Knockout of tusA confers cationic antimicrobial resistance via fur and omp genes in Escherichia coli

tRNA 2-thiouridine synthesizing protein A (TusA), a sulfur-carrier protein, plays a crucial role in tRNA sulfur modification. Several studies have reported that tusA deficiency affects iron–sulfur (Fe–S) homeostasis in addition to tRNA sulfur modification, resulting in pleiotropic phenotypes. In this study, we analyzed the phenotype of tusA-deficient Escherichia coli and its underlying mechanisms. Although the Keio tusA knockout strain showed increased swimming motility and flagellar biosynthesis, these phenotypes were not restored by tusA complementation. Genome resequencing of the original Keio tusA knockout strain identified an unintended secondary mutation in lrhA, a transcriptional regulator of flagellar and chemotaxis genes. This secondary mutation impaired lrhA function, contributing to enhanced flagellar synthesis and swimming motility, as well as altered global gene expression. A tusA knockout strain in which the secondary mutation was corrected (ΔtusA) exhibited reduced swimming motility compared with the wild-type strain, despite showing no abnormalities in flagellar formation. Furthermore, ΔtusA displayed increased resistance to cationic antibacterial agents, including cetyltrimethylammonium bromide, cetylpyridinium chloride, and protamine sulfate. The reduced motility caused by tusA deletion was observed independently of Fur, a global regulator of iron homeostasis, whereas resistance to cationic antibacterial agents was abolished in the fur knockout background. In addition, altered expression of outer membrane protein (omp) genes was observed in ΔtusA, and deletion of these omp genes abolished resistance to cationic antibacterial agents. Together, these results indicate that, by disentangling the phenotypic effects of tusA deletion from those of the unintended secondary mutation, loss of tusA confers resistance to cationic antibacterial agents through a Fur-dependent and Omp-dependent mechanism.