Biology subjects

Bayan, N.

Publications and source records attributed to Bayan, N..

4 recordsLinked to original sources

bioRxiv2026

Uncoupling mycomembrane biogenesis from mycolic acid synthesis reveals a distinct role for mycoloyltransferases in mycobacterial cell division.

Bacteria of the order Mycobacteriales, including the genera Mycobacterium and Corynebacterium, possess a unique outer membrane, termed the mycomembrane, which is structurally and chemically distinct from the lipopolysaccharide-containing outer membrane of Gram-negative bacteria. A defining feature of the mycomembrane is its enrichment in mycolic acids, long-chain -branched, {beta}-hydroxylated fatty acids that occur as trehalose monomycolate (TMM), trehalose dimycolate (TDM), or are esterified to arabinogalactan, an unusual polymer that is itself covalently linked to peptidoglycan (PG). Mycoloyltransferases are Mycobacteriales-specific enzymes described to catalyze the transfer of mycolic acids from trehalose monomycolate (TMM) to various cell envelope acceptors, including trehalose and arabinogalactan. In several species, including Mycobacterium tuberculosis, these proteins are essential for viability; however, their occurrence as multiple paralogs with partially redundant functions has hindered the precise assignment of their cellular roles. Previously, we showed that Corynebacterium glutamicum remains viable in the absence of mycolic acids, and thus without a mycomembrane, following deletion of pks, the gene required for mycolic acid biosynthesis. Building on this finding, we systematically deleted all genes encoding mycoloyltransferases in C. glutamicum to further disclose their collective function in the cell. The resulting {Delta}myts mutant lacked arabinogalactan-bound mycolates and TDM, yet continued to synthesize TMM. Despite the high abundance of this major glycolipid, the {Delta}myts strain failed to assemble a mycomembrane and displayed pronounced cell aggregation. Unexpectedly, deletion of mycoloyltransferases also caused very severe defects in cell division and morphogenesis that are not observed in a {Delta}pks strain unable to synthesize mycolic acids. Together, these results demonstrate that mycoloyltransferases are essential for mycomembrane assembly but dispensable for TMM biosynthesis, and reveal an unexpected role for these enzymes in cell division that is independent of their canonical mycolic acid transfer activity. SIGNIFICANCEHow the mycomembrane is assembled and anchored to the cell wall remains a central question in Mycobacteriales, where this outer membrane is necessary for envelope integrity and intrinsic antibiotic resistance. By genetically separating mycolic acid synthesis from their incorporation into the outer membrane, we identify arabinogalactan-linked mycolates as the critical determinant for initiating membrane assembly. Unexpectedly, we also uncover a role for mycoloyltransferases beyond their canonical function in lipid metabolism, revealing a functional link with cell division. These findings point to a critical role of Mycoloyltransferases in the coordination between outer membrane biogenesis and bacterial cytokinesis.

microbiology↗

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

Cryo-EM structure and polar assembly of the PS2 S-layer of Corynebacterium glutamicum

The polar-growing Corynebacteriales have a complex cell envelope architecture characterized by the presence of a specialized outer membrane composed of mycolic acids. In some Corynebacteriales, this mycomembrane is further supported by a proteinaceous surface layer or S-layer, whose function, structure and mode of assembly remain largely enigmatic. Here, we isolated ex vivo PS2 S-layers from the industrially important Corynebacterium glutamicum and determined its atomic structure by 3D cryoEM reconstruction. PS2 monomers consist of a six-helix bundle core, a three-helix bundle arm, and a C-terminal transmembrane (TM) helix. The PS2 core oligomerizes into hexameric units anchored in the mycomembrane by a channel-like coiled-coil of the TM helices. The PS2 arms mediate trimeric lattice contacts, crystallizing the hexameric units into an intricate semipermeable lattice. Using pulse-chase live cell imaging, we show that the PS2 lattice is incorporated at the poles, coincident with the actinobacterial elongasome. Finally, phylogenetic analysis shows a paraphyletic distribution and dispersed chromosomal location of PS2 in Corynebacteriales as a result of multiple recombination events and losses. These findings expand our understanding of S-layer biology and enable applications of membrane-supported self-assembling bioengineered materials.

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↗