Alteration of the repressor activity of MarR, the negative regulator of the Escherichia coli marRAB locus, by multiple chemicals in vitro - PubMed (original) (raw)

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

M N Alekshun et al. J Bacteriol. 1999 Aug.

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.

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Figures

FIG. 1

FIG. 1

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

FIG. 2

FIG. 2

Binding of MarR to marO assayed by a restriction enzyme site protection assay. In the absence of MarR, the _Ssp_I 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 Ssp_I restriction site results in fragment A (4,576 bp) and indicates protection of the _Ssp_I 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 _Bam_HI (linear form); D, pSup-Test digested with _Ssp_I (double-cut form).

FIG. 3

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 _Ssp_I, 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 _Ssp_I. (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.

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