1887

Abstract

Previous studies identified five -like genes ( through ) in (strain W5), where the gene encodes the nitrogenase iron protein. Transcripts of these genes, with the exception of , were detected in molybdenum-sufficient nitrogen-fixing cells. However, the size of the transcripts, the level of transcription and the presence of polypeptides encoded by the -like genes were not reported. The and genes were extremely similar, as they seemed to differ by only two bases in a span of 2481 bp, one in the coding region and another in the upstream region. Re-examination of the DNA sequences revealed that the coding region of and was identical, whereas the difference in the upstream region was confirmed. Results from the authors' ongoing study of the genes of single-colony isolates of suggest that the designation should be eliminated. Here the size of mRNA from and the detection of the NifH2 polypeptide in nitrogen-fixing cells of are reported. Northern blot analysis of periodically collected nitrogen-fixing cells showed that the and mRNAs were present throughout growth. Addition of ammonium acetate repressed the transcription of both these genes similarly. Using an antiserum raised against NifH of , two NifH-related bands were detected by Western blot analysis after electrophoretic separation of proteins in extracts of nitrogen-fixing cells. After separation of proteins by preparative SDS-PAGE, the NifH polypeptides were characterized by MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) and by ES-MS/MS (electrospray tandem mass spectrometry) analyses. The results confirmed the presence of NifH2, in addition to NifH1, in nitrogen-fixing cells.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27931-0
2005-07-01
2019-10-22
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/7/mic1512353.html?itemId=/content/journal/micro/10.1099/mic.0.27931-0&mimeType=html&fmt=ahah

References

  1. Bishop, P. E. & Premakumar, R. ( 1992; ). Alternative nitrogen fixation systems. In Biological Nitrogen Fixation, pp. 736–762. Edited by G. Stacey, R. H. Burris & H. J. Evans. New York: Chapman & Hall.
  2. Bradford, M. M. ( 1976; ). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.[CrossRef]
    [Google Scholar]
  3. Burris, R. H. ( 1988; ). 100 years of discoveries in biological N2 fixation. In Nitrogen Fixation: Hundred Years After, pp. 2121–2130. Edited by H. Bothe, F. J. de Bruijn & W. E. Newton. Stuttgart: Gustav Fischer.
  4. Carnahan, J. E., Mortenson, L. E., Mower, H. F. & Castle, J. E. ( 1960; ). Nitrogen fixation in cell-free extracts of Clostridium pasteurianum. Biochim Biophys Acta 44, 520–535.[CrossRef]
    [Google Scholar]
  5. Chen, J.-S. ( 2004; ). Nitrogen fixation in the clostridia. In Genetics and Regulation of Nitrogen Fixation in Free-Living Bacteria, pp. 53–64. Edited by W. Klipp, B. Masepohl, J. R. Gallon & W. E. Newton. Dordrecht: Kluwer.
  6. Chen, J.-S. & Johnson, J. L. ( 1993; ). Molecular biology of nitrogen fixation in the clostridia. In The Clostridia and Biotechnology, pp. 371–392. Edited by D. R. Woods. Boston, MA: Butterworth-Heinemann.
  7. Chen, K. C.-K., Chen, J.-S. & Johnson, J. L. ( 1986; ). Structural features of multiple nifH-like sequences and very biased codon usage in nitrogenase genes of Clostridium pasteurianum. J Bacteriol 166, 162–172.
    [Google Scholar]
  8. Chen, J.-S., Wang, S.-Z. & Johnson, J. L. ( 1990; ). Nitrogen fixation genes of Clostridium pasteurianum. In Nitrogen Fixation: Achievements and Objectives, pp. 483–490. Edited by P. M. Gresshoff, L. E. Roth, G. Stacey & W. E. Newton. New York: Chapman & Hall.
  9. Chen, J.-S., Toth, J. & Kasap, M. ( 2001; ). Nitrogen-fixation genes and nitrogenase activity in Clostridium acetobutylicum and Clostridium beijerinckii. J Ind Microbiol Biotechnol 27, 281–286.[CrossRef]
    [Google Scholar]
  10. Choo, Q.-C., Samian, M.-R. & Najimudin, N. ( 2003; ). Phylogeny and characterization of three nifH-homologous genes from Paenibacillus azotofixans. Appl Environ Microbiol 69, 3658–3662.[CrossRef]
    [Google Scholar]
  11. Clarke, T. A., Maritano, S. & Eady, R. R. ( 2000; ). Formation of a tight 1 : 1 complex of Clostridium pasteurianum Fe protein-Azotobacter vinelandii MeFe protein: evidence for long-range interactions between the Fe protein binding sites during catalytic hydrogen evolution. Biochemistry 39, 11434–11440.[CrossRef]
    [Google Scholar]
  12. Daesch, G. & Mortenson, L. E. ( 1972; ). Effect of ammonia on the synthesis and function of the N2-fixing enzyme system in Clostridium pasteurianum. J Bacteriol 110, 103–109.
    [Google Scholar]
  13. Emerich, D. W. & Burris, R. H. ( 1978; ). Complementary functioning of the component proteins of nitrogenase from several bacteria. J Bacteriol 134, 936–943.
    [Google Scholar]
  14. Emerich, D. W., Hageman, R. V. & Burris, R. H. ( 1981; ). Interactions of dinitrogenase and dinitrogenase reductase. Adv Enzymol 52, 1–22.
    [Google Scholar]
  15. George, H. A. & Chen, J.-S. ( 1983; ). Acidic conditions are not obligatory for the onset of butanol formation by Clostridium beijerinckii (synonym, C. butylicum). Appl Environ Microbiol 46, 321–327.
    [Google Scholar]
  16. Georgiadis, M. M., Komiya, H., Chakrabarti, P., Woo, D., Kornuc, J. J. & Rees, D. C. ( 1992; ). Crystallographic structure of the nitrogenase iron protein from Azotobacter vinelandii. Science 257, 1653–1659.[CrossRef]
    [Google Scholar]
  17. Johnson, J. L. ( 1994; ). Similarity analysis of rRNAs. In Methods for General and Molecular Bacteriology, pp. 683–701. Edited by P. Gerhardt, R. G. E. Murray, W. A. Woods & N. R. Krieg. Washington, DC: American Society for Microbiology.
  18. Kim, J. D. & Rees, D. C. ( 1993; ). X-ray crystal structure of the nitrogenase molybdenum-iron protein from Clostridium pasteurianum at 3·0-Å resolution. Biochemistry 32, 7104–7115.[CrossRef]
    [Google Scholar]
  19. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef]
    [Google Scholar]
  20. Martinez, E., Pardo, M. A., Palacios, R. & Cevallos, M. A. ( 1985; ). Reiteration of nitrogen fixation gene sequences and specificity of Rhizobium in nodulation and nitrogen fixation in Phaseolus vulgaris. J Gen Microbiol 131, 1779–1786.
    [Google Scholar]
  21. Minamisawa, K., Nishioka, K., Miyaki, T. & 9 other authors ( 2004; ). Anaerobic nitrogen-fixing consortia consisting of clostridia isolated from gramineous plants. Appl Environ Microbiol 70, 3096–3102.[CrossRef]
    [Google Scholar]
  22. Morett, E., Moreno, S. & Espin, G. ( 1988; ). Transcription analysis of the three nifH genes of Rhizobium phaseoli with gene fusions. Mol Gen Genet 213, 499–504.[CrossRef]
    [Google Scholar]
  23. Mortenson, L. E. ( 1964; ). Ferredoxin and ATP requirements for nitrogen fixation in cell-free extracts of Clostridium pasteurianum. Proc Natl Acad Sci U S A 52, 272–279.[CrossRef]
    [Google Scholar]
  24. Norel, F. & Elmerich, C. ( 1987; ). Nucleotide sequence and functional analysis of the two nifH copies of Rhizobium ORS571. J Gen Microbiol 133, 1563–1576.
    [Google Scholar]
  25. Schlessman, J. L., Woo, D., Joshua-Tor, L., Howard, J. B. & Rees, D. C. ( 1998; ). Conformational variability in structures of the nitrogenase iron proteins from Azotobacter vinelandii and Clostridium pasteurianum. J Mol Biol 280, 669–685.[CrossRef]
    [Google Scholar]
  26. Seto, B. & Mortenson, L. E. ( 1974; ). In vivo kinetics of nitrogenase formation in Clostridium pasteurianum. J Bacteriol 120, 822–830.
    [Google Scholar]
  27. Shevchenko, A., Jensen, O. N., Podtelejnikov, A. V. & 7 other authors ( 1996; ). Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proc Natl Acad Sci U S A 93, 14440–14445.[CrossRef]
    [Google Scholar]
  28. Tanaka, M., Haniu, M., Yasunobu, T. & Mortenson, L. E. ( 1977; ). The amino acid sequence of Clostridium pasteurianum iron protein, a component of nitrogenase. J Biol Chem 252, 7089–7092.
    [Google Scholar]
  29. Tso, M.-Y., Ljones, T. & Burris, R. H. ( 1972; ). Purification of the nitrogenase proteins from Clostridium pasteurianum. Biochim Biophys Acta 267, 600–604.[CrossRef]
    [Google Scholar]
  30. Ueda, T., Suga, Y., Yahiro, N. & Matsuguchi, T. ( 1995; ). Remarkable N2-fixing bacterial diversity detected in rice roots by molecular evolutionary analysis of nifH gene sequences. J Bacteriol 177, 1414–1417.
    [Google Scholar]
  31. Vichitphan, K. ( 2001; ). Azotobacter vinelandii nitrogenase: effect of amino-acid substitutions at the αGln-191 residue of the MoFe protein on substrate reduction and CO inhibition. PhD thesis, Virginia Polytechnic Institute and State University, Blacksburg, VA.
  32. Wang, S.-Z., Chen, J.-S. & Johnson, J. L. ( 1988; ). The presence of five nifH-like sequences in Clostridium pasteurianum: sequence divergence and transcriptional properties. Nucleic Acids Res 16, 439–453.[CrossRef]
    [Google Scholar]
  33. Winogradsky, S. ( 1895; ). Recherches sur l'assimilation de l'azote libre de l'atmosphere par les microbes. Arch Sci Biol 3, 297–352.
    [Google Scholar]
  34. Yan, R. T. & Chen, J.-S. ( 1990; ). Coenzyme A-acetylating aldehyde dehydrogenase from Clostridium beijerinckii NRRL B592. Appl Environ Microbiol 56, 2591–2599.
    [Google Scholar]
  35. Zinoni, F., Robson, R. M. & Robson, R. L. ( 1993; ). Organization of potential alternative nitrogenase genes from Clostridium pasteurianum. Biochim Biophy Acta 1174, 83–86.[CrossRef]
    [Google Scholar]
  36. Zumft, W. G. & Mortenson, L. E. ( 1973; ). Evidence for a catalytic-centre heterogeneity of molybdoferredoxin from Clostridium pasteurianum. Eur J Biochem 35, 401–409.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27931-0
Loading
/content/journal/micro/10.1099/mic.0.27931-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error