Titrating Levels of TolC in E. coli: A Sensitive Approach to Quantifying Efflux

The susceptibility of small molecules to Gram-negative bacterial efflux is typically evaluated using an antibacterial activity-based efflux ratio, which is computed as the ratio of the antibacterial activity for a wild-type strain and its isogenic efflux mutant (typically lacking genes encoding major efflux pumps). The magnitude of the ratio is often used as an efflux index. However, early in drug discovery, hits with suboptimal physicochemical properties often lack whole cell inhibition against wild-type strains, which makes efflux ratios indeterminable. To address this gap, we developed an assay to titrate levels of total efflux by varying the TolC expression using an arabinose-inducible promoter (pBAD) in an Escherichia coli Δ tolC strain. We provide a proof of concept for the assay using sets of related compounds from two antibiotic classes and show that the TolC titration provides a sensitive method for rank ordering compounds with respect to their efflux susceptibility.