Control of transcription and gene regulation in mercuric ion and oregano-mercurial resistance operons of plasmids from Gram positive and Gram negative bacteria will be studied. The regulatory genes from the broad spectrum mercurial resistance operons of the Serratia marcescens plasmid pDU1358 and Staphylococcus aureus plasmid pI258 will be cloned into a hyperexpression vector (pI258) in Escherichia coli. Overexpressed regulatory proteins will be purified and their interactions with mercurial resistance operons will be studied by gel binding, nitrocellulose filter binding and hydroxyl radical footprinting techniques. Start sites of the transcriptions of the regulatory and structural genes will be determined. The effects of the regulatory protein on the in vitro transcription of mRNA transcribed the structural genes will be studied in the presence and absence of and organomercurials using run-off transcription assays. Interaction of the predicted helix- turn-helix structure of the regulatory proteins with the operator DNA will be tested and absolute requirement of such a structure for specific binding with the DNA will be confirmed by site-directed mutagenesis studies. Specific nucleotide sequences that make contact with the DNA binding domain of the regulatory proteins will be determined. Genes involved in the transport of mercuric ion and organomercurials in Staphylococcus aureus plasmid pI258 will be characterized by molecular genetic studies using oligonucleotide site directed and sodium bisulfite induced localized mutations.
