Isolation and characterization of hydrogenase-negative mutants of Azorhizobium caulinodans ORS571

W. de Vries, J. Ras, H. Stam, M. M.A. van Vlerken, U. Hilgert, F. J. de Bruijn, A. H. Stouthamer

Research output: Contribution to journalArticlepeer-review

5 Scopus citations


Hydrogenase-negative (Hup-) mutants of Azorhizobium caulinodans ORS571 were isolated by means of Tn5 mutagenesis. The colony test used for screening for Hup- strains was based on the absence of reduction of triphenyltetrazolium chloride with hydrogen. Suspensions from cultures of the mutant strains grown under derepressing conditions did not use hydrogen with methylene blue or oxygen as the hydrogen acceptor. The mutants were shown to carry single Tn5 insertions at different locations in the A. caulinodans genome. Molar growth yields (corrected for poly-β-hydroxybutyrate formation) in chemostat cultures of the mutants were similar to those of the wild type. Molar growth yields of the mutants were not increased by passing additional hydrogen through chemostat cultures, which is in agreement with the hydrogenase-negative phenotype of the mutants. H2/N2 ratios (mol H2 formed per mol N2 fixed) were calculated from the hydrogen content of the effluent gas and the N-content of the bacterial dry weight. Low H2/N2 ratios (between 1.2 and 1.9) were found in both energy-limited (oxygen or succinate) cultures and in cultures limited by the supply of an anabolic substrate (Mg2+). ATP/2e values (mol ATP used at the transport of 2e to nitrogen or H+) were calculated from the H2/N2 ratios and the molar growth yields of nitrogen-fixing and ammonia-assimilating cultures. ATP/2e values were between 7 and 11. It was concluded that the calculated ATP/2e values comprise not only 4 mol ATP used at the transport of 2e through nitrogenase but also energy equivalents needed for reversed electron flow from NADH to the low-potential hydrogen donor used by nitrogenase.

Original languageEnglish (US)
Pages (from-to)595-599
Number of pages5
JournalArchives of Microbiology
Issue number6
StatePublished - Oct 1988
Externally publishedYes


  • ATP/2e value
  • Azorhizobium caulinodans ORS571
  • Chemostat cultures
  • H/N ratio
  • Hydrogenase
  • Nitrogen fixation

ASJC Scopus subject areas

  • Microbiology
  • Biochemistry
  • Molecular Biology
  • Genetics


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