JOURNAL OF BACTERIOLOGY, Sept. 1975, p. 1265-1268 Copyright 0 1975 American Society for Microbiology
Vol. 123, No. 3 Printed in U.S.A.
NOTES Acetylene Reduction by Pure Cultures of Rhizobial DONALD L. KEISTER Charles F. Kettering Research Laboratory, Yellow Springs, Ohio 45387 Received for publication 2 May 1975
Acetylene reduction has been demonstrated in pure cultures of rhizobia. The requirements and conditions necessary for the activity in Rhizobium sp. 32H1 are described. The most important factors are a low cell density and a very low oxygen concentration.
The genus Rhizobium is characterized by its ability to elicit nodules and fix nitrogen in the roots oI legumes. No species of Rhizobium has previously been shown to fix nitrogen or to reduce acetylene independently of the host plant. This apparent dependence on the plant led to the concept that some factor(s) supplied by the host plant, possibly even the genetic information, is required for development of the nitrogenase enzyme system. However, such a concept has been questioned in several recent publications (2, 5, 11). Other recent reports on the association of rhizobia with plant cell cultures have questioned the likelihood that it is genetic information which is supplied by the host plant but concluded that an unknown diffusible factor is necessary (3, 12). I have found that several strains of Rhizobium sp. and R. japonicum can reduce acetylene. No plant materials are required and the characteristics of the reaction are similar to those found for many free-living nitrogen-fixing bacteria. The reduction of acetylene to ethylene is a characteristic of nitrogenase (6, 7) and has been used to assay for activity in these experiments. Although '5N 2-incorporation studies need to be done to confirm nitrogen fixation in these bacteria, the following experiments constitute evidence that these free-living bacteria are capable of nitrogenase formation. Stock cultures of rhizobia were maintained on a yeast extract-mannitol-soil extract-agar medium. They were routinely cultured in the following liquid medium: Na-KPO4, 7.4 mM; MgSO4, 0.8 mM; CaCl2, 0.5 mM; FeSO,, 48 AM; NaMoO,, 11,M; mannitol, 0.5%; sodium gluconate, 0.5%; yeast extract, 0.1%; Casamino 1 Contribution no. 542 from the Charles F. Kettering Research Laboratory.
Acids, 0.01%; and trace metals, pH 7.2. The validity of the conclusions in this paper of course depends upon the use of pure rhizobial cultures. Therefore all reactions were performed under aseptic conditions for the entire term of the experiments. This included the use of sterile gasses and sterile syringes for removing samples for the gas chromatographic assays. Purity of the cultures was assured by the repeated use of single colony isolates. At various times during the experiments, 12 separate cultures of 32H1 which originated from single colony isolates were used. All were determined to be Rhizobium sp. by inoculation into Phaseolus aureus (mung bean). Mung bean seeds were sterilized and grown in sterilized modified Leonard jars according to Vincent (13). Inoculated plants were well nodulated, whereas no nodules were found on uninoculated controls. We have isolated bacteria from the resultant nodules and found them to have the same cultural characteristics, colony morphology, and acetylenereducing ability as the original. We have not positively identified the nodule isolate as 32H1, but there is no doubt that it is a slow-growing rhizobium. Other criteria of purity included plating of cultures at the termination of the experiments on the media described above plus 1.5% agar. The resultant colonies appeared after 3 to 4 days and reached a 1.5-mm diameter only after 7 days, and all had the uniform morphological appearance of the original culture. Another indication that the observed C2H2 reduction does not arise from a dinitrogen-fixing contaminant is that I have found the activity in six additional strains of Rhizobium sp. (41A1 and 41F2) and R. japonicum 31Ib83, 3I1b125, 61A76, and ATCC 10324). Good activity was found with 41A1 and 61A76, whereas low activi-
1265
1266
NOTES
J. BACTERIOL.
ties were observed in the others. We have not found any activity so far in several other strains of R. japonicum, Rhizobium sp., nor in any of three fast-growing rhizobia that we have cultured. Rhizobial strains were kindly provided by Joe Burton of the Nitragin Co. and Deane Weber of the United States Department of Agriculture. Figure 1 shows the time course of cell growth and acetylene reduction under an argon-C2H, atmosphere (In some experiments, oxygen was rigorously removed from the gas phase using an "OXY-TRAP" obtained from Alltech Associates, Arlington Heights, Ill. However, the serum bottles were capped with rubber serum caps and sealed with RTV silicone rubber. Some oxygen may diffuse slowly through this seal, and the experiments should be interpreted with the possibility that this small, but unknown, amount of oxygen may have an effect. Experiments are in progress which bear on this question.) using Rhizobium sp. 32H1. A lag period is always observed before C2H,, reduction begins, but once initiated the reduction continues for several days. In this experiment the inoculum contained approximately 107 cells/ml (optical density at 680 nm 0.003). The final optical =
La.
density
was
0.089, and cell densities much
greater than this cannot be attained under these
conditions even when a larger inoculum is used. Some cell division, as measured by an increase in optical density, is apparently required for obtaining good C,H, reduction. With high initial cell densities (0.25 or greater), little or no cell division occurs, and little activity develops. Experiments which will be presented elsewhere show that a nitrogen source and a carbon source are required for the development of good activity. Sugars or sugar derivatives are the most effective carbon source. With glutamate as the nitrogen source, specific activities of 600 nmol of ethylene/mg of protein per h have been observed, but rates of 50 to 100 are more typical. As a comparison, bacteroids isolated from lupin nodules have been reported to have a specific activity of 300 to 1,500 (9). In most experiments, C2H, was present from time zero so that C2H, formation could be monitored continually. Separate experiments in which C,H2 was added periodically, after the removal of oxygen, have shown that C 2H,2 not required for the formation of the enzyme system.
The effect of various inhibitors on acetylene reduction in Rhizobium sp. 32H1 is illustrated in Fig. 2. The inhibitors were added after the reduction was well initiated so that the effects observed can be presumed to be on the enzyme
I0
O.D.
I
200
wo
I
CONTROL/
CZ
*ONH +
iL
/00 f4
-
0
48
96
144
192
240
//
100
-
HOURS 0
FIG. 1. Time course of anaerobic growth and ethylene formation by Rhizobium sp. 32HI. Aerobically grown cells were harvested and inoculated into fresh culture medium to about 107 cells/ml. Casamino Acids replaced yeast extract in the medium. Samples (3 ml) were aseptically transferred to 30-ml serum bottles followed by evacuation and flushing four times with a 90% argon-10% C,H, mixture. The bottles were incubated at 28 C with gentle rotary shaking. Samples of the gas phase were analyzed for C1H, by gas chromatography using a Porapak N column. The acetylene was used as an internal standard. All reactions were performed in triplicate, and aseptic technique was rigidly followed throughout the experiments. Optical density measurements were made at 680 nm in a 1-cm cuvette with a Beckman DU. No acetyleneindependent ethylene production has ever been observed under an argon atmosphere.
CON
-CO,NO 3
CN-,N3-,2
/
0
0
E
2
c
0
L
.
0
40
. 1%
I
.
80 120 HOURS
,
160
200
#%^ _
FIG. 2. Effect of inhibitors on acetylene reduction. Aerobically grown cells were diluted to 1.2 x 108 cells/ml in the following medium: K,HPO,, 50 mM; MgSO,, 0.4 mM; mannitol, q.5%; sodium gluconate, 0.5%; Casamino Acids, 0.025%; NaMoO,, 30 ,gM; and iron citrate, 0.1 mM, pH 7.2. The experiment was as indicated in the legend to Fig. 1. The inhibitors were added after 94 h or as indicated by the arrow. Concentrations were as follows: NH,Cl, 2 mg/ml; KNO,, 6.6 mM; NaN,, I mM; KCN, I mM; CO, 0.8%; and 02, 2.7%.
VOL. 123, 1975
activity rather than on enzyme formation. The inhibition by carbon monoxide, cyanide, azide, and 0, are characteristic of nitrogenase-catalyzed acetylene reduction. Oxygen at concentrations greater than 2% totally inhibited the development of activity. Concentrations lower than 2% were partially tolerated, but maximum specific activity was obtained with no added oxygen. This is in contrast to the relatively high oxygen requirement for acetylene reduction by bacteroids isolated from legume nodules (1). Therefore future experiments will be designed to explore and hopefully resolve this paradox. Nitrate was inhibitory and cannot be used as a terminal electron acceptor for studies with nitrogenase with Rhizobium sp. 32H1. Ammonium chloride had no effect on the enzyme activity. More interestingly, NH4+ up to 37 mM had little effect on enzyme formation. This is in contrast to the effect of ammonia on other free-living nitrogen-fixing microorganisms where repression by ammonia seems to be a general characteristic of wild-type organisms. Lack of ammonia repression may not be a general characteristic of rhizobia, however, for preliminary results indicate that nitrogenase synthesis is repressed by NH,Cl in R. japonicum 61A76. The parameters which appear most important in inducing nitrogenase in these experiments are in order of importance, are as follows. (i) The use of low cell densities in small volumes is the most important. Some division is apparently required, and with initial densities greater than 6 x 10' cells/ml little or no growth occurs and little acetylene reduction is observed. Typically, cultures of less than 5 ml in 15- or 30-ml serum bottles have been used for these experiments. For unknown reasons, attempts to scale up to larger volumes have given only sporadic success. (ii) Removal of oxygen is important. although the cells tolerate some oxygen, the presence of oxygen increases the lag period for nitrogenase development and decreases the specific activity. R. japonicum 505 has been reported to use nitrate as a terminal electron acceptor (4). I have found that several other rhizobia can utilize nitrate as a terminal electron acceptor; however, nitrate inhibited nitrogenase activity with Rhizobium sp. 32H1 and thus could not be used as a terminal electron acceptor in the present experiments. (iii) The strain of Rhizobium used is important. Rhizobium sp. 32H1 has given the most consistent results and highest specific activities of ethylene formation. However, we have also observed acetylene reduction with Rhizobium
NOTES
1267
sp. 41A1 and 41F2, and R. japonicum 3Ilb83, 3Ilb125, 61A76, and ATCC 10324. Thus, it is likely that nitrogenase development in the absence of plant materials is possible for many rhizobia and that with the development of optimal conditions the enzyme can be demonstrated in other species. (iv) A source of combined nitrogen is required, with glutamate or aspartate giving the best results. (v) With some strains a high phosphate concentration is stimulatory but not required. Many past attempts have been made to induce nitrogenase in cultures of rhizobia. The use of plant cell cultures (3, 8, 10) or material diffusing from such cultures (M. Reporter, Biochem. Biophys. Res. Commun., in press; 12) has given positive results. This paper is the first report of the induction of nitrogenase activity in rhizobia with a defined media without plant materials. Thus it appears probable that the host plant is supplying an appropriate environment and the necessary substrates for nitrogen fixation in symbiosis but not the genetic information. I thank Gerald Peters, Minocher Reporter, Robert Darrow, and Marvin Lamborg for helpful discussion and criticism, and Norma Jean Raveed for technical assistance.
ADDENDUM Since this paper was submitted it has been found that oxygen is required for the cell growth and acetylene reduction reported. When the reaction flasks containing an argon-C,H, atmosphere were incubated in an external oxygen-free atmosphere, little ethylene formation was observed. When the external oxygen concentration was increased above 0.2 atmosphere, higher rates were found. Thus it appears that diffusion of oxygen through the rubber septum is required. Gas chromatographic analysis of the reaction vessels indicated that the oxygen concentration during the active acetylene-reducing period was less than 0.001 atmosphere. The rate of oxygen diffusion, however, is not known. Preliminary experiments carried out with small liquid culture volumes in large-volume flasks, so that the oxygen concentration can be maintained for a reasonable period of time, indicate that the optimal oxygen concentration of the gas phase is very low, about 0.001 atmosphere. LITERATURE CITED 1. Bergersen, F. J., and G. L. Turner. 1968. Comparative studies of nitrogen fixation by soybean root nodules, bacterial suspension and cell-free extracts. J. Gen. Microbiol. 53:205-220. 2. Bishop, P. E., H. J. Evans, R. M. Daniel, and R. 0. Hampton. 1975. Immunological evidence for the capability of free-living Rhizobium japonicum to synthesize a portion of a nitrogenase component. Biochim. Bio-
phys. Acta 381:248-256. 3. Child, J. J. 1975. Nitrogen fixation by a Rhizobium sp. in association with non-leguminous plant cell cultures.
1268
NOTES
Nature (London) 253:350-351. 4. Daniel, R. M., and C. A. Appleby. 1972. Anaerobicnitrate, symbiotic and aerobic growth of Rhizobium japonicum: effect on cytochrome P4,O, other haemoproteins, nitrate and nitrite reductases. Biochim. Biophys. Acta 275:347-354. 5. Dunican, L. K., and A. B. Tierney. 1974. Genetic transfer of nitrogen fixation from Rhizobium trifolii to Klebsiella aerogenes. Biochem. Biophys. Res. Commun. 57:62-72. 6. Hardy, R. W. F., R. C. Burns, and R. D. Holsten. 1973. Applications of the acetylene-ethylene assay for measurement of nitrogen fixation. Soil Biol. Biochem. 5:47-81. 7. Hardy, R. W. F., D. Holsten, E. K. Jackson, and R. C. Burns. 1968. The acetylene-ethylene assay for N, fixation: laboratory and field evaluation. Plant Physiol. 43:1185-1207. 8. Holsten, R. D., R. C. Burns, R. W. F. Hardy, and R. R.
J. BACTERIOL.
9. 10. 11.
12. 13.
Herbert. 1971. Establishment of symbiosis between Rhizobium and plant cells in vitro. Nature (London) 232:173-176. Kennedy, I. R. 1970. Kinetics of acetylene and CNreduction by the N,-fixing system of Rhizobium lupini. Biochim. Biophys. Acta 222:135-144. Phillips, D. A. 1974. Factors affecting the reduction of acetylene by Rhizobium-soybean cell associations in vitro. Plant Physiol. 53:67-72. Phillips, D. A., R. L. Howard, and H. J. Evans. 1973. Studies on the genetic control of a nitrogenase component in leguminous root nodules. Plant Physiol. 28:248-253. Scowcroft, W. R., and A. H. Gibson. 1975. Nitrogen fixation by Rhizobium associated with tobacco and cowpea cell cultures. Nature (London) 253:351-352. Vincent, J. M. 1970. A Manual for the practical study of root nodule bacteria. IBP Handbook No. 15. Blackwell Science Publishers, Oxford.
Vol. 123, No. 3 Printed in U.S.A.
NOTES Acetylene Reduction by Pure Cultures of Rhizobial DONALD L. KEISTER Charles F. Kettering Research Laboratory, Yellow Springs, Ohio 45387 Received for publication 2 May 1975
Acetylene reduction has been demonstrated in pure cultures of rhizobia. The requirements and conditions necessary for the activity in Rhizobium sp. 32H1 are described. The most important factors are a low cell density and a very low oxygen concentration.
The genus Rhizobium is characterized by its ability to elicit nodules and fix nitrogen in the roots oI legumes. No species of Rhizobium has previously been shown to fix nitrogen or to reduce acetylene independently of the host plant. This apparent dependence on the plant led to the concept that some factor(s) supplied by the host plant, possibly even the genetic information, is required for development of the nitrogenase enzyme system. However, such a concept has been questioned in several recent publications (2, 5, 11). Other recent reports on the association of rhizobia with plant cell cultures have questioned the likelihood that it is genetic information which is supplied by the host plant but concluded that an unknown diffusible factor is necessary (3, 12). I have found that several strains of Rhizobium sp. and R. japonicum can reduce acetylene. No plant materials are required and the characteristics of the reaction are similar to those found for many free-living nitrogen-fixing bacteria. The reduction of acetylene to ethylene is a characteristic of nitrogenase (6, 7) and has been used to assay for activity in these experiments. Although '5N 2-incorporation studies need to be done to confirm nitrogen fixation in these bacteria, the following experiments constitute evidence that these free-living bacteria are capable of nitrogenase formation. Stock cultures of rhizobia were maintained on a yeast extract-mannitol-soil extract-agar medium. They were routinely cultured in the following liquid medium: Na-KPO4, 7.4 mM; MgSO4, 0.8 mM; CaCl2, 0.5 mM; FeSO,, 48 AM; NaMoO,, 11,M; mannitol, 0.5%; sodium gluconate, 0.5%; yeast extract, 0.1%; Casamino 1 Contribution no. 542 from the Charles F. Kettering Research Laboratory.
Acids, 0.01%; and trace metals, pH 7.2. The validity of the conclusions in this paper of course depends upon the use of pure rhizobial cultures. Therefore all reactions were performed under aseptic conditions for the entire term of the experiments. This included the use of sterile gasses and sterile syringes for removing samples for the gas chromatographic assays. Purity of the cultures was assured by the repeated use of single colony isolates. At various times during the experiments, 12 separate cultures of 32H1 which originated from single colony isolates were used. All were determined to be Rhizobium sp. by inoculation into Phaseolus aureus (mung bean). Mung bean seeds were sterilized and grown in sterilized modified Leonard jars according to Vincent (13). Inoculated plants were well nodulated, whereas no nodules were found on uninoculated controls. We have isolated bacteria from the resultant nodules and found them to have the same cultural characteristics, colony morphology, and acetylenereducing ability as the original. We have not positively identified the nodule isolate as 32H1, but there is no doubt that it is a slow-growing rhizobium. Other criteria of purity included plating of cultures at the termination of the experiments on the media described above plus 1.5% agar. The resultant colonies appeared after 3 to 4 days and reached a 1.5-mm diameter only after 7 days, and all had the uniform morphological appearance of the original culture. Another indication that the observed C2H2 reduction does not arise from a dinitrogen-fixing contaminant is that I have found the activity in six additional strains of Rhizobium sp. (41A1 and 41F2) and R. japonicum 31Ib83, 3I1b125, 61A76, and ATCC 10324). Good activity was found with 41A1 and 61A76, whereas low activi-
1265
1266
NOTES
J. BACTERIOL.
ties were observed in the others. We have not found any activity so far in several other strains of R. japonicum, Rhizobium sp., nor in any of three fast-growing rhizobia that we have cultured. Rhizobial strains were kindly provided by Joe Burton of the Nitragin Co. and Deane Weber of the United States Department of Agriculture. Figure 1 shows the time course of cell growth and acetylene reduction under an argon-C2H, atmosphere (In some experiments, oxygen was rigorously removed from the gas phase using an "OXY-TRAP" obtained from Alltech Associates, Arlington Heights, Ill. However, the serum bottles were capped with rubber serum caps and sealed with RTV silicone rubber. Some oxygen may diffuse slowly through this seal, and the experiments should be interpreted with the possibility that this small, but unknown, amount of oxygen may have an effect. Experiments are in progress which bear on this question.) using Rhizobium sp. 32H1. A lag period is always observed before C2H,, reduction begins, but once initiated the reduction continues for several days. In this experiment the inoculum contained approximately 107 cells/ml (optical density at 680 nm 0.003). The final optical =
La.
density
was
0.089, and cell densities much
greater than this cannot be attained under these
conditions even when a larger inoculum is used. Some cell division, as measured by an increase in optical density, is apparently required for obtaining good C,H, reduction. With high initial cell densities (0.25 or greater), little or no cell division occurs, and little activity develops. Experiments which will be presented elsewhere show that a nitrogen source and a carbon source are required for the development of good activity. Sugars or sugar derivatives are the most effective carbon source. With glutamate as the nitrogen source, specific activities of 600 nmol of ethylene/mg of protein per h have been observed, but rates of 50 to 100 are more typical. As a comparison, bacteroids isolated from lupin nodules have been reported to have a specific activity of 300 to 1,500 (9). In most experiments, C2H, was present from time zero so that C2H, formation could be monitored continually. Separate experiments in which C,H2 was added periodically, after the removal of oxygen, have shown that C 2H,2 not required for the formation of the enzyme system.
The effect of various inhibitors on acetylene reduction in Rhizobium sp. 32H1 is illustrated in Fig. 2. The inhibitors were added after the reduction was well initiated so that the effects observed can be presumed to be on the enzyme
I0
O.D.
I
200
wo
I
CONTROL/
CZ
*ONH +
iL
/00 f4
-
0
48
96
144
192
240
//
100
-
HOURS 0
FIG. 1. Time course of anaerobic growth and ethylene formation by Rhizobium sp. 32HI. Aerobically grown cells were harvested and inoculated into fresh culture medium to about 107 cells/ml. Casamino Acids replaced yeast extract in the medium. Samples (3 ml) were aseptically transferred to 30-ml serum bottles followed by evacuation and flushing four times with a 90% argon-10% C,H, mixture. The bottles were incubated at 28 C with gentle rotary shaking. Samples of the gas phase were analyzed for C1H, by gas chromatography using a Porapak N column. The acetylene was used as an internal standard. All reactions were performed in triplicate, and aseptic technique was rigidly followed throughout the experiments. Optical density measurements were made at 680 nm in a 1-cm cuvette with a Beckman DU. No acetyleneindependent ethylene production has ever been observed under an argon atmosphere.
CON
-CO,NO 3
CN-,N3-,2
/
0
0
E
2
c
0
L
.
0
40
. 1%
I
.
80 120 HOURS
,
160
200
#%^ _
FIG. 2. Effect of inhibitors on acetylene reduction. Aerobically grown cells were diluted to 1.2 x 108 cells/ml in the following medium: K,HPO,, 50 mM; MgSO,, 0.4 mM; mannitol, q.5%; sodium gluconate, 0.5%; Casamino Acids, 0.025%; NaMoO,, 30 ,gM; and iron citrate, 0.1 mM, pH 7.2. The experiment was as indicated in the legend to Fig. 1. The inhibitors were added after 94 h or as indicated by the arrow. Concentrations were as follows: NH,Cl, 2 mg/ml; KNO,, 6.6 mM; NaN,, I mM; KCN, I mM; CO, 0.8%; and 02, 2.7%.
VOL. 123, 1975
activity rather than on enzyme formation. The inhibition by carbon monoxide, cyanide, azide, and 0, are characteristic of nitrogenase-catalyzed acetylene reduction. Oxygen at concentrations greater than 2% totally inhibited the development of activity. Concentrations lower than 2% were partially tolerated, but maximum specific activity was obtained with no added oxygen. This is in contrast to the relatively high oxygen requirement for acetylene reduction by bacteroids isolated from legume nodules (1). Therefore future experiments will be designed to explore and hopefully resolve this paradox. Nitrate was inhibitory and cannot be used as a terminal electron acceptor for studies with nitrogenase with Rhizobium sp. 32H1. Ammonium chloride had no effect on the enzyme activity. More interestingly, NH4+ up to 37 mM had little effect on enzyme formation. This is in contrast to the effect of ammonia on other free-living nitrogen-fixing microorganisms where repression by ammonia seems to be a general characteristic of wild-type organisms. Lack of ammonia repression may not be a general characteristic of rhizobia, however, for preliminary results indicate that nitrogenase synthesis is repressed by NH,Cl in R. japonicum 61A76. The parameters which appear most important in inducing nitrogenase in these experiments are in order of importance, are as follows. (i) The use of low cell densities in small volumes is the most important. Some division is apparently required, and with initial densities greater than 6 x 10' cells/ml little or no growth occurs and little acetylene reduction is observed. Typically, cultures of less than 5 ml in 15- or 30-ml serum bottles have been used for these experiments. For unknown reasons, attempts to scale up to larger volumes have given only sporadic success. (ii) Removal of oxygen is important. although the cells tolerate some oxygen, the presence of oxygen increases the lag period for nitrogenase development and decreases the specific activity. R. japonicum 505 has been reported to use nitrate as a terminal electron acceptor (4). I have found that several other rhizobia can utilize nitrate as a terminal electron acceptor; however, nitrate inhibited nitrogenase activity with Rhizobium sp. 32H1 and thus could not be used as a terminal electron acceptor in the present experiments. (iii) The strain of Rhizobium used is important. Rhizobium sp. 32H1 has given the most consistent results and highest specific activities of ethylene formation. However, we have also observed acetylene reduction with Rhizobium
NOTES
1267
sp. 41A1 and 41F2, and R. japonicum 3Ilb83, 3Ilb125, 61A76, and ATCC 10324. Thus, it is likely that nitrogenase development in the absence of plant materials is possible for many rhizobia and that with the development of optimal conditions the enzyme can be demonstrated in other species. (iv) A source of combined nitrogen is required, with glutamate or aspartate giving the best results. (v) With some strains a high phosphate concentration is stimulatory but not required. Many past attempts have been made to induce nitrogenase in cultures of rhizobia. The use of plant cell cultures (3, 8, 10) or material diffusing from such cultures (M. Reporter, Biochem. Biophys. Res. Commun., in press; 12) has given positive results. This paper is the first report of the induction of nitrogenase activity in rhizobia with a defined media without plant materials. Thus it appears probable that the host plant is supplying an appropriate environment and the necessary substrates for nitrogen fixation in symbiosis but not the genetic information. I thank Gerald Peters, Minocher Reporter, Robert Darrow, and Marvin Lamborg for helpful discussion and criticism, and Norma Jean Raveed for technical assistance.
ADDENDUM Since this paper was submitted it has been found that oxygen is required for the cell growth and acetylene reduction reported. When the reaction flasks containing an argon-C,H, atmosphere were incubated in an external oxygen-free atmosphere, little ethylene formation was observed. When the external oxygen concentration was increased above 0.2 atmosphere, higher rates were found. Thus it appears that diffusion of oxygen through the rubber septum is required. Gas chromatographic analysis of the reaction vessels indicated that the oxygen concentration during the active acetylene-reducing period was less than 0.001 atmosphere. The rate of oxygen diffusion, however, is not known. Preliminary experiments carried out with small liquid culture volumes in large-volume flasks, so that the oxygen concentration can be maintained for a reasonable period of time, indicate that the optimal oxygen concentration of the gas phase is very low, about 0.001 atmosphere. LITERATURE CITED 1. Bergersen, F. J., and G. L. Turner. 1968. Comparative studies of nitrogen fixation by soybean root nodules, bacterial suspension and cell-free extracts. J. Gen. Microbiol. 53:205-220. 2. Bishop, P. E., H. J. Evans, R. M. Daniel, and R. 0. Hampton. 1975. Immunological evidence for the capability of free-living Rhizobium japonicum to synthesize a portion of a nitrogenase component. Biochim. Bio-
phys. Acta 381:248-256. 3. Child, J. J. 1975. Nitrogen fixation by a Rhizobium sp. in association with non-leguminous plant cell cultures.
1268
NOTES
Nature (London) 253:350-351. 4. Daniel, R. M., and C. A. Appleby. 1972. Anaerobicnitrate, symbiotic and aerobic growth of Rhizobium japonicum: effect on cytochrome P4,O, other haemoproteins, nitrate and nitrite reductases. Biochim. Biophys. Acta 275:347-354. 5. Dunican, L. K., and A. B. Tierney. 1974. Genetic transfer of nitrogen fixation from Rhizobium trifolii to Klebsiella aerogenes. Biochem. Biophys. Res. Commun. 57:62-72. 6. Hardy, R. W. F., R. C. Burns, and R. D. Holsten. 1973. Applications of the acetylene-ethylene assay for measurement of nitrogen fixation. Soil Biol. Biochem. 5:47-81. 7. Hardy, R. W. F., D. Holsten, E. K. Jackson, and R. C. Burns. 1968. The acetylene-ethylene assay for N, fixation: laboratory and field evaluation. Plant Physiol. 43:1185-1207. 8. Holsten, R. D., R. C. Burns, R. W. F. Hardy, and R. R.
J. BACTERIOL.
9. 10. 11.
12. 13.
Herbert. 1971. Establishment of symbiosis between Rhizobium and plant cells in vitro. Nature (London) 232:173-176. Kennedy, I. R. 1970. Kinetics of acetylene and CNreduction by the N,-fixing system of Rhizobium lupini. Biochim. Biophys. Acta 222:135-144. Phillips, D. A. 1974. Factors affecting the reduction of acetylene by Rhizobium-soybean cell associations in vitro. Plant Physiol. 53:67-72. Phillips, D. A., R. L. Howard, and H. J. Evans. 1973. Studies on the genetic control of a nitrogenase component in leguminous root nodules. Plant Physiol. 28:248-253. Scowcroft, W. R., and A. H. Gibson. 1975. Nitrogen fixation by Rhizobium associated with tobacco and cowpea cell cultures. Nature (London) 253:351-352. Vincent, J. M. 1970. A Manual for the practical study of root nodule bacteria. IBP Handbook No. 15. Blackwell Science Publishers, Oxford.
