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
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ABSTRACT |
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Keywords: malate transport, nitrogen fixation, respiration, Bradyrhizobium japonicum
Abbreviations: [O2 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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INTRODUCTION |
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It is commonly assumed, when constructing simulations of nodule function, that bacteroid respiration rates are governed by the [O2 free] to which they are exposed (e.g. Thumfort et al., 1994 ; Bergersen, 1999
). Whilst this assumption is supported by results obtained with isolated bacteroids under certain experimental conditions, when energy-yielding substrates were not limiting (Bergersen & Turner, 1993
; Bergersen, 1997b
), when no exogenous substrates were supplied, utilization of endogenous reserves by bacteroids was regulated by the rate of supply of O2 (Bergersen & Turner, 1992
), which in turn regulated bacteroid respiration. In the present paper we report experiments with a different strain of B. japonicum showing that bacteroid respiratory O2 demand appears to be regulated by rates of O2 supply when the bacteroids are supplied with limiting (50 µM) exogenous malate and that there is a parallel regulation of rates of transport of malate into the bacteroids.
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METHODS |
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Flow chamber experiments
Reaction solutions.
These were prepared in deionized water and contained MOPS/KOH buffer (25 mM, pH 7·4), 2 mM MgCl2, sodium DL-malate (0·052 mM) and 1015 ml leghaemoglobin solution (prepared as above) per 300 ml. The solutions, in reservoir flasks connected to the reaction chamber by a peristaltically pumped capillary line, were stirred at 23 °C for 1 h before use, in air (0·21 atm O2) at 1 atm (93·32595·992 N m-2 or 95·3398·06 kPa pressure at the elevation of Canberra), or in closed reservoirs under atmospheres containing 0·125 or 0·25 atm O2 in N2. Concentrations of [O2 free] in these solutions were calculated from tables of solubility. Actual concentrations of leghaemoglobin were measured by the pyridine haemochromogen method and the proportional oxygenation of leghaemoglobin by spectrophotometry (Appleby & Bergersen, 1980 ) of the solutions passing into and out of the reaction chamber.
Flow reaction chamber and methods.
The flow chamber, associated equipment and methods were as described previously (Bergersen & Turner, 1985 , 1990
, 1992
, 1993
). The water-jacketed (30 °C), mechanically stirred conical chamber (volume 12 ml) is closed at its base by a bacteroid-retaining, microporous membrane filter (pore size 0·45 µm), supported on a porous plastic disc. Beneath the disc is an annular collecting space connected to a spectrophotometer flow cuvette by stainless steel capillary (0·3 mm i.d.) tubing. Bacteroids in the chamber are perfused by reaction solution pumped from the reservoirs described above. Spent reaction solution emerging from the cuvette is collected as fractions for determination of NH3 in solution, considered to be the principal product of N2 fixation by bacteroids in the chamber (Bergersen & Turner, 1990
). The apparatus incorporates facilities for recording automatically, at intervals of 1 min, values of
A (the difference between optical absorbances at 576 and 560 nm) of effluent solution and the time of the beginning and end of each effluent fraction collected for analysis. Values of
A are converted to concentrations of [O2 free] and rates of consumption of O2 having regard to (a) the
At values for effluent solution at t min, the
Aoxy value for fully oxygenated (in air) reaction solution and Ared, the value for reaction solution reduced with a few crystals of dithionite; (b) the concentrations of total leghaemoglobin and [O2 free] in the reservoir reaction solution; (c) the kinetics of O2 binding by soybean leghaemoglobin (Keq=0·047 µM O2; Appleby, 1984
), and (d) the flow rate of solution through the chamber and the dry mass of bacteroids present there. At t min, the proportional oxygenation of leghaemoglobin in the chamber is:
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Experimental.
The assembled flow chamber and connecting tubes were filled with N2 before being completely filled with reaction solution which had been degassed under vacuum before storage under N2. Bacteroids, prepared as above, were finally resuspended under N2 in reaction solution which had been degassed under vacuum; 0·51·0 ml of the suspension was injected slowly into the stirred chamber. When the chamber contents became visibly purplish (leghaemoglobin partially deoxygenated), the reaction solution pump was started at a flow of about 0·5 ml min-1 and data recording commenced. After several periods of different flow rates, the flow was switched to a different reservoir and another series of flow rates was run.
Measurement of malate transport.
In preliminary experiments, the effects of method of preparation of bacteroids and of conditions imposed during the assays were examined. In these tests, uptake of [14C]malate was measured essentially as described by Udvardi et al. (1988) but with modifications to determine the effects of [O2 free] during preparation of bacteroids and during assay of uptake. From these tests, the following protocol was developed for application to bacteroids recovered from the flow chamber.
Samples (3·5 ml) were withdrawn from the flow chamber into argon-flushed syringes at intervals in experiments in which the air-saturated reaction solutions contained only 50 µM malate at a flow rate of 1·0 ml min-1. The first sample in each experiment was taken after the period during which O2 demand gradually increased (020 min; Holtzapffel & Bergersen, 1998 ). Taking of each sample removed 25·4% of the bacteroids present in the chamber when sampling commenced. Thus with constant flow, after each sampling the O2 flux to each bacteroid in the chamber increased. The time courses of uptake from 1 mM L-[U14C]malate ([1,4(2,3)-14C]malic acid, 2·04 GBq mmol-1, Amersham; diluted with unlabelled sodium malate to give suitable concentrations and specific radioactivities) by bacteroids in these samples were measured in standard microaerobic assays in duplicate in rubber-capped argon-flushed tubes, into each of which was injected 1·0 ml of the reaction mixture taken from the chamber by argon-flushed syringe. Into each tube, air-saturated reaction solution (100 µl) plus 10 µl 100 mM [14C]malate was injected (bringing the tube contents to >1·0 mM malate and approx. 10 µM O2) and timing was started. Samples (150 µl) were taken from the tubes at intervals of 2 min into microfuge tubes, the bacteroids centrifuged through 100 µl of silicone oil into 10 µl 40% (v/v) HClO4 and radioactivity in the pellets measured by scintillation counting (Udvardi et al., 1988
).
Analytical.
Ammonia in samples of effluent reaction solution was measured by the Chaney-Marbach method (Bergersen, 1980 ). Radioactivity was measured by scintillation methods. Calculations of relative oxygenation of leghaemoglobin (Y) and of [O2 free] from Y were as described previously (Appleby & Bergersen, 1980
; Bergersen & Turner 1990
).
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RESULTS |
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Uptake of malate by bacteroids as affected by method of preparation and assay conditions
The initial rate of uptake of [14C]malate by bacteroids was greatest in aerobically treated bacteroids which were also assayed aerobically (Fig. 4) but the rate declined after 5 min. Usually, rates declined earlier in all aerobic uptake assays than in microaerobic assays. Although rates of uptake were lower for microaerobic than for aerobic assays, they were usually maintained for at least 9 min. Preparation of bacteroids anaerobically suppressed malate uptake rates, even when uptake was measured in the presence of O2. It was considered that the microaerobic assays most closely resembled conditions prevailing in flow-chamber experiments. Other experiments (data not presented) showed that rates of malate uptake by bacteroids recovered from the flow chamber were independent of malate concentration at concentrations up to 1 mM.
Malate uptake by flow-chamber bacteroids
Bacteroid N2 fixation in vivo is supported by respiration, coupled with the consumption of substrates (e.g. malate uptake; Udvardi et al., 1988 ) provided by the host plant. Also, bacteroids supplied with malate accumulate poly-ß-hydroxybutyrate, which serves as an endogenous reserve of respirable substrate. Utilization of this endogenous reserve is regulated in relation to the rate of O2 supply (Bergersen & Turner, 1992
). We investigated uptake of malate by bacteroids taken from the flow chamber and in separate experiments in which batches of bacteroids were exposed simultaneously to different oxygen environments. In the flow-chamber experiments, bacteroids were withdrawn from the flow chamber and uptake of [14C]malate measured in standard microaerobic assays. The results showed that concomitantly with increased bacteroid respiration preceding each sample, maximum (malate un-limited) rates of transport of [14C]malate into bacteroids increased (Table 2
). These results suggest that rates of malate uptake may be tightly coupled with bacteroid respiration and thus with N2 fixation. In other experiments (data not shown), we found that the Vmax but not the apparent Km for malate uptake changed during exposure to higher respiratory demand. These changes were not affected by the inclusion of chloramphenicol (at concentrations shown independently to inhibit protein synthesis) in the flow-chamber medium.
To investigate whether the higher rates of malate uptake were simply the result of an increased demand for malate upon an increase in respiration, bacteroids were prepared anaerobically, incubated either under anaerobic conditions or in air-saturated medium for 17 min, and malate uptake measured under both aerobic and microaerobic conditions (Fig. 4). Quite clearly, the exposure to higher O2 preconditioned the bacteroids to take up malate at a faster rate, regardless of the assay conditions.
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DISCUSSION |
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The results also suggest that malate transport is regulated by a related mechanism since rates of malate uptake by bacteroids increased according to respiration rates preceding sampling (Table 2). The rate of supply of malate to bacteroids in the chamber (e.g. 40 nmol min-1 mg-1 at a flow of 1·0 ml min-1; Table 1
) greatly exceeded the rate of O2 consumption (2·2 nmol min-1 mg-1 in the same conditions; Table 1
), so it is unlikely that the increase in malate uptake resulted simply from an increase in malate supply to the chamber; this was certainly not the case in the standard uptake assays of Fig. 4
. Nor was this simply a case of a stronger sink for malate (resulting from increased respiration) stimulating uptake, because (i) significant differences in rate of uptake were recorded when aerobically and anaerobically prepared bacteroids were assayed at the same oxygen concentration (Fig. 4
). Also, (ii) with successive increases in bacteroid respiration preceding each sampling (Fig. 3
), when malate uptake was measured under standard conditions in which respiratory rates were uniform and malate concentrations were non-limiting, specific rates of malate uptake also increased (Table 2
). Rather, the respiratory demand during the period preceding the transport assays seemed to feedback and affect the uptake of malate. Since Vmax but not Km of uptake increased upon exposure to higher oxygen supply, it appears that more malate carriers were engaged following the increase in respiratory demand, but the lack of effect of chloramphenicol indicates that this was not the result of synthesis of new carrier proteins. Instead, the respiratory demand seemed to feedback and affect the uptake of malate directly. The nature of this conditioning process is not known but it suggests that both organic acid transport and metabolism in the bacteroid are controlled by a common factor, such as pyridine nucleotide redox poise.
It has long been known that the bacteroids of soybean root nodules have a branched respiratory electron-transport pathway (e.g. Appleby, 1969 ). Inflected curves of respiration vs [O2] were interpreted as reflecting differences in the affinities for O2 of the terminal oxidases of the branches (e.g. Bergersen & Turner, 1975
). Respiration at low [O2] (<0·1 µM) was more CO-sensitive and more efficiently coupled to ATP production and N2 fixation (Appleby et al., 1975
; Bergersen & Turner, 1975
) than was respiration at [O2] >0·5 µM. Supply of O2 to bacteroids, when mediated by the presence of partially oxygenated leghaemoglobin, supported respiration in the well-coupled range of [O2] (Wittenberg et al., 1974
; Appleby et al., 1975
; Bergersen & Turner, 1975
). More recently (Preisig et al., 1996
), it has been proposed that there are four terminal oxidases for the electron-transport branches in B. japonicum and that in bacteroids it is the cbb3-type cytochrome oxidase that is the principal oxidase operating in the microaerobic environment of soybean nodules. This oxidase has an extremely high affinity for O2 (Km=7 nM). The results of the flow-chamber experiments reported in the present paper show that, when bacteroids were challenged with increasing supply of O2, high steady rates of respiration [>11 nmol O2 min-1 (mg dry mass)-1] and N2 fixation [>2 nmol NH3 min-1 (mg dry mass)-1] were achieved at about 6 nM O2 (Table 1
, Fig. 1
), close to the Km of the cbb3-type cytochrome oxidase (Preisig et al., 1996
). These results imply that Vmax for respiration via this oxidase increases with rates of O2 supply, perhaps up to values as high as 30 nmol O2 min-1 (mg dry mass) -1 (i.e. twice the rate at the Km) as transport of malate into bacteroids (Table 2
) increases to meet the respiratory demand. In the flow-chamber experiments, the principal product of N2 fixation was NH3 in the effluent solution as reported previously with a different strain of B. japonicum (Bergersen & Turner 1990
). In the present work, no more than traces of alanine (as described by Waters et al., 1998
) were found by enzymic assay or by HPLC (data not presented), even when density-gradient-purified bacteroids were used (Waters et al., 1998
). Allaway et al. (2000)
suggested that in the flow chamber, NH3 was swept away, thus preventing the concentration from rising sufficiently for alanine to be synthesized in detectable amounts. This seems a reasonable explanation.
Conclusion
The results presented here show that under certain conditions (low substrate and low oxygen supply), bacteroid metabolism, including nitrogenase activity, is regulated by the rate of oxygen supply instead of the concentration of free dissolved oxygen. Regulatory mechanisms apparently exist which coordinate respiratory demand with rates of substrate transport into the bacteroid. This has important implications for maintenance of N2 fixation rates in vivo and needs to be further investigated with intact symbiosomes. However, since bacteroid demand for malate strongly influences the rate of malate uptake into symbiosomes (Ou Yang et al., 1991 ), it is likely that this regulation will be sensed also at the peribacteroid membrane.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 29 August 2000;
revised 20 November 2000;
accepted 1 December 2000.