The objectives of this study are to use genetic and physiological analyses to investigate the roles of periplasmic and membrane proteins encoded by the mer operon of Tn21 in mercury transport. We have previously shown by analysis of deletion and frameshift mutations that the merP-encoded periplasmic protein and the merT-encoded membrane protein are required both for mercury resistance and for mercury-inducible mercury uptake. We will use both oligonucleotide-directed, site-specific mutagenesis and localized in vitro hydroxylamine mutagenesis to identify specific amino acid residues that are involved in mercury binding and mercury resistance. Molecular topology of membrane proteins will be investigated by making fusions with alkaline phosphatase, which is active only if secreted into the periplasm. Basic physiological characterization of the mercury transport system will include investigation of: sensitivity to osmotic shock, dependence on various host function, requirement for sulfhydryl reagents, and sensitivity to inhibitors and uncoulers of energy metabolism.
