Integrating classical genetics and whole-genome sequencing to reveal the chromosomal basis of hygromycin resistance in Escherichia coli

Hygromycin B (HygB), a broad-spectrum antibiotic, is widely used in molecular biology, agriculture, and veterinary medicine. It inhibits protein synthesis by binding to ribosomes, and its plasmid-borne resistance gene serves as a selectable marker for applications in gene manipulation technologies. The binding site of the P1 phage-borne toxin Doc, which induces bacterial apoptosis, overlaps with the binding site of HygB in helix h44 of 16S rRNA. Hence isolation and characterization of chromosomal HygB resistance would largely serve as gateway to understand the less studied but imperative and emerging modes of drug resistance like bacterial multicellularity and heteroresistance. In this study we have investigated the chromosomal origin of HygB resistance in E. coli through a combination of classical genetics and whole-genome sequencing. Eight HygB-resistant mutants were analyzed, and co-transduction experiments revealed a narrow region (71.8–75.8 min) to have conferred resistance. Whole-genome sequencing confirmed a single base pair change in the fusA gene (A1754 to G1754) as the cause. This is a maiden report on a missense mutation of fusA leading to HygB resistance and these findings provide valuable insights into the mechanisms of HygB resistance.