Motility defects in Campylobacter jejuni defined gene deletion mutants caused by second-site mutations. Whole genome sequence of mutants in Campylobacter jejuni strain M1

Genetic variation due to mutation and phase-variation has a considerable impact on the commensal and pathogenic behaviours of Campylobacter jejuni. In this study, we provide an example of how second-site mutations can interfere with gene function analysis in C. jejuni. Deletion of the flagellin B gene (flaB) in C. jejuni M1 resulted in mutant clones with inconsistent motility phenotypes. From the flaB mutant clones picked for further analysis, two were motile, one showed intermediate motility, and two displayed severely attenuated motility. To determine the molecular basis of this differential motility, a genome re-sequencing approach was used. Second-site mutations were identified in the severely attenuated and intermediate motility flaB mutant clones: a TA-dinucleotide deletion in fliW and an A deletion in flgD, respectively. Restoration of wild-type fliW, using a newly developed genetic complementation system, confirmed that the second-site fliW mutation caused the motility defect as opposed to the primary deletion of flaB. This study highlights the importance of i) screening for second-site mutations that might interfere with the pathways/mechanisms under study and ii) performing genetic complementation of the gene deletion mutants.