Gene disruption and replacement as a feasible approach for mutagenesis of Campylobacter jejuni

Abstract

Campylobacter jejuni and Campylobacter coli are important causes of human enteric infections. Several determinants of pathogenicity have been proposed based on the clinical features of diarrheal disease and on the phenotypic properties of Campylobacter strains. To facilitate an understanding of the genetic determinants of Campylobacter virulence, we have developed a method for constructing C. jejuni mutants by shuttle mutagenesis. In the example described here, a kanamycin resistance gene was inserted into Campylobacter DNA fragments encoding 16S rRNA cloned in Escherichia coli. These disrupted, modified sequences were returned to C. jejuni via conjugation. Through the apparent process of homologous recombination, the kanamycin resistance-encoding sequences were rescued by chromosomal integration, resulting in the simultaneous gene replacement of one of the 16S sequences of C. jejuni and the loss of the vector. We propose that Campylobacter isogenic mutants could be developed by using this system of shuttle mutagenesis.