Molecular and General Genetics MGG

, Volume 206, Issue 2, pp 207–219 | Cite as

Cloning and characterization of nifA and ntrC genes of the stem nodulating bacterium ORS571, the nitrogen fixing symbiont of Sesbania rostrata: Regulation of nitrogen fixation (nif) genes in the free living versus symbiotic state

  • K. Pawlowski
  • P. Ratet
  • J. Schell
  • F. J. de Bruijn
Article

Summary

A cosmid bank of ORS571, a diazotrophic bacterium capable of inducing aerial stem and root nodules on Sesbania rostrata, was constructed in the vector pLAFR1. A DNA probe carrying the Klebsiella pneumoniae nifA gene was used to identify nifA-and ntrC-like regions of ORS571 in the cosmid bank by colony hybridization. Cosmids carrying these regions were mapped by restriction endonuclease analysis, Southern blotting and transposon Tn5 mutagenesis. Selected Tn5 insertion mutations in the nifA/ntrC homologous regions were used for gene-replacement experiments and the resulting ORS571 mutants were examined for Nif, Fix and Ntr phenotypes. Two clearly distinct regulatory loci were thus identified and named nifA and ntrC. Plasmids carrying gene fusions of the ORS571 nifH and nifD genes to lacZ were constructed and the regulation of the ORS571 nifHDK promoter, and of the Rhizobium meliloti nifHDK promoter, was studied under varying physiological conditions in ORS571, ORS571 nifA::Tn5 and ORS571 nitrC::Tn5 strains. A model for the role of nifA and ntrC in the regulation of ORS571 nif and other nitrogen assimilation genes is proposed.

Key words

Azorhizobium sesbaniae ORS571 Nitrogen fixation Regulation Tn5 mutagenesis lacZ fusions 

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References

  1. Alvarez-Morales A, Hennecke H (1985) Expression of Rhizobium japonicum nifH and nifDK operons can be activated by the Klebsiella pneumoniae nifA protein but not by the product of ntrC. Mol Gen Genet 199:306–314Google Scholar
  2. Ausubel FM (1984) Regulation of nitrogen fixation genes. Cell 37:5–6Google Scholar
  3. Ausubel FM, Buikema WJ, Earl CD, Klingensmith JA, Nixon BT, Szeto WW (1985) Organization and regulation of Rhizobium meliloti and Parasponia Bradyrhizobium nitrogen fixation genes. In: Evans HJ, Bottomley PJ, Newton WE (eds) Nitrogen fixation research progress. Martinus Nijhoff, Dordrecht Boston Lancaster, pp 165–171Google Scholar
  4. Backman K, Chen YM, Magasanik B (1981) Physical and genetic characterization of the glnA-glnG region of the Escherichia coli chromosome. Proc Natl Acad Sci USA 78:3743–4747Google Scholar
  5. Bender RA, Janssen KA, Resnick AD, Blumenberg M, Foor F, Magasanik B (1977) Biochemical parameters of glutamine synthetase from Klebsiella aerogenes. J Bacteriol 129:1001–1009Google Scholar
  6. Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188–189Google Scholar
  7. Beringer JE (1978) Transfer of the drug-resistant transposon Tn5 to Rhizobium. Nature 276:633–634Google Scholar
  8. Beynon J, Cannon M, Buchanan-Wollaston V, Cannon F (1983) The nif promoters of Klebsiella pneumoniae have a characteristic primary structure. Cell 34:665–671Google Scholar
  9. Bolivar F, Rodrigues RL, Greene PJ, Betlach H, Heynecker HL, Boyer HW, Crossa JH, Falkow S (1977) Construction and characterisation of new cloning vehicles. A multipurpose cloning system. Gene 2:95–100Google Scholar
  10. Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in E. coli. J Mol Biol 41:459–472Google Scholar
  11. Broughton WJ, Dilworth MY (1971) Control of leghaemoglobin synthesis in snake beans. Biochem J 125:1075–1080Google Scholar
  12. Broughton WJ, John CK (1979) Rhizobia in tropical legumes. III Experimentation and supply in Malaysia 1927–1976. In: Broughton WJ, John CK, Rajarao JC, Lin B (eds) Soil microbiology and plant nutrition. University of Malaysia Press. Kuala Lumpur, Malaysia, pp 113–136Google Scholar
  13. de Bruijn FJ (1987) Tn5 mutagenesis to map genes. Methods Enzymol (in press)Google Scholar
  14. de Bruijn FJ, Ausubel fM (1983) The cloning and characterization of the glnF (ntrA) gene of Klebsiella pneumoniae: Role of glnF (ntrA) in the regulation of nitrogen fixation (nif) and other nitrogen assimilation genes. Mol Gen Genet 192:342–353Google Scholar
  15. de Bruijn FJ, Lupski JR (1984) The use of transposon Tn5 mutagenesis in the rapid generation of correlation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids — a review. Gene 27:131–149Google Scholar
  16. de Bruijn FJ, Sundaresan V, Szeto WW, Ow DW, Ausubel FM (1984) Regulation of the nitrogen fixation (nif) genes of Klebsiella pneumoniae and Rhizobium meliloti: role of nitrogen regulation (ntr) genes. In: Veeger C, Newton WE (eds) Advances in nitrogen fixation research. Nijhoff/Junk, The Hague, pp 627–633Google Scholar
  17. Buck M, Khan H, Dixon R (1985) Site-directed mutagenesis of the Klebsiella pneumoniae nifL and nifH promoters and in vivo analysis of promoter activity. Nucleic Acids Res 13:7621–7637Google Scholar
  18. Buikema WJ, Szeto WW, Lemley PV, Orme-Johnson WH, Ausubel FM (1985) 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 13:4539–4555Google Scholar
  19. Casadaban M (1975) Fusion of the E. coli lac genes to the ara promoter: a general technique using bacteriophage Mu-1 insertions. Proc Natl Acad Sci USA 72:4399–4403Google Scholar
  20. Casadaban M (1976) Transposition and fusion of the lac genes to selected promoters in E. coli using bacteriophages lambda and Mu. J Mol Biol 104:541–555Google Scholar
  21. Castilho B, Olfson P, Casadaban M (1984) Plasmid insertion mutagenesis and lac gene fusion with mini-Mu bacteriophage transposon. J Bacteriol 158:488–495Google Scholar
  22. Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156Google Scholar
  23. Chen YM, Backman K, Magasanik B (1981) Characterization of a gene, glnL, the product of which is involved in the regulation of nitrogen utilization in Escherichia coli. J Bacteriol 150:214–220Google Scholar
  24. Ditta G, Stanfield S, Corbin D, Helinski D (1980) Broad host range DNA cloning system for gram negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77:7347–7351Google Scholar
  25. Dixon R (1984a) The genetic complexity of nitrogen fixation. The ninth Fleming lecture. J Gen Microbiol 130:2745–2755Google Scholar
  26. Dixon R (1984b) Tandem promoters determine regulation of the Klebsiella pneumoniae glutamine synthetase (glnA) gene. Nucleic Acids Res 12:7811–7830Google Scholar
  27. Donald RGK, Ludwig RA (1984) Rhizobium sp. strain ORS571 ammonium assimilation and nitrogen fixation. J Bacteriol 158:1144–1151Google Scholar
  28. Donald RGK, Nees, DW, Raymond CK, Loroch AL, Ludwig RA (1986) Characterization of three genomic loci encoding Rhizobium sp. strain ORS571 N2 fixation genes. J Bacteriol 165:72–81Google Scholar
  29. Dreyfus BL, Dommergues YR (1981) Nitrogen-fixing nodules induced by Rhizobium on the stem of the tropical legume Sesbania rostrata. FEMS Microbiol Lett 10:313–317Google Scholar
  30. Dreyfus BL, Elmerich C, Dommergues YR (1983) Free-living Rhizobium strain able to grow under N2 as a sole nitrogen source. Appl Environ Microbiol 45:711–713Google Scholar
  31. Drummond M, Whitty P, Wooton J (1986) Sequence and domain relationships of ntrC and nifA from Klebsiella pneumoniae: homologies to other regulatory proteins. EMBO J 5:441–447Google Scholar
  32. Elmerich C, Dreyfus B, Reysset G, Aubert JP (1982) Genetic analysis of nitrogen fixation in a tropical fast-growing Rhizobium. EMBO J 1:499–503Google Scholar
  33. Fischer H-M, Alvarez-Morales A, Hennecke H (1986) The pleiotropic nature of symbiotic regulatory mutants: Bradyrhizobium japonicum nifA is involved in control of nif gene expression and formation of determinate symbiosis. EMBO J 5:1165–1173Google Scholar
  34. Friedman AM, Long SR, Brown SE, Buikema WJ, Ausubel FM (1982) Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene 18:289–296Google Scholar
  35. Fuchs RL, Keister DL (1980) Comparative properties of glutamine synthetases I and II in Rhizobium and Agrobacterium spp. J Bacteriol 144:641–648Google Scholar
  36. Hirschmann J, Wong PK, Sei K, Keener J, Kustu S (1985) Products of nitrogen regulatory genes ntrA and ntrC of enteric bacteria activate glnA transcription in vitro: evidence that the ntrA product is a sigma factor. Proc Natl Acad Sci USA 82:7525–7529Google Scholar
  37. Hunt TP, Magasanik B (1985) Transcription of glnA by purified Escherichia coli components: core RNA polymerase and the products of glnF, glnG and glnL. Proc Natl Acad Sci USA 82:8453–8357Google Scholar
  38. Ish-Horowicz D, Burke JF (1981) Rapid and efficient cosmid cloning. Nucleic Acids Res 9:2989–2998Google Scholar
  39. Jarvis BDW, Gillis M, De Ley J (1986) Intra-and intergeneric similarities between the ribosomal ribonucleic acid cistrons of Rhizobium and Bradyrhizobium species and some related bacteria. Int J Syst Bacteriol 36:129–138Google Scholar
  40. Kush A, Elmerich C, Aubert JP (1985) Nitrogenase of Sesbania Rhizobium strain ORS571: purification, properties and ‘switch off’ by ammonia. J Gen Microbiol 131:1765–1777Google Scholar
  41. Legocki RP, Yun AC, Szalay AA (1984) Expression of β-galactosidase controlled by a nitrogenase promoter in stem nodules of Aeschynomene scabra. Proc Natl Acad Sci USA 81:5806–5810Google Scholar
  42. Ludwig RA (1986) Rhizobium sp. strain ORS571 grows synergistically on N2 and Nicotinate as N sources. J Bacteriol 165:304–307Google Scholar
  43. Maas R (1983) An improved colony hybridization method with significantly increased sensitivity for detection of single genes. Plasmid 10:296–298Google Scholar
  44. Magasanik B (1982) Genetic control of nitrogen assimilation in bacteria. Annu Rev Genet 16:135–168Google Scholar
  45. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  46. Meade HM, Long SR, Ruvkun GB, Brown SE, Ausubel FM (1982) Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol 149:114–122Google Scholar
  47. Merrick M (1983) Nitrogen control of the nif regulon in Klebsiella pneumoniae: involvement of the ntrA gene and analogies between ntrC and nifA. EMBO J 2:39–44Google Scholar
  48. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  49. Norel F, Desnoues N, Elmerich C (1985) Characterization of DNA sequences homologous to Klebsiella pneumoniae nifH, D, K and E in the tropical Rhizobium ORS571. Mol Gen Genet 199:352–356Google Scholar
  50. Ow DW, Ausubel FM (1983) Regulation of nitrogen metabolism genes by the nifA gene product in Klebsiella pneumoniae. Nature 301:307–313Google Scholar
  51. Ow DW, Sundaresan V, Rothstein DM, Brown SE, Ausubel FM (1983) Promoters regulated by the glnG (ntrC) and nifA gene products share a heptameric consensus sequence in the-15 region. Proc Natl Acad Sci USA 80:2524–2528Google Scholar
  52. Ratet P and Richaud F (1986) Construction and uses of a new transposable element whose insertion is able to produce gene fusions with the neomycin-phosphotransferase-coding region of Tn903. Gene 42:185–192Google Scholar
  53. Riedel GE, Brown SE, Ausubel FM (1983) Nitrogen fixation by Klebsiella pneumoniae is inhibited by certain multicopy hybrid nif plasmids. J Bacteriol 153:45–46Google Scholar
  54. Rossen L, Ma QS, Mudd EA, Johnston AWB, Downie JA (1984) Identification and DnA sequence of fixZ, a nifB-like gene from Rhizobium leguminosarum. Nucleic Acids Res 12:7123–7134Google Scholar
  55. Ruvkun GB, Ausubel FM (1981) A general method for site-directed mutagenesis in prokaryotes. Nature 289:85–88Google Scholar
  56. Schetgens R, Hontelez J, van den Bos R, van Kammen A (1985) Identification and phenotypical characterization of a cluster of fix genes, including a nif regulatory gene, from Rhizobium leguminosarum PRE. Mol Gen Genet 200:368–374Google Scholar
  57. Scott DB, Court CB, Ronson CW, Scott KF, Watson JM, Schofield PR, Shine J (1984) Organization of nodulation and nitrogen fixation genes on a Rhizobium trifolii symbiotic plasmid. Arch Microbiol 139:151–157Google Scholar
  58. Shapira SK, Chou J, Richaud F, Casadaban MJ (1983) New versatile hybrid protein β-galactosidase gene fusion plasmid vectors. Gene 25:71–82Google Scholar
  59. Sundaresan V, Jones JDG, Ow DW, Ausubel FM (1983a) Klebsiella pneumoniae nifA product activates the Rhizobium meliloti nitrogenase promoter. Nature 301:728–731Google Scholar
  60. Sundaresan V, Ow DW, Ausubel FM (1983b) Activation of Klebsiella pneumoniae and Rhizobium meliloti nitrogenase promoters by gln (ntr) regulatory proteins. Proc Natl Acad Sci USA 80:4030–4034Google Scholar
  61. Szeto W, Zimmermann L, Sundaresan V, Ausubel FM (1984) A Rhizobium meliloti symbiotic regulatory gene. Cell 36:1035–1043Google Scholar
  62. Weber G, Reilander H, Pühler A (1985) Mapping and expression of a regulatory nitrogen fixation gene (fixD) of Rhizobium meliloti. EMBO J 4:2751–2756Google Scholar

Copyright information

© Springer-Verlag 1987

Authors and Affiliations

  • K. Pawlowski
    • 1
  • P. Ratet
    • 1
  • J. Schell
    • 1
  • F. J. de Bruijn
    • 1
  1. 1.Max-Planck-Institut für ZüchtungsforschungKöln 30Federal Republic of Germany

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