AdeR protein regulates adeABC expression by binding to a direct-repeat motif in the intercistronic spacer

Abstract

Overexpression of the efflux pump AdeABC is associated with tigecycline resistance of multi-drug resistant Acinetobacter baumannii (MDRAB). A two-component regulatory system, sensor AdeS and regulator AdeR proteins regulate the pump. However, the detailed mechanism of the AdeR protein to enhance the expression of adeABC operon is not well defined. We illustrated the biological characteristics of AdeR proteins by comparing a mutant AdeR protein of a tigecycline resistant MDRAB to the wild AdeR protein. By analyzing a series of deletion constructs, a minimal gene cassette of the intercistronic spacer DNA fragment specifically bound with the adeR protein and resulted in band shifting in electrophoresis mobility shifting assays (EMSA). A conserve direct repeat motif was observed in the intercistronic spacer DNA. We demonstrated the AdeR protein was a direct-repeat-binding protein. Two common residue mutations on the AdeR proteins of tigecycline resistant MDRAB isolates could reduce their binding affinity with the intercistronic spacer. The free intercistronic spacer may then more efficiently support the read-through of the adeABC operon during the co-transcriptional translation in tigecycline resistant MDRAB isolates.

Mechanistic insight into how multidrug resistant Acinetobacter baumannii response regulator AdeR recognizes an intercistronic region

“…The molecular weight determination was cross-validated using small angle X-ray scattering ( Supplementary Table S2 ). A DNA fragment consisting of the direct repeat sequence in the intercistronic region between adeR and the RND efflux pump adeABC was previously identified to interact with AdeR ( 23 ). Here, we show that both AdeR_FL and AdeR_DBD alone can bind a 25 bp DNA fragment containing this direct repeat sequence, forming a complex with a distinct chromatographic behavior (Figure 1A ).…”

“…The response regulator AdeR consists of a CheY-like receiver domain (RD, amino acid residues 1–127) and an OmpR/PhoB type DNA binding domain (DBD, residues 138–247). Unlike most OmpR/PhoB type response regulators which bind to the promoter region of their target genes ( 21 , 22 ), AdeR recognizes a 10bp perfect direct-repeat DNA sequence in a 168bp intercistronic region between adeR and adeABC ( 23 ).…”

Resistance to pentamidine is mediated by AdeAB, regulated by AdeRS, and influenced by growth conditions in Acinetobacter baumannii ATCC 17978

“…The genetic arrangement of the AdeABC system places adeABC in an operon that is divergently transcribed to the regulatory adeRS two component signal transduction system (TCSTS). Expression of adeABC occurs by binding of AdeR to a ten base-pair direct repeat motif found within the intercistronic region separating these operons [ 18 , 19 ]. Many clinical A .…”

Structure and mechanism of bacterial tripartite efflux pumps

“…Other examples for two component systems that regulate efflux pump expression include AmgRS in Pseudomonas aeruginosa, CpxAR in Enterobacteriaceae, and AdeSR in Acinetobacter baumannii [134]. In the latter case, AdeR (the response regulator) activates the expression of the adeABC tripartite system genes by binding to a direct-repeat motif in the intercistronic spacer [135]. Interestingly, at least some two component systems seem to be capable of multisystem regulation: The BaeSR system in Salmonella typhimurium, for instance, has been shown to regulate both acrD and mdtABC expression [136].…”