Functional promiscuity of small multidrug resistance transporters from Staphylococcus aureus, Pseudomonas aeruginosa, and Francisella tularensis

Small multidrug resistance transporters efflux toxic compounds from bacteria and are a minimal system to understand multidrug transport. Most previous studies have focused on EmrE, the model SMR from Escherichia coli, finding that EmrE has a broader substrate profile than previously thought and that EmrE may perform multiple types of transport, resulting in substrate‐dependent resistance or susceptibility. Here, we performed a broad screen to identify potential substrates of three other SMRs: PAsmr from Pseudomonas aeruginosa; FTsmr from Francisella tularensis; and SAsmr from Staphylococcus aureus. This screen tested metabolic differences in E. coli expressing each transporter versus an inactive mutant, for a clean comparison of sequence and substrate‐specific differences in transporter function, and identified many substrates for each transporter. In general, resistance compounds were charged, and susceptibility substrates were uncharged, but hydrophobicity was not correlated with phenotype. Two resistance hits and two susceptibility hits were validated via growth assays and IC50 calculations. Susceptibility is proposed to occur via substrate‐gated proton leak, and the addition of bicarbonate antagonizes the susceptibility phenotype, consistent with this hypothesis. Three SMRs from high‐priority pathogens were heterologously expressed in Escherichia coli and compared to transport‐dead mutants. A broad screen revealed that these transporters can confer both resistance and susceptibility to many compounds, consistent with the behavior of the model SMR EmrE. As EmrE and these SMRs were previously thought to only confer resistance, this implies greater functional promiscuity than previously understood across the Qac subfamily of SMRs.