The next generation of the Efflux Platform: A genetic toolkit to study compound transport across the Escherichia coli cell envelope

Overcoming intrinsic antimicrobial resistance requires an in-depth understanding of compound permeation and transport across the bacterial cell envelope. The outer membrane (OM) is a formidable barrier for entry, which synergizes with the activities of drug efflux pump systems. To facilitate the study of drug efflux pump function, we previously reported the generation of EKO-35 (Efflux Knockout-35), a highly susceptible Escherichia coli K-12 (BW25113) mutant devoid of 35 known or putative drug efflux pumps (BioProject ID PRJNA838981). Here, we report the next-generation of this mutant, EKO-35-2.0. Efflux pump-encoding genes were disrupted using CRISPR-Cas9 mediated counter selection, producing a pan efflux-deficient mutant that harbours only one secondary nonsynonymous mutation compared to the parental strain. Additionally, E. coli K-12 laboratory-associated cell envelope mutations were repaired including restoration of the wbbL locus, enabling production of O-antigen, and the BW25113-associated fabR missense mutation. To enable the study of compound permeation, we also integrated an open and nonselective variant of the OM siderophore transporter FhuA, referred to as a Pore, in each of these strains.