Characterization of Campylobacter jejuni asd gene cloned in Escherichia coli

asd gene of the human enteric pathogen, Campylobacter jejuni Dz72/92, has been isolated from a genomic library constructed in the expression plasmid vector pUC8-2 in Escherichia coli. The gene has been fished out by complementation of the asd gene deletion present in the genome of E. coli chi 6097. The smallest recombinant plasmid (pUWM3) able to confer Asd+ phenotype contains a 1.8 kb insert cloned into HindIII site located within the multi-cloning site of the pUC8-2 vector. The origin of the insert has been confirmed by hybridization. Several pieces of evidence indicate that the expression of the cloned house-keeping gene is driven from its own promoter, which can be recognised by E. coli RNA polymerase. The asd gene promoter has been located on 300 kb HindIII-DraI fragment of pUWM3. Recombinant plasmid pUWM3 specifies a new 38 kDa protein which we believe is the asd gene product. Overproduction of the 38 kDa protein due to the transcription originating from the vector lacZ gene promoter is toxic for the cells.