No abstract

The genus Mycobacterium contains several slow-growing human pathogens, including Mycobacterium tuberculosis , Mycobacterium leprae , and Mycobacterium avium . Mycobacterium smegmatis is a nonpathogenic and fast growing species within this genus. In 1990, a mutant of M. smegmatis , designated mc 2 155, that could be transformed with episomal plasmids was isolated, elevating M. smegmatis to model status as the ideal surrogate for mycobacterial research.

Lateral partitioning of proteins and lipids shapes membrane function. In model membranes, partitioning can be influenced by interactions with other membranes and solid supports. While cellular membranes can departition in response to various perturbations, including disruption of bilayer-extrinsic structures, the mechanisms by which they partition de novo are largely unknown. The plasma membrane of Mycobacterium smegmatis can be spatially and biochemically departitioned by the fluidizing agent benzyl alcohol. By screening for mutants that are sensitive to benzyl alcohol, we show that the bifunctional cell wall synthase PonA2 promotes membrane partitioning and cell growth upon fluidizer washout. The role of PonA2 in membrane repartitioning and regrowth depends solely on its conserved transglycosylase domain. We find that the cell wall polymer, but not active cell wall polymerization, is critical for membrane partitioning. Our work highlights a key initiating role for bilayer-extrinsic structures in patterning cellular membranes.

Bacterial cell division requires identification of the division site, assembly of the division machinery, and constriction of the cell envelope. These processes are regulated in response to several cellular and environmental signals. Here, we use small molecule iron chelators to characterize the surprising connections between bacterial iron homeostasis and cell division. We demonstrate that iron starvation downregulates the transcription of genes encoding proteins involved in cell division, reduces protein biosynthesis, and prevents correct positioning of the division machinery at the division site. These combined events arrest the constriction of the cell during late stages of cytokinesis in a manner distinct from known mechanisms of inhibiting cell division. Overexpression of genes encoding cell division proteins or iron transporters partially suppresses the biological activity of iron chelators and restores growth and division. We propose a model demonstrating the effect of iron availability on the regulatory mechanisms coordinating division in response to the nutritional state of the cell.

The intracellular membrane domain (IMD) is a metabolically active and laterally discrete membrane domain initially discovered in Mycobacterium smegmatis. The IMD correlates both temporally and spatially with the polar cell envelope elongation in M. smegmatis. Whether or not a similar membrane domain exists in pathogenic species remains unknown. Here we show that the IMD is a conserved membrane structure found in Mycobacterium tuberculosis. We used two independent approaches, density gradient fractionation of membrane domains and visualization of IMD-associated proteins through fluorescence microscopy, to determine the characteristics of the plasma membrane compartmentalization in M. tuberculosis. Proteomic analysis revealed that the IMD is enriched in metabolic enzymes that are involved in the synthesis of conserved cell envelope components such as peptidoglycan, arabinogalactan, and phosphatidylinositol mannosides. Using a fluorescent protein fusion of IMD-associated proteins, we demonstrated that this domain is concentrated in the polar region of the rod-shaped cells, where active cell envelope biosynthesis is taking place. Proteomic analysis further revealed the enrichment of enzymes involved in synthesis of phthiocerol dimycocerosates and phenolic glycolipids in the IMD. We validated the IMD association of two enzymes, α1,3-fucosyltransferase and fucosyl 4-O-methyltransferase, which are involved in the final maturation steps of phenolic glycolipid biosynthesis. Taken together, these data indicate that functional compartmentalization of membrane is an evolutionarily conserved feature found in both M. tuberculosis and M. smegmatis, and M. tuberculosis utilizes this membrane location for the synthesis of its surface-exposed lipid virulence factors.