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Supply of O₂ regulates demand for O₂ and uptake of malate by N₂-fixing bacteroids from soybean nodules

Division of Biochemistry and Molecular Biology, Faculty of Science, The Australian National University, ACT 0200, Australia¹

Author for correspondence: David A. Day. Tel: +61 8 9380 3324. Fax: +61 8 9380 1148. e-mail: dday{at}cyllene.uwa.edu.au

Bacteroids, prepared anaerobically from soybean root nodules by fractional centrifugation or by sucrose or Percoll density-gradient methods, were retained within a stirred, flow-through reaction chamber and used to determine rates of respiration and N₂ fixation at various rates of O₂ supply. Liquid reaction solutions containing malate, oxyleghaemoglobin, dissolved N₂ and various levels of dissolved O₂ were passed through the reaction chamber at measured rates of flow. The relative oxygenation of leghaemoglobin in the chamber was determined automatically by spectrophotometry of the effluent solution, and the concentrations of free, dissolved O₂ ([O_{2 free}]) and the rates of O₂ consumption were calculated. N₂ fixation was measured by analysis of fractions of effluent. The principal finding was that stepwise increases in the flow rate (increasing the supply of O₂ and malate) induced an increase in O₂ demand (respiration) resulting in a decrease in [O_{2 free}] and increased N₂ fixation. In some experiments, samples of bacteroids were withdrawn from the flow chamber during steady states and the rates of malate uptake were measured in standard, microaerobic assays. Progressive taking of samples from the flow chamber whilst maintaining constant flow rates (increasing the supply of O₂ and malate per bacteroid) also resulted in increased O₂ demand and declines in [O_{2 free}]. With increased bacteroid respiration, transport of malate into bacteroids (linear with time between 1 and 5 min after starting each assay) increased proportionally. This suggests that the rate of malate transport is tightly coupled with bacteroid respiration. Thus, bacteroid respiration, coupled with malate uptake, must be regulated by the rate of O₂ supply, rather than by the [O_{2 free}] prevailing in the stirred chamber as found or assumed in previous work. These features are discussed in relation to N₂ fixation by anaerobically isolated bacteroids.

Keywords: malate transport, nitrogen fixation, respiration, Bradyrhizobium japonicum

Abbreviations: [O_{2 free}]; free; dissolved O₂

^b Present address: Biochemistry Department, University of Missouri, Columbia, MO, USA.

^a Present address: Biochemistry Department, The University of Western Australia, Nedlands, WA 6907, Australia.

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