1887

Abstract

The response regulator PrrA directly activates transcription of genes necessary for energy conservation at low O tensions and under anaerobic conditions. It is proposed that PrrA homologues contain a C-terminal DNA-binding domain (PrrA-CTD) that lacks significant amino acid sequence similarity to those found in other response regulators. To test this hypothesis, single amino acid substitutions were created at 12 residues in the PrrA-CTD. These mutant PrrA proteins were purified and tested for the ability to be phosphorylated by the low-molecular-mass phosphate donor acetyl phosphate, to activate transcription and to bind promoter DNA. Each mutant PrrA protein accepted phosphate from P-labelled acetyl phosphate. At micromolar concentrations of acetyl phosphate-treated wild-type PrrA, a single 20 bp region in the PrrA-dependent P2 promoter was protected from DNase I digestion. Of the mutant PrrA proteins tested, only acetyl phosphate-treated PrrA-N168A and PrrA-I177A protected P2 from DNase I digestion at similar protein concentrations compared to wild-type PrrA. The use of transcription assays with the PrrA-dependent P2 and promoters showed that acetyl phosphate-treated PrrA-N168A produced transcript levels similar to that of wild-type PrrA at comparable protein concentrations. Using concentrations of acetyl phosphate-treated PrrA that are saturating for the wild-type protein, PrrA-H170A and PrrA-I177A produced <45 % as much transcript as wild-type PrrA. Under identical conditions, the remaining mutant PrrA proteins produced little or no detectable transcripts from either promoter . Explanations are presented for why these amino acid side chains in the PrrA-CTD are important for its ability to activate transcription.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.28300-0
2005-12-01
2019-11-14
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/12/4103.html?itemId=/content/journal/micro/10.1099/mic.0.28300-0&mimeType=html&fmt=ahah

References

  1. Anthony, J., Green, H. & Donohue, T. J. ( 2003; ). Rhodobacter sphaeroides RNA polymerase and its sigma factors. Methods Enzymol 370, 54–65.
    [Google Scholar]
  2. Bauer, E., Kaspar, T., Fischer, H. M. & Hennecke, H. ( 1998; ). Expression of the fixR-nifA operon in Bradyrhizobium japonicum depends on a new response regulator, RegR. J Bacteriol 180, 3853–3863.
    [Google Scholar]
  3. Bauer, C. E., Elsen, S. & Bird, T. H. ( 1999; ). Mechanisms for redox control of gene expression. Annu Rev Microbiol 53, 495–523.[CrossRef]
    [Google Scholar]
  4. Bird, T. H., Du, S. & Bauer, C. E. ( 1999; ). Autophosphorylation, phosphotransfer, and DNA-binding properties of the RegB/RegA two-component regulatory system in Rhodobacter capsulatus. J Biol Chem 274, 16343–16348.[CrossRef]
    [Google Scholar]
  5. Bowman, W. C., Du, S., Bauer, C. E. & Kranz, R. G. ( 1999; ). In vitro activation and repression of photosynthesis gene transcription in Rhodobacter capsulatus. Mol Microbiol 33, 429–437.[CrossRef]
    [Google Scholar]
  6. Brennan, R. G. & Matthews, B. W. ( 1989; ). The helix-turn-helix DNA binding motif. J Biol Chem 264, 1903–1906.
    [Google Scholar]
  7. Comolli, J. C. & Donohue, T. J. ( 2002; ). Pseudomonas aeruginosa RoxR, a response regulator related to Rhodobacter sphaeroides PrrA, activates expression of the cyanide-insensitive terminal oxidase, CioAB. Mol Microbiol 45, 755–768.[CrossRef]
    [Google Scholar]
  8. Comolli, J., Carl, A., Hall, C. & Donohue, T. J. ( 2002; ). Transcriptional activation of the Rhodobacter sphaeroides cytochrome c 2 gene P2 promoter by the response regulator PrrA. J Bacteriol 184, 390–399.[CrossRef]
    [Google Scholar]
  9. Du, S., Bird, T. H. & Bauer, C. E. ( 1998; ). DNA binding characteristics of RegA. A constitutively active anaerobic activator of photosynthesis gene expression in Rhodobacter capsulatus. J Biol Chem 273, 18509–18513.[CrossRef]
    [Google Scholar]
  10. Dubbs, J. M., Bird, T. H., Bauer, C. E. & Tabita, F. R. ( 2000; ). Interaction of CbbR and RegA* transcription regulators with the Rhodobacter sphaeroides cbbI promoter-operator region. J Biol Chem 275, 19224–19230.[CrossRef]
    [Google Scholar]
  11. Elsen, S., Swem, L. R., Swem, D. L. & Bauer, C. E. ( 2004; ). RegB/RegA, a highly conserved redox-responding global regulatory system. Microbiol Mol Biol Rev 68, 263–279.[CrossRef]
    [Google Scholar]
  12. Eraso, J. M. & Kaplan, S. ( 1994; ). prrA, a putative response regulator involved in oxygen regulation of photosynthesis gene expression in Rhodobacter sphaeroides. J Bacteriol 176, 32–43.
    [Google Scholar]
  13. Eraso, J. M. & Kaplan, S. ( 1995; ). Oxygen-insensitive synthesis of the photosynthetic membranes of Rhodobacter sphaeroides: a mutant histidine kinase. J Bacteriol 177, 2695–2706.
    [Google Scholar]
  14. Eraso, J. M. & Kaplan, S. ( 1996; ). Complex regulatory activities associated with the histidine kinase PrrB in expression of photosynthesis genes in Rhodobacter sphaeroides 2 4 1. J Bacteriol 178, 7037–7046.
    [Google Scholar]
  15. Eraso, J. M. & Kaplan, S. ( 2000; ). From redox flow to gene regulation: role of the PrrC protein of Rhodobacter sphaeroides 2 4 1. Biochemistry 39, 2052–2062.[CrossRef]
    [Google Scholar]
  16. Hakenback, R. & Stock, J. B. ( 1996; ). Analysis of two-component signal transduction systems involved in transcriptional regulation. Methods Enzymol 273, 281–300.
    [Google Scholar]
  17. Joshi, H. M. & Tabita, F. R. ( 1996; ). A global two component signal transduction system that integrates the control of photosynthesis, carbon dioxide assimilation, and nitrogen fixation. Proc Natl Acad Sci U S A 93, 14515–14520.[CrossRef]
    [Google Scholar]
  18. Karls, R. K., Wolf, J. R. & Donohue, T. J. ( 1999; ). Activation of the cycA P2 promoter for the Rhodobacter sphaeroides cytochrome c 2 gene by the photosynthesis response regulator. Mol Microbiol 34, 822–835.[CrossRef]
    [Google Scholar]
  19. Kenney, L. J. ( 2002; ). Structure/function relationships in OmpR and other winged-helix transcription factors. Curr Opin Microbiol 5, 135–141.[CrossRef]
    [Google Scholar]
  20. Laguri, C., Phillips-Jones, M. K. & Williamson, M. P. ( 2003; ). Solution structure and DNA binding of the effector domain from the global regulator PrrA (RegA) from Rhodobacter sphaeroides: insights into DNA binding specificity. Nucleic Acids Res 31, 6778–6787.[CrossRef]
    [Google Scholar]
  21. Lee, J. K. & Kaplan, S. ( 1992; ). cis-acting regulatory elements involved in oxygen and light control of puc operon transcription in Rhodobacter sphaeroides. J Bacteriol 174, 1146–1157.
    [Google Scholar]
  22. McCleary, W. R. & Stock, J. B. ( 1994; ). Acetyl phosphate and the activation of two-component response regulators. J Biol Chem 269, 31567–31572.
    [Google Scholar]
  23. Newlands, J. T., Ross, W., Gosink, K. K. & Gourse, R. L. ( 1991; ). Factor-independent activation of Escherichia coli rRNA transcription. II. Characterization of complexes of rrnB P1 promoters containing or lacking the upstream activator region with Escherichia coli RNA polymerase. J Mol Biol 220, 569–583.[CrossRef]
    [Google Scholar]
  24. Oh, J. I. & Kaplan, S. ( 2000; ). Redox signaling: globalization of gene expression. EMBO J 19, 4237–4247.[CrossRef]
    [Google Scholar]
  25. Oh, J. I. & Kaplan, S. ( 2001; ). Generalized approach to the regulation and integration of gene expression. Mol Microbiol 39, 1116–1123.[CrossRef]
    [Google Scholar]
  26. Oh, J. I., Ko, I. J. & Kaplan, S. ( 2004; ). Reconstitution of the Rhodobacter sphaeroides cbb 3-PrrBA signal transduction pathway in vitro. Biochemistry 43, 7915–7923.[CrossRef]
    [Google Scholar]
  27. Pan, C., Feng, J., Finkel, S., Landgraf, R., Sigman, D. & Johnson, R. ( 1994; ). Structure of the Escherichia coli Fis-DNA complex probed by protein conjugated with 1,10-phenanthroline copper(I) complex. Proc Natl Acad Sci U S A 91, 1721–1725.[CrossRef]
    [Google Scholar]
  28. Pan, C. Q., Finkel, S. E., Cramton, S. E., Feng, J.-A., Sigman, D. S. & Johnson, R. C. ( 1996; ). Variable structures of Fis-DNA complexes determined by flanking DNA-protein contacts. J Mol Biol 264, 675–695.[CrossRef]
    [Google Scholar]
  29. Patschkowski, T., Bates, D. M. & Kiley, P. J. ( 2000; ). Mechanisms for sensing and responding to oxygen deprivation. In Bacterial Stress Responses, pp. 61–78. Edited by G. S. A. R. Hengge-Aronis. Washington, DC: American Society for Microbiology.
  30. Potter, C. A., Ward, A., Laguri, C., Williamson, M. P., Henderson, P. J. F. & Phillips-Jones, M. K. ( 2002; ). Expression, purification and characterisation of full-length histidine protein kinase RegB from Rhodobacter sphaeroides. J Mol Biol 320, 201–213.[CrossRef]
    [Google Scholar]
  31. Qian, Y. & Tabita, F. R. ( 1996; ). A global signal transduction system regulates aerobic and anaerobic CO2 fixation in Rhodobacter sphaeroides. J Bacteriol 178, 12–18.
    [Google Scholar]
  32. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  33. Stock, A. M., Robinson, V. L. & Goudreau, P. N. ( 2000; ). Two-component signal transduction. Annu Rev Biochem 69, 183–215.[CrossRef]
    [Google Scholar]
  34. Swem, L. R., Elsen, S., Bird, T. H., Swem, D. L., Koch, H. G., Myllykallio, H., Daldal, F. & Bauer, C. E. ( 2001; ). The RegB/RegA two-component regulatory system controls synthesis of photosynthesis and respiratory electron transfer components in Rhodobacter capsulatus. J Mol Biol 309, 121–138.[CrossRef]
    [Google Scholar]
  35. Yuan, H. S., Finkel, S. E., Feng, J. A., Kaczor-Grzeskowiak, M., Johnson, R. C. & Dickerson, R. E. ( 1991; ). The molecular structure of wild-type and a mutant Fis protein: relationship between mutational changes and recombinational enhancer function or DNA binding. Proc Natl Acad Sci U S A 88, 9558–9562.[CrossRef]
    [Google Scholar]
  36. Zeilstra-Ryalls, J., Gomelsky, M., Eraso, J. M., Yeliseev, A., O'Gara, J. & Kaplan, S. ( 1998; ). Control of photosystem formation in Rhodobacter sphaeroides. J Bacteriol 180, 2801–2809.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.28300-0
Loading
/content/journal/micro/10.1099/mic.0.28300-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error