Advertisement

Generation of Nonpolar Deletion Mutants in Listeria monocytogenes Using the “SOEing” Method

  • Kathrin RychliEmail author
  • Caitriona M. Guinane
  • Karen Daly
  • Colin Hill
  • Paul D. Cotter
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1157)

Abstract

The ability to manipulate chromosomally encoded genes is a fundamental biological tool for the analysis of gene function. Here, we provide in greater depth protocols for the creation of nonpolar unlabeled gene deletions in Listeria (L.) monocytogenes that are facilitated by the splicing overlap extension PCR technique. For mutagenesis in L. monocytogenes, we describe two different plasmid-based approaches, which facilitate the introduction of this spliced amplicon in place of the corresponding segment of chromosomal DNA: the pKSV7 system and the pORI280/pVE6007 system.

Keywords

Nonpolar deletion mutants Listeria monocytogenes SOEing method pKSV7 vector pORI280/pVE6007 system 

Notes

Acknowledgement

This work was supported by the EU FOODSEG grant.

References

  1. 1.
    Arnaud M, Chastanet A, Debarbouille M (2004) New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria. Appl Environ Microbiol 70:6887–6891PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Li G, Kathariou S (2003) An improved cloning vector for construction of gene replacements in Listeria monocytogenes. Appl Environ Microbiol 69:3020–3023PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Monk IR, Gahan CG, Hill C (2008) Tools for functional postgenomic analysis of Listeria monocytogenes. Appl Environ Microbiol 74:3921–3934PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Horton RM, Cai ZL, Ho SN (1990) Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques 8:528–535PubMedGoogle Scholar
  5. 5.
    Horton RM (1995) PCR-mediated recombination and mutagenesis. SOEing together tailor-made genes. Mol Biotechnol 3:93–99PubMedCrossRefGoogle Scholar
  6. 6.
    Cotter PD, Gahan CG, Hill C (2001) A glutamate decarboxylase system protects Listeria monocytogenes in gastric fluid. Mol Microbiol 40:465–475PubMedCrossRefGoogle Scholar
  7. 7.
    Wiedmann M, Arvik TJ, Hurley RJ (1998) General stress transcription factor sigmaB and its role in acid tolerance and virulence of Listeria monocytogenes. J Bacteriol 180:3650–3656PubMedCentralPubMedGoogle Scholar
  8. 8.
    Smith K, Youngman P (1992) Use of a new integrational vector to investigate compartment-specific expression of the Bacillus subtilis spoIIM gene. Biochimie 74:705–711PubMedCrossRefGoogle Scholar
  9. 9.
    Law J, Buist G, Haandrikman A (1995) A system to generate chromosomal mutations in Lactococcus lactis which allows fast analysis of targeted genes. J Bacteriol 177:7011–7018PubMedCentralPubMedGoogle Scholar
  10. 10.
    Maguin E, Duwat P, Hege T (1992) New thermosensitive plasmid for gram-positive bacteria. J Bacteriol 174:5633–5638PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Kathrin Rychli
    • 1
    Email author
  • Caitriona M. Guinane
    • 2
  • Karen Daly
    • 3
  • Colin Hill
    • 3
    • 4
  • Paul D. Cotter
    • 2
    • 4
  1. 1.University of Veterinary Medicine ViennaViennaAustria
  2. 2.Teagasc Food Research Centre, MooreparkFermoy, County CorkIreland
  3. 3.Department of MicrobiologyUniversity College CorkCorkIreland
  4. 4.Alimentary Pharmabiotic CentreUniversity College CorkCorkIreland

Personalised recommendations