Antonie van Leeuwenhoek

, Volume 50, Issue 5–6, pp 505–524 | Cite as

Hydrogen oxidation and nitrogen fixation in rhizobia, with special attention focused on strain ORS 571

  • Wytske de Vries
  • Hein Stam
  • Adriaan H. Stouthamer
Physiology And Growth

Abstract

In this survey we describe the influence of hydrogen oxidation on the physiology ofRhizobium ORS 571. The presence of hydrogen is required for the synthesis of hydrogenase. Carbon substrates do not repress the synthesis of hydrogenase. The respiratory system contains cytrochromes of theb- andc-type. Cytochromea600 is present after growth at high oxygen tensions. The nature of the terminal oxidases functioning at low oxygen tensions has not been established yet. → H+/O values with endogenous substrates are between 6 and 7. The results show the presence of two phosphorylation sites: site 1 (ATP/2e=1.0) and site 2(ATP/2e=1.33). By measuring molar growth yields it has been demonstrated that carbon-limited, nitrogen-fixing cultures obtain additional ATP from hydrogen oxidation, and that site 2 of oxidative phosphorylation is passed during hydrogen oxidation. A method is described to calculate ATP/N2 values (the total amount of ATP used by nitrogenase during the fixation of 1 mol N2) and H2/N2 ratios (mol hydrogen formed per mol N2 fixed) in aerobic organisms. ForRhizobium ORS 571 the ATP/N2 value is about 40 and the H2/N2 ratio is between 5 and 7.5. Cells obtained from oxygen-limited nitrogen-fixing cultures contain 30–40% poly-β-hydroxybutyrate, which explains the high molar growth yields found. Hydrogen has not been detected in the effluent gas of these cultures, which may point to reoxidation of the hydrogen formed at nitrogen fixation. Calculations show that the effect of hydrogen reoxidation on the efficiency of nitrogen fixation (g N fixed × mol−1 substrate converted) is not very large and that the actual H2/N2 ratio is of much more importance.

After addition of hydrogen to succinate-limited, ammonia-assimilating cultures, an initial increase of the Ysuccinate value (g dry wt × mol−1 succinate) is followed by a gradual decrease. This is accompanied by a large decrease of the\(Y_{O_2 } \) value, and an increased permeability of the cytoplasmic membrane to protons. The results may be explained by a transition of the culture from an energy-limited state to a carbon-limited state.

Keywords

Nitrogen Fixation Oxygen Tension Endogenous Substrate Hydrogen Oxidation Aerobic Organism 

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References

  1. Albrecht, S. L., Maier, R. J., Hanus, F. J., Russell, S. A., Emerich, D. W. andEvans, H. J. 1979. Hydrogenase inRhizobium japonicum increases nitrogen fixation by nodulated soybeans. — Science203: 1255–1257.Google Scholar
  2. Appleby, C. A. 1969. Electron transport systems ofRhizobium japonicum II.Rhizobium haemoglobin, cytochromes and oxidases in free-living (cultured) cells. — Biochim. Biophys. Acta172: 88–105.PubMedGoogle Scholar
  3. Babel, W., Müller, R. H. andMarkuske, K. D. 1983. Improvement of growth yield of yeast on glucose to the maximum by using an additional energy source. — Arch. Microbiol.136: 203–208.CrossRefGoogle Scholar
  4. Bauchop, T. andElsden, S. R. 1960. The growth of micro-organisms in relation to their energy supply. — J. Gen. Microbiol.23: 457–469.PubMedGoogle Scholar
  5. Bergersen, F. J. andTurner, G. L. 1975. Leghaemoglobin and the supply of O2 to nitrogen-fixing root nodule bacteroids: presence of two oxidase systems and ATP production at low free O2 concentration. — J. Gen. Microbiol.91: 345–354.PubMedGoogle Scholar
  6. Bergersen, F. J. andTurner, G. L. 1978. Activity of nitrogenase and glutamine synthetase in relation to availability of oxygen in continuous cultures of a strain of cowpeaRhizobium sp. supplied with excess ammonium. — Biochim. Biophys. Acta538: 406–416.PubMedGoogle Scholar
  7. Bergersen, F. J. andTurner, G. L. 1980. Properties of terminal oxidase systems of bacteroids from root nodules of soybean and cowpea and of N2-fixing bacteria grown in continuous culture. — J. Gen. Microbiol.118: 235–252.Google Scholar
  8. Bergersen, F. J., Turner, G. L., Gibson, A. H. andDudman, W. F. 1976. Nitrogenase activity and respiration of cultures ofRhizobium spp. with special reference to concentration of dissolved oxygen. — Biochim. Biophys. Acta444: 164–174.PubMedGoogle Scholar
  9. Bethlenfalvay, G. J. andPhillips, D. A. 1979. Variation in nitrogenase and hydrogenase activity of Alaska pea root nodules. — Plant Physiol.63: 816–820.Google Scholar
  10. Boogerd, F. C., Van Verseveld, H. W. andStouthamer, A. H. 1981. Respiration-driven proton translocation with nitrite and nitrous oxide inParacoccus denitrificans. — Biochim. Biophys. Acta638: 181–191.PubMedGoogle Scholar
  11. Boogerd, F. C., Van Verseveld, H. W. andStouthamer, A. H. 1983. Dissimilatory nitrate uptake inParacoccus denitrificans via a\(\Delta \widetilde\mu _H + \) -dependent system and a nitrate-nitrite antiport system. — Biochim. Biophys. Acta723: 415–427.Google Scholar
  12. Carter, K. R., Jennings, N. T., Hanus, J. andEvans, H. J. 1978. Hydrogen evolution and uptake by nodules of soybeans inoculated with different strains ofRhizobium japonicum. — Can. J. Microbiol.24: 307–311.PubMedGoogle Scholar
  13. Daesch, G. andMortenson, L. E. 1968. Sucrose catabolism inClostridium pasteurianum and its relation to N2 fixation. — J. Bacteriol.96: 346–351.PubMedGoogle Scholar
  14. Dalton, H. andPostgate, J. R. 1969. Growth and physiology ofAzotobacter chroococcum in continuous culture. — J. Gen. Microbiol.56: 307–319.Google Scholar
  15. De Hollander, J. A. 1981. Studies on the physiology ofRhizobium trifolii. — Ph. D. Thesis, Vrije Universiteit, Amsterdam.Google Scholar
  16. De Hollander, J. A. andStouthamer, A. H. 1980. The electron transport chain ofRhizobium trifolii. — Eur. J. Biochem.111: 473–478.PubMedGoogle Scholar
  17. DeJong, T. M., Brewin, N. J., Johnston, A. W. B. andPhillips, D. A. 1982. Improvement of symbiotic properties inRhizobium leguminosarum by plasmid transfer. — J. Gen. Microbiol.128: 1829–1838.Google Scholar
  18. Dixon, R. O. D. 1972. Hydrogenase in legume root nodule bacteroids: occurrence and properties. — Arch. Microbiol.85: 193–201.Google Scholar
  19. Dreyfus, B. L. andDommergues, Y. R. 1981. Nitrogen-fixing nodules induced byRhizobium on the stem of the tropical legumeSesbania rostrata. — FEMS Microbiol. Lett.10: 313–317.Google Scholar
  20. Dreyfus, B. L., Elmerich, C. andDommergues, Y. R. 1983. Free-livingRhizobium strain able to grow under N2 as the sole nitrogen source. — Appl. Environ. Microbiol.45: 711–713.PubMedGoogle Scholar
  21. Eisbrenner, G. andEvans, H. J. 1982. Carriers in electron transport from molecular hydrogen to oxygen inRhizobium japonicum bacteroids. — J. Bacteriol.149: 1005–1012.PubMedGoogle Scholar
  22. Emerich, D. W., Ruiz-Argüeso, T., Ching, T. M. andEvans, H. J. 1979. Hydrogen-dependent nitrogenase activity and ATP formation inRhizobium japonicum bacteroids. — J. Bacteriol.137: 153–160.PubMedGoogle Scholar
  23. Gebhardt, C., Turner, G. L., Gibson, A. H., Dreyfus, B. L. andBergensen, F. J. 1984. Nitrogenfixing growth in continuous culture of a strain ofRhizobium sp. isolated from stem nodules onSesbania rostrata. — J. Gen. Microbiol.130: 843–848.Google Scholar
  24. Gibson, A. H., Scowcroft, W. R., Child, J. J. andPagan, J. D. 1976. Nitrogenase activity in culturedRhizobium sp. strain 32H1. Nutritional and physical considerations. — Arch. Microbiol.108: 45–54.PubMedCrossRefGoogle Scholar
  25. Hardy, R. W. F. andHavelka, U. D. 1976. Photosynthate as a major factor limiting nitrogen fixation by field-grown legumes with emphasis on soybeans. p. 421–439.In P. S. Nutman (ed.), Symbiotic Nitrogen Fixation in Plants. — Cambridge University Press, Cambridge.Google Scholar
  26. Hill, S. 1976. The apparent ATP requirement for nitrogen fixation in growingKlebsiella pneumoniae. — J. Gen. Microbiol.95: 297–312.Google Scholar
  27. Karr, D. B., Waters, J. K. andEmerich, D. W. 1983. Analysis of poly-β-hydroxybutyrate inRhizobium japonicum bacteroids by ion-exclusion high-pressure liquid chromatography and UV detection. — Appl. Environ. Microbiol.46: 1339–1344.PubMedGoogle Scholar
  28. Kashket, E. R. 1982. Stoichiometry of the H+-ATPase of growing and resting, aerobicEscherichia coli. — Biochemistry21: 5534–5538.PubMedCrossRefGoogle Scholar
  29. Keister, D. L. 1975. Acetylene reduction by pure cultures of rhizobia. — J. Bacteriol.123: 1265–1268.PubMedGoogle Scholar
  30. Kretovich, W. L., Romanov, V. I. andKorolyov, A. V. 1973.Rhizobium leguminosarum cytochromes (Vicia faba). — Plant Soil39: 619–634.CrossRefGoogle Scholar
  31. Lambers, H. andDe Visser, R. 1984. Energy metabolism in nodulated roots. p. 453–460.In C. Veeger and W. E. Newton (eds), Advances in Nitrogen Fixation Research. — Martinus Nijhoff/Dr W. Junk Publ., The Hague and Pudoc, Wageningen.Google Scholar
  32. Larue, T. A., Peterson, J. B. andTajima, S. 1984. Carbon metabolism in the legume nodule. p. 437–443.In C. Veeger and W. E. Newton (eds), Advances in Nitrogen Fixation Research. — Martinus Nijhoff/Dr W. Junk Publ., The Hague and Pudoc, Wageningen.Google Scholar
  33. Law, J. H. andSlepecky, R. A. 1961. Assay of poly-β-hydroxybutyric acid. — J. Bacteriol.82: 33–36.PubMedGoogle Scholar
  34. Lepo, J. E., Hickok, R. E., Cantrell, M. A., Russell, S. A. andEvans, H. J. 1981. Revertible hydrogen uptake-deficient mutants ofRhizobium japonicum. — J. Bacteriol.146: 614–620.PubMedGoogle Scholar
  35. Lim, S. T. 1978. Determination of hydrogenase in free-living cultures ofRhizobium japonicum and energy efficiency of soybean nodules. — Plant Physiol.62: 609–611.Google Scholar
  36. Lim, S. T. andUratsu, S. L. 1983. Repression of growth inRhizobium japonicum 3I1b 110 by molecular hydrogen. — FEMS Microbiol. Lett.18: 109–112.Google Scholar
  37. Linton, J. D. andStephenson, R. J. 1978. A preliminary study on growth yields in relation to the carbon and energy content of various organic growth substrates. — FEMS Microbiol. Lett.3: 95–98.Google Scholar
  38. Lopez, M., Carbonero, V., Cabrera, E. andRuiz-Argüeso, T. 1983. Effects of host on the expression of the H2-uptake hydrogenase ofRhizobium in legume nodules. — Plant Sci. Lett.29: 191–199.Google Scholar
  39. Maier, R. J., Campbell, N. E. R., Hanus, F. J., Simpson, F. B., Russell, S. A. andEvans, H. J. 1978. Expression of hydrogenase activity in free-livingRhizobium japonicum. — Proc. Natl Acad. Sci. USA75: 3258–3262.PubMedGoogle Scholar
  40. Maier, R. J., Hanus, F. J. andEvans, H. J. 1979. Regulation of hydrogenase inRhizobium japonicum. — J. Bacteriol.137: 824–829.Google Scholar
  41. Merberg, D., O’Hara, E. B. andMaier, R. J. 1983. Regulation of hydrogenase inRhizobium japonicum: analysis of mutants altered in regulation by carbon substrates and oxygen. — J. Bacteriol.156: 1236–1242.PubMedGoogle Scholar
  42. Nelson, L. M. 1983. Hydrogen recycling byRhizobium leguminosarum isolates and growth and nitrogen contents of pea plants (Pisum sativum L.). — Appl. Environ. Microbiol.45: 856–861.PubMedGoogle Scholar
  43. Nelson, L. M. andSalminen, S. O. 1982. Uptake hydrogenase activity and ATP formation inRhizobium leguminosarum bacteroids. — J. Bacteriol.151: 989–995.PubMedGoogle Scholar
  44. Neijssel, O. M. andTempest, D. W. 1975. The regulation of carbohydrate metabolism inKlebsiella aerogenes NCTC 418 organisms, growing in chemostat culture. — Arch. Microbiol.106: 251–258.PubMedCrossRefGoogle Scholar
  45. O’Brian, M. R. andMaier, R. J. 1982. Electron transport components involved in hydrogen oxidation in free-livingRhizobium japonicum. — J. Bacteriol.152: 422–430.PubMedGoogle Scholar
  46. Papa, S. 1976. Proton translocation reactions in the respiratory chains. — Biochim. Biophys. Acta456: 39–84.PubMedGoogle Scholar
  47. Reibach, P. H. andStreeter, J. G. 1983. Metabolism of14C-labeled photosynthate and distribution of enzymes of glucose metabolism in soybean nodules. — Plant Physiol.72: 634–640.Google Scholar
  48. Robson, R. L. andPostgate, J. R. 1980. Oxygen and hydrogen in biological nitrogen fixation. — Annu. Rev. Microbiol.34: 183–207.PubMedCrossRefGoogle Scholar
  49. Roels, J. A. 1980. Simple model for the energetics of growth on substrates with different degrees of reduction. — Biotechnol. Bioeng.22: 33–53.Google Scholar
  50. Ruiz-Argüeso, T., Cabrera, E. andDe Bertalmio, M. B. 1981. Selection of symbiotically energy efficient strains ofRhizobium japonicum by their ability to induce a H2-uptake hydrogenase in the free-living state. — Arch. Microbiol.128: 275–279.CrossRefGoogle Scholar
  51. Ruiz-Argüeso, T., Hanus, J. andEvans, H. J. 1978. Hydrogen production and uptake by pea nodules as affected by strains ofRhizobium leguminosarum. — Arch. Microbiol.116: 113–118.CrossRefGoogle Scholar
  52. Schubert, K. R. andEvans, H. J. 1976. Hydrogen evolution: a major factor affecting the efficiency of nitrogen fixation in nodulated symbionts. — Proc. Natl Acad. Sci. USA73: 1207–1211.PubMedGoogle Scholar
  53. Schubert, K. R., Jennings, N. T. andEvans, H. J. 1978. Hydrogen reactions of nodulated leguminous plants. II. Effects of dry matter accumulation and nitrogen fixation. — Plant Physiol.61: 398–401.Google Scholar
  54. Senior, P. J. andDawes, E. A. 1971. Poly-β-hydroxybutyrate biosynthesis and the regulation of glucose metabolism inAzotobacter beijerinckii. — Biochem. J.125: 55–66.PubMedGoogle Scholar
  55. Stam, H., Van Verseveld, H. W., De Vries, W. andStouthamer, A. H. 1984. Hydrogen oxidation and efficiency of nitrogen fixation in succinate-limited chemostat cultures ofRhizobium ORS 571. — Arch. Microbiol.139: 53–60.CrossRefGoogle Scholar
  56. Stam, H., Van Verseveld, H. W. andStouthamer, A. H. 1983. Derepression of nitrogenase in chemostat cultures of the fast growingRhizobium leguminosarum. — Arch. Microbiol.135: 199–204.CrossRefGoogle Scholar
  57. Stouthamer, A. H. 1977. Theoretical calculations on the influence of the inorganic nitrogen source on parameters for aerobic growth of microorganisms. — Antonie van Leeuwenhoek43: 351–367.PubMedCrossRefGoogle Scholar
  58. Stouthamer, A. H. 1984. Energy generation and hydrogen metabolism inRhizobium. p. 189–197.In C. Veeger and W. E. Newton (eds), Advances in Nitrogen Fixation Research. — Martinus Nijhoff/Dr W. Junk Publ., The Hague and Pudoc, Wageningen.Google Scholar
  59. Stouthamer, A. H. andVan Verseveld, H. W. 1984. Stoichiometry of bacterial growth.In A. T. Bull (ed.), Comprehensive Biotechnology, Principles and Practice, Vol. 1, Section 1. — Pergamon Press, Oxford (in press).Google Scholar
  60. Van Verseveld, H. W., Krab, K. andStouthamer, A. H. 1981. Proton pump coupled to cytochrome c oxidase inParacoccus denitrificans. — Biochim. Biophys. Acta635: 525–534.PubMedGoogle Scholar
  61. Zablotowicz, R. M., Russell, S. A. andEvans, H. J. 1980. Effect of the hydrogenase system inRhizobium japonicum on the nitrogen fixation and growth of soybeans at different stages of development. — Agron. J.72: 555–559.Google Scholar
  62. Zumft, W. G. andMortenson, L. E. 1975. The nitrogen-fixing complex of bacteria. — Biochim. Biophys. Acta416: 1–52.PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1984

Authors and Affiliations

  • Wytske de Vries
    • 1
  • Hein Stam
    • 1
  • Adriaan H. Stouthamer
    • 1
  1. 1.Biological LaboratoryVrije UniversiteitAmsterdamThe Netherlands

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