CONSTRUCTION OF NEW CAMPYLOBACTER CLONING VECTORS AND A NEW MUTATIONAL CAT CASSETTE

We have developed new Campylobacter shuttle vectors which are 6.5-6.8-kb plasmids carrying Campylobacter and Escherichia coli replicons, a multiple cloning site (MCS), the lacZalpha gene, oriT and either a kanamycin or chloramphenicol resistance-encoding gene (Km(R) or Cm(R)) from Campylobacter which functions in both hosts. These vectors can be mobilized efficiently from E. coli into C. jejuni or C. coli, and stably maintained in these hosts. Plasmids pRY107 and pRY108 carry a Km(R) marker and 17 unique cloning sites in two different orientations in lacZalpha, allowing easy blue/white color selection. Plasmids pRY111 and pRY112 contain a Cm(R) gene and 17 unique sites in both orientations. In addition, MCS are flanked by T7 and T3 late promoters and M13 forward and reverse primer sites, facilitating expression in T7 or T3 expression systems and sequence analysis. A Campylobacter CM(R) gene cartridge, bracketed by six restriction sites, has been developed for use in site-specific mutagenesis of Campylobacter genes.