Bacteroids from soybean root nodules: respiration and N 2 -fixation in flow-chamber reactions with oxyleghaemoglobin

Abstract

Suspensions of bacteroids prepared from root nodules of glasshouse-grown soybeans were studied in a stirred chamber supplied with variable flows of solutions containing dissolved air, oxyleghaemoglobin and various energy sources. In experiments of up to 6 h duration, several steady states were established in which frequent measurements were made of the concentration of free, dissolved O$_2$ in the range 5-200 nM and of rates of O$_2$ consumption. CO$_2$ efflux and N$_2$ fixation. The principal findings were. (i) bacteroids were capable of efficient N$_2$ fixation without the supply of exogenous energy sources when respiring at 10-50 nM free O$_2$. This endogenous respiration was enhanced after periods of supply of succinate or malate. (ii) Exogenous energy sources were of three types, those that were poorly utilized (glucose), those that enhanced endogenous respiration and supported efficient N$_2$ fixation (glutamate. 2-oxoglutarate) and succinate and malate, which promoted respiration but supported N$_2$ fixation mefficiently or in some circumstances inhibited it. It is proposed that succinate and malate act primarily to increase endogenous reserves that become the principal source of reducing power for N$_2$ fixation. (iii) Bacteroids have a terminal oxidase system with very high apparent affinity for O$_2$ (s$_{0.5}$ $\approx$ 5-8 nM) and complex kinetics (plots of v against s are sigmoidal n$_{app}$ > 1.8). This system was active with endogenous substrates and when glutamate or 2-oxoglutarate were supplied. With succinate or malate, respiration appeared to be the sum of endogenous activity plus O$_2$ consumption by a system with lower affinity for O$_2$ (K$_s$ = 38 nM) and simple kinetics (n$_{app}$ $\approx$ 1). (iv) During the first hour of reactions there were changes in O$_2$ demand and oscillations in O$_2$ demand and CO$_2$ efflux followed supply or withdrawal of exogenous substrates. It is proposed that these changes are examples of phenotypic plasticity in these symbiotic bacteria.