Legionella pp 197-212

Part of the Methods in Molecular Biology book series (MIMB, volume 954)

Constructing Unmarked Gene Deletions in Legionella pneumophila

  • Andrew Bryan
  • Zachary D. Abbott
  • Michele S. Swanson


The ability to construct recombinant alleles efficiently in strains of interest, particularly unmarked deletions that reduce the potential for polar effects, is essential to studies of both pathogenesis and basic bacterial physiology. Here we describe a three-phase approach for generating unmarked deletions in Legionella pneumophila by constructing a mutant allele in E. coli using λ-Red recombination, so-called recombineering; transferring the allele onto the L. pneumophila chromosome by natural transformation; and then removing the selectable marker by utilizing the Flp site-specific recombinase. This strategy can decrease the amount of clone screening required while also increasing the percentage of the time the desired allele is obtained on the first attempt. The approach is particularly suited for constructing multiple unmarked deletions in a single strain in fewer steps than traditional methods.

Key words

Legionella pneumophila Flp recombinase Recombination Recombineering Unmarked deletions Counter-selection 


  1. 1.
    Heckman KL, Pease LR (2007) Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc 2:924–932PubMedCrossRefGoogle Scholar
  2. 2.
    Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68PubMedCrossRefGoogle Scholar
  3. 3.
    Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59PubMedCrossRefGoogle Scholar
  4. 4.
    Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci USA 97:5978–5983PubMedCrossRefGoogle Scholar
  5. 5.
    Court DL, Sawitzke JA, Thomason LC (2002) Genetic engineering using homologous recombination. Annu Rev Genet 36:361–388PubMedCrossRefGoogle Scholar
  6. 6.
    Thomason L, Court DL, Bubunenko M, Costantino N, Wilson H, Datta S, Oppenheim A (2007) Recombineering: genetic engineering in bacteria using homologous recombination. Curr Protoc Mol Biol 78:1.16.11–11.16.24Google Scholar
  7. 7.
    Thomason LC, Costantino N, Shaw DV, Court DL (2007) Multicopy plasmid modification with phage lambda red recombineering. Plasmid 58:148–158PubMedCrossRefGoogle Scholar
  8. 8.
    Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645PubMedCrossRefGoogle Scholar
  9. 9.
    Sharan SK, Thomason LC, Kuznetsov SG, Court DL (2009) Recombineering: a homologous recombination-based method of genetic engineering. Nat Protoc 4:206–223PubMedCrossRefGoogle Scholar
  10. 10.
    Stone BJ, Kwaik YA (1999) Natural competence for DNA transformation by Legionella pneumophila and its association with expression of type IV pili. J Bacteriol 181:1395–1402PubMedGoogle Scholar
  11. 11.
    Sexton JA, Vogel JP (2004) Regulation of hypercompetence in Legionella pneumophila. J Bacteriol 186:3814–3825PubMedCrossRefGoogle Scholar
  12. 12.
    Bryan A, Harada K, Swanson MS (2011) Efficient generation of unmarked deletions in Legionella pneumophila. Appl Environ Microbiol 77:2545–2548PubMedCrossRefGoogle Scholar
  13. 13.
    Bryan A, Swanson MS (2011) Oligonucleotides stimulate genomic alterations of Legionella pneumophila. Mol Microbiol 80:231–247PubMedCrossRefGoogle Scholar
  14. 14.
    Reyrat JM, Pelicic V, Gicquel B, Rappuoli R (1998) Counterselectable markers: untapped tools for bacterial genetics and pathogenesis. Infect Immun 66:4011–4017PubMedGoogle Scholar
  15. 15.
    Goodwin A, Kersulyte D, Sisson G, Veldhuyzen van Zanten SJ, Berg DE, Hoffman PS (1998) Metronidazole resistance in Helicobacter pylori is due to null mutations in a gene (rdxA) that encodes an oxygen-insensitive NADPH nitroreductase. Mol Microbiol 28:383–393PubMedCrossRefGoogle Scholar
  16. 16.
    LeBlanc JJ, Davidson RJ, Hoffman PS (2006) Compensatory functions of two alkyl hydroperoxide reductases in the oxidative defense system of Legionella pneumophila. J Bacteriol 188:6235–6244PubMedCrossRefGoogle Scholar
  17. 17.
    Ott M (1994) Genetic approaches to study Legionella pneumophila pathogenicity. FEMS Microbiol Rev 14:161–176PubMedCrossRefGoogle Scholar
  18. 18.
    Merriam JJ, Mathur R, Maxfield-Boumil R, Isberg RR (1997) Analysis of the Legionella pneumophila fliI gene: intracellular growth of a defined mutant defective for flagellum biosynthesis. Infect Immun 65:2497–2501PubMedGoogle Scholar
  19. 19.
    Berger KH, Isberg RR (1993) Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol Microbiol 7:7–19PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Andrew Bryan
    • 1
  • Zachary D. Abbott
    • 1
  • Michele S. Swanson
    • 1
  1. 1.Department of Microbiology and ImmunologyUniversity of Michigan Medical SchoolAnn ArborUSA

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