Eukaryotic-like gephyrin and cognate membrane receptor coordinate corynebacterial cell division and polar elongation

The order Corynebacteriales includes major industrial and pathogenic Actinobacteria such as Corynebacterium glutamicum or Mycobacterium tuberculosis. These bacteria have multi-layered cell walls composed of the mycolyl-arabinogalactan-peptidoglycan complex and a polar growth mode, thus requiring tight coordination between the septal divisome, organized around the tubulin-like protein FtsZ, and the polar elongasome, assembled around the coiled-coil protein Wag31. Here, using C. glutamicum, we report the discovery of two divisome members: a gephyrin-like repurposed molybdotransferase (Glp) and its membrane receptor (GlpR). Our results show how cell cycle progression requires interplay between Glp/GlpR, FtsZ and Wag31, showcasing a crucial crosstalk between the divisome and elongasome machineries that might be targeted for anti-mycobacterial drug discovery. Further, our work reveals that Corynebacteriales have evolved a protein scaffold to control cell division and morphogenesis, similar to the gephyrin/GlyR system that mediates synaptic signalling in higher eukaryotes through network organization of membrane receptors and the microtubule cytoskeleton.Agencia Nacional de Investigación e InnovaciónECOS-Sud France-Uruguay U20B02FOCEM-COF 03/11Agence Nationale de la Recherche ANR-18-CE11-0017/ANR-21-CE11-000

A Bacterial Ras-Like Small GTP-Binding Protein and Its Cognate GAP Establish a Dynamic Spatial Polarity Axis to Control Directed Motility

Directional control of bacterial motility is regulated by dynamic polarity inversions driven by pole-to-pole oscillation of a Ras family small G-protein and its associated GTPase-activating protein

Emergence and Modular Evolution of a Novel Motility Machinery in Bacteria

Bacteria glide across solid surfaces by mechanisms that have remained largely mysterious despite decades of research. In the deltaproteobacterium Myxococcus xanthus, this locomotion allows the formation stress-resistant fruiting bodies where sporulation takes place. However, despite the large number of genes identified as important for gliding, no specific machinery has been identified so far, hampering in-depth investigations. Based on the premise that components of the gliding machinery must have co-evolved and encode both envelope-spanning proteins and a molecular motor, we re-annotated known gliding motility genes and examined their taxonomic distribution, genomic localization, and phylogeny. We successfully delineated three functionally related genetic clusters, which we proved experimentally carry genes encoding the basal gliding machinery in M. xanthus, using genetic and localization techniques. For the first time, this study identifies structural gliding motility genes in the Myxobacteria and opens new perspectives to study the motility mechanism. Furthermore, phylogenomics provide insight into how this machinery emerged from an ancestral conserved core of genes of unknown function that evolved to gliding by the recruitment of functional modules in Myxococcales. Surprisingly, this motility machinery appears to be highly related to a sporulation system, underscoring unsuspected common mechanisms in these apparently distinct morphogenic phenomena

Adaptation and Preadaptation of Salmonella enterica to Bile

Bile possesses antibacterial activity because bile salts disrupt membranes, denature proteins, and damage DNA. This study describes mechanisms employed by the bacterium Salmonella enterica to survive bile. Sublethal concentrations of the bile salt sodium deoxycholate (DOC) adapt Salmonella to survive lethal concentrations of bile. Adaptation seems to be associated to multiple changes in gene expression, which include upregulation of the RpoS-dependent general stress response and other stress responses. The crucial role of the general stress response in adaptation to bile is supported by the observation that RpoS− mutants are bile-sensitive. While adaptation to bile involves a response by the bacterial population, individual cells can become bile-resistant without adaptation: plating of a non-adapted S. enterica culture on medium containing a lethal concentration of bile yields bile-resistant colonies at frequencies between 10−6 and 10−7 per cell and generation. Fluctuation analysis indicates that such colonies derive from bile-resistant cells present in the previous culture. A fraction of such isolates are stable, indicating that bile resistance can be acquired by mutation. Full genome sequencing of bile-resistant mutants shows that alteration of the lipopolysaccharide transport machinery is a frequent cause of mutational bile resistance. However, selection on lethal concentrations of bile also provides bile-resistant isolates that are not mutants. We propose that such isolates derive from rare cells whose physiological state permitted survival upon encountering bile. This view is supported by single cell analysis of gene expression using a microscope fluidic system: batch cultures of Salmonella contain cells that activate stress response genes in the absence of DOC. This phenomenon underscores the existence of phenotypic heterogeneity in clonal populations of bacteria and may illustrate the adaptive value of gene expression fluctuations

A Microscope Automated Fluidic System to Study Bacterial Processes in Real Time

Most time lapse microscopy experiments studying bacterial processes ie growth, progression through the cell cycle and motility have been performed on thin nutrient agar pads. An important limitation of this approach is that dynamic perturbations of the experimental conditions cannot be easily performed. In eukaryotic cell biology, fluidic approaches have been largely used to study the impact of rapid environmental perturbations on live cells and in real time. However, all these approaches are not easily applicable to bacterial cells because the substrata are in all cases specific and also because microfluidics nanotechnology requires a complex lithography for the study of micrometer sized bacterial cells. In fact, in many cases agar is the experimental solid substratum on which bacteria can move or even grow. For these reasons, we designed a novel hybrid micro fluidic device that combines a thin agar pad and a custom flow chamber. By studying several examples, we show that this system allows real time analysis of a broad array of biological processes such as growth, development and motility. Thus, the flow chamber system will be an essential tool to study any process that take place on an agar surface at the single cell level

Rules Governing Selective Protein Carbonylation

BACKGROUND:Carbonyl derivatives are mainly formed by direct metal-catalysed oxidation (MCO) attacks on the amino-acid side chains of proline, arginine, lysine and threonine residues. For reasons unknown, only some proteins are prone to carbonylation. METHODOLOGY/PRINCIPAL FINDINGS:we used mass spectrometry analysis to identify carbonylated sites in: BSA that had undergone in vitro MCO, and 23 carbonylated proteins in Escherichia coli. The presence of a carbonylated site rendered the neighbouring carbonylatable site more prone to carbonylation. Most carbonylated sites were present within hot spots of carbonylation. These observations led us to suggest rules for identifying sites more prone to carbonylation. We used these rules to design an in silico model (available at http://www.lcb.cnrs-mrs.fr/CSPD/), allowing an effective and accurate prediction of sites and of proteins more prone to carbonylation in the E. coli proteome. CONCLUSIONS/SIGNIFICANCE:We observed that proteins evolve to either selectively maintain or lose predicted hot spots of carbonylation depending on their biological function. As our predictive model also allows efficient detection of carbonylated proteins in Bacillus subtilis, we believe that our model may be extended to direct MCO attacks in all organisms

Viabilité et cultivabilité de L. pneumophila (étude des mécanismes impliqués dans la récupération de l'aptitude à former des colonies)

Legionella pneumophila est une bactérie pathogène responsable de la légionellose qui se transmet par voies aériennes à partir de sites industriels ou naturels générateurs d'aérosols (installation d'eau chaude sanitaire, tours aéroréfrigérantes, ...). Actuellement le dénombrement et l'isolement de L. pneumophila sont régis par une norme basée sur l'utilisation de culture gélosés. Cependant, comme de très nombreuses bactéries Gram négatif, Legionella pneumophila serait capable de passer, en condition de carence ou de stress, dans un état viable mais non cultivable (VBNC). Au cours de cette étude, nou avons montré que les différents traitements biocides classiquement utilisés pour éradiquer L. pneumophila dans les installations industrielles, induisaient la formation de cellules VBNC, déterminées à travers l'utilisation de marqueurs de viabilité et du milieu de culture de référence. Cependant, l'analyse fine des marqueurs de viabilité estimée pour chaque cellule viable détectée montre que celles-ci présentent une altération progressive des différents descripteurs de viabilité utilisés à mesure que la concentration en biocide augmente. En supposant dans un premier temps, que la perte de cultivabilité des cellules VBNC soit potentiellement due au stress oxydant généré lors que l'étalement, nous avons cherché à optimiser le milieu de référence. Au cours de cette étude de nombreux composés (antioxudants, métaboliques...) ont ainsi été identifiés comme étant bénéfiques à la restauration de la cultivabilité d'une fonction de la population après un stress mais aussi, et de manière intéressante, au cours de la croissance. La co-existence de deux sous population cultivables (sur le milieu de référence ou supplémenté) dont la proportion relative évolue au cours du temps, soulève donc à ce jour, un certain nombre de questions quant à l'origine et à la pertinence physiologique de chacune des deux populations. Pour des raisons techniques, ces questions ne pourrons être résolues qu'à travers l'utilisation de méthodes centrées sur l'individu et non plus sur la réponse globale d'une population. Dans ce sens, nous avons initié le développement d'une chambre d'observation et d'un système de fluidique qui permet aujourd'hui un suivi en temps réel de la viabilité des cellules observé et de leur devenir au cours du temps ou au cours d'un stress tout en observant in fine leur capacité respective à former une colonieLegionella pneumophila is the causative agent of Legionellosis transmitted by air from industrial or natural aerosols (installation of hot water, cooling towers ...) Currently standard procedure that uses the agar cultur media governs the detection and isolation of L. Pneumophila. However, like many Gram-negative bacteria, Legionella pneumophila is able to enter in a viable state but non culturable (VBNC) during starvation or stress conditions. In this study, we showed that the different treatment biocides traditionally used to eradicate. L. pneumophila in industrial plants, induced the formation of VBNC cells, determined using viability markers and reference agar medium (BCYE). However, detailled analysis of the viability markers shows that they have a progressive detetioration of the viability of different descriptors used as the biocide concentration increases. Assuming initially that the loss of culturability of VBNC cells is potentially due to oxidative stress generated during spreading, we sought to optimize the reference medium. During the study many compounds (antioxidants, metabolic ...) have been identified as benefical to the restoration of culturability for a fraction of the population after stress but also, interestingly, during growth. The co-existence of two populations (culturable on the reference medium or the supplemented medium) with the relative proportion changes over time, raises to date, a number of questions about the origin and physiological relevance of each of the two populations. For technical reasons, these issues can be resolved only with methods focused on the individual rather than on the overall response of a population. In this sense, we initiated the development of an observation chamber and a fluidic system that now allows as realtime monitoring of cell viability observed and their evolution over time or during a stress while observing ultimately their respective ability to form a colonyAIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Recent progress in our understanding of peptidoglycan assembly in Firmicutes

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CO2 exacerbates oxygen toxicity

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