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Chinese Science Bulletin

, Volume 45, Issue 18, pp 1673–1677 | Cite as

Construction and characterization of partiallyntrC-deleted mutants inAlcaligenes faecalis

  • Hongmei Cheng
  • Min Lin
  • Shuzheng Ping
  • Shirong Jia
Notes
  • 19 Downloads

Abstract

To study the effect ofntrC gene product on the expression and regulation of other important nitrogen-fixing genes inAlcaligenes faecalis, partiallyntrC-deleted mutants ofA. faecalis have been generated. To start with, thentrC gene ofA. faecalis was cloned into a suicide plasmid pSUP202 to create a recombinant plasmid pSUM1. ThentrC gene in pSUM1 was then replaced by alacZ-Kmr fragment resulted in the generation of a plasmid pSUM2. ThelacZ fragment in pSUM2 was further removed and a plasmid pSUM3 produced. As a second step, the plasmid pSUM2 or pSUM3 was introduced into the wild type ofA. faecalis A1501 by conjugation and two partiallyntrC-deleted mutants A15CM1 (ntrC::lacZ) and A15CM2 (ntrC -) were obtained. To understand the regulatory effect of the NtrC on the expression ofnifH andnifA, anifH-lacZ gene or anifA-lacZ gene was introduced into the ntrC- mutant by conjugation. The results indicated that: (i) although the ntrC- mutant was nif+, its nitrogen fixation activity was only 20% that of the wild type; (ii) the ntrC- mutant failed to grow on the medium containing nitrate as a sole nitrogen source; (iii) the regulation ofntrC gene expression did not require its own product; (iv) the expression ofnifH inA. faecalis was positively regulated by thentrC. Deletion of thentrC resulted in the reduction ofnifH expression or even totally inactivated nitrogen fixation; (v) there was no obvious influence on the expression ofnifA inA. faecalis if thentrC gene was deleted.

Keywords

Alcaligenes faecalis ntrC-mutant expression and regulation ofnif genes nitrogen fixation 

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References

  1. 1.
    Kustu, S., Santera, E., Keener, J. et al., Expression of σ54 (ntrA)-dependent genes is probably united by a common mechanism, Microbiol. Rev., 1989, 53:367.Google Scholar
  2. 2.
    Liang, Y. Y., Arsene, F., Eimerich, C., Characterization of thentrBC genes ofAzzospirillum brasilense Sp7: their involvement in the regulation of nitrogenade synthesis and activity, Mol. Gen. Genet., 1993, 240: 188.CrossRefGoogle Scholar
  3. 3.
    Hai, W. L., You, C. B., Construction ofntrC-nifA gene transconjugant ofAlcaligenes faecalis, in Agricultural Biotechnology, Beijing: China Science and Technology Press, 1992, 758.Google Scholar
  4. 4.
    Cheng, H. M., Lin, M., Ping, S. Z. et al., The construction ofAlcaligenes faecalis ntrC-lacZ fusion gene and its expression during association with rice roots, Acta Microbiologica Sinica (in Chinese), 1998, 38(3): 168.Google Scholar
  5. 5.
    Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory Press, 1989, 16.Google Scholar
  6. 6.
    Fannche, C., Elmerich, C., Moblization of the chromosome ofAzosirllum brasilense by plasmid R68-45, FEMs Microbiol. Lett., 1981, 10: 199.CrossRefGoogle Scholar
  7. 7.
    Lin, M., You, C. B., Denitrification and nitrogen fixation byAlcaligenes faecalis, Acta Agriculturae Nucleatae (in Chinese), 1987, 1: 1.Google Scholar
  8. 8.
    Miller, J. H., Assay of β-galactosidase, in Experiment in Molecular Genetics, New York: Cold Spring Harbor Laboratory Press, 1972, 352.Google Scholar

Copyright information

© Science in China Press 2000

Authors and Affiliations

  • Hongmei Cheng
    • 1
    • 2
  • Min Lin
    • 1
  • Shuzheng Ping
    • 1
  • Shirong Jia
    • 2
  1. 1.Institute of Application of Atomic EnergyChinese Academy of Agricultural SciencesBeijingChina
  2. 2.Biotechnology Research CenterChinese Academy of Agricultural SciencesBeijingChina

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