Random Transposon Mutagenesis of Vibrio cholerae

  • Jyl S. MatsonEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1839)


Transposon-based random mutagenesis of bacterial genomes has proven to be a powerful genetic tool for the identification of genes and regulatory elements that contribute to specific phenotypes. One such approach that has been used in Vibrio cholerae for many years is the introduction of mariner transposons to generate random libraries of mutants. These libraries have been successfully used for a wide variety of genetic screens and selections in this important bacterial pathogen. Here we present a detailed protocol for the use of plasmid pFD1 (containing the mariner transposon magellan3) to create mutant libraries in V. cholerae.

Key words

Random mutagenesis Transposon mutagenesis Random-primed sequencing Nested PCR Mariner transposon 


  1. 1.
    Lampe DJ, Churchill ME, Robertson HM (1996) A purified mariner transposase is sufficient to mediate transposition in vitro. EMBO J 15:5470–5479PubMedPubMedCentralCrossRefGoogle Scholar
  2. 2.
    Rubin EJ, Akerley BJ, Novik VN, Lampe DJ, Husson RN, Mekalanos JJ (1999) In vivo transposition of mariner-based elements in enteric bacteria and mycobacteria. Proc Natl Acad Sci U S A 96:1645–1650CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Curtis PD, Klein D, Brun YV (2013) Effect of a ctrA promoter mutation, causing a reduction in CtrA abundance, on the cell cycle and development of Caulobacter crescentus. BMC Microbiol 13:166CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Heckel BC, Tomlinson AD, Morton ER, Choi JH, Fuqua C (2014) Agrobacterium tumefaciens exoR controls acid response genes and impacts exopolysaccharide synthesis, horizontal gene transfer, and virulence gene expression. J Bacteriol 196:3221–3233CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Pan NJ, Brady MJ, Leong JM, Goguen JD (2009) Targeting type III secretion in Yersinia pestis. Antimicrob Agents Chemother 53:385–392CrossRefPubMedGoogle Scholar
  6. 6.
    Guo BP, Mekalanos JJ (2001) Helicobacter pylori mutagenesis by mariner in vitro transposition. FEMS Immunol Med Microbiol 30:87–93CrossRefPubMedGoogle Scholar
  7. 7.
    Hendrixson DR, Akerley BJ, DiRita VJ (2001) Transposon mutagenesis of Campylobacter jejuni identifies a bipartite energy taxis system required for motility. Mol Microbiol 40:214–224CrossRefPubMedGoogle Scholar
  8. 8.
    Wong SM, Mekalanos JJ (2000) Genetic footprinting with mariner-based transposition in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 97:10191–10196CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Matson JS, DiRita VJ (2005) Degradation of the membrane-localized virulence activator TcpP by the YaeL protease in Vibrio cholerae. Proc Natl Acad Sci U S A 102:16403–16408CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Matson JS, Yoo HJ, Hakansson K, DiRita VJ (2010) Polymyxin B resistance in El Tor Vibrio cholerae requires lipid acylation catalyzed by MsbB. J Bacteriol 192:2044–2052CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Hase CC, Mekalanos JJ (1999) Effects of changes in membrane sodium flux on virulence gene expression in Vibrio cholerae. Proc Natl Acad Sci U S A 96:3183–3187CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Van Dellen KL, Houot L, Watnick PI (2008) Genetic analysis of Vibrio cholerae monolayer formation reveals a key role for DeltaPsi in the transition to permanent attachment. J Bacteriol 190:8185–8196CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Minato Y, Fassio SR, Wolfe AJ, Hase CC (2013) Central metabolism controls transcription of a virulence gene regulator in Vibrio cholerae. Microbiology 159:792–802CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Medical Microbiology and ImmunologyUniversity of Toledo College of Medicine and Life SciencesToledoUSA

Personalised recommendations