Abstract/Summary

Campylobacter jejuni, the major cause of bacterial gastroenteritis, is widespread in farmyard animals and poultry. The main source of human disease is through the food chain, often through contaminated chickens, but C. jejuni and C. coli are also present in the environment. C. jejuni is a strict microaerophile with an essentially asaccharolytic metabolism. These bacteria are naturally competent, allowing uptake of naked DNA from lysed cells, which contributes to enhanced genome variation and potentially ability to survive in varied environments and niche adaptation. This study has focused on the unusual ability of some C. jejuni and C. coli strains to metabolise glucose via the Entnerdouderoff (ED) pathway with an aim of shedding light on the benefit of possession of the glc locus by a subset of ‘environmental’ strains. Addition of glucose to minimal media, DMEMf, had no impact on exponential growth but prevented rapid decline in viability of C. jejuni Dg275 from 24 h. Reverse transcription- quantitative polymerase chain reaction (RT-qPCR) demonstrated a 7-8 fold increase in expression of edd and zwf (encoding enzymes of the ED pathway) and a small but significant increase in glcP (encodes glucose transporter) between late exponential (18 h) to stationary phase (24 h), correlating with initiation of glucose utilisation. cDNA PCR analysis across gene junctions of transcripts and promoter predictions provided preliminary evidence of a working map of this divergent seven gene glc locus. Ribosomal (r)MLST and core genome (cg)MLST highlighted phylogenetic clustering of glc positive C. jejuni strains close to but distinct from glc negative ST45CC45 strains isolated from Norway rats. Further comparisons of available genome sequences revealed that most glycolytic C. jejuni and C. coli strains, also carried the fuc locus encoding genes for fucose metabolism. In DMEMf with glucose and fucose, growth of Dg275 followed the characteristic diauxic profile of growth on fucose, indicating fucose as a preferred substrate. Deletion of fucR, encoding the FucR repressor, together with the main fuc regulatory region, in Dg275ΔfucR::cat, indicated that loss of FucR regulator and products of fucose metabolism did not impact glucose-driven growth, but that in the wild-type Dg275 either FucR or products of fucose metabolism somehow represses the characteristic glucose promoted survival. Although assumed to occur, transfer of the glc locus between C. jejuni strains, in vitro, has never been demonstrated. Attempts to transfer the glc locus, via natural transformation, from genomic DNA of the glycolytic Dg275 strain to the related nonglycolytic strain, Dg200, were unsuccessful. The presence of duplicate copies of the glc locus, one within rrnA and the other within rrnB complicated construction of isogenic knockout mutant and wild-type strains. Ultimately, the mutated strains Dg275ΔglcP::KANrrnAΔglcP::catrrnB (Dg275ΔglcP) and Dg275Δedd::KANrrnAΔedd::catrrnB (Dg275Δedd) were created in two steps. Hybrid genome sequencing was used to confirm sequences of single and double knockout mutants. While inactivation of both copies of the genes resulted in the inability to grow on glucose, possession of a single functional copy of glcP or edd, was shown to be sufficient for glucose utilisation and enhanced survival. These results and knockout mutants will provide the foundation and tools for future studies to further investigate control of expression and function of the glc locus with a view to understanding the significance of the Campylobacter glc locus to niche adaptation.