Targeted allele replacement mutagenesis of Corynebacterium pseudotuberculosis

Caray A. Walker, Willie Donachie, David George Emslie Smith, Michael C. Fontaine

    Research output: Contribution to journalArticle

    Abstract

    A two-step allele replacement mutagenesis procedure, using a conditionally replicating plasmid, was developed to allow the creation of targeted, marker-free mutations in Corynebacterium pseudotuberculosis. The relationship between homologous sequence length and recombination frequency was determined, and enhanced plasmid excision was observed due to the rolling-circle replication of the mutagenesis vector. Furthermore, an antibiotic enrichment procedure was applied to improve the recovery of mutants. Subsequently, as proof of concept, a marker-free, cp40-deficient mutant of C. pseudotuberculosis was constructed.

    Original languageEnglish
    Pages (from-to)3532-3535
    Number of pages4
    JournalApplied and Environmental Microbiology
    Volume77
    Issue number10
    DOIs
    Publication statusPublished - May 2011

    Fingerprint

    Corynebacterium pseudotuberculosis
    Mutagenesis
    Plasmids
    Alleles
    Homologous Recombination
    Sequence Homology
    Anti-Bacterial Agents
    Mutation

    Keywords

    • Alleles
    • Anti-Bacterial Agents
    • Corynebacterium pseudotuberculosis
    • DNA, Bacterial
    • Gene Deletion
    • Gene Targeting
    • Genetic Vectors
    • Molecular Sequence Data
    • Mutagenesis, Insertional
    • Plasmids
    • Selection, Genetic
    • Sequence Analysis, DNA

    Cite this

    Walker, Caray A. ; Donachie, Willie ; Smith, David George Emslie ; Fontaine, Michael C. / Targeted allele replacement mutagenesis of Corynebacterium pseudotuberculosis. In: Applied and Environmental Microbiology. 2011 ; Vol. 77, No. 10. pp. 3532-3535.
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    Targeted allele replacement mutagenesis of Corynebacterium pseudotuberculosis. / Walker, Caray A.; Donachie, Willie; Smith, David George Emslie; Fontaine, Michael C.

    In: Applied and Environmental Microbiology, Vol. 77, No. 10, 05.2011, p. 3532-3535.

    Research output: Contribution to journalArticle

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    T1 - Targeted allele replacement mutagenesis of Corynebacterium pseudotuberculosis

    AU - Walker, Caray A.

    AU - Donachie, Willie

    AU - Smith, David George Emslie

    AU - Fontaine, Michael C.

    PY - 2011/5

    Y1 - 2011/5

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    KW - Alleles

    KW - Anti-Bacterial Agents

    KW - Corynebacterium pseudotuberculosis

    KW - DNA, Bacterial

    KW - Gene Deletion

    KW - Gene Targeting

    KW - Genetic Vectors

    KW - Molecular Sequence Data

    KW - Mutagenesis, Insertional

    KW - Plasmids

    KW - Selection, Genetic

    KW - Sequence Analysis, DNA

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