Signature-tagged transposon mutagenesis studies demonstrate the dynamic nature of cecal colonization of 2-week-old chickens by Campylobacter jejuni.

We have constructed plasmids to be used for in vitro signature-tagged mutagenesis (STM) of Campylobacter jejuni and used these to generate STM libraries in three different strains. Statistical analysis of the transposon insertion sites in the C. jejuni NCTC 11168 chromosome and the plasmids of strain 81-176 indicated that their distribution was not uniform. Visual inspection of the distribution suggested that deviation from uniformity was not due to preferential integration of the transposon into a limited number of hot spots but rather that there was a bias towards insertions around the origin. We screened pools of mutants from the STM libraries for their ability to colonize the ceca of 2-week-old chickens harboring a standardized gut flora. We observed high-frequency random loss of colonization proficient mutants. When cohoused birds were individually inoculated with different tagged mutants, random loss of colonization-proficient mutants was similarly observed, as was extensive bird-to-bird transmission of mutants. This indicates that the nature of campylobacter colonization in chickens is complex and dynamic, and we hypothesize that bottlenecks in the colonization process and between-bird transmission account for these observations.

Transposon mutagenesis in a hyper-invasive clinical isolate of Campylobacter jejuni reveals a number of genes with potential roles in invasion.

Transposon mutagenesis has been applied to a hyper-invasive clinical isolate of Campylobacter jejuni, 01/51. A random transposon mutant library was screened in an in vitro assay of invasion and 26 mutants with a significant reduction in invasion were identified. Given that the invasion potential of C. jejuni is relatively poor compared to other enteric pathogens, the use of a hyper-invasive strain was advantageous as it greatly facilitated the identification of mutants with reduced invasion. The location of the transposon insertion in 23 of these mutants has been determined; all but three of the insertions are in genes also present in the genome-sequenced strain NCTC 11168. Eight of the mutants contain transposon insertions in one region of the genome (approximately 14 kb), which when compared with the genome of NCTC 11168 overlaps with one of the previously reported plasticity regions and is likely to be involved in genomic variation between strains. Further characterization of one of the mutants within this region has identified a gene that might be involved in adhesion to host cells.

Identification of Cj1051c as a Major determinant for the restriction barrier of Campylobacter jejuni StrainNCTC11168

Campylobacter jejuni is a leading cause of human diarrheal illness in the world, and research on it has benefitted greatly by the completion of several genome sequences and the development of molecular biology tools. However, many hurdles remain for a full understanding of this unique bacterial pathogen. One of the most commonly used strains for genetic work with C. jejuni is NCTC11168. While this strain is readily transformable with DNA for genomic recombination, transformation with plasmids is problematic. In this study, we have identified a determinant of this to be cj1051c, predicted to encode a restriction-modification type IIG enzyme. Knockout mutagenesis of this gene resulted in a strain with a 1,000-fold-enhanced transformation efficiency with a plasmid purified from a C. jejuni host. Additionally, this mutation conferred the ability to be transformed by plasmids isolated from an Escherichia coli host. Sequence analysis suggested a high level of variability of the specificity domain between strains and that this gene may be subject to phase variation. We provide evidence that cj1051c is active in NCTC11168 and behaves as expected for a type IIG enzyme. The identification of this determinant provides a greater understanding of the molecular biology of C. jejuni as well as a tool for plasmid work with strain NCTC11168. © 2012, American Society for Microbiology.