Skip to main content
SpringerLink
Log in
Book cover

Gene Essentiality pp 97–123Cite as

Essential Genes in the Infection Model of Pseudomonas aeruginosa-PCR-Based Signature-Tagged Mutagenesis

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1279))

Abstract

PCR-based signature tagged mutagenesis is an “en masse” screening technique based upon unique oligonucleotide tags (molecular barcodes) for identification of genes that will diminish or enhance maintenance of an organism in a specific ecological niche or environment. PCR-based STM applied to Pseudomonas aeruginosa permitted the identification of genes essential for in vivo maintenance by transposon insertion and negative selection in a mixed population of bacterial mutants. The innovative adaptations and refinement of the technology presented here with P. aeruginosa STM mutants selected in the rat model of chronic lung infection have given critical information about genes essential for causing a chronic infection and a wealth of information about biological processes in vivo. The additional use of competitive index analysis for measurement of the level of virulence in vivo, microarray-based screening of selected prioritized STM mutants coupled to metabolomics analysis can now be attempted systematically on a genomic scale. PCR-based STM and combined whole-genome methods can also be applied to any organism having selectable phenotypes for screening.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Juhas M, Eberl L, Glass JI (2011) Essence of life: essential genes of minimal genomes. Trends Cell Biol 21:562–568

    Article  CAS  PubMed  Google Scholar 

  2. Handfield M, Levesque RC (1999) Strategies for isolation of in vivo expressed genes from bacteria. FEMS Microbiol Rev 23:69–91

    Article  CAS  PubMed  Google Scholar 

  3. Levesque RC (2006) In vivo functional genomics of Pseudomonas: PCR-based signature-tagged mutagenesis. Springer, Boston

    Google Scholar 

  4. Handfield M, Lehoux DE, Sanschagrin F et al (2000) In vivo-induced genes in Pseudomonas aeruginosa. Infect Immun 68:2359–2362

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Rusmini R, Vecchietti D, Macchi R et al (2014) A shotgun antisense approach to the identification of novel essential genes in Pseudomonas aeruginosa. BMC Microbiol 14:24

    Article  PubMed Central  PubMed  Google Scholar 

  6. Dotsch A, Klawonn F, Jarek M et al (2010) Evolutionary conservation of essential and highly expressed genes in Pseudomonas aeruginosa. BMC Genomics 11:234

    Article  PubMed Central  PubMed  Google Scholar 

  7. Skurnik D, Roux D, Aschard H et al (2013) A comprehensive analysis of in vitro and in vivo genetic fitness of Pseudomonas aeruginosa using high-throughput sequencing of transposon libraries. PLoS Pathog 9:e1003582

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Balasubramanian D, Mathee K (2009) Comparative transcriptome analyses of Pseudomonas aeruginosa. Hum Genomics 3:349–361

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Hensel M, Shea JE, Gleeson C et al (1995) Simultaneous identification of bacterial virulence genes by negative selection. Science 269:400–403

    Article  CAS  PubMed  Google Scholar 

  10. Lehoux DE, Sanschagrin F, Levesque RC (1999) Defined oligonucleotide tag pools and PCR screening in signature-tagged mutagenesis of essential genes from bacteria. Biotechniques 26:473–480

    CAS  PubMed  Google Scholar 

  11. Autret N, Charbit A (2005) Lessons from signature-tagged mutagenesis on the infectious mechanisms of pathogenic bacteria. FEMS Microbiol Rev 29:703–717

    Article  CAS  PubMed  Google Scholar 

  12. Potvin E, Lehoux DE, Kukavica-Ibrulj I et al (2003) In vivo functional genomics of Pseudomonas aeruginosa for high-throughput screening of new virulence factors and antibacterial targets. Environ Microbiol 5:1294–1308

    Article  CAS  PubMed  Google Scholar 

  13. Kukavica-Ibrulj I, Sanschagrin F, Peterson A et al (2008) Functional genomics of PycR, a LysR family transcriptional regulator essential for maintenance of Pseudomonas aeruginosa in the rat lung. Microbiology 154:2106–2118

    Article  CAS  PubMed  Google Scholar 

  14. Lehoux DE, Sanschagrin F, Levesque RC (2001) Discovering essential and infection-related genes. Curr Opin Microbiol 4:515–519

    Article  CAS  PubMed  Google Scholar 

  15. Lemieux A-A, Jeukens J, Kukavica-Ibrulj I et al (2014) Genes Required for free phage production are essential for Pseudomonas aeruginosa chronic lung infections. JID (submitted for publication)

    Google Scholar 

  16. Wiehlmann L, Salunkhe P, Larbig K et al (2002) Signature-tagged mutagenesis of Pseudomonas aeruginosa. Genome Lett 1:131–139

    Article  CAS  Google Scholar 

  17. Winstanley C, Langille MG, Fothergill JL et al (2009) Newly introduced genomic prophage islands are critical determinants of in vivo competitiveness in the Liverpool Epidemic Strain of Pseudomonas aeruginosa. Genome Res 19:12–23

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Stover CK, Pham XQ, Erwin AL et al (2000) Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406:959–964

    Article  CAS  PubMed  Google Scholar 

  19. Winsor GL, Lo R, Sui SJ et al (2005) Pseudomonas aeruginosa Genome Database and PseudoCAP: facilitating community-based, continually updated, genome annotation. Nucleic Acids Res 33:D338–D343

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. De Lorenzo V, Herrero M, Jakubzik U et al (1990) Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in Gram-negative eubacteria. J Bacteriol 172:6568–6572

    PubMed Central  PubMed  Google Scholar 

  21. Matthysse AG, Stretton S, Dandie C et al (1996) Construction of GFP vectors for use in gram-negative bacteria other than Escherichia coli. FEMS Microbiol Lett 145:87–94

    Article  CAS  PubMed  Google Scholar 

  22. Choi KH, Schweizer HP (2005) An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants. BMC Microbiol 5:30

    Article  PubMed Central  PubMed  Google Scholar 

  23. Schweizer HP (1991) Improved broad-host-range lac-based plasmid vectors for the isolation and characterization of protein fusions in Pseudomonas aeruginosa. Gene 103:87–92

    Article  CAS  PubMed  Google Scholar 

  24. Dewar K, Sabbagh L, Cardinal G et al (1998) Pseudomonas aeruginosa PAO1 bacterial artificial chromosomes: strategies for mapping, screening, and sequencing 100 kb loci of the 5.9 Mb genome. Microb Comp Genomics 3:105–117

    Article  CAS  PubMed  Google Scholar 

  25. Kwok S, Kellogg DE, Mckinney N et al (1990) Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res 18:999–1005

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Rychlik W (1993) Selection of primers for polymerase chain reaction. Humana Press, Totowa, NJ

    Google Scholar 

  27. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  28. Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. BioTechnology 1:784–791

    Article  CAS  Google Scholar 

  29. Cash HA, Woods DE, Mccullough B et al (1979) A rat model of chronic respiratory infection with Pseudomonas aeruginosa. Am Rev Respir Dis 119:453–459

    CAS  PubMed  Google Scholar 

  30. Van Heeckeren AM, Schluchter MD (2002) Murine models of chronic Pseudomonas aeruginosa lung infection. Lab Anim 36:291–312

    Article  PubMed  Google Scholar 

  31. Schweizer HP (1992) Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker. Mol Microbiol 6:1195–1204

    Article  CAS  PubMed  Google Scholar 

  32. Beuzon CR, Holden DW (2001) Use of mixed infections with Salmonella strains to study virulence genes and their interactions in vivo. Microbes Infect 3:1345–1352

    Article  CAS  PubMed  Google Scholar 

  33. Hava DL, Camilli A (2002) Large-scale identification of serotype 4 Streptococcus pneumoniae virulence factors. Mol Microbiol 45:1389–1406

    CAS  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank members of the bioinformatics and next-generation sequencing and genomics analysis platforms at IBIS for their excellent support. RCL is funded by the Canadian Institute of Health Research (CIHR), Cystic Fibrosis Canada, by the Fonds de Recherche du Québec—Nature et technologies (FQR-NT), by Génome Québec and Génome Canada. RCL is a member of the FQR-Santé-Respiratory Health Network.

Author information

Authors and Affiliations

  1. Institut de biologie intégrative et des systèmes (IBIS), Faculté de Médecine, Université Laval, Pavillon Charles-Eugène Marchand, 1030 Avenue de la médecine, Québec, QC, Canada, G1K 7P4

    Irena Kukavica-Ibrulj & Roger C. Levesque

Authors
  1. Irena Kukavica-Ibrulj
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Roger C. Levesque
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Roger C. Levesque .

Editor information

Editors and Affiliations

  1. Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA

    Long Jason Lu

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Kukavica-Ibrulj, I., Levesque, R.C. (2015). Essential Genes in the Infection Model of Pseudomonas aeruginosa-PCR-Based Signature-Tagged Mutagenesis. In: Lu, L. (eds) Gene Essentiality. Methods in Molecular Biology, vol 1279. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2398-4_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2398-4_7

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2397-7

  • Online ISBN: 978-1-4939-2398-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation