Biology subjects

Lablaine, A.

Publications and source records attributed to Lablaine, A..

6 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↗

bioRxiv2024

Real-Time Nanoscale Investigation of Spore Coat Assembly in Bacillus subtilis

Spores of Bacillaceae, ubiquitous in soil, can withstand extreme conditions virtually indefinitely. The spore coat, one of the most sophisticated multiprotein complexes built by bacteria, protects the spore from environmental stresses and predators. In Bacillus subtilis, assembly of the multilayered coat initiates at the forespore pole and is perceived as a stable process that progresses continuously until covering the entire forespore surface. In contrast, in Bacillus cereus, coat formation initiates in the midspore region and extends outward to the spore poles. Using Structured Illumination Microscopy, we monitored B. subtilis coat development in real-time at the single-sporangium level with lateral resolution of 70 nm. We found that late-synthesized proteins from the innermost coat layers first assemble in the midspore region and are subsequently displaced towards the poles. This process is coupled with a unique redistribution of pre-assembled coat material across the forespore surface and influenced by outer coat development, highlighting a dynamic interplay in coat layers co-construction.

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

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↗

bioRxiv2020

The morphogenetic protein CotE drives exosporium formation by positioning CotY and ExsY during sporulation of Bacillus cereus

The exosporium is the outermost spore layer of some Bacillus and Clostridium species and related organisms. It mediates interactions of spores with their environment, modulates spore adhesion and germination and could be implicated in pathogenesis. The exosporium is composed of a crystalline basal layer, formed mainly by the two cysteine-rich proteins CotY and ExsY, and surrounded by a glycoprotein hairy nap. The morphogenetic protein CotE is necessary for Bacillus cereus exosporium integrity, but how CotE directs exosporium assembly remains unknown. Here, we followed the localization of SNAP-tagged CotE, -CotY and -ExsY during B. cereus sporulation, using super-resolution fluorescence microscopy and evidenced interactions among these proteins. CotE, CotY and ExsY are present as complexes at all sporulation stages and follow a similar localization pattern during endospore formation that is reminiscent of the localization of Bacillus subtilis CotE. We show that B. cereus CotE drives the formation of one cap at both forespore poles by positioning CotY and then guides forespore encasement by ExsY, thereby promoting exosporium elongation. By these two actions, CotE ensures the formation of a complete exosporium. Importantly, we demonstrate that the assembly of the exosporium is not a unidirectional process as previously proposed but it is performed through the formation of two caps, as observed during B. subtilis coat morphogenesis. It appears that a general principle governs the assembly of the spore surface layers of Bacillaceae. IMPORTANCESpores of Bacillaceae are enveloped in a glycoprotein outermost layer. In the B. cereus group, encompassing the B. anthracis and B. cereus pathogens, this layer is easily recognizable by a characteristic balloon-like appearance separated from the underlying coat by an interspace. In spite of its importance for the environmental interactions of spores, including those with host cells, the mechanism of assembly of the exosporium is poorly understood. We used super-resolution fluorescence microscopy to directly visualize formation of the exosporium during sporulation of B. cereus and we studied the localization and interactions of proteins essential for exosporium morphogenesis. We discovered that these proteins form a morphogenetic scaf-fold, before a complete exosporium or coat are detectable. We describe how the different proteins localize to the scaffold and how they subsequently assemble around the spore and we present a model for the assembly of the exosporium.

microbiology↗

bioRxiv2019

Multiple activities of the MatP protein are involved in post-replicative pairing of sister chromosomes in Escherichia coli

The ter region of the bacterial chromosome, where replication terminates, is the last to be segregated before cell division in Escherichia coli. Delayed segregation is controlled by the MatP protein, which binds to specific sites (matS) within ter, and interacts with other proteins such as ZapB. Here, we investigate the role of MatP by combining short-time mobility analyses of the ter locus with biochemical approaches. We find that ter mobility is similar to that of a non-ter locus, except when sister ter loci are paired after replication. This effect depends on MatP, the persistence of catenanes, and ZapB. We characterise MatP/DNA complexes and conclude that MatP binds DNA as a tetramer, but bridging matS sites in a DNA-rich environment remains infrequent. We propose that tetramerisation of MatP links matS sites with ZapB and/or with non-specific DNA to promote optimal pairing of sister ter regions until cell division.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗