The arsenical resistance (ars) operon of the conjugative plasmid R773 encodes an ATP-driven anion extrusion pump, conferring bacterial resistance to arsenicals. The operon contains a regulatory gene, arsR, and three structural genes, arsA, arsB, and arsC. The hydrophilic ArsA and ArsC proteins are produced in large amounts, but the hydrophobic ArsB protein, an integral membrane polypeptide, is synthesized in limited quantities. Northern (RNA-DNA) hybridizations provide evidence that the inducible operon is regulated at the level of transcription. The genes were transcribed in the presence of an inducer (arsenite) as a single polycistronic mRNA with an approximate size of 4.4 kilobases (kb). This transcript was processed to generate relatively stable mRNA species: one of 2.7 kb, encoding the ArsR and ArsA proteins, and a second of 0.5 kb, encoding the ArsC protein. Segmental differences in stability within the polycistronic transcript are proposed to account for the differential expression of the ars genes. In addition, analysis of the mRNA structure at the 5' end of arsB suggests a potential translational block to the synthesis of this membrane protein.The arsenical resistance (ars) operon of resistance plasmid R773 confers resistance to arsenite, arsenate, and antimonite on Escherichia coli cells by the synthesis of an anion pump (23). This unique oxyanion-translocating ATPase, induced by the presence of its substrates, mediates their active extrusion from cells with energy derived from ATP (18,22,23,28). Thus, resistance results from a lowering of the intracellular concentration of the toxic oxyanion.A 4.3-kilobase (kb) HindIll fragment from R factor R773 was cloned into the vector pBR322 to produce a recombinant plasmid which produces constitutive resistance to arsenicals (17). Analysis of the nucleotide sequence of this fragment reveals three structural genes: arsA, arsB, and arsC (6). From the genetic evidence (7,22) and from the nucleotide sequence (6), the oxyanion pump was predicted to be composed of a complex of the 63-kilodalton ArsA and the 45.5-kilodalton ArsB proteins. The 16-kilodalton ArsC protein appears to act as a modifier subunit and is not necessary for arsenite resistance or transport (22). The ArsA protein was purified from the cytosol and shown to be an oxyanionstimulated ATPase (23). The hydrophobic ArsB protein has been identified as an inner membrane protein by creation of a gene fusion of the arsB gene with lacZ (26). It can be visualized as a [35S]methionine-labeled membrane protein when made in a T7 expression vector but is not present in amounts sufficient to be visible as a Coomassie blue-or silver-stained band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even though the operon is transcribed in high amounts by using the T7 expression system (31).Recently, the regulatory gene, arsR, has been identified on a 0.73-kb EcoRI-HindIII fragment contiguous with the 4. quence of the arsR gene has been determined, and its product, the ArsR protein, has been identified.Alt...
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