Recherche - Nouveaux entrants - 2024

Lanthanides (Ln) also known as Rare earth elements (REE) possess chemical, magnetic, luminescent and electric properties of high interest in the industry, medicine and renewable energy. REE are critical resources for the high technologies of ever-growing interest (Figure1). REE are common metals (0.5 to 60 ppm) of the Earth’s crust but are most often found in mixture. Due to their close physicochemical properties, their selective extraction is a challenge. Classic extraction and separation methods required several steps (up to 100) and generate a lot of pollutant wastes. Alternative methods to obtain more efficient and more sustainable REE extraction that selective are highly needed. Bio-inspired tools are the actual most promising options. Indeed, in the past decades, studies on bacterial system suggest that REE biochemistry could solve the selectivity issue. Only the light REE (La to Eu) are naturally used by bacteria, indicating that bacteria are able to selectively discriminate between REE. More broadly, those discoveries open a new field of research for which much is to be uncovered.

The first REE-interacting protein identified was the methanol dehydrogenase (MDH) of a methylotroph bacteria. Further investigations revealed that methylotroph bacteria also possess other REE interacting proteins, consequently most of the work focused on methylotroph’s proteins. To date three binding sites of REE-interacting protein have been characterized (XoxF, LanM and LanD), some present a preference trend toward light REE but limit of the selectivity is not fully understood yet. Conservation analysis revealed that MDH like proteins are more widespread among bacteria than initially thought. This opened the way for new investigations in other bacteria. Interestingly, for at least some organisms carrying the two forms of methanol/alcohol desydrogenases, i.e. calcium (Ca²⁺) or REE dependent. The regulation allowing the bacterial adaptation leading to the expression switch in between the two forms of MDH/ADH, is under the control of a complex regulatory network involving two-component systems (TCS), and in some case other proteins. Importantly, TCS involved in the REE adaptation seems to be among the most selective and vary from organisms revealing that selective detection mechanisms might differ between bacteria. None of those detailed molecular mechanism involved in the REE detection has been uncover.

Few recent studies done by IPM collaborator P. Billard (Univ. Lorraine) in the non-methylotroph bacteria Pseudomonas putida, confirmed the broader conservation of REE proteins. Among them the two-component system (TCS) called PedS2/PedR2, in charge of the regulation switch between the two alcohol deshydrogenases (REE or Ca²⁺ dependent), for which the detailed REE detection mechanism is not fully understood (Figure2). The regulon of this TCS is not identified either. Deciphering the molecular mechanism used by this TCS to first sense the REE, either indirectly or directly, discriminate and then regulate the targeted genes is key to broaden the knowledge on REE adaptation. 

The aim of this project is to characterize for the first time the molecular mechanism involved in the bacterial REE sensing using P. putida as model. To this end, the strategy is to purify PedS2 protein and identify the REE detection mechanism using a combination of approaches to provide insights on the potential key residues involved in the REE sensing. Following the PedS2 characterization, the goal is to further investigate the PedR2 regulon and its binding properties. Thanks to the IM2B grant, we have been able to test in vivo potential PedS2 interacting partner, validate the PedS2 topology (Figure3), notably the sensor loop, purified the PedS2 sensor loop, initiate the in vitro characterization of PedS2 REE detection mechanism with preliminary results suggesting a direct binding. In other proteins, REE binding is coordinated by carboxylate residues. Next, we realized an in silico analysis to predict potential residues involved in the binding (Figure3). 

Arteriviruses are one of 14 families that make up the highly diverse order of positive-stranded RNA viruses known as the Nidovirales. Certain members of this family, such as Equine Arterivirus (EAV) and Porcine Reproductive and Respiratory Syndrome Virus (PPRSV) represent significant animal pathogens which present a large economic burden. However they have been relatively understudied, especially in comparison with viruses of the related Coronaviridae (CoV) family (notably SARS-CoV-2), which have attracted significant attention over the past few years. This project was aimed at bridging this knowledge-gap, by investigating key arterivirus viral enzymes: including the RNA-dependent RNA polymerase (RdRp) and its unique N−terminal domain, known as the NiRAN. These two enzymes are at the center of the viral replication transcription complex (RTC), responsible for copying and amplifying the genomic information for passage to progeny virions. As such, they represent extremely attractive antiviral targets. The project, which is still ongoing, involves the structural and functional characterization of these two essential enzymes for viruses of the Arteriviridae family. This will expand our understanding of understudied viruses with epidemic potential, and guide drug design strategies against key viral targets.

Water scarcity in agriculture is worsening, leading to plant drought stress with direct impacts on physiology and productivity. Abscisic acid (ABA), a key plant stress hormone, plays a central role in drought response pathways. Recent research has revealed that nicotinamide adenine dinucleotide (NAD), a metabolic cofactor, is also involved in drought stress. Plants impaired in NAD metabolism display differential drought responses depending on NAD⁺ content with a direct correlation with ABA levels and signaling. However, the mechanisms underlying the NAD-ABA interaction and its validation as essential for plant responses to drought remain to be explored. The project explores the connection between plant metabolism and hormone signaling, with a focus on its consequences in drought responses. Through a combination of genetics, physiology, and metabolic approaches, we aim to gain fresh insights into the regulation of the interaction between the metabolic cofactor NAD and the plant stress hormone ABA.

2,4-diacetylphloroglucinol (DAPG) is a potent fungicide produced by Pseudomonas isolates (https://doi.org/10.3389/fmicb.2017.01218). Indeed, suppression of Take-all disease, caused by the ascomycete Gaeumannomyces graminis var. tritici, in disease suppressive soils has been repeatedly associated with an increased abundance of DAPG-producing bacterial strains (https://doi.org/10.1016/j.soilbio.2012.05.012). Accordingly, DAPG-producing bacteria have potential as biocontrol agents. In this project, we aimed to evaluate the direct use of DAPG as a biosourced fungicide to circumvent drawbacks of products based on living bacteria.

A major aim was to investigate possible bioproduction of DAPG. To identify a suitable bacterial chassis, we screened twenty-one Pseudomonas strains, selected from a previous large-scale screen, for antifungal properties. We showed, by mutations in the gene coding for the enzyme catalyzing the committing step in DAPG biosynthesis, phlD, that antifungal properties in three best-performing strains were based on DAPG production. We further improved DAPG production, by approximately a factor four, by inactivation of a repressor (PhlF) and a hydrolase (PhlG). In yet another approach to manipulate DAPG production, we designed and assembled a synthetic gene cluster encompassing the phl genes for DAPG biosynthesis. The synthetic gene cluster, when inserted into a non DAPG-producing Pseudomonas strain, confers antifungal properties. The modular design of the synthetic cluster will allow us to further manipulate DAPG production and content, as promoters driving individual transcription units can be exchanged with ease following standardized assembly reactions. Last, we assayed DAPG biocontrol capacities in plate assays, and observed that DAPG doses efficient as fungicide (≥ 20 µM) also impede plant growth and development. Considering the natural biocontrol capacities of DAPG-producing bacteria in the field, we will address the feasibility and efficacy of DAPG in seed coatings to protect from fungal diseases in the future.