Site directed chromosomal marking of a fluorescent pseudomonad isolated from the phytosphere of sugar beet; stability and potential for marker gene transfer.

Authors

  • M. J. BAILEY,

    Corresponding author
    1. Natural Environment Research Council, Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, UK
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  • A. K. LILLEY,

    1. Natural Environment Research Council, Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, UK
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  • I. P. THOMPSON,

    1. Natural Environment Research Council, Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, UK
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  • P. B. RAINEY,

    Corresponding author
    1. Natural Environment Research Council, Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, UK
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  • R. J. ELLIS

    1. Natural Environment Research Council, Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, UK
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  • This work was undertaken as a part of our ongoing investigations into the ecology, community succession, gene flux and persistence of plant associated micrmrganisms. The work presented here is a part of an interdisciplinary research programme supported in part by the UK Department of the Environment and the European Community investigating the impact and dispersal of GMMs released to the environment. The design strategy selected for the GMh4 was based on our understanding of the microbiology of the plant surface and provides a means with which to undertake realistic environmental evaluations of the fate of inocula, bacterial population genetics and gene mobility.

†Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK.

Tel.: +44 (0)1865 512361. Fax: +44 (0)1865 59962. E-mail: MBJ@mail.nerc.-oxford.ac.uk

Abstract

A plasmid-free, non-pathogenic, ribosomal RNA group 1 fluorescent pseudomonad, Pseudomonas fluorescens SBW25, was selected from the microflora of sugar beet (Beta vulgaris) and modified to contain constitutively expressed marker genes. By site directed homologous recombination a KX cassette [kanamycin resistance (kanτ) and catechol 2,3 dioxygenase (xylE)] and a ZY cassette [lactose utilization (lacZY, β-galactosidase, lactose permease)] were introduced at least 1 Mbp apart on the 6.6 Mbp bacterial chromosome. Separate sites were selected to provide sensitive detection methods and allow assessments of marker gene stability of the genetically modified micro-organism (GMM), SBW25EeZY6KX, when it colonized the leaves and roots of sugar beet plants following seed inoculation.

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