Clostridium pasteurianum

Transcription

1 JOURNAL OF BACTERIOLOGY, Apr. 1972, p Copyright American Society for Microbiology Vol. 110, No. 1 Printed in U.S.A. Effect of Ammonia on the Synthesis and Function of the N2-Fixing Enzyme System in Clostridium pasteurianum GERALDINE DAESCH AND LEONARD E. MORTENSON Department of Biological Sciences, Purdue University, Lafayette, Indiana Received for publication 1 November 1971 The N2-fixing system of Clostridium pasteurianum operates under regulatory controls; no activity is found in cultures growing on excess NH2. The conditions which are necessary for the synthesis and function of this system were studied in whole cells by using acetylene reduction as a sensitive assay for the presence of the N2-fixing system. Nitrogenase of N2-fixing cultures normally can fix twice as much N2 as is needed to maintain the growth rate. When cultures that have grown for four or more generations on NH3 exhaust NH3 from the medium, a diauxic lag of about 90 min ensues before growth is resumed on N2. Neither N 2-fixing nor acetylene reduction activity can be detected before growth is resumed on N2. N2 is not a necessary requirement for this synthesis since under argon that contains less than 10-8 M N2, the N2-fixing system is made. If NH3 is added to N2-dependent cultures, synthesis of the enzyme system is abruptly stopped, but the enzyme already present remains stable and functional for at least 6 hr (over three generations). Cultures grown under argon in a chemostat controlled by limiting ammonia have derepressed nitrogenase synthesis. If the argon is removed and replaced by N2, partial repression of nitrogenase occurs. Previous studies (8, 12, 14, 16) have shown that there is a lag in growth that occurs when a potential N 2-fixing bacterium growing on ammonia exhausts its ammonia supply and prepares to fix N 2. Assay for nitrogenase showed that little or no N2 was fixed during this lag period and that the nitrogenase components, Fe protein and MoFe protein, are not detected until the end of the lag period (10). Also it was reported that nitrogenase was synthesized at a derepressed rate if cells were cultured in a chemostat on limiting ammonia under argon and that partial repression was restored if N2 replaced the argon (3). Cell-free extracts of Clostridium pasteurianum which have N2-reducing activity are also capable of reducing acetylene to ethylene (4). This reaction has the same requirements as the reduction of nitrogen but is much simpler and more sensitive than other techniques for measuring N fixation. It has been used as an assay for N 2-fixing activity in excised nodules, bacteroids, and cell-free extracts from soybean roots (5, 6) and in soils and natural waters (13). Intact cells of C. pasteurianum also reduce acetylene to ethylene, provided an oxidizable carbohydrate is supplied. Since only small amounts of the intact cells are needed for the assay and since the assay can be performed immediately after sampling with almost no preliminary manipulation, it has been of great value in the study of regulatory mechanisms operating on the N 2-fixing complex. The latter is the subject of this publication. MATERIALS AND METHODS The cultures were grown on a modified Winogradsky medium containing the following: 2 x 10-4 M MgSO4 and FeCls; 2 x 10-' M MnSO4 and Na- MoO4; 2 x 10-6 M ZnSO4, CuSO4, and CoCl2; 2% sucrose; 6.5 x 10-3 M phosphate and traces of biotin. The 160-liter culture was grown in a stainless-steel fermentor under a constant flow of N2; the ph was maintained at 6.0 by the addition of 10 N KOH with an automatic ph controller. Small cultures (200 ml) were grown under high purity N2 at constant flow of less than 200 ml/min in 500-ml aspirator bottles, the tubulation of which was fitted with thick-walled rubber tubing closed with a screw clamp. Samples were withdrawn anaerobically by a hypodermic needle and syringe through this tubing while mag- 103

2 104 DAESCH AND MORTENSON J. BACTERIOL. netic stirrers were used to keep the cells evenly suspended. These 200-ml cultures, as well as the 8-liter inoculum for the 160-liter culture, were buffered with solid CaCO, (3 g/liter). The same medium was used for chemostat cultures except that instead of adding CaCO, it was made 0.15 M in potassium phosphate, ph 6.9. This buffer maintained the ph of the cultures above 6.2 if the optical density (OD) at 660 nm (see below) of the culture was kept below 2.0. Vacuum flasks (500 ml) were used for the growth vessel. The overflow exited from the side arm. A 9-liter serum bottle was used for the reservoir medium which was introduced to the growth vessel through tygon tubing by an adjustable Sigma motor pump. (NHJ 2S90 was used for the NH, source in all NH,-supplemented medium. Acetylene reduction assays were performed by a modification of the method of Koch and Evans (5) as described by Moustafa and Mortenson (11). No additions were made to the 2-ml samples of continuous cultures assayed since sufficient quantities of buffer and sucrose already were present. For assys of noncontinuous cultures, 1.9 ml of the culture and 0.1 ml of anaerobic 1 M phosphate buffer, ph 7, containing 2% sucrose were added to the reaction tube. OD measurements for following cell density were made at 660 nm in a 1-cm cuvette with a Bausch and Lomb Spectronic 20 colorimeter. Samples were diluted to give readings between 0.1 and 0.3 with 4% acetic acid to solubilize interfering precipitated salts in the medium. One liter of culture at an OD of 1.00 at 660 nm contained 600 mg of dry cells; 1 ml of cells at an OD of 1.00 contained about 300 pg of protein. NH, analysis was by the microdiffusion method of Conway (2). A previously reported procedure was used for the preparation of cell-free extracts and the manometric assay for N, fixation (9). Protein was assayed by the biuret method (7). RESULTS AND DISCUSSION Synthesis of the N,-fixing system following NH, exhaustion. The enzyme system of C. pasteurianum that reduces N, to NH, is not present in extracts of cells that have been grown on media supplemented with excess NH, (10). To see if N,-fixing activity could be detected before growth began on N, when a culture was switched from NH, to N,-dependent growth, the following experiment was performed. A medium that contained 4 mm NH, was inoculated to an OD of 0.09 with a culture growing exponentially on NH3. The total volume of the culture was 160 liters. After three doublings and while NH, was still present in the medium, 40 liters was centrifuged and dried under vacuum. Approximately one-half generation later, NH, was exhausted from the medium and 20 min later a second 40 liters was harvested. The lag phase lasted 1 hr. Twenty minutes after the resumption of growth, a third 40-liter portion. was harvested, and, after the culture had completed another doubling, the remaining 40 liters was centrifuged and dried. Extracts were made of all four samples, and these were tested for N,-flxing activity (9). The activities measured are given in Fig. 1. Nitrogen was fixed only in extracts from cells obtained after the lag phase. Addition of either the Fe or the MoFe protein components of clostridial nitrogenase (10) did not give activity, so one can presume that neither nitrogenase component was present. Extracts of cells harvested 2 hr and 20 min after the lag had three times the activity of cells taken 20 min after the lag. These results were confirmed using the more sensitive assay, acetylene reduction. For this experiment, we used a 200-ml culture buffered with CaCO, and supplemented with 4 mm NH,. The inoculum was from a culture 4._ too* 'a 0.60 C) Q40- Q201 N ȧ a ao E 'a w- z U, 4.0 E I a o Time (hours) FIG. 1. N.-fixing activity in cultures of C. pasteurianum changed from NHs-dependent to N.-dependent growth. Forty liters of culture was harvested at the times indicated by the open arrows. T7he solid arrow indicates the point at which NH, in the medium (2 mm) was consumed. The cells were dried, extracted, and assayed for N,-fixing activity as described in Materials and Methods. T7e first point indicating the presence of N,-fixing activity is of questionable significance since the extremely low activity obtained is within the experimental error of the technique. 2D E

3 VOL. 10, 1972 NH, AND N, FIXATION 105 growing exponentially with NH, as the nitrogen source. At the times indicated in Fig. 2, samples were removed anaerobically with a syringe for OD measurement, determination of residual NH,, and acetylene reduction analysis. Acetylene reduction assays were performed immediately after sampling and were started, as indicated in the figure, at the time that NH, was exhausted from the medium. No activity was observed until growth had resumed on N,. Crude extracts of cells harvested in the mid- and late-diauxic lag phase also had no acetylene-reducing activity even if supplemented by either the Fe or MoFe proteins of C. pasteurianum (10). Since a specific activity of 1 nmole of N2- fixed per min per mg of protein is equivalent to about 3 to 4 nmoles of acetylene reduced to ethylene per min per mg of protein, the activities in Fig. 1 and 2 are comparable. A threefold increase in N.-fixing activity from 7.2 to 22.0 nmoles of N, fixed per min per mg of protein in the first generation of growth on N, (Fig. 1) TireAhours) FIG. 2. Acetylene-reducing activity in cultures of C. pasteurianum changed from NH, to N,-dependent growth. Samples to be tested for acetylenereducing activity were removed anaerobically from a 200-ml culture growing under an atmosphere of N, with (NH.),S04 present at an initial concentration of 2 mm. The arrow pointing downward indicates that this sample and all subsequent samples were assayed for acetylene-reducing activity; the arrow pointing upward indicates the time at which NH, was exhausted from the medium. The first sample was taken just before NH, was exhausted from the medium; no activity was observed before N,-dependent growth began. suggests excessive synthesis of nitrogenase. The OD increase was logarithmic, whereas the capacity of a cell to fix nitrogen, based on cellfree activity, increased. The same conclusion can be drawn from Fig. 2 in which the rate of acetylene reduction increased from 9.5 to 23.0 nmoles per OD per min (31 to 76 nmoles of ethylene produced per min per mg of protein) during the first hour. The dramatic increase in nitrogenase (measured by acetylene reduction) was followed by a drop in nitrogenase activity (Fig. 2). This suggests that a surge in nitrogenase synthesis and hence N, fixation occurred during the first 90 min after the lag. This produced a pool of ammonia large enough to decrease the rate of further nitrogenase synthesis and the concentration of nitrogenase per OD decreased. Relationship of length of diauxic lag to the number of generations cells were grown on NH,. The growth curve shown in Fig. 1 is typical of cultures supplied with limiting NH, under an atmosphere of N, and inoculated from an NH,-dependent culture; that is, the density of the culture increases until shortly after the NH, is exhausted, the culture then goes through a diauxic lag of greater than 1 hr, and then it resumes growth at the rate characteristic of a N2-fixing culture. It was observed, however, that if the NH,-grown inoculum was replaced by a N.-fixing inoculum, the diauxic lag was shortened to about 30 min. This suggested that the length of the lag was affected by the number of generations during which the culture was exposed to NH,. One could also ask why there is a lag at all since nitrogenase is present in the inoculum. To examine this further, in two separate but identical experiments, two flasks were simultaneously inoculated from a single N -fixing culture. The first was supplemented with enough NH, to permit one generation of growth and the second with enough for two generations of growth. Two additional flasks, with the higher amount of NH, present, were inoculated from a culture grown for four generations on excess NH,. All were under an atmosphere of N,. Samples were removed at appropriate times for OD measurement and NH, analysis. The results (Fig. 3) show that the cultures exposed to NH, for one and two generations had shortened diauxic lag periods in the order of 36 min, whereas those which had grown on NH, for six generations (four before inoculation) had lag periods of 75 min. The culture that had grown a total of six generations on NH, stopped growth abruptly when the NH, was exhausted from the me-

4 106 DAESCH AND MORTENSON J. BACTERIOL. TIME (Hours) FIG. 3. Effect of the number of generations of exposure to NH, on the length of the NH, to N2 diauxic lag. Symbols: 0, one generation growth of NH3; 0, two generations growth on NH,; A, six generations growth on NH3. All cultures were grown with a low flow of N2 through the standard medium containing 2No sucrose. Flask 0 was supplemented with (NH),2SO4 at a concentration of 0.75 gmole/ml; flasks 0 and A were supplemented with 1.5 gmoles of (NH4)2SO4 per ml. Flasks * and 0 were inoculated from a N2-fixing culture and flask A was from a culture grown four generations on excess NH3. Samples were withdrawn anaerobically and OD was measured. Arrows indicate points at which NH, was no longer detectable in the medium. dium. In contrast, the cultures that had grown only one or two generations on NH3 continued to increase for a quarter of a generation and a full generation, respectively, after the NH3 was depleted from the medium before they entered the diauxic lag. These findings suggest that the N 2-fixing system remains stable and functional in the presence of NH3 and that the lag is shortened because the cell is still capable of providing itself, via limited N2 fixation, with NH3 for the synthesis of additional N2-fixing system. The fact that the culture, which had been given only enough NH3 to support one generation of growth, continued to grow for another generation after the NH3 was exhausted suggests that there is a critical level of the N2-fixing system that is necessary for N 2-dependent growth, and, after two generations of growth in the absence of nitrogenase synthesis, the concentration of the N2-fixing system is below the level. This suggestion is supported by the facts that (i) after two generations of growth without enzyme synthesis, growth stops after onequarter generation until more enzyme is synthesized, whereas, after one generation without enzyme synthesis, the cells continue to divide until a second doubling occurs, and (ii), after six generations without synthesis of the N2- fixing system, there is no further growth when NH, is exhausted. Complete repression of synthesis of the N,-fixing system by NH,. An experiment was performed to investigate if the N2-fixing system is immediately stopped by the presence of NH, and to further examine the stability of the N2-fixing system by a more direct and quantitative method. First, a continuous culture of C. pasteurianum growing on NH3- free medium under N, was equilibrated at an OD of 0.60 with a generation time of 2 hr. The culture then was assayed at 0 and 15 hr for its acetylene-reducing activity. The average maximal rate of ethylene formation for duplicate determinations at each time was 110 i 3 nmoles per min per mg of cell protein. Ten mmoles of (NH4XSO4 was then added to the 500 ml of culture in the growth vessel, and the flow of medium was stopped so that the cells containing the N2-fixing system would not be washed out. Samples were taken at 10 and 50 min and at hourly intervals thereafter and assayed for acetylene-reducing activity. To prevent the acid products of an excessive population density from decreasing the ph, it was necessary to introduce fresh medium to keep the culture at a density less than OD 2.5. The results shown in Fig. 4 were corrected for these dilutions, but after 6 hr the N2-fixing system was so diluted by cell growth in the absence of nitrogenase synthesis that the activity was too low to be measured. Therefore the drop in activity seen in Fig. 4, at 6 hr, is questionable. Figure 4 shows that there was no significant decrease or increase in the activity of the N2- fixing system already present when NH3 was added to the medium but that further synthesis of the N 2-fixing system was abruptly stopped by the addition of NH,. The N2-fixing system was shown (17) to fix 16N2 in the presence of NHs.. The above experiment confirms and extends those results. Effect of NH3 on acetylene reduction. The manometric assay for N2 fixation by cell-free extracts is insensitive to the addition of rather high levels of NH3 to the assay mixture (1). To establish that acetylene reduction activity was not affected by the presence of NH3, a reaction tube in which acetylene reduction had been measured at a constant rate over a period of 9 min was then made 0.05 M with respect to (NH4) 2S04. Like the control reaction tube, there was no change in the reaction rate during the next 12 min. Effect of N2 on the synthesis of the N2- fixing system. The experiments described strongly suggest that NH3 acts to cause repres-

5 VOL. 10, 1972 NHS AND N2 FIXATION Time (hours) FIG. 4. Stability of the N2-fixing enzyme system in the presence of ammonia. (NHJ ISO4 (20 mm) was added to a N2-fixing culture of C. pasteurianum and samples were assayed for acetylene reduction at the times indicated. In each assay, ethylene production was followed for at least 15 min and the activity was calculated as nmoles of ethylene produced per OD per min. All values were adjusted to the total that would be present if the culture had not been diluted. sion of enzyme synthesis and that the loss of the function of N3 fixation during growth on NH. is a result of simple dilution of the cellular enzyme level rather than of active destruction of the existing proteins. To determine if the synthesis of the N 2-fixing system was controlled solely by the level of NH3 present, several experiments were performed. In one, a medium containing NH3, but in a limiting concentration, was inoculated from an NH3-grown culture and kept under argon for up to 2 hr after the NH3 was exhausted; the usual lag period in growth observed by measuring OD increase when N2 was present instead of argon was 1 hr and 15 min. Since there was no source of nitrogen for growth, no increase (or decrease) in cell density occurred. Two hours after the beginning of the lag, N, was introduced, and without a lag period the cell density of the culture increased. Obviously, the N2-fixing system was synthesized during the period in which argon was being sparged through the culture, and its synthesis did not appear to require induction by N2, but only the absence of NH3. In a second experiment, a chemostat culture growing under a continuous flow of argon on media containing limiting NH3 (2 mm), was 107 equilibrated overnight at an OD of 0.60 and a generation time of 3.3 hr. Its generation time with NH, in excess was 72 min. The atmosphere of the growth vessel was then changed from argon to N2, and cell density measurements were made on the effluent during the next 8 hr. There was no drop in OD as expected. Instead the OD in the vessel increased from 0.60 to 1.68 even when the generation time was decreased from 3.3 hr to 2.25 hr by changing the flow rate. When the gas phase was again changed to argon and the generation time restored to 3.3 hr, the OD retumed to its former value, Again it was returned to a N2 atmosphere, and the OD increase followed for 5 hr at which time the reservoir medium was changed to an NH3-free one. A small but significant drop in OD from 1.32 to 1.20 followed the change to the NH 3-free medium. This experiment was repeated twice with good reproducibility. It was concluded that with limiting NH, present, a high concentration of the N2-fixing system is present. In a third experiment (Fig. 5), acetylene reduction was used as an assay for the presence of the N2-fixing system before and after the culture was placed under N2. The culture was equilibrated overnight at an OD of 0.54 under argon on a medium limited in NH3 (1.5 mm). It was tested for acetylene reduction and found to have higher activity than is found in cultures growing completely on N2, 300 nmoles of ethylene formed per min per mg of protein compared with 101. The culture was then Time (hours) FIG. 5. Acetylene reduction activity (nitrogenase) of C. pasteurianum grown on a limited NH, medium under argon and then put under N2. A chemostat culture was equilibrated on a medium that was 1 mm in (NH.)$SO, under argon and with the flow rate of the medium such that the generation time of the culture was 3.5 hr. Samples were assayed for acetylene-reducing activity and then the culture was placed under an atmosphere of N2. The increase in OD because of N2-dependent growth was measured at the indicated times, and assays for acetylene-reducing activity were performed.

6 108 DAESCH AND MORTENSON J. BACTERIOL. changed to a N atmosphere and acetylene reduction assays were performed at 15-min intervals. An increase in activity was observed over the first 45 min which then began to decline. The increase suggests that, because NH. was limiting, nitrogenase was not made at its maximum rate, but, when N2 was fixed and NH8 became available, the rate of nitrogenase synthesis increased. When a larger NH, pool per cell was produced by N2 fixation, nitrogenase synthesis decreased until a new level was established. At 45 min an increase in OD was detected. The rapid drop in acetylene reduction observed from 30 to 60 min is greater than one would expect from simple dilution. This could represent a decay in active nitrogenase during the period although it seems unlikely that nitrogenase would decay when obviously N, fixation is occurring. More likely it reflects a change in metabolism of the organism during the period when NH, is in excess, i.e., at 30 min. This change in metabolism would be expected to increase the adenosine diphosphate (ADP)-adenosine triphosphate (ATP) ratio in the cell because of a large increase in synthesis of NH, acceptors. Increase in ADP/ATP would inhibit nitrogen fixation (11). Thus, if during the 30- to 60-min period nitrogenase activity (measured by acetylene reduction) was partially inhibited, the drop in measured acetylene reduction would be greater than expected by dilution. When the chemostat culture (Fig. 5) was later switched to an NHr-free medium and acetylene reduction activity was measured two hr later, the activity was 160 nmole per min per mg of protein, a result approaching the 101 nmole per min per mg of protein characteristic of a N2-dependent culture in a chemostat. The high level of acetylene-reducing activity, observed in cultures in the absence of N, but in the presence of a limiting supply of NH3, steadily declines when N, is supplied because the "corepressor," NH3, produced by N2 fixation, accumulates. This is probably a result of limiting ATP supply which would also result in an insufficient production of NH3 acceptors for NH3 assimilation. It is known that cultures grown under conditions where N2 is limiting excrete NH, when excess N, is supplied but then readjust so that no additional excretion occurs and the previously excreted NH3 is assimilated (unpublished results). This suggests that the high level of acetylene-reducing activity in the nitrogen-starved cells is the end result of a compensatory mechanism which raises the level of the Nr-fixing system (even in the absence of No) as the level of available nitrogen (NH ) declines. The production of the N2-fixing system, regulated by the concentration of NH,, in turn may be regulated by the rate of production of NH3 acceptors since NH3 accumulates without them. The decline in the acetylene-reducing activity during the first 2.25 hr of growth after N2 was supplied indicates that such a large excess of NH3 was produced that decreased synthesis of the N2-fixing system occurred until this corepressor pool (NH3) decreased by assimilation to a level normal for a N2-fixing culture. Thus the acetylene-reducing activity dropped from 300 to 120 over a period of 8 hr (Fig. 5). Under these conditions the cell which is nitrogen starved is in the curious position of utilizing a previously limiting substrate (N) to synthesize its product (NH3) rapidly in such an excess that it in turn stops the synthesis of the N 2-fixing system which only minutes before was "limiting." Is N2 required as an inducer? A culture under argon is not under an absolutely N2-free environment since trace amounts of N2 are probably present in the argon. Since N2 in trace amounts cannot be excluded with assurance because there is no method to detect its absence, the upper limit of N2 contamination was calculated to see if the concentration was comparable to the required concentration of inducers for known inducible systems. With the assumption that the reservoir medium was saturated with air (this could not be so since it was kept under an argon atmosphere during the experiment), the upper limit of N2 contamination was calculated to be 5 x 10-8 M, approximately 3 x 1016 molecules per liter or about 3 x 105 molecules per cell, when the OD was about 1.0. This is to be contrasted to an inducer of the,b-galactosidase system in which the minimal concentration of isopropyl-fl-dthiogalactoside that will induce enzyme formation is 2 x 10-I M with the cell density of Escherichia coli at 0.1 to 0.15 (15). Although it is conceivable that a substance at this concentration could act as an inducer, it is not reasonable to suppose that the organism would have evolved a working control system that would depend on an artificial situation. That is, it is highly unlikely that any natural terrestrial environment would be less free of N, than the conditions used here, and, if N2 were an inducer at these low concentrations, nitrogenase would always be present. Argon was the preferred choice of gases for these experiments since the N2 content was low. The cultures could not be grown under vacuum to eliminate

7 VOL. 10, 1972 NH3 AND N2 FIXATION 109 traces of N2 since C. pasteurianum has a definite growth requirement for CO2. In vivo rate of N2 fixation. The acetylene reduction assay provides a mean of estimating the in vivo rate of N, fixation. If the assumptions are made (i) that about one-third the rate of acetylene reduction to ethylene is represent- Ative of the rate at which N2 is reduced to 2 NH2 and (ii) that under the nongrowing conditions used when measuring acetylene reduction (no nitrogen source) ATP is nonlimiting and therefore no mechanism controlling the N 2- fixing system is operating, the rate of acetylene reduction of about 67 nmoles per min per OD would equal a rate of N fixation of 22 nmoles per min per OD. Since 1 OD unit of culture contains about 1,500 nmoles of N2, a growing culture at an initial OD of 1.0 should be able to fix enough N, to double its OD in about 47 min. Since the shortest generation time observed for a N2-fixing culture is 110 min, the capacity of the N2-fixing system is considerably greater than that needed. This is consistent with evidence that the rate of energy supply is the growth-rate-limiting factor for these N2-fixing organisms, rather than the rate of N, fixation (3). ACKNOWLEDGMENTS We wish to thank Esam Moustafa for his help in the measurement of ethylene by gas chromatography. This work was supported by National Science Foundation grant GB LITERATURE CITED 1. Carnahan, J. E., L. E. Mortenson, J. F. Mower, and J. E. Castle Nitrogen fixation in cell-free extracts of Clostridium pasteurianum. Biochim. Biophys. Acta 44: Conway, F. J Microdiffusion analysis and volumetric error. Crosby, Lockwood and Son, Ltd., London. 3. Daesch, G., and L. E. Mortenson Sucrose catabolism in Clostridium pasteurianum and its relation to N, fixation. J. Bacteriol. 96: Dilworth, M. J Acetylene reduction by nitrogenfixing preparations from Clostridium pasteurianum. Biochim. Biophys. Acta 127: Koch, B., and H. J. Evans Reduction of acetylene to ethylene by soybean root nodules. Plant Physiol. 41: Koch, B., H. J. Evans, and S. Russell Reduction of acetylene and nitrogen gas by breis and cell-free extracts of soybean root nodules. Plant Physiol. 42: Layne, E Spectrophotometric and turbidimetric methods for measuring proteins, p In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 3. Academic Press Inc., New York. 8. Mahl, M. C., and P. W. Wilson Nitrogen fixation by cell-free extracts of Klebsiella pneumoniae. Can. J. Microbiol. 14: Mortenson, L. E Ferredoxin and ATP, requirements for nitrogen fixation in cell-free extracts of Clostridium pasteurianum. Proc. Nat. Acad. Sci. U.S.A. 52: Mortenson, L. E., J. Morris, and D. Y. Jeng Purification, metal composition and properties of molybdoferredoxin and azoferredoxin, two of the components of the nitrogen-fixing system of Clostridium pasteurianum. Biochim. Biophys. Acta 141: Moustafa, E., and L. E. Mortenson Acetylene reduction by nitrogen-fixing extracts of Clostridium pasteurianum: ATP requirement and inhibition by ATP. Nature (London) 216: Pengra. R. M., and P. W. Wilson Physiology of nitrogen fixation by Aerobacter aerogenes. J. Bacteriol. 75: Stewart, W. D. P., G. P. Fitzgeral, and R. H. Burris In situ studies on N2 fixation using the acetylene reduction technique. Proc. Nat. Acad. Sci. U.S.A. 58: Strandberg, G. W., and P. W. Wilson Formation of the nitrogen-fixing enzyme system in Azotobacter vinelandii. Can. J. Microbiol. 14: Tonomura, B., and J. C. Rabinowitz An investigation of the induction of,8-galactosidase in a broken spheroplast preparation of E. coli. J. Mol. Biol. 24: Yoch, D. C., and R. M. Pengra Effect of amino acids on the nitrogenase system of Klebsiella pneumoniae. J. Bacteriol. 92: Zelitch, I Simultaneous use of molecular nitrogen and ammonia by Clostridium pasteurianum. Proc. Nat. Acad. Sci. U.S.A. 37: