Genetic variation due to mutation and phase variation has a considerable impact on the commensal and pathogenic behaviours of . In this study, we provide an example of how second-site mutations can interfere with gene function analysis in . Deletion of the flagellin B gene () in M1 resulted in mutant clones with inconsistent motility phenotypes. From the 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 mutant clones: a TA-dinucleotide deletion in and an A deletion in , respectively. Restoration of WT , using a newly developed genetic complementation system, confirmed that the second-site mutation caused the motility defect as opposed to the primary deletion of . 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.


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  1. Balaban M., Joslin S. N., Hendrixson D. R. (2009). FlhF and its GTPase activity are required for distinct processes in flagellar gene regulation and biosynthesis in Campylobacter jejuni J Bacteriol 19166026611 [View Article][PubMed]. [Google Scholar]
  2. Barrero-Tobon A. M., Hendrixson D. R. (2014). Flagellar biosynthesis exerts temporal regulation of secretion of specific Campylobacter jejuni colonization and virulence determinantsMol Microbiol 93957974 [View Article][PubMed]. [Google Scholar]
  3. Cingolani P., Platts A., Wang L., Coon M., Nguyen T., Wang L., Land S. J., Lu X., Ruden D. M. (2012). A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3Fly (Austin) 68092 [View Article][PubMed]. [Google Scholar]
  4. Coward C., van Diemen P. M., Conlan A. J., Gog J. R., Stevens M. P., Jones M. A., Maskell D. J. (2008). Competing isogenic Campylobacter strains exhibit variable population structures in vivoAppl Environ Microbiol 7438573867 [View Article][PubMed]. [Google Scholar]
  5. Friis C., Wassenaar T. M., Javed M. A., Snipen L., Lagesen K., Hallin P. F., Newell D. G., Toszeghy M., Ridley A., other authors. (2010). Genomic characterization of Campylobacter jejuni strain M1PLoS One 5e12253[PubMed].[CrossRef] [Google Scholar]
  6. Gaasbeek E. J., van der Wal F. J., van Putten J. P., de Boer P., van der Graaf-van Bloois L., de Boer A. G., Vermaning B. J., Wagenaar J. A. (2009). Functional characterization of excision repair and RecA-dependent recombinational DNA repair in Campylobacter jejuni J Bacteriol 19137853793 [View Article][PubMed]. [Google Scholar]
  7. Gao B., Lara-Tejero M., Lefebre M., Goodman A. L., Galán J. E. (2014). Novel components of the flagellar system in epsilonproteobacteriaMBio 5e01349e14 [View Article][PubMed]. [Google Scholar]
  8. Guerry P. (2007). Campylobacter flagella: not just for motilityTrends Microbiol 15456461 [View Article][PubMed]. [Google Scholar]
  9. Guerry P., Alm R. A., Power M. E., Logan S. M., Trust T. J. (1991). Role of two flagellin genes in Campylobacter motilityJ Bacteriol 17347574764[PubMed]. [Google Scholar]
  10. Hendrixson D. R. (2006). A phase-variable mechanism controlling the Campylobacter jejuni FlgR response regulator influences commensalismMol Microbiol 6116461659 [View Article][PubMed]. [Google Scholar]
  11. Hendrixson D. R. (2008). Restoration of flagellar biosynthesis by varied mutational events in Campylobacter jejuni Mol Microbiol 70519536 [View Article][PubMed]. [Google Scholar]
  12. Hendrixson D. R., DiRita V. J. (2003). Transcription of sigma54-dependent but not sigma28-dependent flagellar genes in Campylobacter jejuni is associated with formation of the flagellar secretory apparatusMol Microbiol 50687702 [View Article][PubMed]. [Google Scholar]
  13. Hendrixson D. R., DiRita V. J. (2004). Identification of Campylobacter jejuni genes involved in commensal colonization of the chick gastrointestinal tractMol Microbiol 52471484 [View Article][PubMed]. [Google Scholar]
  14. Hendrixson D. R., Akerley B. J., DiRita V. J. (2001). Transposon mutagenesis of Campylobacter jejuni identifies a bipartite energy taxis system required for motilityMol Microbiol 40214224 [View Article][PubMed]. [Google Scholar]
  15. Holt J. P., Grant A. J., Coward C., Maskell D. J., Quinlan J. J. (2012). Identification of Cj1051c as a major determinant for the restriction barrier of Campylobacter jejuni strain NCTC11168Appl Environ Microbiol 7878417848 [View Article][PubMed]. [Google Scholar]
  16. Howard S. L., Jagannathan A., Soo E. C., Hui J. P., Aubry A. J., Ahmed I., Karlyshev A., Kelly J. F., Jones M. A., other authors. (2009). Campylobacter jejuni glycosylation island important in cell charge, legionaminic acid biosynthesis, and colonization of chickensInfect Immun 7725442556 [View Article][PubMed]. [Google Scholar]
  17. Humphrey S., Chaloner G., Kemmett K., Davidson N., Williams N., Kipar A., Humphrey T., Wigley P. (2014). Campylobacter jejuni is not merely a commensal in commercial broiler chickens and affects bird welfareMBio 5e01364e14 [View Article][PubMed]. [Google Scholar]
  18. Jerome J. P., Bell J. A., Plovanich-Jones A. E., Barrick J. E., Brown C. T., Mansfield L. S. (2011). Standing genetic variation in contingency loci drives the rapid adaptation of Campylobacter jejuni to a novel hostPLoS One 6e16399 [View Article][PubMed]. [Google Scholar]
  19. Joslin S. N., Hendrixson D. R. (2009). Activation of the Campylobacter jejuni FlgSR two-component system is linked to the flagellar export apparatusJ Bacteriol 19126562667 [View Article][PubMed]. [Google Scholar]
  20. Karlyshev A. V., Linton D., Gregson N. A., Wren B. W. (2002). A novel paralogous gene family involved in phase-variable flagella-mediated motility in Campylobacter jejuni Microbiology 148473480 [View Article][PubMed]. [Google Scholar]
  21. Lertsethtakarn P., Ottemann K. M., Hendrixson D. R. (2011). Motility and chemotaxis in Campylobacter and Helicobacter Annu Rev Microbiol 65389410 [View Article][PubMed]. [Google Scholar]
  22. Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G., Durbin R., 1000 Genome Project Data Processing Subgroup. (2009). The Sequence Alignment/Map format and SAMtoolsBioinformatics 2520782079 [View Article][PubMed]. [Google Scholar]
  23. Lunter G., Goodson M. (2011). Stampy: a statistical algorithm for sensitive and fast mapping of Illumina sequence readsGenome Res 21936939 [View Article][PubMed]. [Google Scholar]
  24. Mohawk K. L., Poly F., Sahl J. W., Rasko D. A., Guerry P. (2014). High frequency, spontaneous motA mutations in Campylobacter jejuni strain 81-176PLoS One 9e88043 [View Article][PubMed]. [Google Scholar]
  25. Neal-McKinney J. M., Konkel M. E. (2012). The Campylobacter jejuni CiaC virulence protein is secreted from the flagellum and delivered to the cytosol of host cellsFront Cell Infect Microbiol 231 [View Article][PubMed]. [Google Scholar]
  26. Nuijten P. J., van Asten F. J., Gaastra W., van der Zeijst B. A. (1990). Structural and functional analysis of two Campylobacter jejuni flagellin genesJ Biol Chem 2651779817804[PubMed]. [Google Scholar]
  27. Nuijten P. J., van den Berg A. J., Formentini I., van der Zeijst B. A., Jacobs A. A. (2000). DNA rearrangements in the flagellin locus of an flaA mutant of Campylobacter jejuni during colonization of chicken cecaInfect Immun 6871377140 [View Article][PubMed]. [Google Scholar]
  28. Pearson B. M., Gaskin D. J., Segers R. P., Wells J. M., Nuijten P. J., van Vliet A. H. (2007). The complete genome sequence of Campylobacter jejuni strain 81116 (NCTC11828)J Bacteriol 18984028403 [View Article][PubMed]. [Google Scholar]
  29. Ruiz-Palacios G. M. (2007). The health burden of Campylobacter infection and the impact of antimicrobial resistance: playing chickenClin Infect Dis 44701703 [View Article][PubMed]. [Google Scholar]
  30. Seemann T. (2014). Prokka: rapid prokaryotic genome annotationBioinformatics 3020682069 [View Article][PubMed]. [Google Scholar]
  31. Tatusov R. L., Koonin E. V., Lipman D. J. (1997). A genomic perspective on protein familiesScience 278631637 [View Article][PubMed]. [Google Scholar]
  32. Thomas D. K., Lone A. G., Selinger L. B., Taboada E. N., Uwiera R. R., Abbott D. W., Inglis G. D. (2014). Comparative variation within the genome of Campylobacter jejuni NCTC 11168 in human and murine hostsPLoS One 9e88229 [View Article][PubMed]. [Google Scholar]
  33. Titz B., Rajagopala S. V., Ester C., Häuser R., Uetz P. (2006). Novel conserved assembly factor of the bacterial flagellumJ Bacteriol 18877007706 [View Article][PubMed]. [Google Scholar]
  34. van Alphen L. B., Wuhrer M., Bleumink-Pluym N. M., Hensbergen P. J., Deelder A. M., van Putten J. P. (2008). A functional Campylobacter jejuni maf4 gene results in novel glycoforms on flagellin and altered autoagglutination behaviourMicrobiology 15433853397 [View Article][PubMed]. [Google Scholar]
  35. van Vliet A. H., Wood A. C., Henderson J., Wooldridge K. G., Ketley J. M. (1998). Genetic manipulation of enteric Campylobacter species . In Methods in Microbiology, pp. 405419. Edited by Williams P., Ketley J., Salmond G. London, UKAcademic Press. [Google Scholar]
  36. Wassenaar T. M. (2011). Following an imaginary Campylobacter population from farm to fork and beyond: a bacterial perspectiveLett Appl Microbiol 53253263 [View Article][PubMed]. [Google Scholar]
  37. Wassenaar T. M., Bleumink-Pluym N. M., van der Zeijst B. A. (1991). Inactivation of Campylobacter jejuni flagellin genes by homologous recombination demonstrates that flaA but not flaB is required for invasionEMBO J 1020552061[PubMed]. [Google Scholar]
  38. Wassenaar T. M., Bleumink-Pluym N. M., Newell D. G., Nuijten P. J., van der Zeijst B. A. (1994). Differential flagellin expression in a flaA flaB+ mutant of Campylobacter jejuni Infect Immun 6239013906[PubMed]. [Google Scholar]
  39. Wassenaar T. M., Fry B. N., van der Zeijst B. A. (1995). Variation of the flagellin gene locus of Campylobacter jejuni by recombination and horizontal gene transferMicrobiology 14195101 [View Article][PubMed]. [Google Scholar]
  40. Wilson D. J., Gabriel E., Leatherbarrow A. J., Cheesbrough J., Gee S., Bolton E., Fox A., Hart C. A., Diggle P. J., Fearnhead P. (2009). Rapid evolution and the importance of recombination to the gastroenteric pathogen Campylobacter jejuni Mol Biol Evol 26385397 [View Article][PubMed]. [Google Scholar]
  41. Yanisch-Perron C., Vieira J., Messing J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectorsGene 33103119 [View Article][PubMed]. [Google Scholar]
  42. Yao R., Alm R. A., Trust T. J., Guerry P. (1993). Construction of new Campylobacter cloning vectors and a new mutational cat cassetteGene 130127130 [View Article][PubMed]. [Google Scholar]
  43. Yao R., Burr D. H., Doig P., Trust T. J., Niu H., Guerry P. (1994). Isolation of motile and non-motile insertional mutants of Campylobacter jejuni: the role of motility in adherence and invasion of eukaryotic cellsMol Microbiol 14883893 [View Article][PubMed]. [Google Scholar]
  44. Young K. T., Davis L. M., Dirita V. J. (2007). Campylobacter jejuni: molecular biology and pathogenesisNat Rev Microbiol 5665679 [View Article][PubMed]. [Google Scholar]

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