Alanine dependence of trans-translation contributes to riboregulation of mycobacterial antibiotic recalcitrance genes

Antibiotic recalcitrance refers to a slower rate of death for either a bacterial population or a subpopulation of cells upon antibiotic exposure. It complicates treatment of many bacterial infections by contributing to treatment length, treatment failure, disease recurrence, and the emergence of antimicrobial resistance (AMR). Thus, blocking antibiotic recalcitrance could be a powerful strategy for improving treatment outcomes and reducing AMR rates. Here, using a forward genetic method for the isolation of antibiotic-recalcitrant mutants, we isolated two Mycobacterium smegmatis strains with mutations in the tRNA-modifying enzyme adenine-N(1)-methyltransferase. Both mutants were recalcitrant to proteostasis-perturbing antibiotics. We linked these phenotypes to upregulation of the transcriptional regulator WhiB7, highlighting its role as a point of convergence in the regulation of multiple mechanisms of antibiotic recalcitrance and resistance. Further, we identified a mechanism by which the amino acid alanine couples trans -translation to ribosome regulation-dependent, WhiB7-mediated expression of antibiotic resistance and recalcitrance genes, allowing bacterial cells to engage seemingly mutually exclusive mechanisms of survival upon exposure to stress.