Molecular and General Genetics MGG

, Volume 213, Issue 2–3, pp 499–504 | Cite as

Transcription analysis of the three nifH genes of Rhizobium phaseoli with gene fusions

  • Enrique Morett
  • Soledad Moreno
  • Guadalupe Espin
Article

Summary

Rhizobium phaseoli CFN42 possesses three regions containing genes coding for the nitrogenase polypeptides: region a and region b contain copies of the three nitrogenase structural genes nifH, nifD and nifK and region c contains only a nifH gene. Gene fusions in which the Escherichia coli lacZ gene is under the control of each of the three nifH promoters were constructed. These fusions have been used to study expression of these genes. All mutant strains carrying any of the three nifH::lacZ fusions as well as double mutants in which only regions a or b remain functional showed β-galactosidase and acetylene reduction activites, indicating that the three nifH genes were transcribed and that transcription occurred simultaneously. Compared with the wild type, mutant strains carrying nifHa ::lacZ or nifHb::lacZ showed a reduced ability to support plant growth in the absence of added nitrogen, suggesting a gene dosage effect of the reiterated nifH genes. We also report a genetic rearrangement that occurs in strains with the nifHc::lacZ fusion that generates a new nifHDK operon.

Key words

Rhizobium phaseoli nifH genes lacZ fusions DNA rearrangements 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Beynon J, Cannon M, Buchanan-Wollaston AV, Cannon F (1983) The nif promoters of Klebsiella pneumoniae have a characteristic primary structure. Cell 34:665–675Google Scholar
  2. Bishop PE, Jarlenski DML, Hetherington DR (1980) Evidence for an alternative nitrogen fixation system in Azotobacter vinelandii. Proc Natl Acad Sci USA 77:7342–7346Google Scholar
  3. Buck M, Miller S, Drummond D, Dixon R (1986) Upstream activator sequences are present in the promoters of nitrogen fixation genes. Nature 320:374–378Google Scholar
  4. Chem NA, Chen JS, Johnson JL (1985) Nitrogenase structural genes and multiple nifH-like sequences in Clostridium pasteurianum W5. In: Evans HJ, Bottomley RP, Newton N (eds) Nitrogen fixation research progress. Martinus Nijhoff, Dordrecht, p 512Google Scholar
  5. Figurski OH, Helinski DR (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA 76:1648–1652Google Scholar
  6. Inouye S, Ike Y, Inouye M (1983) Tandem repeat of the genes for protein S, a development-specific protein of Myxococcus xanthus. J Biol Chem 258:38–40Google Scholar
  7. Jacobson MR, Premakumar R, Bishop PE (1986) Transcriptional regulation of nitrogen fixation by molybdenum in Azotobacter vinelandii. J Bacteriol 167:480–486Google Scholar
  8. Jones R, Woodley P, Bobson R (1984) Cloning and organization of some genes for nitrogen fixation from Azotobacter chroococcum and their expression in Klebsiella pneumoniae. Mol Gen Genet 197:318–327Google Scholar
  9. Kallas T, Rebiere MC, Rippka R, Tandeau de Marsac N (1983) The structural nif genes of the cyanobacteria Gloeothece sp and Calothrix sp. share homology with those of Anabaena sp. but the Gloeothece genes have a different arrangement. J Bacteriol 155:427–431Google Scholar
  10. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  11. Martinez E, Pardo MA, Palacios R, Cevallos MA (1985) Reiteration of nitrogen fixation gene sequences and specificity of Rhizobium in nodulation and nitrogen fixation in Phaseolus vulgaris. J Gen Microbiol 131:1779–1786Google Scholar
  12. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  13. Morrison NA, Hau CY, Trinick MJ, Shine J, Rolfe BG (1983) Heat curingof a sym plasmid in fast-growing Rhizobium sp. that is able to nodulate legumes and the non legume Parasponia sp. J Bacteriol 153:527–531Google Scholar
  14. Newcomb W (1981) Nodule morphogenesis and differentiation. In: Gilest KL, Atherly AG (eds) Biology of the Rhizobiaceae. Academic Press, New York, pp 247–298Google Scholar
  15. Noel KD, Sanchez A, Fernandez L, Leemans J, Cevallos MA (1984) Rhizobium phaseoli symbiotic mutants with transposon Tn5 insertions. J Bacteriol 158:148–155Google Scholar
  16. Norel F, Elmerich C (1987) Nucleotide sequence and functional analysis of the two nifH copies of Rhizobium ORS571. J Gen Microbiol 133:1563–1576Google Scholar
  17. 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
  18. Ow DW, Sundaresan V, Rothstein DM, Brown SE, Ausubel FM (1983) Promoters regulated by glnG (ntrC) and nifA gene products share an heptameric consensus sequence in the-15 region. Proc Natl Acad Sci USA 80:2524–2528Google Scholar
  19. Prakash RK, Atherly AF (1984) Reiteration of genes involved in symbiotic nitrogen fixation by fast-growing Rhizobium japonicum. J Bacteriol 160:785–787Google Scholar
  20. Quinto C, de la Vega H, Flores M, Fernández L, Ballado T, Soberón G, Palacios R (1982) Reiteration of nitrogen fixation gene sequences in Rhizobium phaseoli. Nature 299:724–726Google Scholar
  21. Quinto C, de la Vega H, Flores M, Leemans J, Cevallos MA, Pardo MA, Azpiroz R, Girard ML, Calva E, Palacios R (1985) Nitrogenase reductase: A functional multigene family in Rhizobium phaseoli. Proc Natl Acad Sci USA 82:1170–1174Google Scholar
  22. Rice P, Mazur BJ, Haselkorn R (1982) Isolation and physical mapping of nitrogen fixation genes from the cyanobacterium Anabaena 7120. J Biol Chem 257:13157–13162Google Scholar
  23. Riley M, Anilionis A (1978) evolution of the bacterial genome. Annu Rev Microbiol 32:519–560Google Scholar
  24. Robson R, Woodley P, Jones R (1986) Second gene (nifH*) coding for a nitrogenase iron protein in Azotobacter chroococcum is adjacent to a gene coding for a ferredoxin-like protein. EMBO J 5:1159–1163Google Scholar
  25. Scolnik PA, Haselkorn R (1984) Activation of extra copies of genes coding for nitrogenase in Rhodopseudomonas capsulata. Nature 307:289–292Google Scholar
  26. Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria. Bio Technology 1:784–791Google Scholar
  27. Soberón-Chávez G, Nájera R, Olivera H, Segovia L (1986) Genetic rearrangement of a Rhizobium phaseoli symbiotic plasmid. J Bacteriol 167:487–491Google Scholar
  28. Sundaresan V, Jones JDG, Ow DW, Ausubel FM (1983) Conservation of nitrogenase promoters from Rhizobium meliloti and Klebsiella pneumoniae. Nature 301:728–732Google Scholar
  29. Vincent JM (1980) Factors controlling the legume Rhizobium symbosis. In: Newton WE, Orme-Johnson WH (eds) Nitrogen fixation 2. University Park Press, Baltimore, pp 103–129Google Scholar
  30. Wacek T, Brill WJ (1976) Simple, rapid assay for screening nitrogen fixing ability in soybean. Crop Sci 16:519–522Google Scholar

Copyright information

© Springer-Verlag 1988

Authors and Affiliations

  • Enrique Morett
    • 1
  • Soledad Moreno
    • 1
  • Guadalupe Espin
    • 1
  1. 1.Depto. de Biologia Molecular de Plantas, Centro de Investigación sobre Fijación de NitrógenoUniversidad Nacional Autónoma de MéxicoCuernavacaMorelos México

Personalised recommendations