Synergistic effects of tRNA modification defects inEscherichia coliK12

Abstract Transfer RNAs (tRNAs) are essential components of the translation machinery and carry numerous post-transcriptional modifications that contribute to decoding accuracy, efficiency, and cellular fitness. In Escherichia coli K 12, all tRNA modification pathways have been identified, yet the functional interactions between these pathways remain largely unexplored. Here, we systematically analyze genetic interactions between 29 non essential tRNA modification genes using a pairwise synthetic lethal screen based on P1 transduction. Most combinations of tRNA modification gene deletions are tolerated during growth in rich medium; however, we identify five synthetically lethal pairs and fifteen additional combinations that display negative genetic interactions. Deletions of truA, which encodes the pseudouridine synthase responsible for modifications at positions 38 to 40 of multiple tRNAs, show the highest frequency of negative epistasis. Synthetic lethality associated with truA can be complemented by expression of truA in trans and, in specific cases, partially suppressed by overexpression of its tRNA substrates, indicating substrate specific functional dependencies. Analysis of tRNA abundance by northern blotting and AQRNA seq demonstrates that the loss of individual tRNA modification enzymes does not generally lead to widespread tRNA destabilization. Instead, further phenotypic characterization of viable double mutants reveals condition-dependent growth defects influenced by carbon source, temperature, and metabolic stress, as well as toxicity associated with overexpression of specific tRNAs. Together, these results reveal a limited but distinct set of genetic interactions among bacterial tRNA modification pathways and highlight the importance of physiological context in uncovering their cellular roles. Competing Interest Statement The authors have declared no competing interest. Footnotes I had forgotten one author in the list https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE331519