Identification of Cj1051c as a Major Determinant for the Restriction Barrier of Campylobacter jejuni Strain NCTC11168

Abstract

cCampylobacter 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.

Heat Shock-Enhanced Conjugation Efficiency in Standard Campylobacter jejuni Strains

“…This finding suggests that the heat shock-enhanced conjugation might be caused by other C. jejuni RM systems or the synergistic effect of the four tested RM systems. A previous study has showed that Cj1051c is a major restriction barrier for C. jejuni NCTC 11168 to acquire pMW10-derived plasmid through electroporation (17). However, Cj1051c is absent in the C. jejuni 81-176 genome, which suggests that Cj1051c may not be critical for heat shock-enhanced conjugation.…”

“…In Corynebacterium glutamicum ATCC 13032, a stress-sensitive type II restriction system has been identified to be responsible for the heat shock-enhanced conjugation (20). C. jejuni NCTC 11168 possesses up to seven sets of RM genes (17). Since both C. jejuni NCTC 11168 and 81-176 displayed heat shock-enhanced conjugation (Fig.…”

“…jejuni NCTC 11168, a widely used standard C. jejuni strain and the first C. jejuni isolate for which the whole genome has been sequenced (15), displays high efficiency for natural transformation with donor DNA from the same species (16) but low transformation efficiency for a plasmid derived from different bacterial species (e.g., Escherichia coli) (17). In addition, it has been widely observed that C. jejuni NCTC 11168 displays extremely low conjugation efficiency, posing a challenge for molecular manipulation of this organism.…”

Gene Loss and Lineage-Specific Restriction-Modification Systems Associated with Niche Differentiation in the Campylobacter jejuni Sequence Type 403 Clonal Complex

“…Strain-specific R-M systems have been reported previously in C. jejuni strains 81116 (30), ATCC 43431 (31), and 81176 (32) and are thought to contribute to the apparent recombination and transformation restriction that has hindered genetic manipulation of the organism for some time. Mutagenesis of the type IIG R-M enzyme Cj1051c in NCTC11168 increased the strain's ability to take up plasmid DNA, including that from Escherichia coli (33). Single nucleotide polymorphisms in known R-M systems in the Japanese ST4526 clone are thought to be responsible for the reduced uptake of plasmid DNA compared to NCTC11168 (34), as well as contributing to the ability of the clone to thrive in Japan.…”

Genome‐wide association of functional traits linked with Campylobacter jejuni survival from farm to fork

“…Defined gene deletion mutants were obtained after allelic replacement of the target gene with a chloramphenicol (cat) resistance cassette, as described earlier (De Vries et al, 2015). The overlap PCR product was subsequently used for electroporation (Holt et al, 2012) of C. jejuni M1 to obtain first generation defined gene deletion mutants. The PCR primers used to amplify the target gene flanking regions contain extensions complementary to the cat cassette.…”