Abstract:

Halorhodospira halophila is a strict anaerobe and obligate phototroph using sulfur compounds oxidation as electron donors to supply its anoxygenic photosynthesis. Very little information on its photosynthetic pathway is currently available. The previous works propose a metabolic network with a high degree of "redundancy", with multiple soluble electron carriers, multiple soluble and membranous enzymes, funnelling the central photosynthetic reaction Centre (RC) in electrons. One of the questions is therefore the respective role of the different electron carriers and enzymes. The previous works also suggest unusual properties, in H. halophila, for three enzymes: the RC, the Sulfur OXidation system SOX and the alternative arsenite oxidase Arx. The very low redox potential of the first one raises the question of its function, the possible role of the second one in oxidation of thioarsenate would make it unique while the inability of the third one to sustain photosynthetic growth under arsenite is questioning. The PhD project aims to address these points in a multidisciplinary approach combining biochemistry, molecular biology, and light induced/UV-Vis/EPR spectroscopies for both structural and functional studies.

Co-supervisors :

• Jean Alric (BIAM)
• Barbara Schoepp-Cothenet (BIP)

Abstract:

The photosynthetic purple bacterium Rubrivivax (R.) gelatinosus possesses an original copper resistance system thanks to an operon encoding 3 periplasmic proteins: CopI, CopJ and CopH. These proteins have homologs in other environmental as well as pathogenic bacteria, and some of these bacteria lack systems such as Cu oxidase or the Cus export complex. In order to gain a better understanding of this mechanism, and to complement the in vitro studies carried out at BIP, it is important to carry out in vivo studies as well. In this context, proteomic studies are ideally suited to determine which part of the R. gelatinosus proteome is affected by copper or other metal stress. However, difficulties can be encountered in detecting certain expressed proteins. This is particularly true of the CopH protein, whose sequence has very few trypsin cleavage sites (K (0), R (3), on 97 AA) and the only R residues are located at the end of the sequence, generating only 1 trypsin peptide that can be analyzed under conventional conditions.
In order to improve this, we have established new protocols using trypsin/chymotrypsin to digest CopH in peptides identifiable by LC- MS/MS. This biochemical method combined with targeted proteomics experiments allowed a significant improvement in the detection of CopH. We applied those methods to study the effect of a Cu stress in vivo on the whole proteome of R. gelatinosus under photosynthetic or microaerobic conditions. Overexpression of several proteins is detected under Cu stress including all those of the Cop operon except CopH. The results obtained led us to hypothezise that either CopH is used as a primary barrier against Cu and is then degraded or that it is exported outside of the cell. Further analyses of the global proteomic experiments in vivo are underway to help deciphering the Cu resistance mechanism of R. gelatinosus.

Co-supervisors :

• Régine LEBRUN (IMM)
• Pierre DORLET (BIP)

Abstract:

Lipid droplets (LDs) are dynamic organelles that play a pivotal role in the cellular response to environmental stress by facilitating energy storage and lipid metabolism. In Chlamydomonas reinhardtii, the degradation of triacylglycerols (TAGs) stored in LDs during nitrogen (N) resupply represents a critical metabolic switch, yet the mechanisms driving LD remodelling and interaction with other organelles remain poorly understood. The objective of this study was to elucidate the role of the DGTT3 gene, which encodes a type-2 diacylglycerol acyltransferase, in TAG resynthesis during LD turnover. In addition, the study sought to developing imaging and microfluidic tools to study the dynamics of LD degradation in real time. We conducted a study to investigate the physiological characteristics of dgtt3 knock-out mutants (dgtt3-1 and dgtt3-2). Our findings revealed that dgtt3-1 exhibited a greater accumulation of larger lipid droplets (LDs) during the process of triacylglycerol (TAG) remobilisation. In addition, dgtt3-1 displayed a divergent phenotype compared to dgtt3-2, suggesting the presence of alternative metabolic pathways that may contribute to these observed differences. Fluorescence microscopy in conjunction with BODIPY staining, in addition to quantitative image analysis employing an ImageJ macro, demonstrated a correlation between the number of LDs per cell and the TAG content quantitated by HP-TLC. However, this analysis did not reveal any discernible indications of the micro-LDs phenotype. It will be necessary to generate our own knock-out mutant to investigate the real impact of knock out, given the lack of consistency between the two mutants. In order to further explore organelle interactions during LD breakdown, reporter constructs targeting peroxisomes (GFY5) and mitochondria (CAG1), fused with fluorescent markers, were developed. Despite the initial transformation success being low due to challenges with large plasmid size and random genomic insertion, optimisation efforts, including testing in the UVM4 strain known to be efficient in expressing transgenes, are ongoing. A novel microfluidic setup was initiated. This setup was based on a design previously described by literature to enable long-term imaging of single cells. While preliminary results were limited by dye sensitivity and PDMS absorption, this platform still needs optimization. However, it showed promise for future real-time observation of lipid remodelling.

Supervisor :

• Marie BERTRAND (BIAM)

Abstract:

Bacteria have evolved numerous specialized multiprotein membrane nanomachines to interact with their environment. Among them, Type IV Filaments (TFF) superfamily has emerged as the "Swiss Army knife" of surface molecular machines, capable of mediating an array of cellular functions, including protein secretion, DNA uptake, host-cell adhesion, biofilm formation, prey killing, sensing and various forms of motility.

TFF are ubiquitous nanomachines essential for bacterial survival, however the mechanism triggering active pili synthesis remains unknown. Using a combination of in vivo single-cell analyses and high-resolution NMR spectroscopy, we showed that PilM (a cytoplasmic component of the machinery) consists of a molecular gate requiring a lock and key mechanism to insert the PilB extension motor and activate TFF machineries.

Using two evolutionary distant model systems, Myxococcus xanthus and Vibrio cholerae, we demonstrated that distinct activators, respectively the SgmX protein and the secondary messenger c-di-GMP, both act on the same binding interface situated in the N1D subdomain of PilB to facilitate its interaction with PilM.

Our findings reveal a conserved regulatory hub for TFF machineries, explaining how these nanomachines were co-opted into diverse cellular processes across evolution.

Co-supervisors :

• Romain MERCIER (LCB)
• Latifa ELANTAK (LISM)

Summary:

The development of renewable energy sources is of paramount importance in meeting the energy challenge of the ^21st century. Molecular hydrogen's ability to store and transport energy makes it an ideal energy carrier. It is at the heart of research aimed at replacing fossil fuels with responsible, environmentally-friendly alternatives. However, its production method generates significant carbon dioxide emissions, hindering its large-scale integration. A more environmentally-friendly alternative would be to produce it by electrolysis of water, although this method remains costly due to the materials used, which are generally platinum- and iridium-based. Current research is aimed at replacing these expensive noble metals with affordable, sustainable alternatives. The aim of this thesis was to develop molecular catalysts based on non-noble and abundant metals for hydrogen production under mild environmental conditions. The approach taken was to draw inspiration from hydrogenases, biological systems capable of reversibly converting hydrogen, to effectively reproduce natural processes in artificial systems.

Thesis work thus focused on the design of original bio-inspired catalysts, based on non-innocent thiosemicarbazone ligands, with the aim of understanding the impact of their structure on catalytic properties. By playing on the nature and position of the substituents decorating the thiosemicarbazone ligand, several metal complexes, based on nickel and cobalt, were prepared and evaluated for their ability to promote the electrocatalytic reduction of protons to hydrogen. Our studies were also extended to the exploration of different experimental conditions, including the choice of proton source and solvent, with the aim of identifying the most favorable parameters for hydrogen production. This work has enabled us to gain a better understanding of the relationship between molecular structure and catalytic activity, and to identify promising combinations for the development of high-performance molecular catalysts for the electrocatalytic conversion of protons into hydrogen. Our results pave the way for the integration of these catalysts in solid matrices to improve their performance, stability and durability in electrocatalytic hydrogen production.

Co-supervisors :

• Maylis ORIO (iSm2)
• Renaud HARDRE (iSm2)

Abstract:

Apoptosis is a tightly regulated form of programmed cell death essential for development and tissue homeostasis. A key event in this process is the permeabilization of the mitochondrial outer membrane, driven by the pro-apoptotic BCL-2 family of proteins; Bax and Bak. VDAC, the major channel of the mitochondrial outer membrane, has long been proposed to play a central role in apoptosis through interactions with BCL-2 family proteins. However, despite extensive cellular studies, the molecular basis of these interactions remains poorly understood, and direct structural and biochemical characterization of VDAC-Bax complexes is lacking. Here, we address these questions by a structure-function in vitro approach. We produced and purified the VDAC2-Bax complex in a minimal membrane system. Using complementary biochemical, biophysical, and structural approaches, we demonstrate that Bax directly forms a stable complex with VDAC2. We identify the C-terminal helix α9 as a key determinant of the interaction and show that VDAC2-associated Bax adopts a membrane-embedded, 'primed' conformation in which the N-terminal region and BH3 domain remain accessible. Finally, we establish the specificity of this interaction for the isoform VDAC2 over VDAC1 and propose a mechanistic framework by which VDAC2 defines a distinct intermediate in the Bax activation pathway.

Co-supervisors :

• Lucie BERGDOLL (LISM)
• Guillaume GERBAUD (BIP)
• James STURGIS (LISM)

Summary:

Microorganisms live in complex communities, perceiving and responding to their environment by exchanging signals and resources. This complexity and interconnectedness endow the entire community with new functions and enhanced performance that cannot be predicted simply by analyzing the genomes of the species present. Synthetic consortia, inspired by natural ecosystems, offer a valuable model for exploring, deciphering and controlling for potential applications inter-species interactions under simplified conditions. This work focuses on a model consortium associating Nitratidesulfovibrio vulgaris Hildenborough(NvH) and Clostridium acetobutylicum. Within this system, under conditions of nutritional stress, metabolic and physical interactions are set in motion, leading to higher bio-hydrogen production than in pure culture. While the presence of AI-2 quorum-sensing molecules has been demonstrated to be essential for these interactions, the aim of this thesis was to decipher the molecular basis of this interaction. An in silico approach was defined and implemented to identify potential AI-2 QS players in C. acetobutylicum and NvH. The results suggested the existence of previously uncharacterized, non-canonical Lsr-type QS pathways in these bacteria. In vitro approaches demonstrated the function of LsrK-like and LsrR-like proteins in NvH, and thus their role in AI-2-type signaling. LsrK-like is a kinase with the dual function of phosphorylating AI-2 and glycerol, while LsrR-like is a DNA-binding regulator that responds exclusively to AI-2 phosphorylation under the conditions tested. These results reflect an adaptation of the NvHbacterium to perceive an environmental signal through remodeling of the glycerol signaling pathway. Indeed, this hypothesis is supported by a transcriptomic study which identified the NvHgenes regulated by AI-2. This study identified a transporter that may be involved in AI-2 import, thus completing the QS signalling pathway, as well as a metabolic remodelling of NvHbacteria in the presence of AI-2, such as activation of the thiamine synthesis pathway. In parallel, a meta-transcriptomic analysis was used to study the genes regulated within the consortium, pointing to metabolic pathways significantly impacted by the interaction between the two bacteria. The results show modifications in the production of certain amino acids and in other quorum sensing pathways in C. acetobutylicum. This work necessitated the development of a new absolute quantification method using digital droplet PCR to assess the cell ratio within a synthetic consortium. This work highlights the importance of intra- and interspecific communication systems in regulating interactions within the consortium, and opens up avenues for future studies on the biocontrol of microbial systems.

Co-supervisors :

• Marie-Thérèse Giudici-Orticoni (BIP)
• Christophe Bordi (LISM)

Abstract:

Bacteria produce small molecules known as secondary metabolites, which interest research for their numerous therapeutic properties and their role within the biotope. Myxobacteria are Gram-negative bacteria described as multi-producers of secondary metabolites. However, their identification and production in the laboratory is complex. During my thesis, I used the myxobacterium, M. xanthus, to develop a 2PRIM-BOOST approach, which combines optimization of the M. xanthus culture protocol and synthetic biology. The advantages of this approach are an accumulation of compounds involved in the biosynthesis of secondary metabolites, and a simplified metabolome facilitating structural identification and the demonstration of biological activities. As a proof of concept, the combination of the different tools enabled us to significantly increase myxoprincomide production. Once validated, this method enabled me to discover a new metabolite, and analogues, which remain to be characterized. For their biosynthesis, some metabolites require proteins with an Fe-S cofactor. I have studied the process of Fe-S protein biogenesis in M. xanthus and discovered a genetic context in which the expression of Fe-S cluster biogenesis machineries is maximal. All my work will be used to identify unknown secondary metabolites of M. xanthus, and more broadly those of myxobacteria, whether or not they depend on Fe-S proteins.

Co-supervisors :

• Corinne AUBERT (LCB)
• Marc MARESCA (iSm2)
• Béatrice PY (LCB)