Biology subjects

Brenner, J. R.

Publications and source records attributed to Brenner, J. R..

3 recordsLinked to original sources

bioRxiv2022

Compartmentalized Cell Envelope Biosynthesis in Mycobacterium tuberculosis

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, 1,3-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. IMPORTANCEM. tuberculosis remains an important public health threat, with more than one million deaths every year. The pathogens ability to survive in the human host for decades highlights the importance of understanding how this bacterium regulates and coordinates its metabolism, cell envelope elongation, and growth. The IMD is a membrane structure that associates with the subpolar growth zone of actively growing mycobacteria, but its existence is only known in a non- pathogenic model, M. smegmatis. Here, we demonstrated the presence of the IMD in M. tuberculosis, making the IMD an evolutionarily conserved plasma membrane compartment in mycobacteria. Furthermore, our study revealed that the IMD is the factory for synthesizing phenolic glycolipids, virulence factors produced by slow-growing pathogenic species.

microbiology↗

bioRxiv2021

Arginine methylation helps SepIVA balance regulation of septation and elongation in Mycobacterium smegmatis

Growth of mycobacterial cells requires successful coordination between elongation and septation of the cell wall. However, it is not clear which factors directly mediate this coordination. Here, we studied the function and post-translational modification of an essential division factor, SepIVA, in Mycobacterium smegmatis. We find that SepIVA is arginine methylated, and that alteration of these methylation sites affects both septation and polar elongation of Msmeg. Furthermore, we show that SepIVA regulates the localization of MurG, and that this regulation may impact polar elongation. Finally, we map SepIVAs two regulatory functions to different sites on the protein: the N-terminus regulates elongation while the C-terminus regulates division. These results establish SepIVA as a regulator of both elongation and division and characterize a physiological role for protein arginine methylation sites for the first time in mycobacteria.

microbiology↗

bioRxiv2021

Fluorescence Imaging-Based Discovery of Membrane Domain-Associated Proteins in Mycobacterium smegmatis

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 envelope biosynthesis. While we have identified more than 300 IMD-associated proteins by proteomic analyses, only a handful of these have been verified by other experimental methods. Furthermore, we speculate that 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 expressing a Dendra2-FLAG-tagged recombinant protein. We identified 29 fusion proteins that showed fluorescence patterns similar to those of IMD proteins and, consistent with this co-localization, we had previously identified 20 of these using a proteomics approach. Of the nine remaining IMD candidate proteins, three were confirmed to be associated with the IMD while some others appear to be lipid droplet-associated. Taken together, our newly devised strategy is effective in verifying the IMD association of proteins found by proteomic analyses, while facilitating the discovery of additional IMD-associated proteins. ImportanceThe 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, that likely contribute to the function of the IMD. How can we find more IMD-associated proteins that escaped proteomic detection? Here, as an alternative approach, fluorescence microscope images of 523 proteins were screened to identify IMD-associated proteins. We confirmed the IMD association of previously identified proteins and discovered three additional proteins associated with the IMD. Together, subcellular fractionation, proteomics, and fluorescence microscopy form a robust combination to more rigorously define IMD proteins, which will aid future investigations to decipher the synthesis, maintenance and functions of this membrane domain.

microbiology↗