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. 1984 Jan;157(1):143–147. doi: 10.1128/jb.157.1.143-147.1984

Mutations affecting regulation of the Klebsiella pneumoniae nifH (nitrogenase reductase) promotor.

S E Brown, F M Ausubel
PMCID: PMC215143  PMID: 6360993

Abstract

Starting with plasmid pSA30 which contains an intact nifHDKY operon, we selected mutants that no longer inhibited nitrogen fixation in Klebsiella pneumoniae. Three categories of mutants were found among eight mutant plasmids examined in detail. Three mutant plasmids carried a single-base-pair (bp) change at position -12 or -14 relative to the nifH transcription start site. These mutations were located in a previously described consensus sequence found in the -10 to -15 region of nif promoters. Four of the mutant plasmids contain lesions considerably upstream from the start point of transcription. Two of these upstream mutations are identical 112-bp deletions of nucleotides -72 to -184, and two others are a single-bp change at position -136. The final plasmid does not contain a mutation within a 525-bp region which includes 289 bp upstream from the nifH ATG initiation codon and which extends 69 codons into the nifH gene. All eight of the mutant pSA30 plasmids failed to complement a chromosomal nifH mutation, suggesting that the mutations that block inhibition of nif expression also prevent transcription of the nifHDKY operon.

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Selected References

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

  1. Beynon J., Cannon M., Buchanan-Wollaston V., Cannon F. The nif promoters of Klebsiella pneumoniae have a characteristic primary structure. Cell. 1983 Sep;34(2):665–671. doi: 10.1016/0092-8674(83)90399-9. [DOI] [PubMed] [Google Scholar]
  2. Bitoun R., Berman J., Zilberstein A., Holland D., Cohen J. B., Givol D., Zamir A. Promoter mutations that allow nifA-independent expression of the nitrogen fixation nifHDKY operon. Proc Natl Acad Sci U S A. 1983 Oct;80(19):5812–5816. doi: 10.1073/pnas.80.19.5812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  4. Buchanan-Wollaston V., Cannon M. C., Beynon J. L., Cannon F. C. Role of the nifA gene product in the regulation of nif expression in Klebsiella pneumoniae. Nature. 1981 Dec 24;294(5843):776–778. doi: 10.1038/294776a0. [DOI] [PubMed] [Google Scholar]
  5. Buchanan-Wollaston V., Cannon M. C., Cannon F. C. The use of cloned nif (nitrogen fixation) DNA to investigate transcriptional regulation of nif expression in Klebsiella pneumoniae. Mol Gen Genet. 1981;184(1):102–106. doi: 10.1007/BF00271203. [DOI] [PubMed] [Google Scholar]
  6. Cannon F. C., Riedel G. E., Ausubel F. M. Overlapping sequences of Klebsiella pneumoniae nifDNA cloned and characterized. Mol Gen Genet. 1979 Jul 2;174(1):59–66. doi: 10.1007/BF00433306. [DOI] [PubMed] [Google Scholar]
  7. Cannon F. C., Riedel G. E., Ausubel F. M. Recombinant plasmid that carries part of the nitrogen fixation (nif) gene cluster of Klebsiella pneumoniae. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2963–2967. doi: 10.1073/pnas.74.7.2963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clewell D. B. Nature of Col E 1 plasmid replication in Escherichia coli in the presence of the chloramphenicol. J Bacteriol. 1972 May;110(2):667–676. doi: 10.1128/jb.110.2.667-676.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dixon R., Eady R. R., Espin G., Hill S., Iaccarino M., Kahn D., Merrick M. Analysis of regulation of Klebsiella pneumoniae nitrogen fixation (nif) gene cluster with gene fusions. Nature. 1980 Jul 10;286(5769):128–132. doi: 10.1038/286128a0. [DOI] [PubMed] [Google Scholar]
  11. Drummond M., Clements J., Merrick M., Dixon R. Positive control and autogenous regulation of the nifLA promoter in Klebsiella pneumoniae. Nature. 1983 Jan 27;301(5898):302–307. doi: 10.1038/301302a0. [DOI] [PubMed] [Google Scholar]
  12. Espin G., Alvarez-Morales A., Cannon F., Dixon R., Merrick M. Cloning of the glnA, ntrB and ntrC genes of Klebsiella pneumoniae and studies of their role in regulation of the nitrogen fixation (nif) gene cluster. Mol Gen Genet. 1982;186(4):518–524. doi: 10.1007/BF00337959. [DOI] [PubMed] [Google Scholar]
  13. Espin G., Alvarez-Morales A., Merrick M. Complementation analysis of glnA-linked mutations which affect nitrogen fixation in Klebsiella pneumoniae. Mol Gen Genet. 1981;184(2):213–217. doi: 10.1007/BF00272907. [DOI] [PubMed] [Google Scholar]
  14. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Higgins C. F., Ames G. F. Regulatory regions of two transport operons under nitrogen control: nucleotide sequences. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1083–1087. doi: 10.1073/pnas.79.4.1083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  18. Kennedy C., Robson R. L. Activation of nif gene expression in Azotobacter by the nifA gene product of Klebsiella pneumoniae. Nature. 1983 Feb 17;301(5901):626–628. doi: 10.1038/301626a0. [DOI] [PubMed] [Google Scholar]
  19. Leonardo J. M., Goldberg R. B. Regulation of nitrogen metabolism in glutamine auxotrophs of Klebsiella pneumoniae. J Bacteriol. 1980 Apr;142(1):99–110. doi: 10.1128/jb.142.1.99-110.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Magasanik B. Genetic control of nitrogen assimilation in bacteria. Annu Rev Genet. 1982;16:135–168. doi: 10.1146/annurev.ge.16.120182.001031. [DOI] [PubMed] [Google Scholar]
  21. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Merrick M. J. Nitrogen control of the nif regulon in Klebsiella pneumoniae: involvement of the ntrA gene and analogies between ntrC and nifA. EMBO J. 1983;2(1):39–44. doi: 10.1002/j.1460-2075.1983.tb01377.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ow D. W., Ausubel F. M. Conditionally replicating plasmid vectors that can integrate into the Klebsiella pneumoniae chromosome via bacteriophage P4 site-specific recombination. J Bacteriol. 1983 Aug;155(2):704–713. doi: 10.1128/jb.155.2.704-713.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ow D. W., Ausubel F. M. Regulation of nitrogen metabolism genes by nifA gene product in Klebsiella pneumoniae. Nature. 1983 Jan 27;301(5898):307–313. doi: 10.1038/301307a0. [DOI] [PubMed] [Google Scholar]
  26. Ow D. W., Sundaresan V., Rothstein D. M., Brown S. E., Ausubel F. M. Promoters regulated by the glnG (ntrC) and nifA gene products share a heptameric consensus sequence in the -15 region. Proc Natl Acad Sci U S A. 1983 May;80(9):2524–2528. doi: 10.1073/pnas.80.9.2524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Riedel G. E., Ausubel F. M., Cannon F. C. Physical map of chromosomal nitrogen fixation (nif) genes of Klebsiella pneumoniae. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2866–2870. doi: 10.1073/pnas.76.6.2866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Riedel G. E., Brown S. E., Ausubel F. M. Nitrogen fixation by Klebsiella pneumoniae is inhibited by certain multicopy hybrid nif plasmids. J Bacteriol. 1983 Jan;153(1):45–56. doi: 10.1128/jb.153.1.45-56.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Roberts G. P., Brill W. J. Genetics and regulation of nitrogen fixation. Annu Rev Microbiol. 1981;35:207–235. doi: 10.1146/annurev.mi.35.100181.001231. [DOI] [PubMed] [Google Scholar]
  30. Roberts G. P., MacNeil T., MacNeil D., Brill W. J. Regulation and characterization of protein products coded by the nif (nitrogen fixation) genes of Klebsiella pneumoniae. J Bacteriol. 1978 Oct;136(1):267–279. doi: 10.1128/jb.136.1.267-279.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sundaresan V., Jones J. D., Ow D. W., Ausubel F. M. Klebsiella pneumoniae nifA product activates the Rhizobium meliloti nitrogenase promoter. Nature. 1983 Feb 24;301(5902):728–732. doi: 10.1038/301728a0. [DOI] [PubMed] [Google Scholar]
  32. Sundaresan V., Ow D. W., Ausubel F. M. Activation of Klebsiella pneumoniae and Rhizobium meliloti nitrogenase promoters by gln (ntr) regulatory proteins. Proc Natl Acad Sci U S A. 1983 Jul;80(13):4030–4034. doi: 10.1073/pnas.80.13.4030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. de Bruijn F. J., Ausubel F. M. The cloning and transposon Tn5 mutagenesis of the glnA region of Klebsiella pneumoniae: identification of glnR, a gene involved in the regulation of the nif and hut operons. Mol Gen Genet. 1981;183(2):289–297. doi: 10.1007/BF00270631. [DOI] [PubMed] [Google Scholar]

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