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. 1989 May;171(5):2485–2498. doi: 10.1128/jb.171.5.2485-2498.1989

Novel transcriptional control of the pyruvate formate-lyase gene: upstream regulatory sequences and multiple promoters regulate anaerobic expression.

G Sawers 1, A Böck 1
PMCID: PMC209925  PMID: 2651404

Abstract

The sequence of the 5' regulatory region of the gene encoding pyruvate formate-lyase is presented together with a detailed analysis of the transcriptional signals required for its expression. The sequence data revealed that a gene coding for an open reading frame (orf) of unknown function is situated just upstream of the pfl gene. Analysis of RNA transcripts by Northern blot hybridization demonstrated that the genes for orf and pfl were cotranscribed as an operon but that the pfl gene was also transcribed alone. S1 nuclease protection analysis, primer extension, and construction of lacZ fusions with sequential deletions in the pfl 5' regulatory sequence revealed that transcription initiated from at least six promoters which spanned 1.2 kilobases of DNA. Three of these lay within the orf structural gene and were responsible for the high expression of pfl. All transcripts originating from these promoters terminated in the 3' untranslated region of the pfl gene at a strong rho-independent transcription terminator. All of the promoters were coordinately regulated by anaerobiosis, pyruvate, nitrate, and the fnr gene product, and the sequences thought to be responsible for this regulation lay 0.8 to 1.3 kilobases upstream of the translational initiation codon of the pfl gene. There were two sequences within this region which showed strong homology with that proposed to be required for recognition by the Fnr protein.

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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., Adhya S., de Crombrugghe B. Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem. 1981 Nov 25;256(22):11905–11910. [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. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [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. Birkmann A., Zinoni F., Sawers G., Böck A. Factors affecting transcriptional regulation of the formate-hydrogen-lyase pathway of Escherichia coli. Arch Microbiol. 1987 Jun;148(1):44–51. doi: 10.1007/BF00429646. [DOI] [PubMed] [Google Scholar]
  6. Båga M., Göransson M., Normark S., Uhlin B. E. Processed mRNA with differential stability in the regulation of E. coli pilin gene expression. Cell. 1988 Jan 29;52(2):197–206. doi: 10.1016/0092-8674(88)90508-9. [DOI] [PubMed] [Google Scholar]
  7. Casadaban M. J., Cohen S. N. Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4530–4533. doi: 10.1073/pnas.76.9.4530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen C. Y., Beatty J. T., Cohen S. N., Belasco J. G. An intercistronic stem-loop structure functions as an mRNA decay terminator necessary but insufficient for puf mRNA stability. Cell. 1988 Feb 26;52(4):609–619. doi: 10.1016/0092-8674(88)90473-4. [DOI] [PubMed] [Google Scholar]
  9. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  10. Christiansen L., Pedersen S. Cloning, restriction endonuclease mapping and post-transcriptional regulation of rpsA, the structural gene for ribosomal protein S1. Mol Gen Genet. 1981;181(4):548–551. doi: 10.1007/BF00428751. [DOI] [PubMed] [Google Scholar]
  11. Cole S. T. Nucleotide sequence coding for the flavoprotein subunit of the fumarate reductase of Escherichia coli. Eur J Biochem. 1982 Mar 1;122(3):479–484. doi: 10.1111/j.1432-1033.1982.tb06462.x. [DOI] [PubMed] [Google Scholar]
  12. Dixon R. The xylABC promoter from the Pseudomonas putida TOL plasmid is activated by nitrogen regulatory genes in Escherichia coli. Mol Gen Genet. 1986 Apr;203(1):129–136. doi: 10.1007/BF00330393. [DOI] [PubMed] [Google Scholar]
  13. Dorman C. J., Barr G. C., Ni Bhriain N., Higgins C. F. DNA supercoiling and the anaerobic and growth phase regulation of tonB gene expression. J Bacteriol. 1988 Jun;170(6):2816–2826. doi: 10.1128/jb.170.6.2816-2826.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Grisolia V., Riccio A., Bruni C. B. Structure and function of the internal promoter (hisBp) of the Escherichia coli K-12 histidine operon. J Bacteriol. 1983 Sep;155(3):1288–1296. doi: 10.1128/jb.155.3.1288-1296.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gussin G. N., Ronson C. W., Ausubel F. M. Regulation of nitrogen fixation genes. Annu Rev Genet. 1986;20:567–591. doi: 10.1146/annurev.ge.20.120186.003031. [DOI] [PubMed] [Google Scholar]
  16. Higgins C. F., Dorman C. J., Stirling D. A., Waddell L., Booth I. R., May G., Bremer E. A physiological role for DNA supercoiling in the osmotic regulation of gene expression in S. typhimurium and E. coli. Cell. 1988 Feb 26;52(4):569–584. doi: 10.1016/0092-8674(88)90470-9. [DOI] [PubMed] [Google Scholar]
  17. Horowitz H., Platt T. Initiation in vivo at the internal trp p2 promoter of Escherichia coli. J Biol Chem. 1983 Jul 10;258(13):7890–7893. [PubMed] [Google Scholar]
  18. Ikemura T., Dahlberg J. E. Small ribonucleic acids of Escherichia coli. I. Characterization by polyacrylamide gel electrophoresis and fingerprint analysis. J Biol Chem. 1973 Jul 25;248(14):5024–5032. [PubMed] [Google Scholar]
  19. Jayaraman P. S., Gaston K. L., Cole J. A., Busby S. J. The nirB promoter of Escherichia coli: location of nucleotide sequences essential for regulation by oxygen, the FNR protein and nitrite. Mol Microbiol. 1988 Jul;2(4):527–530. doi: 10.1111/j.1365-2958.1988.tb00059.x. [DOI] [PubMed] [Google Scholar]
  20. Jayaraman P. S., Peakman T. C., Busby S. J., Quincey R. V., Cole J. A. Location and sequence of the promoter of the gene for the NADH-dependent nitrite reductase of Escherichia coli and its regulation by oxygen, the Fnr protein and nitrite. J Mol Biol. 1987 Aug 20;196(4):781–788. doi: 10.1016/0022-2836(87)90404-9. [DOI] [PubMed] [Google Scholar]
  21. Jones H. M., Gunsalus R. P. Transcription of the Escherichia coli fumarate reductase genes (frdABCD) and their coordinate regulation by oxygen, nitrate, and fumarate. J Bacteriol. 1985 Dec;164(3):1100–1109. doi: 10.1128/jb.164.3.1100-1109.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. King T. C., Sirdeskmukh R., Schlessinger D. Nucleolytic processing of ribonucleic acid transcripts in procaryotes. Microbiol Rev. 1986 Dec;50(4):428–451. doi: 10.1128/mr.50.4.428-451.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kleckner N., Barker D. F., Ross D. G., Botstein D. Properties of the translocatable tetracycline-resistance element Tn10 in Escherichia coli and bacteriophage lambda. Genetics. 1978 Nov;90(3):427–461. doi: 10.1093/genetics/90.3.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kornberg H. L., Smith J. Genetic control of the uptake of pyruvate by Escherichia coli. Biochim Biophys Acta. 1967 Nov 28;148(2):591–592. doi: 10.1016/0304-4165(67)90167-5. [DOI] [PubMed] [Google Scholar]
  25. Krikos A., Mutoh N., Boyd A., Simon M. I. Sensory transducers of E. coli are composed of discrete structural and functional domains. Cell. 1983 Jun;33(2):615–622. doi: 10.1016/0092-8674(83)90442-7. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  29. Melefors O., von Gabain A. Site-specific endonucleolytic cleavages and the regulation of stability of E. coli ompA mRNA. Cell. 1988 Mar 25;52(6):893–901. doi: 10.1016/0092-8674(88)90431-x. [DOI] [PubMed] [Google Scholar]
  30. Mizuno T., Mizushima S. Characterization by deletion and localized mutagenesis in vitro of the promoter region of the Escherichia coli ompC gene and importance of the upstream DNA domain in positive regulation by the OmpR protein. J Bacteriol. 1986 Oct;168(1):86–95. doi: 10.1128/jb.168.1.86-95.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Müllner E. W., Kühn L. C. A stem-loop in the 3' untranslated region mediates iron-dependent regulation of transferrin receptor mRNA stability in the cytoplasm. Cell. 1988 Jun 3;53(5):815–825. doi: 10.1016/0092-8674(88)90098-0. [DOI] [PubMed] [Google Scholar]
  32. Newbury S. F., Smith N. H., Higgins C. F. Differential mRNA stability controls relative gene expression within a polycistronic operon. Cell. 1987 Dec 24;51(6):1131–1143. doi: 10.1016/0092-8674(87)90599-x. [DOI] [PubMed] [Google Scholar]
  33. Nilsson G., Lundberg U., von Gabain A. In vivo and in vitro identity of site specific cleavages in the 5' non-coding region of ompA and bla mRNA in Escherichia coli. EMBO J. 1988 Jul;7(7):2269–2275. doi: 10.1002/j.1460-2075.1988.tb03067.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Padan E., Arbel T., Rimon A., Shira A. B., Cohen A. Biosynthesis of the lactose permease in Escherichia coli minicells and effect of carrier amplification on cell physiology. J Biol Chem. 1983 May 10;258(9):5666–5673. [PubMed] [Google Scholar]
  35. Pecher A., Blaschkowski H. P., Knappe K., Böck A. Expression of pyruvate formate-lyase of Escherichia coli from the cloned structural gene. Arch Microbiol. 1982 Oct;132(4):365–371. doi: 10.1007/BF00413390. [DOI] [PubMed] [Google Scholar]
  36. Platt T. Transcription termination and the regulation of gene expression. Annu Rev Biochem. 1986;55:339–372. doi: 10.1146/annurev.bi.55.070186.002011. [DOI] [PubMed] [Google Scholar]
  37. Ponnambalam S., Busby S. RNA polymerase molecules initiating transcription at tandem promoters can collide and cause premature transcription termination. FEBS Lett. 1987 Feb 9;212(1):21–27. doi: 10.1016/0014-5793(87)81549-1. [DOI] [PubMed] [Google Scholar]
  38. Ptashne M. Gene regulation by proteins acting nearby and at a distance. Nature. 1986 Aug 21;322(6081):697–701. doi: 10.1038/322697a0. [DOI] [PubMed] [Google Scholar]
  39. Raibaud O., Schwartz M. Positive control of transcription initiation in bacteria. Annu Rev Genet. 1984;18:173–206. doi: 10.1146/annurev.ge.18.120184.001133. [DOI] [PubMed] [Google Scholar]
  40. Reitzer L. J., Magasanik B. Transcription of glnA in E. coli is stimulated by activator bound to sites far from the promoter. Cell. 1986 Jun 20;45(6):785–792. doi: 10.1016/0092-8674(86)90553-2. [DOI] [PubMed] [Google Scholar]
  41. Ronson C. W., Nixon B. T., Albright L. M., Ausubel F. M. Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions. J Bacteriol. 1987 Jun;169(6):2424–2431. doi: 10.1128/jb.169.6.2424-2431.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ronson C. W., Nixon B. T., Ausubel F. M. Conserved domains in bacterial regulatory proteins that respond to environmental stimuli. Cell. 1987 Jun 5;49(5):579–581. doi: 10.1016/0092-8674(87)90530-7. [DOI] [PubMed] [Google Scholar]
  43. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  44. Rödel W., Plaga W., Frank R., Knappe J. Primary structures of Escherichia coli pyruvate formate-lyase and pyruvate-formate-lyase-activating enzyme deduced from the DNA nucleotide sequences. Eur J Biochem. 1988 Oct 15;177(1):153–158. doi: 10.1111/j.1432-1033.1988.tb14356.x. [DOI] [PubMed] [Google Scholar]
  45. Saint Girons I., Margarita D. Evidence for an internal promoter in the Escherichia coli threonine operon. J Bacteriol. 1985 Jan;161(1):461–462. doi: 10.1128/jb.161.1.461-462.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sawers G., Böck A. Anaerobic regulation of pyruvate formate-lyase from Escherichia coli K-12. J Bacteriol. 1988 Nov;170(11):5330–5336. doi: 10.1128/jb.170.11.5330-5336.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schmidt C., Schmieger H. Selective transduction of recombinant plasmids with cloned pac sites by Salmonella phage P22. Mol Gen Genet. 1984;196(1):123–128. doi: 10.1007/BF00334103. [DOI] [PubMed] [Google Scholar]
  48. Shaw D. J., Guest J. R. Molecular cloning of the fnr gene of Escherichia coli K12. Mol Gen Genet. 1981;181(1):95–100. doi: 10.1007/BF00339011. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Simons R. W., Houman F., Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987;53(1):85–96. doi: 10.1016/0378-1119(87)90095-3. [DOI] [PubMed] [Google Scholar]
  52. Spiro S., Guest J. R. Regulation and over-expression of the fnr gene of Escherichia coli. J Gen Microbiol. 1987 Dec;133(12):3279–3288. doi: 10.1099/00221287-133-12-3279. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. Subbarao M. N., Kennell D. Evidence for endonucleolytic cleavages in decay of lacZ and lacI mRNAs. J Bacteriol. 1988 Jun;170(6):2860–2865. doi: 10.1128/jb.170.6.2860-2865.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  56. Wek R. C., Hatfield G. W. Examination of the internal promoter, PE, in the ilvGMEDA operon of E. coli K-12. Nucleic Acids Res. 1986 Mar 25;14(6):2763–2777. doi: 10.1093/nar/14.6.2763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wich G., Hummel H., Jarsch M., Bär U., Böck A. Transcription signals for stable RNA genes in Methanococcus. Nucleic Acids Res. 1986 Mar 25;14(6):2459–2479. doi: 10.1093/nar/14.6.2459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Yamamoto N., Droffner M. L. Mechanisms determining aerobic or anaerobic growth in the facultative anaerobe Salmonella typhimurium. Proc Natl Acad Sci U S A. 1985 Apr;82(7):2077–2081. doi: 10.1073/pnas.82.7.2077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Zinoni F., Birkmann A., Stadtman T. C., Böck A. Nucleotide sequence and expression of the selenocysteine-containing polypeptide of formate dehydrogenase (formate-hydrogen-lyase-linked) from Escherichia coli. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4650–4654. doi: 10.1073/pnas.83.13.4650. [DOI] [PMC free article] [PubMed] [Google Scholar]

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