Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Dec;178(23):6817–6823. doi: 10.1128/jb.178.23.6817-6823.1996

Mechanism of coordinated synthesis of the antagonistic regulatory proteins NifL and NifA of Klebsiella pneumoniae.

F Govantes 1, J A Molina-López 1, E Santero 1
PMCID: PMC178581  PMID: 8955302

Abstract

The nifLA operon of Klebsiella pneumoniae codes for the two antagonistic regulatory proteins which control expression of all other nitrogen fixation genes. NifA is a transcriptional activator, and NifL inhibits NifA. The importance of a correct NifL-NifA stoichiometry for efficient regulation of nitrogen fixation genes has been investigated by constructing a strain with an altered nifL-nifA gene dosage ratio, resulting from the integration of an extra copy of nifA. Results showed that a balanced synthesis of both gene products is essential for correct regulation. Effects of mutations provoking translation termination of nifL upstream or downstream of its natural stop codon, combined with overproduction of both proteins when the genes are transcribed and translated from signals of the phi10 gene of the phage T7, showed that, in addition to the previously reported transcriptional polarity, there is translational coupling between nifL and nifA. In spite of the apparently efficient ribosome binding site of nifA, its rate of independent translation is very low. This is due to a secondary structure masking the Shine-Dalgarno sequence of nifA, which could be melted by ribosomes translating nifL. Mutational analysis confirmed the functional significance of the secondary structure in preventing independent translation of nifA. Translational coupling between the two cistrons is proposed as an efficient mechanism to prevent production of an excess of NifA, which would affect the normal regulation of nitrogen fixation genes.

Full Text

The Full Text of this article is available as a PDF (341.6 KB).

Selected References

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

  1. Adhin M. R., van Duin J. Translational regulation of the lysis gene in RNA bacteriophage fr requires a UUG initiation codon. Mol Gen Genet. 1989 Jul;218(1):137–142. doi: 10.1007/BF00330576. [DOI] [PubMed] [Google Scholar]
  2. Berg J. M. Potential metal-binding domains in nucleic acid binding proteins. Science. 1986 Apr 25;232(4749):485–487. doi: 10.1126/science.2421409. [DOI] [PubMed] [Google Scholar]
  3. Buck M., Cannon W., Woodcock J. Transcriptional activation of the Klebsiella pneumoniae nitrogenase promoter may involve DNA loop formation. Mol Microbiol. 1987 Sep;1(2):243–249. doi: 10.1111/j.1365-2958.1987.tb00518.x. [DOI] [PubMed] [Google Scholar]
  4. Buck M. Deletion analysis of the Klebsiella pneumoniae nitrogenase promoter: importance of spacing between conserved sequences around positions -12 and -24 for activation by the nifA and ntrC (glnG) products. J Bacteriol. 1986 May;166(2):545–551. doi: 10.1128/jb.166.2.545-551.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cali B. M., Micca J. L., Stewart V. Genetic regulation of nitrate assimilation in Klebsiella pneumoniae M5al. J Bacteriol. 1989 May;171(5):2666–2672. doi: 10.1128/jb.171.5.2666-2672.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Casadaban M. J., Chou J., Cohen S. N. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Csonka L. N., Ikeda T. P., Fletcher S. A., Kustu S. The accumulation of glutamate is necessary for optimal growth of Salmonella typhimurium in media of high osmolality but not induction of the proU operon. J Bacteriol. 1994 Oct;176(20):6324–6333. doi: 10.1128/jb.176.20.6324-6333.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eydmann T., Söderbäck E., Jones T., Hill S., Austin S., Dixon R. Transcriptional activation of the nitrogenase promoter in vitro: adenosine nucleotides are required for inhibition of NIFA activity by NIFL. J Bacteriol. 1995 Mar;177(5):1186–1195. doi: 10.1128/jb.177.5.1186-1195.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Govantes F., Santero E. Transcription termination within the regulatory nifLA operon of Klebsiella pneumoniae. Mol Gen Genet. 1996 Mar 7;250(4):447–454. [PubMed] [Google Scholar]
  10. Hellmuth K., Rex G., Surin B., Zinck R., McCarthy J. E. Translational coupling varying in efficiency between different pairs of genes in the central region of the atp operon of Escherichia coli. Mol Microbiol. 1991 Apr;5(4):813–824. doi: 10.1111/j.1365-2958.1991.tb00754.x. [DOI] [PubMed] [Google Scholar]
  11. Hill S., Austin S., Eydmann T., Jones T., Dixon R. Azotobacter vinelandii NIFL is a flavoprotein that modulates transcriptional activation of nitrogen-fixation genes via a redox-sensitive switch. Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):2143–2148. doi: 10.1073/pnas.93.5.2143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hill S., Kennedy C., Kavanagh E., Goldberg R. B., Hanau R. Nitrogen fixation gene (nifL) involved in oxygen regulation of nitrogenase synthesis in K. pneumoniae. Nature. 1981 Apr 2;290(5805):424–426. doi: 10.1038/290424a0. [DOI] [PubMed] [Google Scholar]
  13. Kleiner D., Paul W., Merrick M. J. Construction of multicopy expression vectors for regulated over-production of proteins in Klebsiella pneumoniae and other enteric bacteria. J Gen Microbiol. 1988 Jul;134(7):1779–1784. doi: 10.1099/00221287-134-7-1779. [DOI] [PubMed] [Google Scholar]
  14. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Lee H. S., Narberhaus F., Kustu S. In vitro activity of NifL, a signal transduction protein for biological nitrogen fixation. J Bacteriol. 1993 Dec;175(23):7683–7688. doi: 10.1128/jb.175.23.7683-7688.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. MacNeil T., MacNeil D., Roberts G. P., Supiano M. A., Brill W. J. Fine-structure mapping and complementation analysis of nif (nitrogen fixation) genes in Klebsiella pneumoniae. J Bacteriol. 1978 Oct;136(1):253–266. doi: 10.1128/jb.136.1.253-266.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Macián F., Pérez-Roger I., Armengod M. E. An improved vector system for constructing transcriptional lacZ fusions: analysis of regulation of the dnaA, dnaN, recF and gyrB genes of Escherichia coli. Gene. 1994 Jul 22;145(1):17–24. doi: 10.1016/0378-1119(94)90317-4. [DOI] [PubMed] [Google Scholar]
  19. McCarthy J. E., Gualerzi C. Translational control of prokaryotic gene expression. Trends Genet. 1990 Mar;6(3):78–85. doi: 10.1016/0168-9525(90)90098-q. [DOI] [PubMed] [Google Scholar]
  20. Merrick M. J., Edwards R. A. Nitrogen control in bacteria. Microbiol Rev. 1995 Dec;59(4):604–622. doi: 10.1128/mr.59.4.604-622.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Miller V. L., Mekalanos J. J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. doi: 10.1128/jb.170.6.2575-2583.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Molina-López J. A., Govantes F., Santero E. Geometry of the process of transcription activation at the sigma 54-dependent nifH promoter of Klebsiella pneumoniae. J Biol Chem. 1994 Oct 14;269(41):25419–25425. [PubMed] [Google Scholar]
  23. Morett E., Buck M. In vivo studies on the interaction of RNA polymerase-sigma 54 with the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters. The role of NifA in the formation of an open promoter complex. J Mol Biol. 1989 Nov 5;210(1):65–77. doi: 10.1016/0022-2836(89)90291-x. [DOI] [PubMed] [Google Scholar]
  24. Morett E., Buck M. NifA-dependent in vivo protection demonstrates that the upstream activator sequence of nif promoters is a protein binding site. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9401–9405. doi: 10.1073/pnas.85.24.9401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Morett E., Kreutzer R., Cannon W., Buck M. The influence of the Klebsiella pneumoniae regulatory gene nifL upon the transcriptional activator protein NifA. Mol Microbiol. 1990 Aug;4(8):1253–1258. doi: 10.1111/j.1365-2958.1990.tb00704.x. [DOI] [PubMed] [Google Scholar]
  26. Narberhaus F., Lee H. S., Schmitz R. A., He L., Kustu S. The C-terminal domain of NifL is sufficient to inhibit NifA activity. J Bacteriol. 1995 Sep;177(17):5078–5087. doi: 10.1128/jb.177.17.5078-5087.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ringquist S., Shinedling S., Barrick D., Green L., Binkley J., Stormo G. D., Gold L. Translation initiation in Escherichia coli: sequences within the ribosome-binding site. Mol Microbiol. 1992 May;6(9):1219–1229. doi: 10.1111/j.1365-2958.1992.tb01561.x. [DOI] [PubMed] [Google Scholar]
  28. Santero E., Hoover T. R., North A. K., Berger D. K., Porter S. C., Kustu S. Role of integration host factor in stimulating transcription from the sigma 54-dependent nifH promoter. J Mol Biol. 1992 Oct 5;227(3):602–620. doi: 10.1016/0022-2836(92)90211-2. [DOI] [PubMed] [Google Scholar]
  29. Schmidt B. F., Berkhout B., Overbeek G. P., van Strien A., van Duin J. Determination of the RNA secondary structure that regulates lysis gene expression in bacteriophage MS2. J Mol Biol. 1987 Jun 5;195(3):505–516. doi: 10.1016/0022-2836(87)90179-3. [DOI] [PubMed] [Google Scholar]
  30. Schmitz R. A., He L., Kustu S. Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae. J Bacteriol. 1996 Aug;178(15):4679–4687. doi: 10.1128/jb.178.15.4679-4687.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Silberstein Z., Cohen A. Synthesis of linear multimers of OriC and pBR322 derivatives in Escherichia coli K-12: role of recombination and replication functions. J Bacteriol. 1987 Jul;169(7):3131–3137. doi: 10.1128/jb.169.7.3131-3137.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  33. Valentin-Hansen P., Albrechtsen B., Løve Larsen J. E. DNA-protein recognition: demonstration of three genetically separated operator elements that are required for repression of the Escherichia coli deoCABD promoters by the DeoR repressor. EMBO J. 1986 Aug;5(8):2015–2021. doi: 10.1002/j.1460-2075.1986.tb04458.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES