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

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.

Deletion mutants ΔmarA, ΔyadG, ΔyadH, 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.

MIC assays revealed that single deletions of yadG or yadH had negligible effects on most antibiotics. However, in the ΔacrB background, deleting yadGH (ΔacrBΔyadG and ΔacrBΔyadH) 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 ΔmarA 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.

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.