Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1980 Jul;143(1):274–284. doi: 10.1128/jb.143.1.274-284.1980

In vivo energetics and control of nitrogen fixation: changes in the adenylate energy charge and adenosine 5'-diphosphate/adenosine 5'-triphosphate ratio of cells during growth on dinitrogen versus growth on ammonia.

R G Upchurch, L E Mortenson
PMCID: PMC294225  PMID: 6995432

Abstract

The effects of the intracellular energy balance and adenylate pool composition on N2 fixation were examined by determining changes in the energy charge (EC) and the ADP/ATP (D/T) ratio of cells in chemostat and batch cultures of Clostridium pasteurianum, Klebsiella pneumoniae, and Azotobacter vinelandii. When cells of C. pasteurianum, K. pneumoniae, and A. vinelandii in sucrose-limited chemostats were examined, in all cases the EC increased greater than or equal to 15% when the nitrogen source was switched from N2 to NH3 and decreased greater than or equal to 15% when the nitrogen source was switched from NH3 to N2. The D/T ratio of the same cultures decreased greater than or equal to 70% when they were switched from N2 to NH3. In such cultures the adenylate pools remained constant when the cells were grown on either NH3 or N2. In nitrogen (NH3)-limited cultures, the adenylate pool was two- to threefold higher than the adenylate pool in sucrose-limited cultures, and the nitrogenase content of such cells was two- to threefold greater than the nitrogenase content of sucrose-limited N2-fixing cells. The EC and D/T ratio of cells from batch cultures of C. pasteurianum growing on NH3 in the presence of N2 were 0.82 and 0.83, respectively, but when the NH3 was consumed and the cells were switched to a nitrogen-fixing metabolism, the EC and D/T ratio changed to 0.70 and 0.90, respectively. Conversely, when NH3 was added to N2-fixing cultures the EC and D/T ratio changed within 1.5 h the EC and D/T ratio of NH3-grown cells. The nitrogen content of N2-fixing cells to which NH3 was added decreased at a rate greater could be accounted for by cell growth in the absence of further synthesis. This decay of nitrogenase activity (with a half-life about 1.2 to 1.4 h) suggests that some type of inactivation of nitrogenase occurs during repression. The nitrogenase of whole cells was estimated to be operating at about 32% of its theoretical maximum activity during steady-state N2-fixing conditions. Similarities in the data from chemostat and batch cultures of both aerobic and anaerobic N2-fixing organisms suggest that low EC and high D/T ratio are normal manifestations of an N2-fixing physiology.

Full text

PDF
274

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Atkinson D. E. Regulation of enzyme function. Annu Rev Microbiol. 1969;23:47–68. doi: 10.1146/annurev.mi.23.100169.000403. [DOI] [PubMed] [Google Scholar]
  2. Ball W. J., Jr, Atkinson D. E. Adenylate energy charge in Saccharomyces cerevisiae during starvation. J Bacteriol. 1975 Mar;121(3):975–982. doi: 10.1128/jb.121.3.975-982.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bui P. T., Mortenson L. E. Mechanism of the enzymic reduction of N2: the binding of adenosine 5'-triphosphate and cyanide to the N2-reducing system. Proc Natl Acad Sci U S A. 1968 Nov;61(3):1021–1027. doi: 10.1073/pnas.61.3.1021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. CHANEY A. L., MARBACH E. P. Modified reagents for determination of urea and ammonia. Clin Chem. 1962 Apr;8:130–132. [PubMed] [Google Scholar]
  5. Carithers R. P., Yoch D. C., Arnon D. I. Two forms of nitrogenase from the photosynthetic bacterium Rhodospirillum rubrum. J Bacteriol. 1979 Feb;137(2):779–789. doi: 10.1128/jb.137.2.779-789.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chapman A. G., Fall L., Atkinson D. E. Adenylate energy charge in Escherichia coli during growth and starvation. J Bacteriol. 1971 Dec;108(3):1072–1086. doi: 10.1128/jb.108.3.1072-1086.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Daesch G., Mortenson L. E. Effect of ammonia on the synthesis and function of the N 2 -fixing enzyme system in Clostridium pasteurianum. J Bacteriol. 1972 Apr;110(1):103–109. doi: 10.1128/jb.110.1.103-109.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Daesch G., Mortenson L. E. Sucrose catabolism in Clostridium pasteurianum and its relation to N2 fixation. J Bacteriol. 1968 Aug;96(2):346–351. doi: 10.1128/jb.96.2.346-351.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dalton H., Postgate J. R. Effect of oxygen on growth of Azotobacter chroococcum in batch and continuous cultures. J Gen Microbiol. 1968 Dec;54(3):463–473. doi: 10.1099/00221287-54-3-463. [DOI] [PubMed] [Google Scholar]
  10. Dietzler D. N., Lais C. J., Magnani J. L., Leckie M. P. Maintenance of the energy charge in the presence of large decreases in the total adenylate pool of Escherichia coli and concurrent changes in glucose-6-p, fructose-p2 and glycogen synthesis. Biochem Biophys Res Commun. 1974 Oct 8;60(3):875–881. [PubMed] [Google Scholar]
  11. Eady R. R., Smith B. E., Cook K. A., Postgate J. R. Nitrogenase of Klebsiella pneumoniae. Purification and properties of the component proteins. Biochem J. 1972 Jul;128(3):655–675. doi: 10.1042/bj1280655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Haaker H., Veeger C. Involvement of the cytoplasmic membrane in nitrogen fixation by Azotobacter vinelandii. Eur J Biochem. 1977 Jul 1;77(1):1–10. doi: 10.1111/j.1432-1033.1977.tb11634.x. [DOI] [PubMed] [Google Scholar]
  13. Haaker H., Veeger C. Regulation of respiration and nitrogen fixation in different types of Azotobacter vinelandii. Eur J Biochem. 1976 Apr 1;63(2):499–507. doi: 10.1111/j.1432-1033.1976.tb10253.x. [DOI] [PubMed] [Google Scholar]
  14. Haaker H., de Kok A., Veeger C. Regulation of dinitrogen fixation in intact Azotobacter vinelandii. Biochim Biophys Acta. 1974 Sep 20;357(3):344–357. doi: 10.1016/0005-2728(74)90024-3. [DOI] [PubMed] [Google Scholar]
  15. Kimmich G. A., Randles J., Brand J. S. Assay of picomole amounts of ATP, ADP, and AMP using the luciferase enzyme system. Anal Biochem. 1975 Nov;69(1):187–206. doi: 10.1016/0003-2697(75)90580-1. [DOI] [PubMed] [Google Scholar]
  16. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  17. Laane C., Haaker H., Veeger C. Involvement of the cytoplasmic membrane in nitrogen fixation by Rhizobium leguminosarum bacteroids. Eur J Biochem. 1978 Jun 1;87(1):147–153. doi: 10.1111/j.1432-1033.1978.tb12361.x. [DOI] [PubMed] [Google Scholar]
  18. Mortenson L. E. Regulation of nitrogen fixation. Curr Top Cell Regul. 1978;13:179–232. doi: 10.1016/b978-0-12-152813-3.50010-0. [DOI] [PubMed] [Google Scholar]
  19. Mortenson L. E., Zumpft W. G., Palmer G. Electron paramagnetic resonance studies on nitrogenase. 3. Function of magnesium adenosine 5'-triphosphate and adenosine 5'-diphosphate in catalysis by nitrogenase. Biochim Biophys Acta. 1973 Feb 22;292(2):422–435. doi: 10.1016/0005-2728(73)90048-0. [DOI] [PubMed] [Google Scholar]
  20. Mustafa E., Mortenson L. E. Acetylene reduction by nitrogen fixing extracts of Clostridium pasteurianum: ATP requirement and inhibition by ADP. Nature. 1967 Dec 23;216(5121):1241–1242. doi: 10.1038/2161241a0. [DOI] [PubMed] [Google Scholar]
  21. Scherings G., Haaker H., Veeger C. Regulation of nitrogen fixation by Fe-S protein II in Azotobacter vinelandii. Eur J Biochem. 1977 Aug 1;77(3):21–30. doi: 10.1111/j.1432-1033.1977.tb11706.x. [DOI] [PubMed] [Google Scholar]
  22. Seto B. L., Mortenson L. E. Mechanism of carbamyl phosphate inhibition of nitrogenase of Clostridium pasteurianum. J Bacteriol. 1974 Feb;117(2):805–812. doi: 10.1128/jb.117.2.805-812.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Seto B., Mortenson L. E. In vivo kinetics of nitrogenase formation in Clostridium pasteurianum. J Bacteriol. 1974 Nov;120(2):822–830. doi: 10.1128/jb.120.2.822-830.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shah V. K., Davis L. C., Brill W. J. Nitrogenase. I. Repression and derepression of the iron-molybdenum and iron proteins of nitrogenase in Azotobacter vinelandii. Biochim Biophys Acta. 1972 Feb 28;256(2):498–511. doi: 10.1016/0005-2728(72)90078-3. [DOI] [PubMed] [Google Scholar]
  25. Strandberg G. W., Wilson P. W. Formation of the nitrogen-fixing enzyme system in Azotobacter vinelandii. Can J Microbiol. 1968 Jan;14(1):25–31. doi: 10.1139/m68-005. [DOI] [PubMed] [Google Scholar]
  26. Swedes J. S., Sedo R. J., Atkinson D. E. Relation of growth and protein synthesis to the adenylate energy charge in an adenine-requiring mutant of Escherichia coli. J Biol Chem. 1975 Sep 10;250(17):6930–6938. [PubMed] [Google Scholar]
  27. Walker-Simmons M., Atkinson D. E. Functional capacities and the adenylate energy charge in Escherichia coli under conditions of nutritional stress. J Bacteriol. 1977 May;130(2):676–683. doi: 10.1128/jb.130.2.676-683.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Williams J. C., Weiss E. Energy metabolism of Rickettsia typhi: pools of adenine nucleotides and energy charge in the presence and absence of glutamate. J Bacteriol. 1978 Jun;134(3):884–892. doi: 10.1128/jb.134.3.884-892.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zumft W. G., Mortensson L. E. Evidence for a catalytic-centre heterogeneity of molybdoferredoxin from Clostridium pasteurianum. Eur J Biochem. 1973 Jun 15;35(3):401–409. doi: 10.1111/j.1432-1033.1973.tb02852.x. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES