Etude du recrutement de la nucléase EndA au sein du complexe de transport de l'ADN transformant chez Streptococcus pneumoniae

La transformation est un mécanisme largement répandu dans le monde bactérien permettant des échanges génétiques via la capture d'ADN exogène. Elle requiert généralement le développement d'un état physiologique transitoire, la compétence, au cours de laquelle sont néo-synthétisées les protéines essentielles à la capture de l'ADN du milieu extérieur, à sa traversée de la paroi cellulaire, à son internalisation dans le cytoplasme sous forme simple brin et à son intégration dans le génome par recombinaison homologue. Collectivement, ces protéines définissent l'appareil de transformation génétique, l'ADN Transformasome, dont la caractérisation est la plus avancée pour les bactéries modèles Streptococcus pneumoniae et Bacillus subtilis. Mon projet de thèse visait à caractériser la dynamique de mise en place de la partie du transformasome en charge du transport de l'ADN exogène au travers de la membrane cytoplasmique chez S. pneumoniae. Pour cela, j'ai principalement utilisé (et adapté au pneumocoque) des techniques de biologie cellulaire permettant de suivre par épifluorescence l'assemblage de ce pore d'entrée de l'ADN transformant dans des cellules vivantes. Ainsi, j'ai pu mettre en évidence un retard du processus de septation cellulaire lors du premier événement de division qui suit l'induction de la compétence. Pour localiser le pore d'entrée du transformasome, j'ai étudié la localisation de l'endonucléase membranaire EndA, en charge de la dégradation à l'extérieur de la cellule du brin complémentaire de l'ADN transformant internalisé. Contrairement à toutes les autres protéines du Transformasome, EndA est exprimée de manière constitutive. Mes résultats montrent que, d'une part, EndA localise de manière homogène dans la membrane des cellules non compétentes et, d'autre part, forme des foci dans les cellules compétentes. La formation des foci d'EndA est dépendante de la protéine membranaire ComEA du Transformasome, dont le rôle est de recevoir l'ADN double-brin exogène au niveau du pore pore d'entrée. Lorsque la capacité de transformation est maximale, EndA et ComEA sont préférentiellement localisées au niveau de la zone équatoriale des cellules. Par ailleurs, j'ai pu montrer que l'ADN transformant marqué par des fluorophores se concentre aussi principalement à la zone équatoriale des cellules compétentes. Dans l'ensemble, ce travail suggère que l'assemblage du pore d'entrée de l'ADN transformant chez S. pneumoniae se ferait à la zone équatoriale des cellules compétentes. Chez B. subtilis il a été montré que la fixation de l'ADN a lieu aux pôles des cellules compétentes. Ainsi, bien que les composantes des transformasomes de B. subtilis et de S. pneumoniae soient très conservées, il semble qu'une (ou des) caractéristique(s) propre(s) à ces deux espèces bactériennes déterminent le site d'assemblage du pore d'entrée de l'ADN transformasome dans la membrane.Natural genetic transformation is widely distributed in bacteria, allowing genetics exchange. Transformation requires a specialized membrane-associated complex which forms a DNA entry pore allowing internalization of exogenous single-stranded DNA (ssDNA). It also requires dedicated cytosolic proteins to integrate internalised ssDNA into the chromosome by homologous recombination. This complex, named DNA Transformasome, has been intensively studied in the human pathogen S. pneumoniae and the soil bacteria B. subtilis. The aim of my project was to characterize the membrane-associated complex in S. pneumoniae. I adapted two complementary cellular biology methods. To identify physical links between Transformasome components, I developed a method to purify membrane complexes in S. pneumoniae. Moreover, I performed fluorescence microscopy, which allows us to track and record individual bacterial cells of S. pneumoniae upon competence induction. Using GFP (Green Fluorescent Protein) fused to a key division protein FtsZ, I demonstrated a delayed septation process in competent cells. To visualize the integration of components of the DNA entry pore, I focused on EndA. EndA is a sequence non-specific endonuclease bound to the membrane which is responsible for processing double-stranded DNA into single-stranded fragments that in turn enter the microbial cell. This nuclease has been identified only in the streptococcal system. Remarkably, EndA is the only known component of the DNA entry pore that is constitutively expressed in non-competent cells and is not induced in competence. I showed that EndA concentrates into localized foci upon competence induction. This specific localization depends on the presence of the DNA-receptor, ComEA. Moreover, EndA and ComEA appear preferentially located at midcell in cultures exhibiting optimal transformation efficiency. Similarly, binding of extracellular DNA was localized to the cell centre. Together, our results are consistent with a model in which the active entry pore for DNA transformation is located at the septum in pneumococcal cells. In contrast, it appears that the entire process of DNA transformation localizes at the cell poles in B. subtilis. Although components of Transformasome are conserved between species, it appears that bacteria have adapted the transformation process according to their own physiology

Streptococcus pneumoniae, le transformiste

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Genus-Specific Interactions of Bacterial Chromosome Segregation Machinery Are Critical for Their Function

Most bacteria use the ParABS system to segregate their newly replicated chromosomes. The two protein components of this system from various bacterial species share their biochemical properties: ParB is a CTPase that binds specific centromere-like parS sequences to assemble a nucleoprotein complex, while the ParA ATPase forms a dimer that binds DNA non-specifically and interacts with ParB complexes. The ParA-ParB interaction incites the movement of ParB complexes toward the opposite cell poles. However, apart from their function in chromosome segregation, both ParAB may engage in genus-specific interactions with other protein partners. One such example is the polar-growth controlling protein DivIVA in Actinomycetota, which binds ParA in Mycobacteria while interacts with ParB in Corynebacteria. Here, we used heterologous hosts to investigate whether the interactions between DivIVA and ParA or ParB are maintained across phylogenic classes. Specifically, we examined interactions of proteins from four bacterial species, two belonging to the Gram positive Actinomycetota phylum and two belonging to the Gram-negative Pseudomonadota. We show that while the interactions between ParA and ParB are preserved for closely related orthologs, the interactions with polarly localised protein partners are not conferred by orthologous ParABs. Moreover, we demonstrate that heterologous ParA cannot substitute for endogenous ParA, despite their high sequence similarity. Therefore, we conclude that ParA orthologs are fine-tuned to interact with their partners, especially their interactions with polarly localised proteins are adjusted to particular bacterial species demands

Smartphone-Based 3D Navigation Technique for Use in a Museum Exhibit

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Modularity and determinants of a (bi-)polarization control system from free-living and obligate intracellular bacteria

Although free-living and obligate intracellular bacteria are both polarized it is unclear whether the underlying polarization mechanisms and effector proteins are conserved. Here we dissect at the cytological, functional and structural level a conserved polarization module from the free living α-proteobacterium Caulobacter crescentus and an orthologous system from an obligate intracellular (rickettsial) pathogen. The NMR solution structure of the zinc-finger (ZnR) domain from the bifunctional and bipolar ZitP pilus assembly/motility regulator revealed conserved interaction determinants for PopZ, a bipolar matrix protein that anchors the ParB centromere-binding protein and other regulatory factors at the poles. We show that ZitP regulates cytokinesis and the localization of ParB and PopZ, targeting PopZ independently of the previously known binding sites for its client proteins. Through heterologous localization assays with rickettsial ZitP and PopZ orthologs, we document the shared ancestries, activities and structural determinants of a (bi-)polarization system encoded in free-living and obligate intracellular α-proteobacteria.ISSN:2050-084

A programmed cell division delay preserves genome integrity during natural genetic transformation in Streptococcus pneumoniae

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Midcell Recruitment of the DNA Uptake and Virulence Nuclease, EndA, for Pneumococcal Transformation

<div><p>Genetic transformation, in which cells internalize exogenous DNA and integrate it into their chromosome, is widespread in the bacterial kingdom. It involves a specialized membrane-associated machinery for binding double-stranded (ds) DNA and uptake of single-stranded (ss) fragments. In the human pathogen <i>Streptococcus pneumoniae</i>, this machinery is specifically assembled at competence. The EndA nuclease, a constitutively expressed virulence factor, is recruited during competence to play the key role of converting dsDNA into ssDNA for uptake. Here we use fluorescence microscopy to show that EndA is uniformly distributed in the membrane of noncompetent cells and relocalizes at midcell during competence. This recruitment requires the dsDNA receptor ComEA. We also show that under ‘static’ binding conditions, i.e., in cells impaired for uptake, EndA and ComEA colocalize at midcell, together with fluorescent end-labelled dsDNA (Cy3-dsDNA). We conclude that midcell clustering of EndA reflects its recruitment to the DNA uptake machinery rather than its sequestration away from this machinery to protect transforming DNA from extensive degradation. In contrast, a fraction of ComEA molecules were located at cell poles post-competence, suggesting the pole as the site of degradation of the dsDNA receptor. In uptake-proficient cells, we used Cy3-dsDNA molecules enabling expression of a GFP fusion upon chromosomal integration to identify transformed cells as GFP producers 60–70 min after initial contact between DNA and competent cells. Recording of images since initial cell-DNA contact allowed us to look back to the uptake period for these transformed cells. Cy3-DNA foci were thus detected at the cell surface 10–11 min post-initial contact, all exclusively found at midcell, strongly suggesting that active uptake of transforming DNA takes place at this position in pneumococci. We discuss how midcell uptake could influence homology search, and the likelihood that midcell uptake is characteristic of cocci and/or the growth phase-dependency of competence.</p></div

Respiratory tissue-associated commensal bacteria offer therapeutic potential against pneumococcal colonization.

Under eubiotic conditions commensal microbes are known to provide a competitive barrier against invading bacterial pathogens in the intestinal tract, on the skin or on the vaginal mucosa. Here, we evaluate the role of lung microbiota in Pneumococcus colonization of the lungs. In eubiosis, the lungs of mice were dominantly colonized by Lactobacillus murinus. Differential analysis of 16S rRNA gene sequencing or L. murinus-specific qPCR of DNA from total organ homogenates vs.broncho alveolar lavages implicated tight association of these bacteria with the host tissue. Pure L. murinus conditioned culture medium inhibited growth and reduced the extension of pneumococcal chains. Growth inhibition in vitro was likely dependent on L. murinus-produced lactic acid, since pH neutralization of the conditioned medium aborted the antibacterial effect. Finally, we demonstrate that L. murinus provides a barrier against pneumococcal colonization in a respiratory dysbiosis model after an influenza A virus infection, when added therapeutically

Localization and stability of GFP-ComEA.

<p>(<b>A</b>) Transformation proficiency (open diamonds), fraction of cells containing GFP-ComEA foci and focus position (histograms). See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003596#ppat-1003596-g001" target="_blank">Figure 1D</a> legend for details. (<b>B</b>) GFP-ComEA stability. Western-blot analysis of R2940 (<i>gfp-comEA</i>) extracts used anti-GFP antibodies. FL, full length fusion protein; asterisks, degradation products. (<b>C</b>) Persistence of Cy3-DNA fluorescence signal post-competence. Images of R3606 (<i>gfp-comEA endA</i>⁻) cells 60 min after addition of CSP and 285 bp Cy3-DNA. Yellow arrows point to polar DNA molecules. (<b>D</b>) Stabilizing effect of persistent DNA on GFP-ComEA protein. Western-blot analysis of R3606 (<i>gfp-comEA</i>, <i>endA⁻</i>) cells incubated with or without R304 chromosomal DNA. Samples were collected at the indicated time points (see legend of panel B for details).</p