Metalloregulated expression of the ars operon

Area of Science:

• Molecular Biology
• Microbiology
• Biochemistry

Background:

• The plasmid-borne arsenical resistance (ars) operon in Escherichia coli confers resistance to toxic metalloids like arsenicals and antimonials.
• This resistance is achieved through the action of an arsenical-translocating ATPase that extrudes these compounds from the cytosol.
• The trans-acting regulatory ArsR protein plays a crucial role in controlling the expression of the ars operon.

Purpose of the Study:

• To elucidate the mechanism of ArsR protein binding to the ars promoter.
• To identify the specific DNA sequence recognized by the ArsR repressor.
• To determine the in vivo and in vitro induction mechanism of the ars operon by arsenic compounds.

Main Methods:

• DNA-protein binding assays, including gel retardation and DNase I footprinting.
• Hydroxyl radical footprinting to define the ArsR binding site.
• Construction and analysis of an operator mutant to assess in vivo function.

Main Results:

• The ArsR repressor binds to a specific DNA sequence upstream of the -35 promoter site, characterized by imperfect dyad symmetry.
• The active form of the ArsR repressor is a dimer.
• Arsenite (As(III)) and other +3 oxidation state oxyions induce the operon in vivo, while arsenate (As(V)) requires intracellular reduction to As(III) for induction.

Conclusions:

• The ArsR repressor directly binds to its operator sequence to regulate ars operon expression.
• Arsenate is a pro-inducer, necessitating its reduction to arsenite for ars operon induction.
• Understanding this regulatory mechanism is key to microbial resistance against metalloid toxicity.