Abstract
Construction of gene disruption mutants and analysis of the resultant phenotypes are an important strategy to study gene function. A simple and high-throughput method developed for microorganisms combines two different types of transposons, direct genomic DNA amplification and thermal asymmetric interlaced-PCR. The considerable utility of this approach is demonstrable in Corynebacterium glutamicum, where 18,000 transposon disruptants enabled the generation of an insertion library covering nearly 80% of the organism’s 2,990 ORFs.
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References
Dean F. B., Nelson J. R., Giesler T. L., and Lasken R. S. (2001) Rapid amplification of plasmid and phage DNA using Phi29 DNA polymerase and 5 multiply-primed rolling circle amplification. Genome Res. 11, 1095–1099.
Knobloch J. K., Nedelmann M., Kiel K., Bartscht K., Horstkotte M. A., Dobinsky S., Rohde H., and Mack D. (2003) Establishment of an arbitrary PCR for rapid identification of Tn917 insertion sites in Staphylococcus epidermidis: characterization of biofilm-negative and nonmucoid mutants. Appl. Environ. Microbiol. 69, 5812–5818.
Liu Y. G., Mitsukawa N., Oosumi T., and Whittier R. F. (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J. 8, 457–463.
Vertès A. A., Asai Y., Inui M., Kobayashi M., Kurusu Y., and Yukawa H. (1994) Transposon mutagenesis of coryneform bacteria. Mol. Gen. Genet. 245, 397–405.
Vertès A. A., Inui M., Kobayashi M., Kurusu Y., and Yukawa H. (1994) Isolation and characterization of IS31831, a transposable element from Corynebacterium glutamicum. Mol. Microbiol. 11, 739–746.
Goryshin I. Y. and Reznikoff W. S. (1998) Tn5 in vitro transposition. J. Biol. Chem. 273, 7367–7374.
Goryshin I. Y., Miller J. A., Kil Y. V., Lanzov V. A., and Reznikoff W. S. (1998) Tn5/IS50 target recognition. Proc. Natl. Acad. Sci. U. S. A. 95, 10716–10721.
Oram D. M., Avdalovic A., and Holmes R. K. (2002) Construction and characterization of transposon insertion mutations in Corynebacterium diphtheriae that affect expression of the diphtheria toxin repressor (DtxR). J. Bacteriol. 184, 5723–5732.
Goryshin I. Y., Jendrisak J. L., Hoffman M., Meis R., and Reznikoff W. S. (2000) Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes. Nat. Biotechnol. 18, 97–100.
Herron P. R., Hughes G., Chandra G., Fielding S., and Dyson P. J. (2004) Transposon Express, a software application to report the identity of insertions obtained by comprehensive transposon mutagenesis of sequenced genomes: analysis of the preference for in vitro Tn5 transposition into GC-rich DNA. Nucleic. Acids. Res. 32, e113.
Suzuki N., Okai N., Nonaka H., Tsuge Y., Inui M., and Yukawa H. (2006) High throughput transposon mutagenesis of Corynebacterium glutamicum and construction of a single-gene disruptant mutant library. Appl. Environ. Microbiol. 72, 3750–3755.
Vertès A. A., Inui M., Kobayashi M., Kurusu Y., Yukawa H. (1993) Presence of mrr- and mcr-like restriction systems in coryneform bacteria. Res. Microbiol. 144, 181–185.
Acknowledgments
We wish to thank Dr. C. Omumasaba for critical reading of the manuscript. This research was partly supported by New Energy and Industrial Technology Development Organization (NEDO), Japan.
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Suzuki, N., Inui, M., Yukawa, H. (2011). High-Throughput Transposon Mutagenesis of Corynebacterium glutamicum . In: Williams, J. (eds) Strain Engineering. Methods in Molecular Biology, vol 765. Humana Press. https://doi.org/10.1007/978-1-61779-197-0_24
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DOI: https://doi.org/10.1007/978-1-61779-197-0_24
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