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Sporulation in Bacillus subtilis is governed by a cascade of alternative RNA polymerase sigma factors. We previously identified a small protein Fin that is produced under the control of the sporulation sigma factor σ F to create a negative feedback loop that inhibits σ F -directed gene transcription. Cells deleted for fin are defective for spore formation and exhibit increased levels of σ F -directed gene transcription. Based on pull-down experiments, chemical crosslinking, bacterial two-hybrid experiments and nuclear magnetic resonance chemical shift analysis, we now report that Fin binds to RNA polymerase and specifically to the coiled-coil region of the β' subunit. The coiled-coil is a docking site for sigma factors on RNA polymerase, and evidence is presented that the binding of Fin and σ F to RNA polymerase is mutually exclusive. We propose that Fin functions by a mechanism distinct from that of classic sigma factor antagonists (anti-σ factors), which bind directly to a target sigma factor to prevent its association with RNA polymerase, and instead functions to inhibit σ F by competing for binding to the β' coiled-coil., (© 2017 John Wiley & Sons Ltd.)

Sigma (σ) factors direct gene transcription by binding to and determining the promoter recognition specificity of RNA polymerase (RNAP) in bacteria. Genes transcribed under the control of alternative sigma factors allow cells to respond to stress and undergo developmental processes, such as sporulation in Bacillus subtilis , in which gene expression is controlled by a cascade of alternative sigma factors. Binding of sigma factors to RNA polymerase depends on the coiled-coil (or clamp helices) motif of the β' subunit. We have identified an amino acid substitution (L257P) in the coiled coil that markedly inhibits the function of σ H , the earliest-acting alternative sigma factor in the sporulation cascade. Cells with this mutant RNAP exhibited an early and severe block in sporulation but not in growth. The mutant was strongly impaired in σ H -directed gene expression but not in the activity of the stress-response sigma factor σ B Pulldown experiments showed that the mutant RNAP was defective in associating with σ H but could still associate with σ A and σ B The differential effects of the L257P substitution on sigma factor binding to RNAP are likely due to a conformational change in the β' coiled coil that is specifically detrimental for interaction with σ H This is the first example, to our knowledge, of an amino acid substitution in RNAP that exhibits a strong differential effect on a particular alternative sigma factor. IMPORTANCE In bacteria, all transcription is mediated by a single multisubunit RNA polymerase (RNAP) enzyme. However, promoter-specific transcription initiation necessitates that RNAP associates with a σ factor. Bacteria contain a primary σ factor that directs transcription of housekeeping genes and alternative σ factors that direct transcription in response to environmental or developmental cues. We identified an amino acid substitution (L257P) in the B. subtilis β' subunit whereby RNAP L257P associates with some σ factors (σ A and σ B ) and enables vegetative cell growth but is defective in utilization of σ H and is consequently blocked for sporulation. To our knowledge, this is the first identification of an amino acid substitution within the core enzyme that affects utilization of a specific sigma factor., (Copyright © 2017 American Society for Microbiology.)

Unlabelled: SpoIIQ is an essential component of a channel connecting the developing forespore to the adjacent mother cell during Bacillus subtilis sporulation. This channel is generally required for late gene expression in the forespore, including that directed by the late-acting sigma factor σ(G) Here, we present evidence that SpoIIQ also participates in a previously unknown gene regulatory circuit that specifically represses expression of the gene encoding the anti-sigma factor CsfB, a potent inhibitor of σ(G) The csfB gene is ordinarily transcribed in the forespore only by the early-acting sigma factor σ(F) However, in a mutant lacking the highly conserved SpoIIQ transmembrane amino acid Tyr-28, csfB was also aberrantly transcribed later by σ(G), the very target of CsfB inhibition. This regulation of csfB by SpoIIQ Tyr-28 is specific, given that the expression of other σ(F)-dependent genes was unaffected. Moreover, we identified a conserved element within the csfB promoter region that is both necessary and sufficient for SpoIIQ Tyr-28-mediated inhibition. These results indicate that SpoIIQ is a bifunctional protein that not only generally promotes σ(G)activity in the forespore as a channel component but also specifically maximizes σ(G)activity as part of a gene regulatory circuit that represses σ(G)-dependent expression of its own inhibitor, CsfB. Finally, we demonstrate that SpoIIQ Tyr-28 is required for the proper localization and stability of the SpoIIE phosphatase, raising the possibility that these two multifunctional proteins cooperate to fine-tune developmental gene expression in the forespore at late times., Importance: Cellular development is orchestrated by gene regulatory networks that activate or repress developmental genes at the right time and place. Late gene expression in the developing Bacillus subtilis spore is directed by the alternative sigma factor σ(G) The activity of σ(G)requires a channel apparatus through which the adjacent mother cell provides substrates that generally support gene expression. Here we report that the channel protein SpoIIQ also specifically maximizes σ(G)activity as part of a previously unknown regulatory circuit that prevents σ(G)from activating transcription of the gene encoding its own inhibitor, the anti-sigma factor CsfB. The discovery of this regulatory circuit significantly expands our understanding of the gene regulatory network controlling late gene expression in the developing B. subtilis spore., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)