Deep mutational scan of a drug efflux pump reveals its structure-function landscape

Drug efflux is a common resistance mechanism found in bacteria and cancer cells. Although several structures of drug efflux pumps are available, they provide only limited functional information on the phenomenon of drug efflux. Here, we performed deep mutational scanning (DMS) on the bacterial ATP binding cassette (ABC) transporter EfrCD from Enterococcus faecalis to determine the drug efflux activity profile of more than 1400 single variants Plasmid libraries (triplicates rep1-3) of EfrCD variants were expressed in Lactococcus lactis NZ9000 ΔlmrCD ΔlmrA and subjected to one round of growth competition in the presence of drugs (Daunorubicin, Ethidium and Hoechst 33342). Plasmids from input library (t0) and after one round of growth competition (t1) were extracted. The coding region of EfrCD was sequenced by Illumina sequencing and reads for individual variants were counted.