Bacterial resistance to arsenicals occurs by an extrusion and is encoded by the inducible arsenical resistance operon which is carried on conjugative R-factors or plasmids. The arsenical resistance system is a good model system for understanding antibiotic resistance including multidrug resistance. The overall goal of this investigation is the elucidation of the molecular mechanism of arsenical transport. This proposal will focus on a structural and functional characterization of the arsB gene product. The ArsB protein is an integral membrane protein predicted to be an anion channel. A topological model for the ArsB protein is an integral membrane protein predicted to be an anion channel. A topological model for the ArsB protein has been predicted from the nucleotide sequence. This model will be tested by a molecular genetic approach using lacZ and phoA gene fusions. In parallel, with the above study, a biochemical approach will also be used for the identification and purification of the ArsB protein. A fusion protein will be constructed between the arsB gene product and an N-terminal tag of six histidine residues. The resultant His-tagged ArsB protein will be solubilized and purified by affinity chromatography. Alternatively, the ArsB protein will be fused with the biotinylated domain of oxaloacetate decarboxylase from Klebsiella pneumoniae and purified by affinity chromatography. The resultant purified protein will be physiologically and biochemically characterized. Either in parallel with or subsequent to the above studies a collection of arsB point mutants will be generated in vitro by the use of oligonucleotide-directed random mutagenesis. Understanding the molecular mechanisms of anion transport will greatly increase our understanding of antibiotic resistances in human health. In addition to the potential health benefits, this study will make a significant contribution toward the infrastructure of biomedical research and development. Both graduate and undergraduate students will play an active role in this research project. Their research experience and training will help ensure a future generation of biomedical scientists for both the public and private sectors.
