Synthesis of the [NiFe] hydrogenases 1 and 2 of is induced in response to anaerobiosis and is repressed when nitrate is present in the growth medium. The hydrogenase 1 and hydrogenase 2 enzymes are encoded by the polycistronic and operons, respectively. Primer extension analysis was used to determine the initiation site of transcription of both operons. This permitted the construction of single-copy operon fusions, which were used to examine the transcriptional regulation of the two operons. Expression of both was induced by anaerobiosis and repressed by nitrate, which is in complete accord with earlier biochemical studies. Anaerobic induction of the operon was only partially dependent on the FNR protein and, surprisingly, was enhanced by an mutation. This latter result indicated that ArcA suppresses anaerobic expression and that a further factor, which remains to be identified, is involved in controlling anaerobic induction of operon expression. Nitrate repression of expression was mediated by the NarL/NarX and NarP/NarQ two-component regulatory systems. Remarkably, a mutant lacked anaerobic induction of expression, even in the absence of added nitrate. Anaerobic induction of expression was dependent on the ArcA and AppY regulators, which confirms earlier observations by other authors. Nitrate repression of the operon was mediated by both NarL and NarP. Taken together, these data indicate that although the and operons share common regulators, there are important differences in the control of expression of the individual operons.


Article metrics loading...

Loading full text...

Full text loading...



  1. Andrews, S. C., Berks, B. C., McClay, J., Ambler, A., Quail, M. A., Golby, P. & Guest, J. R. (1997). A 12-cistron Escherichia coli operon (hyf) encoding a putative proton-pumping formate hydrogenlyase system. Microbiology 143, 3633-3647.[CrossRef] [Google Scholar]
  2. Atlung, T. & Bronsted, L. (1994). Role of the transcriptional activator AppY in regulation of the cpx appA operon of Escherichia coli by anaerobiosis, phosphate starvation, and growth phase. J Bacteriol 176, 5414-5422. [Google Scholar]
  3. Atlung, T., Knudsen, K., Heerfordt, L. & Bronsted, L. (1997). Effects of σs and the transcriptional activator AppY on induction of the Escherichia coli hya and cbd–appA operons in response to carbon and phosphate starvation. J Bacteriol 179, 2141-2146. [Google Scholar]
  4. Ballantine, S. P. & Boxer, D. H. (1985). Nickel-containing hydrogenase isoenzymes from anaerobically grown Escherichia coli K-12. J Bacteriol 163, 454-459. [Google Scholar]
  5. Ballantine, S. P. & Boxer, D. H. (1986). Isolation and characterisation of a soluble active fragment of hydrogenase isoenzyme 2 from the membranes of anaerobically grown Escherichia coli. Eur J Biochem 156, 277-284.[CrossRef] [Google Scholar]
  6. Bell, A. I., Gaston, K. L., Cole, J. A. & Busby, S. W. J. (1989). 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 17, 3865-3874.[CrossRef] [Google Scholar]
  7. Birkmann, A., Sawers, G. & Böck, A. (1987). Involvement of the ntrA gene product in the anaerobic metabolism of Escherichia coli. Mol Gen Genet 210, 535-542.[CrossRef] [Google Scholar]
  8. Böhm, R., Sauter, M. & Böck, A. (1990). Nucleotide sequence and expression of an operon in Escherichia coli coding for formate hydrogenlyase. Mol Microbiol 4, 231-243.[CrossRef] [Google Scholar]
  9. Bronsted, L. & Atlung, T. (1994). Anaerobic regulation of the hydrogenase 1 (hya) operon of Escherichia coli. J Bacteriol 176, 5423-5428. [Google Scholar]
  10. Busby, S. & Kolb, A. (1996). The Cap modulon. In Regulation of Gene Expression in Escherichia coli, pp. 255-279. Edited by A. S. Lynch & E. C. C. Lin. New York: Chapman & Hall.
  11. Casadaban, M. J. & Cohen, S. N. (1979). Lactose genes fused to exogenous promoters in one step using Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. Proc Natl Acad Sci USA 76, 4530-4533.[CrossRef] [Google Scholar]
  12. 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 A. S. Lynch & E. C. C. Lin. New York: Chapman & Hall.
  13. Darwin, A. J., Tyson, K. L., Busby, S. J. W. & Cole, J. A. (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.[CrossRef] [Google Scholar]
  14. Darwin, A. J., Ziegelhoffer, E. C., Kiley, P. J. & Stewart, V. (1998). Fnr, NarP, and NarL regulation of Escherichia coli K-12 napF (periplasmic nitrate reductase) operon transcription in vitro. J Bacteriol 180, 4192-4198. [Google Scholar]
  15. Drapal, N. & Sawers, G. (1995). Purification of ArcA and analysis of its specific interaction with the pfl promoter-regulatory region. Mol Microbiol 16, 597-607.[CrossRef] [Google Scholar]
  16. 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 A. S. Lynch & E. C. C. Lin. New York: Chapman & Hall.
  17. Hopper, S., Korsa, I. & Böck, A. (1996). The nucleotide concentration determines the specificity of in vitro transcription activation by the σ54-dependent activator FhlA. J Bacteriol 178, 199-203. [Google Scholar]
  18. Ingmer, H., Miller, C. A. & Cohen, S. N. (1998). Destabilised inheritance of pSC101 and other Escherichia coli plasmids by DpiA, a novel two-component system regulator. Mol Microbiol 29, 49-59.[CrossRef] [Google Scholar]
  19. Jamieson, D. J., Sawers, R. G., Rugman, P. A., Boxer, D. H. & Higgins, C. F. (1986). Effects of anaerobic regulatory mutations and catabolite repression on regulation of hydrogen metabolism and hydrogenase isoenzyme composition in Salmonella typhimurium. J Bacteriol 168, 405-411. [Google Scholar]
  20. Kaiser, M. & Sawers, G. (1995). Nitrate regulation of the Escherichia coli pfl operon is mediated by the dual sensors NarQ and NarX and the dual regulators NarL and NarP. J Bacteriol 177, 3647-3655. [Google Scholar]
  21. Lutz, S., Böhm, R., Beier, A. & Böck, A. (1990). Characterisation of divergent NtrA-dependent promoters in the anaerobically expressed gene cluster encoding hydrogenase 3 components of Escherichia coli. Mol Microbiol 4, 13-20.[CrossRef] [Google Scholar]
  22. Lynch, A. S. & Lin, E. C. C. (1996a). Regulation of aerobic and anaerobic metabolism by the Arc system. In Regulation of Gene Expression in Escherichia coli, pp. 361-381. Edited by A. S. Lynch & E. C. C. Lin. New York: Chapman & Hall.
  23. Lynch, A. S. & Lin, E. C. C. (1996b). Transcriptional control mediated by the ArcA two-component response regulator protein of Escherichia coli: characterization of DNA binding at target sites. J Bacteriol 178, 6238-6249. [Google Scholar]
  24. Menon, N. K., Robbins, J., Peck, H. D.Jr, Chatelus, C. Y., Choi, E.-S. & Przybyla, A. E. (1990). Cloning and sequencing of a putative Escherichia coli [NiFe] hydrogenase-1 operon containing six open reading frames. J Bacteriol 172, 1969-1977. [Google Scholar]
  25. Menon, N. K., Chatelus, C. Y., Dervartanian, M., Wendt, J. C., Shanmugam, K. T., Peck, H. D.Jr & Przybyla, A. E. (1994). Cloning, sequencing, and mutational analyses of the hyb operon encoding Escherichia coli hydrogenase 2. J Bacteriol 176, 4416-4423. [Google Scholar]
  26. Miller, J. H. (1972).Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  27. Rodrigue, A., Boxer, D. H., Mandrand-Berthelot, M. A. & Wu, L.-F. (1996). Requirement for nickel of the transmembrane translocation of NiFe-hydrogenase 2 in Escherichia coli. FEBS Lett 392, 81-86.[CrossRef] [Google Scholar]
  28. Rossmann, R., Sawers, G. & Böck, A. (1991). Mechanism of regulation of the formate-hydrogenlyase pathway by oxygen, nitrate and pH: definition of the formate regulon. Mol Microbiol 5, 2807-2814.[CrossRef] [Google Scholar]
  29. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989).Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  30. Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74, 5463-5467.[CrossRef] [Google Scholar]
  31. Sargent, F., Ballantine, S. P., Rugman, P. A., Palmer, T. & Boxer, D. H. (1998). Reassignment of the gene encoding the Escherichia coli hydrogenase 2 small subunit: identification of a soluble precursor of the small subunit in a hypB mutant. Eur J Biochem 255, 746-754.[CrossRef] [Google Scholar]
  32. Sauter, M., Böhm, R. & Böck, A. (1992). Mutational analysis of the operon (hyc) determining hydrogenase 3 formation in Escherichia coli. Mol Microbiol 6, 1523-1532.[CrossRef] [Google Scholar]
  33. Sawers, G. & Böck, A. (1989). Novel transcriptional control of the pyruvate formate-lyase gene: upstream regulatory sequences and multiple promoters regulate anaerobic expression. J Bacteriol 171, 2485-2498. [Google Scholar]
  34. Sawers, R. G. & Boxer, D. H. (1986). Purification and properties of membrane-bound hydrogenase isoenzyme 1 from anaerobically grown Escherichia coli. Eur J Biochem 156, 265-275.[CrossRef] [Google Scholar]
  35. 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]
  36. Sawers, R. G., Ballantine, S. P. & Boxer, D. H. (1985). Differential expression of hydrogenase isoenzymes in Escherichia coli K-12: evidence for a third isoenzyme. J Bacteriol 164, 1324-1331. [Google Scholar]
  37. Sawers, R. G., Jamieson, D. J., Higgins, C. F. & Boxer, D. H. (1986). Characterization and physiological roles of membrane-bound hydrogenase isoenzymes from Salmonella typhimurium. J Bacteriol 168, 398-404. [Google Scholar]
  38. Schlensog, V. & Böck, A. (1990). Identification and sequence analysis of the gene encoding the transcriptional activator of the formate hydrogenlyase system of Escherichia coli. Mol Microbiol 4, 1319-1327.[CrossRef] [Google Scholar]
  39. Schlensog, V., Lutz, S. & Böck, A. (1994). Purification and DNA-binding properties of FhlA, the transcriptional activator of the formate-hydrogenlyase system from Escherichia coli. J Biol Chem 269, 19590-19596. [Google Scholar]
  40. Simons, R. W., Houman, F. & Kleckner, N. (1987). Improved single and multicopy lac-based protein and operon fusion cloning tools. Gene 53, 85-96.[CrossRef] [Google Scholar]
  41. Unden, G. & Schirawski, J. (1997). The oxygen-responsive transcriptional regulator FNR of Escherichia coli: the search for signals and reactions. Mol Microbiol 25, 205-210.[CrossRef] [Google Scholar]
  42. Wu, L.-F., Mandrand-Berthelot, M.-A., Waugh, R., Edmonds, C. J., Holt, S. E. & Boxer, D. H. (1989). Nickel deficiency gives rise to the defective hydrogenase phenotype of hydC and fnr mutants in Escherichia coli. Mol Microbiol 3, 1709-1718.[CrossRef] [Google Scholar]

Data & Media loading...

Most cited this month Most Cited RSS feed

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