Construction of mutant strains of Neisseria gonorrhoeae lacking new antibiotic resistance markers using a two gene cassette with positive and negative selection.

The pathogenesis of infections caused by Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, can be studied using experimental infection of human male volunteers. The desire to avoid introducing new antibiotic resistance markers into strains to be used in human experimental infection has complicated the construction of genetically defined mutants in which expression of potential virulence factors is inactivated. To facilitate construction of such mutants, we have used a two-step mutagenesis strategy that allows for gene replacements without introducing new selectable markers into the final strain. The method uses a two-gene cassette containing both a selectable marker (ermC') and a counterselectable marker (rpsL). The cassette is cloned into the gene of interest and used to replace the wild-type gene on the chromosome by allelic exchange. A second transformation replaces the cassette-containing version of the gene with an engineered version with an unmarked deletion or other mutation. The rpsL gene of Escherichia coli functioned for the counterselection in the gonococcus, albeit with low efficiency. To improve the efficiency of the counterselection, we cloned the gonococcal rpsL gene and incorporated it into the cassette. This technique has been successful in creating defined mutants for human challenge, and also circumvents the limitation in the number of different selectable markers that are useful in Neisseria species.