Upregulation of the AcrAB2NodT efflux pump confers antibiotic resistance at the cost of collateral metal sensitivity

Bacterial resistance to antibiotics (AB) such as {beta}-lactams, fluoroquinolones, and aminoglycosides often emerges through mutations that alter AB targets, reduce membrane permeability, or increase the activity of AB-modifying enzymes and efflux pumps. Yet the physiological costs associated with AB resistance remain poorly understood. In Caulobacter vibrioides, a {Delta}tipR mutant that which constitutively upregulates the RND pump AcrAB2nodT, displays heightened sensitivity to copper (Cu), revealing a physiological vulnerability driven by the energetic burden imposed by excessive efflux activity. Deletion of acrAB2nodT in the {Delta}tipR background restored Cu resistance to wild-type levels, confirming the role of pump overexpression in metal sensitivity. Morphological and microscopy analyses revealed that pump overexpression leads to cell envelope defects and compromised fitness. To disentangle the effects of pump abundance from efflux activity, we engineered an AcrB2 transport-impaired mutant. This variant also rescued Cu resistance, demonstrating that high expression and active transport contribute to the observed toxicity. Notably, this sensitivity was not limited to Cu; the {Delta}tipR mutant also exhibited increased susceptibility to other transition metals, including zinc (Zn), nickel (Ni) and cadmium (Cd), suggesting a broader vulnerability linked to metal stress. Mechanistically, pump overexpression depleted the proton motive force, reduced ATP levels, and impaired motility, all of which are essential for Cu stress adaptation. This physiological tradeoff highlights the importance of precise efflux regulation and reveals a potential therapeutic vulnerability: targeting the cost of pump upregulation could enhance the efficacy of antimicrobial treatments. ImportanceWhile efflux pumps primarily protect bacteria from AB, their excessive activity can impose a fitness cost. This study underscores a crucial tradeoff between resistance and cellular fitness by connecting Cu sensitivity to heightened AcrAB2NodT efflux pump expression in Caulobacter vibrioides. This work challenges the assumption that more efflux always benefits the cell and shows that unregulated pump activity can undermine survival under metal stress. These findings broaden our understanding of bacterial stress physiology and suggest new ways to combat antimicrobial resistance by targeting the hidden costs of resistance mechanisms. Understanding this balance between resistance and fitness opens new perspectives for combination therapy against multidrug-resistant bacteria.