Shuttle mutagenesis is a system we developed for producing stable transposon insertions in Saccharomyces cerevisiae [Scifert et al, Proc. Natl. Acad. Sci. USA 83 (1986) 735-739; Hoekstra et al., Methods Enzymol. 194 (1991) 329-342] and Neisseria gonorrhoeae (Gc) [Scifert 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 mTnJ which confer resistance to chloramphenicol in Gc (mTn Cm) (Scifert et al., 1990). Herein, we describe the creation of two mTnCm derivatives for use in Gc. One of these transposons, mTnCmNS, contains the infrequently occurring NheI and SpeI restriction sites to localize genes on the gonococcal macro-restriction map which was recently developed using these restriction sites [Bihlmaier et al., Mol. Microbiol. 5 (1991) 2529-2539; Dempsey et al., J. Bacteriol. 173 (1991) 5476-5486]. The mTnCmLac was developed to generate lacZ transcriptional fusions using transposition. It contains at its end a promoterless lacZ gene which is expressed once the element has transposed downstream from a promoter in a cloned gene. In adapting the use of mTnCmLac to the shuttle mutagenesis system, we have identified some factors which affect the transformation of Gc using cloned chromosomal fragments containing the large heterologous insertion, mTnCmLac. Using mTnCmLac, we have created Gc variants containing a pilE::mTnCmLac fusion to determine that pilE transcription in Gc is not auto-regulated.
- DNA transformation
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