Abstract
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 resequencing 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 WT 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 multiple defined gene deletion mutant clones, (ii) genetic complementation of the gene deletion and ideally (iii) screening for second-site mutations that might interfere with the pathways/mechanisms under study.
Highlights
Campylobacter jejuni is the leading bacterial cause of foodborne gastroenteritis worldwide
The genome sequence data for the C. jejuni defined gene deletion mutant clones are deposited at the European Nucleotide Archive under study accession number PRJEB10223
C. jejuni M1 was routinely cultured on brain heart infusion (BHI, Oxoid) agar plates supplemented with 5 % defibrinated horse blood (Oxoid) and 5 mg trimethoprim ml21 (TrM)
Summary
Campylobacter jejuni is the leading bacterial cause of foodborne gastroenteritis worldwide. The impact of C. jejuni infections is significant due to their high incidence, duration of infection and possible post-infection sequelae (Ruiz-Palacios, 2007). C. jejuni has a broad range of environmental reservoirs including water, birds and other domestic animals, with chickens representing the largest source of human infection (Young et al, 2007). C. jejuni is considered as commensal in chickens, it has a significant impact on animal welfare in certain breeds of bird (Humphrey et al, 2014). The genome of C. jejuni is Abbreviations: cat, chloramphenicol resistance cassette; CDS, coding sequence; EM, electron microscopy; flaB, flagellin B; INDEL, insertion and deletion; TrM, trimethoprim. The genome sequence data for the C. jejuni defined gene deletion mutant clones are deposited at the European Nucleotide Archive (http://www.ebi.ac.uk/ena) under study accession number PRJEB10223
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