Methods for investigating functional interplay between efflux pumps in Escherichia coli.

Efflux pumps play a critical role in antimicrobial resistance (AMR) and contribute to diverse physiological processes in Escherichia coli. Diderm bacteria, such as E. coli, employ two structural types of efflux pumps-single-component inner membrane pumps and multi-component cell envelope-spanning systems-to detoxify the cell. These distinct types of efflux pumps can exhibit cooperative and often synergistic impacts on resistance phenotypes, particularly when removing compounds with cytoplasmic targets. This coordinated action, also known as functional interplay, ensures efficient removal of environmental stressors. To facilitate a deeper understanding of these complex resistance phenotypes, we describe a genetic platform and methodological workflows for investigating the functional interplay amongst E. coli efflux pumps. These approaches rely on efflux-deficient E. coli mutants to overcome well-described AMR redundancies within the efflux network. We detail protocols for assessing the effects of dual efflux pump combinations on resistance phenotypes in E. coli, including strain construction and phenotypic assessment to quantify the impacts of functional interplay. These methods could be adapted to the study of other bacterial species.