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Functional analysis of the integration host factor site of the σ54Pu promoter of Pseudomonas putida by in vivo UV imprinting

Authors

  • Marc Valls,

    1. Dept. de Genètica, Facultat de Biologia, Universitat de Barcelona, 08028 Barcelona, Spain
    2. Systems and Synthetic Biology Program, Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco, 28049 Madrid, Spain
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  • Rafael Silva-Rocha,

    1. Systems and Synthetic Biology Program, Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco, 28049 Madrid, Spain
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  • Ildefonso Cases,

    1. Systems and Synthetic Biology Program, Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco, 28049 Madrid, Spain
    2. Institute of Predictive and Personalized Medicine of Cancer (IMPPC), 08916 Barcelona, Spain
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  • Amalia Muñoz,

    1. Systems and Synthetic Biology Program, Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco, 28049 Madrid, Spain
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    • Present address: Institute for Reference Materials and Measurements, Joint Research Centre, European Commission, Geel, Belgium.

  • Víctor de Lorenzo

    Corresponding author
    1. Systems and Synthetic Biology Program, Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco, 28049 Madrid, Spain
      E-mail vdlorenzo@cnb.csic.es; Tel. (+34) 91 585 45 36; Fax (+34) 91 585 45 06.
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E-mail vdlorenzo@cnb.csic.es; Tel. (+34) 91 585 45 36; Fax (+34) 91 585 45 06.

Summary

The integration host factor (IHF) of Pseudomonas putida connects cell growth to transcriptional activity of distinct promoters. The IHF site of the σ54 promoter Pu of the TOL (m-xylene biodegradation) plasmid pWW0 of P. putida has been examined to define experimentally a relationship between occupation of the promoter by this factor, the biological activity of the protein and the tolerance of the target site to single-base changes through the bound DNA core sequence. The use of an in vivo high-intensity UV imprinting procedure to examine such an occupation of Pu by IHF allowed inspection of the interplay between the factor and cognate site variants under the physiologically relevant conditions of monocopy gene dosage. The resulting data were merged in a structural model for establishing key features of the IHF–DNA interaction. A functional consensus for first-order IHF binding was instrumental for a genome-wide survey of sequences with potential regulatory value. This search revealed that very few, if any, of the maximum 330 sites within intergenic regions were placed in locations controlling expression of central metabolic genes. It thus seems that the IHF regulon of P. putida has a degree of functional specialization that is not evenly distributed through all gene categories.

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