A member of the cAMP receptor protein family of transcription regulators in Mycobacterium tuberculosis is required for virulence in mice and controls transcription of the rpfA gene coding for a resuscitation promoting factor

Authors

  • Lisa Rickman,

    1. Division of Mycobacterial Research, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
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    • Present address: The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK;

  • Colin Scott,

    1. The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
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    • CSIRO Entomology, Canberra, ACT 2601, Australia;

  • Debbie M. Hunt,

    1. Division of Mycobacterial Research, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
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  • Thomas Hutchinson,

    1. The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
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  • M. Carmen Menéndez,

    1. Division of Mycobacterial Research, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
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    • CSIRO Entomology, Canberra, ACT 2601, Australia;

  • Rachael Whalan,

    1. Division of Mycobacterial Research, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
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  • Jason Hinds,

    1. Bacterial Microarray Group, Department of Medical Microbiology, St. George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK.
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  • M. Joseph Colston,

    1. Division of Mycobacterial Research, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
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    • §

      Departamento de Medicina Preventiva, Facultad de Medicina, Universidad Autonoma de Madrid, Arzobispo Morcillo, s/n. 28029-Madrid, Spain.

    • Deceased 20 February 2003.

  • Jeffrey Green,

    1. The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
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  • Roger S. Buxton

    Corresponding author
    1. Division of Mycobacterial Research, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
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E-mail rbuxton@nimr.mrc.ac.uk; Tel. (+44) 020 8816 2225; Fax (+44) 020 8906 4477.

Summary

Deletion of gene Rv3676 in Mycobacterium tuberculosis coding for a transcription factor belonging to the cAMP receptor protein (CRP) family caused growth defects in laboratory medium, in bone marrow-derived macrophages and in a mouse model of tuberculosis. Transcript profiling of M. tuberculosis grown in vitro identified 16 genes with significantly altered expression in the mutant compared with the wild type. Analysis of the DNA sequences upstream of the corresponding open reading frames revealed that 12 possessed sequences related to a consensus CRP binding site that could represent the sites of action of Rv3676. These included rpfA, lprQ, whiB1 and ahpC among genes with enhanced expression in the wild type, and Rv3616c-Rv3613c, Rv0188 and lipQ among genes exhibiting enhanced expression in the mutant. The activity of an rpfA::lacZ promoter fusion was lowered in the Rv3676 mutant and by mutation of the predicted Rv3676 binding site. Moreover, the product of Rv3676 (isolated as a TrxA fusion protein) interacted specifically with the rpfA promoter, and binding was inhibited by mutation of the Rv3676 site. Although Rv3676 retains four of the six amino acid residues that bind cAMP in Escherichia coli CRP addition of cAMP did not enhance Rv3676 binding at the rpfA promoter in vitro. In summary, it has been shown that Rv3676 is a direct regulator of rpfA expression, and because rpfA codes for a resuscitation promoting factor this may implicate Rv3676 in reactivation of dormant M. tuberculosis infections.

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