The Wobble Uridine tRNA Writer MnmA Shapes Codon-Dependent Stress Response Systems

<jats:p>Escherichia coli uses wobble uridine (U34) modifications to tune codon decoding, but how individual tRNA writer enzymes shape gene expression remains unclear. Here, we identify MnmA, the U34 thiolation enzyme for tRNALys, tRNAGln, and tRNAGlu, as a central regulator linking codon-directed translation to regulon control and stress response. Loss of MnmA depleted s2U-dependent wobble modifications, preventing geranyl- (ges2U) and seleno- (se2U)- based modifications, causing growth defects, reduced catalase activity, and multi-level gene expression dysregulation. The ∆mnmA cells showed broad adaptive transcriptional reprogramming associated with RpoS- and OxyR-regulated pathways, which was accompanied by compromised protein output. Endogenous and tagged-protein analyses revealed specific impairment of transcriptional regulators, adaptive and detoxification proteins, including RpoS, OxyR, FliA, KatE, and KatG. Polysome profiling and polysome-associated RNA sequencing showed that MnmA deficiency globally reduces translational capacity and uncouples mRNA abundance from translational efficiency, which is exacerbated during oxidative stress. We developed genome-wide codon-usage mapping analytics to identify five codon-defined gene clusters, with specific clusters enriched for Lys, Gln, and Glu codons disproportionately affected by MnmA loss. Together, these findings support that wobble uridine thiolation and downstream modifications pair with corresponding codon architecture to coordinate the translation of regulon controllers and stress-response networks linked to bacterial fitness.</jats:p>