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A lactococcal pWV01-based integration toolbox for bacteria

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Methods for studying the genetics, molecular biology, physiology, and pathogenesis of the streptococci

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Abstract

A conditionally replicating lactococcal vector system is described, based on pWV01, that is used for chromosomal integration in Lactococcus lactis. The system consists of plasmids that are all based on the broad-host-range lactococcal replicon pWV01 which has been deprived of its gene encoding the replication initiation protein RepA. These so-called pORI plasmids can only replicate if RepA is provided in trans. Special Escherichia coli, Bacillus subtilis and L. lactis helper strains, producing RepA in trans (RepA+), are used as intermediate hosts for the construction of pORI integration plasmids. The presence of a lactococcal chromosomal DNA fragment in a pORI plasmid enables its chromosomal integration by homologous recombination in an L. lactis strain that does not produce RepA (RepA−). A set of special purpose pORI vectors are available: (i) pOR1280, designed to mutate or delete genes or to insert new genes in the chromosome in such a way that no heterologous DNA or antibiotic resistance markers are left in the recombinant strain, here designated as silent gene replacement; (ii) pORIl9, suitable for random mutagenesis of the chromosome, and (iii) pORI13, constructed to make random single copy transcriptional fusions in the chromosome to search for (environmentally regulated) promoters. Some of these vectors have also been succesfully applied for integration in E. coli and B. subtilis. We strongly believe that the systems described here can be used in various other bacterial species.

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Author information

Authors and Affiliations

  1. Department of Genetics, Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands

    Kees Leenhouts & Gerard Venema

  2. Department of Genetics, Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 NN, Haren, The Netherlands

    Dr Jan Kok

Authors
  1. Kees Leenhouts
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  2. Gerard Venema
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  3. Dr Jan Kok
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Corresponding author

Correspondence to Jan Kok .

Editor information

Editors and Affiliations

  1. Department of Microbiology and Molecular Genetics, University of Vermont, Stafford Building, 05405, Burlington, Vermont, USA

    Paula M. Fives-Taylor

  2. Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana, USA

    Donald J. LeBlanc

  3. Department of Oral Biology, Indiana University School of Dentistry, Indianapolis, IN, USA

    Donald J. LeBlanc

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© 1998 Springer Science+Business Media Dordrecht

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Leenhouts, K., Venema, G., Kok, J. (1998). A lactococcal pWV01-based integration toolbox for bacteria. In: Fives-Taylor, P.M., LeBlanc, D.J. (eds) Methods for studying the genetics, molecular biology, physiology, and pathogenesis of the streptococci. Springer, Dordrecht. https://doi.org/10.1007/978-94-017-2258-2_4

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  • DOI: https://doi.org/10.1007/978-94-017-2258-2_4

  • Publisher Name: Springer, Dordrecht

  • Print ISBN: 978-90-481-5262-9

  • Online ISBN: 978-94-017-2258-2

  • eBook Packages: Springer Book Archive

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