Insertion-duplication mutagenesis of Neisseria: Use in characterization ofDNA transfer genes in the gonococcal genetic island

We created plasmids for use in insertion-duplication mutagenesis (IDM) of N eisseria gonorrhoeae. This mutagenesis method has the advantage that it req uires only a single cloning step prior to transformation into gonococci. Ch romosomal DNA cloned into the plasmid directs insertion into the chromosome at the site of homology by a single-crossover (Campbell-type) recombinatio n event. Two of the vectors contain an erythromycin resistance gene, ermC, with a strong promoter and in an orientation such that transcription will p roceed into the cloned insert. Thus, these plasmids can be used to create i nsertions that are effectively nonpolar on the transcription of downstream genes. In addition to the improved ermC, the vector contains two copies of the neisserial DNA uptake sequence to facilitate high-frequency DNA uptake during transformation. Using various chromosomal DNA insert sizes, we have determined that even small inserts can target insertion mutation by this me thod and that the insertions are stably maintained in the gonococcal chromo some. We have used IDM to create knockouts in two genes in the gonococcal g enetic island (GGI) and to clone additional regions of the GGI by a chromos ome-walking procedure. Phenotypic characterization of traG and traH mutants suggests a role for the encoded proteins in DNA secretion by a novel type IV secretion system.