Our ultimate objective is to characterize the genetic organization and regulation of the lactose and galactose genes of S. aureus. The map positions on the staphylococcal chromosome will be determined. The genes will be isolated by recombinant DNA techniques either in S. aureus or E. coli. The cloned DNA will be physically characterized with a variety of restriction endonucleases and the physical map correlated with the genetic map. The number of transcription units contained within the cloned sequences will be determined using an in vitro transcription assay. Restriction fragments of the cloned DNA will be used as templates for RNA synthesis directed by E. coli or B. subtilis RNA polymerase. DNA fragments containing putative promoter sequences will be examined by nucleotide sequence analysis. The sequence analysis will provide information as to the nature of staphylococcal promoters, ribosome binding sites, and ultimately the primary structure of the phosphobetagalactosidase gene. The nucleotide sequence is a necessary prerequisite for future studies on the interaction of the repressor molecule with the lac DNA. Isolation of the phosphobetagalactosidase gene will result in the construction of a vehicle for monitoring gene expression in S. aureus. This gene can be fused by in vitro or in vivo manipulations to the regulatory regions of other staphylococcal determinants. Advantage can then be taken of the easily assayable p-Beta-gal activity to monitor the expression of genes whose phenotype is difficult to assay. This latter group would include the enterotoxin genes, which currently require rather laborous immunological assays in order to follow their expression.
