The overall objective of this project is to study the basis of regulation for plasmid-borne fusidic acid and streptomycin resistance in Staphylococcus aureus. In order to fulfill this objective, we will: 1) Isolate Staphylococcus aureus strains with plasmid-borne resistance to streptomycin and fusidic acid; 2) Produce restriction maps of the isolated plasmids; 3) Determine if the mechanism of regulation for resistance is at the level of transcription or translation or both; 4) Begin to study regulation at the translational level if this is proven to be a major mechanism of regulation. This study will contribute to our overall understanding of the regulation of antibiotic resistance and the molecular biology of regulation at the translational level. This in turn could have impact on public health in the control of antibiotic resistance disease causing microorganisms. Clinical isolates of Staphylococcus aureus will serve as the source of fusidic acid and streptomycin resistance bearing plasmids. These plasmids will be isolated and restriction mapped. The gene in the plasmid which specifies resistance will be located by a pTV53 insertion. The resultant gene fusion will be used to sequence the upstream portion of the gene specifying resistance and its regulatory region. Using the restriction maps of the original plasmids and the sequences for their regulatory regions, the vector, pSKl0 Delta 6, will be used to clone a lacZ gene minus its promoter and ribosome binding site into a site downstream from the ribosome binding site for the gene specifying antibiotic resistance. These constructs will be used to study regulation of resistance by following Beta-galactosidase activity. Transcriptional versus translational regulation will be examined by following the resistance gene's m-RNA level in the presence and absence of antibiotic. Translational regulation will be studied through deletions and insertions within the regulatory regions for the resistance genes.
