As a cost of civilization, the massive usage of nitrogen−containing fertilizers ultimately leading to detrimental environmental impacts. One reason behind is being the nitrification (biological oxidation of NH₃ to NO₂⁻ and NO₃⁻). These anions ultimately leached into water bodies, leading to downstream eutrophication. Another pathway is denitrification (reduction of NO₂⁻ and NO₃⁻ to N₂O and N₂). During the denitrification process, substantial amounts of greenhouse gas, N₂O escape into the atmosphere. In line with such an emergency, this proposal is set to tackle this grand challenge to convert oxidized form of nitrogen usually abbreviated as NOx to its ultimate reduced form i.e., NH₃. This project is constructed on a two−legged strategy; development of functional artificial proteins to replicate to its best the active sites of the natural enzymes involved in the NOx transformations. The second part deals with the development of cost efficient synthetic systems holding the appropriate chemical functionalities to reproduce the unmatched catalytic power of natural enzymes.

Plants have evolved complex structures, like hydathodes and stomata, that regulate water balance and gas exchange, essential for survival and growth. Hydathodes, specialised structures located at leaf edges, allow guttation, where excess water is expelled as droplets, especially in high humidity and low transpiration. In contrast, stomata on the leaf's abaxial surface regulate gas exchange, allowing CO₂ in for photosynthesis and releasing O₂ and water vapor. Hydathode pores (HPs) are formed by pairs of guard cells, like stomata. However, unlike stomata, HPs remain predominantly open and thus help prevent excess turgor pressure but on the other hand can allow pathogen entry, especially for moisture-loving bacteria. Examining the differing roles of hydathodes and stomata in pathogen susceptibility is key to understanding plant resilience to environmental stress and diseases. Although HPs and stomata are crucial in plant physiology, their comparative roles are still under-explored. While initial studies identified cell clusters in Arabidopsis leaves encompassing guard cells and hydathodes, the scarcity of hydathode cells has limited their in-depth characterisation. Transcriptomics at the single cell level is revolutionising plant studies and is particularly suited for the characterisation of rare cell populations and hence could help reveal unique gene expression profiles in HPs versus stomata. My project aims to use single cell transcriptomics to deeply characterise HPs versus stomata and to analyse the impact of HPs on host-pathogen interaction.

In the three domains of life, there is a great diversity of metabolisms. This diversity is the consequence of multiple evolutionary events, linked to changes in the ecological niches and constrained by the ancestral metabolic network topology. Methanogenesis, one of the most ancient pathways in Archaea, represents a particularly suitable model pathway for the analysis of dynamics of metabolism. Although being very ancient and central in metabolism, it has been lost several times during the diversification of Archaea. Due to the place occupied by the methanogenesis, it is thought that its loss has induced a drastic reshuffle of the central metabolism, such as losses of ancient metabolic blocks associated to methanogenesis (MBAM), acquisitions of alternative enzymes to methanogenesis and MBAM, and substantial modifications of related enzymes.

The aim of my research is to identify the key events of MBAM losses that have profoundly influenced the evolution of Archaea, using comparative genomics and phylogenomics. This work also holds potential for biotechnological advancements, particularly through the discovery of novel enzymes. Furthermore, it will provide critical insights into the dynamics of metabolism at the scale of an entire domain of life and reveal the evolutionary constraints tied to the structure of metabolic networks. This enhanced understanding will shed light on the physiology of the earliest living organisms.

Résumé:

STOP1 from the model plant Arabidopsis thaliana is a master gene for tolerance to low-pH and Al. STOP1 (Sensitive To Proton rhizotoxicity1) is a transcription factor (TF) responsible for tolerance not only to low-pH and Al but also to several other abiotic stresses. A. thaliana mutants lacking STOP1 activity are extremely sensitive to H⁺ and Al³⁺, with this being mainly due to a deficit in the STOP1-dependent expression of root efflux transporters of small metal-chelating organic acids. pH and metals post-transcriptionally regulate the abundance of the STOP1 protein. The mechanism by which acidic pH and Al stimulate the STOP1 signaling pathway remain elusive. STOP1 contains three predicted intrinsically disordered regions (IDRs) accounting for 55 % of the STOP1 sequence. The high conformational flexibility and multivalency of IDRs render them highly prone to undergo liquid-liquid phase separation (LLPS). In eukaryotes, including A. thaliana, TFs are enriched in IDRs. Transcriptional regulators are known to sub-compartmentalize via LLPS. Their condensation can either stimulate their activity by concentrating them with components of the transcription machinery or, on the contrary, inactivates them by sequestration. Importantly, phase-separation of certain IDR-containing proteins depends on the cellular pH. In silico analysis of the STOP1 sequence indicates that its three IDRs contain multiple droplet-promoting regions that could drive LLPS. Our working hypothesis is that STOP1 undergoes LLPS in response to a decrease in pH and that the resulting condensates trigger expression of the target gene(s). The aim of this project is to experimentally ascertain the disordered nature of STOP1 IDRs and to assess their ability to undergo pH-mediated LLPS both in vitro and in planta. The three IDRs will be expressed in E. coli and the resulting purified proteins will be characterized in vitro in terms of their conformational properties and phase-separating abilities. The ability of STOP1 to undergo pH- and Al-induced LLPS in planta will be assessed by confocal microscopy using STOP1-GFP fusions. STOP1 mutants will also be designed, transiently expressed in N.benthamiana and then assessed for their ability to undergo LLPS. This project, by shedding light on the potential phase-separating abilities of STOP1, will constitute a breakthrough in our understanding of the molecular mechanisms underlying the activity of this class of plant TFs with the potential to unveil a new regulatory mechanism.

Résumé:

Protein folding is crucial for proper function, and chaperones play a key role in preventing protein aggregation and facilitating folding. Within the bacterial envelope, the periplasmic space is a site of passage and activity for many proteins, whose proper folding depends on a network of chaperones essential for maintaining proteostasis. Unexpectedly, we have discovered that one of the periplasmic chaperones of E. coli, though already well studied, binds to a heme. This observation, based on solid preliminary results, raises new questions about the role of a holo-chaperone form in periplasmic proteostasis. To this end, we have formed a multidisciplinary consortium bringing together microbiologists from the LCB, biochemists from the BIAM, and physico-chemists from the BIP.

Résumé:

Oxygenic photosynthesis was a pivotal event in Earth's history, enabling organisms to convert light into chemical energy, fixing CO2 and producing O₂, which supported the evolution of aerobic life. Cyanobacteria are unique as the only prokaryotes capable of both oxygenic photosynthesis and nitrogen fixation, making them key models for research and biotechnology. Nitrogen fixation, catalyzed by nitrogenase, is highly sensitive to O2, creating challenges for cyanobacteria, which solve this through specialized heterocysts that maintain an anaerobic environment for nitrogen fixation while neighboring vegetative cells handle photosynthesis.

In heterocysts, sugars from vegetative cells are broken down via the oxidative pentose phosphate pathway, generating NADPH and ATP for nitrogen fixation. This balance is essential for survival, but the transfer of carbon and reductants can limit efficiency. A promising bioengineering approach involves heterocysts producing hydrogen by introducing hydrogenase enzymes. Previous experiments raised bioenergetic questions about how reductants are supplied to heterocysts during hydrogen production, highlighting the need to understand carbon and electron transport in engineered strains.

The REBONC project aims to deepen understanding of bioenergetic trade-offs in cyanobacteria by studying photosynthesis and nitrogen fixation in engineered strains, with the goal of enhancing productivity for biofertilizer and bioenergy applications. The project aligns with Horizon Europe’s goals of renewable nitrogen use and advancing agricultural resilience