Biology subjects

Collet, J.-F.

Publications and source records attributed to Collet, J.-F..

14 recordsLinked to original sources

bioRxiv2026

Polysaccharide release factors drive lipid-carrier recycling in bacteria

Gram-negative bacteria build their cell envelope by transporting various sugar-based precursors across the inner membrane on the shared lipid carrier undecaprenyl (also known as C55). Because the cellular pool of undecaprenyl is finite and used by multiple competing pathways, it must be efficiently regenerated to sustain envelope assembly. Yet the enzymes that release polysaccharide cargo from the lipid carrier have remained elusive, leaving a major gap in our understanding of bacterial envelope biogenesis. Here we identify three periplasmic enzymes in Escherichia coli - UprA, UprB, and UprC - that hydrolyze the bond linking polysaccharide precursors to undecaprenyl. UprA releases elongating peptidoglycan glycan strands from the inner membrane, catalyzing the long-sought terminal step of peptidoglycan synthesis. UprB cleaves the undecaprenyl-linked initiating unit common to the enterobacterial common antigen and O-antigen pathways, while UprC is the likely release factor for the colanic acid pathway. These enzymes define a widespread Upr-like protein family (formerly DUF218) as polysaccharide release factors, revealing a unifying mechanism by which bacteria coordinate envelope assembly. Because all three Upr-like proteins share a conserved catalytic core, this family represents a potential target for antibiotics that simultaneously disrupt multiple envelope pathways.

microbiology↗

bioRxiv2026

Stress-induced Membrane Insertion at the β-Barrel Assembly Machinery Complex Regulates BepA Metalloprotease Activity

Proteases must be tightly regulated to prevent uncontrolled degradation, yet the mechanisms ensuring such control remain poorly understood. Members of the widespread M48 metalloprotease family are kept inactive by an autoinhibitory plug that blocks catalytic water activation, but how this plug is released was unknown. Here, using genetic, biochemical and cryo-EM approaches, we discover the activation mechanism of BepA, a quality-control protease that preserves outer membrane integrity by surveilling the {beta}-barrel assembly machinery (BAM) in Gram-negative bacteria. Our cryo-EM analysis of BepA engaged with a stalled BAM-substrate assembly complex revealed that a flexible, unstructured 6-lid covering the active site in the latent protein functions as a molecular harpoon, inserting into the outer membrane when a substrate stalls at BAM and thereby docking BepA at the complex. This membrane anchoring promotes displacement of the autoinhibitory plug and unlocks protease activity precisely where and when it is needed. Thus, a dual enzyme activation mechanism is coupled to membrane association under stress, ensuring that BepA remains inactive until properly localized. Our findings reveal how membranes themselves can license protease activation, a principle that may extend beyond M48 metalloproteases.

microbiology↗

bioRxiv2026

Extreme envelope plasticity drives temperature-dependent morphogenesis in the LPS-free bacterium Sphingobium yanoikuyae

Bacterial growth patterns are generally considered to be constrained and species-specific. Here, we show that Sphingobium yanoikuyae exhibits exceptional plasticity in envelope architecture and morphogenesis supported by a glycosphingolipid (GSL)-based outer membrane and an atypical peptidoglycan structure. At 30{degrees}C, cells expand asymmetrically via a rare bipolar envelope synthesis mode, whereas growth at 37{degrees}C triggers a transition toward longitudinal elongation accompanied by increased outer membrane vesiculation, indicating a reversible reprogramming of morphogenesis under host-relevant temperature. This switch is supported by rapid outer membrane dynamics, including high membrane fluidity and fast redistribution of envelope components. In addition to these pronounced morphological changes, peptidoglycan contains unusually short glycan strands and displays specific temperature-dependent remodeling suggesting that growth plasticity is not only governed by spatial regulation but also by changes in cell wall cross-linking pattern. Genetic analyses further identify essential roles for proteins involved in outer membrane-peptidoglycan and outer membrane-inner membrane coupling, as well as GSL transport systems, and core cell wall synthesis machinery, while revealing extensive redundancy in envelope remodeling enzymes. Together, these results establish S. yanoikuyae as a model for extreme envelope adaptability, where a highly fluid outer membrane and structurally unconventional peptidoglycan enable reversible transitions between distinct growth programs, potentially shaping environmental fitness and host-associated interactions.

microbiology↗

bioRxiv2026

A genetically-encoded cysteine biosensor to monitor cysteine dynamics across life domains

Cysteine is a central metabolite in cellular redox regulation and iron-sulfur cluster assembly. Despite its critical role, monitoring cysteine dynamics in living systems has remained a challenge due to the lack of tools that avoid cysteine oxidation and/or do not destroy the cell in the process. Here, we report the development of Cystector (from Cysteine Detector), a genetically encoded, ratiometric green fluorescent biosensor for cysteine that exhibits an exceptional selectivity, minimal pH sensitivity in the physiological range, and a dynamic range of up to 4500%. Furthermore, the sensor retains functionality in the presence of physiological glutathione concentrations. We demonstrate the live-cell functionality of Cystector by monitoring intracellular and extracellular cysteine dynamics in different organisms. In E. coli, we show how cystine reduction in Escherichia coli is dependent on glutathione and glutaredoxins, and that the reduced cysteine is then exported into the extracellular environment. In yeast, we demonstrate how energy metabolism and oxidative stress determine cysteine homeostasis. In mammalian cells, we show how Cystector effectively monitors cysteine depletion in response to treatments such as H2O2, erastin2, and glutamate. Finally, we demonstrate, via a mitochondrially targeted variant, that Cystector can be used to monitor subcellular cysteine dynamics. These results together establish Cystector as a robust tool to unravel cysteine metabolism and transport in live cells across life domains.

molecular biology↗

bioRxiv2025

Fine-tuning of outer membrane-peptidoglycan tethering by the redox-active lipoprotein LppB from Salmonella enterica

The bacterial envelope is an essential compartment for survival, acting as a protective and permeability barrier. In diderm bacteria, it comprises an inner membrane, a thin peptidoglycan layer, and an outer membrane. A stable outer membrane-peptidoglycan connection is crucial for envelope integrity, preventing outer membrane detachment and enabling the generation of periplasmic turgor that counterbalances cytoplasmic pressure, thereby limiting susceptibility to antibiotics and environmental stressors. In Escherichia coli, the major lipoprotein Lpp (Brauns lipoprotein) serves as the only covalent linker between the outer membrane and peptidoglycan, through its unique C-terminal lysine residue. Homologs of Lpp are widely conserved among Enterobacteriaceae, including Salmonella enterica serovar Typhimurium, which encodes LppA and LppB. While Lpp and LppA function similarly, the role of LppB remains unclear. Using E. coli as a surrogate model, we investigated the structural and functional properties of Salmonella LppB. Here, we demonstrate that LppB forms disulfide-linked dimers via its unique C-terminal cysteine and crosslinks to peptidoglycan through its terminal lysine, albeit with significantly lower efficiency than Lpp. Notably, LppB interacts with Lpp to form heterotrimers, thereby reducing overall Lpp-peptidoglycan crosslinking. These findings suggest that LppB modulates outer membrane-peptidoglycan tethering by fine-tuning the bacterial envelope architecture in response to environmental conditions. Given the established link between outer membrane-peptidoglycan uncoupling and outer membrane vesicles (OMV) formation, a key mechanism in bacterial pathogenicity, our results suggest a model in which LppB regulates outer membrane-peptidoglycan attachment, potentially influencing OMV production. This study highlights a modulating role for LppB in envelope remodeling, providing new insights into how Salmonella adapts its envelope structure in response to environmental cues to enhance fitness and virulence.

microbiology↗

bioRxiv2025

Identification of receptor-binding domains of Bacteroidales antibacterial pore-forming toxins

Bacteroidales are abundant Gram-negative bacteria present in the gut microbiota of most animals, including humans, where they carry out vital functions for host health. To thrive in this competitive environment, Bacteroidales use sophisticated weapons to outmatch competitors. Among these, BSAPs (Bacteroidales Secreted Antimicrobial Proteins) represent a novel class of bactericidal pore-forming toxins that are highly specific to their receptor, typically targeting only a single membrane protein or lipopolysaccharide. The molecular determinants conferring this high selectivity remain unknown. In this study, we therefore investigated the model protein BSAP-1 and determined which of its domains is involved in providing receptor specificity. We clearly demonstrate that receptor recognition is entirely driven by the C-terminal domain (CTD) of BSAP-1 using a combination of in vivo competition assays and in vitro protein binding studies. Specifically, we show that deletion of the CTD abrogates BSAP-1 bactericidal activity by preventing receptor binding, while grafting the CTD to unrelated carrier proteins enables CTD-driven interaction with the BSAP-1 receptor. Building upon this discovery, we show that BSAPs can be categorized according to the structure of their CTD and that BSAPs within the same cluster are likely to target the same type of receptor. Additionally, we show that the CTD of BSAP-1 can be repurposed to generate probes for fluorescent labelling of membrane proteins in live cells. In summary, our research demonstrates that BSAP receptor recognition is driven by their CTD and that these can be engineered to develop novel tools for the investigation of Bacteroidales biology.

microbiology↗

bioRxiv2024

Deep plasma proteomics with data-independent acquisition: A fastlane towards biomarkers identification.

Plasma proteomic is a precious tool in human disease research, but requires extensive sample preparation in order to perform in-depth analysis and biomarker discovery using traditional Data-Dependent Acquisition (DDA). Here, we highlight the efficacy of combining moderate plasma prefractionation and Data-Independent Acquisition (DIA) to significantly improve proteome coverage and depth, while remaining cost- and time-efficient. Using human plasma collected from a 20-patient COVID-19 cohort, our method utilises commonly available solutions for depletion, sample preparation, and fractionation, followed by 3 LC-MS/MS injections for a 360-minutes DIA run time. DIA-NN software was then used for precursor identification, and the QFeatures R package was used for protein aggregation. We detect 1,321 proteins on average per patient, and 2,031 unique proteins across the cohort. Filtering precursors present in under 25% of patients, we still detect 1,230 average proteins and 1,590 unique proteins, indicating robust protein identification. Differential analysis further demonstrates the applicability of this method for plasma proteomic research and clinical biomarker identification. In summary, this study introduces a streamlined, cost- and time-effective approach to deep plasma proteome analysis, expanding its utility beyond classical research environments and enabling larger-scale multi-omics investigations in clinical settings.

systems biology↗

bioRxiv2023

The outer membrane and peptidoglycan layer form a single mechanical device balancing turgor

Bacteria are subject to a substantial concentration differential of osmolytes between the interior and exterior of the cell, which results in cytoplasmic turgor pressure. Failure to mechanically balance turgor pressure causes cells to burst. Here, we show that in Gram-negative bacteria, the outer membrane and peptidoglycan layer function together to resist turgor: when attached to each other, these two layers form a robust mechanical unit that allows pressure build-up in the periplasmic compartment, which in turn balances cytoplasmic turgor across the inner membrane, preventing cell death. Thus, the peptidoglycan layer is necessary but not sufficient to maintain turgor, which challenges the general view that protecting cells from bursting is the specific task of the peptidoglycan cell wall. ONE-SENTENCE SUMMARYThe peptidoglycan and outer membrane are interconnected layers that cooperate to balance cytoplasmic turgor.

microbiology↗

bioRxiv2022

Bacterial cell widening alters periplasmic size and activates envelope stress responses

The Rcs signal transduction system is a phosphorelay responsible for sensing a wide variety of enterobacterial cell envelope stresses. In Escherichia coli, the Rcs system is required to survive A22 and mecillinam treatment, two drugs that perturb cell size. To test whether cell size changes might be correlated with envelope damage and thereby sensed by the Rcs system, we tuned E. coli cell size via drug inhibition with A22, point mutations to the cell-shape determinant MreB, and mechanically confined growth. In all conditions, cell width was strongly correlated with Rcs activation, with wider cells exhibiting more activation than wild-type. In all conditions, RcsF, the outer membrane-localized upstream component of the Rcs system, was essential for responding to cell width changes. Consistently, several envelope gene deletions known to induce the Rcs system via RcsF resulted in cells that were wider than wild-type. Cryo- electron microscopy revealed that the periplasm of a wide MreB mutant was on average [~]3 nm thinner than wild-type, thereby bringing RcsF closer to the downstream components of the signaling cascade in the inner membrane. Conversely, extending the flexible linker region of RcsF by [~]3 nm increased Rcs activity in wild-type cells. In summary, we propose that the Rcs system responds to changes in cell width because of altered periplasmic thickness.

microbiology↗

bioRxiv2022

A molecular plugin rescues GroEL/ES substrates from pre-folding oxidation

Hsp60 chaperonins and their Hsp10 cofactors assist protein folding in all living cells, constituting the paradigmatic example of molecular chaperones. Despite extensive investigations of their structure and mechanism, crucial questions regarding how these chaperonins promote folding remain unsolved. Here, we report that the bacterial Hsp60 chaperonin GroEL forms a stable, functionally relevant complex with the chaperedoxin CnoX, a protein combining a chaperone and a redox function. Binding of GroES (Hsp10) to GroEL induces CnoX release. Cryo-electron microscopy provided crucial structural information on the GroEL-CnoX complex, showing that CnoX binds GroEL outside the substrate-binding site via a highly conserved C-terminal -helix. Furthermore, the identification of complexes in which CnoX, bound to GroEL, forms mixed-disulfides with GroEL substrates indicates that CnoX likely functions as a redox quality-control plugin for GroEL. Proteins sharing structural features with CnoX exist in eukaryotes, which suggests that Hsp60 molecular plugins have been conserved through evolution.

biochemistry↗

bioRxiv2021

YhcB coordinates peptidoglycan and LPS biogenesis with phospholipid synthesis during Escherichia coli cell growth.

The cell envelope is essential for viability in all kingdoms of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the most well studied organisms, Escherichia coli, there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell division. Here, we used a whole genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB, a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies we report the complete genetic interaction network of yhcB, revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a {Delta}yhcB background. Subsequent analyses suggest that YhcB sits at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.

microbiology↗

bioRxiv2021

Disorder is a critical component of lipoprotein sorting in Gram-negative bacteria

Gram-negative bacteria express structurally diverse lipoproteins in their envelope. Here we found that approximately half of lipoproteins destined to the Escherichia coli outer membrane display an intrinsically disordered linker at their N-terminus. Intrinsically disordered regions are common in proteins, but establishing their importance in vivo has remained challenging. Here, as we sought to unravel how lipoproteins mature, we discovered that unstructured linkers are required for optimal trafficking by the Lol lipoprotein sorting system: linker deletion re-routes three unrelated lipoproteins to the inner membrane. Focusing on the stress sensor RcsF, we found that replacing the linker with an artificial peptide restored normal outer membrane targeting only when the peptide was of similar length and disordered. Overall, this study reveals the role played by intrinsic disorder in lipoprotein sorting, providing mechanistic insight into the biogenesis of these proteins and suggesting that evolution can select for intrinsic disorder that supports protein function.

biochemistry↗

bioRxiv2020

Defining the function of OmpA in the Rcs stress response

OmpA, a protein commonly found in the outer membrane of Gram-negative bacteria, has served as a paradigm for the study of {beta}-barrel proteins for several decades. In Escherichia coli, OmpA was previously reported to form complexes with RcsF, a surface-exposed lipoprotein that triggers the Rcs stress response when damage occurs in the outer membrane and the peptidoglycan. How OmpA interacts with RcsF and whether this interaction allows RcsF to reach the surface has remained unclear. Here, we integrated in vivo and in vitro approaches to establish that RcsF interacts with the C-terminal, periplasmic domain of OmpA, not with the N-terminal {beta}-barrel, thus implying that RcsF does not reach the bacterial surface via OmpA. Our results reveal a novel function for OmpA in the cell envelope: OmpA competes with the inner membrane protein IgaA, the downstream Rcs component, for RcsF binding across the periplasm, thereby regulating the Rcs response.

microbiology↗

bioRxiv2019

Structural insight into the formation of lipoprotein-β-barrel complexes by the β-barrel assembly machinery

The {beta}-barrel assembly machinery (BAM) inserts outer membrane {beta}-barrel proteins (OMPs) in the outer membrane of Gram-negative bacteria. In Enterobacteriacea, BAM also mediates export of the stress sensor lipoprotein RcsF to the cell surface by assembling RcsF-OMP complexes. Although BAM has been the focus of intense research due to its essential activity in generating and maintaining the outer membrane, how it functions remains poorly understood. Here, we report the crystal structure of the key BAM component BamA in complex with RcsF. BamA adopts an inward-open conformation, with the lateral gate to the membrane closed. The globular domain of RcsF is lodged deep inside the lumen of the BamA barrel, binding regions proposed to undergo an outward and lateral opening during OMP insertion. Our structural and biochemical data indicate a push-and-pull mechanism for RcsF export upon conformational cycling of BamA and provide a mechanistic explanation for how RcsF uses its interaction with BamA to detect envelope stress. Our data also suggest that the flux of incoming OMP substrates is involved in the control of BAM activity. Overall, the structural insights gleaned here elucidate a fundamental biological process and suggest a new avenue for antibiotic development.

microbiology↗