A rogue, plasmid-encoded sigma factor that kills Bacillus subtilis is the focus of a new study by A. T. Burton, D. Pospíšilová, 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.
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.
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.
During spore formation in Bacillus subtilis, σE-directed gene expression in the mother-cell compartment of the sporangium triggers the activation of σG 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 (SpoIIIA A through SpoIIIA H) produced in the mother cell under the control of σE are ordinarily required for intercellular signalling. In contrast, an anti-σG 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 σG. We therefore propose that under certain genetic conditions, SpoIIIAH and SpoIIQ can constitute a minimal pathway for the activation of σG. 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.
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.
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,
, and present an X-ray crystal structure of a region of
SigE, a sigma factor that plays a critical role in the developmental process of spore formation.
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.
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 β-barrel that forms a large inner channel encircled by two concentric rings, one β-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 β-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.
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.
Display omitted
•The structure of CsfB is unique among anti-sigma factors•CsfB assembles into a tight homodimer of treble-clef zinc finger domains•CsfB dimerization is essential for inhibition of two alternative sigma factors
Martínez-Lumbreras, Alfano et al. have solved the structure of the anti-sigma factor CsfB and explored its role in inhibiting two alternative sigma factors during Bacillus subtilis spore formation. The results provide insight into the molecular mechanism underlying a gene expression switch in bacteria.
PDF
