1887

Abstract

FNR (regulator for fumarate and nitrate reduction) and CRP (cAMP receptor protein) are global regulators which regulate the transcription of overlapping modulons of target genes in response to anaerobiosis and carbon source in An ORF, designated because it encodes an FNR-like protein of the FNR-CRP family, has been found in The product of the coding region (FLP) was overproduced in , purified and crystallized. FLP is a homodimeric protein in which each subunit can form an intramolecular disulphide bond. The isolated protein also contains non-stoichiometric amounts of Cu and Zn. Although the DNA recognition helix of FLP resembles that of FNR, the gene failed to complement the anaerobic respiratory deficiency of an mutant when expressed in and it neither activated nor interfered with transcription from FNR- or CRP-dependent promoters in Site-specific DNA binding by oxidized FLP (the form containing intrasubunit disulphide bonds) was abolished by reduction. The interconversion between disulphide and dithiol forms thus provides the basis for a novel redox-mediated transcriptional switch. Two non-identical FLP-binding sites, distinct from FNR- and CRP-binding sites, were identified in the region of by gel-retardation analysis. A further eight FLP-binding sites were selected from a random library. A synthetic oligonucleotide conforming to a putative FLP site consensus, C/TGA-N-TCA/G (the most significant bases are underlined), was retarded by FLP. Functional tests showed that FLP represses the aerobic transcription of a semi-synthetic promoter in A C5S variant of FLP lacking the ability to form intramolecular disulphide bonds was unable to bind to FLP sites and failed to repress transcription

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1998-03-01
2021-08-04
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References

  1. Almiron M., Link A.J., Furlong D., Kolter R. 1992; A novel DNA-binding protein with regulatory properties and protective roles in starved Escherichia coli. . Genes Dev 6:2646–2654
    [Google Scholar]
  2. Blackwell T.K. 1995; Selection of protein binding sites from random nucleic acid sequences.. Methods Enzymol 254:604–618
    [Google Scholar]
  3. Blake M. S., Johnson K. H., Rassec-Jones G. W., Golschlick E. C. 1984; A rapid, sensitive method for detection of alkaline- phosphatase conjugated anti-antibody on Western blots.. Anal Biochem 136:175–179
    [Google Scholar]
  4. Bougerie S.J., Michán C.M., Thomas M.S., Busby S.J.W., Hyde E.I. 1997; DNA binding and DNA bending by the MelR transcription activator protein from Escherichia coli. . Nucleic Acid Res 25:1685–1693
    [Google Scholar]
  5. Bowie J.U., Lüthy R., Eisenberg D. 1991; A method of identifying protein sequences that fold into a known threedimensional structure.. Science 253:164–170
    [Google Scholar]
  6. 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 Biochem 72:248–254
    [Google Scholar]
  7. Busby S., Kolb A. 1996; The CAP modulon. . In Regulation of Gene Expression in Escherichia coli pp. 255–279 Lin E.C.C., Lynch S.A. Edited by Austin, TX: R. G. Landes Company;
    [Google Scholar]
  8. Chen L., Helmann J.D. 1995; Bacillus subtilis MrgA is a Dps(PexB) homologue: evidence for metalloregulation of an oxidative-stress gene.. Mol Microbiol 18:295–300
    [Google Scholar]
  9. Condon S. 1987; Responses of lactic acid bacteria to oxygen.. FEMS Microbiol Rev 46:269–280
    [Google Scholar]
  10. Gasson M.J. 1993; Progress and potential in the biotechnology of lactic acid bacteria.. FEMS Microbiol Rev 12:3–20
    [Google Scholar]
  11. Gaston K., Kolb A., Busby S. 1989; Binding of the Escherichia coli cyclic-AMP receptor protein to DNA fragments containing consensus nucleotide-sequences.. Biochem J 261:649–653
    [Google Scholar]
  12. Gilles-Gonzalez M.A., Gonzalez G., Perutz M.F., Kiger L., Poyart C. 1994; Heme-based sensors, exemplified by kinase FixL, are a new class of heme protein with distinctive ligandbinding and autoxidation.. Biochemistry 33:8067–8073
    [Google Scholar]
  13. Green J., Sharrocks A.D., Green B., Geisow M., Guest J.R. 1993; Properties of FNR proteins substituted at each of the five cysteine residues.. Mol Microbiol 8:61–68
    [Google Scholar]
  14. Green J., Bennett B., Jordan P., Ralph E.T., Thomson A.J., Guest J.R. 1996; Reconstitution of the [4Fe-4S] cluster in FNR and demonstration of the aerobic-anaerobic transcription switch in vitro. . Biochem J 316:887–892
    [Google Scholar]
  15. Guest J.R., Green J., Irvine A.S., Spiro S. 1996; The FNR modulon and FNR-regulated gene expression. . In Regulation of Gene Expression in Escherichia coli pp. 317–342 Lin E.C.C., Lynch S.A. Edited by Austin, TX: R. G. Landes Company;
    [Google Scholar]
  16. Hildago E., Ding H., Demple B. 1997; Redox signal transduction via iron-sulfur clusters in the SoxR transcription activator.. Trends Biochem Sci 22:207–210
    [Google Scholar]
  17. Hill S., Austin S., Eydmann T., Jones T., Dixon R. 1996; Azotobacter vinelandii NifL is a flavoprotein that modulates transcriptional activation of nitrogen-fixation genes via a redox- sensitive switch.. Proc Natl Acad Sci USA 932143–2148
    [Google Scholar]
  18. Irvine A.S., Guest J.R. 1993; Lactobacillus casei contains a member of the CRP-FNR family.. Nucleic Acid Res 21:753
    [Google Scholar]
  19. Kerr L.D. 1995; Electrophoretic mobility shift assay.. Methods Enzymol 254:619–631
    [Google Scholar]
  20. Kullik I., Toledano M.B., Yartaglia L.A., Storz G. 1995; Mutational analysis of the redox-sensitive transcriptional regulator OxyR: regions important for oxidation and transcriptional activation.. J Bacteriol 177:1275–1284
    [Google Scholar]
  21. Laemmli U.K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4.. Nature 227:680–685
    [Google Scholar]
  22. Lazazzera B.A., Beinert H., Khoroshilova N., Kennedy M.C., Kiley P.J. 1996; DNA-binding and dimerization of the Fe-S- containing FNR protein from Escherichia coli are regulated by oxygen.. J Biol Chem 271:2762–2768
    [Google Scholar]
  23. Lodge J., Williams R., Bell A., Chan B., Busby S. 1990; Comparison of promoter activities in Escherichia coli and Pseudomonas aeruginosa - use of a new broad-host-range promoter-probe plasmid.. FEMS Microbiol Lett 67:221–225
    [Google Scholar]
  24. Lynch S.A., Lin E. C. C. 1996a; Responses to molecular oxygen. . In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, 2nd edn. pp. 1526–1538 Neidhardt E.C.C. Edited by others Washington, DC: American Society for Microbiology;
    [Google Scholar]
  25. Lynch S.A., Lin E. C. C. 1996b; Regulation of aerobic and anaerobic metabolism by the Arc system. . In Regulation of Gene Expression in Escherichia coli pp. 361–381 Lin E.C.C., Lynch S.A. Edited by Austin, TX: R. G. Landes Company;
    [Google Scholar]
  26. Marsh P. 1986; ptac-85, an Escherichia coli vector for expression of non-fusion proteins.. Nucleic Acids Res 14:3603
    [Google Scholar]
  27. Miller J.H. 1972 Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  28. Monson E.K., Ditta G.S., Helinski D.R. 1995; The oxygen sensor protein, FixL, of Rhizobium meliloti. Role of histidine residues in heme-binding, phosphorylation, and signal-transduction.. J Biol Chem 270:5243–5250
    [Google Scholar]
  29. Natori Y., Kano Y., Imamoto F. 1990; Nucleotide sequences and genomic constitution of five tryptophan genes of Lacto-bacillus casei. . J Bacteriol 107:248–255
    [Google Scholar]
  30. Pandey A., Bringel F., Meyer J.M. 1994; Iron requirement and search for siderophores in lactic acid bacteria.. Appl Microbiol Biotechnol 40:735–739
    [Google Scholar]
  31. Rallu F., Gruss A., Maguin E. 1996; Lactococcus lactis and stress.. Antonie Leeuwenhoek 70:99–110
    [Google Scholar]
  32. Sambrook J., Fritsch E.F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  33. Sanders J.W., Leenhouts K.J., Haandrikman A.J., Venema G., Kok J. 1995; Stress response in Lactococcus lactis: cloning, expression analysis, and mutation of the lactococcal superoxide dismutase gene.. J Bacteriol 177:5254–5260
    [Google Scholar]
  34. Shaw D.J., Guest J.R. 1982; Amplification and product identification of the fnr gene of Escherichia coli. . J Gen Microbiol 128:2221–2228
    [Google Scholar]
  35. Silhavy T.J., Barman M.L., Enquist L.W. 1984 Experiments with Gene Fusions. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  36. Spiro S. 1994; The FNR family of transcriptional regulators.. Antonie Leeuwenhoek 66:23–36
    [Google Scholar]
  37. Spiro S., Guest J.R. 1987; Regulation and overexpression of the fnr gene of Escherichia coli. . J Gen Microbiol 133:3279–3288
    [Google Scholar]
  38. Spiro S., Guest J. R. 1990; FNR and its role in oxygen- regulated gene expression in Escherichia coli. . FEMS Microbiol Rev 75:399–428
    [Google Scholar]
  39. Spiro S., Gaston K.L., Bell A.I., Roberts R.E., Busby S.J.W., Guest J.R. 1990; Interconversion of the DNA-binding specificities of two related transcription regulators, CRP and FNR.. Mol Microbiol 4:1831–1838
    [Google Scholar]
  40. Takagi T., Isemura T. 1965; Accelerating effect of copper ion on the reactivation of reduced Taka-amylase A through catalysis of the oxidation of thiol groups.. J Biochem 56:344–350
    [Google Scholar]
  41. Thannhauser T.W., Konishi Y., Scheraga H.A. 1987; Analysis for disulphide bonds in peptides and proteins.. Methods Enzymol 143:115–119
    [Google Scholar]
  42. Thelander L. 1973; Physicochemical characterization of ribo- nucleoside diphosphate reductase from Escherichia coli. . J Biol Chem 248:4591–1601
    [Google Scholar]
  43. Unden G., Becker S., Bongaerts J., Six S. 1994; Oxygen- regulated gene expression in facultatively anaerobic bacteria.. Antonie Leeuwenhoek 66:3–23
    [Google Scholar]
  44. Vishniac W., Santer M. 1957; The Thiobacilli.. Bacteriol Rev 21:95–215
    [Google Scholar]
  45. Vogel H., Bonner D.M. 1956; A convenient growth medium for E. coli and some other organisms.. Microbial Genet Bull 13:43
    [Google Scholar]
  46. de Vos W.M. 1996; Metabolic engineering of sugar catabolism in lactic acid bacteria.. Antonie Leeuwenhoek 70:223–242
    [Google Scholar]
  47. Webster G, Kempsall K., Booth I., Busby S. 1987; Organisation of the regulatory region of the Escherichia coli melibiose operon.. Gene 59:253–263
    [Google Scholar]
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