Understanding the mechanism that controls space-time coordination of elongation and division of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is critical for fighting the tubercle bacillus. Most of the numerous enzymes involved in the synthesis of Mycolic acid - Arabinogalactan-Peptidoglycan complex (MAPc) in the cell wall are essential in vivo. Using a dynamic approach, we localized Mtb enzymes belonging to the fatty acid synthase-II (FAS-II) complexes and involved in mycolic acid (MA) biosynthesis in a mycobacterial model of Mtb: M. smegmatis. Results also showed that the MA transporter MmpL3 was present in the mycobacterial envelope and was specifically and dynamically accumulated at the poles and septa during bacterial growth. This localization was due to its C-terminal domain. Moreover, the FAS-II enzymes were co-localized at the poles and septum with Wag31, the protein responsible for the polar localization of mycobacterial peptidoglycan biosynthesis. The dynamic localization of FAS-II and of the MA transporter with Wag31, at the old-growing poles and at the septum suggests that the main components of the mycomembrane may potentially be synthesized at these precise foci. This finding highlights a major difference between mycobacteria and other rod-shaped bacteria studied to date. Based on the already known polar activities of envelope biosynthesis in mycobacteria, we propose the existence of complex polar machinery devoted to the biogenesis of the entire envelope. As a result, the mycobacterial pole would represent the Achilles' heel of the bacillus at all its growing stages.

Summary: Despite the existence of efficient chemotherapy, tuberculosis remains a leading cause of mortality worldwide. New drugs are urgently needed to reduce the potential impact of the emergence of multidrug‐resistant strains of the causative agent Mycobacterium tuberculosis (Mtb). The front‐line antibiotic isoniazid (INH), and several other drugs, target the biosynthesis of mycolic acids and especially the Fatty Acid Synthase‐II (FAS‐II) elongation system. This biosynthetic pathway is essential and specific for mycobacteria and still represents a valuable system for the search of new anti‐tuberculous agents. Several data, in the literature, suggest the existence of protein–protein interactions within the FAS‐II system. These interactions themselves might serve as targets for a new generation of drugs directed against Mtb. By using an extensive in vivo yeast two‐hybrid approach and in vitro co‐immunoprecipitation, we have demonstrated the existence of both homotypic and heterotypic interactions between the known components of FAS‐II. The condensing enzymes KasA, KasB and mtFabH interact with each other and with the reductases MabA and InhA. Furthermore, we have designed and constructed point mutations of the FAS‐II reductase MabA, able to disrupt its homotypic interactions and perturb the interaction pattern of this protein within FAS‐II. Finally, we showed by a transdominant genetic approach that these mutants are dominant negative in both non‐pathogenic and pathogenic mycobacteria. These data allowed us to draw a dynamic model of the organization of FAS‐II. They also represent an important step towards the design of a new generation of anti‐tuberculous agents, as being inhibitors of essential protein–protein interactions.

Mycobacterium smegmatisis often used as a surrogate host for pathogenic mycobacteria, especially since the isolation of the transformable smooth morphotype strain mc²155 from the isogenic rough wild-type strain ATCC 607. Biochemical analysis of the cell envelope components revealed a lack of polar glycolipids, namely the lipooligosaccharides and the polar subfamilies of glycopeptidolipids, in the mc²155 strain. In addition, the latter strain differs from its parent by the distribution of various species of glycolipids and phospholipids between the outermost and deeper layers of the cell envelope. The presence of filamentous and rope-like structures at the cell surface of mc²155 cells grown in complex media further supported an ultrastructural change in the cell envelope of the mutant. Importantly, a significantly more rapid uptake of the hydrophobic chenodeoxycholate was observed for the mutant compared to wild-type cells. Taken together, these data indicate that the nature of the surface-exposed and envelope constituents is crucial for the surface properties, cell wall permeability and bacterial phenotype, and suggest that the transformable character of the mc²155 strain may be in part explained by these profound modifications of its cell envelope.