Biology subjects

Carballido-Lopez, R.

Publications and source records attributed to Carballido-Lopez, R..

4 recordsLinked to original sources

bioRxiv2023

Comprehensive and Comparative Transcriptional Profiling of the Cell Wall Stress Response in Bacillus subtilis

The bacterial cell wall (CW) is an essential protective barrier and the frontline of cellular interactions with the environment and also a target for numerous antimicrobial agents. Accordingly, its integrity and homeostasis are closely monitored and rapid adaptive responses by transcriptional reprogramming induce appropriate counter-measures against perturbations. Here, we report a comprehensive and comparative transcriptional profiling of the primary cell envelope stress responses (CESR), based on combining RNAseq and high-resolution tiling array studies of the Gram-positive model bacterium Bacillus subtilis exposed to a range of antimicrobial compounds that interfere with cytoplasmic, membrane-coupled or extracellular steps of peptidoglycan (PG) biosynthesis. It revealed the complexity of the CESR of B. subtilis and unraveled the contribution of extracytoplasmic function sigma factors (ECFs) and two-component signal transduction systems (TCSs) to protect the cell envelope. While membrane-anchored steps are tightly controlled, early cytoplasmic and late extracellular steps of PG biosynthesis are hardly monitored at all. The ECF {sigma} factors {sigma}W and particularly {sigma}M provide a general CESR, while {sigma}V is almost exclusively induced by lysozyme, against which it provides specific resistance. Remarkably, {sigma}X was slightly repressed by most antibiotics, pointing towards a role in envelope homeostasis rather than CESR. It shares this role with the WalRK TCS, which balances CW growth with controlled autolysis. In contrast, all remaining TCSs are envelope stress-inducible systems. LiaRS is induced by a wide range of PG synthesis inhibitors, while the three paralogous systems BceAB, PsdRS and ApeRS are more compound-specific detoxification modules. Induction of the CssRS TCS by all antibiotics interfering with membrane-anchored steps of PG biosynthesis points towards a physiological link between CESR and secretion stress. Based on the expression signatures, a suite of CESR-specific B. subtilis whole cell biosensors were developed and carefully evaluated. This is the first comprehensive transcriptomic study focusing exclusively on the primary effects of envelope perturbances that shall provide a reference point for future studies on Gram-positive CESR.

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↗

bioRxiv2022

Polymerization cycle of actin homolog MreB from a Gram-positive bacterium

In most rod-shaped bacteria, the actin homologue MreB is an essential component of the protein complex effecting cell wall elongation. The polymerization cycle and filament properties of eukaryotic actin have studied for decades and are well characterized. However, purification and in vitro work on MreB proteins have proven very difficult. Current knowledge of MreB biochemical and polymerization properties remains limited and is based on MreB proteins from Gram-negative species. In this study, we report the first observation of organized filaments and the first 3D-structure of MreB from a Gram-positive bacterium. We have purified MreB from the thermophilic Geobacillus stearothermophilus and shown that it forms straight pairs of protofilaments in vitro, and that polymerization depends on the presence of both lipids and nucleotide triphosphate. Two spatially close short hydrophobic sequences mediate membrane anchoring. Importantly, we demonstrate that unlike eukaryotic actin, nucleotide hydrolysis is a prerequisite for MreB interaction with the membrane, and that binding to lipids then triggers polymerization. Based on our results, we propose a molecular model for the mechanism of MreB polymerization.

biochemistry↗

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↗