Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Jan;179(1):41–45. doi: 10.1128/jb.179.1.41-45.1997

Characterization of fhlA mutations resulting in ligand-independent transcriptional activation and ATP hydrolysis.

I Korsa 1, A Böck 1
PMCID: PMC178659  PMID: 8981978

Abstract

The FhlA protein belongs to the NtrC family of transcriptional regulators. It induces transcription from the -12/-24 promoters of the genes of the formate regulon by sigma54 RNA polymerase. FhlA is activated by binding of the ligand formate and does not require phosphorylation. A mutational analysis of the fhLA gene portion coding for the A and C domains was conducted with the aim of gaining information on the interaction between formate binding and ATP hydrolysis plus transcription activation. Four mutations were identified, all located in the A domain; one of them rendered transcription completely independent from the presence of formate, and the others conferred a semiconstitutive phenotype. The FhlA protein of one of the semiconstitutive variants was purified. Catalytic efficiency of ATP hydrolysis of the mutant FhlA was increased in the absence of formate in the same manner as formate influences the activity of wild-type FhlA. Moreover, in vitro transcription occurred at much lower threshold concentrations of the mutant protein and of nucleoside triphosphates than with the wild-type FhlA.

Full Text

The Full Text of this article is available as a PDF (277.6 KB).

Selected References

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

  1. Austin S., Dixon R. The prokaryotic enhancer binding protein NTRC has an ATPase activity which is phosphorylation and DNA dependent. EMBO J. 1992 Jun;11(6):2219–2228. doi: 10.1002/j.1460-2075.1992.tb05281.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Balch W. E., Wolfe R. S. New approach to the cultivation of methanogenic bacteria: 2-mercaptoethanesulfonic acid (HS-CoM)-dependent growth of Methanobacterium ruminantium in a pressureized atmosphere. Appl Environ Microbiol. 1976 Dec;32(6):781–791. doi: 10.1128/aem.32.6.781-791.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Birkmann A., Böck A. Characterization of a cis regulatory DNA element necessary for formate induction of the formate dehydrogenase gene (fdhF) of Escherichia coli. Mol Microbiol. 1989 Feb;3(2):187–195. doi: 10.1111/j.1365-2958.1989.tb01807.x. [DOI] [PubMed] [Google Scholar]
  4. Birkmann A., Sawers R. G., Böck A. Involvement of the ntrA gene product in the anaerobic metabolism of Escherichia coli. Mol Gen Genet. 1987 Dec;210(3):535–542. doi: 10.1007/BF00327209. [DOI] [PubMed] [Google Scholar]
  5. Böhm R., Sauter M., Böck A. Nucleotide sequence and expression of an operon in Escherichia coli coding for formate hydrogenlyase components. Mol Microbiol. 1990 Feb;4(2):231–243. doi: 10.1111/j.1365-2958.1990.tb00590.x. [DOI] [PubMed] [Google Scholar]
  6. Delgado A., Ramos J. L. Genetic evidence for activation of the positive transcriptional regulator Xy1R, a member of the NtrC family of regulators, by effector binding. J Biol Chem. 1994 Mar 18;269(11):8059–8062. [PubMed] [Google Scholar]
  7. Dixon R., Eady R. R., Espin G., Hill S., Iaccarino M., Kahn D., Merrick M. Analysis of regulation of Klebsiella pneumoniae nitrogen fixation (nif) gene cluster with gene fusions. Nature. 1980 Jul 10;286(5769):128–132. doi: 10.1038/286128a0. [DOI] [PubMed] [Google Scholar]
  8. Drummond M., Whitty P., Wootton J. Sequence and domain relationships of ntrC and nifA from Klebsiella pneumoniae: homologies to other regulatory proteins. EMBO J. 1986 Feb;5(2):441–447. doi: 10.1002/j.1460-2075.1986.tb04230.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fernández S., de Lorenzo V., Pérez-Martín J. Activation of the transcriptional regulator XylR of Pseudomonas putida by release of repression between functional domains. Mol Microbiol. 1995 Apr;16(2):205–213. doi: 10.1111/j.1365-2958.1995.tb02293.x. [DOI] [PubMed] [Google Scholar]
  10. Fiedler S., Wirth R. Transformation of bacteria with plasmid DNA by electroporation. Anal Biochem. 1988 Apr;170(1):38–44. doi: 10.1016/0003-2697(88)90086-3. [DOI] [PubMed] [Google Scholar]
  11. Fiedler U., Weiss V. A common switch in activation of the response regulators NtrC and PhoB: phosphorylation induces dimerization of the receiver modules. EMBO J. 1995 Aug 1;14(15):3696–3705. doi: 10.1002/j.1460-2075.1995.tb00039.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Flashner Y., Weiss D. S., Keener J., Kustu S. Constitutive forms of the enhancer-binding protein NtrC: evidence that essential oligomerization determinants lie in the central activation domain. J Mol Biol. 1995 Jun 16;249(4):700–713. doi: 10.1006/jmbi.1995.0330. [DOI] [PubMed] [Google Scholar]
  13. Hopper S., Babst M., Schlensog V., Fischer H. M., Hennecke H., Böck A. Regulated expression in vitro of genes coding for formate hydrogenlyase components of Escherichia coli. J Biol Chem. 1994 Jul 29;269(30):19597–19604. [PubMed] [Google Scholar]
  14. Hopper S., Böck A. Effector-mediated stimulation of ATPase activity by the sigma 54-dependent transcriptional activator FHLA from Escherichia coli. J Bacteriol. 1995 May;177(10):2798–2803. doi: 10.1128/jb.177.10.2798-2803.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hopper S., Korsa I., Böck A. The nucleotide concentration determines the specificity of in vitro transcription activation by the sigma 54-dependent activator FhlA. J Bacteriol. 1996 Jan;178(1):199–203. doi: 10.1128/jb.178.1.199-203.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Huala E., Stigter J., Ausubel F. M. The central domain of Rhizobium leguminosarum DctD functions independently to activate transcription. J Bacteriol. 1992 Feb;174(4):1428–1431. doi: 10.1128/jb.174.4.1428-1431.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Inouye S., Nakazawa A., Nakazawa T. Nucleotide sequence of the regulatory gene xylR of the TOL plasmid from Pseudomonas putida. Gene. 1988 Jun 30;66(2):301–306. doi: 10.1016/0378-1119(88)90366-6. [DOI] [PubMed] [Google Scholar]
  18. Klose K. E., North A. K., Stedman K. M., Kustu S. The major dimerization determinants of the nitrogen regulatory protein NTRC from enteric bacteria lie in its carboxy-terminal domain. J Mol Biol. 1994 Aug 12;241(2):233–245. doi: 10.1006/jmbi.1994.1492. [DOI] [PubMed] [Google Scholar]
  19. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  20. Lutz S., Böhm R., Beier A., Böck A. Characterization of divergent NtrA-dependent promoters in the anaerobically expressed gene cluster coding for hydrogenase 3 components of Escherichia coli. Mol Microbiol. 1990 Jan;4(1):13–20. doi: 10.1111/j.1365-2958.1990.tb02010.x. [DOI] [PubMed] [Google Scholar]
  21. Lutz S., Jacobi A., Schlensog V., Böhm R., Sawers G., Böck A. Molecular characterization of an operon (hyp) necessary for the activity of the three hydrogenase isoenzymes in Escherichia coli. Mol Microbiol. 1991 Jan;5(1):123–135. doi: 10.1111/j.1365-2958.1991.tb01833.x. [DOI] [PubMed] [Google Scholar]
  22. Maier T., Binder U., Böck A. Analysis of the hydA locus of Escherichia coli: two genes (hydN and hypF) involved in formate and hydrogen metabolism. Arch Microbiol. 1996 May;165(5):333–341. doi: 10.1007/s002030050335. [DOI] [PubMed] [Google Scholar]
  23. Pecher A., Zinoni F., Böck A. The seleno-polypeptide of formic dehydrogenase (formate hydrogen-lyase linked) from Escherichia coli: genetic analysis. Arch Microbiol. 1985 May;141(4):359–363. doi: 10.1007/BF00428850. [DOI] [PubMed] [Google Scholar]
  24. Pérez-Martín J., de Lorenzo V. In vitro activities of an N-terminal truncated form of XylR, a sigma 54-dependent transcriptional activator of Pseudomonas putida. J Mol Biol. 1996 May 17;258(4):575–587. doi: 10.1006/jmbi.1996.0270. [DOI] [PubMed] [Google Scholar]
  25. Rossmann R., Maier T., Lottspeich F., Böck A. Characterisation of a protease from Escherichia coli involved in hydrogenase maturation. Eur J Biochem. 1995 Jan 15;227(1-2):545–550. doi: 10.1111/j.1432-1033.1995.tb20422.x. [DOI] [PubMed] [Google Scholar]
  26. Rossmann R., Sawers G., Böck A. Mechanism of regulation of the formate-hydrogenlyase pathway by oxygen, nitrate, and pH: definition of the formate regulon. Mol Microbiol. 1991 Nov;5(11):2807–2814. doi: 10.1111/j.1365-2958.1991.tb01989.x. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Sauter M., Böhm R., Böck A. Mutational analysis of the operon (hyc) determining hydrogenase 3 formation in Escherichia coli. Mol Microbiol. 1992 Jun;6(11):1523–1532. doi: 10.1111/j.1365-2958.1992.tb00873.x. [DOI] [PubMed] [Google Scholar]
  29. Schlensog V., Birkmann A., Böck A. Mutations in trans which affect the anaerobic expression of a formate dehydrogenase (fdhF) structural gene. Arch Microbiol. 1989;152(1):83–89. doi: 10.1007/BF00447016. [DOI] [PubMed] [Google Scholar]
  30. Schlensog V., Böck A. Identification and sequence analysis of the gene encoding the transcriptional activator of the formate hydrogenlyase system of Escherichia coli. Mol Microbiol. 1990 Aug;4(8):1319–1327. doi: 10.1111/j.1365-2958.1990.tb00711.x. [DOI] [PubMed] [Google Scholar]
  31. Schlensog V., Lutz S., Böck A. Purification and DNA-binding properties of FHLA, the transcriptional activator of the formate hydrogenlyase system from Escherichia coli. J Biol Chem. 1994 Jul 29;269(30):19590–19596. [PubMed] [Google Scholar]
  32. Shingler V., Bartilson M., Moore T. Cloning and nucleotide sequence of the gene encoding the positive regulator (DmpR) of the phenol catabolic pathway encoded by pVI150 and identification of DmpR as a member of the NtrC family of transcriptional activators. J Bacteriol. 1993 Mar;175(6):1596–1604. doi: 10.1128/jb.175.6.1596-1604.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shingler V., Moore T. Sensing of aromatic compounds by the DmpR transcriptional activator of phenol-catabolizing Pseudomonas sp. strain CF600. J Bacteriol. 1994 Mar;176(6):1555–1560. doi: 10.1128/jb.176.6.1555-1560.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Shingler V., Pavel H. Direct regulation of the ATPase activity of the transcriptional activator DmpR by aromatic compounds. Mol Microbiol. 1995 Aug;17(3):505–513. doi: 10.1111/j.1365-2958.1995.mmi_17030505.x. [DOI] [PubMed] [Google Scholar]
  35. Shingler V. Signal sensing by sigma 54-dependent regulators: derepression as a control mechanism. Mol Microbiol. 1996 Feb;19(3):409–416. doi: 10.1046/j.1365-2958.1996.388920.x. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Wedel A., Kustu S. The bacterial enhancer-binding protein NTRC is a molecular machine: ATP hydrolysis is coupled to transcriptional activation. Genes Dev. 1995 Aug 15;9(16):2042–2052. doi: 10.1101/gad.9.16.2042. [DOI] [PubMed] [Google Scholar]
  38. Weiss V., Claverie-Martin F., Magasanik B. Phosphorylation of nitrogen regulator I of Escherichia coli induces strong cooperative binding to DNA essential for activation of transcription. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5088–5092. doi: 10.1073/pnas.89.11.5088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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

RESOURCES