The project aimed to study the complex regulatory network involving the secondary messenger cyclic di-GMP and its effectors, diguanylate cyclases (DGCs) and the central regulator of chemotaxis CheY. The latter plays a dual role in the aquatic bacterium Shewanella oneidensis, controlling chemotaxis (classical) and biofilm biogenesis (unexpected). The project also aimed to develop deep-learning approaches to better define the players of c-di-GMP (synthesis by DGCs, degradation by phosphodiesterases (PDEs), and detection by effectors) and to reclassify the CheY and CheY-like proteins. It was structured around 3 main tasks : i) Generation of a database grouping DGCs, PDEs and c-di-GMP effectors; ii) Characterization of the interactions of CheY with its DGC and c-di-GMP effector partners; iii) Classification of CheY and CheY-like proteins and definition of a signature specific to CheY proteins presenting, like CheY of S. oneidensis, a dual role in chemotaxis and in another process (biofilm, or other).

Résumé:

About 1.8 Ga ago, cyanobacteria were phagocyted by eukaryotes, leading to primary endosymbiosis and giving rise to chloroplast. Since then, other eukaryotes have included photosynthetic eukaryotes (secondary/tertiary endosymbiosis), which appear to have evolved many times independently. Despite the ubiquity of photo-endosymbiosis in the environment, these early mechanisms remain widely unknown. The aim is to study the first steps leading to the maintenance of a photosynthetic organism within a eukaryote. To this end, we are using unicellular models, the host ciliate Tetrahymena thermophila and the photosynthetic alga Chlorella variabilis and the cyanobacterium Synechococcus elongatus. These models allowed us to study the selective advantage that phagocytosis of a photosynthetic organism can bring to its host under unfavorable conditions. The results show the importance of O₂ for a better survival of T. thermophila having phagocytosed photosynthetic organisms in anaerobic conditions.

Mots clés:

 Photosymbiosis, evolution, oxygen, phagocytosis.

Résumé:

Filamentous phages are viruses that behave like bacterial symbionts that are an underestimated component of microbiota. They notably improve antibiotic resistance and virulence in human and plant pathogens. Yet, the infection process remains poorly understood at the molecular level, and the dynamic parameters of the process are unknown, as real-time visualization of viral infection was not yet available. In this project, we combined our expertise in the biology of filamentous phages and single cell fluorescent microscopy to decipher the dynamic of the infection process for the fd/E. coli couple. We validated individual fluorescent labeling strategies on the different macromolecular structures involved : the phage capsid, the pilus and the phage DNA and started the monitoring their dynamics during filamentous phage infection at the cell level. We expect these tools to be helpful for structural and biochemical characterization of key infection steps, and to open up prospects for future studies of other filamentous phages associated with pathogens.

Mots clés:

Filamentous phages, infection, dynamics, single cell microscopy

Résumé:

Magnetotactic bacteria (MTBs) are microaerophilic aquatic bacteria that can swim (using their flagella) toward their optimal development zone, the oxic-anoxic transition zone (OATZ). For that, they are helped by magnetotaxis (detection of the Earth's magnetic field) and chemotaxis (detection of the oxygen gradient). These three components (magnetotaxis, chemotaxis, and swimming) define an unique property of MTBs: the magneto-aerotaxis. We demonstrated that the flagellin (the protein that forms the flagellar filament) of MTBs is glycosylated, and that this glycosylation is crucial for the set-up of the filament and therefore for the motility. The synergy of our respective expertise in bacterial genetics, protein crystallography, and glycobiology bioinformatics has allowed us to identify three new types of bacterial glycosyltransferases found in MTBs, as well as in many other bacterial species, and to demonstrate their essential roles in the mechanism of forming a functional flagellum.

Mots clés: 

Magnetotactic bacteria, motility, flagella structure, glycosylation, glycosyltransferases

Résumé:

Cancer is a major health problem and important efforts are devoted to find out novel and effective treatments. Despite the progresses achieved in targeted- and immune-therapies, reality shows that in a clinical setting at least one potent cytotoxic compound is required in the therapeutic regimen. Metal-based chemotherapeutics (e.g. cisplatin, carboplatin and oxaliplatin) have shown high clinical relevance becoming first-line treatment for several cancer types. However, the non-physiological nature of the metal often leads to undesired side-effects. This situation has awaked a quest for the development of transition biometal-based therapeutics.

In this project we are developing cytotoxic redox-active Cu complexes and deciphering their location and mechanisms of action inside cells to improve their therapeutic efficiency.

Résumé:

This work aims to understand how the CO₂ fixation is driven by the reducing power, in bacteria assimilating carbon by the reverse Krebs cycle. This cycle is powered by low potential ferredoxins, which are reduced by energy sources with a higher redox potential. Deciphering how carbon fixation is linked to energy metabolism in Aquifex aeolicus, an hyperthermophilic bacterium, and identifying enzymes that can bypass this thermodynamic bias was the objective of this project. Using biochemistry, we identified a putative enzyme that uses the electron bifurcation mechanism to reduce low potential ferredoxins from NADPH. This work has made also possible to undertake the essential task of developing the CrispR-Cas9 tool in a hyperthermophilic bacterium which is particularly exciting. This will be used to study the role of genes in vivo not only as part of this project but also to open up the field of possibilities for research on this organism in general.

Mots clés:

Bacteria, Carbon assimilation, Energy metabolism, Low-potential Ferredoxin, CrispR-Cas9