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 gonorrhoene, the causati ve agent of the sexually transmitted disease gonorrhea, can be studied usin g experimental infection of human male volunteers. The desire to avoid intr oducing new antibiotic resistance markers into strains to be used in human experimental infection has complicated the construction of genetically defi ned mutants in which expression of potential virulence factors is inactivat ed. To facilitate construction of such mutants, we have used a two-step mut agenesis strategy that allows for gene replacements without introducing new selectable markers into the final strain. The method uses a two-gene casse tte containing both a selectable marker (ermC') and a counterselectable mar ker (rpsL). The cassette is cloned into the gene of interest and used to re place the wild-type gene on the chromosome by allelic exchange. A second tr ansformation replaces the cassette-containing version of the gene with an e ngineered version with an unmarked deletion or other mutation. The rpsL gen e of Escherichia coli functioned for the counterselection in the gonococcus , albeit with low efficiency. To improve the efficiency of the counterselec tion, we cloned the gonococcal rpsL gene and incorporated it into the casse tte. This technique has been successful in creating defined mutants for hum an challenge, and also circumvents the limitation in the number of differen t selectable markers that are useful in Neisseira species. (C) 1999 Elsevie r Science B.V. All rights reserved.