\H

\

|

w
W

|\\

‘\

*\

1
\
\

   

.xl’

M

LEVELS 0F PYRIDENE NUCLEOTEDE
TRANSHYDROGENASE DURWG
ENCYSTMENT IN AZOTOBACTER
VNELANDH

      
     
  

1

‘\

    

4%;
I030
(DUI-P-

‘s‘hesis for the Degree of M. S.
MECHEGAN STATE UMVERSITY
PAU Wé‘NE SHELF
1975

912 -~ «AHW - m-
5v.,_
"
v ‘f ." '
. .~' 1- ' ~-‘ I: f. Y
, - - .. :x

i

I;-
l
n
C
,
‘~§I

. .. . . .
9‘: a} ‘ ¢ H :‘l‘.’~ ' b
~ . 1 M‘ 5 fl
rd.

.‘-“._w._v_ r‘
Wig-17'

go ‘vn L. u.

gnaw!)

‘ r

. ‘5' magma silk.
"DAG & SDNS’
‘ . 8071K BINDERY INC.

‘ L 3mm BINDERS

r.“ manna! Il'm'“ ..

   

    
    

 

    

 

 

 

ABSTRACT

LEVELS OF PYRIDINE NUCLEOTIDE TRANSHYDROGENASE
DURING ENCYSTMENT IN AZOTOBACTER VINELANDII

 

BY

Paulanne Chelf

Azotobacter vinelandii, when grown with glucose

 

as a carbon source, contains measurable levels of pyri-
dine nucleotide transhydrogenase activity. The Specific
activity of the enzyme increases 7-fold within a 4 h
period after the glucose in the medium is replaced with
B-hydroxybutyrate (BHB) to induce encystment. Vegetative

cells of A. vinelandii grown with acetate as a carbon

 

source have no measurable pyridine nucleotide trans-
hydrogenase activity. When acetate is replaced with

BHB, very little increase in specific activity of the
transhydrogenase occurs. Microsc0pic observation and
plate counting indicate that less than 0.2% of the cells
grown in acetate and transferred to BHB form desiccation-
resistant cysts. In contrast, more than 70% of the cells
grown in glucose form cysts when transferred to BHB.

We propose that pyridine nucleotide transhydrogenase

is responsible for a shift in the levels of reduced

Paulanne Chelf

pyridine nucleotides, and that this shift is a critical

step in the encystment process.

LEVELS OF PYRIDINE NUCLEOTIDE TRANSHYDROGENASE

DURING ENCYSTMENT IN AZOTOBACTER VINELANDII

 

BY

Paulanne Chelf

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Microbiology and Public Health

1975

DEDICATION

To my mother

ii

ACKNOWLEDGMENTS

I wish to extend my sincere appreciation to
Dr. Harold Sadoff for his never-ending patience and
infinite wisdom. Thanks are also due to Dr. James
Tiedje and Dr. Clarence Suelter for their interest in
this work as members of my guidance committee and to
Dr. Ralph Costilow for his excellent review of the
manuscript.

This research was supported in part by Public
Health Training Grant GM-Ol9ll of the National Institute

of Health.

iii

TABLE OF CONTENTS

Page
LIST OF FIGURES . . . . . . . . . . . . v
INTRODUCTION . . . . . . . . . . . . . 1
MATERIALS AND METHODS . . . . . . . . . . 6
Strain and Cultivation. . . . . . . . . 6
Preparation of Cell Extracts. . . . . . . 6
Materials . . . . . . . . . . . . . 8
Assay of the Enzyme. . . . . . . . . . 8
RESULTS 0 O O O O O O O O O O O O O O 9
Optimal Method of Cell Breakage. . . . . . 9
Transhydrogenase Levels During Encystment
and Abortive Encystment. . . . . . . . 10
DISCUSSION. . . . . . . . . . . . . . l7
LITEMTUM CITED. 0 O O O O O O O O O O 21

iv

LIST OF FIGURES

Figure Page

1. Schematic diagram of the electron trans-
port system of Azotobacter . . . . . . 4

2. Comparison of transhydrogenase activity in
cultures grown in glucose or acetate con-
taining medium and induced to encyst with
BHB. . . . . . . . . . . . . . 12

3. Transhydrogenase activity in cells grown in
glucose or acetate and transferred at
time 0 h to Burk's buffer plus 0.2% BHB. . l4

INTRODUCTION

Bacterial pyridine nucleotide transhydrogenase
was first described by Colowick et al. in 1952 (8),
and subsequently it also has been found in plant and
animal tissues (11, 12). The transhydrogenase of 552:

tobacter vinelandii has been purified to homogeneity

 

and its kinetic properties evaluated (6, 19, 21). The
bacterial enzyme catalyzes the transfer of electrons
between the reduced and oxidized forms of nicotinamide
adenine dinucleotide (NAD) and nicotinamide adenine
dinucleotide phosphate (NADP) as illustrated in the

following equation:

 

 

NADPH + NAD+ NADP+ + NADH

The reaction in yitrg occurs rapidly from left to right
as indicated, but the reverse reaction is relatively
slow (6, 19, 20). The enzyme is capable of carrying

out this reaction using nicotinamide hypoxanthine‘
dinucleotide or -dinucleotide phosphate (deamino-NAD+(P))
or thiol-NAD+(P), and also catalyzes the interconversion
of the oxidized and reduced forms of these pyridine

nucleotides:

NADH + deamino-NAD+ <————r NAD+ + deamino-NADH;

NADPH + deamino-NADP+ # NADP+ + deamino-NADPH.

The physiological function of the transhydrogenase
enzyme with respect to the overall metabolic activity of
pyridine nucleotide coenzymes is not well defined. When
this enzyme was first discovered, Colowick et al. (8)
thought that it might play an important role in regulat-
ing the pathway of electron transport. They further
suggested that if NADH and NADPH systems were found to
differ in their phosphorylation ability, transhydrogenase
might serve to regulate the conversion of oxidative
energy into phosphate bond energy. Work with the respira-

tory system of 5. vinelandii in more recent years has

 

indeed demonstrated a difference in the phosphorylation
efficiency of the NADPH and the NADH respiratory chains
(1, 10, 22). The respiratory system of Azotobacter is
shown in Figure l (Yates & Jones, 22). Thus entry into
the respiratory chain at the level of NADH results in
greater phosphorylation than entry at the level of
malate or NADPH. Transhydrogenase is therefore in a
position to exert control over both the intracellular
NAD(P)H/NAD(P)+ ratio and the extent of oxidative phos-
phorylation.

g. vinelandii cells are capable of undergoing a

 

cyclic process of differentiation to produce metabolically

dormant, thick-walled cells known as cysts. The cell

 

.HHH new .HH .H an pmumsmflmmp mum mmuwm coaumamuonmmosm .uwuomnouo~¢

mo Empmmm uuommsmuu couuomam man no EMHmMMU oaumEmsom .a musmflm

wommcomotgcoo ooxc__-+n_o<z .m_n:_ow

mm oEoEooio 3522... .md... All-I Ido<z
\ 4

 

 

o mEoEooKo Al mo mEoEoogo Allwn oEoEoogo Al 0 Al. Eva/Eon. All BEN—2 can
@ \ AH / 47
Pm mEoEooSO T we DEOEQOSQ 22”. I we“; I 10.42

8

8858628 8xc__.+o<z .938

undergoes this process in the laboratory in reaponse to
specific inducers, which include n-butanol, B-hydroxybuty-
rate (BHB), and crotonate (13). Vegetative cells of

this organism possess "a very high level of pyridine
nucleotide transhydrogenase" (6) ranging from 0.3 -

1.0 unit/mg of soluble protein. Vegetative cells of

g. vinelandii are capable of using atmospheric nitrogen,

 

but cessation of nitrogen fixation occurs rapidly upon
the induction of encystment with BHB, suggesting that
pool levels of NADPH, the primary reductant (4), may be
depleted. We have therefore measured the levels of

transhydrogenase in A. vinelandii during the course of

 

encystment with the thought that this enzyme may promote

shifts in energetic and synthetic pathways.

MATERIALS AND METHODS

Strain and Cultivation

 

g. vinelandii ATCC 12837, which was used through-

 

out the course of these experiments, was cultivated in
Burk's nitrogen-free medium (5) at 30 C. Vegetative
cells were grown to mid-exponential phase (0.6 O.D.)
with 1% glucose or 0.2% sodium acetate as the carbon
source. To prepare cysts, cells were harvested asepti-
cally, washed twice with sterile Burk's buffer (Burk's
medium without carbon source), resuspended in sterile
Burk's buffer with half the normal phosphate concentra-
tion and 0.2% BHB as the carbon source, and incubated
with aeration at 30 C. All carbon sources were prepared
as aqueous solutions and were autoclaved separately
from the Burk's buffer.

Abortive encystment (13) was initiated by culti-
vating cells in the presence of 1% glucose, washing twice
with sterile Burk's buffer, and resuspending the cells

in fresh medium containing 0.2% BHB and 0.2% glucose.

Preparation of Cell Extracts

 

Vegetative and encysting cells of g. vinelandii

 

from 50 ml of culture were harvested by centrifugation

and resuspended in 5 ml of 0.05 M tris(hydroxymethyl)
amino methane (Tris) buffer, pH 8.0, containing 10.3 M
ethylenediamine tetraacetic acid (EDTA). The cells were
subjected to sonic oscillation (Measuring & Scientific
Equipment, Ltd.) for 15 sec periods followed by 45 sec
cooling until 2.5 min treatment was achieved. Cellular
debris was removed by centrifugation at 10,000 x g at
4 C in a Sorvall RC2-B refrigerated centrifuge. This
supernatant solution was spun again at 35,000 x g for
30 min, and the clarified extract served as the source
of the enzyme.

Proteolytic activity was measured by combining
1.0 m1 of 0.5% azoalbumin with 0.5 ml of cell free
extract. Distilled water was added to the blank, and
all tubes were incubated at 30° C for 20 min. At the
end of incubation, 2.0 ml of 10% trichloroacetic acid
was added to each tube. Tubes were Spun at high speed
on a clinical centrifuge for 15 min, and the supernatant
was read at 340 nm.

Protein was determined by the method of Lowry
et a1. (14). Desiccation resistant cysts were enumerated
according to the method of Socolofsky and Wyss (17),
but using Whatman #4 filter paper in place of membrane
filters. The Whatman paper disintegrates upon vigorous
agitation in solution and therefore allows more accurate

quantitation of cysts.

 

III III" III] l‘l‘l‘ll‘f‘ 1|! Ill til" ' 1 I!

Materials

 

Deamino-NAD+ and glucose-6-phosphate dehydro-
genase (NADP+-specific) were obtained from Sigma Chemical
Co. All other reagents were analytical grade and were

obtained from various commercial sources.

Assay of the Enzyme

 

The enzyme activity was measured at 30 C by
modifications of existing assays (8, 19). The conditions

were: 0.05 M Tris buffer, pH 8.0, with 10-2 M MgClz,

10'4 M NADP+, 6 x 10-3

M glucose-6-phosphate (final
concentrations in the assay volume), and 10 to 20 ul

of a one unit/ml stock solution of glucose-6-ph08phate
dehydrogenase (one unit oxidizes one umole of glucose-6-
phosphate per min at 25 C, pH 7.4) in a total volume of
2.0 ml. This reaction was monitored at 340 nm until

no further absorbance change occurred, and then

4 M) and 100 ul

deamino-NAD+ (final concentration of 10-
of transhydrogenase enzyme preparation were added to
initiate the reaction. One enzyme unit is defined as
the amount which catalyzes the reduction of one umole
of deamino-NAD+ in one min under the above assay con-

ditions. The specific activity is expressed in enzyme

units per milligram of protein.

RESULTS

Optimal Method of Cell Breakage

 

Preliminary experiments were carried out to
select a method of cell and cyst breakage which would
permit reproducible enzyme recovery. Vegetative cells
were easily ruptured using lysozyme, sonic treatment,
or extrusion through a French pressure cell. Cysts
were very resistant to all of these methods of cell
breakage unless the cyst coats were initially disrupted
by use of a chelating agent such as EDTA prior to
breakage (17).

Enzyme assays of lysate fractions of the vege-
tative cells indicated that sonication resulted in the
greatest yield of enzyme activity. This activity was
found in the supernatant fraction resulting from cen-
trifugation of extracts at 78,000 x g for 4.5 h in a
Beckman Model L3-50 ultracentrifuge. Cell extracts
clarified at 35,000 x g for 30 min had equivalent
activities and thus the lower speed was used throughout
this work. Cyst lysates prepared by sonication and by
extrusion through a French pressure cell possessed

comparable levels of enzyme activity. However, lysates

10

prepared by extrusion through the French pressure cell
contained transhydrogenase in both the high speed super—
natant and pellet, whereas activity in the sonicated
lysates was found only in the supernatant fraction.
Sonication seemed to be more effective in solubilizing
the transhydrogenase and was therefore used for prepar-
ation of all cell and cyst lysates.

Transhydrogenase Levels Durinngncystment
and Abortive Encystment

 

Cultures which had been grown to mid-exponential
phase with glucose or acetate were washed free of carbon
source and resuspended in 0.2% BHB. Samples for assay
of transhydrogenase activity were taken at this time
and again after 1, 2, 3, and 4 h of incubation (Fig. 2).
Transhydrogenase specific activity in cultures grown in
glucose and undergoing normal encystment underwent a
progressive 7-fold increase by 4 h while those cells
grown initially in acetate were almost devoid of the
enzyme. In abortive encystment the transhydrogenase
levels remained at approximately the same as those in
glucose-grown vegetative cells. In longer term experi-
ments, Figure 3, it was repeatedly observed that the
initial peak of transhydrogenase activity occurred and
then fell to zero at 6 h. A smaller peak of activity
was detected at 9 h into the encystment process which

fell to zero by 18 h and then rose to pre-induction

ll

.mmoosam ca GBOHm maamo ucmmmummu mmmum muH£3 xucmsummocm

m>wuuonm msflwso muw>fluom 303m mmmum ammo .muMDmom GA czoum maamo ca muw>fluom
unmmmummu mmmum pmsmxomHn one .mmm wm.o msam ummmsn m.xusm ou pounmmmcmuu
new Umcmmz mum3 maamo a o mafia pd .nusoum m>fiumummm> musmmmummu a mu mafia
.mmm sufl3 unmoam on because use Edwpme mswsflmusoo mbmuoom Ho mmoosam cw

szoum mmuduado cw >ua>auom mmmsmmouomSmsmuu mo sOmHHmmEOU .m musmflm

12

 

 

 

 

q 0.
l‘) I
(EN/SLIM“!

 

 

 

 

ALI/\ILOV‘

Houna

13

.mHHmo cBOHmlmumumom .0 “maamo czonmlmmoozam .o "maonfimm
.mmm mm.o mafia Hmmmsn m.xusm on a o mEHu um Umuummmsmnu paw mumnmom

no mmoosam ca :3oum maamo cH >ua>fluom mmmsmmoupmsmcmue .m musmfim

(SW/BLIND)

14

 

ALIAILOV

 

HOURS

15

levels until the completion of encystment. Intracellular
protease could not be detected in extracts at any time
during encystment. The specific activity of trans-
hydrogenase in the acetate-grown cells was very low,

and additional activity was not induced in cells upon
their resuspension in BHB. Levels remained very low
throughout the 48 h period after BHB addition.

Microscopic observations and plate counting
indicated that less than 0.2% of the acetate-grown
culture formed desiccation resistant cysts after
resuspension in BHB. In contrast, 48 h after the
induction of encystment the glucose-grown culture which
had been resuspended in BHB had undergone greater than
70% encystment.

The addition of BHB to the transhydrogenase
assay mixture produced no measurable effect and thus the
fatty acid is probably not an allosteric effector of the
enzyme. NAD+-linked BHB dehydrogenase which is also
induced during encystment (9) was present in these
cell-free extracts, but was inactive with deamino-NAD+.

Glucose-G-phosphate dehydrogenase from A. vinelandii was

 

also inactive with this dinucleotide.

In a separate set of experiments, cells were
grown to mid-exponential phase with glucose as the
carbon source, washed free of glucose, and transferred

to an acetate containing medium. Ten hours after this

16

transfer had occurred, the specific activity of trans-
hydrogenase in these cells had fallen to one-third that
in the initial glucose-grown culture. é. vinelandii has
a generation time under these conditions of approximately
3 h, so if acetate was responsible for the repression
of transhydrogenase synthesis one would expect approxi-
mately one-tenth the original activity to remain after
10 h. The results suggest that the synthesis of the
enzyme proceeds more slowly in the presence of acetate
or that synthesis continues until the concentration of
some metabolic product of acetate represses further

transhydrogenase synthesis.

DISCUSSION

Pyridine nucleotide transhydrogenase is in a
unique position in the cell to regulate both the intra-
cellular NAD(P)H/NAD(P)+ ratio and the extent of oxi-
dative phosphorylation. Since reduced nucleotide levels
may have a profound effect on the metabolic processes
which are operative during encystment, consideration
must be taken of the metabolic processes which contribute
to the levels of these molecules during encystment.

Differentiation in Azotobacter involves the
functioning of the tricarboxylic acid (TCA) cycle, the
glyoxylate cycle, and gluconeogenesis (9). An important

TCA cycle enzyme in g. vinelandii is the highly active

 

NADP+-linked isocitrate dehydrogenase. This enzyme con-
stitutes about 2% of the total cellular protein (7) and
is probably the principal generator of NADPH in the cell.
Barrera and Jurtshuk (3) have proposed that isocitrate
oxidation is the primary source of electrons (NADPH)

for nitrogen fixation in this organism because the
specific activity of the isocitrate dehydrogenase is

10- to lOO-fold greater than any other dehydrogenase

17

18

in the cell. Nitrogen fixation, however, ceases within
one hour after the induction of encystment with BHB (9),
and this could result in an accumulation of NADPH.

High reduced pyridine nucleotide levels in Azotobacter

 

specifically inhibit citrate synthase, isocitrate
dehydrogenase, and enzymes involved in glucose cata-
bolism (16). Inhibition of the TCA cycle would reduce
available levels of intermediates and thus affect the
glyoxylate cycle and gluconeogenesis as well. It is
apparent that reduced nucleotide levels must be closely
regulated during the encystment process. Excess NADPH
may be oxidized via the NADPH dehydrogenase or may be
transhydrogenated to NADH and oxidized via the NADH
oxidase system. Ackrell et al. (2) feel that a major
fate of NADPH in the cell is transhydrogenation, and
that NADPH dehydrogenase functions only at very high
substrate concentrations.

The results presented here are consistent with
the hypothesis that pyridine nucleotide transhydrogenase
is an important regulatory enzyme during encystment.
Comparison of transhydrogenase activity in an encysting
culture and a nonencysting culture gives support to
this argument. In cells grown in glucose containing
medium and transferred to BHB, the rise in trans-
hydrogenase activity is accompanied by encystment.

In cells grown in acetate and transferred to BHB,

19

the failure to induce transhydrogenase activity is
accompanied by failure to encyst. It appears that the
physiological signal which induces transhydrogenase is
lacking in acetate-grown cells. This signal may be

the level of NADPH. Van den Broek and Veeger (20) have
demonstrated two binding sites for NADPH on the enzyme:
one catalytic and one regulatory. Cells growing in
acetate must utilize the anaplerotic glyoxylate cycle
in addition to the TCA cycle. Nagai et al. (15) have
presented evidence which suggests that the glyoxylate
cycle predominates under these conditions. Since cells
utilizing the glyoxylate cycle by-pass the NADPH-
generating isocitrate dehydrogenase, pool levels of
this reduced nucleotide are probably lower in acetate-
grown cells. This is supported by the fact that the
rate of nitrogen fixation (for which NADPH is the primary
reductant [4]) during vegetative growth on acetate is
lower than that of glucose-grown cells (9).

The precipitous drOp in transhydrogenase activity
early in encystment was repeatedly observed and pre-
sumably represents some mode of cellular control of
this activity. It is unlikely that this enzyme is
degraded extensively and resynthesized since no pro-
teolytic activity could be detected in encysting cells.

This does not exclude the possibility of other types

20

of covalent modification. Alternatively the enzyme may
undergo association-dissociation reactions with a

tightly bound molecule.

LITERATURE CITED

 

l.'{lll|1.' 11 .II‘IIIK'I‘III'I‘III ' i1!

LITERATURE CITED

I
Ackrell) B. A. C., and C. W. Jones. 1971. The
respiratory system of Azotobacter vinelandii.
l. iPrOperties of phosphorylating membranes.
Eurfl J. Biochem. 22: 22-28.

 

Ackrell,EB. A. C., S. K. Erickson, and C. W. Jones.
1972; The respiratory-chain NADPH dehydrogenase
of Azotobacter vinelandii. Eur. J. Biochem. 26:
387-392.

 

Barrera, C. R., and P. Jurtshuk. 1970. Characteri-
zation of the highly active isocitrate (NADP+)
dehydrogenase of Azotobacter vinelandii. Biochim.
Biophys. Acta. 220: 416-429.

 

Benemann, J. R., D. C. Yoch, R. C. Valentine, and
D. I. Arnon. 1971. The electron transport
system in nitrogen fixation by Azotobacter.
Biochim. Biophys. Acta. 226: 205-212.

 

Burk, 0., J. w. Newton, P. w. Wilson, and R. H.
Burris. 1953. Direct demonstration of ammonia
as an intermediate in nitrogen fixation by
Azotobacter. J. Biol. Chem. 294: 445-451.

 

Chung, A. E. 1970. Pyridine nucleotide trans-
hydrogenase from Azotobacter vinelandii.
J. Bacteriol. 102: 438-447.

 

Chung, A. E., and J. S. Franzen. 1969. Oxidized
triphosphopyridine nucleotide specific isocitrate
dehydrogenase from Azotobacter vinelandii.

Isolation and characterization. Biochemistry.
8: 3175-3184.

 

Colowick, S. P., N. 0. Kaplan, E. F. Neufeld, and
M. M. Ciotti. 1952. Pyridine nucleotide trans-
hydrogenase. I. Indirect evidence for the
reaction and purification of the enzyme. J.
Biol. Chem. 125: 95-106.

21

10.

ll.

12.

13.

14.

15.

l6.

17.

18.

19.

22

Hitchins, V. M., and H. L. Sadoff. 1972. Sequential
metabolic events during encystment of Azotobacter
vinelandii. J. Bacteriol. 113: 1273-1279.

 

 

Jones, C. W., and E. R. Redfearn. 1967a. The cyto-
chrome system of Azotobacter vinelandii. Biochim.
Biophys. Acta. 143: 340-353.

 

Kaplan, N. 0., S. P. Colowick, and E. F. Neufeld.
1952. Pyridine nucleotide transhydrogenase.
III. Animal tissue transhydrogenase. J. Biol.
Chem. ‘205: l-16.

Keister, D. L., and R. B. Hemmes. 1966. Pyridine
nucleotide transhydrogenase from Chromatium.
J. Biol. Chem. 241: 2820-2825.

 

Lin, L. P., and H. L. Sadoff. 1968. Encystment
and polymer production by Azotobacter vinelandii
in the presence of B-hydroxybutyrate. J. Bac-
teriol. 95: 2336-2343.

 

Lowry, O. H., N. J. Rosebrough, A. L. Farr, and
R. J. Randall. 1951. Protein measurement with
the Folin phenol reagent. J. Biol. Chem. 193:
265-275.

Nagai, S., Y. Nishizawa, P. Doin, and S. Aiba. 1972.
Energy difference in metabolism between glucose
and acetate in chemostat culture of Azotobacter
vinelandii. J. E22: App . Microbiol. ‘18:
201-208. ,

 

 

 

Senior, P. J., and E. A. Dawes. 1971. Poly-B-
hydroxybutyrate biosynthesis and the regulation
of glucose metabolism in Azotobacter beiierinckii.
Biochem. J. 125: 55-66.

 

Socolofsky, M. D., and O. Wyss. 1961. Cysts of
Azotobacter. J. Bacteriol. 81: 946-954.

 

Socolofsky, M. D., and O. Wyss. 1962. Resistance
of the Azotobacter cyst. J. Bacteriol. 84:
119-124.

 

Van den Broek, H. W. J., J. S. Santema, J. H.
Wassink, and C. Veeger. 1971. Pyridine nucleo-
tide transhydrogenase. I. Isolation, purifica-
tion, and characterization of the transhydro-
genase from Azotobacter vinelandii. Eur. J.
Biochem. 24; 31-45.

 

23

20. Van den Broek, H. W. J., and C. Veeger. 1968.
Regulation of NADH and NADPH metabolism in
Azotobacter vinelandii. FEBS Lett. ‘1:
301-304.

 

21. Van den Broek, H. W. J., and C. Veeger. 1971.
Pyridine nucleotide transhydrogenase. V.
Kinetic studies on transhydrogenase from

Azotobacter vinelandii. Eur. J. Biochem. 24:
72-82.

 

 

22. Yates, M. G., and C. W. Jones. 1974. Respiration
and nitrogen fixation in Azotobacter, pp. 97-
135. £2.A° H. Rose and D. W. Tempest. Advances
in Microbial Physiology. Vol. 11. Academic
Press, Inc. London.

 

   

M'TlTIi‘fiflllefllfljfibflfijujfflfiflflflflWWI”

24