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. 1996 Oct;178(19):5579–5585. doi: 10.1128/jb.178.19.5579-5585.1996

Characterization of the genes and proteins of a two-component system from the hyperthermophilic bacterium Thermotoga maritima.

P J Lee 1, A M Stock 1
PMCID: PMC178394  PMID: 8824600

Abstract

As a step towards studying representative members of the two-component family of signal transduction proteins, we have cloned genes encoding a histidine protein kinase and a response regulator from the hyperthermophilic bacterium Thermotoga maritima. The genes have been designated HpkA and drrA, respectively. The deduced HpkA sequence contains all five characteristic histidine protein kinase motifs with the same relative order and spacing found in the mesophilic bacterial proteins. A hydropathy profile indicates that HpkA possesses only one membrane-spanning segment located at the extreme N terminus. The N-terminal region of DrrA exhibits all of the characteristics of the conserved domains of mesophilic bacterial response regulators, and the C-terminal region shows high similarity to the OmpR-PhoB subfamily of DNA-binding proteins. Recombinant T. maritima proteins, truncated HpkA lacking the putative membrane-spanning N- terminal amino acids and DrrA, were expressed in Escherichia coli. Partial purification of T. maritima proteins was achieved by heat denaturation of E. coli host proteins. In an in vitro assay, truncated HpkA protein was autophosphorylated in the presence of ATP. Thus, the N-terminal hydrophobic region is not required for kinase activity. Phosphotransfer between truncated HpkA and DrrA was demonstrated in vitro with the partially purified proteins. The phosphorylation reactions were strongly temperature dependent. The results indicate that the recombinant T. maritima two-component proteins overexpressed in E. coli are stable as well as enzymatically active at elevated temperatures.

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Selected References

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  1. Adams M. W. Enzymes and proteins from organisms that grow near and above 100 degrees C. Annu Rev Microbiol. 1993;47:627–658. doi: 10.1146/annurev.mi.47.100193.003211. [DOI] [PubMed] [Google Scholar]
  2. Arthur M., Molinas C., Courvalin P. The VanS-VanR two-component regulatory system controls synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147. J Bacteriol. 1992 Apr;174(8):2582–2591. doi: 10.1128/jb.174.8.2582-2591.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fisher S. L., Kim S. K., Wanner B. L., Walsh C. T. Kinetic comparison of the specificity of the vancomycin resistance VanSfor two response regulators, VanR and PhoB. Biochemistry. 1996 Apr 16;35(15):4732–4740. doi: 10.1021/bi9525435. [DOI] [PubMed] [Google Scholar]
  5. Kim C. W., Markiewicz P., Lee J. J., Schierle C. F., Miller J. H. Studies of the hyperthermophile Thermotoga maritima by random sequencing of cDNA and genomic libraries. Identification and sequencing of the trpEG (D) operon. J Mol Biol. 1993 Jun 20;231(4):960–981. doi: 10.1006/jmbi.1993.1345. [DOI] [PubMed] [Google Scholar]
  6. Kozak M. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev. 1983 Mar;47(1):1–45. doi: 10.1128/mr.47.1.1-45.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  8. Lee J. W., Hulett F. M. Nucleotide sequence of the phoP gene encoding PhoP, the response regulator of the phosphate regulon of Bacillus subtilis. Nucleic Acids Res. 1992 Nov 11;20(21):5848–5848. doi: 10.1093/nar/20.21.5848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lee T. Y., Makino K., Shinagawa H., Amemura M., Nakata A. Phosphate regulon in members of the family Enterobacteriaceae: comparison of the phoB-phoR operons of Escherichia coli, Shigella dysenteriae, and Klebsiella pneumoniae. J Bacteriol. 1989 Dec;171(12):6593–6599. doi: 10.1128/jb.171.12.6593-6599.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Makino K., Shinagawa H., Amemura M., Nakata A. Nucleotide sequence of the phoB gene, the positive regulatory gene for the phosphate regulon of Escherichia coli K-12. J Mol Biol. 1986 Jul 5;190(1):37–44. doi: 10.1016/0022-2836(86)90073-2. [DOI] [PubMed] [Google Scholar]
  11. Makino K., Shinagawa H., Amemura M., Nakata A. Nucleotide sequence of the phoR gene, a regulatory gene for the phosphate regulon of Escherichia coli. J Mol Biol. 1986 Dec 5;192(3):549–556. doi: 10.1016/0022-2836(86)90275-5. [DOI] [PubMed] [Google Scholar]
  12. Nakano M. M., Zuber P., Glaser P., Danchin A., Hulett F. M. Two-component regulatory proteins ResD-ResE are required for transcriptional activation of fnr upon oxygen limitation in Bacillus subtilis. J Bacteriol. 1996 Jul;178(13):3796–3802. doi: 10.1128/jb.178.13.3796-3802.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ogasawara N., Nakai S., Yoshikawa H. Systematic sequencing of the 180 kilobase region of the Bacillus subtilis chromosome containing the replication origin. DNA Res. 1994;1(1):1–14. doi: 10.1093/dnares/1.1.1. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rees D. C., Adams M. W. Hyperthermophiles: taking the heat and loving it. Structure. 1995 Mar 15;3(3):251–254. doi: 10.1016/s0969-2126(01)00155-1. [DOI] [PubMed] [Google Scholar]
  17. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Saxild H. H., Andersen L. N., Hammer K. Dra-nupC-pdp operon of Bacillus subtilis: nucleotide sequence, induction by deoxyribonucleosides, and transcriptional regulation by the deoR-encoded DeoR repressor protein. J Bacteriol. 1996 Jan;178(2):424–434. doi: 10.1128/jb.178.2.424-434.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Seki T., Yoshikawa H., Takahashi H., Saito H. Cloning and nucleotide sequence of phoP, the regulatory gene for alkaline phosphatase and phosphodiesterase in Bacillus subtilis. J Bacteriol. 1987 Jul;169(7):2913–2916. doi: 10.1128/jb.169.7.2913-2916.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Seki T., Yoshikawa H., Takahashi H., Saito H. Nucleotide sequence of the Bacillus subtilis phoR gene. J Bacteriol. 1988 Dec;170(12):5935–5938. doi: 10.1128/jb.170.12.5935-5938.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sorokin A., Zumstein E., Azevedo V., Ehrlich S. D., Serror P. The organization of the Bacillus subtilis 168 chromosome region between the spoVA and serA genetic loci, based on sequence data. Mol Microbiol. 1993 Oct;10(2):385–395. doi: 10.1111/j.1365-2958.1993.tb02670.x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Stock J. B., Stock A. M., Mottonen J. M. Signal transduction in bacteria. Nature. 1990 Mar 29;344(6265):395–400. doi: 10.1038/344395a0. [DOI] [PubMed] [Google Scholar]
  24. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  25. Sun G., Sharkova E., Chesnut R., Birkey S., Duggan M. F., Sorokin A., Pujic P., Ehrlich S. D., Hulett F. M. Regulators of aerobic and anaerobic respiration in Bacillus subtilis. J Bacteriol. 1996 Mar;178(5):1374–1385. doi: 10.1128/jb.178.5.1374-1385.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Swanson R. V., Alex L. A., Simon M. I. Histidine and aspartate phosphorylation: two-component systems and the limits of homology. Trends Biochem Sci. 1994 Nov;19(11):485–490. doi: 10.1016/0968-0004(94)90135-x. [DOI] [PubMed] [Google Scholar]
  27. Swanson R. V., Sanna M. G., Simon M. I. Thermostable chemotaxis proteins from the hyperthermophilic bacterium Thermotoga maritima. J Bacteriol. 1996 Jan;178(2):484–489. doi: 10.1128/jb.178.2.484-489.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Volz K. Structural conservation in the CheY superfamily. Biochemistry. 1993 Nov 9;32(44):11741–11753. doi: 10.1021/bi00095a001. [DOI] [PubMed] [Google Scholar]
  30. Walter M. R., Cook W. J., Cole L. B., Short S. A., Koszalka G. W., Krenitsky T. A., Ealick S. E. Three-dimensional structure of thymidine phosphorylase from Escherichia coli at 2.8 A resolution. J Biol Chem. 1990 Aug 15;265(23):14016–14022. doi: 10.2210/pdb1tpt/pdb. [DOI] [PubMed] [Google Scholar]
  31. Wang S. P., Sharma P. L., Schoenlein P. V., Ely B. A histidine protein kinase is involved in polar organelle development in Caulobacter crescentus. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):630–634. doi: 10.1073/pnas.90.2.630. [DOI] [PMC free article] [PubMed] [Google Scholar]

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