1887

Abstract

BlcR represses transcription of the operon, which is involved in metabolism of γ-butyrolactone, and this repression is alleviated by succinate semialdehyde (SSA). BlcR exists as a homodimer, and the promoter DNA contains two BlcR-binding sites (IR1 and IR2) that correspond to two BlcR dimers. In this study, we established an system to examine the SSA-responsive control of BlcR transcriptional regulation. The endogenous , encoded in the pAtC58 plasmid of C58, was not optimal for investigating the effect of SSA on BlcR repression, probably due to the SSA degradation mediated by the pAt-encoded . We therefore introduced (and the promoter DNA, separately) exogenously into a strain of C58 cured of pAtC58 (and pTiC58). We applied this system to interrogate BlcR–DNA interactions and to test predictions from our prior structural and biochemical studies. This analysis confirmed the previously mapped SSA-binding site and supported a model by which DNA coordinates formation of a BlcR tetramer. In addition, we identified a specific lysine residue (K59) as an important determinant for DNA binding. Moreover, based on isothermal titration calorimetry analysis, we found IR1 to play the dominant role in binding to BlcR, relative to IR2. Together, these results expand the biochemical findings and provide new mechanistic insights into BlcR–DNA interactions.

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2013-04-01
2020-01-26
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References

  1. Brune I., Jochmann N., Brinkrolf K., Hüser A. T., Gerstmeir R., Eikmanns B. J., Kalinowski J., Pühler A., Tauch A..( 2007;). The IclR-type transcriptional repressor LtbR regulates the expression of leucine and tryptophan biosynthesis genes in the amino acid producer Corynebacterium glutamicum. J Bacteriol189:2720–2733 [CrossRef][PubMed]
    [Google Scholar]
  2. Carlier A., Chevrot R., Dessaux Y., Faure D..( 2004;). The assimilation of γ-butyrolactone in Agrobacterium tumefaciens C58 interferes with the accumulation of the N-acyl-homoserine lactone signal. Mol Plant Microbe Interact17:951–957 [CrossRef][PubMed]
    [Google Scholar]
  3. Chai Y., Tsai C. S., Cho H., Winans S. C..( 2007;). Reconstitution of the biochemical activities of the AttJ repressor and the AttK, AttL, and AttM catabolic enzymes of Agrobacterium tumefaciens. J Bacteriol189:3674–3679 [CrossRef][PubMed]
    [Google Scholar]
  4. Guazzaroni M. E., Krell T., Felipe A., Ruiz R., Meng C., Zhang X., Gallegos M. T., Ramos J. L..( 2005;). The multidrug efflux regulator TtgV recognizes a wide range of structurally different effectors in solution and complexed with target DNA: evidence from isothermal titration calorimetry. J Biol Chem280:20887–20893 [CrossRef][PubMed]
    [Google Scholar]
  5. Guazzaroni M. E., Gallegos M. T., Ramos J. L., Krell T..( 2007a;). Different modes of binding of mono- and biaromatic effectors to the transcriptional regulator TTGV: role in differential derepression from its cognate operator. J Biol Chem282:16308–16316 [CrossRef][PubMed]
    [Google Scholar]
  6. Guazzaroni M. E., Krell T., Gutiérrez del Arroyo P., Vélez M., Jiménez M., Rivas G., Ramos J. L..( 2007b;). The transcriptional repressor TtgV recognizes a complex operator as a tetramer and induces convex DNA bending. J Mol Biol369:927–939 [CrossRef][PubMed]
    [Google Scholar]
  7. Hanahan D..( 1983;). Studies on transformation of Escherichia coli with plasmids. J Mol Biol166:557–580 [CrossRef][PubMed]
    [Google Scholar]
  8. Jiang H., Kendrick K. E..( 2000;). Characterization of ssfR and ssgA, two genes involved in sporulation of Streptomyces griseus. J Bacteriol182:5521–5529 [CrossRef][PubMed]
    [Google Scholar]
  9. Krell T., Molina-Henares A. J., Ramos J. L..( 2006;). The IclR family of transcriptional activators and repressors can be defined by a single profile. Protein Sci15:1207–1213 [CrossRef][PubMed]
    [Google Scholar]
  10. Lu D., Fillet S., Meng C., Alguel Y., Kloppsteck P., Bergeron J., Krell T., Gallegos M. T., Ramos J., Zhang X..( 2010;). Crystal structure of TtgV in complex with its DNA operator reveals a general model for cooperative DNA binding of tetrameric gene regulators. Genes Dev24:2556–2565 [CrossRef][PubMed]
    [Google Scholar]
  11. Mersereau M., Pazour G. J., Das A..( 1990;). Efficient transformation of Agrobacterium tumefaciens by electroporation. Gene90:149–151 [CrossRef][PubMed]
    [Google Scholar]
  12. Miller J. H..( 1972;). Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  13. Molina-Henares A. J., Krell T., Eugenia Guazzaroni M., Segura A., Ramos J. L..( 2006;). Members of the IclR family of bacterial transcriptional regulators function as activators and/or repressors. FEMS Microbiol Rev30:157–186 [CrossRef][PubMed]
    [Google Scholar]
  14. Pan Y., Fiscus V., Meng W., Zheng Z., Zhang L. H., Fuqua C., Chen L..( 2011;). The Agrobacterium tumefaciens transcription factor BlcR is regulated via oligomerization. J Biol Chem286:20431–20440 [CrossRef][PubMed]
    [Google Scholar]
  15. Reverchon S., Nasser W., Robert-Baudouy J..( 1991;). Characterization of kdgR, a gene of Erwinia chrysanthemi that regulates pectin degradation. Mol Microbiol5:2203–2216 [CrossRef][PubMed]
    [Google Scholar]
  16. Romero-Steiner S., Parales R. E., Harwood C. S., Houghton J. E..( 1994;). Characterization of the pcaR regulatory gene from Pseudomonas putida, which is required for the complete degradation of p-hydroxybenzoate. J Bacteriol176:5771–5779[PubMed]
    [Google Scholar]
  17. Sciaky D., Montoya A. L., Chilton M. D..( 1978;). Fingerprints of Agrobacterium Ti plasmids. Plasmid1:238–253 [CrossRef][PubMed]
    [Google Scholar]
  18. Sunnarborg A., Klumpp D., Chung T., LaPorte D. C..( 1990;). Regulation of the glyoxylate bypass operon: cloning and characterization of iclR. J Bacteriol172:2642–2649[PubMed]
    [Google Scholar]
  19. Tempé J., Petit A., Holsters M., Montagu M., Schell J..( 1977;). Thermosensitive step associated with transfer of the Ti plasmid during conjugation: possible relation to transformation in crown gall. Proc Natl Acad Sci U S A74:2848–2849 [CrossRef][PubMed]
    [Google Scholar]
  20. Traag B. A., Kelemen G. H., Van Wezel G. P..( 2004;). Transcription of the sporulation gene ssgA is activated by the IclR-type regulator SsgR in a whi-independent manner in Streptomyces coelicolor A3(2). Mol Microbiol53:985–1000 [CrossRef][PubMed]
    [Google Scholar]
  21. Tsoi T. V., Plotnikova E. G., Cole J. R., Guerin W. F., Bagdasarian M., Tiedje J. M..( 1999;). Cloning, expression, and nucleotide sequence of the Pseudomonas aeruginosa 142 ohb genes coding for oxygenolytic ortho dehalogenation of halobenzoates. Appl Environ Microbiol65:2151–2162[PubMed]
    [Google Scholar]
  22. Wang C., Zhang H. B., Wang L. H., Zhang L. H..( 2006;). Succinic semialdehyde couples stress response to quorum-sensing signal decay in Agrobacterium tumefaciens. Mol Microbiol62:45–56 [CrossRef][PubMed]
    [Google Scholar]
  23. Yamazaki H., Ohnishi Y., Horinouchi S..( 2003;). Transcriptional switch on of ssgA by A-factor, which is essential for spore septum formation in Streptomyces griseus. J Bacteriol185:1273–1283 [CrossRef][PubMed]
    [Google Scholar]
  24. Zhang H. B., Wang L. H., Zhang L. H..( 2002;). Genetic control of quorum-sensing signal turnover in Agrobacterium tumefaciens. Proc Natl Acad Sci U S A99:4638–4643 [CrossRef][PubMed]
    [Google Scholar]
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