MarA binds and activates the ABC efflux pump YadGH to confer conditional macrolide resistance in AcrB-deficient Escherichia coli

Background The global transcriptional regulator MarA drives multidrug resistance in Escherichia coli . MarA regulation of Resistance-Nodulation-Cell Division (RND) efflux pumps such as AcrAB-TolC is well established, but its control over ATP-binding cassette (ABC)-type efflux pumps remains unclear. The ABC efflux pump YadGH exports macrolides, yet its upstream transcriptional regulation is unknown. Methods Deletion mutants Δ marA , Δ yadG , Δ yadH , and double mutants were constructed in E. coli K-12 MG1655. Minimum inhibitory concentrations (MICs) of 15 antibiotics were measured by broth microdilution. YadGH transcription was assessed by qRT-PCR. MarA binding to the yadGH promoter was verified by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation quantitative real-time PCR (ChIP-qPCR). Biofilm formation and swarming motility were assayed. Results MIC assays revealed that single deletions of yadG or yadH had negligible effects on most antibiotics. However, in the Δ acrB background, deleting yadGH (Δ acrB Δ yadG and Δ acrB Δ yadH ) increased sensitivity to erythromycin and tylosin, indicating that YadGH conditionally contributes to macrolide resistance specifically when the major efflux pump AcrB is absent. qRT-PCR showed that yadGH transcription is induced in a MarA-dependent manner in E. coli K-12. The Δ marA mutant phenocopied this susceptibility profile, supporting the role of yadGH as a downstream effector of MarA within this genetic context. ChIP-qPCR confirmed MarA binding to the yadGH promoter, and EMSA mapped a degenerate marbox motif upstream of the yadG start codon. Genome annotation lists yadG and yadH as a two-gene operon. Conclusion MarA binds to the yadGH promoter and positively regulates its transcription. YadGH serves as a conditional auxiliary efflux pump for macrolides when AcrAB-TolC is absent. Deletion of yadGH also affects biofilm formation and motility.