Cloning of Campylobacter jejuni genes required for leucine biosynthesis, and construction of leu-negative mutant of C. jejuni by shuttle transposon mutagenesis

Abstract

Campylobacter jejuni is a Gram-negative pathogen responsible for diarrhoeal diseases in humans. To date, very little is known about the genetic organization and molecular biology of this microorganism. The cosmid vector pHC79 was used to construct a genomic library from the total genomic DNA of C. jejuni strain C31 in Escherichia coli and recombinant cosmids capable of complementing the auxotrophic defect in leucine biosynthesis of E. coli HB101 were identified. Three of 400 clones tested were found to be capable of complementing the nutritional defect of E. coli HB101 as well as those of independent leuB mutants of E. coli strains. These results indicated that the cloned genes responsible for leucine complementation encoded an enzyme analogous to the beta-isopropylmalate dehydrogenase specified by the leuB gene in E. coli strains. The sizes of the recombinant cosmids which became stabilized in E. coli cells ranged from 12.9 to 15.4 kb compared to the expected, originally packaged, 45- to 50-kb molecules, attesting to major rearrangements occurring in this background. The recombinant plasmid pILL547 was shown to carry genes that were analogous to the leuB gene and also to the leuC and leuD genes of E. coli. The gene required for leuB complementation was subcloned on a 1.6-kb restriction fragment and was mapped more precisely by insertional mutagenesis using as transposon a newly constructed (MiniTn3-Km) element engineered to mutagenize Campylobacter genes. The leuB gene of C. jejuni was shown to be expressed from its own promoter in E. coli cells. In E. coli minicells, the cloned insert encoded a polypeptide with an apparent molecular weight of 40 kDa. A leucine auxotrophic mutant of C. jejuni strain C31 was constructed in vitro by allelic exchange, replacing the original copy of the leucine gene by an allele mutated by the insertion of the kanamycin transposable element.