MSMEG_1353 is a critical determinant of metabolic and cell envelope homeostasis in mycobacteria

The mycobacterial cell envelope is central to antibiotic resistance and stress adaptation, maintained by a network of essential proteins. Owing to their evolutionary divergence from other model organisms, mycobacteria encode numerous genus-specific genes that remain functionally uncharacterized, including the network of these essential genes. Here, we define MSMEG_1353 as a previously uncharacterized, essential ATP binding protein that plays a role in mycobacterial metabolism and cell envelope integrity. Conditional depletion of MSMEG_1353 resulted in severe growth attenuation, and the loss of characteristic mycobacterial cording. Further, the knockdown strain exhibited altered cell envelope permeability and an unusual antibiotic susceptibility profile, marked by isoniazid resistance accompanied with hypersensitivity to vancomycin. Functional analysis revealed pronounced membrane depolarisation and severe envelope defects, including distortion of outer membrane layers. Concomitantly, integrated transcriptomic and metabolomic profiling demonstrated widespread suppression of carbon metabolism accompanied by cofactor imbalance and redox perturbation, indicating a global metabolic rerouting upon MSMEG_1353 depletion. At the level of cell envelope composition, the lipid profiles indicate depletion of phthiocerol dimycocerosate (PDIM), trehalose polyphleate (TPP), and elevated levels of mycolic acid in the knockdown strain. Further, localisation studies revealed that MSMEG_1353 forms discrete clusters along the cell, with preferential enrichment at the old pole. Collectively, our findings establish MSMEG_1353 as a critical determinant of metabolic homeostasis and envelope permeability in Mycobacterium smegmatis, linking central carbon metabolism, and adaptive cell envelope remodeling.