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. 1991 Jan;173(1):16–22. doi: 10.1128/jb.173.1.16-22.1991

Fnr mutants that activate gene expression in the presence of oxygen.

P J Kiley 1, W S Reznikoff 1
PMCID: PMC207150  PMID: 1898918

Abstract

The regulatory protein Fnr is required for anaerobic expression of several anaerobic respiratory enzymes in Escherichia coli. To gain insight into how Fnr activity is regulated by oxygen, we have isolated Fnr mutants that increase expression of the nitrate reductase operon in the presence of oxygen (Fnr* mutants). Seven single-amino-acid substitutions that mapped within two regions of Fnr have been characterized. Two mutants mapped adjacent to two Cys residues in the N-terminal Cys cluster. Five Fnr* substitutions mapped to a region of Fnr that is similar to the cyclic AMP-binding domain of the catabolite activator protein (CAP). Within this group, four mutants were clustered in a region analogous to the CAP C helix, which is important in CAP dimer subunit interactions. Taken together, these data implicate regions in Fnr that may be important either in sensing oxygen deprivation or in the conformational change proposed to be necessary for Fnr activation under anaerobic conditions.

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Selected References

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

  1. Aiba H., Nakamura T., Mitani H., Mori H. Mutations that alter the allosteric nature of cAMP receptor protein of Escherichia coli. EMBO J. 1985 Dec 1;4(12):3329–3332. doi: 10.1002/j.1460-2075.1985.tb04084.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bagg A., Neilands J. B. Ferric uptake regulation protein acts as a repressor, employing iron (II) as a cofactor to bind the operator of an iron transport operon in Escherichia coli. Biochemistry. 1987 Aug 25;26(17):5471–5477. doi: 10.1021/bi00391a039. [DOI] [PubMed] [Google Scholar]
  3. Bell A. I., Gaston K. L., Cole J. A., Busby S. J. Cloning of binding sequences for the Escherichia coli transcription activators, FNR and CRP: location of bases involved in discrimination between FNR and CRP. Nucleic Acids Res. 1989 May 25;17(10):3865–3874. doi: 10.1093/nar/17.10.3865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bonnefoy V., Fons M., Ratouchniak J., Pascal M. C., Chippaux M. Aerobic expression of the nar operon of Escherichia coli in a fnr mutant. Mol Microbiol. 1988 May;2(3):419–425. doi: 10.1111/j.1365-2958.1988.tb00047.x. [DOI] [PubMed] [Google Scholar]
  5. Castilho B. A., Olfson P., Casadaban M. J. Plasmid insertion mutagenesis and lac gene fusion with mini-mu bacteriophage transposons. J Bacteriol. 1984 May;158(2):488–495. doi: 10.1128/jb.158.2.488-495.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fischer H. M., Bruderer T., Hennecke H. Essential and non-essential domains in the Bradyrhizobium japonicum NifA protein: identification of indispensable cysteine residues potentially involved in redox reactivity and/or metal binding. Nucleic Acids Res. 1988 Mar 25;16(5):2207–2224. doi: 10.1093/nar/16.5.2207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fowler R. G., Degnen G. E., Cox E. C. Mutational specificity of a conditional Escherichia coli mutator, mutD5. Mol Gen Genet. 1974;133(3):179–191. doi: 10.1007/BF00267667. [DOI] [PubMed] [Google Scholar]
  9. Garges S., Adhya S. Cyclic AMP-induced conformational change of cyclic AMP receptor protein (CRP): intragenic suppressors of cyclic AMP-independent CRP mutations. J Bacteriol. 1988 Apr;170(4):1417–1422. doi: 10.1128/jb.170.4.1417-1422.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Garges S., Adhya S. Sites of allosteric shift in the structure of the cyclic AMP receptor protein. Cell. 1985 Jul;41(3):745–751. doi: 10.1016/s0092-8674(85)80055-6. [DOI] [PubMed] [Google Scholar]
  11. Harman J. G., McKenney K., Peterkofsky A. Structure-function analysis of three cAMP-independent forms of the cAMP receptor protein. J Biol Chem. 1986 Dec 15;261(35):16332–16339. [PubMed] [Google Scholar]
  12. Iuchi S., Lin E. C. The narL gene product activates the nitrate reductase operon and represses the fumarate reductase and trimethylamine N-oxide reductase operons in Escherichia coli. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3901–3905. doi: 10.1073/pnas.84.11.3901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jayaraman P. S., Cole J. A., Busby S. J. Mutational analysis of the nucleotide sequence at the FNR-dependent nirB promoter in Escherichia coli. Nucleic Acids Res. 1989 Jan 11;17(1):135–145. doi: 10.1093/nar/17.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jones H. M., Gunsalus R. P. Regulation of Escherichia coli fumarate reductase (frdABCD) operon expression by respiratory electron acceptors and the fnr gene product. J Bacteriol. 1987 Jul;169(7):3340–3349. doi: 10.1128/jb.169.7.3340-3349.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lambden P. R., Guest J. R. Mutants of Escherichia coli K12 unable to use fumarate as an anaerobic electron acceptor. J Gen Microbiol. 1976 Dec;97(2):145–160. doi: 10.1099/00221287-97-2-145. [DOI] [PubMed] [Google Scholar]
  16. Li S. F., DeMoss J. A. Location of sequences in the nar promoter of Escherichia coli required for regulation by Fnr and NarL. J Biol Chem. 1988 Sep 25;263(27):13700–13705. [PubMed] [Google Scholar]
  17. Li S. F., DeMoss J. A. Promoter region of the nar operon of Escherichia coli: nucleotide sequence and transcription initiation signals. J Bacteriol. 1987 Oct;169(10):4614–4620. doi: 10.1128/jb.169.10.4614-4620.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mizusawa S., Ward D. F. A bacteriophage lambda vector for cloning with BamHI and Sau3A. Gene. 1982 Dec;20(3):317–322. doi: 10.1016/0378-1119(82)90200-1. [DOI] [PubMed] [Google Scholar]
  19. Moore G. R., Pettigrew G. W., Rogers N. K. Factors influencing redox potentials of electron transfer proteins. Proc Natl Acad Sci U S A. 1986 Jul;83(14):4998–4999. doi: 10.1073/pnas.83.14.4998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mousset S., Thomas R. Ter, a function which generates the ends of the mature lambda chromosome. Nature. 1969 Jan 18;221(5177):242–244. doi: 10.1038/221242a0. [DOI] [PubMed] [Google Scholar]
  21. Prentki P., Krisch H. M. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. doi: 10.1016/0378-1119(84)90059-3. [DOI] [PubMed] [Google Scholar]
  22. SISTROM W. R. A requirement for sodium in the growth of Rhodopseudomonas spheroides. J Gen Microbiol. 1960 Jun;22:778–785. doi: 10.1099/00221287-22-3-778. [DOI] [PubMed] [Google Scholar]
  23. Shaw D. J., Guest J. R. Amplification and product identification of the fnr gene of Escherichia coli. J Gen Microbiol. 1982 Oct;128(10):2221–2228. doi: 10.1099/00221287-128-10-2221. [DOI] [PubMed] [Google Scholar]
  24. Shaw D. J., Guest J. R. Nucleotide sequence of the fnr gene and primary structure of the Enr protein of Escherichia coli. Nucleic Acids Res. 1982 Oct 11;10(19):6119–6130. doi: 10.1093/nar/10.19.6119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shaw D. J., Rice D. W., Guest J. R. Homology between CAP and Fnr, a regulator of anaerobic respiration in Escherichia coli. J Mol Biol. 1983 May 15;166(2):241–247. doi: 10.1016/s0022-2836(83)80011-4. [DOI] [PubMed] [Google Scholar]
  26. Spiro S., Guest J. R. Activation of the lac operon of Escherichia coli by a mutant FNR protein. Mol Microbiol. 1987 Jul;1(1):53–58. doi: 10.1111/j.1365-2958.1987.tb00526.x. [DOI] [PubMed] [Google Scholar]
  27. Spiro S., Guest J. R. Inactivation of the FNR protein of Escherichia coli by targeted mutagenesis in the N-terminal region. Mol Microbiol. 1988 Nov;2(6):701–707. doi: 10.1111/j.1365-2958.1988.tb00080.x. [DOI] [PubMed] [Google Scholar]
  28. Spiro S., Roberts R. E., Guest J. R. FNR-dependent repression of the ndh gene of Escherichia coli and metal ion requirement for FNR-regulated gene expression. Mol Microbiol. 1989 May;3(5):601–608. doi: 10.1111/j.1365-2958.1989.tb00207.x. [DOI] [PubMed] [Google Scholar]
  29. Stewart V. Nitrate respiration in relation to facultative metabolism in enterobacteria. Microbiol Rev. 1988 Jun;52(2):190–232. doi: 10.1128/mr.52.2.190-232.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Trageser M., Spiro S., Duchêne A., Kojro E., Fahrenholz F., Guest J. R., Unden G. Isolation of intact FNR protein (Mr 30,000) of Escherichia coli. Mol Microbiol. 1990 Jan;4(1):21–27. doi: 10.1111/j.1365-2958.1990.tb02011.x. [DOI] [PubMed] [Google Scholar]
  31. Trageser M., Unden G. Role of cysteine residues and of metal ions in the regulatory functioning of FNR, the transcriptional regulator of anaerobic respiration in Escherichia coli. Mol Microbiol. 1989 May;3(5):593–599. doi: 10.1111/j.1365-2958.1989.tb00206.x. [DOI] [PubMed] [Google Scholar]
  32. Unden G., Duchene A. On the role of cyclic AMP and the Fnr protein in Escherichia coli growing anaerobically. Arch Microbiol. 1987 Mar;147(2):195–200. doi: 10.1007/BF00415284. [DOI] [PubMed] [Google Scholar]
  33. Unden G., Guest J. R. Isolation and characterization of the Fnr protein, the transcriptional regulator of anaerobic electron transport in Escherichia coli. Eur J Biochem. 1985 Jan 2;146(1):193–199. doi: 10.1111/j.1432-1033.1985.tb08638.x. [DOI] [PubMed] [Google Scholar]
  34. Weber I. T., Gilliland G. L., Harman J. G., Peterkofsky A. Crystal structure of a cyclic AMP-independent mutant of catabolite gene activator protein. J Biol Chem. 1987 Apr 25;262(12):5630–5636. [PubMed] [Google Scholar]
  35. Weber I. T., Steitz T. A. Structure of a complex of catabolite gene activator protein and cyclic AMP refined at 2.5 A resolution. J Mol Biol. 1987 Nov 20;198(2):311–326. doi: 10.1016/0022-2836(87)90315-9. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Zhang X. P., Ebright R. H. Substitution of 2 base pairs (1 base pair per DNA half-site) within the Escherichia coli lac promoter DNA site for catabolite gene activator protein places the lac promoter in the FNR regulon. J Biol Chem. 1990 Jul 25;265(21):12400–12403. [PubMed] [Google Scholar]

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