No abstract

As its common name implies, tuberculostearic acid is an abundant and genus-specific branched-chain fatty acid in mycobacterial membranes. This fatty acid, 10-methyl octadecanoic acid, has been an intense focus of research, particularly as a diagnostic marker for tuberculosis.

Mycobacteria spatially organize their plasma membrane, and many enzymes involved in envelope biosynthesis associate with a membrane compartment termed the intracellular membrane domain (IMD). The IMD is concentrated in the polar regions of growing cells and becomes less polarized under non-growing conditions. Because mycobacteria elongate from the poles, the observed polar localization of the IMD during growth likely supports the localized biosynthesis of envelope components. While we have identified more than 300 IMD-associated proteins by proteomic analyses, only a handful of these have been verified by independent experimental methods. Furthermore, some IMD-associated proteins may have escaped proteomic identification and remain to be identified. Here, we visually screened an arrayed library of 523 Mycobacterium smegmatis strains, each producing a Dendra2-FLAG-tagged recombinant protein. We identified 29 fusion proteins that showed polar fluorescence patterns characteristic of IMD proteins. Twenty of these had previously been suggested to localize to the IMD based on proteomic data. Of the nine remaining IMD candidate proteins, three were confirmed by biochemical methods to be associated with the IMD. Taken together, this new co-localization strategy is effective in verifying the IMD association of proteins found by proteomic analyses, while facilitating the discovery of additional IMD-associated proteins. Importance The intracellular membrane domain (IMD) is a membrane subcompartment found in Mycobacterium smegmatis cells. Proteomic analysis of purified IMD identified more than 300 proteins, including enzymes involved in cell envelope biosynthesis. However, proteomics on its own is unlikely to detect every IMD-associated protein because of technical and biological limitations. Here, we describe fluorescent protein co-localization as an alternative, independent approach. Using a combination of fluorescence microscopy, proteomics, and subcellular fractionation, we identified three new proteins associated with the IMD. Such a robust method to rigorously define IMD proteins will benefit future investigations to decipher the synthesis, maintenance and functions of this membrane domain, and help delineate a more general mechanisms of subcellular protein localization in mycobacteria.

Mycobacteria share an unusually complex, multilayered cell envelope, which contributes to adaptation to changing environments. The plasma membrane is the deepest layer of the cell envelope and acts as the final permeability barrier against outside molecules. There is an obvious need to maintain the plasma membrane integrity, but the adaptive responses of plasma membrane to stress exposure remain poorly understood. Using chemical treatment and heat stress to fluidize the membrane, we show here that phosphatidylinositol (PI)-anchored plasma membrane glycolipids known as PI mannosides (PIMs) rapidly remodel their structures upon membrane fluidization in Mycobacterium smegmatis. Without membrane stress, PIMs are predominantly in a tri-acylated form: two acyl chains of PI moiety plus one acyl chain modified at one of the mannose residues. Upon membrane fluidization, the fourth fatty acid is added to the inositol moiety of PIMs, making them tetra-acylated variants. PIM inositol acylation is a rapid response independent of de novo protein synthesis, representing one of the fastest mass conversions of lipid molecules found in nature. Strikingly, we found that M. smegmatis is more resistant to the bactericidal effect of a cationic detergent after benzyl alcohol preexposure. We further demonstrate that fluidization-induced PIM inositol acylation is conserved in pathogens such as Mycobacterium tuberculosis and Mycobacterium abscessus. Our results demonstrate that mycobacteria possess a mechanism to sense plasma membrane fluidity change. We suggest that inositol acylation of PIMs is a novel membrane stress response that enables mycobacterial cells to resist membrane fluidization.

Mycobacteria diverge in a basic way from other bacterial and eukaryotic cells based on their distinct membrane structures. Here we report genome-wide transposon sequencing to discover the controllers of membrane compartmentalization in Mycobacterium smegmatis. cfa, a gene that encodes a putative cyclopropane-fatty-acyl-phospholipid synthase, shows the most significant effect on recovery from a membrane destabilizer, dibucaine. Lipidomic analysis of cfa deletion mutants demonstrates an essential role of Cfa in the synthesis of specific membrane lipids containing a C19:0 monomethyl-branched stearic acid. This molecule, also known as tuberculostearic acid (TBSA), has been intensively studied for decades due to its high level and genus-specific expression in mycobacteria. The proposed Cfa-mediated conversion of an unsaturation to a methylation matched well with its proposed role in lateral membrane organization, so we used new tools to determine the non-redundant effects of Cfa and TBSA in mycobacterial cells. cfa expression regulated major classes of membrane lipids including phosphatidylinositols, phosphatidylethanolamines and phosphatidylinositol mannosides. Cfa localized within the intracellular membrane domain (IMD), where it controls both cellular growth and recovery from membrane fluidization by facilitating subpolar localization of the IMD. Overall, cfa controls lateral membrane partitioning but does not detectably alter orthogonal transmembrane permeability. More generally, these results support the proposed role of the subpolar IMD as a subcellular site of mycobacterial control of membrane function.