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NsrR targets in the Escherichia coli genome: new insights into DNA sequence requirements for binding and a role for NsrR in the regulation of motility

Authors

  • Jonathan D. Partridge,

    1. Department of Molecular and Cell Biology, The University of Texas at Dallas, 800 W Campbell Road, Richardson, TX 75080, USA.
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  • Diane M. Bodenmiller,

    1. School of Biology, Georgia Institute of Technology, 310 Ferst Drive, Atlanta, GA 30332, USA.
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    • Present addresses: Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA.

  • Michael S. Humphrys,

    1. School of Biology, Georgia Institute of Technology, 310 Ferst Drive, Atlanta, GA 30332, USA.
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    • Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA.

  • Stephen Spiro

    Corresponding author
    1. Department of Molecular and Cell Biology, The University of Texas at Dallas, 800 W Campbell Road, Richardson, TX 75080, USA.
      *E-mail stephen.spiro@utdallas.edu; Tel. (+1) 972 883 6896; Fax (+1) 972 883 2409.
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*E-mail stephen.spiro@utdallas.edu; Tel. (+1) 972 883 6896; Fax (+1) 972 883 2409.

Summary

The Escherichia coli NsrR protein is a nitric oxide-sensitive repressor of transcription. The NsrR-binding site is predicted to comprise two copies of an 11 bp motif arranged as an inverted repeat with 1 bp spacing. By mutagenesis we confirmed that both 11 bp motifs are required for maximal NsrR repression of the ytfE promoter. We used chromatin immunoprecipitation and microarray analysis (ChIP-chip) to show that NsrR binds to 62 sites close to the 5′ ends of genes. Analysis of the ChIP-chip data suggested that a single 11 bp motif (with the consensus sequence AANATGCATTT) can function as an NsrR-binding site in vivo. NsrR binds to sites in the promoter regions of the fliAZY, fliLMNOPQR and mqsR-ygiT transcription units, which encode proteins involved in motility and biofilm development. Reporter fusion assays confirmed that NsrR negatively regulates the fliA and fliL promoters. A mutation in the predicted 11 bp NsrR-binding site in the fliA promoter impaired repression by NsrR and prevented detectable binding in vivo. Assays on soft-agar confirmed that NsrR is a negative regulator of motility in E. coli K12 and in a uropathogenic strain; surface attachment assays revealed decreased levels of attached growth in the absence of NsrR.

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