HYDROGENASE AND NITROGEN FIXATION BY AZOTOBACTER

HYDROGENASE AND NITROGEN FIXATION BY AZOTOBACTER BY S. B. LEE? AND P. W. WILSON (From the Department of Agricultural Bacteriology, University of Wisconsin, Madison) (Received for publication, August 5, 1943) Previous studies (14) have demonstrated that cultures of ilzotobacter possess an active hydrogenase, the enzyme which catalyzes the oxidation of hydrogen. Some evidence has been uncovered which suggests that the occurrence of this enzyme is correlated with nitrogen fixation by this organism. If true, this has great significance for studies of the mechanism of biological nitrogen fixation. A detailed investigation was accordingly undertaken in which we measured the hydrogenase activity of Azotobacter cells grown or maintained under conditions which would vary the rate or extent of the nitrogen-fixing reaction. These included (a) the use of various forms of combined nitrogen; (b) variation in the level of combined nitrogen; (c) adaptation of cells to combined nitrogen; (d) growing cells in the presence of hydrogen and absence of free nitrogen. The general methods have been described in earlier reports; any necessary details will be furnished in the text. Effect of ph of Medium on Hydrogenase in Axotobacter-In the initial experiments with combined nitrogen (2) ammonium phosphate usually served as a source of this element. During growth a pronounced decrease in the ph occurred because of selective utilization of the ammonium ion. As is shown in Table I, however, it is the presence of combined nitrogen in the medium and not the alteration of ph which is accompanied by a decrease in hydrogenase. The data demonstrate that the hydrogenase content of Axotobacter drops in the presence of ammonium ion, whet,her the ph rises, falls, or remains constant. Effect of Combined Nitrogen on Hydrogenase in Various Species of Azotobacter-Cultures of Axotobacter vinelandii, A. agile, and,4. chroococcum were grown on Burk s agar medium and on the same medium plus combined nitrogen in various forms (50 mg. of N per 100 ml.). Table II gives the results of these experiments. As noted previously with A. vinelandii (2), NHk-N is much more effective in reducing the hydrogenase content than is NOS-N, whereas glutamate N has little if any effect. Experiments by Burris (unpublished data) in this laboratory with isotopic * This research was supported in part by grants from the Rockefeller Foundation and from the Wisconsin Alumni Research Foundation. t Present address, General Mills, Inc., Research Laboratories, Xnneapolis. 377

378 NITROGEN FIXATION W5 have shown that fixation of free nitrogen by a culture of Azotobacter transferred previously on an medium is not markedly inhibited by nitrate and even less by glutamate. If hydrogenase and nitrogenase are related, a marked reduction in the hydrogenase content of organisms grown TABLE Effect of ph and Nitrogen Source on Hydrogenase in Azotobacter I N\Tp NH~NOB NH4 acetate NS (NH,),HPO~$ (NH&HP04 NH4NOz NH1 acetate Ng - Relative hydrogenaset 7.0 6.9 3980 100 7.0 6.6 1480. 37.2 7.0 7.8 1920 48.2 6.5 6.5 4170 104.8 7.6 5.9 1510 38.0 7.0 5.8 1530 38.2 7.0 6.4 1500 37.7 7.0 6.7 1970 49.5 7.0 6.8 3970 99.8 * &(N) is the cmm. of total gas uptake (H,-02) per hour per mg. of N in the cells. i Per cent of Q=(N) for suspension transferred and grown on NI. $ Nitrogen added aseptically after sterilization of the medium. TABLE Effect of Various Nitrogen Sources on Hydrogenase Content of Azotobacter Species Azotobacter vinelandii Organism Azotobacter chroococcum Azotobacter agile II Medium Q#) Relative hydrogenas? 4415 100 (NH&HP04 1240 28.1 KNO, 2680 60.8 Glutamate 4395 99.5 2535 100 NHa acetate 790 31.2 KNO, 2020 81.0 Glutamate 2589 101.8 NH,NOs 348 13.7 3900 100 NH4 acetate 655 16.8 KN03 1275 32.7 Glutamate 3290 84.5 NHdNOz 1700 43.6 * The hydrogenase in cells grown on an medium equals 100. in the presence of these combined nitrogen sources would therefore not be expected. Adaptation of Azotobacter vinelandii to Various Sources of Combined Nitrogen--The results in Tables I and II were obtained with cultures carried in

S. B. LEE AND P. W. WILSON 379 stock on the medium. Similar experiments have been made which include cultures Lacclimatized or adapted, by previous serial transfer, to the source of nitrogen used. Smmonium Acetate-50 ml. of Burk s K-free liquid medium were sterilized in Roux bottles. T arying quantities of nitrogen as ammonium acetate were then added aseptically to the flasks. One set of flasks was inoculat,ed with a culture of Axotobacter vinelandii carried on the medium and a similar set with a culture which had undergone six transfers on Burk s TABLE E$ect of Different Concentrations of NH4-N on Nitrogen Fixation and Hydrogenase Content of Azotobacter vinelandii Cultures Grown Previously on N-Free Medium and on Medium Containing NH,-N. NH1 Culture acetatt NHa acetate N in medium nzf. per 100 ml 0 5 10 20 30 50 70 100 0 5 10 20 30 50 70 100 @K(N) - Relative hydrogenase 4740 100 4235 89.5 4380 92.5 3035 64.0 2395 50.6 1930 40.7 2025 42.8 2155 49.4 4945 104 4520 95.5 4950 105.0 3150 66.4 2475 52.2 1950 41.2 1245 26.2 1035 21.8 III Nitrogen balance Cells Medium Recovery* totoz mg. 10.4 8.8 10.2 16.6 21.4 25.8 17.8 10.2 10.4 7.6 10.4 17.0 19.4 27.4 18.8 14.0 m rg. per 100 ml per cent 0.3 182.5 3.4 136.0 4.1 103.7 7.9 97.7 26.1 103.5 49.1 95.5 85.4 95.6 0.0 152.0 1.2 116.0 3.7 103.6 8.1 91.7 19.7 94.4 49.7 97.8 83.1 97.1 - * When the recovery of N exceeds 100 f 10 per cent, the excess represents fixation of free N2. medium plus 500 parts per million of N as ammonium acetate. After 34 hours the cells were removed by centrifugation, washed, and the hydrogenase determined. The nitrogen in the supernatant was estimated by aeration of aliquots into standard acid after addition of NaOH. Table III presents the data from a typical experiment. As the quantity of combined nitrogen added to the medium decreases until it is equal to or is less than the total nitrogen in the cells, increased hydrogenase in the cells is observed. This means that an increase in the nitrogen-fixing enzyme system (as indicated by a recovery of nitrogen exceeding 100 per cent) is accompanied by an increase in hydrogenase. The hydrogenase

380 NITROGEN FIXATION activity of cells in the presence of lower levels of NHd-X is the same whether the culture had been previously grown on Ns or on NHJ. It is concluded that Axotobacter utilizes NHI-N at a rate that is independent of previous contact with this source of N. At higher levels (above 500 p.p.m.) the reduction in hydrogenase apparently is less with the non-adapted culture. This result is not clear cut, however, since the observed reduction in hydrogenase of the culture kept in air in this experiment was not as great as is usually noted (see, for example, Tables II and V). Potassium Nitrate-In this experiment the source of nitrogen was KNOs; the culture was acclimatized to it by being transferred serially eight times TABLE Effect of Different Concentrations of KNOS-N on Nitrogen Fixation andhydrogenase Content of Azotobacter vinelandii Cultures Grown Previously on N-Free Medium and on Medium Containing KNOZ-N Culture g. per 100 ml Q#) Relative hydrogenase IV Cells?Ctrogen balance Recovery total mg. 1 rtg. per 100 ml per cenf 0 4130 100 12.2 5 3610 87.3 9.4 3.5 258.0 10 3150 76.3 12.0 7.2 192.0 25 2920 70.7 12.0 14.0 104.0 35 2600 62.8 11.0 23.7 99.2 50 2400 58.2 10.2 36.8 93.6 100 2690 65.2 15.0 79.2 94.2 K1\T03 0 2870* 69.5* 10.0 5 10 25 35 50 100 2250 1350 1520 1710 1820 1630 54.4 32.7 36.8 41.4 44.0 39.4 10.6 0.0 212.0 9.0 1.1 101.0 9.4 14.4 95.3 8.6 30.3 111.0 9.0 44.5 106.8 9.6 98.0 107.6 * These low values may be explained by the carry-over of X0,-N in the inoculum. in the usual medium plus 3000 p.p.m. of KNOX-N. The residual nitrogen in the supernatant was determined by reduction with Devarda s alloy and distillation into standard acid. The results in Table IV show that with the non-adapted cultures KNOa, especially at low levels, has relatively little effect on the hydrogenase content, in agreement with the fact that it does not readily compete with the nitrogen-fixing reaction (5, 6). In contrast, the hydrogenase content of the adapted culture was quite markedly reduced by low levels of KNOS. The analytical methods were not sufficiently reliable to make it possible to determine from these data whether free nitrogen was fixed by either the adapted or non-adapted culture in the presence of excessive KNOZ.

S. 33. LEE AND I. W. WILSON 381 Fig. 1 illustrates the results which are relevant for the argument that hydrogenase activity is associated with the functioning of nitrogen fixation in Azotobacter. Whenever NH4 is the source of combined nitrogen, the hydrogenase activity is associated with the functioning of nitrogen fixation in Azotobncter. Also, if NH, is the source of combined nitrogen, the hydrogenase act,ivity decreases with an increase in the supply of fixed nitrogen whether or not the culture has been previously adapted to NH,. This agrees with the fact that Axotobacter does not require an adaptation RELATIVE WDROGENASE i4*~:t9i~y Bp lol m FIG. 1. Effect of different concentrations of NH*-N and NOS-N on nitrogen fixation and on the hydrogenase content of Azotobacter vinelandii cultures grown previously on an medium and on a medium containing NH,-N and NOI-N respectively. to NH, before this source of nitrogen successfully competes with Nz. If KOa-N is supplied the organism, however, the quantitative aspects of the decrease in hydrogenase depend on whether the culture has been adapted to nitrate N. With non-adapted cultures nitrogen fixation is not entirely suppressed by NOa-K (5, 6); as can be seen in the figure, the formation of hydrogenase likewise is not markedly reduced. Effect of Growth in Hz-02 Gas Mixture on Hydrogenase Activity of Axotobatter vinelandii-the effect of the specific substrate on the hydrogenase content of Szotobacter was determined by growing the organisms in an Hz-O2 mixture which forces the cells to obtain all nitrogen from the sub-

382 NITROGE?rT FIXATION &rate, as fixation cannot occur. There should be little, if any, nitrogenfixing enzyme formed; consequently, if hydrogenase and nitrogenase are closely related, such cells should have less hydrogenase activity. The technique for growing the cells is essentially that of Wyss and Wilson (7). 50 ml. of Burl& medium are placed in liter Pyrex bottles, in the center of each of which is a test-tube (24 X 100 mm.) containing 15 ml. of 20 per cent KOH. Each bottle is closed with a sterile, gas-tight rubber stopper through which is passed a gas inlet tube that terminates inside the KOH well. A sterile cotton filter in the line also aids in preventing contamination from the incoming gas. The desired gas mixture (ph2 0.8, TABLE Eflect of Growth in Hz-02 Gas Mixture on Hydrogenase Activity of Azotobacter vinelandii Medium NH4 acetate ICNO, I NH,NOa NH, acetate ICNO3 NH,NOs V Gas phase QKW) Relative ydrogenase Air 3310 100 990 29.9 Hz-02 623 18.8 Air 1160 35.0 H&h 632 19.1 Air 900 2i.2 Hz-02 324 9.8 Air 3070 92.8 1040 31.4 IL-02 782 23.6 Air 2540 77.0 I 422 12.6 Hz-02 95 2.9 Air 2470 74.8 460 13.9 Hz- & 290 8.8 poz 0.2 atmosphere) is added to the bottles by drawing a vacuum of 2G inches of mercury and filling it with hydrogen. This is repeated three times; then a vacuum of 5.8 to 6.0 inches is drawn and 02 added to zero pressure. The bottles are connected through a water seal to a manifold which leads to a reservoir of OS. During the test CO2 from respiration by the bacteria is absorbed in the KOH with a consequent pressure deficit which draws in more oxygen to replace that respired. Table V gives data from an experiment in which two types of inoculum were used, one serially transferred on an medium and a second on media containing the homologous combined nitrogen sources. All combined nitrogen was added to the bottles at the rate of 500 p.p.m., and the cells were grown for 34 hours.

S. B. LEE AND I. W. WILSON 383 In agreement with previous results, adaptation to ammonium acetate had little effect on the reduction of hydrogenase by this source of nitrogen, but adaptation to KNOB or NHhNOs was followed by a greater reduction in hydrogenase when supplied with these sources of fixed nitrogen. With either adapted or non-adapted cultures, growth in the Hz-O2 gas mixture, far from increasing the hydrogenase content, actually resulted in a decrease. This was especially marked in the adapted KNOs culture in which the hydrogenase was practically eliminated, even though the specific substrate, HZ, was present. DISCUSSION The evidence concerned with the association of hydrogenase activity and the nitrogen nutrition of Axotobacter provided in this report may be summarized as follows : 1. In the presence of low levels of combined nitrogen hydrogenase activity parallels, at least roughly, nitrogen fixation by Azotobacter. Initiation of nitrogen fixation through depletion of the combined nitrogen in the medium is accompanied by a sharp rise in the hydrogenase activity of the organisms. 2. It has been demonstrated with several species of Axotobacter that sources of combined nitrogen which do not readily inhibit nitrogen fixation also do not materially lower the hydrogenase content of the organism. 3. When organisms are acclimatized to sources of combined nitrogen not readily utilizable (for example, NOS), the nitrogen source not only inhibits nitrogen fixation more effectively, but also reduces the activity of hydrogenase. 4. The presence of the specific substrate for hydrogenase during the growth of Axotobacter does not cause a rise in the formation of the enzyme. Cells grown in the presence of HZ-O2 and combined nitrogen must obtain Oheir nitrogen from the substrate; since fixation is not possible, a minimum nitrogenase activity would be expected. Coincidental with this, a minimum hydrogenase content occurs in the cells. The major weakness of the data is the apparent lack of quantitative agreement between t,he activity of the nitrogen-fixing system and hydrogenase. In several of the experiments conditions were such that nitrogen fixation was entirely suppressed, but in only a few instances was hydrogenase activity completely absent. It must be realized, however, that whereas a quantitative estimation of hydrogenase is possible, only qualitative statements can be made about the nitrogen fixation system. In the presence of high concentrations of combined nitrogen, nitrogen fixation probably ceases, but whether this is due to the absence of the enzyme or to competitive inhibition by the combined nitrogen with Nz cannot be stat,ed

384 NITROGEN FIXATION with certainty. It may be that there occurs merely a reduction, not an elimination, of the nitrogenase and that this reduced content of enzyme is completely inhibited by the combined nitrogen source. The activity of the hydrogenase need not be affected by the competition, since it is known to differ from nitrogenase in many of its properties. In the complete absence of Nz we can be sure that no fixation occurs, but not so certain that this implies complete absence of the nitrogen-fixing enzyme. If the latter, it appears that nitrogenase is a highly adaptive enzyme formed only in the presence of the particular substrate. It is significant that hydrogenase likewise appears to be adaptive but responds to the presence of Nz and not HZ. Another observation which apparently conflicts with the view that the enzyme system responsible for nitrogen fixation in Axotobacter may be concerned with the oxidation of Hz is the inability to demonstrate an active hydrogenase in the components of the symbiotic nitrogen-fixing system (4). This opposing evidence may not be serious, however, when it is realized that the root nodule bacteria alone are unable to fix nitrogen and that results with excised nodules are erratic (8). Hydrogenase would not be expected, therefore, to be found in pure cultures of the bacteria and would probably be weak in excised nodules. Its detection with present methods may not be feasible, especially in view of the fact that demonstration of the enzyme, even in Azotobacter, is sometimes difficult in the presence of organic sources of carbon. The use of heavy hydrogen may supply a more sensit.ive method (4). The failure of the intact symbiotic system to utilize measurable quantities of hydrogen gas might also be explained by the observation that hydrogenase frequently appears to be inhibited by the presence of organic carbon substrates. SUMMARY Sources of combined nitrogen which readily inhibit nitrogen fixation by Axotobacter also inhibit formation of hydrogenase. This result does not arise because of concomitant changes in ph. Adaptation of the organism to nitrate nitrogen so that the latt,er is more readily assimilated causes an increase in the effectiveness of NO3 to inhibit both nitrogen fixation and hydrogenase formation. Inhibition of nitrogen fixation by combined nitrogen in an Hz-O2 atmosphere is accompanied by a marked decrease in hydrogenase, even though its specific substrate, Hz, is present. Its formation appears to respond more to the presence of NP than to HZ. From these results it is concluded that hydrogenase is closely related to the nitrogen-fixing system in Azotobacter.

S. B. LEE AND P. W. WILSON 385 BIBLIOGRAPHY 1. Wilson, J. B., Lee, S. B., and Wilson, P. W., J. Biol. Chem., 144, 265 (1942). 2. Lee, S. B., Wilson, J. B., and Wilson, P. W., J. Biol. Chem., 144, 273 (1942). 3. Wilson, J. B., and Wilson, P. W., J. Gen. Physiol., 26, 277 (1943). 4. Wilson, P. W., Burris, R. H., and Coffee, W. B., J. Biol. Chem., 147, 475 (1943). 5. Lind, C. J., and Wilson, P. W., Arch. Biochem., 1, 59 (1942). 6. Wilson, P. W., and Lind, C. J., J. Bat., 45, 219 (1943). 7. Wyss, O., and Wilson, P. W., Proc. Nut. Acad. SC., 27, 162 (1941). 8. Burris, R. H., Eppling, F. J., Wahlin, H. B., and Wilson, P. W., J. BioZ. Chem., 148, 349 (1943).

HYDROGENASE AND NITROGEN FIXATION BY AZOTOBACTER S. B. Lee and P. W. Wilson J. Biol. Chem. 1943, 151:377-385. Access the most updated version of this article at http://www.jbc.org/content/151/2/377.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/151/2/377.citation.full.h tml#ref-list-1