Formation of the nitrogen-fixing enzyme system in Azotobacter vinelandii

G. W. STRAND BERG^ AND P. W. WILSON

Department of Bacteriology, University of Wisconsin, Madison, Wisconsin
Received January 30, 1967

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)

Introduction nitrogen-could not be unequivocally demon-

Some 25 years ago, Burris, Phelps, and
Wilson (1942) studied the formation of
"adaptive" enzymes concerned with the res-
piration of carbon sources by the root nodule
bacteria and by Azotobncter vinelnndii. The
results suggested that many of the enzyme
systems were "adaptive" in the sense that
resting suspensions of the organism would
oxidize a given carbon source at a higher rate
if the organism had been grown 011 the same
source.
The more interesting study of whether or
not the nitrogen fixing system in the azoto-
bacter is adaptive had to be postponed until
the end of World War 11, but in the meantime
a much more critical tool for this purpose had
been developed, the use of 15N2 as a tracer.
Wit11 this, we could readily demonstrate that
little, if any, fixation occurred in the presence
of NH4+ and that cells grown on N2 would
immediately assimilate added NH4+ without a
period of lag, but the reverse-immediate
uptake of N2 on exhaustion of the con~bined
1Present address : Northern Regional Laboratory,
United States Department of Agriculture, Peoria,
Illinois.
zCLNitrogenase"is used in this paper and in the
following one by Mahl and Wilson in a n operational
sense as the enzyme system(s) that lead to the forma-
tion of NH3 in the assay. At least two protein fractions
are now known to be involved in the complex.
strated. Also, the technique introduced by
Monod-the diauxie phenomenon-failed to
~ r o v i d eclear-cut results.
In the meantime, however, several new
agents of fixation have been discovered, and
these do exhibit a definite diauxic .nrowth ,
pattern when cultured in the presence of N2
with limiting quantities of NH4+ (Pengra and
Wilson 1958; Proctor and Wilson 1961; Witz
1964). Strandberg (1963) re-examined the
behavior of the azotobacter when it was grow-
ing on the two sources of nitrogen and ob-
tained what amears . to be a definite diauxic
growth curve, but the period of lag was often
short and might readily have been missed in
the earlier work. The developnlent of a tech-
nique for preparing cell-free extracts that
would fix N2 (Bulen, Burns, and LeComte
1964) provided a new tool for investigating
the formation of the responsible enzyme sys-
tein as will be described in this paper.
Materials and Methods
Growth
Azotobncter virzelnndiiii O P was used throughout
these studies. The organism was grown in a modified
Burk's nitrogen-free medium of the following compo-
sition: KH2P04, 0.2 g; K2HP04, 0.8 g; MgS04.7H20,
0.2 g; CaCl2.2H70, 0.09 g; sucrose, 20 g; 1 ml of
Fe-Mo solntion (1 mg F e and 0.1 mg Molml); H20,
1000 ml. T o prevent the formation of a precipitate
which would interfere with turbidity readings, the
26 CANADIAN JOURNAL O F M ICROBIOLOGY. VOL. 14, 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
The generation time of carboy-grown cultures
is generally longer than that of a culture in a
shake flask, probably because of insufficient
oxygen. Also, although the cultures were kept
at 30 "C, the high rate of aeration caused a
slight cooling to 28 "C. The slower rate of
growth probably accounts for the longer
period required for the detection of nitroge-
nase activity.
Although it is evident that nitrogenase is
absent in extracts from cells grown on ainmo-
nia in the presence of N2, it is necessary t o
0 4 8 12 16
T i m e (hours)
FIG.1. Growth pattern of Azotobacter vinelnndii in
presence of limited quantity of NH4'-N. @ Cells were
grown on ammonium acetate (20 pg/ml) in an atmos-
phere of 20y0 0 2 - 80% H?. 0 Cells were grown on
ammonium acetate (20 pg/ml) in an atmosphere of
20% 0 2 - 20% N2 - 60% He. A. Arrow indicates
cells transferred to an atmosphere of 20% 0 2 - 20%
N2 - 60% He after exhaustion of NH4+. B. Arrow
indicates that cells were kept in same gas mixture but
additional ammonium supplied.
27
grow the organisms in the absence of N2 t o
determine whether the substrate is required
for the formation of nitrogenase. Initial
attempts t o grow the organisms in a closed
system were ui~successfulbecause of the prob-
lem of supplying adequate oxygen t o obtain
utilization of the ammonia. This problem was
later solved by supplying tank 0 2 directly t o
the growth flask, which allowed utilization of
enough ammonia t o get a final yield of cells
(more than 1 gram) t o break and assay for
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
8 10 12 14 16
TIME (h)
FIG. 2. Growth curve and formation of nitrogenase
in Azotobacter vinelanrlii OP. The culture was grown
in heavily aerated, 15-liter Burk's medium containing
87 p g N/ml as ammonium acetate. One-liter samples
were harvested at intervals and assayed for cell-free
nitrogen-fixing activity as described in Methods.
During harvesting and washing, the cells were exposed
to 5 pg/ml chloramphenicol. The unit of specific
activity is nanomoles N2 fixed /mg protein per min.
28 CANADIAN JOURNAL O F
TABLE I
Nitrogen fixation by cell-free extracts of Azotobacter
vinela~ldiiOP g o w n on ammonium acetate
Specific activity,
Time after nanomoles Nz
exhaustion of fixed /mg
Experiment NH4+ protein per min
1 0 hours 0 experiment.
2 0.75 hours 0.75
3 2.25 hours 5.15
NOTE:One-liter cultures were grown on 75 ,tg N/ml as amrno-
nium acetate under a flowing gas phase of 20' 0 2 . 80% He. At
the time of harvest, 10 ~ g / r n chloramphen~col
l was added to the
culture. The cells were harvested at 0-5 "C. The cells were washed
once in cold C.015 M phosphate buffer, p H 7.0, before being
broken.
times after exhausting their ammonia supply.
Table I shows that no activity was present at
the time when the ammonia was exhausted,
but activity was detected 45 min later and was
high after 2.25 hours.
Other experiments were made in a closed
system by connecting a tank of 0 2 to the
growth flask fitted with a mercury manom-
eter. When 0 2 was required, as indicated by
a measurable vacuum on the manometer,
oxygen was added to atn~osphericpressure.
Although complete utilization of aminonia
was obtained, no nitrogenase was formed in
the presence of 40% 0 2 and 60y0 He or H2.
When the level of 0 2 was lowered to 20%,
enzyme formation did occur. Apparently, the
high oxygen level inhibited the formation of
the enzyme. Significantly, hydrogen did not
inhibit the formation of nitrogenase even at a
concentration of 800/,, which would com-
pletely inhibit nitrogen fixation.
The results of the experiments with flowing
and closed gas systems indicated that nitroge-
nase was forming in the apparent absence of
N2. This would suggest that nitrogenase is not
an inducible enzyme, but that its synthesis is
repressed by the presence of its product,
ammonia. We were spared drawing this pos-
sibly erroneous conclusion by checking the
composition of the gases used. The supplier
(Badger Welding Supply, Madison) gave the
level of N2 in 0 2 (National Cylinder Gas,
Chicago), He (Bureau of Mines, Amarillo,
Texas), and Hz (National Cylinder Gas,
Chicago) as 100, 15, 25 p.p.m. respectively.
The level of nitrogen present in these gases,
especially in the oxygen, could be sufficient to
serve as an inducer for the nitrogenase.
MICROBIOLOGY. VOL. 14, 1968
During growth in a flowing system, a contin-
uous supply of this contaminating nitrogen
would be made available. A closed system was
even worse, because it is the oxygen that is
used; as additional oxygen is added to replace
it, the N2 contaminant remains in the flask
and increases in concentration throughout the
To circumvent this problem, an attempt
was made to produce oxygen of a higher
purity by electrolysis. Even though not all of
the N2 could be eliminated, sufficiently low
levels of N2 might be obtained so that at least
an idea of the minimum level of N2 resulting
in the formation of nitrogenase might be
determined. Oxygen, generated from alkaline
water (25% KOH) by electrolysis at a rate of
20 cc/min, was used to grow cultures of A.
vinelnndii OP at room temperature. It was
necessary to use tank He or H2 as the carrier
gases (180 cc/min), as the electrolytic appa-
ratus would not generate sufficient H2 or 0 2
to maintain an adequate gas flow.
As in the experiments with tank oxygen,
nitrogenase was again formed with electro-
lytically produced oxygen. Gas chroinato-
graphic analysis of the gases by the inethod of
Szulczewski and Higuchi (1957) using a silica
gel column at dry ice - ethanol temperatures
for separation, indicated about as much N2
was present in the electrolytically prepared 0 2
as in the tank 0 2 . Control analysis on tank H2
showed very small 0 2 and N2 peaks, indicat-
ing that our method of sample collecting was
sufficiently free from air contamination. The
supplier quoted values of O2 and N2 impurities
of 5 and 25 p.p.m. respectively in tank hydro-
gen. Whether the N2 may have been due to
some uncontrolled air contamination in the
electrolytic system is not known, but what-
ever the source of nitrogen, its level was
consistent, eliminating the possibility of ran-
dom air contamination of samples. Its pres-
ence also led to the conclusion that the ques-
tion of whether N2 is required as an inducer
for nitrogenase may be difficult to answer
with the aerobic azotobacter. However, a s
will be described in a second paper, Mahl and
Wilson (1968) have found definite evidence
for the requirement of N2 for the formation
of ilitrogenase in the facultative anaerobe,
Klebsiella pneunzoniae.
 STRANDBERG AND WILSON: NITROGENASE IN AZOTOBACTER
In the course of these experiments, we con-
sistently observed a rise of 5-7 Klett units in
the culture during the period of nitrogenase
formation. After this rise the culture would
again maintain a steady turbidity reading as
long as the He-O2 (or H2-02) gas mixture was
supplied. On admittance of air, the turbidity
would increase rapidly without a noticeable
lag. Because small increases in turbidity do
not necessarily indicate growth, total nitrogen
determinations were made after the ammonia
was exhausted. The nlaximunl gain was about
3 pg N/ml, just at the level of significance by
the method used. It appears that a pN2 of the
order of 0.0001 atm in the gases may be
sufficient to cause the formation of the
enzyme, but not enough to allow nitrogen
fixation to occur by the enzyme that has a
KN?of at least 0.01 atm (Wilson et al. 1942).
It is probable, however, that this rise in
turbidity is an optical artifact which is caused
by the change in color of the cells during the
formation of nitrogenase. Cells and extracts
of A. vitzelatzdii grown on combined nitrogen
are reddish brown whereas those of N2-grown
cultures are dark brown. When the organism
was switched from one source of nitrogen to
the other, the change in color could be
observed. This color difference is of particular
interest because it could arise from compo-
nents associated with nitrogen-fixing activity.
No difference in the spectra of extracts from
cells grown on NH4+ and those grown on N2,
however, could be observed. The crude
extracts were centrifuged at 35 000 X g for 1 11
and spectrophotometric examination made in
a Cary Model 15 recording spectrophotom-
eter (Applied Physics Corp., Monrovia,
Calif.). Spectra from both types of cells had
sharp peaks at 521 and 550 mp with shoulders
at 530 and 559 mp. Broad peaks were present
around 510-515 mp, 590-600mp, and 630
mp. Cytochromes a2, a l , and bl are associated
with absorption bands at 630 mp, 590 mp,
and 560 inp (Smith 1954). Cytochromes c4
and c5 exhibit a single peak at 552 m p
(Tissikres and Burris 1956). Difference spectra
on oxidized and reduced extracts gave spectra
similar to that obtained by Bruemmer et al.
(1957) with A . vinelandii 0. Again, no differ-
ence between the extracts was observed.
29
This apparent laclc of any distinguishable
difference probably arises for a number of
reasons. The high concentration of protein
and other components could be masking the
particular component being sought: however,
the difference is visible with the naked eye.
There could also be a difference in quantity
of the component between the two types of
cells. Jones and Redfearn (1966) have suc-
ceeded in separating red and green electron
transport particles from A . vinelandii which
differ in cytochrome content and enzyme
activity. It would be of interest to determine
if nitrogen fixation is associated with either
of these particles.
The eventual purification of nitrogenase, a
project of considerable interest in a number
of laboratories at the present time, may help
to solve this problem. Purification should
enable one to determine if this color difference
is associated with the nitrogen-fixing system
and whether any distinguishable spectral
properties are associated with any particular
component (See Bulen et al. 19653).
Finally, it was observed that the formation
of the enzyme was materially reduced in the
presence of 25 pg/ml of chloran~phenicoland
completely suppressed by 75 pg/ml. However,
as much as 100 pg/ml chloramphenicol had
no effect on the specific activity of the enzyme
in cell-free extracts. With cell suspensions
taken from the exponential phase, an expo-
sure of 2 h to 10 pg/ml in an atmosphere
containing 30.4 atom yo excess 15N2resulted
in a decrease in the uptake of 15N2from 0.977
to 0.288 atom O/, excess (Strandberg 1963).
One possible explanation of this inhibition is
that chlorampl~enicolprevents the utilization
of ammonia, which builds up and inhibits
fixation of N2.
Effect of Combined Nitrogetz on Activity of
Nitrogenase
Various levels of ammonia and nitrate were
added to extracts of nitrogen-fixing cells.
Neither nitrate nor ammonia had an appre-
ciable effect on cell-free nitrogenase activity
at levels up to 40 pg N/ml, i.e., in excess of
the level of ammonia produced by the
enzyme. In another experiment, NH4+-N
(150 pg N/ml) was added to a 15-liter cul-
ture actively fixing nitrogen. Samples were
removed periodically, the cells harvested,
30 CANADIAN JOURNAL OF
FIG. 3. Effect of ammonium acetate on the level of
cell-free nitrogenase activity in Azotobncter vitlelnndii
OP. A 15-liter culture was grown under nitrogen-fixing
conditions. At 13.5 h, 150 pg N/ml as ammonium
acetate was added to the culture. One-liter samples
were taken and the cells assayed for cell-free nitroge-
nase activity.
washed, and broken, and the extract tested
for activity. Figure 3 shows that activity falls
off at a fairly rapid rate and is almost coin-
pletely eliminated in one generation. In a
second experiment of this type, about 1070
activity remained after one generation. The
difference between the two experiments may
be a reflection of the growth rate, the former
growing at 25 K-S units/h during its fastest
growth on NH4+-N, the latter at 22 K-S
units /h.
The drop in activity over several hours
indicates that the effect of NH4+-N on the
enzyme is not a direct inactivation, as activity
can be found in the extracts for several hours.
The synthesis of the enzyme is being inhibited
and the enzyme is diluted out. One would
predict that after one generation, the specific
activity would be reduced by one-half, by a
MICROBIOLOGY. VOL. 14. 1968
doubling of total protein while the total
amount of enzvme remained the same. The
much more rapid disappearance of the enzyme
could be due to the degradation of the
enzvme since. when cells were shaken in air.
at room temperature, the specific activity fell
from 5.7 to 0.2 in 4 h (about one generation
time under the conditions of the experiment
in Fig. 3). In an extract, the specific activity
dropped from 7.2 to 2.6 ill 3 h.
Acknowledgments
Supported in part by National Science
Foundation grant GB-483 and National
Institutes of Health USPHS grant AI-1417-11.
BRUEMMER, J. H., WILSON,P. W., GLENN,J. L., and
CRANE,F. L. 1957. Electron transporting particle
from Azotobacter vitlelntldii. J. Bacteriol. 73:
113-116.
BULEN,W. A,, BURNS,R. C., and LECOMPTE,J. R.
1964. Nitrogen fixat~on: cell-free system with
extracts of Azotobncter. Biochem. Biophys. Res.
Commun. 17: 265-271.
19650. Nitrogen fixation: hydrosulfite as elec-
tron donor with cell-free ~ r e ~ a r a t i o nofs Azoto-
bncter vinelntldii and ~ l ~ o d ~ s ~ & i rsrbrut~z.
l l s r t ~ ~Proc.
Nati. Acad. Sci. U.S. 53: 532-539.
BULEN,W. A., LECOMPTE,J. R., BURNS,R . C., and
HINKSON.J. 19656. Nitrogen fixation studies with
aerobic and photosynthetk bacteria. Itz Non-heme
iron proteins: role in energy conversion. Edited b y
A. San Pietro. The Antioch Press, Yellow Springs,
Ohio. pp. 261-274.
BURRIS, R. H., PHELPS, A. S., and WILSON,J. B. 1942.
Adaptation of Rhizobisr~nand Azotobacter. Soil Sci.
Soc. Am. Proc. 7: 272-275.
ENNOR,A. H. 1957. Determination and preparation
of N-phosphates of biological origin. In Methods
in enzymology. Vol. 3. Edited b y S. P. Colowick
and N. 0. Kaplan. Academic Press, Inc., New York.
pp. 850-862.
JONES.C. W. and REDFEARN. E. R . 1966. The prepa-
ratibn of red and green electron transport ~articles
from Azotobncter vinelnndii. Biochem. J. 99: 33 p.
LOWRY,0. H., ROSEBROUGH, N. S., FARR,A. L., and
RANDALL, R. J. 1951. Protein measurement with
the folin phenol reagent. J. Biol. Chem. 193:
265-275.
MAHL. M. C. and WILSON.P. W. 1968. Nitroeen
fixation by cell-free extraits of Klebsiella pnesrrio-
niae. Can. J. Microbial. 1 4 : 33-38.
PENGRA, R. M. and WILSON,P. W. 1958. Physiology
of nitrogen fixation by ~ e r o b a c t e rnerogenes. J.
Bacteriol. 75: 21-25.
PROCTOR,M. H. and WILSON,P. W. 1961. Biotin in
nitrogen fixation by a pseudomonad. Z. Allgem.
Mikrobiol. 1 : 175.
SMITH,L. 1954. Bacterial cytochromes. Bacteriol.
Rev. 18: 106-130.
STRANDBERG, G. W. 1963. Physiological studies o n
nitrogen fixation by Azotobncter vinelandii. M.S.
Thesis, University of Wisconsin, Madison, Wis.
SZULCZEWSKI, D. H. and HlGUCHl, T. 1957. Gas
 STRANDBERG AND WILSON: NITROGENASE IN AZOTOBACTER 31

chromatographic separation of some perinanent
gases on silica gcl a t reduced temperatures. Anal.
Chem. 29: 1541-1543.
TISSI~RES,A. and BURRIS,R. H. 1956. Purification and
properties of cytochromes C J and cj from Azoto-
bacter vbzeln~~rlii.Biochim. Biophys. Acta, 20:
436-437.
WILSON,P. W., BURRIS,R. H., and LIND,C. J. 1942.
The dissociation constant in nitrogen fixation by
Azotobacter. Proc. Natl. Acad. Sci. U.S. 28:
243-250.
WITZ, D. F. 1964. Studies on nitrogen fixation by
spccies of Bacillirs. M.S. Thesis, University of
Wisconsin, Madison, Wis.