Development of Experimental Genetic Tools for Campylobacter fetus

Abstract

Molecular analysis of the virulence mechanisms of the emerging pathogen Campylobacter fetus has been hampered by the lack of genetic tools. We report the development and functional analysis of Escherichia coli-Campylobacter shuttle vectors that are appropriate for C. fetus. Some vectors were constructed based on the known Campylobacter coli plasmid pIP1455 replicon, which confers a wide host range in Campylobacter spp. Versatility in directing gene expression was achieved by introducing a strong C. fetus promoter. The constructions carry features necessary and sufficient to detect the expression of phenotypic markers, including molecular reporter genes in both subspecies of C. fetus, while retaining function in C. jejuni. The capacity to express several gene products from different vectors in a single host can be advantageous but requires distinct plasmid replicons. To this end, replication features derived from a cryptic plasmid of C. fetus subsp. venerealis strain 4111/108, designated pCFV108, were adapted for a compatible series of constructions. The substitution of the C. coli replication elements reduced vector size while apparently limiting the host range to C. fetus. The complementation of a ciprofloxacin-resistant mutant phenotype via vector-driven gyrA expression was verified. Cocultivation demonstrated that shuttle vectors based on the pCFV108 replicon were compatible with pIP1455 replication functions, and the stable maintenance of two plasmids in a C. fetus subsp. venerealis host over several months was observed. The application of both vector types will facilitate the investigation of the genetics and cellular interactions of the emerging pathogen C. fetus.

A Genomic Island Defines Subspecies-Specific Virulence Features of the Host-Adapted Pathogen Campylobacter fetus subsp. venerealis

“…Campylobacter and Escherichia coli strains were grown as previously described (48). The 112 Campylobacter strains used in this study are listed in Table 1 and Table S1 in the supplemental material.…”

“…For each ligation reaction, 16 to 36 independent transformants were analyzed further. About 500 ng of plasmid DNA from the transformants was transferred to nylon membranes with a vacuum manifold (48). Two identical blots were produced for each set of clones.…”

“…The suicide vector pGG5 was brought into C. fetus subsp. venerealis ATCC 19438 via an electrotransformation protocol originally developed for C. jejuni (35,84) and adapted for C. fetus (48). Generally, for each mutagenesis, kanamycin-resistant (Km r ) mutants were analyzed via Southern hybridization with linearized fragments of vector, aphA-3, and the targeted gene to confirm the site specificity and copy number of the integration.…”

“…Probes specific to the genomic island were constructed by PCR amplification with the oligonucleotide primers 3Dv5Ј and 3Dv3Ј (orf21); Top/traE4 and TraE7 (orf27); VirB8-1 and VirB9-1, spanning from virB8 to virB9; VirB10-1 and VirB10-2 (virB10); and TaxB2 and TaxB3 (virB4) ( Table 2). The DNA fragments were radiolabeled internally and hybridized as described previously (48). Additionally, PFGE was used to resolve SmaI-macrorestricted DNA originating from 10 C. fetus subsp.…”

“…Genomic DNA (15 ng) of each subspecies was digested with Sau3AI and radioactively labeled with 2.5 U of Klenow polymerase and 20 Ci of [␣- 32 P]dCTP by using the Prime-a-Gene labeling kit according to the manufacturer's specifications (Promega, Mannheim, Germany). Each pair of blots was hybridized to gene probes as described previously (48). …”