Affinity purification and binding characteristics of Citrobacter freundii AmpR, the transcriptional regulator of the ampC beta-lactamase gene

Abstract

The transcriptional regulator of the Citrobacter freundii ampC beta-lactamase gene, AmpR, was purified as a single SDS/PAGE-gel band by using various techniques, including DNA-Sepharose 4B affinity chromatography, The purified AmpR consisted of a 32.5 kDa monomer that interacted with three operator sequences: two binding sequences, at positions -75 to -70 and -67 to -51 with respect to the transcriptional start site, were located in the LysR motif (-72 to -60), and the third sequence was at position -43 to -38. Equilibrium binding studies raise the possibility that the adjacent operator sequence could exert a positive influence on the ability of AmpR to bind to these sites.