Mémoire
| Résumé : | R-bodies are immense intracellular structures previously described in several unrelated species of Proteobacteria. They undergo telescopic expansion linked to environmental changes, yet their synthesis, mode of action, and biological role remain speculative. The genes responsible for their synthesis were first discovered encoded on a plasmid in endosymbiotic bacteria from genus Caedibacter. These genes are found in a genetic cluster coding for 3 predicted short alpha-helical peptides termed rebA-C. In more recent studies, Reb-like peptides have been found widely distributed across proteobacterial genomes with some species containing up to 9 Reb-homologs per genome. These Reb-homologs are often interrupted by genes of unknown function, indicating variations in the structure or function of R-bodies.In our laboratory, R-body-like structures were recently visualized in Chryseobacterium nematophagum, a bacterium that kills and digests roundworms. Chryseobacterium belongs to phylum Bacteroidota, recognized for degrading complex organic matter. Although genus Chryseobacterium overall presents diverse lifestyles, C. nematophagum species stands out for its worm-killer phenotype. Our laboratory has recently found that a functional type IX secretion system was indispensable for C. nematophagum pathogenicity, however the precise virulence factors remain to be discovered. Intriguingly, Caedibacter R-bodies were shown to be involved in killing of unicellular ciliates from genus Paramecium and could be considered as a potential virulence factor.R-bodies have never been described outside of the Proteobacteria phylum and C. nematophagum thus presents a first example of these structures in phylum Bacteroidota. The genetic cluster responsible for R-body synthesis identified in C. nematophagum comprises multiple predicted Reb-like peptides as well as genes of unknown function. Preliminary data showed that it is extremely simple to purify the R-bodies from C. nematophagum, which is a prerequisite for biochemical studies of its composition but also provides the basis to explore its biotechnological applications. In this work, we have performed single and combined reb gene deletions to study their essentiality for R-body synthesis. We have also constructed fluorescent reporter fusions to learn whether the different reb peptides are incorporated into the R-body structure. We have then analysed the synthesis and fluorescence of the R-body derivatives by light, fluorescence, and electron microscopy. In addition, we have analysed the protein composition of each derivative by SDS-PAGE. These analyses will soon be complemented by mass-spectrometry and cryo-electron microscopy to learn more about the composition of the C. nematophagum R-bodies. Supported by these findings, our laboratory has proposed the possibility of genetically fusing proteins of interest to the peptides composing the R-body. In particular, we have found that chromosomal fusion of reb1 and reb9 to mCherry yielded fluorescent R-bodies. We will now explore the possibility to immobilize the protein of interest, such as a commercially valuable enzyme, onto the R-body structure, allowing for easy purification, recovery, and reuse of the enzyme. |
