1887

Abstract

In K-12 the expression of many genes is controlled by the oxygen-responsive transcription factor FNR and the nitrate- and nitrite-responsive two-component systems NarXL and NarPQ. Here, the gene is shown to be the first gene of a six-gene operon () that encodes proteins predicted to be components of an oxidoreductase. Mapping the transcript start and site-directed mutagenesis confirmed that the genes are transcribed from an FNR-dependent class II promoter and showed that the FNR site is centred at −42.5. In the presence of nitrate or nitrite, NarXL and NarPQ repressed expression. Analysis of the DNA sequence of the promoter region (P) revealed the presence of four heptameric sequences similar to NarL/P binding sites centred at −42, −16, +6 and +15. The latter heptamers are arranged as a 7-2-7 inverted repeat, which is required for recognition by NarP. Accordingly, NarP protected the 7-2-7 region in DNase I footprints, and mutation of either heptamer +6 or heptamer +15 impaired nitrite-mediated repression, whereas mutation of heptamer −42 and heptamer −16 did not affect the response to nitrite. The NarL protein also protected the 7-2-7 region, but in contrast to NarP, the NarL footprint extended further upstream to encompass the −16 heptamer. The extended NarL footprint was consistent with the presence of multiple NarL–P complexes in gel retardation assays. Mutation of heptamer −42, which is located within the FNR binding site, or heptamer +6 (but not heptamers −16 or +15) impaired nitrate-mediated repression. Thus, although the region of the promoter containing the −16 and +15 heptamers was recognized by NarL , mutation of these heptamers did not affect NarL-mediated repression .

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2007/012146-0
2008-02-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/2/608.html?itemId=/content/journal/micro/10.1099/mic.0.2007/012146-0&mimeType=html&fmt=ahah

References

  1. Bearson S. M. D., Albrecht J. A., Gunsalus R. P. 2002; Oxygen and nitrate-dependent regulation of dmsABC operon expression in Escherichia coli : sites for FNR and NarL protein interactions. BMC Microbiol 2:13
    [Google Scholar]
  2. Berks B. C., Palmer T., Sargent F. 2005; Protein targeting by the bacterial twin arginine translocation (Tat) pathway. Curr Opin Microbiol 8:174–181
    [Google Scholar]
  3. Bochner B. R. 2003; New technologies to assess genotype–phenotype relationships. Nat Rev Genet 4:309–314
    [Google Scholar]
  4. Browning D., Lee D., Green J., Busby S. 2003; Secrets of bacterial transcription initiation taught by the Escherichia coli FNR protein. In Signals, Switches, Regulons and Cascades: Control of Bacterial Gene Expression pp 127–142 Edited by Hodgson D. A., Thomas C. M. Cambridge, UK: Cambridge University Press;
    [Google Scholar]
  5. Busby S., Kotlarz D., Buc H. 1983; Deletion mutagenesis of the Escherichia coli galactose operon promoter region. J Mol Biol 167:259–274
    [Google Scholar]
  6. Constantinidou C., Hobman J. L., Griffiths L., Patel M. D., Penn C. W., Cole J. A., Overton T. W. 2006; A reassessment of the FNR regulon and transcriptomic analysis of the effects of nitrate, nitrite, NarXL and NarPQ as Escherichia coli adapts from aerobic to anaerobic growth. J Biol Chem 281:4802–4815
    [Google Scholar]
  7. Darwin A. J., Stewart V. 1996; The Nar modulon systems: nitrate and nitrite regulation of anaerobic gene expression. In Regulation of Gene Expression in Escherichia coli pp 343–359 Edited by Lin E. C. C., Lynch A. S. Austin, TX: R. G. Landes;
    [Google Scholar]
  8. Darwin A. J., Tyson K. L., Busby S. J. W., Stewart V. 1997; Differential regulation by the homologous response regulators NarL and NarP of Escherichia coli K-12 depends on DNA binding site arrangement. Mol Microbiol 25:583–595
    [Google Scholar]
  9. Eiglmeier K., Honore N., Iuchi S., Lin E. C. C., Cole S. T. 1989; Molecular genetic analysis of FNR-dependent promoters. Mol Microbiol 3:869–878
    [Google Scholar]
  10. Evans C. G. T., Herbert D., Tempest D. W. 1970; The continuous cultivation of micro-organisms. 2. Construction of a chemostat. Methods Microbiol 2:277–327
    [Google Scholar]
  11. Greenberg J. T., Monach P., Chou J. H., Josephy D., Demple B. 1990; Positive control of a global antioxidant defense regulon activated by superoxide-generating agents in Escherichia coli . Proc Natl Acad Sci U S A 87:6181–6185
    [Google Scholar]
  12. 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 Edited by Lin E. C. C., Lynch A. S. Austin, TX: R. G. Landes;
    [Google Scholar]
  13. Heinzinger N. K., Fujimoto S. Y., Clark M. A., Moreno M. S., Barrett E. L. 1995; Sequence analysis of the phs operon in Salmonella typhimurium and the contribution of thiosulfate reduction to anaerobic energy metabolism. J Bacteriol 177:2813–2820
    [Google Scholar]
  14. Hensel M., Shea J. E., Baumler A. J., Gleeson C., Blattner F., Holden D. W. 1997; Analysis of the boundaries of Salmonella pathogenicity island 2 and the corresponding chromosomal region of Escherichia coli K-12. J Bacteriol 179:1105–1111
    [Google Scholar]
  15. Jervis A. J., Green J. 2007; In vivo demonstration of FNR dimers in response to lower O2 availability. J Bacteriol 189:2930–2932
    [Google Scholar]
  16. Kang Y., Weber K. D., Qiu Y., Kiley P. J., Blattner F. R. 2005; Genome-wide expression analysis indicates that FNR of Escherichia coli K-12 regulates a large number of genes of unknown function. J Bacteriol 187:1135–1160
    [Google Scholar]
  17. Kiley P. J., Beinert H. 2003; The role of Fe–S proteins in sensing and regulation in bacteria. Curr Opin Microbiol 6:181–185
    [Google Scholar]
  18. Kletzin A., Mukund S., Kelley-Crouse T. L., Chan M. K., Rees D. C., Adams M. W. W. 1995; Molecular characterisation of the genes encoding the tungsten-containing aldehyde ferredoxin oxidoreductase from Pyrococcus furiosus and formaldehyde ferredoxin oxidoreductase from Thermococcus litoralis . J Bacteriol 177:4817–4819
    [Google Scholar]
  19. Koonin E. V., Mushegian A. R., Bork P. 1996; Non-orthologous gene displacement. Trends Genet 12:334–336
    [Google Scholar]
  20. 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]
  21. Lee A. I., Delgado A., Gunsalus R. P. 1999; Signal-dependent phosphorylation of the membrane-bound NarX two-component sensor–transmitter protein of Escherichia coli : nitrate elicits a superior anion ligand response compared to nitrite. J Bacteriol 181:5309–5316
    [Google Scholar]
  22. Lennox E. S. 1955; Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190–206
    [Google Scholar]
  23. Li J., Kustu S., Stewart V. 1994; In vitro interaction of nitrate-responsive regulatory protein NarL with DNA target sequences in fdnG , narG , narK and frdA operon control regions of Escherichia coli K-12. J Mol Biol 241:150–165
    [Google Scholar]
  24. Martinez E., Bartolome B., Delacruz F. 1988; pACYC184-derived cloning vectors containing the multiple cloning site and lacZα reporter gene of pUC8/9 and pUC18/19 plasmids. Gene 68:159–162
    [Google Scholar]
  25. Meganathan R., Schrementi J. 1987; Tetrahydrothiophene 1-oxide as an electron acceptor for Escherichia coli . J Bacteriol 169:2862–2865
    [Google Scholar]
  26. Miller J. H. 1972; Assay of β -galactosidase. In Experiments in Molecular Genetics pp 352–354 Edited by Miller J. H. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press;
    [Google Scholar]
  27. Poteete A. R., Fenton A. C. 1984; λ red -dependent growth and recombination of phage P22. Virology 134:161–167
    [Google Scholar]
  28. Powell B. S., Court D. L., Nakamura Y., Rivas M. P., Turnbough C. L. Jr 1994; Rapid confirmation of single copy lambda prophage integration by PCR. Nucleic Acids Res 22:5765–5766
    [Google Scholar]
  29. Rabin R. S., Stewart V. 1992; Either of two functionally redundant sensor proteins, NarX and NarQ, is sufficient for nitrate regulation in Escherichia coli K-12. Proc Natl Acad Sci U S A 89:8419–8423
    [Google Scholar]
  30. Rabin R. S., Stewart V. 1993; Dual response regulators (NarL and NarP) interact with dual sensors (NarX and NarQ) to control nitrate- and nitrite-regulated gene expression in Escherichia coli K-12. J Bacteriol 175:3259–3268
    [Google Scholar]
  31. Sambrook J. W., Russell D. W. 2001 Molecular Cloning: a Laboratory Manual , 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Press;
  32. Sawers G., Suppmann B. 1992; Anaerobic induction of pyruvate formate-lyase gene expression is mediated by the ArcA and FNR proteins. J Bacteriol 174:3474–3478
    [Google Scholar]
  33. Simons R. W., Houman F., Kleckner N. 1987; Improved single and multicopy lac -based cloning vectors for protein and operon fusions. Gene 53:85–96
    [Google Scholar]
  34. Spiro S., Guest J. R. 1987; Regulation and over-expression of the fnr gene of Escherichia coli . J Gen Microbiol 133:3279–3288
    [Google Scholar]
  35. Stewart V. 1994; Dual interacting two-component regulatory systems mediate nitrate- and nitrite-regulated gene expression in Escherichia coli . Res Microbiol 145:450–454
    [Google Scholar]
  36. Sutton V. R., Mettert E. L., Beinert H., Kiley P. J. 2004; Kinetic analysis of the oxidative conversion of the [4Fe–4S]2+ cluster of FNR to a [2Fe–S]2+ cluster. J Bacteriol 186:8018–8025
    [Google Scholar]
  37. Turner R. J., Papish A. L., Sargent F. 2004; Sequence analysis of bacterial redox enzyme maturation proteins (REMPs. Can J Microbiol 50:225–238
    [Google Scholar]
  38. Unden G., Achebach S., Holighaus G., Tran H.-Q., Wackwitz B., Zeuner Y. 2002; Control of FNR function of Escherichia coli by O2 and reducing conditions. J Mol Microbiol Biotechnol 4:263–268
    [Google Scholar]
  39. Wang H., Gunsalus R. P. 2003; Coordinate regulation of the Escherichia coli formate dehydrogenase fdnGHI and fdhF genes in response to nitrate, nitrite, and formate: roles for NarL and NarP. J Bacteriol 185:5076–5085
    [Google Scholar]
  40. Williams S. B., Stewart V. 1997; Discrimination between structurally related ligands nitrate and nitrite controls autokinase activity of the NarX transmembrane signal transducer of Escherichia coli K-12. Mol Microbiol 26:911–925
    [Google Scholar]
  41. Wing H. J., Williams S. M., Busby S. J. W. 1995; Spacing requirements for transcription activation by Escherichia coli FNR protein. J Bacteriol 177:6704–6710
    [Google Scholar]
  42. Yamamoto K., Hirao K., Oshima T., Aiba H., Utsumi R., Ishihama A. 2005; Functional characterisation in vitro of all two-component signal transduction systems from Escherichia coli . J Biol Chem 280:1448–1456
    [Google Scholar]
  43. Yu D., Ellis H. M., Lee E.-C., Jenkins N. A., Copeland N. G., Court D. L. 2000; An efficient recombination system for chromosome engineering in Escherichia coli . Proc Natl Acad Sci U S A 97:5978–5983
    [Google Scholar]
  44. Zheng M., Doan B., Schneider T. D., Storz G. 1999; OxyR and SoxRS regulation of fur . J Bacteriol 181:4639–4643
    [Google Scholar]
  45. Ziegelhoffer E. C., Kiley P. J. 1995; In vitro analysis of a constitutively active mutant form of the Escherichia coli global transcription factor FNR. J Mol Biol 245:351–361
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2007/012146-0
Loading
/content/journal/micro/10.1099/mic.0.2007/012146-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error