Stages de Master 2 au sein des plateformes technologiques de l’IM2B - 2024

Résumé:

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.

Stagiaire : Mary DE LA SAYETTE, étudiante en master Microbiologie d'Aix-Marseille Université 

Résumé:

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 optimisation. However, it showed promise for future real-time observation of lipid remodelling.

Stagiaire : 

• Maxime BASTIDE, étudiant en Master Biologie, Agrosciences de Université de Bordeaux (B2AS)