Development and Application of an Insertional System for Gene Delivery and Expression in Campylobacter jejuni

Abstract

The genetic investigation of Campylobacter jejuni, an important gastrointestinal pathogen, has been hampered by the lack of an efficient system for introduction of exogenous genetic information, as commonly used vectors designed for Escherichia coli and other bacteria cannot be maintained in Campylobacter cells. Additionally, gene expression in Campylobacter requires the presence of species-specific promoters. In this study we exploited the availability of several conserved copies of rRNA gene clusters for insertion of various genes into the chromosome by homologous recombination. The high conservation of the rRNA sequences means that the procedure can be applied to other Campylobacter strains. The presence of a Campylobacter-derived promoter in this vector ensures expression of exogenous genes in target cells. The efficiency of the procedure was demonstrated by complementation of mutations in two strains of Campylobacter. In addition, we applied the system for introduction and expression of a green fluorescent protein (GFP). GFP-expressing Campylobacter allowed visualization of sessile bacteria attached to a glass surface in stationary liquid culture. The study demonstrated that the attached bacteria contained an assemblage of coccoid and spiral forms with liquid channels preserving viable highly motile cells. We demonstrate a novel universal procedure for gene delivery and expression that can be used as an efficient tool to study this poorly understood pathogen. The principles developed in this study could be more widely applied for the manipulation of other bacteria that are refractory to genetic analysis.

Campylobacter jejuni Glycosylation Island Important in Cell Charge, Legionaminic Acid Biosynthesis, and Colonization of Chickens

“…Plasmid pRRC was used to complement all gene knockouts in C. jejuni strains. A description of the construction of and further details for this plasmid are given elsewhere (30).…”

Campylobacter jejuni proteins Cj0952c and Cj0951c affect chemotactic behaviour towards formic acid and are important for invasion of host cells

“…A single XbaI site is located immediately downstream of the cam R gene cassette, and allows the expression of XbaI-cloned genes under the control of the constitutively expressed cam R gene promoter. After introduction of the plasmid into competent cells of C. jejuni, the cam R gene cassette together with the XbaI-cloned gene was integrated into one of the 16S rRNA loci of the recipient cell via highly efficient double recombination (Karlyshev & Wren, 2005).…”

Contribution of the stereospecific methionine sulphoxide reductases MsrA and MsrB to oxidative and nitrosative stress resistance in the food-borne pathogen Campylobacter jejuni

“…For construction of a complemented msrB strain, a complementation construct was produced by cloning the msrB gene plus the upstream ribosome-binding site into the Campylobacter complementation vector pRRC (Karlyshev & Wren, 2005). The msrB gene product was produced by PCR using primers C-msrB-F (59-GCT CTA GAT ACA AGG GCA GAT CAT GAA AGA-39) and C-msrB-R (59-GCT CTA GAC TAT CAA TCC TTA GTT TTT ACA-39), and cloned into the XbaI site of pRRC to produce plasmid p-msrB-COMP.…”

“…This meant that construction of a complemented msrB strain was essential to correctly attribute any phenotypes seen in the msrB mutant to the loss of this protein alone and not to any polar effects caused by loss of the Cj1111c protein. Complementation was achieved using the integrative vector pRRC (Karlyshev & Wren, 2005), with expression of the wild-type msrB allele under the control of the chloramphenicol-resistance gene on this plasmid.…”