriptional regulator of sporulation, Spo0A. Subsequently, phosphorylated Spo0A regulates the expression of genes required for sporulation to initiate. Here we identified a group of genes whose transcription levels are controlled by Spo0A during exponential growth. Among them, three upregulated genes, termed sivA, sivB (bslA), and sivC, encode factors found to inhibit Spo0A activation. We furthermore show that the Siv factors operate by reducing the activity of histidine kinases located at the top of the sporulation phosphorelay, thereby decreasing Spo0A phosphorylation. Thus, we demonstrate the existence of modulators, positively controlled by Spo0A, which inhibit inappropriate entry into the costly process of sporulation, when conditions are favorable for exponential growth.
Novel mutations that alter the regulation of sporulation in Bacillus subtilis
Journal of Molecular Biology, 1990
Sporulation in Bacillus subtilis is a complex developmental process that occurs in response to nutrient deprivation. To identify components of the mechanism that allows cells to monitor their nutritional status and to understand how this sensory information is 9 transduced into a signal to activate specific sporulation genes, we have isolated mutants that are able to sporulate efficiently under nutritional conditions that strongly inhibit sporulation io wild-type bacteria, a phenotype we refer to as Cot (control of initiation). Four cot mutations were found to be within the coding sequence of spoOA, a gene in wifich null mutations prevent the initiation of sporulation and a gene whose product shares a domain of homology with phosphorylation-aetivated proteins that play signal transduction roles in bacteria. All four of the spoOA mutations were within this conserved domain and in close proximit3; to the presumptive phosphoaeceptor site. The wild-type and one of the mutant SpoOA proteins were purified and shown to be competent to accept phosphoryl groups from a phosphohistidine within a bacterial signal transduction kinase (CheA). The mutant SpoOA protein exhibited enhanced phosphoaceeptor activity compared with the wild-type. This property of the mutant protein, together with additional genetic information, supports a model for regulation of sporulation initiation by control of tile phosphorylation level of SpoOA.
Broadly heterogeneous activation of the master regulator for sporulation in Bacillus subtilis
Proceedings of the National Academy of Sciences, 2010
A model system for investigating how developmental regulatory networks determine cell fate is spore formation in Bacillus subtilis. The master regulator for sporulation is Spo0A, which is activated by phosphorylation via a phosphorelay that is subject to three positive feedback loops. The ultimate decision to sporulate is, however, stochastic in that only a portion of the population sporulates even under optimal conditions. It was previously assumed that activation of Spo0A and hence entry into sporulation is subject to a bistable switch mediated by one or more feedback loops. Here we reinvestigate the basis for bimodality in sporulation. We show that none of the feedback loops is rate limiting for the synthesis and phosphorylation of Spo0A. Instead, the loops ensure a just-in-time supply of relay components for rising levels of phosphorylated Spo0A, with phosphate flux through the relay being limiting for Spo0A activation and sporulation. In addition, genes under Spo0A control did not exhibit a bimodal pattern of expression as expected for a bistable switch. In contrast, we observed a highly heterogeneous pattern of Spo0A activation that increased in a nonlinear manner with time. We present a computational model for the nonlinear increase and propose that the phosphorelay is a noise generator and that only cells that attain a threshold level of phosphorylated Spo0A sporulate. noise | Spo0A | bistable switch | cell fate | heterogeneity A challenge in developmental biology is to understand how cells in an apparently homogeneous population adopt different fates. An attractive organism in which to address this challenge is Bacillus subtilis, which can adopt a variety of alternative fates depending on growth conditions (1-3). In some cases, cell population heterogeneity is generated stochastically. That is, fluctuations in gene expression due to noise can be amplified by feedback loops to lock cells in alternative stable states, resulting in a bimodal distribution of cell types. This is exemplified by genetic competence in which a positive feedback loop acting as a bistable switch creates such a distribution (2, 4). We use bimodal to mean systems that exhibit two discrete states and bistable to specify a class of bimodal systems in which nonlinear reinforcement stabilizes the alternative states. Here we are concerned with bimodality in the capacity of B. subtilis to sporulate.
Temporal regulation of the Bacillus subtilis early sporulation gene spo0F
Journal of Bacteriology
The initiation of sporulation in Bacilus subtilis depends on seven genes of the spoO class. One of these, spoOF, codes for a protein of 14,000 daltons. We studied the regulation of spoOF by using spoOF-lacZ translational fusions and also measured SpoOF protein levels by immunoassays. spoOF-lacZ and SpoOF levels increased as the cells entered the stationary phase, and this effect was repressed by glucose and glutamine. Decoyinine, which lowers GTP levels and allows sporulation in the presence of normally repressing levels of glucose, induced spoOF-lacZ expression and raised SpoOF levels. The expression of spoOF-lacZ was dependent on spoOA, -OB, -OE, -OF, and -OH genes, a spoOH deletion causing the strongest effect. In most respects, the spoOF gene was regulated in a manner similar to that of spoVG. However, the presence of an abrB mutation did not relieve the dependence of spoOF gene expression on spoOA, as it does with spoVG (P.
Molecular Microbiology, 2012
Spore formation in Bacillus subtilis takes place in a sporangium consisting of two chambers, the forespore and the mother cell, which are linked by pathways of cell-cell communication. One pathway, which couples the proteolytic activation of the mother cell transcription factor s E to the action of a forespore synthesized signal molecule, SpoIIR, has remained enigmatic. Signalling by SpoIIR requires the protein to be exported to the intermembrane space between forespore and mother cell, where it will interact with and activate the integral membrane protease SpoIIGA. Here we show that SpoIIR signal activity as well as the cleavage of its N-terminal extension is strictly dependent on the prespore fatty acid biosynthetic machinery. We also report that a conserved threonine residue (T27) in SpoIIR is required for processing, suggesting that signalling of SpoIIR is dependent on fatty acid synthesis probably because of acylation of T27. In addition, SpoIIR localization in the forespore septal membrane depends on the presence of SpoIIGA. The orchestration of s E activation in the intercellular space by an acylated signal protein provides a new paradigm to ensure local transmission of a weak signal across the bilayer to control cell-cell communication during development.
Journal of bacteriology, 1999
SpoIIAA, a phosphorylatable protein, is essential to the regulation of sigmaF, the first sporulation-specific transcription factor of Bacillus subtilis. The solution structure of SpoIIAA has recently been published. Here we examine four mutant SpoIIAA proteins and correlate their properties with the phenotypes of the corresponding B. subtilis mutant strains. Two of the mutations severely disrupted the structure of the protein, a third greatly diminished the rate of its phosphorylation and abolished dephosphorylation, and the fourth left phosphorylation unaffected but reduced the rate of dephosphorylation about 10-fold.