SpoIIIL is a forespore factor required for efficient cell-cell signalling during Bacillus subtilis sporulation

TL;DR

This study shows that SpoIIIL is a forespore factor involved in cell-cell signaling during spore formation in Bacillus subtilis, not part of the A-Q complex.

Contribution

SpoIIIL is redefined as a forespore-specific signaling factor, not an A-Q complex component, and its role in σK activation is revealed.

Findings

SpoIIIL is required for efficient cell-cell signaling leading to late mother cell transcription.

SpoIIIL contributes to the activity of SpoIVB, a protease that processes pro-σK into active σK.

SpoIIIL is restricted to a subset of Bacillales species, indicating evolutionary specialization.

Abstract

During endospore formation, the mother cell and developing spore establish cell-cell signalling pathways that lead to compartment-specific transcription and key steps in morphogenesis. Endospore-forming bacteria also assemble a highly conserved essential membrane complex, called the A-Q complex, that physically connects these cells and may serve as a molecular conduit between them. While SpoIIIL was previously identified as a putative A-Q complex component in Bacillus subtilis, its exact role remains unclear. Here, we found that SpoIIIL does not function in the A-Q complex but instead acts as a forespore-specific factor required for efficient cell-cell signalling that leads to late mother cell transcription. Quantitative image analysis revealed that spoIIIL mutant spores do not exhibit hallmark phenotypes of A-Q complex mutants. Furthermore, unlike well-characterized A-Q complex proteins, SpoIIIL-GFP localizes uniformly in the forespore membrane before dispersing into the forespore cytoplasm. A synthetic sporulation screen identified a genetic relationship between spoIIIL and murAB, a paralog of murAA, required for efficient peptidoglycan precursor synthesis during sporulation. Cytological analysis indicates that the spoIIIL murAB double mutant is severely defective in the assembly of spore cortex peptidoglycan. Investigations into how SpoIIIL affects the cortex suggest it contributes to the activity of SpoIVB, a secreted forespore protease that initiates the signalling pathway required for processing of inactive pro-σK to active σK in the mother cell, which in turn up-regulates peptidoglycan precursor synthesis required for cortex formation. Accordingly, the spoIIIL mutant exhibits delayed and reduced pro-σK processing and decreased accumulation of peptidoglycan precursors. Thus, cortex assembly defects in the spoIIIL murAB double mutant results from alterations in separate pathways contributing to peptidoglycan precursor synthesis. Finally, phylogenetic analyses reveal that SpoIIIL is restricted to a subset of Bacillales species, highlighting evolutionary specialization in the signalling pathway leading to σK activation. Collectively, our findings redefine SpoIIIL as a forespore factor required for efficient cell-cell signalling that controls late mother-cell transcription.

Bacterial endospores allow bacteria to survive stress, persist in the environment and seed new and recurring infections. Cell-cell signalling is a cornerstone of how bacteria develop into spores, and the mother cell and developing spore establish several essential signalling pathways that underlie activation of compartment-specific σ factors at different stages of the development. In addition, communication between the mother cell and forespore involves the highly conserved A-Q complex, a specialized secretion-like system that, rather than directly regulating σ factor activation, plays a crucial role in sustaining forespore transcriptional potential. SpoIIIL was previously identified as a putative component of the A-Q complex, however its precise role remained unclear. Here we demonstrate that SpoIIIL does not function in the A-Q complex but instead contributes to the cell-cell signalling pathway that activates σK in the mother cell. Our findings refine the composition of the A-Q complex and provide new insight into the regulatory mechanisms controlling late mother-cell transcription.

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