Just-in-Time Control of Spo0A Synthesis in Bacillus subtilis by Multiple Regulatory Mechanisms

Abstract

The response regulator Spo0A governs multiple developmental processes in Bacillus subtilis, including most conspicuously sporulation. Spo0A is activated by phosphorylation via a multicomponent phosphorelay. Previous work has shown that the Spo0A protein is not rate limiting for sporulation. Rather, Spo0A is present at high levels in growing cells, rapidly rising to yet higher levels under sporulation-inducing conditions, suggesting that synthesis of the response regulator is subject to a just-in-time control mechanism. Transcription of spo0A is governed by a promoter switching mechanism, involving a vegetative, A -recognized promoter, P v , and a sporulation H -recognized promoter, P s , that is under phosphorylated Spo0A (Spo0AϳP) control. The spo0A regulatory region also contains four (including one identified in the present work) conserved elements that conform to the consensus binding site for Spo0AϳP binding sites. These are herein designated O 1 , O 2 , O 3 , and O 4 in reverse order of their proximity to the coding sequence. Here we report that O 1 is responsible for repressing P v during the transition to stationary phase, that O 2 is responsible for repressing P s during growth, that O 3 is responsible for activating P s at the start of sporulation, and that O 4 is dispensable for promoter switching. We also report that Spo0A synthesis is subject to a posttranscriptional control mechanism such that translation of mRNAs originating from P v is impeded due to RNA secondary structure whereas mRNAs originating from P s are fully competent for protein synthesis. We propose that the opposing actions of O 2 and O 3 and the enhanced translatability of mRNAs originating from P s create a highly sensitive, self-reinforcing switch that is responsible for producing a burst of Spo0A synthesis at the start of sporulation.

Ultrasensitivity of the Bacillus subtilis sporulation decision

“…Surprisingly, despite the presence of positive feedback in the phosphorelay architecture, we have found that the phosphorelay response to KinA induction is graded and nonultrasensitive. This is a unique and crucial design feature, with which the sporulation network avoids the slowdown in the dynamical response that is characteristic of bistable systems (51,54,55). Instead, the sporulation network generates an ultrasensitive response by using a cascade of coherent feed-forward loops combining transcriptional and posttranslational interactions.…”

“…Immediately after forespore formation, Spo0A∼P in the mother cell and active σ F in the forespore act as inputs for the σ E module, and thus control expression of the σ E regulon as the output (8,38). Details of all posttranslational interactions and transcriptional regulation, as well as relevant parameter values (Table S3), were extracted from the literature (5,8,12,16,32,33,36,40,(55)(56)(57).…”

Fifty Years after the Replicon Hypothesis: Cell-Specific Master Regulators as New Players in Chromosome Replication Control

“…The Spo0A activity is also temporally controlled on the transcriptional level by two promoters: a vegetative-state-related A -recognized promoter (Pv) and a sporulation-related H -recognized promoter (Ps) (102). The A housekeeping sigma factor controls a weak Pv promoter, which provides a basal level of Spo0A that is required for efficient firing of a strong Ps promoter (Spo0AϳP and H activated) during the transition to the stationary phase (103)(104)(105). spo0A transcription is autoregulated by Spo0A itself in a manner similar to (but more complex than) that seen with ctrA in C. crescentus.…”

Evidence that Autophosphorylation of the Major Sporulation Kinase in Bacillus subtilis Is Able To Occur in trans

“…Thus, the phosphorelay network is controlled at both the transcriptional and posttranslational levels as a whole, resulting in both temporal and spatial regulation of the activity of Spo0A (Fig. 1) (13,14,18,26,27). Based on the findings and facts described above, it has been proposed that the complexity of the phosphorelay provides multiple entry points to control the precise concentration of the master regulator Spo0A during the development and progression of sporulation (13,27).…”

“…While many of the bacterial two-component systems control rapid and transient gene expression in response to various and specific stimuli (3), the phosphorelay system is involved in the control of the crucial and highly coordinated cell fate decision, which ultimately leads to the development of spores (2,4,(9)(10)(11). Accumulated evidence indicates that a temporal and spatial increase in the level and activity of the master regulator Spo0A is required for sporulation to proceed properly (12)(13)(14). Under nutrient-rich conditions, the DNA-binding protein AbrB represses genes involved in the transition from vegetative growth to stationary phase, and only a basal level of Spo0A is expressed from the relatively weak, constitutive A -controlled promoter Pv (15).…”