Chris L. B. Graham

Bacterial endospore formation begins with a polar septum that compartmentalizes two transcriptionally distinct cells, the mother cell and forespore. In Bacillus subtilis , a complex composed of SpoIIIE, SpoIIIM and PbpG maintains compartmentalization at a septal pore during hydrolysis of septal peptidoglycan (PG), by coordinating two critical functions: chromosome translocation from the mother cell to the forespore through the septal pore and preservation of septal pore stability through SpoIIIE-PG interactions and PG synthesis. Disruption of this mechanism leads to cytoplasmic leakage, failed chromosome transfer, and reduced sporulation. Here, we identify CprV (YteV) as a safeguarding factor that maintains compartmentalization in response to septal pore stability defects. Cells lacking CprV show mild defects in compartmentalization and chromosome translocation; however, in mutants experiencing septal pore instability, loss of CprV significantly worsens these defects. Consistent with this role, CprV accumulates at the septal pore in a SpoIIIE-dependent manner when chromosome translocation is impaired. Computational analyses indicate that CprV exhibits structural similarity to Alba proteins found in Eukaryotes and Archaea and is primarily conserved in the Bacillales . Collectively, our data support a model whereby CprV monitors septal pore stability to safeguard compartmentalization during sporulation, providing insight into how cells monitor compartmental integrity during cellular remodeling.

During endospore (spore) development in bacteria, polar cell division generates two transcriptionally distinct cellular compartments, the mother cell and future spore (forespore). Signalling between these cells leads to sequential and compartmentalized transcription, along with key morphogenetics events, including the phagocytic-like process of engulfment and the recruitment of coat proteins to the engulfing membrane. The SpoIVA ATPase is an essential sporulation protein that assembles into static filaments at the forespore surface during engulfment, where it functions as the basement layer for coat assembly. Here, using Bacillus subtilis, we reveal an additional role for SpoIVA during engulfment. Cytological analysis of a spoIVA null mutant ( Δ spoIVA ) revealed engulfment defects such as septal membrane bulges and asymmetric membrane migration, similar to those typically associated with impaired peptidoglycan remodelling during engulfment. Engulfment defects were exacerbated when Δ spoIVA was combined mutants known to impact engulfment progression and efficiency. Importantly, a spoIVA mutant (K30A) impaired for ATP hydrolysis and filament formation in vitro but partially functional for coat assembly in vivo , closely phenocopies the spoIVA null mutant engulfment defects. Based on these data, we propose a model whereby SpoIVA polymerisation at the spore surface, independently of coat assembly, plays a mechanical and structural role during engulfment, akin to the cytoskeletal proteins that drive phagocytosis in eukaryotic cells. IMPORTANCE: Endospore formation relies on membrane remodeling to generate highly resistant dormant spores in human, animal and insect pathogens. During engulfment, the mother-cell membrane migrates around the forespore in a phagocytic-like process while the spore coat is simultaneously assembled. SpoIVA is widely recognized as the ATPase that polymerizes at the forespore surface to nucleate assembly of the multilayered spore coat. Here, we demonstrate that SpoIVA also performs a distinct function during engulfment that is independent of its role in coat formation. Cells lacking SpoIVA, or carrying a polymerization-defective SpoIVA variant, exhibit membrane deformations characteristic of impaired engulfment. We propose that SpoIVA polymers provide structural and mechanical support for membrane migration, analogous to the role of cytoskeletal systems during eukaryotic phagocytosis. These findings reveal an unexpected function for SpoIVA and demonstrate that bacterial protein polymers can provide mechanical support for membrane remodeling during complex developmental processes, analogous to cytoskeletal systems in eukaryotic cells

Multiple sequence alignment (MSA) data underlies current principles in protein folding and protein-protein interaction prediction, from which large language models (LLMs) in tandem with protein datasets, can predict protein structure. However, what is missing are user-friendly tools that enable researchers to predict and demonstrate coevolution - the principal input which these MSAs infer. Here we present tools to identify and visualize coevolution, through a pipeline (CoEVFold) that uses basic direct coupling algorithms derived from GREMLIN and alignment of sequences from MMSEQs2. The pipeline generates a visual representation of coevolution for a single protein but can also represent coevolution of homomeric or heteromeric protein complexes, as well as coevolution within protein networks. The input for this pipeline can be an amino acid sequence, or user input protein structures from Alphafold their own files or the PDB database. In validation of CoEVFold’s capabilities, and utilising proteins from known prokaryotic and eukaryotic model systems ( Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae) , as well as phage proteins, CoEVFold predicts coevolution between proteins known to interact, proteins known to oligomerise, and coevolution in proteins known to be part of a protein complex. Collectively, these suite of tools, named ‘CoEVFold suite’, have broad applicability and provide a useful toolkit to those interested in dissecting protein-protein interactions and networks. Availability: The code is available online at https://colab.research.google.com/drive/1MSSvNTq7KZ4Lr0XTz89vUuK-J3xOTzwS?usp=sharing and Github. https://github.com/MishterBluesky/CoEVFold Supplementary information: Supplementary data is available via Figshare and supplementary materials.

Vibrio cholerae , the causative agent of cholera, remains a major global health burden despite ongoing initiatives for vaccination and improved sanitation. Much of the V. cholerae lifecycle occurs in aquatic environments, requiring gene functions that enable survival under multiple stresses associated with this niche and during the transition to the human host. Although numerous V. cholerae genome sequences are available, functional annotation across its pan-genome remains incomplete, limiting our understanding of its biology. In this study, we employed a chemical genomics approach to profile fitness across 104 diverse stress conditions and identify growth phenotypes for unannotated genes using 3,026 single-gene deletion mutants of V. cholerae C6706, a widely used research strain. In total, we identified significant growth phenotypes in 1,658 mutants, of which 285 correspond to currently unannotated genes. Together, these data provide a comprehensive resource for exploring gene function in V. cholerae , supported by detailed quality metrics and open-access tools to facilitate community-driven discovery. AUTHOR SUMMARY: Although many bacterial pathogens have been extensively sequenced, we still know relatively little about what many of their genes actually do. In many cases, gene functions are solely predicted by computer algorithms but have not been confirmed in the lab, and we can assume that some of those predictions may be incorrect. Chemical genomics—a method that tests thousands of single-gene mutants under different stresses—can help reveal what these genes are responsible for. Using this approach, we screened a library of Vibrio cholerae C6706 mutants under 104 different conditions. We found that more than half of the mutants showed measurable effects on growth, including many genes that were previously uncharacterized. Here, we describe our experimental pipeline, present key quality checks, and share how other researchers can use this dataset to explore gene function in V. cholerae .

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.

The formation of specialised membrane architectures is fundamental to biological processes. Computational modelling of membrane structures offers a means to unravel complex molecular mechanisms that remain inaccessible through in vitro or in vivo approaches. Among these architectures, toroidal membranes, otherwise known as fusion pores or fission pores, occur during the final stages of cell division, exocytosis, endocytosis, and membrane fission; including during the phagocytic-like engulfment of bacterial endospores. Here we have designed ‘MemTorMD’, a computational simulation pipeline based on the TS2CG methodology, to enable coarse-grained modelling of free toroidal membranes with biologically-relevant dimensions. This approach allowed us to determine the lipid properties required for stable toroidal membrane formation, providing a biologically accurate simulation surface. Furthermore, we demonstrate the applicability of this approach to investigate the function of proteins that localise at toroidal membranes, and drive molecular events such as membrane fission by a prokaryotic fission protein. Collectively, our work provides insight into membrane and protein behaviour at toroidal membranes and provides a platform for the development of more complex simulations involving additional molecular components at membrane toroids present in a range of processes across life.

The use of membrane-specific dyes for in vivo fluorescent microscopy is commonplace. However, most of these reagents are non-specific and cannot track specific lipid species movement, instead often acting as non-covalent lipid-associated probes or requiring the uptake of whole lipids and acyl tails into the membrane. This issue has been solved in eukaryotic cell biology by the use of click-chemistry-liable phospholipid headgroup pulse labels. Here, we describe a method for in vivo phospholipid labelling by fluorescent imaging in Pseudomonas aeruginosa using a phosphatidylcholine mimic, ‘propargyl-choline’ (PCho). This click-chemistry-liable headgroup mimic is visible by microscopy and allows the covalent labelling of lipids. Fluorescence of the cell membranes, visible in heterogeneous patches, is dependent on PCho concentration and is localized in the membrane fraction of cells, demonstrating that it is suitable for membrane labelling and cell imaging.

The use of membrane-specific dyes for in vivo fluorescent microscopy is commonplace. However, most of these reagents are non-specific and cannot track specific lipid species movement, instead often acting as non-covalent lipid associated probes or requiring uptake of whole lipids and acyl tails into the membrane. This issue has been solved in eukaryotic cell biology by use of click-chemistry liable phospholipid headgroup pulse-labels. Here we describe a method for in vivo phospholipid labelling by fluorescent imaging in Pseudomonas aeruginosa using a phosphatidylcholine (PC) mimic, “propargyl-choline”(PCho). This click-chemistry liable headgroup mimic is visible by microscopy and allows the covalent labelling of lipids. Fluorescence of the cell membranes, visible in heterogenous patches, is dependent on PCho concentration and is localised in the membrane fraction of cells, demonstrating that it is suitable for membrane labelling and cell imaging.

Peptidoglycan (PG) is an essential structural component of the bacterial cell wall that is synthetized during cell division and elongation. PG forms an extracellular polymer crucial for cellular viability, the synthesis of which is the target of many antibiotics. PG assembly requires a glycosyltransferase (GT) to generate a glycan polymer using a Lipid II substrate, which is then crosslinked to the existing PG via a transpeptidase (TP) reaction. A Shape, Elongation, Division and Sporulation (SEDS) GT enzyme and a Class B Penicillin Binding Protein (PBP) form the core of the multi-protein complex required for PG assembly. Here we used single particle cryo-electron microscopy to determine the structure of a cell elongation-specific E. coli RodA-PBP2 complex. We combine this information with biochemical, genetic, spectroscopic, and computational analyses to identify the Lipid II binding sites and propose a mechanism for Lipid II polymerization. Our data suggest a hypothesis for the movement of the glycan strand from the Lipid II polymerization site of RodA towards the TP site of PBP2, functionally linking these two central enzymatic activities required for cell wall peptidoglycan biosynthesis.