To contribute to the long term goal of understanding the structure of bacterial chromosomes and their evolution, a complete physical map of ordered restriction fragments will be constructed for the genome of the Staphylococcus aureus and of other species in the genus Staphyloccus Large DNA fragments, averaging 100,000 base pairs, produced by endonucleases that cleave l5 to 30 times in these genomes, (Sma I, Sac II and Bgl I), can be separated by pulsed field gel electrophoresis (PFGE). To produce a physical map using these endonucleases as tools, a transposon derivative from Tn917 with a Not I (GCGGCCGC) site will be constructed and transposed into the genome. Since there are no sites for Not I in the S. aureus genome this introduced site will be unique. It will act as a zero point for the rapid production of an accurate physical map of Sma I, Sac II and Bgl I sites by indirect end-labeling of DNA separated by pulsed field gel electrophoresis. Subchromosomal libraries will be constructed from restriction fragments separated by PFGE. These will be arrayed to produce ordered chromosomal libraries. The physical map will be correlated to the genetic map by probing the plasmid arrays with cloned genes. Differences in the physical order of DNA between species will be compared by hybridizing individual members of a chromosomal library array from one species to all members of an array from the other species.
