Biology subjects

Constantinesco-Becker, F.

Publications and source records attributed to Constantinesco-Becker, F..

3 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↗

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↗

bioRxiv2020

Genome-wide identification of novel genes involved in Corynebacteriales cell envelope biogenesis using Corynebacterium glutamicum as a model.

Corynebacteriales are Actinobacteria that possess an atypical didermic cell envelope. One of the principal features of this cell envelope is the presence of a large complex made up of peptidoglycan, arabinogalactan and mycolic acids. This covalent complex constitutes the backbone of the cell wall and supports an outer membrane, called mycomembrane in reference to the mycolic acids that are its major component. The biosynthesis of the cell envelope of Corynebacteriales has been extensively studied, in particular because it is crucial for the survival of important pathogens such as Mycobacterium tuberculosis and is therefore a key target for anti-tuberculosis drugs. In this study, we explore the biogenesis of the cell envelope of Corynebacterium glutamicum, a non-pathogenic Corynebacteriales, which can tolerate dramatic modifications of its cell envelope as important as the loss of its mycomembrane. For this purpose, we used a genetic approach based on genome-wide transposon mutagenesis. We developed a highly effective immunological test based on the use of anti-arabinogalactan antibodies that allowed us to rapidly identify bacteria exhibiting an altered cell envelope. A very large number (10,073) of insertional mutants were screened by means of this test, and 80 were finally selected, representing 55 different loci. Bioinformatics analyses of these loci showed that approximately 60% corresponded to genes already characterized, 63% of which are known to be directly involved in cell wall processes, and more specifically in the biosynthesis of the mycoloyl-arabinogalactan-peptidoglycan complex. We identified 22 new loci potentially involved in cell envelope biogenesis, 76% of which encode putative cell envelope proteins. A mutant of particular interest was further characterized and revealed a new player in mycolic acid metabolism. Because a large proportion of the genes identified by our study is conserved in Corynebacteriales, the library described here provides a new resource of genes whose characterization could lead to a better understanding of the biosynthesis of the envelope components of these bacteria.

microbiology↗