The chromosomal arsR gene of Escherichia coli encodes a trans-acting metalloregulatory protein

Abstract

Plasmid-encoded arsenical resistance (ars) operons confer high level resistance to arsenicals and antimonisis, while the chromosomally encoded ars operon of Escherichia coli bestows low level resistance. The transcriptional start site of the chromosomal ars mRNA was mapped by primer extension, and putative -10 and -35 promoter recognition sites were identified. The arsR gene, the first gene in this operon, was cloned using polymerase chain reaction. The arsR gene product, the ArsR repressor, was expressed and purified. The results of gel mobility shift assays indicated that the repressor is a DNA binding protein that binds to a fragment of DNA containing the chromosomal ars promoter. The specific binding site, as determined by DNase I footprint analysis, spans 33 nucleotides in the promoter region, including the putative -35 promoter element. By construction and expression of a series of in-frame fusions between truncated arsR genes and the coding region for the mature form of β-lactamase (blaM'), it was shown that ArsR is a trans-acting repressor that regulates expression of the chromosomal ars operon. In addition, the chromosomally-encoded repressor can regulate expression of the ars operon of plasmid R773, and the R773 repressor can cross-regulate expression from the chromosomal operon.