Oxygen, respiration and nitrogen fixation in the facultative anaerobe Klebsiella pneumoniae

Smith, Andrew Trevor (1989) Oxygen, respiration and nitrogen fixation in the facultative anaerobe Klebsiella pneumoniae. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

Biological nitrogen fixation is an oxygen-sensitive energy intensive process; because of the potential benefits of aerobic respiration for ATP production, nitrogen-fixing micro-organisms have adapted to various degrees to cope with and utilize oxygen during N₂ fixation. Klebsiella pneumoniae has been the main `work horse' for research into nitrogen fixation for many years. Although a facultative anaerobe, it too can utilize O₂ during N₂ fixation. The mechanism whereby O₂ regulates expression of the nif genes is not known. In the present work, the hypothesis that haem plays a role in this mechanism is tested by studying the oxygen regulation of nitrogenase synthesis in a Nif⁺ E. coli strain deficient in haem synthesis. The O₂ regulatory mechanism is shown to be independent of haem status. There is good evidence that the respiratory chain of K. pneumoniae has an important physiological role during nitrogen fixation yet little is known about it or its regulation under N₂-fixing conditions. A question of particular interest was the nature of the oxidase(s) responsible for respiration at the very low oxygen concentrations that allow nif gene expression. The cytochrome d oxidase complex was purified from K. pneumoniae and its O₂ affinity determined with leghaemoglobin (Km'21nM). This was consistent with the observed sensitivity to O₂ of nif gene expression; the oxidase was judged to be kinetically competent to support oxidative phosphorylation at low O₂ levels. In support of this, E.coli cytochrome d minus (Cyd^- strains carrying the Nif⁺ plasmid pRD1 failed to support microaerobic nitrogenase activity under conditions where ATP and reductant must have been generated as a result of respiration via cytochrome d. A model is proposed to describe how the oxidation of formate plus lactate supports O₂-dependent nitrogenase activity in Cyd⁺ strains.