Purpose: We attempted to isolate genetic factors which may contribute to the difficulties in plasmid transformation of C. jejuni NCTC11168. Mutagenesis of these factors may yield a strain which can facilitate further research of C. jejuni.
Methods: Random transposon mutants of NCTC11168 were electroporated with the green fluorescent protein (GFP) expressing shuttle plasmid pWM1007 and transposon insertion sites determined by plasmid rescue. Knock out mutagenesis of Cj1051c was performed by overlapping extension PCR creating a construct with the genomic regions flanking Cj1051c cloned around chloramphenicol or tetracycline resistance cassettes followed by electroporation into C. jejuni NCTC11168.
Results: The transposon screen produced 164 colonies which appeared to have successfully taken up the plasmid. Plasmid rescue of two isolates determined the transposon insertion to be in Cj1051c, a gene predicted to be part of a restriction modification system. Knock out strain C. jejuni NCTC11168DCj1051c was found to have an enhanced transformation rate of >600 colonies per microgram of pWM1007 while the wild-type strain had sporadic transformation success with few to no colonies produced from a microgram of pWM1007.
Significance: Strain NCTC11168DCj1051c may be a useful tool in molecular biology research of C. jejuni as it allows enhanced plasmid transformation. The parental strain is one of the most commonly used strains for genetic studies and this mutant will facilitate research that had previously been difficult or impossible without genomic disruptions.