EnvZ/OmpR-dependent OmpF induction contributes to colistin-enhanced plasmid conjugation

Plasmid-mediated horizontal gene transfer plays a pivotal role in accelerating the dissemination of antimicrobial resistance. However, the molecular mechanisms linking antibiotic-induced envelope stress to conjugation remain incompletely understood. Here, we demonstrate that sub-inhibitory colistin significantly enhances conjugative transfer of the RP4 and multiple clinically relevant resistance plasmids in E. coli without affecting bacterial growth. This enhancement was also observed under biofilm-forming conditions. Mechanistically, colistin induces envelope perturbation characterized by increased membrane permeability, elevated lipopolysaccharide release, and structural damage to the membrane. This remodeling is accompanied by selective upregulation of the outer membrane porin OmpF, whereas OmpC remains unchanged. Genetic analyses showed that OmpF is important for the enhancement of plasmid transfer observed under colistin exposure in both donor and recipient strains. Upstream regulatory analysis identified the EnvZ/OmpR two-component system as the principal pathway mediating OmpF induction. Colistin exposure increased envZ and ompR transcription and promoted OmpR phosphorylation, while electrophoretic mobility shift assays confirmed that OmpR binds to the ompF promoter with enhanced activity. Further analyses showed that this activation is not attributable to classical osmotic stress, as neither NaCl nor sucrose induced comparable responses, whereas supplementation with Mg^2 + or Ca²⁺ attenuated colistin-induced gene expression. Collectively, this study uncovers a potential molecular link between colistin-induced envelope stress and horizontal gene transfer in bacteria, providing mechanistic insight into antibiotic-induced horizontal gene transfer.