Biology subjects

Rodrigues, C. D. A.

Publications and source records attributed to Rodrigues, C. D. A..

5 recordsLinked to original sources

bioRxiv2026

SpoIVA contributes to efficient engulfment through a cytoskeletal-like mechanism during Bacillus subtilis sporulation

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 ({Delta}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 {Delta}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. IMPORTANCEEndospore 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

microbiology↗

bioRxiv2025

Computational modelling of toroidal membranes at division and fission sites

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.

microbiology↗

bioRxiv2023

Ultrastructural details of resistance factors of the bacterial spore revealed by in situ cryo-electron tomography

The bacterial spore owes its incredible resistance capacities to various molecular structures that protect the cell content from external aggressions. Among the determinants of resistance are the quaternary structure of the chromosome and an extracellular shell made of proteinaceous layers (the coat), the assembly of which remains poorly understood. Here, in situ cryo-electron tomography (cryo-ET) on bacteria lamellae generated by cryo-focused ion beam micromachining (cryo-FIBM) provides insights into the ultrastructural organization of Bacillus subtilis sporangia, including that of the DNA and nascent coat layers. Analysis of the reconstructed tomograms reveal that rather early during sporulation, the chromosome in the developing spore (the forespore) adopts a toroidal structure harboring 5.5-nm thick fibers. At the same stage, coat proteins at the surface of the forespore form a complex stack of amorphous or structured layers with distinct electron density, dimensions and organization. We investigated the nature of the nascent coat layers in various mutant strains using cryo-FIBM/ET and transmission electron microscopy on resin sections of freeze-substituted bacteria. Combining these two cellular electron microscopy approaches, we distinguish seven nascent coat regions with different molecular properties, and propose a model for the contribution of the morphogenetic proteins SpoIVA, SpoVID, SafA and/or CotE. Significance statementBacterial spores are dormant cells that can resist to multiple stresses, including antibiotics, detergents, irradiation and high temperatures. Such resilience is an asset when spores are used for the benefit of humans, as in the case of probiotics, or a major problem for public health, food safety or biowarfare when it comes to spores of pathogenic bacteria. In this study, we combined state-of-the-art cryo-electron tomography and conventional cellular electron microscopy to provide insights into intermediate stages of spore development. Our data reveal the intracellular reorganization of the chromosome into a toroidal fibrillar structure and the complex assembly of the multi-protein, multilayered extracellular coat, shedding light on the mechanisms by which the spore acquires its incredible resistance capacities.

microbiology↗

bioRxiv2021

Membrane fission during bacterial spore development requires DNA-driven cellular inflation

Bacteria require membrane fission for cell division and endospore formation. FisB catalyzes membrane fission during sporulation, but the molecular basis is unclear as it cannot remodel membranes by itself. Sporulation initiates with an asymmetric division that generates a large mother cell and a smaller forespore that contains only 1/4 of its complete genome. As the mother cell membranes engulf the forespore, a DNA translocase pumps the rest of the chromosome into the small forespore compartment, inflating it due to increased turgor. When the engulfing membranes undergo fission, the forespore is released into the mother cell cytoplasm. Here we show that forespore inflation and FisB accumulation are both required for efficient membrane fission. We suggest that high membrane tension in the engulfment membrane caused by forespore inflation drives FisB-catalyzed membrane fission. Collectively our data indicate that DNA-translocation has a previously unappreciated second function in energizing FisB-mediated membrane fission under energy-limited conditions. HIGHLIGHTS- Membrane fission during endospore formation requires rapid forespore inflation by ATP-driven DNA translocation. - Forespore inflation is fast enough to increase the tension of the engulfment and forespore membranes to near lysis tensions. - FisB catalyzes membrane fission by impeding the flux of lipids that partially support forespore and engulfment membrane growth. - Membrane fission utilizes chemical energy transduced to mechanical energy during DNA-packing into the forespore.

biophysics↗

bioRxiv2020

Requirements for FisB-mediated membrane fission during sporulation

Little is known about mechanisms of membrane fission in bacteria despite their requirement for cytokinesis. The only known dedicated membrane fission machinery in bacteria, FisB, is expressed during sporulation in Bacillus subtilis and is required to release the developing spore into the mother cell cytoplasm. Here we characterized the requirements for FisB-mediated membrane fission. FisB forms mobile clusters of [~]12 molecules that give way to an immobile cluster at the engulfment pole containing [~]40 proteins at the time of membrane fission. Analysis of FisB mutants revealed that binding to acidic lipids and homo-oligomerization are both critical for targeting FisB to the engulfment pole and membrane fission. Experiments using artificial membranes and filamentous cells suggest FisB does not have an intrinsic ability to sense or induce membrane curvature but can bridge membranes. Finally, modeling suggests homo-oligomerization and trans interactions with membranes are sufficient to explain FisB accumulation at the membrane neck that connects the engulfment membrane to the rest of the mother cell membrane during late stages of engulfment. Together, our results show that FisB is a robust and unusual membrane fission protein that relies on homo-oligomerization, lipid-binding and the unique membrane topology generated during engulfment for localization and membrane scission, but surprisingly, not on lipid microdomains, negative-curvature lipids, or curvature-sensing.

biophysics↗