In Vivo Effects of Sporulation Kinases on Mutant Spo0A Proteins in Bacillus subtilis

Abstract

The phosphorylated form of the response regulator Spo0A (Spo0AϳP) is required for the initiation of sporulation in Bacillus subtilis. Phosphate is transferred to Spo0A from at least four histidine kinases (KinA, KinB, KinC, and KinD) by a phosphotransfer pathway composed of Spo0F and Spo0B. Several mutations in spo0A allow initiation of sporulation in the absence of spo0F and spo0B, but the mechanisms by which these mutations allow bypass of spo0F and spo0B are not fully understood. We measured the ability of KinA, KinB, and KinC to activate sporulation of five spo0A mutants in the absence of Spo0F and Spo0B. We also determined the effect of Spo0E, a Spo0AϳP-specific phosphatase, on sporulation of strains containing the spo0A mutations. Our results indicate that several of the mutations relax the specificity of Spo0A, allowing Spo0A to obtain phosphate from a broader group of phosphodonors. In the course of these experiments, we observed mediumdependent effects on the sporulation of different mutants. This led us to identify a small molecule, acetoin, that can stimulate sporulation of some spo0A mutants.In response to starvation and high cell density, Bacillus subtilis can initiate a developmental pathway leading to the formation of dormant endospores (3, 13, 46). The transcription factor Spo0A is a critical regulator of the shift from exponential growth to sporulation. Spo0A is activated by phosphorylation {reviewed in references 3 and 13), and sporulation begins with accumulation of a threshold amount of phosphorylated Spo0A (Spo0AϳP) (5).Spo0A is a response regulator, but unlike most members of this protein family, Spo0A does not obtain phosphate directly from a histidine kinase. Spo0A phosphorylation is controlled by a phosphotransfer pathway, known as the phosphorelay, composed of Spo0F, Spo0B, and at least four histidine kinases, KinA, KinB, KinC, and KinD (1,2,20,21,24,27,36,47). The kinases donate phosphate to Spo0F, a response regulator with no output domain (2, 36). The phosphate from Spo0FϳP is transferred to Spo0B and finally from Spo0BϳP to Spo0A (2). spo0B and spo0F null mutants do not sporulate, indicating that phosphorelay-independent phosphorylation of Spo0A is normally insufficient to activate sporulation.A range of intra-and extracellular signals control sporulation by affecting the flow of phosphate through the phosphorelay. KinA, KinB, KinC, and KinD all have different roles in Spo0A activation and probably respond to distinct stimuli (7,28,47). KinA is the most important kinase for sporulation in rich sporulation media (28,47). KinB is the most important kinase when cells are grown in glucose minimal medium (28). KinC and KinD contribute very little to sporulation under any condition but appear to regulate Spo0A during vegetative growth (20,21,24,27).Spo0AϳP levels are also regulated by phosphatases. Spo0E is a Spo0AϳP-specific phosphatase (33). Several phosphatases indirectly decrease Spo0AϳP levels by dephosphorylating Spo0FϳP. At least three members of the Rap (response regulator asparty...

Mutation inyaaTLeads to Significant Inhibition of Phosphorelay during Sporulation inBacillus subtilis

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Signals, Regulatory Networks, and Materials That Build and Break Bacterial Biofilms

“…When B. subtilis is grown in medium containing 0.1% glucose, a multilayer biofilm forms (302). The stimulatory effect of low glucose concentrations is, in part, due to the metabolism of glucose to acetoin, which stimulates Spo0A, a positive regulator of biofilm formation (260,302). Other pathways for activation of multilayer biofilm formation by glucose may also exist.…”

Functional analysis of 11 putative essential genes in Bacillus subtilis

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