Biology subjects

Billaudeau, C.

Publications and source records attributed to Billaudeau, C..

6 recordsLinked to original sources

bioRxiv2026

Intrafilament nucleotide exchange in a prokaryotic actin homolog

Polymerization and disassembly govern the cellular functions of cytoskeletal proteins. Canonical nucleotide-dependent polymers, including actin and microtubules, renew their nucleotide state through subunit subunit dissociation, nucleotide exchange in solution and repolymerization. Yet the assembly dynamics of the membrane-bound bacterial actin MreB remain unresolved. Here, using total internal reflection fluorescence microscopy and high-speed atomic force microscopy, we visualise Bacillus subtilis MreB assembly on supported lipid bilayers in real time. ATP binding drives polymerization into symmetrically elongating filaments, whereas ATP hydrolysis within filaments promotes disassembly. Unexpectedly, nucleotides continuously exchange within membrane-bound filaments without detectable subunit turnover, coupling filament stability to the surrounding nucleotide pool: ATP exchange stabilizes filaments, whereas ADP exchange triggers rapid fragmentation and disassembly. Monte-Carlo modelling and single-cell in vivo imaging support this mechanism. Thus, intrafilament nucleotide exchange enables MreB filaments to renew their nucleotide state without polymer turnover, revealing nucleotide-state rejuvenation as a new mode of biological polymer regulation.

microbiology↗

bioRxiv2024

Transient inhibition of cell division in competent pneumococcal cells results from deceleration of the septal peptidoglycan complex

Bacterial cells are known to produce inhibitors of cell division in response to stress responses and developmental programs. Knowledge of the underlying molecular mechanisms remains however largely limited. In this study, we investigated the mechanism of transient cell division inhibition observed during the development of competence for transformation in the human pathogen Streptococcus pneumoniae. In this species, ComM, a membrane protein specifically produced during competence, transiently inhibits cell division to preserve genomic integrity during transformation. We show that ComM reduces specifically the dynamics of the septal peptidoglycan synthetic complex FtsW:PBP2x. We also present evidence that ComM interacts with the peptidoglycan precursor synthetic enzyme MurA, and show that overproduction of MurA suppresses FtsW:PBP2x deceleration along the cell division delay in competent cells. Collectively, our data support a model in which ComM interferes with MurA activity to reduce septal peptidoglycan synthesis during competence in S. pneumoniae.

microbiology↗

bioRxiv2023

Quantification of membrane fluidity in bacteria using TIR-FCS

Cell membrane fluidity is an important phenotypic feature that regulates the diffusion, function and folding of transmembrane and membrane-associated proteins. It is particularly interesting to study it in bacteria as variations in membrane fluidity are known to affect fundamental cellular processes such as respiration, transport and antibiotic resistance. As such key parameter, membrane fluidity is regulated to adapt to environmental variations and stresses like temperature fluctuations or osmotic shocks. Membrane fluidity has been however scarcely studied quantitatively in bacterial cells, mostly because of the lack of available tools. Here, we developed an assay based on total internal reflection fluorescence correlation spectroscopy (TIR-FCS) to directly measure membrane fluidity in live bacteria via the diffusivity of fluorescent membrane markers. We used this assay to quantify the fluidity of the cytoplasmic membrane of the Gram-positive model bacterium Bacillus subtilis in response to a cold shock, caused by a shift from 37{degrees}C to 20{degrees}C. In our experimental conditions, steady-state fluidity was recovered within 30 mins, and the steady-state fluidity at 20{degrees}C was about half of that at 37{degrees}C. Our minimally invasive assay opens up exciting perspectives and could be used to study a wide range of phenomena affecting the bacterial membrane, from disruption by antibiotics, antimicrobial peptides, or osmotic shocks. SignificanceUsing fluorescence correlation spectroscopy (FCS) with total internal reflection fluorescence (TIRF) illumination, we measured the diffusion speed of fluorescent membrane markers as a readout for membrane fluidity of growing B. subtilis cells. Quantification of the effect of cold shock provided unique information about the dynamics of the plasma membrane of B. subtilis. The unprecedented capability of TIR-FCS to quantify membrane fluidity in living bacteria opens the door to a whole set of new studies that will shed light on the bacterial plasma membrane and its interactions with the environment.

biophysics↗

bioRxiv2023

A new fluorescence-based approach for direct visualization of coat formation during sporulation in Bacillus cereus

The pathogenic bacteria Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis form spores encased in a protein coat surrounded by a balloon-like exosporium. These structures mediate spore interactions with its environment, including the host immune system, control the transit of molecules that trigger germination and thus are essential for the spore life cycle. Formation of the coat and exosporium has been traditionally visualized by transmission electronic microscopy on fixed cells. Recently, we showed that assembly of the exosporium can be directly observed in live B. cereus cells by super resolution-structured illumination microscopy (SR-SIM) using the membrane MitoTrackerGreen (MTG) dye. Here, we demonstrate that the different steps of coat formation can also be visualized by SR-SIM using MTG and SNAP-cell TMR-star dyes during B. cereus sporulation. We used these markers to characterize a subpopulation of engulfment-defective B. cereus cells that develops at a suboptimal sporulation temperature. Importantly, we predicted and confirmed that synthesis and accumulation of coat material, as well as synthesis of the {sigma}K-dependent protein BxpB, occur in cells arrested during engulfment. These results suggest that, unlike the well-studied model organism Bacillus subtilis, the activity of {sigma}K is not strictly linked to the state of forespore development in B. cereus.

microbiology↗

bioRxiv2023

Peptidoglycan remodeling in response to cell wall acting antibiotics in Bacillus subtilis

Most bacteria are encased into a load-bearing rigid framework, the cell wall (CW). The peptidoglycan (PG) layer, a network composed of glycan strands cross-linked by stem peptides, is the main component of the CW. During PG synthesis, precursors are first synthetized intracellularly, before being incorporated into the existing PG meshwork by transglycosylation (TG) and transpeptidation (TP) reactions. Covalent modifications of the PG meshwork such as amidation and acetylation participate in PG homeostasis by regulating PG-associated enzymes like PG hydrolases. Because of its essential role, PG synthesis represents a primary target for antibiotic action. Here, we investigated the effect on PG composition of antibiotics targeting intracellular and extracellular steps of PG synthesis: inhibitors of PG precursors synthesis (fosfomycin, D-cycloserine, bacitracin and tunicamycin) and TG/TP inhibitors (vancomycin and penicillin G), respectively. Our study revealed interesting correlations between crosslinking and both de-N-acetylation and amidation of the sacculus. A thorough analysis of muropeptides composition put into light an unexpected anti-correlation between the degree of PG crosslinking and accumulation of de-amidated disaccharide-tripeptide monomer subunit (M3) in the presence of TP inhibitors. We confirmed these observations by analyzing mutants of the PG synthesis pathway.

microbiology↗

bioRxiv2021

A high content microscopy screening identifies genes involved in cell width control in Bacillus subtilis

How cells control their shape and size is a fundamental question of biology. In most bacteria, cell shape is imposed by the peptidoglycan (PG) polymeric meshwork that surrounds the cell. Thus, bacterial cell morphogenesis results from the coordinated action of the proteins assembling and degrading the PG shell. Remarkably, during steady-state growth, most bacteria maintain a defined shape along generations, suggesting that error-proof mechanisms tightly control the process. In the rod-shaped model for Gram-positive bacteria Bacillus subtilis, the average cell length varies as a function of the growth rate but the cell diameter remains constant throughout the cell cycle and across growth conditions. Here, in an attempt to shed light on the cellular circuits controlling bacterial cell width, we developed a screen to identify genetic determinants of cell width in B. subtilis. Using high-content screening (HCS) fluorescence microscopy and semi-automated measurement of single-cell dimensions, we screened a library of ~ 4000 single knockout mutants. We identified 13 mutations significantly altering cell diameter, in genes that belong to several functional groups. In particular, our results indicate that metabolism plays a major role in cell width control in B. subtilis.

microbiology↗