Abstract
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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Heckman KL, Pease LR (2007) Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc 2:924–932
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 77:61–68
Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59
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 97:5978–5983
Court DL, Sawitzke JA, Thomason LC (2002) Genetic engineering using homologous recombination. Annu Rev Genet 36:361–388
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 78:1.16.11–11.16.24
Thomason LC, Costantino N, Shaw DV, Court DL (2007) Multicopy plasmid modification with phage lambda red recombineering. Plasmid 58:148–158
Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645
Sharan SK, Thomason LC, Kuznetsov SG, Court DL (2009) Recombineering: a homologous recombination-based method of genetic engineering. Nat Protoc 4:206–223
Stone BJ, Kwaik YA (1999) Natural competence for DNA transformation by Legionella pneumophila and its association with expression of type IV pili. J Bacteriol 181:1395–1402
Sexton JA, Vogel JP (2004) Regulation of hypercompetence in Legionella pneumophila. J Bacteriol 186:3814–3825
Bryan A, Harada K, Swanson MS (2011) Efficient generation of unmarked deletions in Legionella pneumophila. Appl Environ Microbiol 77:2545–2548
Bryan A, Swanson MS (2011) Oligonucleotides stimulate genomic alterations of Legionella pneumophila. Mol Microbiol 80:231–247
Reyrat JM, Pelicic V, Gicquel B, Rappuoli R (1998) Counterselectable markers: untapped tools for bacterial genetics and pathogenesis. Infect Immun 66:4011–4017
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 28:383–393
LeBlanc JJ, Davidson RJ, Hoffman PS (2006) Compensatory functions of two alkyl hydroperoxide reductases in the oxidative defense system of Legionella pneumophila. J Bacteriol 188:6235–6244
Ott M (1994) Genetic approaches to study Legionella pneumophila pathogenicity. FEMS Microbiol Rev 14:161–176
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 65:2497–2501
Berger KH, Isberg RR (1993) Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol Microbiol 7:7–19
Acknowledgments
This work was supported by University of Michigan, Department of Microbiology and Immunology, Novy Fellowship to A.B., and a Rackham Merit Fellowship to Z.D.A.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Bryan, A., Abbott, Z.D., Swanson, M.S. (2013). Constructing Unmarked Gene Deletions in Legionella pneumophila . In: Buchrieser, C., Hilbi, H. (eds) Legionella. Methods in Molecular Biology, vol 954. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-161-5_10
Download citation
DOI: https://doi.org/10.1007/978-1-62703-161-5_10
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-160-8
Online ISBN: 978-1-62703-161-5
eBook Packages: Springer Protocols