Targeting bacterial degradation machinery as an antibacterial strategy.

The exploitation of a cell's natural degradation machinery for therapeutic purposes is an exciting research area in its infancy with respect to bacteria. Here, we review current strategies targeting the ClpCP system, which is a proteolytic degradation complex essential in the biology of many bacterial species of scientific interest. Strategies include using natural product antibiotics or acyldepsipeptides to initiate the up- or down-regulation of ClpCP activity. We also examine exciting recent forays into BacPROTACs to trigger the degradation of specific proteins of interest through the hijacking of the ClpCP machinery. These strategies represent an important emerging avenue for combatting antimicrobial resistance.

From regulation to ruin: a rogue sigma factor causes cell death in Bacillus subtilis.

A rogue, plasmid-encoded sigma factor that kills Bacillus subtilis is the focus of a new study by A. T. Burton, D. Pospísilová, P. Sudzinová, E. V. Snider, A. M. Burrage, L. Krásný, and D. B. Kearns (J Bacteriol 205:e00112-23, 2023, https://doi.org/10.1128/jb.00112-23). The authors demonstrate that SigN is toxic in its own right, causing cell death by potently outcompeting the housekeeping sigma factor for access to RNA polymerase.

Structural Analysis of Bacillus subtilis Sigma Factors.

Bacteria use an array of sigma factors to regulate gene expression during different stages of their life cycles. Full-length, atomic-level structures of sigma factors have been challenging to obtain experimentally as a result of their many regions of intrinsic disorder. AlphaFold has now supplied plausible full-length models for most sigma factors. Here we discuss the current understanding of the structures and functions of sigma factors in the model organism, Bacillus subtilis, and present an X-ray crystal structure of a region of B. subtilis SigE, a sigma factor that plays a critical role in the developmental process of spore formation.

An Adaptable and Modular Set of Laboratory Exercises Connecting Genotype to Phenotype in Sporulating Bacillus subtilis.

Practical lab exercises that help students draw connections between genotype and phenotype, and make and test predictions about the identity of mutants, are invaluable in college-level cell biology, genetics, and microbiology courses. While many bacteria are easy to grow and manipulate within the time and resource constraints of a laboratory course, their phenotypes are not always observable or relevant-seeming to college students. Here, we leverage sporulation by the bacterium Bacillus subtilis, a well-characterized and genetically tractable system, to create 5 adaptable lab exercises that can be implemented in different combinations to suit the needs of a variety of courses and instruction modes. Because phenotypic changes during sporulation are striking morphological changes to cells that are easily observable with basic light microscopy, and because spore-forming bacteria related to B. subtilis have clear applications for human and environmental health, these exercises have the potential to engage students' interest while introducing and reinforcing key concepts in microbiology, cell biology, and genetics.

Transcription and translation of the sigG gene is tuned for proper execution of the switch from early to late gene expression in the developing Bacillus subtilis spore.

A cascade of alternative sigma factors directs developmental gene expression during spore formation by the bacterium Bacillus subtilis. As the spore develops, a tightly regulated switch occurs in which the early-acting sigma factor σF is replaced by the late-acting sigma factor σG. The gene encoding σG (sigG) is transcribed by σF and by σG itself in an autoregulatory loop; yet σG activity is not detected until σF-dependent gene expression is complete. This separation in σF and σG activities has been suggested to be due at least in part to a poorly understood intercellular checkpoint pathway that delays sigG expression by σF. Here we report the results of a careful examination of sigG expression during sporulation. Unexpectedly, our findings argue against the existence of a regulatory mechanism to delay sigG transcription by σF and instead support a model in which sigG is transcribed by σF with normal timing, but at levels that are very low. This low-level expression of sigG is the consequence of several intrinsic features of the sigG regulatory and coding sequence-promoter spacing, secondary structure potential of the mRNA, and start codon identity-that dampen its transcription and translation. Especially notable is the presence of a conserved hairpin in the 5' leader sequence of the sigG mRNA that occludes the ribosome-binding site, reducing translation by up to 4-fold. Finally, we demonstrate that misexpression of sigG from regulatory and coding sequences lacking these features triggers premature σG activity in the forespore during sporulation, as well as inappropriate σG activity during vegetative growth. Altogether, these data indicate that transcription and translation of the sigG gene is tuned to prevent vegetative expression of σG and to ensure the precise timing of the switch from σF to σG in the developing spore.

Structural and Functional Insights into Bacillus subtilis Sigma Factor Inhibitor, CsfB.

Global changes in bacterial gene expression can be orchestrated by the coordinated activation/deactivation of alternative sigma (σ) factor subunits of RNA polymerase. Sigma factors themselves are regulated in myriad ways, including via anti-sigma factors. Here, we have determined the solution structure of anti-sigma factor CsfB, responsible for inhibition of two alternative sigma factors, σG and σE, during spore formation by Bacillus subtilis. CsfB assembles into a symmetrical homodimer, with each monomer bound to a single Zn2+ ion via a treble-clef zinc finger fold. Directed mutagenesis indicates that dimer formation is critical for CsfB-mediated inhibition of both σG and σE, and we have characterized these interactions in vitro. This work represents an advance in our understanding of how CsfB mediates inhibition of two alternative sigma factors to drive developmental gene expression in a bacterium.

Near-atomic resolution cryoelectron microscopy structure of the 30-fold homooligomeric SpoIIIAG channel essential to spore formation in Bacillus subtilis.

Bacterial sporulation allows starving cells to differentiate into metabolically dormant spores that can survive extreme conditions. Following asymmetric division, the mother cell engulfs the forespore, surrounding it with two bilayer membranes. During the engulfment process, an essential channel, the so-called feeding tube apparatus, is thought to cross both membranes to create a direct conduit between the mother cell and the forespore. At least nine proteins are required to create this channel, including SpoIIQ and SpoIIIAA-AH. Here, we present the near-atomic resolution structure of one of these proteins, SpoIIIAG, determined by single-particle cryo-EM. A 3D reconstruction revealed that SpoIIIAG assembles into a large and stable 30-fold symmetric complex with a unique mushroom-like architecture. The complex is collectively composed of three distinctive circular structures: a 60-stranded vertical ß-barrel that forms a large inner channel encircled by two concentric rings, one ß-mediated and the other formed by repeats of a ring-building motif (RBM) common to the architecture of various dual membrane secretion systems of distinct function. Our near-atomic resolution structure clearly shows that SpoIIIAG exhibits a unique and dramatic adaptation of the RBM fold with a unique ß-triangle insertion that assembles into the prominent channel, the dimensions of which suggest the potential passage of large macromolecules between the mother cell and forespore during the feeding process. Indeed, mutation of residues located at key interfaces between monomers of this RBM resulted in severe defects both in vivo and in vitro, providing additional support for this unprecedented structure.

A novel RNA polymerase-binding protein that interacts with a sigma-factor docking site.

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 σF to create a negative feedback loop that inhibits σF -directed gene transcription. Cells deleted for fin are defective for spore formation and exhibit increased levels of σF -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 ß' subunit. The coiled-coil is a docking site for sigma factors on RNA polymerase, and evidence is presented that the binding of Fin and σF 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 σF by competing for binding to the ß' coiled-coil.

A Membrane-Embedded Amino Acid Couples the SpoIIQ Channel Protein to Anti-Sigma Factor Transcriptional Repression during Bacillus subtilis Sporulation.

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.

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

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 among Bacillus species and species of related genera, is transcribed in the forespore under the control of both σ(F) and σ(G). Cells mutant for fin are unable to fully deactivate σ(F) and, conversely, are unable to fully activate σ(G). Consistent with their deficiency in σ(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-σ(G) factor CsfB (also called Gin), we speculate that Fin is an anti-σ(F) factor which, by disabling σ(F), promotes the switch to late developmental gene expression in the forespore.

Small genes under sporulation control in the Bacillus subtilis genome.

Using an oligonucleotide microarray, we searched for previously unrecognized transcription units in intergenic regions in the genome of Bacillus subtilis, with an emphasis on identifying small genes activated during spore formation. Nineteen transcription units were identified, 11 of which were shown to depend on one or more sporulation-regulatory proteins for their expression. A high proportion of the transcription units contained small, functional open reading frames (ORFs). One such newly identified ORF is a member of a family of six structurally similar genes that are transcribed under the control of sporulation transcription factor σ(E) or σ(K). A multiple mutant lacking all six genes was found to sporulate with slightly higher efficiency than the wild type, suggesting that under standard laboratory conditions the expression of these genes imposes a small cost on the production of heat-resistant spores. Finally, three of the transcription units specified small, noncoding RNAs; one of these was under the control of the sporulation transcription factor σ(E), and another was under the control of the motility sigma factor σ(D).

A feeding tube model for activation of a cell-specific transcription factor during sporulation in Bacillus subtilis.

Spore formation by Bacillus subtilis takes place in a sporangium consisting of two chambers, the forespore and the mother cell, which are linked by pathways of intercellular communication. One pathway, which couples the activation of the forespore transcription factor sigma(G) to the action of sigma(E) in the mother cell, has remained mysterious. Traditional models hold that sigma(E) initiates a signal transduction pathway that specifically activates sigma(G) in the forespore. Recent experiments indicating that the mother cell and forespore are joined by a channel have led to the suggestion that a specific regulator of sigma(G) is transported from the mother cell into the forespore. As we report here, however, the requirement for the channel is not limited to sigma(G). Rather, it is also required for the persistent activity of the early-acting forespore transcription factor sigma(F) as well as that of a heterologous RNA polymerase (that of phage T7). We infer that macromolecular synthesis in the forespore becomes dependent on the channel at intermediate stages of development. We propose that the channel is a gap junction-like feeding tube through which the mother cell nurtures the developing spore by providing small molecules needed for biosynthetic activity, including sigma(G)-directed gene activation.

A novel pathway of intercellular signalling in Bacillus subtilis involves a protein with similarity to a component of type III secretion channels.

During spore formation in Bacillus subtilis, sigma(E)-directed gene expression in the mother-cell compartment of the sporangium triggers the activation of sigma(G) in the forespore by a pathway of intercellular signalling that is composed of multiple proteins of unknown function. Here, we confirm that the vegetative protein SpoIIIJ, the forespore protein SpoIIQ and eight membrane proteins (SpoIIIAA through SpoIIIAH) produced in the mother cell under the control of sigma(E) are ordinarily required for intercellular signalling. In contrast, an anti-sigma(G) factor previously implicated in the pathway is shown to be dispensable. We also present evidence suggesting that SpoIIIJ is a membrane protein translocase that facilitates the insertion of SpoIIIAE into the membrane. In addition, we report the isolation of a mutation that partially bypasses the requirement for SpoIIIJ and for SpoIIIAA through SpoIIIAG, but not for SpoIIIAH or SpoIIQ, in the activation of sigma(G). We therefore propose that under certain genetic conditions, SpoIIIAH and SpoIIQ can constitute a minimal pathway for the activation of sigma(G). Finally, based on the similarity of SpoIIIAH to a component of type III secretion systems, we speculate that signalling is mediated by a channel that links the mother cell to the forespore.

gerT, a newly discovered germination gene under the control of the sporulation transcription factor sigmaK in Bacillus subtilis.

We report the identification of a gene, herein designated gerT (formerly yozR), that is involved in germination by spores of Bacillus subtilis. The gerT gene is induced late in sporulation under the positive control of the transcription factor sigma(K) and under the negative control of the DNA-binding protein GerE. The gerT gene product (GerT) is a component of the spore coat, and its incorporation into the coat takes place in two stages. GerT initially assembles into foci, which then spread around the developing spore in a process that is dependent on the morphogenetic protein CotE. Mutant spores lacking GerT respond poorly to multiple germinants and are impaired at an early stage of germination.