Cu(II) and Zn(II) Enhance Antibiotic Resistance Gene Transformation by Regulating Type IV Pili-Mediated DNA Uptake

Natural transformation represents one of the major dissemination pathways of antibiotic resistance genes (ARGs) in the environment. It can be facilitated by heavy metals, which is traditionally attributed to the induction of oxidative stress and an SOS response. Here, we demonstrate an alternative mechanism involving type IV pili (T4P), the molecular machinery for extracellular DNA (eDNA) uptake. Specifically, 2 μM Cu(II) and Zn(II) significantly enhanced transformation in Acinetobacter baylyi by 28% and 46% (p < 0.05) and in soil communities by 51% and 74% (p < 0.05). T4P sensitively responded to the two metals at concentrations as low as 0.5 μM within 2 h, preceding any detectable oxidative stress responses. At the initial phase of T4P-mediated uptake (DNA binding), Cu(II) enhanced extracellular ARGs adhesion by inducing DNA condensation and strengthening T4P-DNA binding via ionic bridging. During the latter phase (DNA pulling), Zn(II) functioned as a cofactor for T4P ATPase, stimulating pilus extension-retraction dynamics to facilitate DNA transformation. At higher concentrations (8 μM), the CpiR-CpiA regulatory axis serves as a molecular brake, curtailing T4P dynamics by derepressing the PilB-inhibitor CpiA and thereby attenuating transformation enhancement. These findings reveal that Cu and Zn interfere with different phases of T4P-mediated DNA uptake, highlighting a previously unrecognized mechanism by which metals accelerate ARG dissemination in the environment.