Alteration of the repressor activity of MarR, the negative regulator of the Escherichia coli marRAB locus, by multiple chemicals in vitro

Abstract

MarR negatively regulates expression of the multiple antibiotic resistance operon (marRAB) in Escherichia coli. In this study, it was demonstrated that sodium salicylate, plumbagin, 2, 4-dinitrophenol, and menadione-inducers of the marRAB operon in whole cells-all interfered with the repressor activity of MarR in vitro. It is proposed that these compounds can interact directly with MarR to affect its repressor activity.

FIG. 1

Map of plasmid pSup-Test showing the positions of the SspI sites within the plasmid.

FIG. 2

Binding of MarR to marO assayed by a restriction enzyme site protection assay. In the absence of MarR, the SspI recognition sequence in marO is not protected and the plasmid is cut into two pieces of 3,472 bp (fragment B) and 1,104 bp (fragment C) in length. A single cut in the non-marO SspI restriction site results in fragment A (4,576 bp) and indicates protection of the SspI site in marO. Serial increases in the concentration of MarR led to increased protection, as indicated by the conversion of the two smaller bands, fragments B and C, to a single 4,756-bp fragment. MW, molecular weight standards; U, uncut pSup-Test from the original plasmid preparation; L, pSup-Test digested with BamHI (linear form); D, pSup-Test digested with SspI (double-cut form).

FIG. 3

Restriction enzyme site protection assays in the presence of structurally dissimilar compounds. In all panels, lane 1 shows molecular weight standards, lane 2 shows the vector (pSup-Test [3.4 nM]) alone digested with SspI, and lane 3 shows pSup-Test in the presence of MarR (2.92 to 3.6 μg or 9.1 to 11.2 μM [assuming the monomeric form of MarR]) digested with SspI. (A) The concentrations of sodium salicylate in lanes 4 to 6 were 0.8, 2, and 5 mM. (B) Lane 4, paraquat at a concentration of 5 mM. (C) Plumbagin (lanes 4 to 6) and 2,4-dinitrophenol (lanes 7 to 9) were tested at concentrations of 0.25, 0.5, and 1 mM; menadione (lanes 10 to 12) was tested at concentrations of 0.8, 2, and 5 mM.