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 a protocol for the creation of nonpolar unlabelled gene deletions in Listeria monocytogenes that are facilitated by the splicing overlap extension PCR technique. For mutagenesis in L. monocytogenes, we describe the pKSV7 plasmid-based approach, which facilitates the introduction of a spliced amplicon in place of the corresponding segment of chromosomal DNA.
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References
Arnaud M, Chastanet A, Débarbouillé M (2004) New vector for efficient allelic replacement in naturally gram-positive bacteria. Appl Environ Microbiol 70:6887–6891. https://doi.org/10.1128/AEM.70.11.6887
Li G, Kathariou S (2003) An improved cloning vector for construction of gene replacements in Listeria monocytogenes. Appl Environ Microbiol 69:3020–3023. https://doi.org/10.1128/AEM.69.5.3020-3023.2003
Monk IR, Gahan CGM, Hill C (2008) Tools for functional postgenomic analysis of Listeria monocytogenes. Appl Environ Microbiol 74:3921–3934. https://doi.org/10.1128/AEM.00314-08
Horton RM (1995) PCR-mediated recombination and mutagenesis. SOEing together tailor-made genes. Mol Biotechnol 3:93–99. https://doi.org/10.1007/BF02789105
Horton RM, Cai Z, Ho SM, Pease LR (1990) Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. BioTechniques 54:129–133. https://doi.org/10.2144/000114017
Cotter PD, Gahan CGM, Hill C (2001) A glutamate decarboxylase system protects Listeria monocytogenes in gastric fluid. Mol Microbiol 40:465–475. https://doi.org/10.1046/j.1365-2958.2001.02398.x
Wiedmann M, Arvik TJ, Hurley RJ, Boor KJ (1998) General stress transcription factor sigmaB and its role in acid tolerance and virulence of Listeria monocytogenes. J Bacteriol 180:3650–3656
Müller A, Rychli K, Muhterem-Uyar M et al (2013) Tn6188 – a novel transposon in Listeria monocytogenes responsible for tolerance to benzalkonium chloride. PLoS One 8:1–11. https://doi.org/10.1371/journal.pone.0076835
Harter E, Wagner EM, Zaiser A et al (2017) Stress survival islet 2, predominantly present in Listeria monocytogenes strains of sequence type 121, is involved in the alkaline and oxidative stress responses. Appl Environ Microbiol 83:e00827-17. https://doi.org/10.1128/AEM.00827-17
Harter E, Lassnig C, Wagner EM et al (2019) The novel internalins InlP1 and InlP4 and the internalin-like protein InlP3 enhance the pathogenicity of Listeria monocytogenes. Front Microbiol 10:1644. https://doi.org/10.3389/fmicb.2019.01644
Cheng C, Jiang L, Ma T et al (2017) Carboxyl-terminal residues N478 and V479 required for the cytolytic activity of listeriolysin O play a critical role in Listeria monocytogenes pathogenicity. Front Immunol 8:1439. https://doi.org/10.3389/fimmu.2017.01439
Smith K, Youngman P (1992) Use of a new integrational vector to investigate compartment-specific expression of the Bacillus subtilis spoIIM gene. Biochimie 74:705–711. https://doi.org/10.1016/0300-9084(92)90143-3
Acknowledgments
This work was supported by the EU FOODSEG grant and the Austrian Science Fund (FWF), grant P27920-B22.
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Rychli, K., Wagner, E., Guinane, C.M., Daly, K., Hill, C., Cotter, P.D. (2021). Generation of Nonpolar Deletion Mutants in Listeria monocytogenes Using the “SOEing” Method. In: Fox, E.M., Bierne, H., Stessl, B. (eds) Listeria Monocytogenes. Methods in Molecular Biology, vol 2220. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0982-8_13
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DOI: https://doi.org/10.1007/978-1-0716-0982-8_13
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