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A small protein required for the switch from {sigma}F to {sigma}G during sporulation in Bacillus subtilis.

ABSTRACT: A cascade of alternative sigma factors governs the program of developmental gene expression during sporulation in Bacillus subtilis. Little is known, however, about how the early-acting sigma factors are inactivated and replaced by the later-acting factors. Here we identify a small protein, Fin (formerly known as YabK), that is required for efficient switching from ?(F)- to ?(G)-directed gene expression in the forespore compartment of the developing sporangium. The fin gene, which is conserved among Bacillus species and species of related genera, is transcribed in the forespore under the control of both ?(F) and ?(G). Cells mutant for fin are unable to fully deactivate ?(F) and, conversely, are unable to fully activate ?(G). Consistent with their deficiency in ?(G)-directed gene expression, fin cells are arrested in large numbers following the engulfment stage of sporulation, ultimately forming 50-fold fewer heat-resistant spores than the wild type. Based in part on the similarity of Fin to the anti-?(G) factor CsfB (also called Gin), we speculate that Fin is an anti-?(F) factor which, by disabling ?(F), promotes the switch to late developmental gene expression in the forespore.

SUBMITTER: Camp AH

PROVIDER: S-EPMC3019954 | biostudies-other | 2011 Jan

REPOSITORIES: biostudies-other

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A small protein required for the switch from {sigma}F to {sigma}G during sporulation in Bacillus subtilis.

Camp Amy H AH Wang Anna F AF Losick Richard R

Journal of bacteriology 20101029 1

A cascade of alternative sigma factors governs the program of developmental gene expression during sporulation in Bacillus subtilis. Little is known, however, about how the early-acting sigma factors are inactivated and replaced by the later-acting factors. Here we identify a small protein, Fin (formerly known as YabK), that is required for efficient switching from σ(F)- to σ(G)-directed gene expression in the forespore compartment of the developing sporangium. The fin gene, which is conserved a ...[more]

PMID: 21037003

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Project description:RNA polymerase sigma factor sigma(F) initiates the prespore-specific program of gene expression during Bacillus subtilis sporulation. sigma(F) governs transcription of spoIIIG, encoding the late prespore-specific regulator sigma(G). However, transcription of spoIIIG is delayed relative to other genes under the control of sigma(F), and after synthesis, sigma(G) is initially kept in an inactive form. Activation of sigma(G) requires the complete engulfment of the prespore by the mother cell and expression of the spoIIIA and spoIIIJ loci. We screened for random mutations in spoIIIG that bypassed the requirement for spoIIIA for the activation of sigma(G). We found a mutation (spoIIIGE156K) that resulted in an amino acid substitution at position 156, which is adjacent to the position of a mutation (E155K) previously shown to prevent interaction of SpoIIAB with sigma(G). Comparative modelling techniques and in vivo studies suggested that the spoIIIGE156K mutation interferes with the interaction of SpoIIAB with sigma(G). The sigma(GE156K) isoform restored sigma(G)-directed gene expression to spoIIIA mutant cells. However, expression of sspE-lacZ in the spoIIIA spoIIIGE156K double mutant was delayed relative to completion of the engulfment process and was not confined to the prespore. Rather, beta-galactosidase accumulated throughout the entire cell at late times in development. This suggests that the activity of sigma(GE156K) is still regulated in the prespore of a spoIIIA mutant, but not by SpoIIAB. In agreement with this suggestion, we also found that expression of spoIIIGE156K from the promoter for the early prespore-specific gene spoIIQ still resulted in sspE-lacZ induction at the normal time during sporulation, coincidently with completion of the engulfment process. In contrast, transcription of spoIIIGE156K, but not of the wild-type spoIIIG gene, from the mother cell-specific spoIID promoter permitted the rapid induction of sspE-lacZ expression. Together, the results suggest that SpoIIAB is either redundant or has no role in the regulation of sigma(G) in the prespore.

Project description:UnlabelledSpoIIQ 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 ?(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 ?(G) The csfB gene is ordinarily transcribed in the forespore only by the early-acting sigma factor ?(F) However, in a mutant lacking the highly conserved SpoIIQ transmembrane amino acid Tyr-28, csfB was also aberrantly transcribed later by ?(G), the very target of CsfB inhibition. This regulation of csfB by SpoIIQ Tyr-28 is specific, given that the expression of other ?(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 ?(G)activity in the forespore as a channel component but also specifically maximizes ?(G)activity as part of a gene regulatory circuit that represses ?(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.ImportanceCellular 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 ?(G) The activity of ?(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 ?(G)activity as part of a previously unknown regulatory circuit that prevents ?(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.

Project description:Bacillus subtilis is a bacterium capable of differentiating into a spore form more resistant to environmental stress. Early in sporulation, each cell possesses two copies of a circular chromosome. A polar FtsZ ring (Z ring) directs septation over one of the chromosomes, generating two cell compartments. The smaller "forespore" compartment initially contains only 25 to 30% of one chromosome, and this transient genetic asymmetry is required for differentiation. Timely assembly of polar Z rings and precise capture of the chromosome in the forespore both require the DNA-binding protein RefZ. To mediate its role in chromosome capture, RefZ must bind to specific DNA motifs (RBMs) that localize near the poles at the time of septation. Cells artificially induced to express RefZ during vegetative growth cannot assemble Z rings, an effect that also requires DNA binding. We hypothesized that RefZ-RBM complexes mediate precise chromosome capture by modulating FtsZ function. To investigate, we isolated 10 RefZ loss-of-function (rLOF) variants unable to inhibit cell division yet still capable of binding RBMs. Sporulating cells expressing the rLOF variants in place of wild-type RefZ phenocopied a ΔrefZ mutant, suggesting that RefZ acts through an FtsZ-dependent mechanism. The crystal structure of RefZ was solved, and wild-type RefZ and the rLOF variants were further characterized. Our data suggest that RefZ's oligomerization state and specificity for the RBMs are critical determinants influencing RefZ's ability to affect FtsZ dynamics. We propose that RBM-bound RefZ complexes function as a developmentally regulated nucleoid occlusion system for fine-tuning the position of the septum relative to the chromosome during sporulation.IMPORTANCE The bacterial nucleoid forms a large, highly organized structure. Thus, in addition to storing the genetic code, the nucleoid harbors positional information that can be leveraged by DNA-binding proteins to spatially constrain cellular activities. During B. subtilis sporulation, the nucleoid undergoes reorganization, and the cell division protein FtsZ assembles polarly to direct septation over one chromosome. The TetR family protein RefZ binds DNA motifs (RBMs) localized near the poles at the time of division and is required for both timely FtsZ assembly and precise capture of DNA in the future spore compartment. Our data suggest that RefZ exploits nucleoid organization by associating with polarly localized RBMs to modulate the positioning of FtsZ relative to the chromosome during sporulation.

Dataset Information

A small protein required for the switch from {sigma}F to {sigma}G during sporulation in Bacillus subtilis.

Publications

A small protein required for the switch from {sigma}F to {sigma}G during sporulation in Bacillus subtilis.

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