SHUTTLE MUTAGENESIS - 2 MINI-TRANSPOSONS FOR GENE-MAPPING AND FOR LACZ TRANSCRIPTIONAL FUSIONS IN NEISSERIA-GONORRHOEAE

Shuttle mutagenesis is a system we developed for producing stable tran sposon insertions in Saccharomyces cerevisiae Seifert et al., Proc. N atl. Acad. Sci. USA 83 (1986) 735-739; Hoekstra et al., Methods Enzymo l. 194 (1991) 329-342! and Neisseria gonorrhoeae (Gc) Seifert et al., J. Bacteriol. 172 (1990) 40-46! by transposition in Escherichia coli and transformation into yeast or Gc. In developing the system for use in Gc, a series of mini-transposons (mTn) were derived from mTn3 which confer resistance to chloramphenicol in Gc (mTnCm) (Seifert et al., 1 990). Herein, we describe the creation of two mTnCm derivatives for us e in Gc. One of these transposons, mTnCmNS, contains the infrequently occurring NheI and SpeI restriction sites to localize genes on the gon ococcal macro-restriction map which was recently developed using these restriction sites Bihlmaier et al., Mol. Microbiol. 5 (1991) 2529-25 39; Dempsey et al., J. Bacteriol. 173 (1991) 5476-5486!. The mTnCmLac was developed to generate lacZ transcriptional fusions using transposi tion. It contains at its end a promoterless lacZ gene which is express ed once the element has transposed downstream from a promoter in a clo ned gene. In adapting the use of mTnCmLac to the shuttle mutagenesis s ystem, we have identified some factors which affect the transformation of Gc using cloned chromosomal fragments containing the large heterol ogous insertion, mTnCmLac. Using mTnCmLac, we have created Gc variants containing a pilE:.mTnCmLac fusion to determine that pilE transcripti on in Gc is not auto-regulated.