Energy supply for dinitrogen fixation by Azotobacter vinelandii and by bacteroids of Rhizobium leguminosarum

N.C.M. Laane

Research output: Thesisinternal PhD, WU


The central issue of this thesis is how obligate aerobes, such as <em>Rhizobium leguminosarum</em> bacteroids and <em>Azotobacter vinelandii,</em> generate and regulate the energy supply (in the form of ATP and reducing equivalents) for nitrogenase.<br/>In an effective <em>Rhizobium</em> -legume symbiosis, <em></em> the actual reduction of dinitrogen into ammonia is carried out in the root nodules by bacteroids. In order to study this intriguing process on the level of the bacteroids, it is clear that bacteroids have to be isolated from the root nodules. The first task to be achieved was, therefore, to develop an isolation procedure for bacteroids with a nitrogenase activity which is comparable with that found in whole plants. Chapter 2 describes such a procedure. The only difference in comparison with conventional procedures is that bacteroids were treated with fatty acid-free bovine serum albumin. By means of this extra treatment, the dinitrogen-fixing capacity of bacteroids could be improved considerably. Using these highly active bacteroids, an introductory survey was performed of the factors controlling nitrogenase activity in this organism. It was observed that aerobic dinitrogen fixation by pea nodule bacteroids in many respects resembles that of the free-living dinitrogen fixer <em>A.vinelandii.</em> As in <em>A.vinelandii,</em> dinitrogen fixation by bacteroids might be controlled by the intracellular ATP/ADP-ratio. Furthermore, evidence was found which suggests that, as in <em>A.vinelandii,</em> the energized state of the cytoplasmic membrane of bacteroids regulates the supply of reducing equivalents to nitrogenase. In addition, the dinitrogen-fixing system of bacteroids, as in <em>Azotobacter,</em> could be inhibited by free oxygen.<br/>In aerobic organisms, membranes are energized by respiration <em>via</em> the electron transfer chain. Thus, the ultimate source of energy for the generation of a low potential reductant in <em>A.vinelandii</em> and bacteroids is provided exclusively by the respiratory chain. However, the so-called energized state of the membrane is a rather unmanageable term. Since the time, it has become known that respiration is accompanied by proton movement across the membrane; the energized state is recognized as a protonmotive force, and can be expressed in real physical parameters, such as Δψ(membrane potential) and ΔpH (pH difference across the membrane) (Chapter 1). In Chapters 4 and 5, these parameters were measured under dinitrogen-fixing conditions. It was observed that both in bacteroids (Chapter 4), as well as in <em>A.vinelandii</em> (Chapter 5), the flow of reducing equivalents to nitrogenase is regulated exclusively by the Δψ-component of the protonmotive force (Δμ <sub>H</sub> +). At this stage of investigation it is difficult to describe a mechanism that explains the Δψ-induced formation of reducing equivalents, nevertheless, one attempt is presented in Chapter 6.<br/>It has been known for some time that ammonium, when added to cultures of <em>A.vinelandii,</em> rapidly inhibits the nitrogenase activity. To date, no reasonable explanation has been given for this phenomenon. In Chapter 5, evidence is presented which shows that uptake of ammonium by <em>A.vinelandii</em> specifically switches off the flow of reducing equivalents to nitrogenase by lowering the AT. In contrast, ammonium had no effect on the rate of dinitrogen fixation by bacteroids. It has been demonstrated that bacteroids do not accumulate added ammonium, but on the contrary excrete the ammonium produced by the nitrogenase system (Chapter 5). Furthermore, it appears that newly-fixed ammonium is excreted in response simply to the ΔpH across the bacterial membrane.<br/>In aerobes, the respiratory chain not only provides energy for the generation of reducing equivalents, but also for the synthesis of ATP <em>via</em> a process called oxidative phosphorylation. During this investigation it became apparent that, in comparison with the ATP-synthesizing system, more respiratory energy is necessary for the generation of reducing equivalents. The rate-limiting step in aerobic dinitrogen fixation therefore seems to be at the level of electron transport to nitrogenase. In Chapter 3, some factors controlling the efficiency of oxidative phosphorylation in <em>A.vinelandii</em> and bacteroids are considered. It was observed that these bacteria are capable to adjust their ATP-synthesizing capacity to the availability of oxygen in their environment. In this way, they are capable of producing ATP most efficiently when the ATP cost for dinitrogen fixation is maximal. In Chapter 6, the flexibility of the ATP-synthesizing system, which results from the considerable flexibility of the proton-translocating activity of the respiratory chain, is discussed.<br/>It is generally accepted that the respiratory chain of <em>A.vinelandii</em> contains three proton-translocating sites. In Chapter 3 evidence is presented which suggests that protons are also translocated at the level of hydrogenase. This implies that <em>A.vinelandii</em> contains four instead of three proton-translocating sites. In contrast, no respiratory chain-linked hydrogenase could be detected in bacteroids of <em>R.Leguminosarum.</em><p/><em></em>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Veeger, C., Promotor
Award date29 Feb 1980
Place of PublicationWageningen
Publication statusPublished - 1980


  • azotobacter
  • pseudomonadaceae
  • biochemistry
  • metabolism
  • microorganisms
  • rhizobiaceae
  • rhizobium
  • synthesis
  • chemical analysis
  • exposure
  • environmental degradation
  • kinetics
  • ecotoxicology
  • cum laude

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