The overall aim is to construct a physical map of the Streptococcus mutans GS5 chromosome, and to locate a number of genes on the physical map. Large DNA restriction fragments will be resolved by pulsed-field gel electrophoresis. Fragments obtained with different restriction endonucleases (yielding 1 to 30 fragments from the chromosome) will be ordered relative to each other. This will be done: (a) by fractionating single and double digests combined with Southern blots using as probes appropriate isolated restricted fragments so as to determine overlaps between fragments obtained with different enzymes; (b) by pulse labeling the DNA of bacteria that have been induced to replicate their chromosome synchronously, and relating restriction fragments to different positions in the replication cycle; (c) by introducing a unique restriction site into the chromosome, and using it as a point of reference to analyze partial digestion patterns obtained with other enzymes. The position on the physical map will be determined for mutations associated with selectable phenotypes, for transposon insertions, and for cloned genes. In this way a genetic map will be obtained. Special emphasis will be given to genes associated with cell surface properties. We will analyze the macro restriction patterns of DNA from other strains of S. mutans and from selected oral streptococci, including S. sobrinus and S. sanguis. This will enable us to assess the usefulness of such an analysis for studies of taxonomy, epidemiology, evolution, and ecology of important oral pathogens.
