From drug export to envelope fitness: characterizing the regulatory landscape of selective Escherichia coli ABC efflux pumps

Abstract

Antimicrobial resistance remains a top global health threat. While ATP-binding cassette (ABC) efflux pumps contribute to multidrug resistance in Gram-negative bacteria, their transcriptional regulation is far less understood than that of the RND and MFS families. We used DNA pull-down followed by LC–MS/MS to identify proteins binding to the promoters of five E. coli ABC efflux pump genes ( msbA , macB , ybhF , yddA , and yadG ). Candidate regulators were analyzed using bioinformatics, domain scanning, and molecular docking. Binding patterns differed sharply across the five genes. MsbA , macB , and ybhF pulled down complex interaction networks, while yddA and yadG bound far fewer regulators. CpxR was among the most highly enriched regulatory proteins, recovered at the msbA , macB , and ybhF promoters with fold changes of 30.57, 57.17, and 17.39, respectively. OmpR and ArcA exhibited significant enrichment at the ybhF promoter (FC = 64.77 and 238.28, respectively). Both regulators are implicated in osmotic and redox signaling, suggesting a potential link to ybhF transcriptional control under such conditions. LrhA was prominently enriched at the yddA promoter (FC = 103.21). This observation raises the possibility of a connection between LrhA and biofilm-related regulatory pathways, which remains to be validated experimentally. Structurally, the ybhFSR operon is organized as a split-type system: ybhF encodes the nucleotide-binding domains (NBDs), while ybhS and ybhR encode the transmembrane domains (TMDs). The system likely assembles with YbhG and TolC into a tripartite complex. By contrast, based on structural prediction, yddA and yddB may form a minimal two-subunit ABC transporter, marked by atypical Walker A (GYSGAGKTT) and ABC signature (LSSGE) motifs. Our data show that ABC pumps are tightly integrated into a regulatory network governing envelope stress, osmotic adaptation, and biofilm formation—not just drug export. These findings identify candidate regulators whose putative promoter binding requires experimental validation.