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Cra regulates the cross-talk between the two branches of the phosphoenolpyruvate : phosphotransferase system of Pseudomonas putida

Authors

  • Max Chavarría,

    1. Systems Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), 28049 Cantoblanco-Madrid, Spain.
    2. Escuela de Química, Universidad de Costa Rica, 2060, San José, Costa Rica.
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  • Tobias Fuhrer,

    1. Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland.
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  • Uwe Sauer,

    1. Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland.
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  • Katharina Pflüger-Grau,

    1. Systems Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), 28049 Cantoblanco-Madrid, Spain.
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    • Present address: Fachgebiet Systembiotechnologie, Technische Universität München, Garching, München, Germany.

  • Víctor de Lorenzo

    Corresponding author
    1. Systems Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), 28049 Cantoblanco-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 gene that encodes the catabolite repressor/activator, Cra (FruR), of Pseudomonas putida is divergent from the fruBKA operon for the uptake of fructose via the phosphoenolpyruvate : carbohydrate phosphotransferase system (PTSFru). The expression of the fru cluster has been studied in cells growing on substrates that change the intracellular concentrations of fructose-1-P (F1P), the principal metabolic intermediate that counteracts the DNA-binding ability of Cra on an upstream operator. While the levels of the regulator were not affected by any of the growth conditions tested, the transcription of fruB was stimulated by fructose but not by the gluconeogenic substrate, succinate. The analysis of the PfruB promoter activity in a strain lacking the Cra protein and the determination of key metabolites revealed that this regulator represses the expression of PTSFru in a fashion that is dependent on the endogenous concentrations of F1P. Because FruB (i.e. the EI-HPr-EIIAFru polyprotein) can deliver a high-energy phosphate to the EIIANtr (PtsN) enzyme of the PTSNtr branch, the cross-talk between the two phosphotransferase systems was examined under metabolic regimes that allowed for the high or low transcription of the fruBKA operon. While fructose caused cross-talk, succinate prevented it almost completely. Furthermore, PtsN phosphorylation by FruB occurred in a Δcra mutant regardless of growth conditions. These results traced the occurrence of the cross-talk to intracellular pools of Cra effectors, in particular F1P. The Cra/F1P duo seems to not only control the expression of the PTSFru but also checks the activity of the PTSNtrin vivo.

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