Lesur, E. | Search bioRxiv

bioRxiv2025

Functional dissection of Wag31 domains for septal recruitment and polar distribution during the cell cycle

Bacterial cell morphogenesis is controlled by the synthesis and organization of peptidoglycan and driven by multi-protein complexes such as the divisome and elongasome. Here we investigate the role of the Corynebacterium glutamicum DivIVA homologue, Wag31, the elongasome scaffold essential for polar growth in Corynebacteriales. Conditional depletion of Wag31 results in viable but coccoid-shaped cells, showing that Wag31 is essential for rod shape maintenance. Our structural phylogenetic analyses of DivIVA homologues revealed that in Actinobacteria, unlike Firmicutes, an intrinsically disordered region spatially separates the N-terminal lipid-binding domain (LBD) from the C-terminal coiled-coil domain (CCD). We show that the LBD is necessary and sufficient for septum localization, independent of its membrane-binding properties, while the CCD domain mediates self-interaction and polar accumulation. Our findings suggest that Wag31 is recruited specifically to the septum through protein-protein interactions, priming the future pole and allowing for a timely divisome-elongasome transition at cytokinesis. Once the pole is formed the self-aggregative properties of the C-terminal CCD dominate and form a stable structure that likely organizes the pole for cell wall biosynthesis.

microbiology↗

bioRxiv2024

Synthetic mycolates derivatives as molecular tools to decipher protein mycoloylation, a unique post-translational modification in bacteria.

Protein mycoloylation is a newly characterized post-translational modification (PTM) specifically found in Corynebacteriales, an order of bacteria that includes numerous human pathogens. Their envelope is composed of a unique outer membrane, the so-called mycomembrane made of very-long chain fatty acids, named mycolic acids. Recently, some mycomembrane proteins including PorA have been unambiguously shown to be covalently modified with mycolic acids in the model organism Corynebacterium glutamicum by a mechanism that relies on the mycoloyltransferase MytC. This PTM represents the first example of protein O-acylation in prokaryotes and the first example of protein modification by mycolic acid. Through the design and synthesis of trehalose monomycolate (TMM) analogs, we prove that i) MytC is the mycoloyltransferase directly involved in this PTM, ii) TMM, but not TDM, is a suitable mycolate donor for PorA mycoloylation, iii) MytC is able to discriminate between an acyl and a mycoloyl chain in vitro unlike other trehalose mycoloyltransferases. We also solved the structure of MytC acyl-enzyme obtained with a soluble short TMM analogs which constitutes the first mycoloyltransferase structure with a covalently linked to an authentic mycolic acid moiety. These data highlight the great conformational flexibility of the active site of MytC during the reaction cycle and pave the way for a better understanding of the catalytic mechanism of all members of the mycoloyltransferase family including the essential Antigen85 enzymes in Mycobacteria.

microbiology↗