Morales Angeles Danae, Coleman Kaitlyn, Progress Odika Chimezie, L B Graham Chris, Chan Helena, Gilmore Michael, Taib Najwa, Bauda Elda, Moriscot Christine, Gallet Benoit, Fisher Hannah, Bullough Per A, Morlot Cécile, Köster Darius, Gribaldo Simonetta, Cava Felipe, Rodrigues Christopher D A
School of Life Sciences, University of Warwick, Coventry, United Kingdom.
Australian Institute for Microbiology & Infection, University of Technology Sydney, Sydney Australia.
PLoS Genet. 2025 Jul 3;21(7):e1011768. doi: 10.1371/journal.pgen.1011768. eCollection 2025 Jul.
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.
在芽孢形成过程中，母细胞和发育中的芽孢建立了细胞间信号通路，这些通路导致特定隔室的转录和形态发生中的关键步骤。形成芽孢的细菌还组装了一种高度保守的必需膜复合物，称为A-Q复合物，它在物理上连接这些细胞，并可能作为它们之间的分子通道。虽然SpoIIIL先前被鉴定为枯草芽孢杆菌中假定的A-Q复合物成分，但其确切作用仍不清楚。在这里，我们发现SpoIIIL不在A-Q复合物中起作用，而是作为一种前芽孢特异性因子，是有效细胞间信号传导所必需的，该信号传导导致后期母细胞转录。定量图像分析表明，spoIIIL突变体芽孢不表现出A-Q复合物突变体的标志性表型。此外，与特征明确的A-Q复合物蛋白不同，SpoIIIL-GFP在分散到前芽孢细胞质之前均匀地定位于前芽孢膜中。一个合成芽孢形成筛选确定了spoIIIL和murAB之间的遗传关系，murAB是murAA的旁系同源物，在芽孢形成过程中高效肽聚糖前体合成所必需。细胞学分析表明，spoIIIL murAB双突变体在芽孢皮层肽聚糖组装方面存在严重缺陷。对SpoIIIL如何影响皮层的研究表明，它有助于SpoIVB的活性，SpoIVB是一种分泌的前芽孢蛋白酶，启动了在母细胞中将无活性的前体σK加工成活性σK所需的信号通路，这反过来又上调了皮层形成所需的肽聚糖前体合成。因此，spoIIIL突变体表现出前体σK加工延迟和减少以及肽聚糖前体积累减少。因此，spoIIIL murAB双突变体中的皮层组装缺陷是由有助于肽聚糖前体合成的不同途径的改变引起的。最后，系统发育分析表明，SpoIIIL仅限于芽孢杆菌属物种的一个子集，突出了导致σK激活的信号通路中的进化特化。总的来说，我们的发现将SpoIIIL重新定义为有效细胞间信号传导所需的前芽孢因子，该信号传导控制后期母细胞转录。
