6
The symbiotic vesicle is a major site for respiration in Frankia from Alnus incana root nodules PER- AKE VIKMAN' Department of Plant Physiology, University of Umeh, S-901 87 Umeh, Sweden Received October 28, 1991 Revision received February 19, 1992 Accepted February 2 1, 1992 VIKMAN, P.-A. 1992. The symbiotic vesicle is a major site for respiration in Frankia from Alnus incana root nodules. Can. J. Microbiol. 38: 779-784. A technique was developed for preparation of Frankia symbiotic vesicles, free of hyphae. The symbiotic vesicles were isolated by isopycnic centrifugation of disrupted Frankia vesicle clusters prepared from root nodules of Alnus incana (L.) Moench. Activities in symbiotic vesicles were compared with activities in intact symbiotic vesicle clusters on a total protein basis. Respiratory capacity was tested with 6-phosphogluconate, malate + glutamate, and NADH as added substrates. With all three substrates, specific respiration was doubled after symbiotic vesicle isolation. Nitrogenase was used as a symbiotic vesicle specific marker and its specific activity increased similarly to respiration. Activities of four respiratory enzymes were assayed on crude cell-free extracts obtained after sonication of symbiotic vesicle prep- arations. According to the increased specific rates after symbiotic vesicle isolation, NAD + :6-phosphogluconate dehy- drogenase (EC 1.1.1.44) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) were mainly localized in symbiotic vesicles. NAD + :malate dehydrogenase (EC 1.1.1.37) and glutamate-oxaloacetate transaminase (EC 2.6.1.1) were also present in symbiotic vesicles, but their specific activities were not increased compared with the symbiotic vesicle clusters. The magnitude of increased activities suggested that the symbiotic vesicle is a major site for hexose respiration in symbiotic Frankia. An apparent Km for 0, between 20 and 30 pM indicated that symbiotic vesicles in symbiotic vesicle clusters have a restricted oxygen diffusion rate. Key words: Frankia, symbiotic vesicles, respiration, nitrogenase, oxygen diffusion. VIKMAN, P.-A. 1992. The symbiotic vesicle is a major site for respiration in Frankia from Alnus incana root nodules. Can. J. Microbiol. 38 : 779-784. Une technique de preparation de vesicules symbiotiques libres d'hyphes de Frankia a ete mise au point. L'isolement de telles vesicules a ete obtenu par centrifugation isopycnique de groupe de vesicules disloquees de Frankia, preparees a partir de nodosites racinaires de Alnus incana (L.) Moench. Les activites dans ces vbicules ont ete comparees a celles des groupes de vesicules intactes, sur la base des proteines totales. Les substrats 6-phosphogluconate, malate + glutamate, et NADH ont servi a tester les capacites respiratoires. Chez les vesicules isolees, la respiration specifique a double avec les trois substrats. La nitrogenase a ete utilisee comme marqueur specifique de vesicules symbiotiques et son activite specifique a augmente la respiration de faqon similaire. Les activites de quatre enzymes reliees a la respiration ont ete testees sur des extraits acellulaires bruts, apres sonication des preparations de vesicules. D'apres les taux specifiques accrus apres l'isolement des vesicules, la NAD + :6-phosphogluconate deshydrogenase (EC 1.1.1.44) et la glucose-6-phosphate deshydrogenase (EC 1.1.1.49) ont ete localisees principalement dans les vesicules symbiotiques. La NAD + :malate deshydrogenase (EC 1.1.1.37) et la glutamate-oxaloacetate transaminase (EC 2.6.1.1) ont aussi ete presentes dans les vesicules isolees, mais leurs activites specifiques n'ont pas ete augmentees par comparaison a celles des groupes de vesicules. L'etendue de l'augmentation des activites suggere que les vesicules sont le siege principal de la respiration des hexoses chez le Frankia en symbiose. Un Km apparent entre 20 et 30 pM pour 1'0, indique que les vesicules des groupes de vesicules symbiotiques ont un taux restreint de diffusion de l'oxygene. Mots elks : Frankia, vesicules symbiotiques, respiration, nitrogenase, oxygene, diffusion. [Traduit par la redaction] Introduction The actinomycete Frankia forms a root nodule symbiosis with certain, so-called actinorhizal plants. With the enzyme nitrogenase, Frankia can reduce atmospheric N2 and supply its host with combined nitrogen. Three morphological forms of Frankia are present in Alnus root nodules (Newcomb and Wood 1987). Multicellular filaments of a vegetative type are called hyphae (i). Symbiotic vesicles (ii) differentiate on hyphal side branches and are subtended by a short stalk. In culture the corresponding structure is referred to as "Frankia vesicle," while the term "symbiotic vesicle" is denoted to those found in root nodules. Sometimes sporangia (iii) are also formed in older parts of the root nodules. In Alnus nodules symbiotic vesicles are spherically 'present address: Department of Plant Science, University of Manitoba, Winnipeg, Man., Canada R3T 2N2. Printed ~n Canada / Imprime au Canada shaped and 3-5 pm in diameter (Lalonde and Knowles 1975), and contain the oxygen-labile nitrogenase (Huss-Dane11 and Bergman 1990). The whole microorganism is surrounded by a multilayered lipid envelope, which appears to have more layers around the symbiotic vesicle and its stalk (Abeysekera et al. 1990). The envelope is believed to function as a barrier against oxygen diffusion. Septa are formed across symbiotic vesicles and the number of septa increase with increasing age and development. Accordingly, the mature symbiotic vesicle appears to be a compartmentalized structure. Inside the root nodules Frankia is completely dependent on host photoassimilates as the source of carbon and energy, but the nature of the compounds transported is not known (Huss-Dane11 1990). So-called symbiotic vesicle clusters, con- sisting of symbiotic vesicles and hyphae, can be obtained from Alnus root nodule homogenates in high purity (Vikman and Huss-Dane11 1987b). In such preparations a Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by 99.250.15.21 on 11/26/14 For personal use only.

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Page 1: The symbiotic vesicle is a major site for respiration in               Frankia               from               Alnus incana               root nodules

The symbiotic vesicle is a major site for respiration in Frankia from Alnus incana root nodules

PER- AKE VIKMAN'

Department of Plant Physiology, University of Umeh, S-901 87 Umeh, Sweden

Received October 28, 1991

Revision received February 19, 1992

Accepted February 2 1, 1992

VIKMAN, P.-A. 1992. The symbiotic vesicle is a major site for respiration in Frankia from Alnus incana root nodules. Can. J. Microbiol. 38: 779-784.

A technique was developed for preparation of Frankia symbiotic vesicles, free of hyphae. The symbiotic vesicles were isolated by isopycnic centrifugation of disrupted Frankia vesicle clusters prepared from root nodules of Alnus incana (L.) Moench. Activities in symbiotic vesicles were compared with activities in intact symbiotic vesicle clusters on a total protein basis. Respiratory capacity was tested with 6-phosphogluconate, malate + glutamate, and NADH as added substrates. With all three substrates, specific respiration was doubled after symbiotic vesicle isolation. Nitrogenase was used as a symbiotic vesicle specific marker and its specific activity increased similarly to respiration. Activities of four respiratory enzymes were assayed on crude cell-free extracts obtained after sonication of symbiotic vesicle prep- arations. According to the increased specific rates after symbiotic vesicle isolation, NAD + :6-phosphogluconate dehy- drogenase (EC 1.1.1.44) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) were mainly localized in symbiotic vesicles. NAD + :malate dehydrogenase (EC 1.1.1.37) and glutamate-oxaloacetate transaminase (EC 2.6.1.1) were also present in symbiotic vesicles, but their specific activities were not increased compared with the symbiotic vesicle clusters. The magnitude of increased activities suggested that the symbiotic vesicle is a major site for hexose respiration in symbiotic Frankia. An apparent Km for 0, between 20 and 30 pM indicated that symbiotic vesicles in symbiotic vesicle clusters have a restricted oxygen diffusion rate.

Key words: Frankia, symbiotic vesicles, respiration, nitrogenase, oxygen diffusion.

VIKMAN, P.-A. 1992. The symbiotic vesicle is a major site for respiration in Frankia from Alnus incana root nodules. Can. J. Microbiol. 38 : 779-784.

Une technique de preparation de vesicules symbiotiques libres d'hyphes de Frankia a ete mise au point. L'isolement de telles vesicules a ete obtenu par centrifugation isopycnique de groupe de vesicules disloquees de Frankia, preparees a partir de nodosites racinaires de Alnus incana (L.) Moench. Les activites dans ces vbicules ont ete comparees a celles des groupes de vesicules intactes, sur la base des proteines totales. Les substrats 6-phosphogluconate, malate + glutamate, et NADH ont servi a tester les capacites respiratoires. Chez les vesicules isolees, la respiration specifique a double avec les trois substrats. La nitrogenase a ete utilisee comme marqueur specifique de vesicules symbiotiques et son activite specifique a augmente la respiration de faqon similaire. Les activites de quatre enzymes reliees a la respiration ont ete testees sur des extraits acellulaires bruts, apres sonication des preparations de vesicules. D'apres les taux specifiques accrus apres l'isolement des vesicules, la NAD + :6-phosphogluconate deshydrogenase (EC 1.1.1.44) et la glucose-6-phosphate deshydrogenase (EC 1.1.1.49) ont ete localisees principalement dans les vesicules symbiotiques. La NAD + :malate deshydrogenase (EC 1.1.1.37) et la glutamate-oxaloacetate transaminase (EC 2.6.1.1) ont aussi ete presentes dans les vesicules isolees, mais leurs activites specifiques n'ont pas ete augmentees par comparaison a celles des groupes de vesicules. L'etendue de l'augmentation des activites suggere que les vesicules sont le siege principal de la respiration des hexoses chez le Frankia en symbiose. Un Km apparent entre 20 et 30 pM pour 1'0, indique que les vesicules des groupes de vesicules symbiotiques ont un taux restreint de diffusion de l'oxygene.

Mots elks : Frankia, vesicules symbiotiques, respiration, nitrogenase, oxygene, diffusion. [Traduit par la redaction]

Introduction The actinomycete Frankia forms a root nodule symbiosis

with certain, so-called actinorhizal plants. With the enzyme nitrogenase, Frankia can reduce atmospheric N2 and supply its host with combined nitrogen. Three morphological forms of Frankia are present in Alnus root nodules (Newcomb and Wood 1987). Multicellular filaments of a vegetative type are called hyphae (i). Symbiotic vesicles (ii) differentiate on hyphal side branches and are subtended by a short stalk. In culture the corresponding structure is referred to as "Frankia vesicle," while the term "symbiotic vesicle" is denoted to those found in root nodules. Sometimes sporangia (iii) are also formed in older parts of the root nodules. In Alnus nodules symbiotic vesicles are spherically

'present address: Department of Plant Science, University of Manitoba, Winnipeg, Man., Canada R3T 2N2. Printed ~n Canada / Imprime au Canada

shaped and 3-5 pm in diameter (Lalonde and Knowles 1975), and contain the oxygen-labile nitrogenase (Huss-Dane11 and Bergman 1990). The whole microorganism is surrounded by a multilayered lipid envelope, which appears to have more layers around the symbiotic vesicle and its stalk (Abeysekera et al. 1990). The envelope is believed to function as a barrier against oxygen diffusion. Septa are formed across symbiotic vesicles and the number of septa increase with increasing age and development. Accordingly, the mature symbiotic vesicle appears to be a compartmentalized structure.

Inside the root nodules Frankia is completely dependent on host photoassimilates as the source of carbon and energy, but the nature of the compounds transported is not known (Huss-Dane11 1990). So-called symbiotic vesicle clusters, con- sisting of symbiotic vesicles and hyphae, can be obtained from Alnus root nodule homogenates in high purity (Vikman and Huss-Dane11 1987b). In such preparations a

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780 CAN. J . MICROBIOL. VOL. 38, 1992

number of metabolic enzymes have been studied. Several enzymes for sugar degradation, like trehalase (Lopez and Torrey 1985a), hexokinase, glucose-6-phosphate dehydro- genase (GPDH, EC 1.1.1.49), 6-phosphogluconate dehydro- genase (PGDH, EC 1.1.1.44), and pyruvate kinase, were present (Lopez and Torrey 198%; Vikman and Huss-Dane11 1987a), as well as glutamate-oxaloacetate transaminase (GOT, EC 2.6.1.1) and some TCA-cycle enzymes like isocitrate dehydrogenase, fumarase, succinate dehydroge- nase, and malate dehydrogenase (MDH, EC 1.1.1.37) (Akkermans et al. 1981).

Respiration of symbiotic vesicle clusters, measured as O2 consumption, was supported by added substrates like sucrose, trehalose, maltose, glucose, fructose (Lopez et al. 1986), 6-phosphogluconate, and glucose-6-phosphate (Vikman and Huss-Dane11 1987a). Respiration was also sup- ported by NADH (Akkermans and Roelofsen 1980; Vikman and Huss-Dane11 1987a) or the combined addition of malate and glutamate (Akkermans et al. 198 1 ; Vikman and Huss- Dane11 1991). Nitrogenase activity in symbiotic vesicle clusters can be supported by added ATP and reductant, but there is presently no report on substantial nitrogenase activ- ity supported by Frankia respiration in symbiotic vesicle clusters. Neither is it known how any of these substrates enters Frankia cells.

Interpretation of the role of observed activities has been complicated, since symbiotic vesicle clusters contain both hyphae and symbiotic vesicles. It is unclear whether aerobic respiration can occur inside the symbiotic vesicle, where nitrogenase must be protected from oxygen. Frankia vesicles have been isolated by isopycnic centrifugation (Noridge and Benson 1986) or differential centrifugation (Tisa and Ensign 1987) from disrupted N2-grown cultures. The Frankia vesicle preparations have been used for studies on nitrogenase and other enzymes for nitrogen metabolism (Noridge and Benson 1986; Tisa and Ensign 1987), devel- opmental potential (Schultz and Benson 1989), fatty acid composition (Tunlid et al. 1989), and oxygen radical scaveng- ing enzymes (Puppo et al. 1989).

However, cultured Frankia is not comparable with the symbiotic stage, e.g., in pathways for lipid degradation (Huss-Dane11 et al. 1982) and respiration of organic acids (Lopez et al. 1986). Symbiotic vesicles were therefore isolated in the present study, and the degradation of phosphorylated hexoses was studied as respiratory capacity and enzyme activities. Because of the suggested operation of a dicarboxylate shuttle in symbiotic Frankia (Akkermans et al. 198 1 ; Vikman and Huss-Dane11 1991) the metabolism of malate and glutamate was also studied in the isolated symbiotic vesicles.

Materials and methods Plant material, Frankia, and growth conditions

Cloned plants of grey alder (Alnus incana (L.) Moench) from southern Sweden (59"37'N, 12'58'E) were inoculated with crushed nodules containing Frankia of a local source, characterized as hav- ing spores but not showing hydrogen uptake activity in symbiosis (Sellstedt and Huss-Dane11 1986). The plants were grown accord- ing to Vikman et al. (1990) and used 11-17 weeks after planting, when the plants were slightly branched and 71-101 cm high.

I<oo~ nodules

SJ mbiotic Sonicat ion Vcsiclc c l~~s tcrs ('entrifugation

1)isrupted s~ mbiotic \ csicle clusters

( dVC )

-+ T,

Sonication - - ('cntrifugation v e

70 '-4

",

FIG. 1. General outline of the preparation procedure and the resulting fractions. T, top band; V, symbiotic vesicle fraction; P, gradient pellet. The subscript e denotes crude cell-free extracts.

anaerobically from root nodules, by homogenization and washing of the homogenate on a 20-pm filter. The protocol was as described by Vikman and Huss-Dane11 (1991), using the two washing buf- fers WBl and WB2 (WBl: 0.05 M Hepes-NaOH (Sigma) pH 8.0, 0.6 M sucrose, 4% (w/v) polyvinylpyrrolidone (PVP) - K25 (Roth), 0.1 M KC1, 2 mM EDTA (Merck), 5 mM dithiothreitol (DTT) (Merck), 10 mM Na2S204; WB2: 0.05 M HEPES-NaOH pH 7.5, 0.25 M sucrose, 4% (w/v) PVP, 0.1 M KCl), with the following modifications. WB2 was supplemented with 2 mM Na2S20, for measurements of respiration, while for cell-free enzyme activity WB2 was replaced by WBl without Na2S204. For assays of nitrogenase activity the concentration of Na2S204 in WBl was increased to 20 mM.

To free the symbiotic vesicles from hyphae, 15-25 mL symbiotic vesicle cluster suspension, obtained from the nodules of one or two plants and corresponding to 0.14-0.33 g (fresh weight) nodules/mL, was passed through a Yeda pressure cell (Yeda Scientific Instruments, Rehovot, Israel) under an N2 pressure of 35 kg/cm2. From the resulting suspension (dVC) 3-4 mL was loaded in 18-mL Ultra-Clear tubes (Beckman) on top of 5.5 mL 70% sucrose (w/w, refractive index (n) = 1.465) and 5.5 mL 60% sucrose (w/w, n = 1.442), both in 0.10 M Hepes-NaOH pH 7.5 with 2 mM EDTA. Refractive index was determined on the sucrose solutions by using a B&S model 60/SR refractometer with a sodium lamp (Bellingham and Stanley, Tunbridge Wells, Great Britain).

For measurements of respiration and nitrogenase activity the sucrose solutions were thoroughly flushed with argon and supplied with 2 mM DTT and 20 mM Na2S204. For further 0, exclusion an overlay consisting of 2 mL 0.5 M Na2S204 (in 50 mM Hepes- NaOH, pH 7.5) and 1 mL mineral oil (Labassco, Gothenburg, Sweden) was loaded on top of the samples. After centrifugation

Isolation of symbiotic vesicles at 100 000 x g for 30 min at 4°C in a Beckman SW 28.1 rotor, An outline of the preparation of symbiotic vesicles is given in fractions were washed twice by centrifugation at 13 000 x g (5 min

Fig. 1. Frankia symbiotic vesicle clusters (VC) were prepared at 4°C) and finally resuspended in 1 mL of appropriate buffers

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VIKMAN 78 1

as described above. The cellular composition of all fractions was examined in an Olympus BH2-PC phase-contrast microscope (Olympus Optical Co. Ltd., Tokyo, Japan).

For assays of cell-free enzyme activity (Fig. 1) the fractions were finally resuspended in buffer (50 mM Hepes-NaOH pH 7.5, 2 mM DTT, and 3 mM MgCl,). All cells were ruptured by 10 rnin sonication on ice with a Branson cell disruptor (Branson Sonic Power, Danbury, Conn., U.S.A.) supplied with a microtip and set at output 7, with 10% pulsed duty cycle. After 15 min centrifuga- tion (15 000 g, 4°C) the clear supernatant was withdrawn (T,, V,, P,), stored at - 20°C for a few days, and diluted 5-250 times with buffer before use.

Measurements of activity Immediately before a measurement of respiration Na,S,O, was

removed from the suspension by centrifugation at 13 000 x g (5 min, 4°C) and resuspending in argon-flushed WB2. Respiration was determined with an oxygen electrode as cyanide-sensitive 0, consumption (Vikman and Huss-Dane11 1987a) supported by three different substrates: (i) 5 mM 6-phosphogluconate plus 2 mM NAD+, (ii) 50 mM glutamatt plus 50 mM malate plus 2 mM NAD +, or (iii) 1.5 mM NADH. These substrate concentrations were well above saturating (Vikman and Huss-Dane11 1991). Varied 0, concentrations were obtained by first equilibrating the suspen- sion around 253 pM 0, with air bubbled a few minutes through the reaction chamber of the oxygen electrode. When the chamber was closed, Frankia respiration gradually reduced the 0, concen- tration to near zero within 15-20 min. Both 0, concentration and the rate of its consumption were calculated from the same oxygen electrode recording.

Substrate-saturated enzyme activity in crude cell-free extracts was determined on a Beckman DU-8 spectrophotometer. MDH and GOT were assayed according to Vikman and Huss-Dane11 (1991) and PGDH and GPDH according to Vikman and Huss- Dane11 (1987a) except that the concentration of 6-phosphogluconate was 4 mM and NADP + 3 mM. Because the GPDH rate was con- tinuously increasing after the addition of substrates, the rate was always determined over the first 5 min.

Nitrogenase activity in isolated symbiotic vesicles and corre- sponding vesicle clusters was determined as C2H2 reduction, as described by Vikman et al. (1990). Rates were linear for at least 45 min of incubation.

Protein determination Total soluble protein in all cell-containing fractions was deter-

mined with a BCA-kit (Pierce) according to Vikman et al. (1990). Since the cell-free extracts contained DTT, which interferes with this method, the following protocol (modified from Hill and Straka, 1988) was used: 50 pL sample was mixed with 50 pL 0.1 M iodoacetamide (Sigma) and incubated 15 min at 37OC before adding 2 mL bicinchoninic acid (BCA) reagent and incubating at 37OC for 75 min. The bovine serum albumin (BSA) protein standards also contained 2 mM DTT and were treated by the same protocol as the samples.

Results - - - . . - - - .

Preparation of symbiotic vesicles When a Frankia symbiotic vesicle cluster preparation was

passed through the Yeda press cell, most symbiotic vesicle clusters were disrupted. Light microscopy revealed that the resulting suspension (dVC) contained free symbiotic vesicles,

NADH O P G I

disrupted symb-iotic symbiotic symb~otic vestcle

vestcle cluster vestcle cluster fraction (VC) (dVC) (V)

FIG. 2. Respiratory capacity with three different substrates added to different fractions of the symbiotic vesicle isolation pro- cedure (cf., Fig. 1). (A) Recovery expressed as the relative respi- ratory rate (%) compared with symbiotic vesicle clusters of the same preparation. (B) Specific respiration on a total protein basis. x k SE (n = 3). ma1 + glu, malate plus glutamate.

was highly enriched in symbiotic vesicles and contained no symbiotic vesicle clusters. Nearly all vesicles in the symbiotic vesicle fraction were phase bright and their interior appeared dark, indicating that the cytoplasmic content was retained. Most symbiotic vesicles were still connected to a short hyphal piece of approximately the same length as the diameter of the symbiotic vesicle, but no other hyphal material was observed in the symbiotic vesicle fraction.

The top band (T) at the interface between WB1 and 60% sucrose mainly contained symbiotic vesicles still held together in clusters of varying size. The gradient pellet (P) contained a mixture of all the structures observed in the disrupted symbiotic vesicle clusters before centrifugation, including free symbiotic vesicles. Most symbiotic vesicles in the pellet appeared similar to those observed in the symbiotic vesicle band, but the pellet also contained symbiotic vesicles that appeared damaged, as judged from their rugged sur- face and dull phase brightness. Replacing the 70% sucrose with 75% sucrose did not further improve the yield or purity of the symbiotic vesicle fraction.

a few symbiotic vesicle clusters, some host cell-wall mate- Respiratory capacity rial, and some starch-like crystals. No free hyphae could be The respiratory capacity on a total protein basis was not recognized. changed by disrupting the symbiotic vesicle clusters (dVC,

After isopycnic sucrose gradient centrifugation of the Fig. 2B). The symbiotic vesicle fraction (V) from anaerobic disrupted symbiotic vesicle clusters, two bands and a pellet sucrose gradients also retained the capacity to respire added were formed. The middle band (V) at the interface between 6-phosphogluconate, NADH, and malate + glutamate. 60 and 70% sucrose (called the symbiotic vesicle fraction) Regardless of which substrate was used, the specific respi-

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CAN. J. MICROBIOL. VOL. 38. 1992

TABLE 1. Nitrogenase activity (x f SE, n = 3) in vitro in fractions of sucrose gradients loaded with ruptured Frankia symbiotic vesicle clusters

Vesicle clusters Vesicle fraction Gradient pellet (VC)" (V)" (PI"

Recovery of nitrogenase activityb 100 12+ 1 44+ 1 Specific nitrogenase activitybc 100 166 + 24 8 7 + 9

'Letters within parentheses refer to Fig. 1. b ~ a t e s expressed as percentage of the rate in intact vesicle clusters from the same preparation. 'Specific activity based on total protein in the fraction; 100% was 2.5-9.3 nmol CzHs.mg protein-'.min-'

O 2 concentra t ion ( p M )

FIG. 3. Substrate-saturated respiration of 6-phosphogluconate plus NAD + by symbiotic vesicle clusters as a function of the con- centration of dissolved oxygen in the surrounding buffer. Rates were measured at different oxygen levels when respiration decreased the oxygen concentration down to zero in two identical experiments (MU). Inset shows Lineweaver-Burk plot of the pooled experiments.

ratory rate was doubled in the symbiotic vesicle fraction compared with symbiotic vesicle clusters (V/VC, Fig. 2B). About 40% of the total respiration in intact symbiotic vesicle clusters was recovered in the symbiotic vesicle fraction (V/VC, Fig. 2A).

Oxygen-limited respiration of 6-phosphogluconate in - -

symbiotic vesicle clusters (VC) showed saturation kinetics for O2 (Fig. 3). The rate was saturated with ca. 120 PM O2 in the suspension medium. A Lineweaver-Burk plot was monophasic without breaks (Fig. 3, inset), which is in accordance with localization of 6-phosphogluconate respira- tion in a compartment with a uniform resistance to the dif- fusion of oxygen. The apparent Km for O2 was estimated to 20-30 pM from the same plot.

Enzyme activities The specific activity of nitrogenase was ca. 1.7 times

higher in the symbiotic vesicle fraction compared with symbiotic vesicle clusters (V/VC, Table 1). About 12% of the total nitrogenase activity in intact symbiotic vesicle clusters was recovered in the symbiotic vesicle fraction (V/VC, Table 1). Cell-free extracts of the symbiotic vesicle fraction (Ve) had substantial activity of all the respiratory enzymes studied (Fig. 4). Similar to nitrogenase, the spe- cific rates of PGDH and GPDH were higher (t-test, p < 0.05) in the symbiotic vesicle fraction compared with

PGDH

GPDH

GOT

MDH

symbiot ic gradient symbiot ic v e s ~ c l e c luster pe l l e t vesic le f ract ion

(VCe 1 ( P e 1 ( V e 1

FIG. 4. Enzyme activities in crude cell-free extracts of symbiotic vesicle clusters and two fractions of the sucrose gradient loaded with ruptured symbiotic vesicle clusters (cf., Fig. 1). (A) Relative rates expressed as percentage of the rate in intact symbiotic vesi- cle clusters from the same preparation. (B) Specific rates on a total protein basis. x + SE (n = 3-10).

the rates obtained with extracts from intact symbiotic vesicle clusters (Ve/VCe, Fig. 4B).

The symbiotic vesicle fraction expressed 9-24% of the enzyme activity in symbiotic vesicle clusters (Ve/VCe), while most of the activity was recovered in the gradient pellet (Fig. 4A). Compared with intact symbiotic vesicle clusters the recovery of enzyme activity and protein in the band formed between WB1 and 60% sucrose was less than 1% (Te/VCe, data not shown). Still about 20-40% of .the activ- ity was not regained in any of the fractions studied, proba- bly as a result of losses in discarded washing buffers.

Discussion Symbiotic vesicles were isolated in high purity, with

increased specific activities of nitrogenase, respiratory O2

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VIKMAN 783

consumption, and the respiratory enzymes PGDH and GPDH. The symbiotic vesicles appeared structurally intact in the light microscope. Higher sucrose densities were needed to isolate symbiotic vesicles in the present work compared with those used in isolation of Frankia CpIl vesicles from culture (Noridge and Benson 1986). The band of symbiotic vesicles was formed where particles with a buoyant density between 1.29 and 1.35 g cm -' should accumulate in the gradient. The gradient pellet also contained many free sym- biotic vesicles, as well as nitrogenase activity (Table 1). This indicates the presence of a subpopulation of symbiotic vesicles with a higher buoyant density, resulting from a dif- ferent developmental stage or more structural damage during the preparation.

No free hyphal material was recognized in any fraction, suggesting that hyphae were selectively ruptured by the Yeda press treatment. This was also found during vesicle isola- tion from cultured Frankia EANI,,, after French press treatment (Tisa and Ensign 1987). In contrast, hyphae accu- mulated in the gradient pellet when a polytron was used for mycelial disruption in Frankia CpIl (Noridge and Benson 1986). Previous workers have reported on a short hyphal piece attached to the isolated Frankia vesicles (Tisa and Ensign 1987; Puppo et al. 1989; Schultz and Benson 1989). This was also observed in the present work with symbiotic material. The length of the attached hyphae corresponds to the so-called vesicle stalk, suggested to be part of the sym- biotic vesicle (Lalonde and Knowles 1975; Newcomb and Wood 1987; Abeysekera et al. 1990). The stalk is surrounded by the same type of lipid envelope as the rest of the sym- biotic vesicle, which could explain why it remains attached during their isolation. The stalk could be used for aerobic metabolism while nitrogenase is held active in more anaerobic parts of the symbiotic vesicle. The correlation between septation of symbiotic vesicles and presence of nitrogenase also suggests a role for compartmentalization of symbiotic vesicles (Newcomb and Wood 1987; Huss-Dane11 and Bergman 1990). This hypothesis is supported by the observa- tion of fewer lipid layers in the envelope surrounding the stalk and basal parts of the symbiotic vesicle (Abeysekera et al. 1990). In addition, Huss-Dane11 and Bergman (1990) found more immunogold labelling of nitrogenase in the apical parts of symbiotic vesicles compared with the basal parts, but stalks were not specifically studied. However, symbiotic vesicles were not separated from their stalks in the present study. The possibility of further Frankia com- partmentalization awaits testing.

Localization of respiration The increased specific respiratory O2 consumption in

symbiotic vesicles compared with vesicle clusters (Fig. 2B) shows that symbiotic vesicles, or some part of them, have the capacity for aerobic respiration. Because this increase was similar with all substrates tested, it is likely that they were all respired in the same compartment, i.e., the symbiotic vesicles. It is unlikely that the increase in respiration was due to altered capacity for uptake of added substrates, since enzyme activities in cell-free extracts of the isolated sym- biotic vesicles also increased (Fig. 4B). Increased permeabil- ity of isolated vesicles could explain why respiration increased slightly more than enzyme activities.

Nitrogenase is presently the only enzyme known as exclu- sively localized in symbiotic vesicles of Frankia from

A . incana (Huss-Dane11 and Bergman 1990). Nitrogenase activity was therefore used in this study as a marker, show- ing how specific enzyme activity in the symbiotic vesicle increases when hyphae are detached. Nitrogenase activity was 1.7 times as high in the symbiotic vesicle fraction as in intact symbiotic vesicle clusters (V/VC, Table 1). The cor- responding increase in activity of PGDH and GPDH (V,/VC,) was 1.3 and 1.6, respectively (Fig. 4B), suggesting that these hexose-degrading enzymes were mainly localized in the symbiotic vesicles.

The observed increase in specific nitrogenase activity (Table 1) was in accordance with what could be expected from an enzyme exclusively localized in symbiotic vesicles. Vikman and Huss-Dane11 (1987b) showed that symbiotic vesicles made up 75% of the particle volume in a symbiotic vesicle cluster. The specific activity in isolated symbiotic vesicles would hence be increased by a factor of 1.33 (= '/0.75) compared with symbiotic vesicle clusters, provided that the protein content is uniformly distributed. Respiration (Fig. 2B) and the activities of hexose-degrading enzymes (Fig. 4B) increased by the same factor or more when hyphae were removed, again suggesting that respira- tion was mainly localized in symbiotic vesicles. In cultured Frankia Cpll specific nitrogenase activity increased as much as 100 times after Frankia vesicle isolation (Noridge and Benson 1986). The lower frequency of Frankia vesicles in cultured Frankia compared with symbiotic vesicle clusters probably explains why the specific activity of a vesicle enzyme increases more when Frankia vesicles are isolated from cultures.

The specific respiration supported by malate + glutamate also increased after isolation of symbiotic vesicles (Fig. 2B). The magnitude of this increase was comparable with that found with 6-phosphogluconate and NADH. This suggests that the respiration of malate + glutamate is also mainly localized in symbiotic vesicles. Still, the specific rates of MDH and GOT were lower in the symbiotic vesicle frac- tion (Fig. 4B). These contradictory results could be an effect of enzyme inactivation during preparation. The excep- tionally high yield and large variation of GPDH activity in the gradient pellet (P,, Fig. 4A) is questionable. Consider- ing both the unstable rates in the GPDH assay and the aber- rant yield, strong conclusions based on GPDH activities should be avoided.

Oxygen diffusion The kinetics of respiratory O2 consumption suggests that

respiration of 6-phosphogluconate was localized in a single type of compartment (Fig. 3), where oxygen diffusion could be limiting. The symbiotic vesicle, or part of it, is likely to be this compartment, since 6-phosphogluconate supported respiration (Fig. 2) and specific activity of PGDH was higher in isolated symbiotic vesicles than in vesicle clusters (Fig. 4B).

The apparent respiratory Km for external O2 has been used to study the cellular localization of respiration. Using mutants of Anabaena, Murry and Wolk (1989) showed that the high Km was due to the presence of an oxygen diffu- sion barrier in the heterocysts. Although no direct data are present, the oxygen diffusion resistance is believed to be higher in Frankia vesicles compared with hyphae. In the present study the apparent Km for O2 was between 20 and 30 pM (Fig. 3), which could reflect the diffusion resistance

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of the symbiotic vesicle envelope in symbiotic Frankia from A . incana. This Km is between the two values, 1 and 215 pM, suggested in cultured Frankia Ar13 to reflect respiration in hyphae and Frankia vesicles, respectively (Murry et al. 1984). The Km for free cytochrome is usually less than 0.1 pM 02. If this is also true for Frankia cytochrome, the observed Km would still reflect a rather high diffusion resistance. The thickness of the symbiotic vesicle envelope, probably corresponding to the diffusion barrier, increases in response to increased surrounding 0 2

both in cultured Frankia HFPCcI3 (Parsons et al. 1987) and in symbiotic Frankia in Alnus nodules (Abeysekera et al. 1990). Since Murry et al. (1984) used stirred cultures adapted to high O2 levels, a high apparent Km was coherent. A lower apparent Km for respiration in symbiotic Frankia is plausible, since for example, nodule respiration may reduce Po2 around Frankia and may result in a less pronounced diffusion barrier.

In conclusion, the present data from symbiotic Frankia show that 6-phosphogluconate and probably also the other tested substrates can be respired in symbiotic vesicles or their stalks. The high apparent Km for O2 may reflect the oxygen diffusion resistance of the symbiotic vesicle. Substantial respiration of these substrates in the hyphae seems unlikely but cannot be excluded.

Acknowledgements This study was financially supported by the Swedish

Natural Science Research Council (grant to Dr. K. Huss- Danell) and the Swedish Council for Forestry and Agricul- tural Research. The author thanks Alf Ekblad for introduc- ing him to factorial experimental designs and Dr. K. Huss- Danell for valuable comments on the manuscript.

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