1887

Abstract

The function of the response regulator DcuR of the DcuSR fumarate two-component sensory system of was analysed . Isolated DcuR protein was phosphorylated by the sensory histidine kinase, DcuS, and ATP, or by acetyl phosphate. In gel retardation assays with target promoters (, , ), phosphoryl DcuR (DcuR-P) formed a high-affinity complex, with an apparent (app. ) of 0·2–0·3 μM DcuR-P, and a low-affinity (app. 0·8–2 μM) complex. The high-affinity complex was formed only with promoters transcriptionally-regulated by DcuSR, whereas low-affinity binding was seen also with some DcuSR-independent promoters. The binding site of DcuR-P at the promoter was determined by DNase I footprinting. One binding site of 42–52 nt (position −359 to −400/−410 nt upstream of the transcriptional start) was identified in the presence of low and high concentrations of DcuR-P. Non-phosphorylated DcuR, or DcuR-D56N mutated in the phosphoryl-accepting Asp56 residue, showed low-affinity binding to target promoters. DcuR-D56N was still able to interact with DcuS. DcuR-D56N increased the phosphorylation of DcuS and competitively inhibited phosphoryl transfer to wild-type DcuR.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.26900-0
2004-04-01
2020-04-03
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/4/mic1500877.html?itemId=/content/journal/micro/10.1099/mic.0.26900-0&mimeType=html&fmt=ahah

References

  1. Abo-Amer E. E., Munn J., Jackson K., Aktas M., Golby P., Kelly D. J., Andrews S. C.. 2004; DNA interaction and phosphotransfer of the C4-dicarboxylate-responsive DcuS-DcuR two-component regulatory system fromEscherichia coli. J Bacteriol186:1879–1889[CrossRef]
    [Google Scholar]
  2. Aizawa S. I., Harwood R. J., Kadner J.. 2000; Signaling components in bacterial locomotion and sensory reception. J Bacteriol182:1459–1471[CrossRef]
    [Google Scholar]
  3. Alting-Mees M. A., Short J. M.. 1989; pBluescript II: gene mapping vectors. Nucleic Acids Res17:9494[CrossRef]
    [Google Scholar]
  4. Bradford M. M.. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem72:248–254[CrossRef]
    [Google Scholar]
  5. Cole S. T.. 1982; Nucleotide sequence coding for the flavoprotein subunit of the fumarate reductase of Escherichia coli. Eur J Biochem122:479–484
    [Google Scholar]
  6. Cotter P. A., Melville S. B., Albrecht J. A., Gunsalus R. P.. 1997; Aerobic regulation of cytochrome c oxidase (cydAB) operon expression in Escherichia coli: roles of Fnr and ArcA in repression and activation. Mol Microbiol25:605–615[CrossRef]
    [Google Scholar]
  7. Davies S. J., Golby P., Omrani D., Guest J. R., Kelly D. J., Andrews S. C.. 1999; Inactivation and regulation of the aerobic C4-dicarboxylate transport (dctA) gene ofEscherichia coli. J Bacteriol181:5624–5635
    [Google Scholar]
  8. Drapal N., Sawers G.. 1995; Purification of ArcA and analysis of its specific interaction with the pfl promoter-regulatory region. Mol Microbiol16:597–607[CrossRef]
    [Google Scholar]
  9. Emmerich R., Panglungtshang K., Strehler P., Hennecke H., Fischer H. M.. 1999; Phosphorylation, dephosphorylation and DNA-binding of the Bradyrhizobium japonicum RegSR two-component regulatory proteins. Eur J Biochem263:455–463[CrossRef]
    [Google Scholar]
  10. Golby P. S., Kelly D. J., Guest J. R., Andrews S. C.. 1998; Transcriptional regulation and organization of the dcuA and dcuB genes, encoding homologous anaerobic C4-dicarboxylate transporters in Escherichia coli. J Bacteriol180:6586–6596
    [Google Scholar]
  11. Golby P., Davies S., Kelly D. J., Guest J. R., Andrews S. C.. 1999; Identification and characterization of a two-component sensor kinase and response regulator system (DcuS-DcuR) controlling gene expression in response to C4-dicarboxylates in Escherichia coli. J Bacteriol181:1238–1248
    [Google Scholar]
  12. Janausch I. G., Zientz E., Kröger, A, Tran Q. H., Unden G.. 2002a; C4-dicarboxylate carriers and sensors in bacteria. Biochim Biophys Acta (Bioenergetics1553:39–56[CrossRef]
    [Google Scholar]
  13. Janausch I. G., Garcia-Moreno I., Unden G.. 2002b; Function of DcuS from Escherichia coli as a fumarate-stimulated histidine protein kinasein vitro. J Biol Chem277:39809–39814[CrossRef]
    [Google Scholar]
  14. Jones H. M., Gunsalus R. P.. 1987; Regulation of Escherichia coli fumarate reductase (frdABCD) operon expression by respiratory electron acceptors and thefnr gene product. J Bacteriol169:3340–3349
    [Google Scholar]
  15. Laemmli U. K.. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227:680–685[CrossRef]
    [Google Scholar]
  16. Lynch A. S., Lin E. C. C.. 1996; Transcriptional control mediated by the ArcA two-component response regulator protein of Escherichia coli: characterization of DNA binding at target promoters. J Bacteriol178:6238–6249
    [Google Scholar]
  17. Mattison K., Kenney L. J.. 2002; Phosphorylation alters the interaction of the response regulator OmpR with its sensor kinase EnvZ. J Biol Chem277:11143–11148[CrossRef]
    [Google Scholar]
  18. Miller J. H.. 1992; A Short Course in Bacterial Genetics Cold Spring Harbor. NY: Cold Spring Harbor Laboratory;
  19. Oshima T. H., Aiba H., Masuda Y., Kanaya S., Suguira M., Wanner B. L., Mori H., Mizuno T.. 2002; Transcriptome analysis of all two-component regulatory system mutants of Escherichia coli. Mol Microbiol46:281–291[CrossRef]
    [Google Scholar]
  20. Pappalardo L., Janausch I. G., Vijayan V., Zientz E., Junker J., Peti W., Zweckstetter M., Unden G., Griesinger C.. 2003; The NMR structure of the sensory domain of the membranous two-component fumarate sensor (histidine protein kinase) DcuS of Escherichia coli. J Biol Chem278:39185–39188[CrossRef]
    [Google Scholar]
  21. Parkinson J. S., Kofoid E. C.. 1992; Communication modules in bacterial signaling proteins. Annu Rev Genet26:71–112[CrossRef]
    [Google Scholar]
  22. Pellicer M. T., Lynch A. S., De Wulf P., Boyd D., Aguilar J., Lin E. C.. 1999; A mutational study of the ArcA-P binding sequences in the aldA promoter of Escherichia coli. Mol Gen Genet261:170–176[CrossRef]
    [Google Scholar]
  23. Sambrook J., Fritsch E. F., Maniatis T.. 1989; Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  24. Shen J., Gunsalus R. P.. 1997; Role of multiple ArcA recognition sites in anaerobic regulation of succinate dehydrogenase (sdhCDAB) gene expression in Escherichia coli. Mol Microbiol26:223–236[CrossRef]
    [Google Scholar]
  25. Tardat B., Touati D.. 1993; Iron and oxygen regulation of Escherichia coli MnSOD expression: competition between the global regulators Fur and ArcA for binding to DNA. Mol Microbiol9:53–63[CrossRef]
    [Google Scholar]
  26. Tuckerman J. R., Gonzalez G., Gilles-Gonzalez M. A.. 2001; Complexation precedes phosphorylation for two-component regulatory system FixL/FixJ of Sinorhizobium meliloti. J Mol Biol308:449–455[CrossRef]
    [Google Scholar]
  27. Wackwitz B., Bongaerts J., Goodman S. D., Unden G.. 1999; Growth phase-dependent regulation of nuoA-N expression in Escherichia coli K-12 by the Fis protein: upstream binding sites and bioenergetic significance. Mol Gen Genet262:876–883 (Corrigendum: Mol Gen Genet (2000) 263, 1070
    [Google Scholar]
  28. Walker M. S., DeMoss J. A.. 1994; NarL-phosphate must bind to multiple upstream sites to activate transcription from narG promoter of Escherichia coli. Mol Microbiol14:633–641[CrossRef]
    [Google Scholar]
  29. Zientz E., Bongaerts J., Unden G.. 1998; Fumarate regulation of gene expression in Escherichia coli by the DcuSR (dcuSR) two-component regulatory system. J Bacteriol180:5421–5425
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.26900-0
Loading
/content/journal/micro/10.1099/mic.0.26900-0
Loading

Data & Media loading...

Most cited this month

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