Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Feb;179(3):968–971. doi: 10.1128/jb.179.3.968-971.1997

Purification and in vitro phosphorylation of HupT, a regulatory protein controlling hydrogenase gene expression in Rhodobacter capsulatus.

S Elsen 1, A Colbeau 1, P M Vignais 1
PMCID: PMC178785  PMID: 9006058

Abstract

The HupT protein of Rhodobacter capsulatus, involved in negative regulation of hydrogenase gene expression, is predicted to be a histidine kinase on the basis of sequence comparisons. The protein was overproduced in Escherichia coli, purified to homogeneity, and demonstrated to autophosphorylate in vitro in the presence of [gamma-32P]ATP. An H217N hupt mutant was constructed, and the mutant protein was shown to have lost kinase activity. This result, and the fact that the phosphoryl group in phosphorylated HupT appeared to be bound to an N atom, support the suggestion from sequence comparisons that HupT is a histidine kinase, which can autophosphorylate on the His217 residue.

Full Text

The Full Text of this article is available as a PDF (240.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barany F. Single-stranded hexameric linkers: a system for in-phase insertion mutagenesis and protein engineering. Gene. 1985;37(1-3):111–123. doi: 10.1016/0378-1119(85)90263-x. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Duclos B., Marcandier S., Cozzone A. J. Chemical properties and separation of phosphoamino acids by thin-layer chromatography and/or electrophoresis. Methods Enzymol. 1991;201:10–21. doi: 10.1016/0076-6879(91)01004-l. [DOI] [PubMed] [Google Scholar]
  4. Elsen S., Colbeau A., Chabert J., Vignais P. M. The hupTUV operon is involved in negative control of hydrogenase synthesis in Rhodobacter capsulatus. J Bacteriol. 1996 Sep;178(17):5174–5181. doi: 10.1128/jb.178.17.5174-5181.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Elsen S., Richaud P., Colbeau A., Vignais P. M. Sequence analysis and interposon mutagenesis of the hupT gene, which encodes a sensor protein involved in repression of hydrogenase synthesis in Rhodobacter capsulatus. J Bacteriol. 1993 Nov;175(22):7404–7412. doi: 10.1128/jb.175.22.7404-7412.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hess J. F., Oosawa K., Kaplan N., Simon M. I. Phosphorylation of three proteins in the signaling pathway of bacterial chemotaxis. Cell. 1988 Apr 8;53(1):79–87. doi: 10.1016/0092-8674(88)90489-8. [DOI] [PubMed] [Google Scholar]
  7. Higuchi R., Krummel B., Saiki R. K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988 Aug 11;16(15):7351–7367. doi: 10.1093/nar/16.15.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Inoue K., Kouadio J. L., Mosley C. S., Bauer C. E. Isolation and in vitro phosphorylation of sensory transduction components controlling anaerobic induction of light harvesting and reaction center gene expression in Rhodobacter capsulatus. Biochemistry. 1995 Jan 17;34(2):391–396. doi: 10.1021/bi00002a002. [DOI] [PubMed] [Google Scholar]
  9. Li Y., Plamann L. Purification and in vitro phosphorylation of Myxococcus xanthus AsgA protein. J Bacteriol. 1996 Jan;178(1):289–292. doi: 10.1128/jb.178.1.289-292.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. McCleary W. R., Zusman D. R. Purification and characterization of the Myxococcus xanthus FrzE protein shows that it has autophosphorylation activity. J Bacteriol. 1990 Dec;172(12):6661–6668. doi: 10.1128/jb.172.12.6661-6668.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Parkinson J. S., Kofoid E. C. Communication modules in bacterial signaling proteins. Annu Rev Genet. 1992;26:71–112. doi: 10.1146/annurev.ge.26.120192.000443. [DOI] [PubMed] [Google Scholar]
  12. Richaud P., Colbeau A., Toussaint B., Vignais P. M. Identification and sequence analysis of the hupR1 gene, which encodes a response regulator of the NtrC family required for hydrogenase expression in Rhodobacter capsulatus. J Bacteriol. 1991 Sep;173(18):5928–5932. doi: 10.1128/jb.173.18.5928-5932.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Stock J. B., Ninfa A. J., Stock A. M. Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev. 1989 Dec;53(4):450–490. doi: 10.1128/mr.53.4.450-490.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  15. Vignais P. M., Dimon B., Zorin N. A., Colbeau A., Elsen S. HupUV proteins of Rhodobacter capsulatus can bind H2: evidence from the H-D exchange reaction. J Bacteriol. 1997 Jan;179(1):290–292. doi: 10.1128/jb.179.1.290-292.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Weiss V., Magasanik B. Phosphorylation of nitrogen regulator I (NRI) of Escherichia coli. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8919–8923. doi: 10.1073/pnas.85.23.8919. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES