Graham L. Turner

Studies of the physiological role of leghaemoglobin in soybean root nodules

Abstract Evolution of H2 by nitrogenase in intact soybean nodules was consistently inhibited by CO when the nodules were equilibrated with argon-CO mixtures for 1 h prior to adding O2 to initiate the reaction. Evolution of H2 by nitrogenase in bacteroid suspensions prepared from nodules, was not inhibited by CO. Dense, slowly shaken suspensions of bacteroids, with 12% O2 in the gas phase maintained slow rates of H2 evolution and acetylene reduction for up to 12 h. Addition of leghaemoglobin to these assays greatly enhanced the nitrogenase-mediated reactions. CO prevented stimulation by leghaemoglobin of H2 evolution by bacteroids. Stimulation of acetylene reduction by bacteroid suspensions was dependent upon leghaemoglobin concentration up to about 1 mM. Increased shaking rates gave greater rates of acetylene reduction and O2 uptake. Stimulation of nitrogenase activity in bacteroid suspensions by leghaemoglobin was much greater than stimulation of O2 uptake. Increasing acetylene reduction in response to increasing agitation was accompanied by increasing oxygenation of the leghaemoglobin. The significance of these results in relation to the physiological role of leghaemoglobin in symbiotic N2 fixation by legume root nodules is discussed.

Bacteroids from soybean root nodules: accumulation of poly-β-hydroxybutyrate during supply of malate and succinate in relation to N2 fixation in flow-chamber reactions.

A liquid reaction medium containing dissolved air and oxyleghaemoglobin was supplied to bacteroids confined in a stirred reaction chamber with no gas phase. The relative oxygenation of leghaemoglobin, dissolved CO2 and NH3 from N2 fixation were monitored before, during (when N2 fixation was diminished) and after the supply of [14C]malate and succinate. The amounts and location of 14C-labelled products were determined in bacteroids recovered from the chamber and 14CO2 was measured in the effluent. During the supply of [14C]succinate, the bacteroids accumulated most 14C from C2 and C3 whereas 14CO2 arose primarily from C1 and C4. This suggested that production of pyruvate from malate via malic enzyme was a central feature. For up to 80 min after removal of [14C]malate and succinate, CO2 arose from stored carbon and N2 fixation was enhanced; during this period 14C continued to accumulate in poly-β-hydroxybutyrate, until this accounted for about 90% of bacteroid 14C. Thereafter CO2 was evolved with steady, low radioactivity and enhanced N2 fixation continued. These results supported the proposal that poly-β-hydroxybutyrate, which is present in large amounts (50-70% of dry mass) in these bacteroids, is a mobilizable, energy-yielding reserve that provides endogenous substrates for support of N2 fixation when exogenous substrates are not available.

Bacteroids from soybean root nodules: respiration and N2-fixation in flow-chamber reactions with oxyleghaemoglobin

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 O2 in the range 5–200 nM and of rates of O2 consumption, CO2 efflux and N2 fixation. The principal findings were: (i) bacteroids were capable of efficient N2 fixation without the supply of exogenous energy sources when respiring at 10–50 nM free O2. 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 N2 fixation (glutamate, 2-oxoglutarate) and succinate and malate, which promoted respiration but supported N2 fixation inefficiently 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 N2 fixation, (iii) Bacteroids have a terminal oxidase system with very high apparent affinity for O2 (s0.5 ≈ 5–8 nM) and complex kinetics (plots of v against s are sigmoidal; napp > 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 O2 consumption by a system with lower affinity for O2 (Ks = 38 nM) and simple kinetics (napp ≈ 1). (iv) During the first hour of reactions there were changes in O2 demand and oscillations in O2 demand and CO2 efflux followed supply or withdrawal of exogenous substrates. It is proposed that these changes are examples of phenotypic plasticity in these symbiotic bacteria.

Nitrogenase activity and respiration of cultures of Rhizobium spp. with special reference to concentration of dissolved oxygen

Studies of nitrogenase in cultures of the cowpea rhizobia (Rhizobium spp.) strains 32H1 and CB756 are reported. Preliminary experiments established that, even when agar cultures were grown in air, suspensions of bacteria prepared anaerobically from them were most active at low concentrations of free dissolved O2. Consequently, assays for activity used low concentrations of O2, stabilized by adding the nodule pigment leghaemoglobin. In continuous, glutamine-limited cultures of 32H1, nitrogenase activity appeared only when the concentration of dissolved O2 in the cultures approached 1 muM. Lowering the glutamine concentration in the medium supplied to the culture from 2 to 1 mM halved the cell yield and nitrogenase activity was also diminished. Omitting succinate from the medium caused the concentration of dissolved O2 to rise and nitrogenase activity was lost. Upon restoration of the succinate supply, the O2 concentration immediately fell and nitrogenase was restored. The activity doubled in about 8 h, whereas the doubling time of this culture was 14 h. Sonic extracts of 32H1 cells from continuous cultures with active nitrogenase contained components reacting with antiserum against nitrogenase Mo-Fe protein from soybean bacteroids. Continuous cultures grown at higher O2 concentration, with only a trace of active nitrogenase, contained less of these antigens and they were not detected in highly aerobic cultures. Nitrogenase activity of a continuous culture was repressed by NH+4; the apparent half-life was about 90 min. Cells of 32H1 from a continuous culture growing at between 30 and 100 muM dissolved O2 possessed a protective mechanism which permitted respiration to increase following exposure to a rapid increase in O2 concentration from low levels (O2 shock). This effect disappeared as the O2 concentration for growth was reduced towards 1 muM.

Effects of concentrations of substrates supplied to N2-fixing soybean bacteroids in flow chamber reactions

A liquid reaction medium containing dissolved air, oxyleghaemoglobin and low concentrations (0-2 mM) of glucose, succinate, malate or ethanol, was supplied to bacteroids prepared from soybean root nodules and confined in a stirred reaction chamber. Measurements of concentrations of O2, CO2 and NH3 from N2 fixation, at various flow rates through the chamber, were used to calculate rates of respiration and N2 fixation. Glucose was utilized poorly under all conditions. Ethanol was required at 0.2-2.0 mM before stimulations of O2 demand and N2 fixation were observed. Demand for O2 was enhanced by 0.05 mM to 0.5 mM succinate and malate at concentrations of O2 from 10 nM to 50 nM. However, the concentrations of C4-dicarboxylate and dissolved O2 determined whether N2 fixation was stimulated or inhibited by these substrates. It is postulated that these effects were associated with the presence in the bacteroids of two malic enzymes and a pyruvate dehydrogenase complex, the resulting acetyl-GoA being channelled through the tricarboxylic acid (TCA) cycle (N2 fixation stimulated) or to poly-β-hydroxybutyrate (PHB) accumulation (N2 fixation inhibited). Contrary to earlier results, the O2 consumption data suggested that there was a single terminal oxidase (Km 20—26 nM and Vmax 20-29 nmol O2 min-1 (mg dry mass)-1) operating in the range of O2 concentration in which N2 fixation occurred.

Activity of nitrogenase and glutamine synthetase in relation to availability of oxygen in continuous cultures of a strain of cowpea Rhizobium sp. supplied with excess ammonium

In samples from nitrogen-fixing continuous cultures of strain CB756 of the cowpea type rhizobia (Rhizobium sp.), newly fixed NH+4 is in equiblibrium with the medium, from where it is assimilated by the glutamine synthetase/glutamate synthase pathway. In samples from steady state cultures with different degrees of oxygen-limitation, nitrogenase activity was positively correlated with the biosynthetic of glutamine synthetase in cell free extracts. Also, activities in biosynthetic assays were positively correlated with activities in gamma-glutamyl transferase assays containing 60 mM Mg2+. Relative adenylylation of glutamine synthetase was conveniently measured in cell free extracts as the ratio of gamma-glutamyl transferase activities without and with addition of 60 mM Mg2+. Automatic control of oxygen supply was used to facilitate the study of transitions between steady-state continuous cultures with high and low nitrogenase activities. Adenylylation of glutamine synthetase and repression of nitrogenase activity in the presence of excess NH+4, were masked when oxygen strongly limited culture yield. Partial relief of the limitation in cultures supplied with 10 mM NH+4 produced early decline in nitrogenase activity and increase in relative adenylylation of glutamine synthetase. Decreased oxygen supply produced a rapid decline in relative adenylylation, followed by increased nitrogenase activity, supporting the concept that control of nitrogenase synthesis is modulated by glutamine synthetase adenylylation in these bacteria.

The role of O2-limitation in control of nitrogenase in continuous cultures of Rhizobium sp

Oxygen-limited continuous cultures of the cowpea Rhizobium sp. strain CB756, had high levels of nitrogenase activity, which were not significantly affected by excess ammonium ions or glutamine. When the growth-restricting O2-limitation was partially relieved, nitrogenase was repressed and this was accompanied by increased adenylylation of glutamine synthetase. It is suggested that the restricted supply of ATP interferes with adenylylation of glutamine synthetase during O2-limited growth, thus preventing repression of nitrogenase in the presence of excess ammonium ions.

LEGHEMOGLOBIN: THE ROLE OF HEMOGLOBIN IN THE NITROGEN‐FIXING LEGUME ROOT NODULE*

A world increasingly short of protein may expend its limited supply of chemical or electrical energy t o fix atmospheric nitrogen into ammonia, or may rely in greater part on plants to harvest solar energy, some part of which is directed t o the support of bacterial nitrogen fixation. The rapid development of the nitrogen-fixing soybean as a major crop suggests that reliance on plants may already have become the more economical way t o regenerate the supply of fixed nitrogen. A hemeprotein, leghemoglobin, is an indispensable component of the system by which leguminous plants support the activity of nitrogen-fixing bacteria. We here inquire into the molecular mechanism by which leghemoglobin augments the oxygen consumption and nitrogen-fixing activity of bacteroids. That mechanism is leghemoglobin-facilitated oxygen diffusion brought about by translational diffusion of the oxygenated protein. The flux of oxygen is enhanced by facilitated diffusion. In addition, we now discover, facilitated diffusion makes oxygen more available to the terminal oxidases of subcellular organelles. We are concerned with the physicochemical definition of what we mean by “available.” Nitrogen-fixing nodules are formed on the roots of legumes in response t o invasion by bacteria of the genus Rhizobium. Rhizobia, modified for symbiotic life, are called bacteroids. The enzyme complex, nitrogenase, which is responsible for nitrogen fixation, is located wholly within the bacteroids. Nitrogenase does not utilize oxygen (in fact it is inhibited by even traces of oxygen) but depends for its activity of a supply of ATP formed (presumably) by bacteroidal oxidative phosphorylation. The bacteroids, which occur within the nodule cell, are in some ways analogous to muscle mitochondria. They occupy about the same fraction, approximately one-third,1,20f the cell volume and are responsible for the largest part of the oxygen consumption. Bacteroidal oxygen demand is vigorous; the oxygen consumption of the intact nodule is about one-tenth as great as that of the most active mammalian muscles. In fact, both

Supply of O2 regulates O2 demand during utilization of reserves of poly- β-hydroxybutyrate in N2-fixing soybean bacteroids

A liquid reaction medium containing dissolved air and oxyleghaemoglobin, but no energy-yielding substrate, was supplied to bacteroids confined in a stirred flow reaction chamber. The relative oxygenation of the leghaemoglobin in the chamber was determined automatically by spectrophotometry of the effluent solution, and the concentrations of free, dissolved O2 ([ O2]) and rates of O2 consumption were calculated. Dissolved CO2 and NH3 from N2 fixation were determined in fractions of the effluent solution. Bacteroids utilized endogenous reserves of poly- β-hydroxybutyrate (PHB), which were depleted by 9.2% during a typical 5 h-long experiment. Stepwise increases in flow rate (increasing supply of O2) initially produced a drop in O2 demand and resulted in a rise in [O2] and a decline in N2 fixation. Subsequently, O2 demand rose (presumably because of increased mobilization of substrate from PHB) and [O2] declined to a low level. N2 fixation was fully restored, or even enhanced, within 15–20 min of establishment of a new, steady [O2. This pattern of regulation by O2 supply was completely eliminated by adding low concentrations (20-50 μm) of oxidizable substrate (succinate, malate, ethanol) to the reaction medium. During endogenous activity, rates of CO2 evolution were proportional to, but less than, rates of O2 consumption up to 5.4 nmol O2 min-1 mg-1, above which CO2 evolution exceeded O2 consumption. These and other features of endogenous activity are discussed in relation to sustaining N2 fixation by nodules in vivo.

Energy status, growth and nitrogenase activity in continuous cultures of Rhizobium sp. strain CB756 supplied with NH+4 and various rates of aeration

Abstract Continuous cultures of the cowpea-type Rhizobium sp., strain CB756, were grown in the presence of NH + 4 at automatically controlled concentrations of dissolved O 2 and rates of aeration. Nitrogenase activity of steady-state cultures was only detected under microaeration conditions (dissolved O 2 typically μ M; aeration rate typically 0.6 μmol O 2 /ml per h), when the cellular ATP pool size was 0.8–1.8 nmol/mg dry wt., (optimum 1.1) and the energy charge 0.6–0.7. At twice this aeration rate and dissolved O 2 concentration of about 0.15 μM, the yield of bacteria doubled, the ATP pool increased and energy charge increased to 0.8. With similar rates of O 2 supply but high concentration of dissolved O 2 (approx. 150 μM), cultures were NH + 4 -limited and the ATP pool and energy charge were slightly reduced. Amongst all of these O 2 supply conditions the total pool of adenosine phosphates was not significantly different (2.6 S.D. 0.7 nmol/mg dry wt.). In steady-state, O 2 -limited cultures, concentrations of cyclic GMP were higher when nitrogenase was present. When rates of O 2 supply to steady-state cultures were changed, oscillations in bacterial energy status and growth rate were induced decreasing in amplitude until a new steady state was reached. This made it difficult to discern precisely the energy status in which nitrogenase activity was derepressed or repressed. However, generally, increases in nitrogenase activity followed decreases in ATP and energy charge and decreased nitrogenase activity accompanied increases in these energy parameters. These results are discussed in relation to the possible involvement of adenylation or deadenylation of glutamine synthetase and to the control of nitrogenase synthesis in the presence of NH + 4 . It is concluded that the small ATP pool size is responsible for failure of adenylylation of glutamine synthetase and is related to nitrogenase synthesis at microaeration rates.