Clinical isolates of Neisseria gonorrhoeae exhibit diverse phenotypes including specific patterns of growth requirements (auxotypes), chromosomally-encoded resistance to antibiotics, and antigenic variations of outer membrane components. To avoid problems inherent in the study of clinical isolates with diverse genetic backgrounds, individual genetic loci responsible for representative phenotypes were placed into the uniform genetic matrix of a single strain. These isogenic strains exhibit mutations which have occurred and been selected in humans, the only natural host of N. gonorrhoeae. They will be used in studies designed to gain a greater understanding of individual mutant loci and their linkage relations on the genetic map of N. gonorrhoeae which currently covers only three-percent of the genome. A system of field-inversion gel electrophoresis (FIGE) has been developed to separate long fragments of parental strain DNA generated by cleavage with restriction endonucleases having rare recognition sites in the genome. The separated DNA fragments will be characterized by transformation of isogenic mutant strains to identify the location of these clinically-relevant mutations and possible linkage relations among them. A specific region which links genes for arginine and pyrimidine synthesis, argA-pyrE-pyrB, to the rif (rifampin resistance) locus on the established map of N. gonorrhoeae will be further characterized by (1) development of an appropriate enzyme assay for the argA gene product to confirm the identity of this locus, (2) classical recombination tests using two- and three-factor crosses and DNA-dilution tests, and (3) further restriction analysis and cloning of the corresponding FIGE-isolated DNA fragments to determine the physical distance between markers in this region of the chromosomal map. These studies are anticipated to add additional genetic loci to the present map of N. gonorrhoeae and to identify other possible linkage groups on the remainder of the largely unmapped genome. A more extensive map is expected to greatly enhance genetic manipulation of this organism and should provide a means of selecting a trait in the absence of a direct test for the gene. Knowledge of specific and easily selected markers that are closely linked to genes determining virulence is anticipated to allow a better understanding of their molecular organization and may give insights into the means of regulation of pathogenesis of N. gonorrhoeae.
