Deletions of recombination genes impair tandem amplification and reshape heteroresistance mechanisms in Escherichia coli

ABSTRACT Heteroresistance is a transient resistance phenotype characterized by the presence of small subpopulations of bacterial cells with elevated antibiotic resistance within a susceptible main population. In gram-negative pathogens, heteroresistance is frequently caused by tandem amplification of genes encoding resistance proteins with low activity toward the antibiotic, a process commonly mediated by homologous recombination between flanking repeated sequences. However, the specific roles of individual recombination proteins in this mechanism remain largely undefined. In this study, we systematically evaluated the contribution of 19 recombination-associated genes to tandem amplification-driven heteroresistance in Escherichia coli . A clinical plasmid causing tobramycin heteroresistance by tandem amplification of the aac(3)-IId gene was conjugated into recombination gene-deficient mutants and the wild-type parental strain. While heteroresistance was observed with all mutants, the frequency of resistant subpopulations was decreased in recA and recB mutants, and a shift in resistance mechanism toward increased plasmid copy number and resistance mutations was observed. Partially reduced frequencies of tandem amplifications and a shift toward other heteroresistance mechanisms were also observed with recC , recJ, ruvA , and ruvC mutants, whereas other deletions of recombination genes had no or little impact on tandem amplifications. These findings identify RecABC as a key pathway in heteroresistance via tandem amplification, but even when these genes are deleted, resistant subpopulations can still be generated by other mechanisms. IMPORTANCE Heteroresistance poses a threat to efficient antibiotic treatment, as the rare resistant subpopulations often go undetected by standard laboratory tests. In Escherichia coli , heteroresistance often arises by tandem gene amplification of a resistance gene with low activity toward the specific antibiotic. This amplification is thought to be mediated by homologous recombination between repeat sequences. However, the specific roles of individual recombination proteins in this process are unclear. Here, we systematically determined the specific roles of individual recombination proteins in this process by the individual deletion of 19 recombination-associated genes. The RecABC pathway was identified as a major contributor to amplification-driven heteroresistance, but even when this pathway was disrupted, resistant subpopulations still emerged through alternative mechanisms, revealing the remarkable adaptability of bacterial populations under antibiotic stress. These findings advance our understanding of the molecular flexibility underlying heteroresistance and highlight that strategies aimed at preventing gene amplification to reduce heteroresistance are unlikely to succeed.