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The formation and activity of nitrogenasez in Azotobacrer vi~~elat~dii O P was examined using a
cell-free assay system. A lag period of about 30 min occurred between the exhaustion of the
combined nitrogen source and growth on N2. Cells grown o n ammonium acetate or potassium
nitrate had no detectable nitrogenase activity. Nitrogenase activity appeared in cells, grown
under a flowing gas phase of 20% 0 2 - 60% He. about 45 min after the exhaustion of ammonia.
Nitrogenase formation was inhibited in a closed system with an atmosphere containing 40% 0 2
but not by one containing 20% Or. Hydrogen did not inhibit enzyme formation. The question of
whether N2 is required for the formation of the enzyme could not be answered as this gas could
not be completely eliminated from the growth system. Chloramphenicol prevented the formation
of the enzyme and inhibited nitrogen fixation in whole cells, but had no effect on cell-Free enzyme
activity. A brief rise in turbidity which occurred during nitrogenase formation appeared to be
due to a color change in the cells from reddish brown to dark brown. Spectrophotometric exami-
nation of extracts from ammonia- and N2-grown cells did not reveal any components responsible
for this color difference, but this result may reflect only the presence of interfering substances in
the crude extract.
Canadian Journal of Microbiology, 14, 25 (1968)
phosphates in distilled water were prepared separately 100 pmoles/ml, prepared anaerobically with 0.2 M
from a solution containing the rest of the salts and cacodylate buffer, was added to the reaction mixture
sucrose. After they were autoclaved, the two solutions with a syringe.
wcre mixed. Unless otherwise noted, all cultures were T o start the reaction, 0.2 ml of the extract, contain-
grown at 30 "C on a rotary shaker. Routine transfers ing approximately 30 mg/ml protein, and kept under
and inocula were grown in 500-mI sidearm Erlenmeyer He, was added to the flasks. The flasks were shaken
flasks containing 100 n11 of medium. Larger cultures for 25 min on a reciprocal shaker at 30°C (approxi-
were grown in 2-1 Erlenmeyer flasks containing 1 1 of mately 200 oscillations/min). The rubber stoppers
medium, or in 15 1 of medium in a 5-gal carboy. The were removed and 1 ml of a saturated K2C03 solution
carboy was aerated using a large gas dispersion stone. added to stop the reaction and to evolve the ammonia
A 2Y0, lo%, 7.5% inoculum was used for the 100-ml, formed. The ammonia was trapped in a drop of 5 N
1-1, and 15-1 culturcs respectively. Growth was fol- H2S04 held on a glass rod supported by a rubber
lowed by measuring turbidity with a Klett-Summerson stopper. The stopper was inserted into the vessel as
pl~otoclectriccalorimeter with a 660 m p filter (1 K-S soon as the K2C03 was added. The flasks were shaken
unit = 0.002 O.D. unit). slowly for at least 90 min before analysis for ammonia.
Ammonium acetate and potassibm nitrate, which The glass rods were washed in 5.0 ml distilled H z 0
were used as combined nitrogen sources, were supplied and the ammonia determined by nesslerization. Trial
from stock solutions (20 nlg N/ml) sterilized by filtra- runs using 15N2 demonstrated that the ammonia
tion. Ammonia util~zationwas followed by spot test- being measured was produced from nitrogen fixation.
ing san~plesof the medium with Nesslcr's solution; In most experiments the phosphocreatine used was
KNO, utilization was determined by spot testing with prepared in our laboratory by the method of Ennor
diphenylamine-H2S04, a test that gives a positive (1957). As the a c t ~ a molecular
l weight varied because
reaction with nitrites as well. of a difference in crystalline forms of the preparations,
The effect of chloramphenicol on the formation of a standard level of 10.5 mg per flask (approximately
nitrogenase was examined and in some experiments 30 pmoles) was used.
this inhibitor was added to prevent protein synthesis Protein was determined by the method of Lowry
during the harvesting procedure. Chloramphenicol et al. (1951).
(Parke Davis and Co., Detroit, Michigan) was pre-
pared in a stock solution containing 100 pg chloram- Results and Discussion
phenicol per milliliter.
Induction oj' Nitrogenase
Etiz.vme Activity Figure 1, taken from Strandberg (1963),
Aftcr 16-18 hours growth, the cultures were har-
vested at 0-5 "C in an International refrigerated centri-
shows a typical growth curve of Azotobacter
fuge at 2000 r.p.m. for 30 min. The cells were washed vinelandii OP when supplied limited combined
once or twice with 3 0 4 0 ml of cold 0.025 M phos- nitrogen. The period of lag is relatively short,
phate bbffer, p H 7.0, centrifuged at 8000 X g for 5 30 to 60 min depending on the rate of growth.
min and the pellet weighed. They were then resus- However, if combined nitrogen is added, an
pended in the same buffer at a ratio of 3 ml of buffer
per gram of cell paste. The cells in this suspension immediate resumption of growth occurs a t
were broken in a precooled (5 "C) French pressure the same rate as before the supply of com-
cell (American Instrument Co., Jnc., Silver Spring, bined nitrogen was exhausted (Fig. 1B). The
Maryland), then centrifuged at 10 000 X g for 10 min. short lag period results in few critical points,
The resulting supernatant fraction containing from
25 to 35 mg protein per milliliter was assayed for
but the ability to de~nonstratesuch a lag in
activity by a modification of the method of Bulen et al. repeated trials supports the view that it is
(19650). Extracts prepared in this manner had a p H real. Nevertheless, the recognition that turbid-
of about 6.8 and were free from whole cells when ity measurements are not always reliable for
examined microscopically. detection of small increases in growth, espe-
Assays were made in 2C-ml serum bottles fitted
with rubber serum stoppers. One-milliliter reaction cially when the conditions change. e.g., source
mixtures were used containing 10.5 mg creatine phos- of nitrogen, led us to the conclusion pre-
phate or approximately 30 pmoles, 0.2 mg phospho- viously mentioned: that the diauxie phenom-
creatine kinase, 5 pmoles adenosine triphosphate, 5 enon fails to provide clear-cut results.
pmoles MgCl2.6Hz0, and 20 pmoles sodium hydro-
sulfite (dithionite). The creatine phosphate, phospho-
Assays for nitrogenase (Fig. 2) furnished
creatinc kinase, and adenosine triphosphate were pre- more convincing support for the conclusion
pared in 44 m M cacodylate buffer, pH 7.0. Three- that the observed lag does exist and represents
tenths of a n~illiliterof this solution was added to each a diauxic pattern of growth. In this experi-
flask, along with 0.05 ml of a MgCI2.6H20 (100 ment, the ammonia supply was exhausted a t
pmoles/ml) solution and 0.25 ml cacodylate buffer
(0.2 M, pH 7.0). The flasks were evacuated and flushed 12.25 h, and definite nitrogenase activity was
four times with helium and finally filled with N2. TWO- not detected until 13.5 h although the turbid-
tenths of a milliliter of a dithionite solution containing ity had started to increase before that time.
STRANDBERG AND WILSON: NITROGENASE IN AZOTOBACTER 27
The generation time of carboy-grown cultures grow the organisms in the absence of N2 t o
is generally longer than that of a culture in a determine whether the substrate is required
shake flask, probably because of insufficient for the formation of nitrogenase. Initial
oxygen. Also, although the cultures were kept attempts t o grow the organisms in a closed
at 30 "C, the high rate of aeration caused a system were ui~successfulbecause of the prob-
slight cooling to 28 "C. The slower rate of lem of supplying adequate oxygen t o obtain
growth probably accounts for the longer utilization of the ammonia. This problem was
period required for the detection of nitroge- later solved by supplying tank 0 2 directly t o
nase activity. the growth flask, which allowed utilization of
Although it is evident that nitrogenase is enough ammonia t o get a final yield of cells
absent in extracts from cells grown on ainmo- (more than 1 gram) t o break and assay for
nia in the presence of N2, it is necessary t o activity. A flowing gas mixture of 2075 0 2
(30 cc /min) and 800,; He (120 cc /min) per-
mitted coillplete utilization of from 40-75 ,ug
N / m l in 7-12 hours. The cultures reached
turbidities of from 90-150 K-S units depend-
ing on the amount of ammoilia added and
how much remained in the inocula, which
were grown in a closed system of 40% 0 2 -
6076 He.
Cells grown in this manner were assayed
for cell-free nitrogellase activity at different
200
KT S Specific activity
unlts P
0
0 4 8 12 16
8 10 12 14 16
T i m e (hours)
TIME (h)
FIG.1. Growth pattern of Azotobacter vinelnndii in
presence of limited quantity of NH4'-N. @ Cells were FIG. 2. Growth curve and formation of nitrogenase
grown on ammonium acetate (20 pg/ml) in an atmos- in Azotobacter vinelanrlii OP. The culture was grown
phere of 20y0 0 2 - 80% H?. 0 Cells were grown on in heavily aerated, 15-liter Burk's medium containing
ammonium acetate (20 pg/ml) in an atmosphere of 87 p g N/ml as ammonium acetate. One-liter samples
20% 0 2 - 20% N2 - 60% He. A. Arrow indicates were harvested at intervals and assayed for cell-free
cells transferred to an atmosphere of 20% 0 2 - 20% nitrogen-fixing activity as described in Methods.
N2 - 60% He after exhaustion of NH4+. B. Arrow During harvesting and washing, the cells were exposed
indicates that cells were kept in same gas mixture but to 5 pg/ml chloramphenicol. The unit of specific
additional ammonium supplied. activity is nanomoles N2 fixed /mg protein per min.
28 CANADIAN JOURNAL O F MICROBIOLOGY. VOL. 14, 1968
In the course of these experiments, we con- This apparent laclc of any distinguishable
sistently observed a rise of 5-7 Klett units in difference probably arises for a number of
the culture during the period of nitrogenase reasons. The high concentration of protein
formation. After this rise the culture would and other components could be masking the
again maintain a steady turbidity reading as particular component being sought: however,
long as the He-O2 (or H2-02) gas mixture was the difference is visible with the naked eye.
supplied. On admittance of air, the turbidity There could also be a difference in quantity
would increase rapidly without a noticeable of the component between the two types of
lag. Because small increases in turbidity do cells. Jones and Redfearn (1966) have suc-
not necessarily indicate growth, total nitrogen ceeded in separating red and green electron
determinations were made after the ammonia transport particles from A . vinelandii which
was exhausted. The nlaximunl gain was about differ in cytochrome content and enzyme
3 pg N/ml, just at the level of significance by activity. It would be of interest to determine
the method used. It appears that a pN2 of the if nitrogen fixation is associated with either
order of 0.0001 atm in the gases may be of these particles.
sufficient to cause the formation of the The eventual purification of nitrogenase, a
enzyme, but not enough to allow nitrogen project of considerable interest in a number
fixation to occur by the enzyme that has a of laboratories at the present time, may help
KN?of at least 0.01 atm (Wilson et al. 1942). to solve this problem. Purification should
It is probable, however, that this rise in enable one to determine if this color difference
turbidity is an optical artifact which is caused is associated with the nitrogen-fixing system
and whether any distinguishable spectral
by the change in color of the cells during the
properties are associated with any particular
formation of nitrogenase. Cells and extracts
component (See Bulen et al. 19653).
of A. vitzelatzdii grown on combined nitrogen
Finally, it was observed that the formation
are reddish brown whereas those of N2-grown
of the enzyme was materially reduced in the
cultures are dark brown. When the organism presence of 25 pg/ml of chloran~phenicoland
was switched from one source of nitrogen to completely suppressed by 75 pg/ml. However,
the other, the change in color could be as much as 100 pg/ml chloramphenicol had
observed. This color difference is of particular no effect on the specific activity of the enzyme
interest because it could arise from compo- in cell-free extracts. With cell suspensions
nents associated with nitrogen-fixing activity. taken from the exponential phase, an expo-
No difference in the spectra of extracts from sure of 2 h to 10 pg/ml in an atmosphere
cells grown on NH4+ and those grown on N2, containing 30.4 atom yo excess 15N2resulted
however, could be observed. The crude in a decrease in the uptake of 15N2from 0.977
extracts were centrifuged at 35 000 X g for 1 11 to 0.288 atom O/, excess (Strandberg 1963).
and spectrophotometric examination made in One possible explanation of this inhibition is
a Cary Model 15 recording spectrophotom- that chlorampl~enicolprevents the utilization
eter (Applied Physics Corp., Monrovia, of ammonia, which builds up and inhibits
Calif.). Spectra from both types of cells had fixation of N2.
sharp peaks at 521 and 550 mp with shoulders Effect of Combined Nitrogetz on Activity of
at 530 and 559 mp. Broad peaks were present Nitrogenase
around 510-515 mp, 590-600mp, and 630 Various levels of ammonia and nitrate were
mp. Cytochromes a2, a l , and bl are associated added to extracts of nitrogen-fixing cells.
with absorption bands at 630 mp, 590 mp, Neither nitrate nor ammonia had an appre-
and 560 inp (Smith 1954). Cytochromes c4 ciable effect on cell-free nitrogenase activity
and c5 exhibit a single peak at 552 m p at levels up to 40 pg N/ml, i.e., in excess of
(Tissikres and Burris 1956). Difference spectra the level of ammonia produced by the
on oxidized and reduced extracts gave spectra enzyme. In another experiment, NH4+-N
similar to that obtained by Bruemmer et al. (150 pg N/ml) was added to a 15-liter cul-
(1957) with A . vinelandii 0. Again, no differ- ture actively fixing nitrogen. Samples were
ence between the extracts was observed. removed periodically, the cells harvested,
30 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 14. 1968
Acknowledgments
Supported in part by National Science
Foundation grant GB-483 and National
Institutes of Health USPHS grant AI-1417-11.
chromatographic separation of some perinanent WILSON,P. W., BURRIS,R. H., and LIND,C. J. 1942.
gases on silica gcl a t reduced temperatures. Anal. The dissociation constant in nitrogen fixation by
Chem. 29: 1541-1543. Azotobacter. Proc. Natl. Acad. Sci. U.S. 28:
TISSI~RES,A. and BURRIS,R. H. 1956. Purification and 243-250.
properties of cytochromes C J and cj from Azoto- WITZ, D. F. 1964. Studies on nitrogen fixation by
bacter vbzeln~~rlii.Biochim. Biophys. Acta, 20: spccies of Bacillirs. M.S. Thesis, University of
436-437. Wisconsin, Madison, Wis.