APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1976, p. 859-863 Copyright © 1976 American Society for Microbiology
Vol. 31, No. 6 Printed in U.S.A.
Improved Method for Preparing Anaerobic Bacteroid Suspensions of Rhizobium leguminosarum for the Acetylene Reduction Assay J. VAN STRATEN*
AND
W. ROELOFSEN
Laboratory of Microbiology, Agricultural University, Wageningen, The Netherlands
Received for publication 20 January 1976
A method using ethylenediaminetetraacetate (EDTA) and toluene-treated bacteroid suspensions for the acetylene reduction assay is described. The high level of acetylene reduction by these bacteroids is comparable to that of intact plants. Reproducibility of the EDTA-toluene treatment is, on the average, within 5%. Preliminary experiments with soybeans indicate that the EDTAtoluene method might be applicable to other legumes as well. pea
It is generally assumed that intact legume plants reduce more acetylene than do detached "parts" such as nodulated roots, detached nodules, or bacteroids. Unfortunately, acetylene reduction data relating these different fractions are very few. Hardy et al. (3) reported that nodulated soybean roots reduced more acetylene than did detached nodules. Mague and Burris (10) compared the acetylene reduction of intact soybean plants (100%) with that of nodulated roots and detached nodules, and found that the nodulated roots and the detached nodules showed only 46 and 23% of the activity of the intact plant, respectively. On the other hand, Akkermans (Ph.D. thesis, Univ. of Leyden, Leyden, The Netherlands, 1971) reported that, after removal of the shoot, the remaining nodulated pea root system reduced as much acetylene as it did before removal. Data comparing nitrogenase activity of anaerobically washed bacteroids with that of whole plants are virtually nonexistent. From work done previously in this laboratory (the late van den Berg, unpublished data), it appears that washed pea bacteroids in the presence of dithionite and adenosine 5'-triphosphate reduce acetylepe at a rate that is about 10% of that of the intact plant or less. It has been suggested that this low level of nitrogenase activity may be due to permeability limitations, possibly of the added adenosine 5'-triphosphate, posed by the membranes of the bacteroids. Treatment with ethylenediaminetetraacetate (EDTA) is known to increase permeability of gram-negative bacteria (2, 6-8). Toluene is also used in enzyme assays (j3-galactosidase: Herzenberg [4]; alkaline phosphatase: Levinthal et al. [9] to improve the accessibility of these enzymes to exogenous substrates.
This paper reports on the use of EDTA and toluene to increase acetylene reduction by washed bacteroid suspensions. MATERIALS AND METHODS Plant material and preparation of bacteroid suspensions. Pea (Pisum sativum L. cv. Rondo) plants, inoculated with Rhizobium leguminosarum strain PRE, were grown in gravel in nitrogen-free nutrient solution in batches of 40 at 18 to 20 C in a growth chamber and harvested after 27 to 31 days. Freshly picked nodules were pressed under anaerobic conditions at 0 to 4 C in 0.05 M tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.2), containing 2.5 mM MgCl2, 20 mM Na2S2O4, and 4% soluble polyvinyl-pyrrolidone, using a Bergersen press (1). Nodule concentration in all experiments was 80 mg of nodule tissue per ml of buffer. Bacteroid suspensions were obtained by centrifuging the nodule brei at 5,000 x g for 10 min at 3 to 5 C, suspending, and washing the pellet with buffer, followed by a second centrifugation. Finally, the pellet was resuspended in the original volume of buffer. The bacteroid suspensions were kept on ice until further use. Nitrogenase activity. Nitrogenase activity was determined with the acetylene reduction assay (3). The reaction was carried out in a shaker bath at 24 C in 5.6-ml vials closed with Suba-seal closures (Rubber B. V., Hilversum, Holland). The shaking rate was 144 strokes/min. The reaction mixture contained 0.5 ml of bacteroid suspension, 50 ,umol of tris(hydroxymethyl)aminomethane - hydrochloride (pH 7.2), 7.5 ,umol of MgCl2 (or the amount indicated), 11.7 ,umol of creatine - P, 2.8 ,mol of adenosine 5'-triphosphate, and 0.03 mg of creatine phosphokinase (Boehringer Mannheim GmbH) in a total volume of 1 ml. The gas phase was 10% acetylene in argon. After 10 min of incubation, 100 Al of the gas phase was removed and analyzed for ethylene on a gas chromatograph (Multigraph-417, Packard, Becker) equipped with a hydrogen flame detector and fitted with a glass column (110 cm by 3 mm) packed with Porapak R (80 to 100 mesh). The column
859
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APPL. ENVIRON. MICROBIOL.
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temperature was 50 C. Results are expressed as micromoles of ethylene per gram of fresh weight nodule per hour.
RESULTS AND DISCUSSION Preliminary experiments with nodule brei showed that treatment with EDTA and especially with toluene or EDTA plus toluene resulted in substantially higher ethylene values (Table 1). Mg2+ concentration. According to Leive (8), divalent cations prevent EDTA action when present in the same or higher concentrations. The buffer used thus far had been 2.5 mM in Mg2+ and, since EDTA was only 1 mM, the effect of Mg2+ on the EDTA-toluene treatment was investigated. In this experiment bacteroid suspensions were prepared at 20 C instead of at 0 to 4 C. Aliquots (1 ml) of bacteroid suspensions were incubated anaerobically with the same volume of a 2 mM EDTA solution in 25-ml test tubes at 24 C. The bacteroid suspensions containing EDTA were 0, 2.5, and 5 mM in Mg2+. The 0 mM Mge+ suspension was attained by treating part of the brei with Mg-free buffer and EDTA. In addition to testing the effect of different Mg2+ concentrations on the EDTAtoluene treatment, the effect of Mg2+ on acetylene reduction itself was studied. After 30 min, 0.2 ml of toluene was added and the mixture was shaken at 190 strokes/min in a 24 C water bath for 1 min. The test tube was removed from the water bath, and the two phases were left to separate. After 3 min, 0.5 ml of the aqueous phase was removed and used for the acetylene reduction test. As can be seen from the results shown in Fig. 1, acetylene reduction should not be carried out at 2.5 mM Mg2+. The positive effect of higher Mg2+ concentrations was much more pronounced with EDTA-toluene-treated bacteroids than with bacteroids without EDTA and toluene treatment. Mg2+ was also necessary for the EDTA treatment, but in this case 2.5 mM was better than 5 mM Mg2+. It was therefore decided to keep the Mg2+ concentration at 2.5 mM during the EDTA treatment and TABLE 1. Effect of EDTA andlor toluene treatment on acetylene reduction by nodule brei AmoI of C2H./g of Brei prepared with:
Buffer
Buffer + EDTA" Buffer + tolueneb Buffer + EDTA" + tolueneb "Final concentration, 1 mM. A total of 0.2 ml/ml of brei.
fresh wt nodule per h 1,438
1,832
14,179 19,057
16 k
0'c
12
-
a
X-------x
_ 8 el
f0
0
0
2
L
Mg'++ (moles/acetylene
6 8 reduction assoy)
10
FIG. 1. Acetylene reduction by EDTA-toluenetreated bacteroid suspensions at varying Mg2+ concentrations during both EDTA treatment and acetylene reduction assay. Mg2+ concentration during EDTA treatment: x, O mM; *, 2.5 mM; A, 5 mM. 0, Bacteroids without EDTA-toluene treatment.
to change to 7.5 mM for the acetylene reduction test. Consequently, our results as to the Mg2+EDTA ratio did not agree with those of Leive (8) for Escherichia coli. The absolute values in
this experiment were rather low, which may have been due to the way the bacteroids were prepared. Reactivation time. Before the bacteroids, which were stored at 0 C, could be used, they had to be kept for some time at the temperature at which the incubation treatments (24 C) were carried out in order to become reactivated. EDTA and toluene were added simultaneously after the reactivation period. No separate EDTA incubation period was included since it had been found that high ethylene values were obtained without such a separate incubation. The time it took the bacteroid suspension to obtain maximum ethylene production (reactivation time) was found to be rather variable (Fig. 2). Also, the response during the first 5 min was quite different. This variability in time to reach maximum activity was not merely a question of reaching 24 C since it took the bacteroid suspension less than 5 min to reach this temperature. Moreover, when the bacteroids were kept at 20 C instead of 0 C, the reactivation time was still 20 min. The variability in reactivation time could, to a large extent, be ascribed to the procedure used to prepare the nodules for pressing. The broken lines (three experiments) represent nodules that were picked, collected on a wet filter paper, blotted dry, weighed, and used for pressing. The solid line (average of four experiments) represents
VOL. 31, 1976
PREPARATION OF ANAEROBIC BACTEROIDS
861
nodules that were rewetted and left for 20 min 30 F under a wet filter paper after being weighed. Rewetting the nodules also influenced the stability of the bacteroid suspension at 0 C with respect to nitrogenase activity (Fig. 3). This 20 procedure was continued in subsequent experiments. The relationship between reactivation 0 time and stability is currently being investigated. EDTA incubation time. Although high eth- -Li210 ylene values could be obtained by adding EDTA together with toluene, it was not known to I what extent the EDTA reaction was completed in such a short time. This was tested by incubating bacteroids with EDTA for different pe180 120 60 riods of time, immediately followed by the addiStorage time at 0 °C (min) tion of toluene. It was found, however, that no FIG. 3. Effect of nodule pretreatment on the acetysignificant increases in ethylene production ability of bacteroids during storage at could be obtained by longer incubations with 0lene-reducing C, as measured after reactivation and EDTA-toluEDTA. As a result, EDTA was added together ene treatment. Symbols: solid line, nodules rewetted, with toluene in subsequent experiments. reactivation 35 min at 24 C; broken line, nodules not Toluene incubation time. Bacteroid samples rewetted, reactivation 20 min at 24 C. containing EDTA (1 mM) and toluene (0.2 ml/2 ml) were placed in a shaker bath at 24 C. The 30 M shaking rate was 190 strokes/min. In addition to the 1-min incubation time used thus far, 0.5-, 2-, 5-, and 10-min incubations with toluene (and EDTA) were studied. The 1-min incubation c 25 F time proved best (Fig. 4). After incubation with EDTA and toluene, the two phases were left to separate for 3 min, after which 0.5 ml of the aqueous phase was removed for the acetylene e 20 z
I
15
30 I
0 c 0
4 6 Shaking time (rnin)
8
10
20
FIG. 4. Effect of EDTA-toluene incubation on acetylene reduction by bacteroid suspensions. Reacti-
10
vation: 35 min at 24 C.
vb
-
2
In
E C-4
I
0
0
60 40 20 Reactivation time(min) FIG. 2. Effect of nodule pretreatment on reactivation time at 24 C of bacteroid suspensions prior to EDTA-toluene treatment. Symbols: solid line, nodules rewetted; * broken line, nodules not rewetted.
reduction test. With different times of phase separation, it appeared that a 1-min separation time gave the highest ethylene production (Fig. 5). For technical reasons no separation times shorter than 1 min were tested. EDTA concentration. A series of five EDTA concentrations was used to determine the most suitable concentration for acetylene reduction. The results in Fig. 6 show that incubation with 0.5 mM EDTA produced maximum ethylene production. The smaller EDTA-Mg2+ ratio, 0.2 instead of 0.4, did not seem to have an inhibitory effect on the action of EDTA. Toluene content. Varying amounts of tolu-
862
APPL. ENVIRON. MICROBIOL.
VAN STRATEN AND ROELOFSEN
ene were added to 1-ml bacteroid samples. After addition of 1 ml of 2 mM EDTA, the mixture was incubated for 1 min. Highest ethylene values were obtained with 0.02 ml of toluene (Fig. 7). In another experiment 0.007 ml was tested as well, but also in this case 0.02 ml of toluene gave maximum acetylene reduction. Linearity. To determine the linearity of the acetylene reduction assay after EDTA-toluene treatment of bacteroid suspensions, a time course experiment was carried out. Instead of a 5.6-ml vial, a 17.5-ml one was used, since more
30
0
25 c
o
_ t 0 20 0Il E
5'
30F 0
z
15
10
0
0
c
20
F
0.1 02 0.3 0.4 Toluene(ml/2ml bocteroid suspension)
FIG. 7. Effect of toluene content on acetylene reduction by EDTA-toluene-treated bacteroid suspensions. Reactivation: 35 min at 24 C.
IA 0-
a
gas samples were drawn from the same vial.
Figure 8 shows that the results obtained after
E i
0
0
2
L
6
Phase separation time(min) FI G. 5. Effect of phase separation time on acetylene reduction by EDTA-toluene-treated bacteroid
susp(ensions. 30
c
25
-i
z
-20
ol
0s I
R
-_
Reactivation: 35 min at 24 C.
10 min of incubation can be used to calculate ethylene production on an hourly basis. Recovery. For the first recovery experiments, 5 plants from every batch of 40 were assayed separately, and the averages, ranging from 26 to 47 ,mol of C2H4g of fresh weight nodule per h, were considered to represent each batch. The other 35 plants were harvested and
used for the preparation of bacteroids. Ethylene production by EDTA-toluene-treated bacteroids was between 26 and 30.8 ,umol of C2H,/g of fresh weight nodule per h, a recovery of 56 to 104%. Bacteroids not treated with EDTA and toluene only gave 3.7 to 4.4 umol of C2H4/g of fresh weight nodule per h. However, within a set of five plants, the variability was often very large. Two- to fourfold differences in ethylene production between two healthy looking plants of the same batch were no exception. Therefore, this method was abandoned and subsequent recovery data were obtained from plants whose activity had been determined first. For this purpose, eight whole plants were assayed together in a 1-liter Erlenmeyer flask. After the assay, the nodules were picked and used for the preparation of washed bacteroids. So far three experiments have been carried out this way. The plant average was 20.4 ,umol of C2H4/g of fresh weight nodule per h, and the average
EDTA (mM) 6. Effect of EDTA concentration on acetylene recovery was 124%, with all three values well reducction by EDTA-toluene-treated bacteroid sus- above 100%. These plant averages were low. A first impression is that in the intact plant the pensiions. Reactivation: 35 min at 24 C. FI
G.
VOL. 31, 1976
PREPARATION OF ANAEROBIC BACTEROIDS
15 .C -
0
Go
A.
0
E I
0
8
16 Time(min)
24
FIG. 8. Time course of acetylene reduction by EDTA-toluene-treated bacteroid suspensions. Reactivation: 35 min at 24 C.
bacteroids are regulated by a system that does not operate once the bacteroids are separated from the plant. More recovery experiments are planned, especially with plants that produce ethylene in the range of 40 to 50 umol/g of fresh weight nodule per h. It was somewhat remarkable that, despite the large variability of intact plants, ethylene production by treated bacteroids was confined to a comparatively narrow range, which had about 30 ,mol of C2H4/g of fresh weight nodule per h as its upper limit. This led to the question of whether the acetylene reduction assay itself might be responsible for this limit. Preliminary experiments have indeed shown that the acetylene reduction assay as described above was not conducted with optimum reagent concentrations. This problem is now being corrected. The EDTA-toluene method is highly reproducible. In a typical experiment with three replicates for each of four treatments, the coefficient of variability was 3.31% (ranging within each treatment from 0.86 to 5.24%). A major source of variability in acetylene reduction between plants was the wetness of the gravel in which the plants were grown. To minimize this variability, plants were last watered 48 h before the assay (5). In contrast, it was very important that, once the plants were harvested, the nodules be kept wet until press-
863
ing and that the weighing period, when the nodules were blotted dry, would be as short as possible. Summarizing the above-described method, the EDTA-toluene treatment should be carried out as follows. To 1 ml of bacteroid suspension, 0.02 ml of toluene and 1 ml of 1 mM EDTA are added. After shaking for 1 min, the mixture is left standing for 1 min and 0.5 ml of the aqueous phase is removed for the acetylene reduction assay. By using the same method for soybean bacteroids, we were unable to measure any ethylene production by untreated bacteroids. EDTA-toluene-treated bacteroids did produce ethylene but only about 0.5 ,umol/g of fresh weight nodule per h, a recovery of approximately 3%. The high recovery data obtained for the pea system indicate that the EDTA-toluene treatment makes the acetylene reduction assay far more sensitive. ACKNOWLEDGMENTS We are indebted to J. C. Kuhn of the Microbiology Department, Technion, Haifa, Israel, for helpful discussions. Thanks are extended to A. Houwers, W. Moller, and E. van Velsen for growing the plant material. LITERATURE CITED 1. Bergersen, F. J. 1966. Some properties of nitrogenfixing breis prepared from soybean root nodules. Biochim. Biophys. Acta 130:304-312. 2. Gray, G. W., and S. G. Wilkinson. 1965. The effect of ethylene-diaminetetra-acetic acid on the cell walls of some Gram-negative bacteria. J. Gen. Microbiol. 39:385-399. 3. Hardy, R. W. F., R. D. Holsten, E. K. Jackson, and R. C. Burns. 1968. The acetylene-ethylene assay for N2 fixation: laboratory and field evaluation. Plant Physiol. 43:1185-1207. 4. Herzenberg, L. A. 1959. Studies on the induction of f-galactosidase in a cryptic strain of Escherichia coli. Biochim. Biophys. Acta 31:525-538. 5. LaRue, T. A. G., and W. G. W. Kurz. 1973. Estimation of nitrogenase in intact legumes. Can. J. Microbiol. 19:304-305. 6. Leive, L. 1965. Actinomycin sensitivity in Escherichia coli produced by EDTA. Biochem. Biophys. Res. Commun. 18:13-17. 7. Leive, L. 1965. A nonspecific increase in permeability in Escherichia coli produced by EDTA. Proc. Natl. Acad. Sci. U.S.A. 53:745-750. 8. Leive, L. 1968. Studies on the permeability change produced in coliform bacteria by ethylenediaminetetraacetate. J. Biol. Chem. 243:2373-2380. 9. Levinthal, C., E. R. Signer, and K. Fetherolf. 1962. Reactivation and hybridization of reduced alkaline phosphatase. Proc. Natl. Acad. Sci. U.S.A. 48:12301237. 10. Mague, T. H., and R. H. Burs. 1972. Reduction of acetylene and nitrogen by fieldgrown soybeans. New Phytol. 71:275-286.
Vol. 31, No. 6 Printed in U.S.A.
Improved Method for Preparing Anaerobic Bacteroid Suspensions of Rhizobium leguminosarum for the Acetylene Reduction Assay J. VAN STRATEN*
AND
W. ROELOFSEN
Laboratory of Microbiology, Agricultural University, Wageningen, The Netherlands
Received for publication 20 January 1976
A method using ethylenediaminetetraacetate (EDTA) and toluene-treated bacteroid suspensions for the acetylene reduction assay is described. The high level of acetylene reduction by these bacteroids is comparable to that of intact plants. Reproducibility of the EDTA-toluene treatment is, on the average, within 5%. Preliminary experiments with soybeans indicate that the EDTAtoluene method might be applicable to other legumes as well. pea
It is generally assumed that intact legume plants reduce more acetylene than do detached "parts" such as nodulated roots, detached nodules, or bacteroids. Unfortunately, acetylene reduction data relating these different fractions are very few. Hardy et al. (3) reported that nodulated soybean roots reduced more acetylene than did detached nodules. Mague and Burris (10) compared the acetylene reduction of intact soybean plants (100%) with that of nodulated roots and detached nodules, and found that the nodulated roots and the detached nodules showed only 46 and 23% of the activity of the intact plant, respectively. On the other hand, Akkermans (Ph.D. thesis, Univ. of Leyden, Leyden, The Netherlands, 1971) reported that, after removal of the shoot, the remaining nodulated pea root system reduced as much acetylene as it did before removal. Data comparing nitrogenase activity of anaerobically washed bacteroids with that of whole plants are virtually nonexistent. From work done previously in this laboratory (the late van den Berg, unpublished data), it appears that washed pea bacteroids in the presence of dithionite and adenosine 5'-triphosphate reduce acetylepe at a rate that is about 10% of that of the intact plant or less. It has been suggested that this low level of nitrogenase activity may be due to permeability limitations, possibly of the added adenosine 5'-triphosphate, posed by the membranes of the bacteroids. Treatment with ethylenediaminetetraacetate (EDTA) is known to increase permeability of gram-negative bacteria (2, 6-8). Toluene is also used in enzyme assays (j3-galactosidase: Herzenberg [4]; alkaline phosphatase: Levinthal et al. [9] to improve the accessibility of these enzymes to exogenous substrates.
This paper reports on the use of EDTA and toluene to increase acetylene reduction by washed bacteroid suspensions. MATERIALS AND METHODS Plant material and preparation of bacteroid suspensions. Pea (Pisum sativum L. cv. Rondo) plants, inoculated with Rhizobium leguminosarum strain PRE, were grown in gravel in nitrogen-free nutrient solution in batches of 40 at 18 to 20 C in a growth chamber and harvested after 27 to 31 days. Freshly picked nodules were pressed under anaerobic conditions at 0 to 4 C in 0.05 M tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.2), containing 2.5 mM MgCl2, 20 mM Na2S2O4, and 4% soluble polyvinyl-pyrrolidone, using a Bergersen press (1). Nodule concentration in all experiments was 80 mg of nodule tissue per ml of buffer. Bacteroid suspensions were obtained by centrifuging the nodule brei at 5,000 x g for 10 min at 3 to 5 C, suspending, and washing the pellet with buffer, followed by a second centrifugation. Finally, the pellet was resuspended in the original volume of buffer. The bacteroid suspensions were kept on ice until further use. Nitrogenase activity. Nitrogenase activity was determined with the acetylene reduction assay (3). The reaction was carried out in a shaker bath at 24 C in 5.6-ml vials closed with Suba-seal closures (Rubber B. V., Hilversum, Holland). The shaking rate was 144 strokes/min. The reaction mixture contained 0.5 ml of bacteroid suspension, 50 ,umol of tris(hydroxymethyl)aminomethane - hydrochloride (pH 7.2), 7.5 ,umol of MgCl2 (or the amount indicated), 11.7 ,umol of creatine - P, 2.8 ,mol of adenosine 5'-triphosphate, and 0.03 mg of creatine phosphokinase (Boehringer Mannheim GmbH) in a total volume of 1 ml. The gas phase was 10% acetylene in argon. After 10 min of incubation, 100 Al of the gas phase was removed and analyzed for ethylene on a gas chromatograph (Multigraph-417, Packard, Becker) equipped with a hydrogen flame detector and fitted with a glass column (110 cm by 3 mm) packed with Porapak R (80 to 100 mesh). The column
859
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APPL. ENVIRON. MICROBIOL.
vAN STRATEN AND ROELOFSEN
temperature was 50 C. Results are expressed as micromoles of ethylene per gram of fresh weight nodule per hour.
RESULTS AND DISCUSSION Preliminary experiments with nodule brei showed that treatment with EDTA and especially with toluene or EDTA plus toluene resulted in substantially higher ethylene values (Table 1). Mg2+ concentration. According to Leive (8), divalent cations prevent EDTA action when present in the same or higher concentrations. The buffer used thus far had been 2.5 mM in Mg2+ and, since EDTA was only 1 mM, the effect of Mg2+ on the EDTA-toluene treatment was investigated. In this experiment bacteroid suspensions were prepared at 20 C instead of at 0 to 4 C. Aliquots (1 ml) of bacteroid suspensions were incubated anaerobically with the same volume of a 2 mM EDTA solution in 25-ml test tubes at 24 C. The bacteroid suspensions containing EDTA were 0, 2.5, and 5 mM in Mg2+. The 0 mM Mge+ suspension was attained by treating part of the brei with Mg-free buffer and EDTA. In addition to testing the effect of different Mg2+ concentrations on the EDTAtoluene treatment, the effect of Mg2+ on acetylene reduction itself was studied. After 30 min, 0.2 ml of toluene was added and the mixture was shaken at 190 strokes/min in a 24 C water bath for 1 min. The test tube was removed from the water bath, and the two phases were left to separate. After 3 min, 0.5 ml of the aqueous phase was removed and used for the acetylene reduction test. As can be seen from the results shown in Fig. 1, acetylene reduction should not be carried out at 2.5 mM Mg2+. The positive effect of higher Mg2+ concentrations was much more pronounced with EDTA-toluene-treated bacteroids than with bacteroids without EDTA and toluene treatment. Mg2+ was also necessary for the EDTA treatment, but in this case 2.5 mM was better than 5 mM Mg2+. It was therefore decided to keep the Mg2+ concentration at 2.5 mM during the EDTA treatment and TABLE 1. Effect of EDTA andlor toluene treatment on acetylene reduction by nodule brei AmoI of C2H./g of Brei prepared with:
Buffer
Buffer + EDTA" Buffer + tolueneb Buffer + EDTA" + tolueneb "Final concentration, 1 mM. A total of 0.2 ml/ml of brei.
fresh wt nodule per h 1,438
1,832
14,179 19,057
16 k
0'c
12
-
a
X-------x
_ 8 el
f0
0
0
2
L
Mg'++ (moles/acetylene
6 8 reduction assoy)
10
FIG. 1. Acetylene reduction by EDTA-toluenetreated bacteroid suspensions at varying Mg2+ concentrations during both EDTA treatment and acetylene reduction assay. Mg2+ concentration during EDTA treatment: x, O mM; *, 2.5 mM; A, 5 mM. 0, Bacteroids without EDTA-toluene treatment.
to change to 7.5 mM for the acetylene reduction test. Consequently, our results as to the Mg2+EDTA ratio did not agree with those of Leive (8) for Escherichia coli. The absolute values in
this experiment were rather low, which may have been due to the way the bacteroids were prepared. Reactivation time. Before the bacteroids, which were stored at 0 C, could be used, they had to be kept for some time at the temperature at which the incubation treatments (24 C) were carried out in order to become reactivated. EDTA and toluene were added simultaneously after the reactivation period. No separate EDTA incubation period was included since it had been found that high ethylene values were obtained without such a separate incubation. The time it took the bacteroid suspension to obtain maximum ethylene production (reactivation time) was found to be rather variable (Fig. 2). Also, the response during the first 5 min was quite different. This variability in time to reach maximum activity was not merely a question of reaching 24 C since it took the bacteroid suspension less than 5 min to reach this temperature. Moreover, when the bacteroids were kept at 20 C instead of 0 C, the reactivation time was still 20 min. The variability in reactivation time could, to a large extent, be ascribed to the procedure used to prepare the nodules for pressing. The broken lines (three experiments) represent nodules that were picked, collected on a wet filter paper, blotted dry, weighed, and used for pressing. The solid line (average of four experiments) represents
VOL. 31, 1976
PREPARATION OF ANAEROBIC BACTEROIDS
861
nodules that were rewetted and left for 20 min 30 F under a wet filter paper after being weighed. Rewetting the nodules also influenced the stability of the bacteroid suspension at 0 C with respect to nitrogenase activity (Fig. 3). This 20 procedure was continued in subsequent experiments. The relationship between reactivation 0 time and stability is currently being investigated. EDTA incubation time. Although high eth- -Li210 ylene values could be obtained by adding EDTA together with toluene, it was not known to I what extent the EDTA reaction was completed in such a short time. This was tested by incubating bacteroids with EDTA for different pe180 120 60 riods of time, immediately followed by the addiStorage time at 0 °C (min) tion of toluene. It was found, however, that no FIG. 3. Effect of nodule pretreatment on the acetysignificant increases in ethylene production ability of bacteroids during storage at could be obtained by longer incubations with 0lene-reducing C, as measured after reactivation and EDTA-toluEDTA. As a result, EDTA was added together ene treatment. Symbols: solid line, nodules rewetted, with toluene in subsequent experiments. reactivation 35 min at 24 C; broken line, nodules not Toluene incubation time. Bacteroid samples rewetted, reactivation 20 min at 24 C. containing EDTA (1 mM) and toluene (0.2 ml/2 ml) were placed in a shaker bath at 24 C. The 30 M shaking rate was 190 strokes/min. In addition to the 1-min incubation time used thus far, 0.5-, 2-, 5-, and 10-min incubations with toluene (and EDTA) were studied. The 1-min incubation c 25 F time proved best (Fig. 4). After incubation with EDTA and toluene, the two phases were left to separate for 3 min, after which 0.5 ml of the aqueous phase was removed for the acetylene e 20 z
I
15
30 I
0 c 0
4 6 Shaking time (rnin)
8
10
20
FIG. 4. Effect of EDTA-toluene incubation on acetylene reduction by bacteroid suspensions. Reacti-
10
vation: 35 min at 24 C.
vb
-
2
In
E C-4
I
0
0
60 40 20 Reactivation time(min) FIG. 2. Effect of nodule pretreatment on reactivation time at 24 C of bacteroid suspensions prior to EDTA-toluene treatment. Symbols: solid line, nodules rewetted; * broken line, nodules not rewetted.
reduction test. With different times of phase separation, it appeared that a 1-min separation time gave the highest ethylene production (Fig. 5). For technical reasons no separation times shorter than 1 min were tested. EDTA concentration. A series of five EDTA concentrations was used to determine the most suitable concentration for acetylene reduction. The results in Fig. 6 show that incubation with 0.5 mM EDTA produced maximum ethylene production. The smaller EDTA-Mg2+ ratio, 0.2 instead of 0.4, did not seem to have an inhibitory effect on the action of EDTA. Toluene content. Varying amounts of tolu-
862
APPL. ENVIRON. MICROBIOL.
VAN STRATEN AND ROELOFSEN
ene were added to 1-ml bacteroid samples. After addition of 1 ml of 2 mM EDTA, the mixture was incubated for 1 min. Highest ethylene values were obtained with 0.02 ml of toluene (Fig. 7). In another experiment 0.007 ml was tested as well, but also in this case 0.02 ml of toluene gave maximum acetylene reduction. Linearity. To determine the linearity of the acetylene reduction assay after EDTA-toluene treatment of bacteroid suspensions, a time course experiment was carried out. Instead of a 5.6-ml vial, a 17.5-ml one was used, since more
30
0
25 c
o
_ t 0 20 0Il E
5'
30F 0
z
15
10
0
0
c
20
F
0.1 02 0.3 0.4 Toluene(ml/2ml bocteroid suspension)
FIG. 7. Effect of toluene content on acetylene reduction by EDTA-toluene-treated bacteroid suspensions. Reactivation: 35 min at 24 C.
IA 0-
a
gas samples were drawn from the same vial.
Figure 8 shows that the results obtained after
E i
0
0
2
L
6
Phase separation time(min) FI G. 5. Effect of phase separation time on acetylene reduction by EDTA-toluene-treated bacteroid
susp(ensions. 30
c
25
-i
z
-20
ol
0s I
R
-_
Reactivation: 35 min at 24 C.
10 min of incubation can be used to calculate ethylene production on an hourly basis. Recovery. For the first recovery experiments, 5 plants from every batch of 40 were assayed separately, and the averages, ranging from 26 to 47 ,mol of C2H4g of fresh weight nodule per h, were considered to represent each batch. The other 35 plants were harvested and
used for the preparation of bacteroids. Ethylene production by EDTA-toluene-treated bacteroids was between 26 and 30.8 ,umol of C2H,/g of fresh weight nodule per h, a recovery of 56 to 104%. Bacteroids not treated with EDTA and toluene only gave 3.7 to 4.4 umol of C2H4/g of fresh weight nodule per h. However, within a set of five plants, the variability was often very large. Two- to fourfold differences in ethylene production between two healthy looking plants of the same batch were no exception. Therefore, this method was abandoned and subsequent recovery data were obtained from plants whose activity had been determined first. For this purpose, eight whole plants were assayed together in a 1-liter Erlenmeyer flask. After the assay, the nodules were picked and used for the preparation of washed bacteroids. So far three experiments have been carried out this way. The plant average was 20.4 ,umol of C2H4/g of fresh weight nodule per h, and the average
EDTA (mM) 6. Effect of EDTA concentration on acetylene recovery was 124%, with all three values well reducction by EDTA-toluene-treated bacteroid sus- above 100%. These plant averages were low. A first impression is that in the intact plant the pensiions. Reactivation: 35 min at 24 C. FI
G.
VOL. 31, 1976
PREPARATION OF ANAEROBIC BACTEROIDS
15 .C -
0
Go
A.
0
E I
0
8
16 Time(min)
24
FIG. 8. Time course of acetylene reduction by EDTA-toluene-treated bacteroid suspensions. Reactivation: 35 min at 24 C.
bacteroids are regulated by a system that does not operate once the bacteroids are separated from the plant. More recovery experiments are planned, especially with plants that produce ethylene in the range of 40 to 50 umol/g of fresh weight nodule per h. It was somewhat remarkable that, despite the large variability of intact plants, ethylene production by treated bacteroids was confined to a comparatively narrow range, which had about 30 ,mol of C2H4/g of fresh weight nodule per h as its upper limit. This led to the question of whether the acetylene reduction assay itself might be responsible for this limit. Preliminary experiments have indeed shown that the acetylene reduction assay as described above was not conducted with optimum reagent concentrations. This problem is now being corrected. The EDTA-toluene method is highly reproducible. In a typical experiment with three replicates for each of four treatments, the coefficient of variability was 3.31% (ranging within each treatment from 0.86 to 5.24%). A major source of variability in acetylene reduction between plants was the wetness of the gravel in which the plants were grown. To minimize this variability, plants were last watered 48 h before the assay (5). In contrast, it was very important that, once the plants were harvested, the nodules be kept wet until press-
863
ing and that the weighing period, when the nodules were blotted dry, would be as short as possible. Summarizing the above-described method, the EDTA-toluene treatment should be carried out as follows. To 1 ml of bacteroid suspension, 0.02 ml of toluene and 1 ml of 1 mM EDTA are added. After shaking for 1 min, the mixture is left standing for 1 min and 0.5 ml of the aqueous phase is removed for the acetylene reduction assay. By using the same method for soybean bacteroids, we were unable to measure any ethylene production by untreated bacteroids. EDTA-toluene-treated bacteroids did produce ethylene but only about 0.5 ,umol/g of fresh weight nodule per h, a recovery of approximately 3%. The high recovery data obtained for the pea system indicate that the EDTA-toluene treatment makes the acetylene reduction assay far more sensitive. ACKNOWLEDGMENTS We are indebted to J. C. Kuhn of the Microbiology Department, Technion, Haifa, Israel, for helpful discussions. Thanks are extended to A. Houwers, W. Moller, and E. van Velsen for growing the plant material. LITERATURE CITED 1. Bergersen, F. J. 1966. Some properties of nitrogenfixing breis prepared from soybean root nodules. Biochim. Biophys. Acta 130:304-312. 2. Gray, G. W., and S. G. Wilkinson. 1965. The effect of ethylene-diaminetetra-acetic acid on the cell walls of some Gram-negative bacteria. J. Gen. Microbiol. 39:385-399. 3. Hardy, R. W. F., R. D. Holsten, E. K. Jackson, and R. C. Burns. 1968. The acetylene-ethylene assay for N2 fixation: laboratory and field evaluation. Plant Physiol. 43:1185-1207. 4. Herzenberg, L. A. 1959. Studies on the induction of f-galactosidase in a cryptic strain of Escherichia coli. Biochim. Biophys. Acta 31:525-538. 5. LaRue, T. A. G., and W. G. W. Kurz. 1973. Estimation of nitrogenase in intact legumes. Can. J. Microbiol. 19:304-305. 6. Leive, L. 1965. Actinomycin sensitivity in Escherichia coli produced by EDTA. Biochem. Biophys. Res. Commun. 18:13-17. 7. Leive, L. 1965. A nonspecific increase in permeability in Escherichia coli produced by EDTA. Proc. Natl. Acad. Sci. U.S.A. 53:745-750. 8. Leive, L. 1968. Studies on the permeability change produced in coliform bacteria by ethylenediaminetetraacetate. J. Biol. Chem. 243:2373-2380. 9. Levinthal, C., E. R. Signer, and K. Fetherolf. 1962. Reactivation and hybridization of reduced alkaline phosphatase. Proc. Natl. Acad. Sci. U.S.A. 48:12301237. 10. Mague, T. H., and R. H. Burs. 1972. Reduction of acetylene and nitrogen by fieldgrown soybeans. New Phytol. 71:275-286.
