Awardees

The OM-PSG project investigates the role of Msr (Methionine Sulfoxide Reductase) enzymes in bacterial responses to oxidative stress. By combining fluorescence microscopy at the single-cell level with microfluidic tools, this project aims at gaining deeper insights into bacterial stress responses and repair mechanisms under oxidative conditions.

The originality of this research lies in the dynamic study of these enzymes under controlled conditions, enabling real-time observation of their role in bacterial physiology. Through the technological developments implemented, the project opens new interdisciplinary perspectives and strengthens collaborations in advanced imaging and oxidative stress biology.

This work is part of a broader effort to decipher bacterial adaptation strategies to oxidative environments and is supported by IM2B for the development of innovative approaches.

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.

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.

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.

Bacteria have evolved sophisticated membrane channels, called secretins, specifically adapted for the assembly or secretion of bulky extracellular structures such as type 4 pili (T4P), needle complexes of type 3 secretion systems (T3SS) or large fully folded effectors in type 2 secretion systems (T2SS) (Review by Barbat et al., 2023b). We have recently shown that T2SS secretins must adopt different conformations to allow the passage of large effectors while maintaining the integrity of the bacterial envelope (Barbat et al., 2023a). In this project, we plan to develop and implement an ambitious in vivo experimental approach aimed not only at demonstrating the need for flexibility of the XcpD secretin during the secretion process, but also at identifying the partners that trigger these conformational changes. Specifically, we will further investigate the protein dynamics that exist between two adjacent secretin subunits by performing in-cell EPR experiments on spin-labelled proteins in their native environment (Review by Pierro et al., 2024) as well as in different genetic backgrounds lacking potential partners. With this approach, we hope to gain a better understanding of the different chronological steps that occur during the secretion process mediated by this singular nanomachine.

References:

Barbat, B., Douzi, B., Ball, G., Tribout, M., El Karkouri, K., Kellenberger, C., and Voulhoux, R. (2023a). Insights into dynamics and gating properties of T2SS secretins. Sci Adv 9, eadg6996.

Barbat, B., Douzi, B., and Voulhoux, R. (2023b). Structural lessons on bacterial secretins. Biochimie 205, 110-116.

Pierro, A., Bonucci, A., Magalon, A., Belle, V., and Mileo, E. (2024). Impact of Cellular Crowding on Protein Structural Dynamics Investigated by EPR Spectroscopy. Chem Rev 124, 9873-9898.

The bio-conversion of_CO2 by the Ribulose 1,5 bisphosphate carboxylase/oxygenase (RuBisCO) reaches 1014 kilograms per year. The carboxylation of Ribulose 1,5 bisphosphate (RuBP) activity of RuBisCO competes with its oxygenation activity. However, the diffusion of_CO2 in water is much less efficient than that of _O2, and the majority of inorganic carbon is in the form of bicarbonate in the aquatic environments. The success of photosynthetic microorganisms relies in their_CO2 concentration mechanisms (CCM) that induces a flux of inorganic carbon near RuBisCO. Contrary to higher plant, eukaryotic microalgae such as the model green algae Chlamydomonas reinhardtii possess biophysical CCMs which involves a peculiar supramolecular organisation and specific location of RuBisCO as well as bicarbonate transporters and carbonic anhydrases. RuBisCO is found in two distinct locations in C. reinhardtii: either diffused in the chloroplast stroma or condensed in a liquid-liquid separated phase (LLSP) named Pyrenoid. The proportion of diffused vs condensed RuBisCO varies from 60%/40% under condition of high partial pressure of_CO2 (_PCO2) to 20%/80% under low _PCO2. While numerous studies have investigated the carboxylation vs oxygenation activities of isolated RuBisCO or in vivo, the effect of condensation on the enzyme activity has been poorly investigated. The objective of this project is to correlate RuBisCO condensation to its activity. This is declined in two main strategies: 1- in-vitro reconstituted LLPS with recombinant RuBisCO and its scaffold protein EPYC1 and a structure-function investigation, and 2- in-vivo quantification of carboxylation and oxygenation activities under conditions in which RuBisCO is mainly diffused or mainly condensed.

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). 

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:

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:

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)