THE CHROMOSOMAL ARSR GENE OF ESCHERICHIA-COLI ENCODES A TRANS-ACTING METALLOREGULATORY PROTEIN

Plasmid-encoded arsenical resistance (ars) operons confer high level r esistance to arsenicals and antimonials, while the chromosomally encod ed ars operon of Escherichia coli bestows low level resistance. The tr anscriptional start site of the chromosomal ars mRNA was mapped by pri mer extension, and putative -10 and -35 promoter recognition sites wer e identified. The arsR gene, the first gene in this operon, was cloned using polymerase chain reaction. The arsR gene product, the ArsR repr essor, was expressed and purified. The results of gel mobility shift a ssays indicated that the repressor is a DNA binding protein that binds to a fragment of DNA containing the chromosomal ars promoter. The spe cific binding site, as determined by DNase I footprint analysis, spans 33 nucleotides in the promoter region, including the putative -35 pro moter element. By construction and expression of a series of in-frame fusions between truncated arsR genes and the coding region for the mat ure form of beta-lactamase (blaM'), it was shown that ArsR is a trans- acting repressor that regulates expression of the chromosomal ars oper on. In addition, the chromosomally-encoded repressor can regulate expr ession of the ars operon of plasmid R773, and the R773 repressor can c ross-regulate expression from the chromosomal operon.