Cotransformation of temperature sensitivity and nutritional markers in Neisseria gonorrhoeae

Cotransformation remains the only tool for establishing linkage in Neisseria gonorrhoeae. Because of the difficulty of inducing auxotrophic markers via mutagenesis in this species, most previous studies have utilized antibiotic resistance and naturally occurring (auxotypic) auxotrophic markers. We have succeeded in isolating auxotrophic and temperature-sensitive mutants. The temperature-sensitive mutants have been characterized by their growth on complex and defined media at 31, 37, and 40 degrees C. Two of the mutants exhibited an unusual pattern of temperature sensitivity--growth on the defined medium but absence of growth on the complex medium at 37 degrees C. Both mutants, however, were temperature-sensitive on the two media at 40 degrees C. We have demonstrated linkages between markers isolated in our laboratory and the auxotypic markers of the clinical isolate RUG208. Ts-2 exhibited 85 to 95% linkage to Arg- and his-2 exhibited 40% linkage to Val-. In addition weak linkages were shown between his-2 and Arg- (2 to 6%) and between Arg- and Val- (3 to 5%). Linkages among his-2, Arg-, and Val- which could be demonstrated when deoxyribonucleic acid from strain F62 was used to transform RUG208 were absent when F62 was used as recipient for RUG208 DNA. Our data are consistent with a tentative map order of his-2, Val-, Arg-, Ts-2.