Recherche - Nouveaux entrants - 2023

The bacterial secretory pathway (Sec) is the main process responsible for the localization of a large number of different essential proteins that need to be translocated across the cytoplasmic membrane into periplasm to be active. However, Sec substrate proteins are capable to assume misfolded conformations or aggregate in stress conditions, compromising the cell viability. Therefore, bacteria have evolved a complex network of various accessory proteins (molecular chaperones (MCs)) that cooperate to maintain in unfolded state the nascent proteins synthetized by the ribosome prior their translocation ( A. Tsirigotaki et al., Nat Rev Microbiol. 2017 15(1):21-36 ; 2) T. Cranford-smith et al. J. Biol. Chem., 295 (2020) 7516–7528).^. In 2020, a new MC related to Sec machinery, called YecA, has been identified in E.coli revealing a role in membrane biogenesis. This MC binds promiscuously various proteins while they are still nascent polypeptides and collaborates with other MC to maintain the translocation activity. The stress conditions required for its overexpression are currently not well understood, even if the iron availability seems to be one of the principal factors². Due to its recent discovery, YecA is yet poorly investigated and essential information are missing. In particular, the molecular mechanism for the recognition and the release of Sec-substrates, as well the interaction with other MCs and components of Sec still need to be investigated.

In this project, a structural and functional characterization of E.coli YecA will be carried out through the development of an innovative SDSL-EPR approach. Specifically, novel experiments based on SDSL-EPR spectroscopy will be carried out to decipher the interactions of YecA with other MCs and components of Sec pathway, focusing on the role of protein dynamics in the complexes formation. Combining EPR results with other biophysical techniques (ITC, DLC, CD, NMR and X-ray cryst.), the role of this MC in the E.coli Sec pathway and in the maintenance of bacterial proteome will be investigated.

As obligate intracellular parasites, viruses are dependent on their hosts for replication. This poses a unique challenge: how can the virus usurp cellular resources whilst avoiding detection by immune surveillance complexes? For viruses with positive sense RNA genomes (+ssRNA), genome replication is coupled to the remodelling of host membranes, yielding organelles that camouflage their RNA within the cell. All such organelles require a point of exchange with the cytosol, allowing for entry of cellular metabolites and exit of the nascently synthesized genome for translation and packaging into new virions. For several viruses including the alphavirus superfamily and coronaviruses, macro pores have been identified within the organelle membrane that may serve as a molecular gates.

This project proposed to structurally characterize these viral pores at high resolution with cryo electron microscopy. Pore complexes were studied following extraction with single particle methods, and a method to purify replication organelles for ex situ analysis of pores in their native membranes was also developed. Characterization of the pores will allow us to understand how they are integrated into viral replication, providing new avenues for therapeutics.

Cyanobacteria were the first organisms to evolve oxygenic photosynthesis, and now comprise a vast number of species, adapted to almost all the Earth’s environments. Some cyanobacteria can do photosynthesis using far-red light, a portion of the solar spectrum that is unused by most other oxygenic photoautotrophs because of its low energy content. These species are of high interest, because they could inspire biotechnological applications aimed at expanding the light usage of crop plants and microalgae into the far-red. Nonetheless, it is still unclear how the low energy content of far-red light affects the photosynthetic efficiency of these cyanobacteria, and which consequences this could have on the production of biomass.  In my previous postdoctoral research at IBPC in Paris, I adapted to cyanobacteria a spectroscopic method based on the ElectroChromic Shift (ECS) of photosynthetic pigments that is widely used in algae and plants to quantify the photosynthetic activity by measuring the trans-thylakoid electric field generated by electron transport. In the model species Synechocystis PCC6803 and Synechococcus elongatus PCC7942, I could show that ECS represents a new tool to measure the photosynthetic activity in vivo in cyanobacteria.

In the project funded by the IM2B, I adapted the ECS method to two far-red absorbing cyanobacteria species, Chroococcidiopsis thermalis PCC7203 and Acaryochloris marina. The funding IM2B was used to upgrade the spectroscopy instruments available in the P&E team at BIAM, and to build a dedicated incubator for the growth of the two far-red cyanobacteria species. Using the newly adapted ECS method, I could show that both Acaryochloris and Chroococcidiopsis use visible and far-red light with similar efficiency to sustain photosynthetic electron transport, despite the significant differences in their light absorption characteristics (https://doi.org/10.1016/j.bbabio.2024.149502 ). This is at variance with the majority of cyanobacteria, which cannot use the far-red portion of the light spectrum efficiently. ECS thus represents a new non-invasive tool to study the performance of naturally occurring far-red photosynthesis, potentially contributing to its future exploitation for sustainable biotechnological applications.