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. 1987 Oct 26;15(20):8479–8499. doi: 10.1093/nar/15.20.8479

The symbiotic nitrogen fixation regulatory operon (fixRnifA) of Bradyrhizobium japonicum is expressed aerobically and is subject to a novel, nifA-independent type of activation.

B Thöny 1, H M Fischer 1, D Anthamatten 1, T Bruderer 1, H Hennecke 1
PMCID: PMC306372  PMID: 3313281

Abstract

The Bradyrhizobium japonicum N2 fixation regulatory gene, nifA, was sequenced and its transcription start site determined. Between the start of transcription and the nifA gene an open reading frame of 278 codons was found and named fixR. A deletion in fixR which allowed transcription into nifA resulted in a 50% reduced Fix activity. The fixRnifA operon was expressed in soybean root nodules, in cultures grown anaerobically with nitrate as terminal electron acceptor, in microaerobic cultures, and in aerobic cultures. The transcription start site (+1) was preceded by a characteristic nif(-24/-12)-type promoter consensus sequence. Double base-pair exchanges in the -12 but not in the -24 region resulted in a 'promoter-down' phenotype. A promoter-upstream DNA region between -50 and -148 was essential for maximal promoter activity. Expression from the promoter was not dependent on nifA. We conclude that the fixRnifA promoter is positively controlled, and that it requires a newly postulated transcriptional factor in order to become activated.

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Selected References

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  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. Alvarez-Morales A., Betancourt-Alvarez M., Kaluza K., Hennecke H. Activation of the Bradyrhizobium japonicum nifH and nifDK operons is dependent on promoter-upstream DNA sequences. Nucleic Acids Res. 1986 May 27;14(10):4207–4227. doi: 10.1093/nar/14.10.4207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alvarez-Morales A., Hennecke H. Expression of Rhizobium japonicum nifH and nifDK operons can be activated by the Klebsiella pneumonia nifA protein but not by the product of ntrC. Mol Gen Genet. 1985;199(2):306–314. doi: 10.1007/BF00330273. [DOI] [PubMed] [Google Scholar]
  4. 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]
  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. Buikema W. J., Szeto W. W., Lemley P. V., Orme-Johnson W. H., Ausubel F. M. Nitrogen fixation specific regulatory genes of Klebsiella pneumoniae and Rhizobium meliloti share homology with the general nitrogen regulatory gene ntrC of K. pneumoniae. Nucleic Acids Res. 1985 Jun 25;13(12):4539–4555. doi: 10.1093/nar/13.12.4539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Burke J. F. High-sensitivity S1 mapping with single-stranded [32P]DNA probes synthesized from bacteriophage M13mp templates. Gene. 1984 Oct;30(1-3):63–68. doi: 10.1016/0378-1119(84)90105-7. [DOI] [PubMed] [Google Scholar]
  8. Casadaban M. J., Martinez-Arias A., Shapira S. K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in escherichia coli and yeast. Methods Enzymol. 1983;100:293–308. doi: 10.1016/0076-6879(83)00063-4. [DOI] [PubMed] [Google Scholar]
  9. Covarrubias L., Bolivar F. Construction and characterization of new cloning vehicles. VI. Plasmid pBR329, a new derivative of pBR328 lacking the 482-base-pair inverted duplication. Gene. 1982 Jan;17(1):79–89. doi: 10.1016/0378-1119(82)90103-2. [DOI] [PubMed] [Google Scholar]
  10. Daniel R. M., Appleby C. A. Anaerobic-nitrate, symbiotic and aerobic growth of Rhizobium japonicum: effects on cytochrome P 450 , other haemoproteins, nitrate and nitrite reductases. Biochim Biophys Acta. 1972 Sep 20;275(3):347–354. doi: 10.1016/0005-2728(72)90215-0. [DOI] [PubMed] [Google Scholar]
  11. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dixon R. A. The genetic complexity of nitrogen fixation. The ninth Fleming lecture. J Gen Microbiol. 1984 Nov;130(11):2745–2755. doi: 10.1099/00221287-130-11-2745. [DOI] [PubMed] [Google Scholar]
  13. Dixon R. Tandem promoters determine regulation of the Klebsiella pneumoniae glutamine synthetase (glnA) gene. Nucleic Acids Res. 1984 Oct 25;12(20):7811–7830. doi: 10.1093/nar/12.20.7811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Dougan G., Sherratt D. The transposon Tn1 as a probe for studying ColE1 structure and function. Mol Gen Genet. 1977 Mar 7;151(2):151–160. doi: 10.1007/BF00338689. [DOI] [PubMed] [Google Scholar]
  16. Favaloro J., Treisman R., Kamen R. Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol. 1980;65(1):718–749. doi: 10.1016/s0076-6879(80)65070-8. [DOI] [PubMed] [Google Scholar]
  17. Ferro-Luzzi Ames G., Nikaido K. Nitrogen regulation in Salmonella typhimurium. Identification of an ntrC protein-binding site and definition of a consensus binding sequence. EMBO J. 1985 Feb;4(2):539–547. doi: 10.1002/j.1460-2075.1985.tb03662.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fischer H. M., Alvarez-Morales A., Hennecke H. The pleiotropic nature of symbiotic regulatory mutants: Bradyrhizobium japonicum nifA gene is involved in control of nif gene expression and formation of determinate symbiosis. EMBO J. 1986 Jun;5(6):1165–1173. doi: 10.1002/j.1460-2075.1986.tb04342.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Inouye S., Nakazawa A., Nakazawa T. Expression of the regulatory gene xylS on the TOL plasmid is positively controlled by the xylR gene product. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5182–5186. doi: 10.1073/pnas.84.15.5182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Johnson K., Parker M. L., Lory S. Nucleotide sequence and transcriptional initiation site of two Pseudomonas aeruginosa pilin genes. J Biol Chem. 1986 Nov 25;261(33):15703–15708. [PubMed] [Google Scholar]
  24. Kassavetis G. A., Geiduschek E. P. Bacteriophage T4 late promoters: mapping 5' ends of T4 gene 23 mRNAs. EMBO J. 1982;1(1):107–114. doi: 10.1002/j.1460-2075.1982.tb01132.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kim C. H., Helinski D. R., Ditta G. Overlapping transcription of the nifA regulatory gene in Rhizobium meliloti. Gene. 1986;50(1-3):141–148. doi: 10.1016/0378-1119(86)90319-7. [DOI] [PubMed] [Google Scholar]
  26. Kim Y. M., Ahn K. J., Beppu T., Uozumi T. Nucleotide sequence of the nifLA operon of Klebsiella oxytoca NG13 and characterization of the gene products. Mol Gen Genet. 1986 Nov;205(2):253–259. doi: 10.1007/BF00430436. [DOI] [PubMed] [Google Scholar]
  27. Kramer W., Drutsa V., Jansen H. W., Kramer B., Pflugfelder M., Fritz H. J. The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 1984 Dec 21;12(24):9441–9456. doi: 10.1093/nar/12.24.9441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Meyer T. F., Billyard E., Haas R., Storzbach S., So M. Pilus genes of Neisseria gonorrheae: chromosomal organization and DNA sequence. Proc Natl Acad Sci U S A. 1984 Oct;81(19):6110–6114. doi: 10.1073/pnas.81.19.6110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Minton N. P., Clarke L. E. Identification of the promoter of the Pseudomonas gene coding for carboxypeptidase G2. J Mol Appl Genet. 1985;3(1):26–35. [PubMed] [Google Scholar]
  32. Mizusawa S., Nishimura S., Seela F. Improvement of the dideoxy chain termination method of DNA sequencing by use of deoxy-7-deazaguanosine triphosphate in place of dGTP. Nucleic Acids Res. 1986 Feb 11;14(3):1319–1324. doi: 10.1093/nar/14.3.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
  34. Regensburger B., Hennecke H. RNA polymerase from Rhizobium japonicum. Arch Microbiol. 1983 Aug;135(2):103–109. doi: 10.1007/BF00408017. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. 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]
  38. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Szeto W. W., Nixon B. T., Ronson C. W., Ausubel F. M. Identification and characterization of the Rhizobium meliloti ntrC gene: R. meliloti has separate regulatory pathways for activation of nitrogen fixation genes in free-living and symbiotic cells. J Bacteriol. 1987 Apr;169(4):1423–1432. doi: 10.1128/jb.169.4.1423-1432.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Szeto W. W., Zimmerman J. L., Sundaresan V., Ausubel F. M. A Rhizobium meliloti symbiotic regulatory gene. Cell. 1984 Apr;36(4):1035–1043. doi: 10.1016/0092-8674(84)90053-9. [DOI] [PubMed] [Google Scholar]

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