MtrA acts as a bifunctional modulator of the GlnR-controlled nitrogen regulon in Mycobacterium smegmatis

Nitrogen metabolism is a crucial factor in bacterial survival and adaptation, its regulation in Mycobacterium spp. remains only partly understood. In this study, we identify a previously unrecognized role for the response regulator MtrA in the regulation of nitrogen metabolism in Mycobacterium smegmatis alongside the well-known master regulator GlnR. Transcriptomic profiling under nitrogen-limiting conditions revealed that MtrA modulates the GlnR regulon by exerting both supportive and opposing effects. For one subset of genes, including those encoding enzymes for proline and histidine utilization, MtrA acts as a positive regulator with GlnR to achieve maximal induction. For another subset, particularly nutrient transport systems such as amino acid permeases and ammonium transporters, MtrA functions antagonistically, repressing expression to prevent metabolic imbalance and maintain carbon-nitrogen balance. Electrophoretic mobility shift assays confirmed that, although MtrA and GlnR share similar binding motifs, they recognize distinct structural features, thereby providing specific control over this regulatory system. Physiological analyses corroborated these findings, showing that the loss of MtrA impaired growth on histidine and proline but enhanced growth on nitrogen sources derived from purines. Together, these data establish a hierarchical model in which GlnR serves as the master nitrogen regulator, while MtrA fine-tunes gene expression to balance nutrient acquisition with cellular state. This dual control mechanism provides an adaptive advantage for mycobacteria in nutrient-limited environments.