Biology subjects

Morlot, C.

Publications and source records attributed to Morlot, C..

10 recordsLinked to original sources

bioRxiv2026

Differential impact of cell wall antibiotics on the Rod complex and aPBPs in Bacillus subtilis: Insights into the peptidoglycan elongation machineries

Bacterial cell wall (CW), primarily composed of the biopolymer peptidoglycan, serve as essential protective barriers against external stresses and the internal turgor pressure. The peptidoglycan (PG) biosynthetic pathway encompasses sequential enzymatic reactions in the cytoplasm and in the membrane that involve critical enzymes susceptible to antibiotic targeting. Virtually each step of the pathway is the target of a known antibiotic. Antibiotic-induced inhibition of PG assembly typically weakens the sacculus, often leading to cell lysis. However, the cascade of events that follow inhibition of a specific enzyme of the pathway, and how these culminate in cell death remain largely unknown. Here, we investigated the effects on growing Bacillus subtilis cells of two categories of CW antibiotics: inhibitors of the synthesis of soluble PG precursors in the cytoplasm (fosfomycin and D-cycloserine) and inhibitors of the polymerisation and crosslinking reactions at the outer leaflet of the membrane, which incorporate newly externalised precursors into the existing network (vancomycin and penicillin). In B. subtilis, the latter reactions are catalysed along the sidewalls by the Rod complex, thought to primarily build the sacculus, and by class A penicillin-binding proteins (aPBPs), thought to add to repair it. Our findings reveal that the two antibiotic groups lead to growth arrest, sacculus thinning, and eventual cell lysis. However, while the impact of vancomycin and penicillin G is rapid, lacking morphological deformation, fosfomycin and D-cycloserine induce cell widening and bulging before lysis. During shortage of PG precursors, dysregulated PG hydrolytic activity contributes to elevated cell lysis but is not responsible of bulging. Instead, dispersed PG synthesis by aPBPs persists while the activity of the Rod system is rapidly arrested, resulting in cell rounding. We propose that this facilitates the redirection of the limited PG precursors to sites of CW repair, thereby preserving cell integrity and allowing for prolonged growth during antibiotic challenge.

microbiology↗

bioRxiv2026

Assembly of the essential SpoIVA coat protein at the surface of developing bacterial spores

Bacterial spores owe their remarkable resistance properties to a multilayered coat, one of the most resilient and durable biological structures on Earth. Assembled at the surface of the outer forespore membrane, the coat comprises dozens of proteins organized into distinct layers. Its formation is initiated by SpoIVA, which is proposed to form a polymeric scaffold for the innermost coat layer. Although SpoIVA has been shown to polymerize into filaments in vitro, there is currently no evidence demonstrating the formation of such assemblies in vivo, and the mechanism underlying its oligomerization remains unresolved. In this study, cryo-focused ion beam milling combined with cryo-electron tomography of sporulating Bacillus subtilis cells reveals that the SpoIVA layer consists of polymers that form track-like structures radiating from the mother cell-proximal forespore pole and extending directionally toward the distal pole. Subtomogram averaging further sheds light on their organized architecture, harboring a straight orientation, uniform spacing, and embedding in the outer forespore membrane. These observations also define SpoIVA spatial orientation relative to the outer forespore membrane. Furthermore, AlphaFold3 predictions, combined with biophysical and functional assays, show that SpoIVA dimerizes through its central and C-terminal regions. We further show that dimerization promotes SpoIVA localization around the forespore but is dispensable for polymer formation, which relies on the ATPase domain. Altogether, these findings suggest a dual oligomerization mechanism, in which SpoIVA transitions from dimers to linear track-like polymers, and reveal that these assemblies play critical roles in coat assembly and spore development.

microbiology↗

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↗

bioRxiv2024

Combining live cell fluorescence imaging with in situ cryo electron tomography sheds light on the septation process in Deinococcus radiodurans

Cell division is a fundamental biological process that allows a single mother cell to produce two daughter cells. In bacteria, different modes of cell division have been reported that are notably associated with distinctive cell shapes, but in all cases, division involves a step of septation, corresponding to the growth of a new dividing cell wall, followed by splitting of the two daughter cells. The radiation-resistant Deinococcus radiodurans is a spherical bacterium protected by a thick and unusual cell envelope. It has been reported to divide using a distinctive mode of septation in which two septa originating from opposite sides of the cell progress with a flat leading edge until meeting and fusing at mid-cell. In the present study, we have combined conventional and super-resolution fluorescence microscopy of live bacteria with in situ cryogenic electron tomography of bacterial lamellae to investigate the septation process in D. radiodurans. This work provides important insight into (i) the complex architecture of the cell envelope of this bacterium, (ii) the sliding doors septation process and (iii) the molecular mechanisms underlying septal growth and closure.

microbiology↗

bioRxiv2024

Characterization of a GpsB-associated regulator of PBP1a reveals the organization of the cell wall remodeling complex of Streptococcus pneumoniae

Class A PBPs (aPBPs) play a key role in the biosynthesis and remodeling of peptidoglycan, the main component of the bacterial cell wall. The human bacterial pathogen Streptococcus pneumoniae produces three aPBPs, which are regulated to maintain the bacteriums ovoid shape. Although their exact functions remain unclear, evidence suggests that PBP1a and PBP2a activities are closely coordinated. In this study, we elucidated the function of an unknown function protein named GarP (GpsB-associated regulator of PBP1a), in the regulation of PBP1a activity. We showed that GarP localizes to the division septum and its absence leads to morphological defects. We further identified a GpsB-binding motif in GarP as well as in PBP2a, the PG deacetylase PgdA and the muramidase MpgA. Our analysis of genetic and protein interactions, combined with cell imaging, supports a model of a molecular complex that coordinates PG remodeling during S. pneumoniae cell division.

microbiology↗

bioRxiv2024

New assessment of teichoic acids in the cell envelope of Streptococcus pneumoniae

Teichoic acids (TA) are linear phospho-saccharidic polymers and important constituents of the cell envelope of Gram-positive bacteria, either bound to the peptidoglycan as wall teichoic acids (WTA) or to the membrane as lipoteichoic acids (LTA). The chemical composition of TA varies greatly but the presence of both WTA and LTA is highly conserved, hinting at an underlying fundamental function that is distinct from their numerous specific roles in diverse organisms. We report here the observation of a periplasmic space in the Gram-positive Streptococcus pneumoniae by cryo-electron microscopy of vitreous sections. The thickness and appearance of this region change upon deletion of genes involved in the attachment of teichoic acids, supporting the role of TA in the maintenance of a periplasmic space in Gram-positive bacteria as a possible universal function. Consequences of these mutations were further examined by super-resolved microscopy (dSTORM), following metabolic and fluorophore coupling by click-chemistry in pulse and pulse-chase experiments. This novel labeling method also enabled in-gel analysis of cell fractions, revealing that LTA-containing membranes sediment at low centrifugal forces. Owing to this easy separation approach, we were able to titrate the actual amount of TA per cell and to determine the ratio of WTA to LTA. In addition, we followed the change of TA length during growth phases, and discovered that a mutant devoid of LTA accumulates the membrane-bound polymerized TA precursor. SignificanceThe existence of a periplasmic space in Gram-positive bacteria has long been debated. The finding that compromising the attachment of teichoic acids changes the appearance and thickness of the periplasm in the pneumococcus indicates a role of these polymers in the maintenance of this space between the membrane and the cell wall. Metabolic labeling and electrophoresis showed that LTA-containing membranes are easily sedimented. This finding indicates that the LTA/WTA ratios reported in previous studies were likely underestimated, since most LTA were probably unknowingly discarded in these studies. Our method of TA analysis opens a new era in the investigation of these important and poorly known bacterial polymers and their role in the periplasmic space of Gram-positive organisms.

microbiology↗

bioRxiv2024

DivIVA controls the dynamics of septum splitting and cell elongation in Streptococcus pneumoniae

Bacterial shape and division rely on the dynamics of cell wall assembly, which involves regulated synthesis and cleavage of the peptidoglycan. In ovococci, these processes are coordinated in an annular mid-cell region with nanometric dimensions. More precisely, the cross-wall that is synthesized by the divisome is split to generate lateral wall, whose expansion is insured by insertion of so-called peripheral peptidoglycan by the elongasome. Septum cleavage and peripheral peptidoglycan synthesis are thus crucial remodeling events for ovococcal cell division and elongation. The structural DivIVA protein has long been known as a major regulator of these processes but its mode of action remains unknown. Here, we integrate click chemistry-based peptidoglycan labeling, direct stochastic optical reconstruction microscopy and in silico modeling, as well as epifluorescence and stimulated emission depletion microscopy to investigate the role of DivIVA in Streptococcus pneumoniae cell morphogenesis. Our work reveals two distinct phases of peptidoglycan remodeling along the cell cycle, that are differentially controlled by DivIVA. In particular, we show that DivIVA ensures homogeneous septum cleavage and peripheral peptidoglycan synthesis around the division site, and their maintenance throughout the cell cycle. Our data additionally suggest that DivIVA impacts the contribution of the elongasome and class A PBPs to cell elongation. We also report the position of DivIVA on either side of the septum, consistent with its known affinity for negatively curved membranes. Finally, we take the opportunity provided by these new observations to propose hypotheses for the mechanism of action of this key morphogenetic protein. IO_SCPLOWMPORTANCEC_SCPLOWThis study sheds light on fundamental processes governing bacterial cell growth and division, using integrated click chemistry, advanced microscopy and computational modeling approaches. More precisely, it addresses mechanisms involved in the regulation of cell wall synthesis and remodeling in Streptococcus pneumoniae. This bacterium belongs to the morphological group of ovococci, which includes many human pathogens, such as streptococci and enterococci. In this study, we have dissected the function of DivIVA, which is a structural protein involved in cell division, cell morphogenesis and chromosome partitioning in Gram-positive bacteria. This work unveils the role of DivIVA in the orchestration of cell division and elongation along the pneumococcal cell cycle. It not only helps understanding how ovoid bacteria proliferate, but also offers an opportunity to consider how DivIVA might serve as a scaffold and sensor for particular membrane regions, and thus be involved in various processes associated with the cell cycle.

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↗

bioRxiv2019

Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors

Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. ClpP protease is a tetradecameric complex that has emerged as a drug target against multiple pathogenic bacteria. During drug development efforts, the activation of different ClpPs by inhibitors was independently reported, but so far, no rationale for inhibitor-induced activation has been proposed. Using an integrated approach that included X-ray crystallography, solid-and solution-state NMR, MD simulations and ITC we show that the proteasome-inhibitor bortezomib binds to the ClpP active site serine mimicking a peptide substrate and induces the concerted allosteric activation of the complex. The bortezomib activated conformation also displays a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association as well as protein processive degradation.

biochemistry↗

bioRxiv2018

Beating Swords into Ploughshares: Domestication of a Phage Lysin for Housekeeping Function

Temperate phages constitute a potentially beneficial genetic reservoir for bacterial innovation despite being selfish entities encoding an infection cycle inherently at odds with bacterial fitness. These phages integrate their genomes into the bacterial host during infection, donating new, but deleterious, genetic material: the phage genome encodes toxic genes, such as lysins, that kill the bacterium during the phage infection cycle. Remarkably, some bacteria have exploited the destructive properties of phage genes for their own benefit by co-opting them as toxins for functions related to bacterial warfare, virulence, and secretion. However, do toxic phage genes ever become raw material for functional innovation? Here we report on a toxic phage gene whose product has lost its toxicity and has become a domain of a core cellular factor, SpmX, throughout the bacterial order Caulobacterales. Using a combination of phylogenetics, bioinformatics, structural biology, cell biology, and biochemistry, we have investigated the origin and function of SpmX and determined that its occurrence is the result of the detoxification of a phage peptidoglycan hydrolase gene. We show that the retained, attenuated activity of the phage-derived domain plays an important role in proper cell morphology and developmental regulation in representatives of this large bacterial clade. To our knowledge, this is the first observation of phage gene domestication in which a toxic phage gene has been co-opted for a housekeeping function.

microbiology↗