California State University, Northridge
NITROGENASE REPRESSION IN Klebsiella pneumoniae
~
'
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
Wayne Walter _Schubert
~
January, 1979
The Thesis of Wayne Walter Schubert is approved:
Charles R. Spotts 7committee Chairman 1/ Date
California State University, Nortl1ridge
January, 1979
;i
ACKNOWLEDGMENTS
To Dr. Charles Spotts I extend my deepest appreciation for his
assistance, guidance, insights and criticisms in the development of
this thesis.
I also thank Dr. Joyce Maxwell and Dr. C.R. Weston for
their assistance, suggestions and for serving on my committee.
I also thank Dr. R.C. Valentine, Dr. K.T. Shanmugam and
Dr. K. Andersen of the University of California, Davis for providing
the bacterial strains used in this study, and useful consultation.
I thank Linh Nguyen and Ruth Jung for preparing media, and
Robert Botts and his staff who searched for the unusual pieces of
equipment used in this study.
I appreciate the interest and co-
operation of the faculty and staff of the CSUN Biology Department.
The Recycling Center provided funds which aided in the procurement of equipment used in this study.
i;;
TABLE OF CONTENTS
iii
/'ACKNOWLEDGEMENTS .
LIST OF TABLES AND FIGURES .
v
ABSTRACT .
vii
CHAPTER I.
CHAPTER II.
INTRODUCTION . .
1
MATERIALS AND METHODS .
13
Microorganisms . . . . . . .
Media . . . . . . . . . . . . . . .
Enzyme Assay and Analytical Methods .
Acetylene Reduction Assay
Ethylene Determination . .
Ammonia Determination .
Protein . . . . . . . .
Microbial Growth Measurement
Population Density . . . . •
Cell Number . . . . . . . .
Reverse Mutation Detection
Cultivation Methods . . . .
Batch Cultures . . . .
Chemostat Cultures . .
Culture Vessel
Pump System . . .
Flow System . . . . . . . .
Atmosphere Control System .
Temperature Control System
Sterilization . .
Sample Collection .
CHAPTER III.
RESULTS
CHAPTER IV.
DISCUSSION
•.
13
13
15
15
17
18
22
22
22
22
23
23
23
24
24
27
28
29
30
30
30
.
....
32
77
BIBLIOGRAPHY
91
iv
LIST OF TABLES AND FIGURES
TABLE
I.
II.
PHENOTYPES OF K. PNEUMONIAE . . . . . . . . . . . . . . .
14
NITROGENASE ACTIVITIES OF A SULFATE LIMITED CHEMOSTAT
71
FIGURES
1.
CONCEPTUAL MODEL OF NON-MUTANT NITROGENASE REGULATION
2.
CHROMATOGRAM FROM THE ACETYLENE REDUCTION ASSAY
FOR NITROGENASE . . . . . . . . . . . . . . .
20
3.
DIAGRAM OF THE CHEMOSTAT USED FOR CONTINUOUS
CULTURE EXPERIMENTS . . . . .
26
4.
KLEBSIELLA PNEUMONIA£ M5a1
........ .
34
5.
KLEBSIELLA PNEUMONIA£ SK-25, GROWTH, AMt~ONIA
AND NITROGENASE . . . . . . . . . . . . . . .
37
6.
GROWTH OF CULTURES SUPPLEMENTED WITH CASEIN
HYDROLYSATE . . .
. . . . . . . . . . . .
39
7.
AMMONIA PRODUCTION IN CULTURES SUPPLEMENTED
WITH CASEIN HYDROLYSATE . . . . . . . . . .
41
8.
NITROGENASE ACTIVITY OF CULTURES SUPPLEMENTED
WITH CASEIN HYDROLYSATE . . . . . . . .
43
9.
RATE OF AMMONIA PRODUCTION IN CULTURES
SUPPLEMENTED WITH CASEIN HYDROLYSATE .
47
10.
AMMONIA PRODUCTION UNDER ARGON AND N2 ATMOSPHERES . .
50
11.
AMMONIA PRODUCED COMPARED TO PROTEIN CONCENTRATION
53
12.
INFLUENCE OF pH ON GROWTH . . . . . .
55
13.
INFLUENCE OF pH ON AMMONIA PRODUCTION
57
14.
GROWTH OF L-GLUTMHNE SUPPLEMENTED CULTURES .
60
15.
NITROGENASE ACTIVITIES OF L-GLUTAMINE
SUPPLEMENTED CULTURES . . . . . . . . . . .
60
v
8
FIGURES
16.
GLUTAMINE CONCENTRATIONS COMPARED TO STEADY
STATE TURBIDITIES . . . . . . . . . . . . .
65
17.
DETERMINATION OF THE CRITICAL DILUTION RATE (De)
67
18.
CHARACTERISTICS OF A L-GLUTAMINE LIMITED
CHEMOSTAT CULTURE . . . . . . . . . . . . . .
......
69
THE REPRESSION OF NITROGENASE ACTIVITY IN A
SULFATE LIMITED CHEMOSTAT IN THE PRESENCE OF
EXCESS GLUTAMINE . . . . . . . . . . . . . .
......
73
CONCEPTUAL MODEL OF NITROGENASE REGULATION
IN THE MUTANT K. PNEUMONIAE SK-25 . . . . .
•
80
19.
20.
vi
a
•
•
•
•
•
ABSTRACT
NITROGENASE REPRESSION IN Klebsiella pneumoniae
by
Wayne Walter Schubert
Master of Science in Biology
January, 1979
Nitrogenase biosynthesis in a mutant strain of Klebsiella.pneumoniae can be fully repressed by L-glutamine alone.
~·
pneumoniae
SK-25 which has no glutamine synthetase activity or immunologically
cross reacting protein, lacks glutamate synthase activity, and has repressed levels of glutamate dehydrogenase cannot directly utilize NH 4+,
and thus requires L-glutamine as a fixed nitrogen source for growth.
When grown in sulfate ion limited chemostat cultures under argon, the
presence of excess L-glutamine will fully repress nitrogenase activity.
In L-glutamine limited chemostat cultures and in batch cultures, nitrogenase was partly derepressed.
These findings indicate that the
presence of L-glutamine as a fixed nitrogen source can affect the regulation of nitrogenase by a mechanism which is independent of the
adenylylation of glutamine synthetase.
vii
CHAPTER I
INTRODUCTION
Nitrogenase is the key enzyme of the earth's nitrogen cycle.
It
is the only enzyme known which will catalyze a reaction with gaseous
dinitrogen (N 2) as the substrate and convert it to a biologically useful form. The atmosphere contains about 78% nitrogen by volume, but
despite its abundance, nitrogen cannot be used by most living organisms
until it has been fixed.
Biological nitrogen fixation is the enzymatic
process mediated by nitrogenase and is defined as the reduction of
molecular nitrogen (N 2 ) to ammonia (NH 3 ).
In addition to catalyzing this unique reaction, nitrogenase has
many unusual features which distinguish it from other enzymes.
It is
a high molecular weight protein made up of dissimilar subunits and
contains molybdenum, iron and sulfur.
It is capable of transferring
both electrons and prote~s to the substrate.
Special electron donors
capable of delivering electrons at a low potential, such as ferredoxin
or flavodoxin are required.
Nitrogenase has a high direct requirement
for energy, with a minimum of 12-15 moles of ATP required to fix one
mole of N2.
JD__y_1.Y.Q• the best symbiotic nitrogen f·ixers require 10-17
kg of carbohydrate to fix l kg of N2 . Nitrogenase also catalyzes a
competing reaction where protons are reduced to molecular hydrogen.
The energy of this reaction is lost, except in those systems possessing
additional specific enzymes (Evans et E_]_., 1977).
Nitrogenase is
coded for· by the .!}j_f genes which are made up of at least 13 complementation groups
(Hact~eil
et al., 1978).
2
H2 is a specific competitive inhibitor of nitrogenase with a Ki of
0.2-0.5 atm, (Hardy et ~., 1975). 02 is an uncompetitive inhibitor
with a Ki of 0.014 atm.
CO and NO also inhibit nitrogenase.
~vitro
the enzyme is inhibited by the accumulation of ADP and is unstable at
temperatures of 0°C and below.
Nitrogenase is extremely sensitive to inactivation by o2 both
in vitro and in vivo. Thus nitrogen fixation occurs only under
anaerobic conditions as in fermentative growth of Klebsiella and
Clostridium, or in microorganisms which have supportive systems to
protect the enzyme from oxygen, such as the high respiration rates
and conformational protection of Azotobacter, the heterocysts of the
blue-green algae, or the leghemoglobin oxygen transport system of the
legume-Rhizobia symbiosis.
The enzyme is repressed by No 3-, NH 4+ and some fixed nitrogen
sources. The functional enzyme system requires special ammonia incorporating pathways which include the enzymes glutamine synthetase,
glutamate synthase and glutamate dehydrogenase.
The most unusual characteristic of nitrogenase is its substrate
promiscuity.
Not only will the enzyme catalyze the reduction of N? to
~
2NH 4+, but the reduction of hydrogen ions to moiecular hydrogen, and
the double and triple bonds of both nitrogen and carbon compounds.
Substrates which are reduced by nitrogenase include; H3o+, N=N,
-NN=N (N 3-), =N+N=o+(N 20), RC=N, RN=C, and RC=CR. The reduction of
acetylene (C2H2) to ethylene (C 2H4)is the basis of the assay used to
determine the activity of the enzyme.
3
Nitrogenase is also unusual in that it has an extremely low turnover rate of only 50-100 molecules per minute, whereas other cellular
enzymes have turnover rates of thousands or tens of thousands per
minute.
The occurence of nitrogenase has been established in 3 orders,
11 families and approximately 26 genera of bacteria (Burns and Hardy,
1975).
The distribution of nitrogenase is limited to procaryotes and
has never been observed in eucaryotic cells.
In nature, nitrogen fix-
ing bacteria occur in diverse environments.
They range from aerobic to
anaerobic, chemoorganotrophic to heterotrophic and photosynthetic, and
from free living to symbiotic associations.
Despite the physiological and ecological differences between the
procaryotic species that possess nitrogenase, the structural and
functional properties of the enzyme are remarkably similar.
The
Klebsiella pneumoniae nitrogenase has a molecular weight of 285,000
daltons and is composed of two proteins.
The largest protein, component
I, contains 2 molybdenum, 32 iron and approximately 32 sulfur atoms.
In K. pneumoniae the molecular weight of component I is 218,000 and in
other species estimates range from 200,000 to 250,000 daltons.
The
component I protein is composed of two dissimilar subunits, each having
two identical units with molecular weights ranging from 50,000 to 60,000
(Zumft and Mortenson, 1975).
The molybdenum and some iron atoms are
arranged into a cofactor which can be isolated from the enzyme.
Component II has a molecular weight of 67,000 daltons and is composed of two identical subunits.
This protein contains 4 iron, 4 sul-
fur and no molybdenum atoms (Eady et
~.,
1972; Ljones, 1974).
4
The reaction catalyzed by nitrogenase is:
N2 + 6e- + 8H+ + nMg·ATP + 2 NH 4+ + nMg·ADP + nPi .
This reaction is apparently nonreversible.
(1)
Electrons generated from catabolic reactions in the cell are
transferred to the iron-sulfur proteins, ferridoxin or flavodoxin
(Klebsiella uses flavodoxin), which in turn reduces the nitrogenase
complex (Burns and Hardy, 1975).
~vitro,
sodium dithionite,
Na2S204, can reduce nitrogenase directly (Bulen et
~.,
1965).
From several lines of evidence it appears that electrons flow
from ferredoxin or flavodoxin to ·a complex of component II and
Mg·ATP.
The protein is conformationally changed from a rhombic to an
axial symmetry and the potential drops from -294 to -400 mv or lower .
. The low potential component II protein transfers electrons to the
component I protein which then reduces the substrate.
Experimental
evidence suggests that the electrons are transferred in pairs.
The
Fe-Mo complex has been identified as the active site of the enzyme.
(See the reviews of Orme-Johnson et
~.,
1977; Winter and Burris,
1976; Skinner 1976).
The number of ATP molecules required to drive the reaction varies
with the experimental conditions.
~vitro
studies with cell free
enzyme preparations indicate that a minimum of 12-15 moles of ATP per
mole of N2 reduced are required, (Winter and Burris, 1976). ~vivo
experiments with f. pneumoniae indicate that as many as 29 moles of ATP
may be required to fix one mole of N2 (Hill, 1976). Andersen et ~.,
(1977) using f. pneumoniae mutant strains, including strain SK-25,
estimate the apparent ATP requirement is 21 - 25 moles ATP/N2.
This
estimate is based on the amount of NH4+ excreted by the cells and
5
includes the ATP required for cell maintenance.
This study suggests
that at least 4.3 pairs of electrons are consumed per N2 reduced. If
the electron requirement value is converted t6 energy in terms of ATP
equivalents, the total
~vivo
energy cost may be as high as 30 ATP/N 2 •
Almost 1/3 of the energy may be lost to the nitrogenase catalyzed H2
evolution.
There are two ammonia assimilation pathways in nitrogen fixing
bacter·ia.
The first pathway incorporates ammonia via the following
reaction:
·2-oxoglutarate + NH 4+ + NADPH t glutamate + NADP+
This reaction is catalyzed by glutamate dehydrogenase (GDH) E.C.
1.4.1 .4.
(2)
This is the predominant pathway of assimilation'when ammonia
concentrations are greater than 1 mf 1 (Nagatani
1
_?t ~·,
1971).
GDH has
a low affinity for NH 4+ and is useless \'lhen concent·r'ations are low
(f'lagasani k ~.t ~·, 1974) .
.~J _(l}_. '
1975).
When N2 is the nitrogen source and NH 4+ concentrations are low,
GDH activity is low and the fixed nitrogen is incorporated via the
second pathway:
L-glutamate + ~"'./ .ll.TP
t
L-g·!uta'"Tine + ADP +Pi
L-glutamine + 2-oxoglutarate + NADPH !
( 3 ),
2 L-glutamate + NADP+ (4)
Reaction #3 is catalyzed by glutamine synthetase (GS) EC 6.3.1.2,
and reaction #4 by glutamate synthase (GOGAT), (L-glutamine: 2-oxoglutarate amino transferase, NADPH oxidizing) EC 2.6.1 .53 (Shanmugam,
Morandi and Valentine, 1977).
affinity for NH 4+ than GDH.
Glutamine synthetase has a much greater
The K
for reaction #3 is about 1 mM,
m(NH4+)
5
(Stewart et
~.,
1975).
Other estimates of the Km(NH +) values for GS
4
and GDH are <0.5 and 5-40 mM respectively (Dilworth, 1974). It is
evident by the Km values that GS is much more effective at low ammonia
levels encountered when N2 is the sole nitrogen source. Glutamate
synthase (GOGAT) has Km values for glutamine and 2-oxoglutarate of
0.2-0.5 and 2-7 respectively.
L-glutamate, the product from both path-
ways of NH 4+ assimilation, is then used for the synthesis of other
cellular intermediates such as amino acids and nucleotides.
Nitrogenase activity and the ammonia assimilation enzymes are
regulated in response to the availabiiity of various nitrogenous compounds.
The addition of ammonium ion to nitrogen fixing cultures
completely represses the biosynthesis of nitrogenase (Yoch and Pengra,
1966; Mahl and Wilson, 1968; Tubb and Postgate, 1973; Streicher et
1974; Shanmugam et
~·,
1975 and 1976).
~.,
In cell free enzyme prepara-
tions from various bacteria, the nitrogenase activity is not inhibited
by the addition of NH 4+, indicating that the repression effect is not
an inhibition of the enzyme, but rather it is a physiologically
mediated process (Mulder and Brotonegoro, 1974).
In order to illustrate the regulatory mechanisms influencing the
biosynthesis of nitrogenase, a conceptual model is presented.
Regula-
tory models have been proposed by Streicher and Valentine (1973),
Shanmugam and Morandi (1976) and Kleiner (1976) and are the basis for
figure 1.
Glutamine synthetase has been implicated as playing a major role
in the regulation of nitrogenase and the ammonia assimilation pathways.
Evidence for the role of GS has been established by investigations
7
FIGURE 1.
Conceptual model of non-mutant nitrogenase regulation. This
model depicts the pathways of incorporation of nitrogenous
compounds as well as nitrogenase regulation in wild type
organisms such as~· pneumoniae M5al. Inside the bacterial
cell the enzymes of the ammonia uptake pathway are encircled
and labeled as follows: N2ase, nitrogenase; GS, glutamine
synthetase, the non-adenylylated form; GS-AMP, adenylylated
glutamine synthetase; AT, adenyl transferase; GDH, glutamate
dehydrogenase; and GOGAT, glutamate synthase.
Biochemical pathway under low NH 4+ concentrations when nitrogen is being fixed.
----~~
Biochemical pathwa¥ operative in high concentrations of NH 4 .
Enzyme regulation; low NH +
4
Enzyme regulation; high NH 4+.
I"
H2C -;-C H2
NH+
4
..,_ -+-· amino acids
nuceotides
GLUTAMATE
HC:=CH
I
\\ <1 ketoglutarate
I
7'
',' \t§:
GS
_. NH 4+
N2
II
II
1
I
mRNA
.
'
'
I
GLUTAMINE
1
.
I
I
f
I
~1~
~-4----- ____ :
I
I
t·---------@
DNA
co
9
utilizing mutants in the regulatory and structural genes coding for the
enzymes of the nitrogen assimilation pathway.
Magasanik et
~-,
(1974) found that glutamine synthetase (in
Klebsiella aerogenes) is of central importance in the regulation of
the enzymes responsible for degrading amino acids into glutamate and
an enzyme responsible for the incorporation of NH 4+ and formation of
glutamate (GDH). Glutamine synthetase possibly regulatesthe inhibition
of its own synthesis.
1eve is.
~·I hen a 10\'1
is initiated.
The intracellular level of NH 4+ controls GS
1eve 1 of NH 4+ is pr'esent an 11 enzyme cascade effect 11
First the deadenylylation of glutamine synthetase occurs.
The deadenylylated form of the enzyme represses the synthesis of GDH
(which cannot use low levels of NH 4+). The GS-GOGAT ammonin uptake
pathway begins to provide glutamate for the cell. Other changes in
If NH 4+ ·is provided in excess these events are reversed and ammonia is assimilated
1evels of enzymes such as pet'meases occur as well.
by the GDH pathv;ay and GS is in the adenylylated form.
The
ad~nylylation
state of glutamine synthetase has been implica-
ted in the regulation of nitrogenase.
It has been suggested that the
non-adenylylated form of GS may act as a positive control for the
synthesis (Tubb, 1974).
Q!leumoni ae
~15a 1
Kleiner (1976), using w·ild type
flebUel~
in cont'i nuous cultur·e found that non-adenylyl a ted
levels of GS occurred when N2 was the sole nitrogen source. This
correlated with high nitrogenase and GOGAT activities and low activity
of GDH.
In the presence of NH 4+, GS was adenylylated which corresponded to low activities of nitrogenase and GOGAT and high activities of
GOH.
10
Gordon and Brill (1974) found that by using glutamate analogues
which are specific inhibitors of GS and GOGAT, nitrogenase was derepressed (i.e. high activities) in the presence of excess NH 4+ By inhibiting GS and GOGAT, the nitrogenase enzyme fixed nitrogen which it
was not able to assimilate, and thus excreted NH 4+ into the medium.
The most convincing evidence implicating GS in the regulation
of nitrogenase is that mutations resulting in the catalytic inactivation or alteration of the glutamine synthetase protein, result in the
continued synthesis of nitrogenase in the presence of NH + (Shanmugam,
4
Morandi and Valentine, 1977; Shanmugan, o•Gara, Andersen, Morandi and
Valentine, 1977).
Derepressed mutants are those that can no longer
repress the biosynthesis of nitrogenase in the presence of excess NH4+.
The mutations result in glutamate or glutamine auxotrophy and the
inability to utilize ammonia.
The ammonia formed by the derepressed
mutants is excreted into the medium.
Complementation of mutants lacking GS and nitrogenase activities
with an
I· coli episome that restores normal GS activity, also brings
about the restoration of nitrogenase (Streicher, 1974).
Nitrogenase derepressed mutants were found to belong to three
classes, none of which has glutamate synthase (GOGAT) activity.
The
majority of mutants in each class are deficient in glutamate dehydrogenase (GDH) activity.
The first class requires glutamate for growth
and synthesizes nitrogenase and GS in the presence of NH 4+ The second
class of mutants requires glutamine for growth, synthesizes nitrogenase
in the presence of NH 4+, and has catalytically inactive glutamine synthetase. The third class, which includes strain SK-25 requires
11
glutamine for growth, produces nitrogenase in the presence of NH 4+ and
has neither glutamine synthetase activity nor immunologically cross
reacting protein (Shanmugam, Morandi and Valentine, 1977).
Fixed nitrogen sources other than NH 4+ can influence the regulation of nitrogenase. Yoch and Pengra (1966) found that when NH 4+ was
exhausted in batch cultures, there was a lag period before the
appearance of nitrogenase activity.
Neither single amino acids nor a
mixture of amino acids was found to delay the appearance of the subsequent nitrogenase activity; in fact, the lag period was shortened and
the nitrogenase activity was enhanced.
In several nitrogen fixing
species of Enterobacteriaceae isolated from paper mill processing
waters, the nitrogenase activity was generally not repressed by amino
acids (Neilson and Sparell, 1976).
On the other hand, Shanmugam and Morandi (1976) observed that
amino acids can repress the biosynthesis of nitrogenase in
~·
pneumon-
iae, including the three classes of derepressed mutant strains which
are not repressed by NH 4+ A mixture of L-glutamine and L-aspartate
repressed nitrogenase biosynthesis completely. A high concentration
of glutamine (1 g/1) plus NH 4+ was able to repress the enzyme·~ biosynthesis, and increase GDH activity in some mutants. L-glutamate
alone was found to be a poor repressor and L-glutamine alone in nitrogen limited batch cultures failed to completely repress nitrogenase.
At concentrations of l g/1, L-glutamine repressed nitrogenase biosynthesis by only 45% at the most in strains SK-25 and SK-55.
These paradoxical reports of the stimulation and repression of
nitrogenase by amino acids warranted further investigation into their
{;
'
12
role as regulators of nitrogenase.
L-glutamine, which is a major
component of the amino acid pool, lies at the cross roads of bioIt is a primary compound in the ammonia uptake
synthetic pathways.
pathway which regulates nitrogenase.
It may provide its amide nitrogen
for the biosynthesis of amino acids, amino sugars, nucleotides and
cofactors.
The carbon skeleton of glutamine may be used in the tri-
carboxylic acid cycle for ATP production.
The nitrogenase derepressed mutants are glutamine (or glutamate)
requiring auxotrophs.
The possibility that L-glutamine could play
two roles, one as a required nutrient for growth and secondly as a
regulator of nitrogenase biosynthesis helped to provide an impetus
for further study.
The availability of nitrogenase derepressed mutants
was a further aid to this study because the products of nitrogen fixation were uncoupled from growth (i.e. glutamine synthetase, a known
regulator of nitrogenase was absent in strain SK-25, as were glutamate
synthase and glutamate dehydrogenase, so that the ammonia fixed from
N2 could not be used). By using a nitrogenase derepressed mutant, the
enzyme activity could be measured by the amount of ammonia excreted
into the medium, thus facilitating physiological studies.
In this series of experiments, evidence is presented that Lglutamine or its metabolic derivatives have a role in the regulation
of nitrogenase activity.
There is also evidence that a regulatory
mechanism exists which is independent of the adenylylation and
deadenylylation of glutamine synthetase for controlling nitrogenase
activity.
CHAPTER II
MATERIALS AND METHODS
Microorganisms
Klebsiella pneumoniae strains SK-25 and M5al were obtained from
Dr. R.C. Valentine at the University of California, Davis.
Klebsiella
pneumoniae M5al is a wild type strain while SK-25 is a mutant strain
showing high nitrogenase activity, even in the presence of ammonia.
Table 1 shows the phenotype of each strain.
The SK-25 mutant was
isolated as a glutamine auxotroph from the parent strain Asm-1.
It
does not have glutamine synthase, glutamine synthetase, or glutamate
dehydrogenase activity.
It is devoid of any immunologically cross
reacting glutamine synthetase protein (Shanmugam, Morandi and Valentine,
1977) and is constitutive for nitrogenase (i.e., nitrogenase is not
repressed in the presence of ammonia).
Klebsiella pneumoniae M5al
synthesizes all four of the above enzymes at normal levels.
Media
All K. pneumoniae strains were maintained on Luria agar (Luria,
et
~·,
1960).
Luria broth was the medium used for growing cells to
be used as inoculum for some experiments.
The composition
~f
the
medium is: 10 g Tryptone, 5 g yeast extract, 10 g NaCl, 1000 ml
distilled water; for solid media 15 g agar was added.
The pH was
adjusted to 7.0, and in some cases 3 g of sucrose was added.
For nitrogen fixing batch cultures, a minimal medium was employed.
The minimal medium of Yoch and Pengra (1966) as modified by Streicher
et
~·,
(1971) was further modified to increase the buffering capacity
13
14
TABLE I
PHENOTYPES OF K. PNEUMONIAE
STRAIN
NITROGENASE
M5a1
+
SK-25
c-
ASM-1
+
+
SYNTHETASE
(GS)
GLUTAMATE
SYNTHASE
(GOGAT}
+
+
GLUTAr~INE
Normal enzyme activity
Lacks enzyme activity
c-
Constitutive enzyme levels
+
GLUTAMATE
DEHYDROGENASE
(GDH)
+
+
15
by
doubling the phosphate concentration.
The minimal medium contains
the following components per liter: 12.5 g Na 2HP0 4 , 1.5 g KH 2Po 4 ,
2.0 g NaCl, 10 mg FeS0 4·?H20, 10 mg Na 2Mo04·2H 20, 0.2 g MgS0 4·7H 20,
15g glucose, and 100 mg L-glutamine (L-a- Amino glutaminic acid,
Sigma Chemical Co.).
The MgS0 4 ·7H 2o was autoclaved separately and
added after the medium cooled. The L-glutamine is heat labile, and
was filter sterilized using a 0.45
~m
Millipore filter.
The pH of the
medium was adjusted to 7.5 before autoclaving.
When used for chemostat studies, the minimal medium was modified
to prevent precipitation by replacing Feso 4 ·7H20 with 25 mg/1 of EDTA
chelated iron (Eastman Chemicals). The phosphates were autoclaved
separately, the glutamine was filter sterilized, and both were added
after the media had cooled.
Some chemostat experiments utilized a medium in which sulfate ion
was the limiting nutrient.
In these experiments, sulfate was supplied
as Na2so 4 at a concentration of 2.5 x lo- 6 M. In place of MgS0 4 ·7H 2o,
0.1 g/1 MgCl 2 ·6H 2o was used. L-glutamine was added at a concentration
of 100 mg/1 or as otherwise indicated .
.Enzyme Assay and Analytical Methods
Nitrogenase activity was determined by two different methods.
The
first measures the quantity of acetylene which has been reduced to
ethylene, and the second measures the quantity of ammonia excreted into
the media by the cells.
Acetylene Reduction Assay
Nitrogenase can catalyze the reduction of acetylene to ethylene
16
and this reaction can be used as a quantitative method of determining
enzyme acti v'ity (Scholl horn and Burris, 1966; Oi 1worth, 1966).
The
product, c2H4 , accumulates in the gas phase of the reaction vessel and
can be directly quantified via gas chromatography (Hardy et ~., 1968).
Nitrogenase activity was determined by the following procedure.
Three
milliliter portions of the cell culture were collected anaerobically
in a plastic syringe and then injected into a 25 ml Delong culture
flask which had been stoppered previously with a 11 Vacutainer 11 serum
stopper, evacuated, and refilled with argon.
The reaction was started
by injecting 3.0 ml of acetylene into the container.
The flasks were
placed on a gyratory shaker and incubated at 30°C for 60 minutes.
The
reaction was terminated by injecting 0.5 ml of 4 NH 2so 4 through the
serum stopper.
Because nitrogenase is inactivated by oxygen, the cells must be
transferred anaerobically and placed in anaerobic assay flasks filled
with argon.
A special apparatus was designed to facilitate the
preparation ofl2 anaerobic flasks at one time.
The main chamber has
12 side arms soldered in place, to which are attached syringe needles
via rubber tubing connectors.
The flasks which had been fitted with
serum stoppers were pierced by the needles of the apparatus.
Two
valve controlled ports, one connected to an argon cylinder and the
other leading to a vacuum pump are manually opened and closed sequentially so that the flasks may be evacuated and then filled with argon.
This evacuation and filling process was repeated three times to remove
any remaining o2 , and then the flasks were filled with argon to the
ambient pressure as indicated by a pressure-vacuum gauge attached to
17
the monitors.
The acetylene used in the assay procedure was generated as
required by placing a few small lumps of calcium carbide (Cac 2 ) in a
100 ml bottle, capping with a serum stopper and then evacuating and
flushing the bottle with argon.
The bottle was evacuated and then
0.5 ml H2o was injected to generate the acetylene. To prevent the
pressure of the evolving gas from popping out the stopper, a 50 ml
syringe was attached.
Excess gas pressure pushed the plunger outward,
and as the acetylene was used, the plunger could be pushed inward to
provide a slight positive pressure.
Positive gas pressures were main-
tained in flasks and during gas transfer to prevent entry of 02 .
Ethylene Determination
The ethylene which was produced was analyzed by a Beckman GC 72-6
gas chromatograph equipped with a flame ionization detector.
The gases
were separated on a l/8" diameter, 1 meter stainless steel column,
packed with Poropak N, supplied by Supelco, Inc.
was set up using the following parameters:
The gas chromatograph
Detector temperature, 70°C;
oven temperature, 50°C; inlet and line temperatures, 60°C; column flow,
Helium 20 ml/min.
Helium make up flow to the detector, 100 ml/min;
Hydrogen 45 ml/min and compressed breathing air, 300 ml/min.
The
electrometer settings were; input, l; suppression, low; peak-; detector
#1, and the range and attenuation were set according to the concentrations of the comoounds being analyzed.
The chart recorder received a
1 rnv input signa1, and the chart speed was set at 111 /minute.
The nitrogenase assay was carried cut in triplicate or quintupli-
18
cate and three or more injections into the GC were made.
gas injected was 100
~1.
A Hamilton 100
~1
The volume of
gas tight syringe was used.
A single injection could be separated and the GC readied for the next
injection in 2-4 minutes, depending on the sensitivity, of the detection
required.
The retention times of ethylene and acetylene were 0.70 and
1.15 minutes, respectively.
A sample chromatogram is given in figure 2.
The amount of ethylene present in the gas phase of the assay flask
was determined by calculating the peak area from the integrator value,
range and attenuation settings, and then calculating the ethylene
present in nM/ml from the slope of a standard curve.
The standard
curve was prepared by making serial dilutions of pure ethylene, and
injecting constant volumes of the known concentrations into the GC.
plot of the peak area vs ethylene concentration in moles was found to
be linear over five orders of magnitude, from lo-10 to lo-5 moles
C2H4/cm3.
The nitrogenase activity is presented herein as the specific
activity of nitrogenase, and is given in terms of nM C2H2 reduced (or
C2H4 produced) per hour, per mg cell protein.
Ammonia Determination
Ammonia in the medium was analyzed by the colorometric Nessler
reaction
(Burris~
1974) following collection by microdiffusion.
ml samples were collected from
fuged to remove the cells.
the~·
Five
pneumoniae cultures and centri-
The supernatant was decanted and 1.0 ml
portions were pipeted into 20 ml microdiffusion vials.
The microdif-
.fusion apparatus consists of a 20 ml vial and a 5 em long glass rod
A
19
FIGURE 2.
Chromatogram from the acetylene reduction assay for nitrogenase. Ethylene elutes from the column first with a retention time of 0.7 minutes, followed by acetylene at 1.15
minutes. The peak areas were integrated and used to calculate enzyme activity.
20
21
inserted into a rubber stopper.
The tip of the glass rod was rounded
and the surface roughened by grinding.
In the procedure, the tip of
the glass rod was dipped into 5N H2so 4 , and then touched to a paper
towel to remove any large drop of H2so 4 and the solution in the vial
was made alkaline by adding 1 ml of saturated K2C03.
The stopper.
holding the glass rod was inserted into the vial immediately after
the sample had been made alkaline.
The apparatus was then rotated to
insure proper mixing of the K2co 3 , and was allowed to stand overnight
at room temperature. The ammonia diffuses out of the alkaline
solution and reacts with the H2so 4 on the glass rod to form (NH4) 2so 4 .
The reaction is complete after three hours, but the microdiffusion may
be allowed to stand overnight or longer without change.
The microdiffusion step is required because media components
interfere with the Nessler reaction.
At the completion of the micro-
diffusion, the stopper was removed and the glass rod was used to stir
a 10 ml working solution of the Nessler reagent.
The working solution
was prepared by diluting the stock reagent tenfold in distilled water.
The Nessler reagent was supplied by Anderson Laboratories Inc., Fort
Worth, TX., or was prepared according to the following formula:
10.0 g Hgi 2 , 7~0 g KI, 16.0 g NaOH and distilled water for a final
volume of 100 ml (Hawk, 1954).
The absorbance of the solution was then measured on a Beckman
model 24 or a Bausch and Lomb Spec 20 spectrophotometer at a wavelength
of 490 nm.
The quantity of ammonia present was calculated by compari-
son with known standards treated in the same way.
The Nessler
reaction follows Beer's law for concentrations between 0.2 and 6.0 mM,
22
at the indicated wavelength.
Protein
Protein was -determined by the method as described by Lowry, et
(1951).
~.,
Bovine serium albumen (Sigma Chemical Co.) was used as the
standard protein.
Microbial Growth Measurement
E,QQ~lation D~ns:!!Y_
The cell population density was measur'ed tur'bidimetrically.
Samples of both batch and continuous cultures were collected by
syringe and measured at 420 nm in a Beckman model 24 spectrophotometer,
with a 1.0 em cuvette path length.
When cultures were at high popula-
tion densities, dilutions were made to yield adsorbancies between 0.1
and 0. 3.
In sorne early
photometer was used.
expei~iments
a Bausch and Lomb Spec 20 spectre--
The absorbance was measured at the above wave-
length with a standard 13 mm cuvette.
Cell Number
The number· of cells per ml of culture \'Jas detenn-i ned by the
standard plate count technique.
A sample of culture was collected and
serially diluted with sterile phosphate buffered water in 100 ml
dilution bottles.
Samples of 1.0 ml from the dilution bottles were
pipeted into plastic petri dishes and mixed with melted Luria 8gar kept
at 42°C.
The plates were incubated aerobically at 37°C for 24-48 hours,
and the colonies were then counted using a New Brunswick colony.counter.
23
Reverse Mutation Detection
Throughout the course of experiments, especially the chemostat
experiments, it was .necessary to check for cells which have undergone
a reverse mutation.
A reverse mutation removes the block in the NH 4+
assimilating pathway, allowing the utilization of the NH 4+ which had
previously been excreted into the medium and thus such mutants are
able to grow without fixing N2 .
Reversions were detected by colony formation on the minimal
medium as described above, supplemented with 1.0 g/1 NH 4Cl. The
plates were streaked with cells from the cultures and then incubated
at 30°C for 24 hours under aerobic atmospheric conditions.
Cultivation Methods
Batch Cultures
The effects of amino acids on nitrogenase activity of
iae strains were studied in batch culture.
500 ml flasks under anaerobic conditions.
of minimal medium.
pneumon-
Cultures were grown in
Each flask contained 500 ml
Cultures were kept anaerobic by sparging with extra
high purity nitrogen.
used.
~·
In some experiments, high purity argon was
Each flask was closed by a rubber stopper with three ports; gas
entrance, exit, and a sampling.port.
The temperature of the cultures
was maintained at 25± l°C by placing the flask in a constant temperature water bath.
The medium consisted of minimal medium as described above, supplemented with various concentrations of L-glutamine and/or casein
hydrolysate (Oifco, pancreatic digest) as indicated for each experi-
24
ment.
The inoculum was prepared by transferring a typical colony
growing on a Luria Agar plate to 10 ml of Luria Broth in a screw cap
test tube.
This culture was incubated 12-18 hours, until late log
phase was reached.
This culture was then used to inoculate the batch
culture by anaerobically transferring the cells via a syringe.
A 1%
(v/0 inoculum was used.
Batch cultures were monitored for a minimum of 18 hours to a maximum of 170 hours.
Cultures were sampled periodically by withdrawing 5
to 10 ml by syringe through the serum stoppered sampling port.
These
samples were routinely analyzed for turbidity, ammonia, reverse mutations, and in some cases, acetylene reduction.
Chemostat Cultures
Continuous culture methods were used to study the effects of
glutamine on nitrogenase activity.
Klebsiella pneumoniae was grown
continuously in a specially-designed chemostat.
The chemostat was
designed and built to provide anaerobic conditions, constant temperature, perfect mixing, a constant supply of nutrients at a given concentration, and a controlled flow rate.
A flow diagram of the chemo-
stat is depicted in figure 3.
Culture Vessel -The chemostat vessel was a 750 ml, water jacketed,
pyrex glass reaction container.
The top of the vessel had a ground
glass surface, upon which a 3/8 stainless steel plate was mounted.
11
The plate was held in place by spring activated clamps which grasped
the glass lip of the vessel.
An 0 ring was seated in the steel
11
11
25
FIGURE 3.
Diagram of the chemostat used for continuous culture experiments.
----
Gas
Medium
-·-·-·
Ce 11 cu 1ture
Temperature control water
1. Chemostat vessel
2. Medium reservoir
3. Medium pump and controller
4. Heat exchanger
5. Drip chamber type medium additi9n port
6. Culture &gas exit port
7. Sample collection flask
8. Waste culture reservoir
9. Gas cylinder with regulator
10. Gas flow meter
11. Liquid trap
12. HCl trap
13. Pressure relief device; hydrostatic back pressure
14. Constant temperature waterbath
15. Circulating pump
16. Magnetic stirrers
17. Glass wool filters
18. Sampling ports
19. Thermometer
26
I
I
I
I
I
I
I
I
I
I
I
I
I
I
......... ···-:
I
I
I
I
I
I
I
I
I
I
I
I
..0
I
, : i.
I
:
:I
:I
II
j··~co
__r-.__.
II
II
I
II
.-
I
I
:
:
:
I
I
I
I
L
t,j,
I
:
-~......_
I
--•
~
L-------~~
~
~
I
I
I
........:-__J
- - - - -------.;..
I'
I
gc__
o-_
I
...,_•-•
-·--•,.
27
plate and coated with silicone vacuum greasG, to give an air-tight seal.
Entrance to the vessel was gained through ports in the steel plate,
fitted with silicone rubber stoppers.
for media entrance, culture
The vessel had separate ports
exit,~pressure
sparge, sampling port, and inoculation.
relief thermometer, argon
The culture was mixed by a
teflon coated stir bar driven by a magnetic stirrer.
Sterile medium was fed into the culture chamber from a reservoir
by means of a positive displacement pumping system.
in a 20 1 pyrex glass bottle.
Medium was stored
The bottle was closed by a rubber
stopper fitted with the following inlets:
medium delivery line from
the bottom of the bottle to the pumping system, argon inlet to a
sintered glass sparging tube, argon exhaust, medium supplement line
for the addition of phosphates or other medium additives, and a
medium addition port capped with a serum stopper.
The medium supple-
ment line and the medium delivery line were equipped with glass tube
connectors with ground glass fittings, so that medium reservoirs
could be changed quickly and aseptically.
All lines open to the
atmosphere were fitted with glass wool filters.
The reservoir was
kept anaer'obic by sparging with argon, and was constantly mixed by a
magnetic stirrer.
Pump System - Medium was pumped from the reservoir by a Masterflex
pump model 7545-10.
The electric motor driven system was equipped
with two pump heads and a speed controller.
Pump head #7013 used
0.0315 x 0.1625" ID x OD tubing and provided flow rates of 30-300 ml/
hour.
Pump head #7014-20 used 0.655 x 0.1945" ID x OD tubing and pro-
28
vided higher flow rates of 150-1650 ml/hr.
Because the medium was
pumped by peristaltic compression of the autoclaved tubing, it never
came into direct contact with the pump surfaces or any other nonsterile environment, thus problems of contamination were avoided.
In
the medium delivery system the stream was split into two peristaltic
pump hoses which were mounted in the two pump heads.
Flow rates were controlled by regulating the pump shaft speed.
Flow rates could be estimated from a curve of flow rate plotted
against pump controller settings.
Actual flow rates were detenmined
by collecting the medium leaving the intact chemostat system, or by a
special method which directly measured the rate of flow.
The latter
method employs a 10 ml pipet positioned in a line tapped off the
medium delivery line, between the reservoir and the pump.
was filled with medium by gravity feed from the reservoir.
The pipet
The
line from the reservoir was then clamped off and medium was pumped from
the pipet.
The change in volume was read directly from the calibra-
tion on the pipet, and by measuring elapsed time, the flow rate could
be calculated from the equation F = dV/dT.
Flow System - From the pump the medium flowed. through a water jacketed
condenser and into the chemostat vessel by means of a drip chamber.
The drip method of adding medium to the chemostat was required to
prevent bacteria from growing back into the lines and reservoir.
The volume of the culture in the chamber was 530 ml, and was
maintained at that constant volume by forcing any excess through a
small diameter glass tube by positive gas pressure.
Another drip
29
chamber was placed on the exit line to prevent any contamination
problems.
flask.
The medium and cells then flowed into a sample collectio·n
A 125 ml sidearm flask was used to collect and sample the
culture.
Excess culture flowed out of the side arm and into a 10 or
20 1 waste collection bottle.
Argon flowed through the same lines,
and finally exited the waste vessel through a glass wool filter, thus
preventing aerosols from escaping into the atmosphere.
Atmosphere Control System - Argon was the atmosphere of choice.
The
requirements for the atmospheric system were the maintenance of
anaerobiosis, inertness, availability and cost.
Nitrogen was
excluded on the basis of reactivity with nitrogenase.
The ammonia
which formed provided a se 1ecti ve advantage for r.evertant ce 11 s
capable of assimilating NH 3.
The flow rate of argon into the chemostat vessel was regulated
at 200 ml/min by a rotameter.
Argon was sparged into the culture
by means of a sintered glass tube.
The gas exited the vessel forcing
excess culture with it, keeping the entire system anaerobic.
A pressure relief device was installed on the chemostat to prevent any change in flow rates or damage to the system.
An open gas
line extended from the chemostat vessel through a trap system (see
figure 3).
the system.
An HCl trap was used to prevent any organisms from entering
The gas from the chemostat if at an elevated pressure,
could bubble through the HCl trap and then through a column of water.
The water level could be adjusted to provide the required hydrostatic
back pressure.
30
Temperature Control System - The temperature of the chemostat was
maintained at 30± l°C by means of a water jacket surrounding the culWater was circulated through the water jacket of the
ture vessel.
culture vessel and the condenser from a constant temperature water
bath by a circulatory pump.
The temperature was measured by a
thermometer extending into the culture.
Sterilization - The chemostat system was autoclaved completely
assembled.
All openings to the atmosphere were fitted with glass
wool filters.
Lines extending into the liquid of the medium reservoir
or the HCl trap were clamped to prevent siphoning of these liquids.
Hoses to be clamped were thick walled rubber tubing, to prevent the
permanent sealing of the hose as
exp~rienced
with thin walled tubing.
The chemostat was autoclaved for 90 minutes at 15 psi and 121°C. This
long period of autoclaving was used when 18-20 1 of medium was to be
sterilized.
While cooling, the medium was sparged with argon to
exclude contaminants and oxygen.
After the system had cooled, the
pump and temperature control systems were set up and medium additions
were made.
The phosphates were autoclaved separately in a 2 1 bottle
and then added to the medium reservoir by displacement of the liquid
through a hose with compressed, filtered argon gas.
Sample Collection - Culture samples were collected from the 125 ml inline flask.
ihe flask was first emptied of any culture by tilting
and allowing any culture to flow through the line into the waste container.
flask.
The flask was then rinsed with new culture flowing into the
The new culture which flowed into the flask was used immediate-
31
ly.
The collection flask was considered to be an expansion of the
chemostat vessel.
This collection flask was used to avoid the high
risk of contaminating the chemostat during sampling procedures.
Samples were withdrawn by syringe and injected into nitrogenase assay
flasks, prefilled with argon.
Samples were also collected for
turbidity measurements, as well as to check for contaminants and
mutants capable of utilizing ammonia.
CHAPTER III
RESULTS
The results of an experiment designed to relate the levels of
nitrogenase activity, and ammonia production with the development of
Klebsiella pneumoniae M5al in batch cultures is shown in figure 4.
This is a wild type strain with no impairments of ammonia assimilating
enzymes.
Since the inoculum was 1% (vjv) of a 12 hour Luria Broth
culture some amino acids were probably carried over from the inoculum
culture to the batch culture.
In this experiment nitrogenase activity
was measured by a colorometric assay for c2H4 and given in terms of nM
c2H4 formed/hour/mg protein. Nitrogenase activity began to appear
early in the log phase and reached a maximum of 2,100 c2H4 units. This
corresponds to a partly derepressed level. The culture grew rapidly
with a doubling time of 1.6 hours and reached a very high level with
640 mg protein/1 when the experiment was terminated.
The nitrogen
which was fixed contributed to the growth of the culture, permitting
high cell densities.
The growth cycle of this culture shows almost no lag phase (<0.5
hours), while very low levels of nitrogenase exist for 4 hours.
This
implies that very low levels of nitrogenase activity existed upon
transfer from the amino acid rich L-Broth, suggesting that high
concentrations of amino acids repress nitrogenase in this strain.
K. pneumoniae M5al does not excrete NH 4+ into the surrounding
medium. Ammonia accumulation as well as growth rates of Klebsiella
pneumoniae were affected by the type and quantity of the nitrogen
32
33
FIGURE 4.
Klebsiella pneumoniae M5al. This is a wild type strain with
normal ammonia assimilating pathways. It utilizes N2 as a
nitrogen source in addition to the 100 mg/l glutamine
present in the minimal medium, and thus can reach high
population densities. All nitrogen fixed is utilized and
no NH 4+ is excreted into the medium.
e- e Turbidity (A420)
• - • Nitrogenase (nM c2H4 produced/hour/mg protein)
• - • NH 4+ concentration ·(mM)
34
(.!J
:E:
1
.J.I
...........
0-
0:::
:X::
...........
.::r
r:t
I
,.-...
0
:X::
S3000
:E:
z
.........
:E
::E:
~
~
C\
. >-
~
-
.
>-
1-
2: 2000
1-
u
.c:::c
l.J..l
t::
1-
C\
u
><
l.J..l
.......
§2
:::J
1-
l.J..l
0:::
0
2.0
I
+.::r
:X::
z:
l.J..l
(/)
<C
z:
.0
~ 1000
0
0:::
1-
.......
:z
0
5
10
15
20
TIME <HOURS)
K.
PNEUMONIAE
M5Al
35
source provided.
Since K. pneumoniae SK-25 cannot grow with Nz as the
soie nitrogen source, cultures were supplemented with L-Glutamine.
The
effects of additional amino acids were determined in cultures supplemented with casein hydrolysate.
The relationship between turbidity, NH 4+ excretion into the medium
and the specific activity of nitrogenase in a culture unsupplemented
with casein hydrolysate is illustrated in figure 5.
Clearly, nitro-
genase activity was present in the lag phase, where no increase in
cell population is occurring.
The nitrogenase activity (as calculated
from the NH 4+ production and growth curves) rose sharply from 1,300 to
4,350 as the population began to grow and ammonia was excreted into
the culture medium.
The nitrogenase activity began to decrease before
the middle of the log phase was reached.
After the culture reached
stationary phase, NH 4+ continued to accumulate and nitrogenase
activity remained constant at a low level. At 115 hours a population
of reverse mutants began to develop.
Ammonia can be used by this sub-
population so the ammonia concentration decreased which corresponded
to an increase in turbidity, and the nitrogenase (NH 4+) specific
activity dropped to zero.
Batch cultures supplemented with casein hydrolysate showed a
decrease in growth lag (figure 6) and greater final cell density
than unsupplemented cultures.
Ammonia in these cultures began to
accumulate earlier than the unsupplemented cultures (figure 7) and
nitrogenase specific activity (NH 4+ units) also increased at an
earlier time (figure 8). The data imply that the supplemented amino
acids were readily used and probably permitted growth without utilizing
)
FIGURE 5.
SK-25, grov-tth, ammonia, and nitro~enase.
This derepressed mutant strain does not utilize the NH 4 fixed
from N2, thus excreting it into the medium. Grown in minimal
medium-containing 100 mg/1 glutamine.
Kle~siel1a_ pneu~oniae
Nitrogenase (nM NH 4+ formed/hour/mg prot?in)
NH 4+ Concentration (mM)
37
•
1.0
5
5
~4
><
>r........
4
0
0
-
>3
r-
w
<C
w
~2
z
i::3
::£
• :cl
0.1
;-
........-
+.:::t"
::c
z2
w
z1
,-.
·1-·
I /
i
<..!J
0
0:::
r.......
6
/,
-·-·;
>r.......
r-l
.......
t:O
0:::
1
=>
r-
..
0.01
,., /\..,.,
6
L
:_.
30
60
90
TIME <HOURS)
120
L
__ _____
....._
~----
.
----~--
'
------------
38
FIGURE 6.
Growth of cultures supplemented with casein hydrolysate.
f. pneumoniae SK-25 grown in 500 ml batch cultures. Minimal
medium with 100 mg/1 L-glutamine was supplemented with casein
hydrolysate.
o-o
0 mg/1 casein hydrolysate
·-·
·-·
10 mg/1 casein hydrolysate
• •
100 mg/1 casein hydrolysate
1000 mg/1 casein hydro·lysate
39
~------4
A
1.0
..........
0
N
.:::r
c::::c::
._...
-- 0.1
>-
IQ
§2
:=>
I-
0.01~~--~----~------~----~----~
30
60
90
TIME <HOURS)
120
150
_;._
__
-------~
-------
-----
40
FIGURE 7.
Ammonia production in cultures supplemented with casein
hydrolysate. !· pneumoniae SK-25 grown in glutamine
minimal medium supplemented with casein hydrolysate.
o-o
·-·
·-·
·-·
0 mg/l casein hydrolysate
10 mg/l casein hydrolysate
100 mg/1 casein hydrolysate
1000 mg/l casein hydrolysate
-~---~-
-
-
41
6.0
5.0
2.0
I
30
90
60
Tir~E
<HOURS)
120
.
I
150
42
FIGURE 8.
Nitrogenase activity of cultures supplemented with casein
hydrolysate. f. pneumoniae SK-25 was grown in glutamine
minimal medium supplemented with casein hydrolysate.
0 mg/l casein hydrolysate
..........
10 mg/1 casein hydrolysate
100 mg/l casein hydrolysate
1000 mg/1 casein hydrolysate
43
--z
w
10
0:::
a_
<..!:>
:E:
...........
0:::
:::c:
5000
...........
+ .:::r
:::c:
z
::E
z
.._.,
--
>1-
4000
>
I
--
~
1-
/!
! ;
u
<C
u
! '
3000
LJ...
u
w
a..
(/)
~
<C
z
w
2000
<..!:>
0
0:::
1-
;; 1000
......
..
".
..
•
--
-~
\
.
'\
\
.... ..
····· ••..
••
r:.~-
j
_}
'\
•
•••••••
·----
••
30
60
90
TIME <HOURS)
120
44
certain biosynthetic pathways that may not have been fully operative
which resulted in lengthy lag times.
Despite this early increase in
nitrogenase activity, the cultures with the highest amount of casein
hydrolysate (1000 mg/1) showed the lowest peak activity while the
cultures with little added casein hydrolysate {0 and 10 mg/1) had
the greatest peak nitrogenase activities.
Although the added amino
acids caused an initial increase in nitrogenase activities (as well
as growth and ammonia production), the activity levels were diminished
throughout the rest of the experiment.
The formation of nitrogenase in a culture supplemented with only
10 mg/1 casein hydrolysate is similar to the unsupplemented culture
(figure 8).
The nitrogenase activity (NH 4+) curve and the maximum
are nearly the same. The nitrogenase activity dropped to low levels
when the culture entered stationary phase.
A very low nitrogenase
activity level was maintained and the NH 4+ concentration continued to
increase slowly.
Cultures supplemented with 100 mg/1 casein hydrolysate show a
higher initial nitrogenase activity (NH4+ formation) with the maximum
of 3,400 nM NH 4+;Hourc/mg protein at 15 hours in the early log phase.
The nitrogenase level continued to decrease steadily until the culture
entered stationary phase at 55 hours.
Slow growth continued to 140
hours, while the specific nitrogenase activity slowly decreased.
Ammonia production which accumulates as a result of nitrogenase
activity, continued albeit at lower rates.
It appears that the
addition of 100 mg/1 casein hydrolysate stimulated growth, eliminated
lengthy lag time and shifted the peak of nitrogenase activity to an
45
earlier time and lower level.
Figure 9 shows the amount of ammonia produced per hour by the
cultures.
Under these conditions NH 4+ production reflects the level
of nitrogenase of the culture. This plot is equivalent to the first
derivative with respect to time of the data in figure 7 and relates
the rate of NH 4+ production at any given moment in the higher concentrations. The effect of supplemental casein hydrolysate is evident
in the increasing NH 4+ concentration at an early time. At about 60
hours the 0 and 100 mg/1 amino acid supplemented cultures increase
ammonia production.
This corresponds to the time of attaining
stationary phase.
In the 1000 mg/1 casein hydrolysate supplemented culture the
initial levels of nitrogenase activity were higher than normally
found in the unsupplemented culture.
The enzyme activity dropped
sharply from the beginning of the experiment and reached extremely
low levels before the culture reached stationary phase.
This culture
excreted NH + earlier and more rapidly than cultures with lower con4
centrations of amino acids. The growth lag and lag in ammonia formation were completely eliminated, and both turbidity and NH 4+ concentration {with one exception) exceeded those found in cultures grown with
lower quantities of amino acids.
Nitrogenase was repressed to a
greater extent in this culture than in those grown with lower quantities of amino acids.
It appears that the added amino acids at first in-
creases the synthesis of nitrogenase but later represses its synthesis.
Nitrogenase activities of other experiments gave similar results
indicating high concentrations of amino acids result in lowered
46
FIGURE 9.
Rate of NH 4+ production in cultures supplemented with casein
hydrolysate. This plot shows the rate at which ammonia was
produced in batch cultures of !· pneumoniae SK-25 grown in
glutamine minimal medium supplemented with various amounts
of casein hydrolysate.
0
0
0 mg/1 casein hydrolysate
e
e
10 mg/1 casein hydrolysate
D
o
100 mg/1 casein hydrolysate
1::::.
1::::.
1000 mg/1 casein hydrolysate
47
100
-- 100
:E
z
'-"'
0:::
:::;::)
0
::c
............
+.::r
:::::r:
z
50
0
30
60
90
TIME CHOURS)
120
48
nitrogenase activities.
In the previous experiments NH 4+ production was assumed to reflect nitrogenase activity. In order to verify that the ammonia
excreted into the medium was actually fixed from N2 , rather than from
some other cellular processes such as the deamination of amino acids,
ammonia production was followed in a batch culture into which argon
was sparged to keep it anaerobic and to exclude nitrogen.
can be seen in figure 10.
The results
The amount of ammonia excreted is small
in comparison to batch cultures run at a different time and sparged
with N2. Argon sparged flasks with only glutamine as the sole
nitrogen source excreted only 0.2 mM NH 4+, and a culture with 1000 mg/1
casein hydrolysate excreted 0.8 mM·NH 4+. Cultures which were grown
under a nitrogen atmosphere produced· 7.25 and 4.0 mM NH 4+ in unsupplemented and supplemented cultures respectively.
Thus, only 2.7% of
the ammonia in the glutamine culture and perhaps as much as 15-20%
in casein hydrolysate supplemented cultures originate from nonnitrogenase sources.
This experiment was repeated with higher concentrations of
glutamine and/or casein hydrolysate.
Batch cultures were set up
containing 100 mg/1 glutamine, 1000 mg/1 glutamine, 100 mg/1 glutamine +
100 mg/1 casein hydrolysate, and 100 mg/1 glutamine + 1000 mg/1 casein
hydrolysate.
In the latter flask with the highest amount of amino
acids was a measurable amount of ammonia excreted (0.37 mM).
It was
noted that flasks which exhibited low levels of NH 4+ in the medium prior
to the start of the experiment (probably from hydrolysis of glutamine)
showed decreases in the ammonia concentration with time indicating that
49
FIGURE 10.
Ammonia production under argon and N2 atmospheres. Batch
cultures of f. pneumoniae SK-25 were grown in glutamine
minimal medium supplemented with casein hydrolysate and
kept anaerobic under a N2 or Ar atmosphere.
•
•
N2; 0 mg/1 casein hydrolysate
A
A
N2; 1000 mg/1 casein hydrolysate
0
0
Ar; 0 mg/1 casein hydrolysate
l::..
/::..
Ar; 1000 mg/1 casei·n hydrolysate
50
7.
6.0
•
5,0
..........
:::E
:E
..._,
4.0
·--~
~
w
1-w
0:::::
u
><
w
+
,.
3.0
-
.;::t"
:::c
" "--
2.0
1.0
-O·--ro
0---+
30
60
90
120
TIME <HOURS)
150
180
51
small quantities of NH 4+ may be utilized.
A differential plot of NH 4+ production as a function of total
protein of the culture (figure 11) shows that the production of NH 4+
is not directly proportional to mass.
Thus, the synthesis of enzyme
(measured in terms of an excreted product) is uncoupled from growth
(i.e., cultures can produce nitrogenase in the absence of growth and
do so near the end of the growth cycle).
A number of factors might be involved in influencing the culture
at the end of growth, such as pH, glutamine, or other nutrient concentrations.
When cultures were started at pH 7.5 acidic waste
products of fermentation accumulated and the pH could drop as low as
pH 6.0.
To determine if pH had any effect on nitrogen fixation, test tube
cultures were inoculated at pH values from 5.0-9.0.
Apparently, pH
variation in this range shows no effect upon the growth rate or final
turbidity (figure 12).
In fact, acidic pH appears to retard nitrogen
fixation (figure 13).
Cultures with starting pH values of 7.0, 8.0 and
9.0 all exhibited similar ammonia production curves with final concentrations of 3.65, 3.75 and 4.2 mM.
Cultures beginning at pH 6 yielded
only 1.25 mM ammonia with a slower rate of formation.
Cultures at pH 5
showed the slowest ammonia formation reaching a concentration of only
0.8 mM.
It appears that the inhibition of nitrogenase is proportional
to the hydrogenion concentration.
In batch cultures where the pH was followed throughout the experiment, the pH did not drop to the inhibitory levels encountered in the
above experiment.
It appeared that the pH of the culture was not
52
FIGURE 11.
Ammonia produced compared to protein concentration. In a
batch culture of f. pneumoniae SK-25 the ammonia concentration present in the medium was plotted against the concentration of protein (based on turbidity values) present in
the culture at the time. This plot eliminates time as a
variable.
53
p
10.0
••..
•
+ .::r
:::::c
z
I I I I I
0 1 ~----~--._--~~~~----~--~I
10
100
PROTEIN <~G/f1L)
'
54
FIGURE 12.
Influence of pH on growth. Batch cultures of f. pneumoniae
SK-25 were grown in glutamine minimal medium starting at
various pH values.
·--·
0--0
pH 6.0
*--tr
pH 7.0
o--o
pH 8.0
.6.-.!:..
pH 9 ..0
pH 5.0
55
0. 01~----J..---;~
20
__.;___..L_ __
TIME CHOURS)
40
.56
FIGURE 13.
Influence of pH on ammonia production.
Batch cultures of
f. pneumoniae SK-25 were grown in glutamine minimal medium
starting at various pH values. The amount of ammonia present
in the medium was measured in the same cultures that appear
in Figure 12.
·--·
o--o
pH 6.0
*:-*:
pH 7.0
o--o
pH 8.0
6.--6.
pH 9.0
pH 5.0
57
5.0
..........
2::
;::1.
...._,
t=l
0
4.0
w
w
1-
e::::
u
><
w
+ .:::r-
3.0
......._
~
z
2.0
1.0
10
20
30
HOURS
40
58
important in the modulation of nitrogenase activity, although because
low pH values could result in lower activities, batch cultures were
usually buffered sufficiently to prevent this possibility.
In experi-
ments where the pH was controlled at i constant value, 7.2 ± 0.3, no
significant differences in the patterns of nitrogenase activity
throughout the experiment were encountered.
In order to determine whether mixtures of glutamine or casamino
acids altered the nitrogenase activity when present in the assay
vessel, experiments were performed where glutamine or casein hydrolysate was added to the assay vessel.
The assay was run for the normal
60 minutes in the presence of glutamine concentrations ranging from 0
to 5000 mg/1 or casein hydrolysate concentrations of 0 to 10,000 mg/1.
Results from these experiments indicate that in most cases nitrogenase
activity is not changed in the 60 minute assay.
To determine if variation in the concentration of glutamine
altered nitrogenase activity, a batch culture experiment was set up in
which !· pneumoniae SK-25 was grown in minimal medium supplemented with
100 and 500 mg/1 glutamine.
incubated at 30°C.
The cultures were sparged with N2 and
Samples of the culture were collected and analyzed
for turbidity, and also nitrogenase activity by the acetylene reduction
method.
The growth of the cultures is shown in figure 14.
The 100 mg/1
culture reached stationary phase at 12 hours while the 500 mg/1 culture
continued growing.
This indicates that nitrogen in the form of gluta-
mine is the limiting nutrient in these experiments.
59
FIGURE 14.
Growth of L-glutamine supplemented cultures. Batch cultures of
pneumoniae SK-25 were grown in minimal medium with 100 and
500 mg/1 glutamine added.
~-
o
100 mg/1 L-glutamine
•
500 mg/1 L-gl utami ne
FIGURE 15.
Nitrogenase activities of L-glutamine supplemented cultures.
The nitrogenase specific activities were measured for the
cultures plotted in figure 14. The nitrogenase values are
given in terms of c2H4 produced/hour/mg protein.
o
100 mg/1 L-glutamine
•
500 mg/l L-glutamine
60
-
0
r-l
w
2:::.:
.__,
1-
C::>
rl
OOOI X AliAilJV
JI~IJJdS 3SVNJ90~1IN
0
N
,-......
U)
n::::
:::::>
0
0
r-i
::c
.........,
w
:2:::
........
1-
61
The nitrogenase activities of these batch cultures are shown in
figure 15.
8 hours.
tn cultures nitrogenase increases in parallel for the first
At 10 hours (5 doublings) the nitrogenase activity reached
a plateau in the 500 mg/1 glutamine culture while nitrogenase in the
100 mg/l glutamine culture cont·i nued to increase.
After 13 hours the
nitrogenase activity in the 500 mg/1 culture began to drop.
This
occurred while the culture was still growing in log phase, although the
growth rate had decreased slightly.
The maximum nitrogenase activity (C 2H4 ) for the 500 mg/1 culture
was 5,200 units while in the 100 mg/1 glutamine culture the activity
was 10,200 and continuing to increase when the experiment was terminated.
This difference in activities suggests that glutamine alone or
its physiological derivatives may repress nitrogenase.
The results
implied that the effect of glutamine is difficult to assess in batch
cultures because it is an essential growth factor and that its concentration is constantly changing.
These results prompted experiments with continuous cultures to
determine if L-glutamine could repress nitrogenase activity independent
of the changes of growth rate, pH turbidity and transient nutrient
concentrations, encountered in batch cultures.
A chemostat is a constant-volume culture vessel designed to grow
cells continuously at constant nutrient levels, cell densities and
growth rates.
The primary advantage of the chemostat in these experi-
ments is that the substrate concentrations (such as glutamine or sulfate) can be closely regulated and kept constant for extended time
periods.
The chemostat used in these experiments is depicted in
62
figure 3.
Chemostat cultures have the advantages of continuously growing
cells at given growth rates under constant environmental conditions
and nutrient supply.
By using glutamine as the limiting nutrient
supplied at known concentrations from the media reservoir, several
steady states were obtained.
It was found that the maximum steady
state turbidity values varied directly with the glutamine concentration.
Figure 17 shows the turbidity of the culture varies linearly
with the glutamine concentration.
The slope is equivalent to the
growth yield (Yx;s), when the turbidity is given in terms of mg
~
protein (X) and the substrate (S) mg/1 glutamine.
conditions Y =XIS= 1.08 mg protein/mg glutamine.
At steady state
This plot .indicates
that glutamine is the limiting nutrient of this media for the range of
concentrations tested.
In the chemostat the growth rate can be set at any physiologically
possible value and kept at that steady state indefinitely, while
maintaining the nutrient concentration at a constant level.
This is
achieved by regulating the flow rate (F) of the medium containing a
limiting concentration of nutrient into the chemostat vessel.
The
dilution rate (D) is the flow rate per unit volume (V) of the chemostat
vessel (D=F/V).
The critical dilution rate (De) is the greatest
dilution rate that can be obtained which does not exceed the maximum
specific growth rate of the bacteria
(~max),
beyond which all of the
cells will eventually wash out of the chemostat (De;
~max).
Unstable conditions can be encountered if the chemostat is operated
near the critical dilution rate.
To avoid this possibility the maximum
63
specific growth rate of Klebsiella pneumoniae SK-25 was determined
Minimal medium with 100
(figure 15).
~g/1
L-glutamine as the limiting
substrate was used and the temperature was 30°C.
The flow rate was
increased in several steps, until the dilution rate (D) exceeded the
critical dilution rate (De), the point at which the dilution rate
exceeds the maximum specific growth rate and wash-out of the cells
occurs.
The wash-out rate was measured by following the decrease in
turbidity.
Figure 17 shows the decrease in protein concentration
(plotted as ln X) over time at the dilution rate of 1.058.
of the line equals the maximum specific growth rate
dilution rate.
The
~max
<~max)
The slope
minus the
value was calculated from the equation
ln X = <~max -D) t + ln X0 , where XQ is the initial biomass cdncentration and t is time given in hours.
found to be 0.798 (volumes/hour).
The critical dilution rate was
By definition, De=
doubling rate of the culture (Td) equals ln
0.868 hours or 52 minutes.
2/~
~max·
The
and the minimum td =
In comparison the maximum specific growth
rate found in batch cultures was 0.55, equivalent to a doubling time
(td) of 1.25 hours.
Batch culture doubling times were often 2.5 hours
and it was not uncommon to find even slower rates.
When the cells were
nitrogen starved before inoculating into minimum media with glutamine,
extremely long doubling times were encountered, often as great as 8 to
16 hours.
The following chemostat experiments used dilution rates well
below De.
With glutamine as the limiting nutrient, high nitrogenase activities were observed.
The maximum activity observed as 7,400 nM c2H4/mg
64
FIGURE 16.
Glutamine concentration compared to steady state turbidities.
Chemostat cultures of f. pneumoniae SK-25 were grown with
various levels of glutamine present in the reservoir. The
maximum turbidity values obtained are plotted for each
glutamine level studied.
65
0.6
,......
0
N
.:::r
0.4
<(
0
••
~
>-
--
1c :l
&::0
0:::
::::::>
1-
0.2
50
L-GLUTAMINE <MG/L)
100
@ '
FIGURE 17.
Determination of the critical dilution rate (Dc). The dilution
rate of a f. pneumoniae SK-25 chemostat culture was increased
in several stages until the dilution rate (D) exceeded the
maximum specific growth rate (~max)· At dilution rates greater
than ~max the culture will wash out and as can be seen in this
graph were lnX (ln mg protein/1) is plotted against time. The
slope of the line equals ~max-D. From this graph the critical
dilution rate which is approximately equal to the maximum
specific growth rate can be calculated.
67
3.6
."'
3.4
·~
__J
.........
::z
........
w
10
0::::
a...
L!:J
:E:
.::1 3.2
10
20
30
40
~liNUTES
50
60
l
68
FIGURE 18.
Characteristics of a L-glutamine limited chemostat culture.
A chemostat culture of K. pneumoniae SK-25 that was limited
with 100 mg/1 L-glutamine and both the nitrogenase activity
(nM CzH4 produced/hr./mg protein) and the turbidity {A4zo)
are plotted separately along the same time axis. The t1me ·
axis is plotted in terms of T/Tr or elapled time in hours
divided by the time of displacing one volume of culture in
the chemostat~ Each integer along this axis is equal to
one volume of the chemostat (in this case 530 ml) replaced
by new media. The total time for the entire experiment was
143 hours.
•
Nitrogenase specific activity
o
Culture density (Turbidity, A4zo)
·•
69
N
.-i
I
/
/
I
J
I
i
•
/
0
II
I
I
I
I
\
\
I
0:::
I...........
I-
0
I
0
•
I
\I
I•
.,&
\••
0
\
• ·-•
00
1.0
-=:!""
N
0
COOOT X) 3S\iN]90(!1IN
-
0
-
0
-
c0Zh\i) AliGIH(!nl
70
protein/hour (figure 18}.
This maximum was obtained just after the
chemostat culture ceased increasing in turbidity.
mained steady between 5,900 and 7,400.
The activities re-
At the end of the experiment
a partly derepressed nitrogenase value of 2,700 was observed.
The
difficulty in these experiments was that glutamine could not be present
in excess, for by theory a steady state continuous culture limited by
a single nutrient, will have a concentration of that limiting nutrient
which is approaching zero, and the small quantities of nutrient entering the chemostat will be utilized instantaneously.
A chemostat experiment was designed to separate the two roles of
glutamine; first, as the limiting nutrient of the culture and second,
as a regulator of nitrogenase activity.
By using sulfate ion as the
limiting nutrient, the culture could be limited at a given population
level, while glutamine would be present in excess.
that the cells limited by
so 4=,
It was hypothesized
and having a greater supply of gluta-
mine than needed would repress the synthesis of nitrogenase.
Concentrations of 0.05 - 0.005 mM
so 4= were
used.
Glutamine con-
centrations were increased in stages throughout the experiment from
values which were limiting to excess.
The nitrogenase levels remained
low <500 throughout the entire experiment (Table II).
In order to establish the effect of excess glutamine under sulfate
limited conditions as well as the effect of the specific growth rate on
the nitrogenase activity, another continuous culture was set up under
more rigorously controlled conditions (figure 19).
experiments Klebsiella pneumoniae SK-25 was used.
As in the previous
Two types of media
were used; the first was the minimal medium with 100 mg/1 glutamine as
71
TABLE II
NITROGENASE ACTIVITIES OF A SULFATE LIMITED CHEMOSTAT
TIME
(HOURS)
93
143
168
234
260
310
337
361
427
430
448
452
457
477
596
625*
696*#
NITROGENASE
SPECIFIC
ACTIVITY
(nM c2H4/hr/
mg Protein)
430
400
280
140
290
250
340
420
380
410
310
290
230
220
380
20
20
TURBIDITY
(A420)
0.088
. 157
.145
.348
.298
.660
.587'
.505
.453
.460
.409
.409
.425
.490
.450
1.368
1. 870
* Reverse Mutation Population Detected
*# Change to N2 atmosphere
GLUTAMINE
CONCENTRATION
(mg/1)
25
SULFATE
CONCENTRATION
(mM)
0.025
II
II
II
II
50
II
II
II
100
II
II
II
II
II
II
.005
II
200
II
II
II
II
1000
II
.050
II
72
FIGURE 19.
The repression of nitrogenase activity in a sulfate limited
chemostat in the presence of excess glutamine. A chemostat
culture of f. pneumoniae SK-25 was grown under both glutamine
limitation and so 4- limitation. Both culture turbidity
and nitrogenase specific activity are plotted separately along
the same time axis. The time axis is plotted in terms of T/T ,
where each integer along the axis is equal to one displacement
of the chemostat culture volume. The advantage of this plot
is that for each unit of T/Tr, roughly one generation of cells
is grown; while most of the cells have washed out in one time
of replacement, a fraction of the original population remains.
A T/Tr value of 4 insures that only a very small fraction of
the original population remains in the chemostat. The nutrient
concentrations and specific growth rate was changed throughout
the course of the experiment and each change is indicated by a
vertical dotted line and a letter, the parameters of which are
as indicated:
•
Nitrogenase specific activity
mg protein)
0
Cell population density (Turbidity, A420)
Period L-glutamine
concentration
mg/1
so 4=
cone.
mM
. A.
B.
1.6
1.6
1.6
1.6
1.6
1.6
0.0025
0.0025
1.6
0.0025
c.
D.
E.
F.
G.
H.
I.
J.
100
100
100
100 .
100
100
100
250
100
250
(nr~
C2H4 produced/hour/
·
Limiting
Substrate
0.048
0.109
0.254
0.492
0.665
0.107
0.107
0.107
0.107
0.107
Glutamine ·
Glutamine
Glutamine
Glutamine
Glutamine
Glutamine
Glutamine
so =
.
G14utam1ne
so 4=
73
0
j
~
... -
- --
..
:c_-
-
-- -
/
0--------
-~ - - -
.
-------
-.
-
-~
-·-·.
!
/
0
- ·-
~ --
}
-
--
/J . i /8----
--·-··-- --,~--~--
I
0
--- . - ·-- ·- - ·- - ·- -- - -- ·- - -e/..::._€>
·-
- - -- - . - .
~~8 - .
- - - . -· -
,j
0~~
~
\
0 /
0
8
I
&
0----~
' - - - - - - - - E)
·- -- ·- ·- - - - - .. - - -- - . - - ~- . ::------...0 .
0
.. . - .. '-.,_ 0
'::!~-- ·- - - -- -- -- - ·- ·- ·- ·- - - --- . -- . ·- ·- -· .. - - - - -- - _./__ - - - -- - - /
-~
o,
a
~~
00
. -· - ·- - - ·- - -- - .. - - - - .. -- - -· - - -- -· :-_ . . --I -- -- -- - --1- -- . - - - .
--------@
f~
<'Iii
u
- - -·.
_,/
/
-------
~~
0
0
\
0
-- - ---- - -- -----_L-
(0001 X)
3SVN390~1IN
AliGiti~nl
0
rl
74
the limiting reagent and second was a sulfate limited medium.
In the
sulfate limited medium, sulfate was provided as Na2S04 at a concentration of 0.0025 mM.
Glutamine in this medium was provided at 100 mg/1
initially which allows a maximum turbidity value of A420 = 0.70 and was
later increased to 250 mg/1.
A low dilution rate (0.048) was used at the start of the experiment.
This is equivalent to a replacement time (Tr) of 20.8 hours.
At Tr=l the nitrogenase specific activity was 5,430 nM c2H4/hour/mg
protein and at Tr=2.7 the nitrogenase activity had increased to 6,930.
These values approach the maximum nitrogenase values observed in the
chemos tat and represents a fully derepressed nitrogenase 1evel.
When the flow rate was increased 2.3 times
to~=
0.109 the
nitrogenase activity dropped sharply to 3,600 and then re-established
at a high value of 7000.
This drop in the nitrogenase activity could
be explained by a sudden increase of glutamine in the medium, which
would tend to repress nitrogenase synthesis.
This phenomenon was seen
several other times in the course of this. experiment.
Each time the
specific growth rate was increased,.the nitrogenase activity decreased,
and subsequently, rose to a high level; but, at specific growth rates
of 0.254 and greater, nitrogenase levels dropped and remained at low
levels without recovery from repression.
When the specific growth rate
was decreased from the high rate of 0.665 to 0.107 the nitrogenase
activity began to increase within one replacement time and had increased to a partly derepressed level of 4,500 within two replacement
times.
Switching from a glutamine limiting medium to a sulfate limiting
75
medium was accomplished by aseptically changing the feedline from one
reservoir to the other.
At Tr=25 when the reservoir was changed, no
change in the nitrogenase activity was noted despite an increase in the
cell population.
given
(~Tr=2.5)
It is probable that within the short amount of time
the sulfate ion was still present in a concentration
which was great enough to make glutamine the limiting reagent.
The
glutamine limited medium had a high so4= limited medium, the high so4=
concentration present in the chemostat vessel was slowly diluted out.
Chemostat theory predicts that nearly 4 replacement times are required
to lower the fraction of original material so it is approaching a
limit of zero.
When the glutamine concentration was then increased to 250 mg/1
and after 2.5 generation times had eJapsed, the nitrogenase activity
dropped to less than 100.
to 70 and then to 46.
The nitrogenase activity decreased slightly
For purposes of interpretation these values
.indicate no detectable nitrogenase activity and are within the range
of the water blanks.
When the reservoir of the chemostat was again changed so that
glutamine again became the limiting nutrient, nitrogenase once more
increased to a moderately high level of 4,600, and finally a change
to so 4= limitation with glutamine in excess once again lowered nitrogenase levels to nearly zero (59 nM c2H4/hr/mg protein).
Thus, when sulfate becomes the limiting nutrient and glutamine
is in excess in the chemostat, the synthesis of nitrogenase is repressed.
In addition, whenever the chemostat parameters are changed
so that there is an excess of glutamine entering the chemostat, de-
76
creases in nitrogenase activity are observed.
The data strongly suggest that nitrogenase activity in Klebsiella
pneumoniae SK-25, can be repressed when an excess of L-glutamine alone
is present in the medium.
CHAPTER IV
DISCUSSION
Ammonia has been well established as a repressor of nitrogenase
biosynthesis in free living nitrogen fixing bacteria.
It became
apparent from genetic experiments that the enzymes of the ammonia
assimilation pathway were involved.
In particular mutants showing
alterations in glutamine synthetase (GS) structure or regulation
continued to produce nitrogenase in the presence of ammonia.
Early research also implicated amino acids in the regulation of
nitrogenase biosynthesis.
High concentrations of amino acids were
found to repress the biosynthesis of nitrogenase.
However, because
amino acids can be deaminated to form free NH 4+ intracellularly, it
was difficult to differentiate the effects of the amino acids from
the possible repressive effects of NH 4+. The derepressed mutants,
which lack glutamine synthetase and no longer show the repression
of nitrogenase biosynthesis by NH 4+ offered a way of differentiating
between the effects of NH 4+ and amino acids.
This prompted another look at the effects of amino acids on the
regulation of nitrogenase.
It was possible that NH 4+ did not repress the biosynthesis of nitrogenase in the mutant bacteria simply
because it could not be assimilated and incorporated into amino
acids and their metabolic products.
In addition, by using a mutant
that did not possess glutamine synthetase, the effects of amino acids
could also be distinguished from the known regulation effects of this
enzyme.
77
78
The data presented in this study make it apparent that GS is
not the only controlling mechanism since nitrogenase is regulated
in the absence of this enzyme.
Since NH + is not utilized by the
4
bacterial strain in question, nor does NH + have any effect directly
4
on the nitrogenase molecule, the regulation of nitrogenase must
also be independent of the influence of NH 4+. Therefore, amino
acids in general, or L-glutamine specifically, may be involved in
the regulation of nitrogenase.
In order to illustrate the possible regulation of nitrogenase
biosynthesis and the biochemical pathway of amino acid assimilation
in the mutant K. pneumoniae SK-25, a second conceptual model is
presented (figure 20).
The three primary enzymes of the ammonia
uptake pathway GS, GOGAT and GDH
ar~
conspicuously absent.
Thus,
the ammonia resulting from the fixation of nitrogen cannot be
assimilated and is then excreted from the cell.
Because the ammonia
cannot be utilized, glutamine auxotrophy results, and a source of
fixed nitrogen in the form of glutamine or a mixture of amino acids
is required for growth.
Glutamate, an intermediate in amino acid
biosynthesis, may be formed by amino transferases or by deaminating
reactions such as those catalyzed by glutaminase A and B which use
glutamine as a substrate and form glutamate and free ammonia.
In this model the major regulatory control for the biosynthesis
of nitrogenase which involves glutamine synthetase is absent.
The hy-
pothesized glutamine synthetase independent regulation of nitrogenase
is schematically represented as being controlled through glutamine
and amino acids.
This proposed influence of L-glutamine as well as
79
FIGURE 20.
Conceptual model of nitrogenase regulation in the
mutant f. pneumoniae SK-25. This model illustrates
the regulation of nitrogenase biosynthesis in the
absence of regulatory control by glutamine synthetase.
The ammonia assimilating enzymes present in non-mutant
bacteria (figure 1) are absent in this mutant resulting
in the excretion of ammonia and glutamine auxotrophy.
Biochemical pathway
----- Enzyme regulation
80
OV)
Zc
~u
<(<(
V)
c
w
w
z
~
<(
1-
z
-+----------!~
<(
~
~
1-
1-
<( ---- <( - - - .
3
C)
3
·C)
u
w
1-
<(
z0
~
3
<(
C)
I
I
I
I
'--------- --,
'
I
+-.:r
I
z
I
I
~~-------------~
$
I
I
1
I
N
<(
'
<(
Z-4)~(-Z
I
u
II
uN
a::::
4--l----------~
E
I
I
u
Ill
u
I
N
z
c
81
amino acids on the activity levels of nitrogenase is supported by
both batch and continuous culture experiments.
It was found that
batch cultures with high concentrations of amino acids had reduced
levels of nitrogenase activity (based on NH 4+ formation). Batch
cultures with glutamine were found to have reduced nitrogenase
activity (based on acetylene reduction), and finally excess glutamine
present in sulfate limited continuous cultures was found to fully
repress nitrogenase activity.
The addition of amino acids to batch cultures affected the rate
and quantity of ammonia formation which reflects the nitrogenase
activity.
High concentrations of a mixture of amino acids resulted
in lower activities of nitrogenase, thus indicating that in the
mutant strain SK-25 a mechanism capable of moderating nitrogenase
activity is present.
The presence of amino acids causes an initial increase in the rate
of ammonia production (figure 9).
The amount of ammonia produced per
hour does not reflect the specific activity of a culture but rather
the product of specific activity and cell number.
Thus, a large
population of cells may produce large amounts of a product while the
actual specific activity or number of enzyme molecules per cell is low.
In the culture supplemented with 1000 mg/1 casein hydrolysate, a
rapid increase in NH 4+ concentration in the medium is observed with a
high cell density and a low nitrogenase specific activity. The
culture supplemented with high concentrations of casein hydrolysate
produces NH 4+ early in the growth cycle but the rate decreases in mid-
82
log phase.
This seems to indicate that although the biosynthesis is
initiated, repression of the enzyme is delayed.
The maximum amount
of NH 4+ produced was the same for both the control culture and the
one supplemented with a high concentration of amino acids, which
indicates that the amino acid supplement does not increase the total
ammonia excreted.
An exception to this occurs when cells are grown
in extremely high concentrations of amino acids which they may use
as substrates for ATP production.
Under these conditions, deamina-
tion occurs and the excess ammonia is excreted into the medium.
The amino acids added to cultures not only influenced ammonia
production but had a dramatic affect on growth.
In general, the
higher the concentration of supplemental amino acids the greater the
growth stimulation and the shorter the lag times.
stimulated growth and increased turbidity.
Casein hydrolysate
The development of nitro-
genase activity in culture may correspond to the initiation of culture
growth.
In several experiments a rapid increase in growth was
accompanied by a rapid acceleration in nitrogenase activity.
However, the nitrogenase activity is not strictly growth linked.
It
is evident from several types of experiments that nitrogenase activity
is not growth linked in the derepressed mutants.
Those enzymes whose
products are directly required for growth are considered to have
growth linked activities.
In the derepressed mutants the ability to
utilize the product of the enzyme has been abolished so that the
nitrogenase has in effect been uncoupled from growth (Shanmugam &
Valentine, 1975).
The batch culture experiments have shown that
the enzyme activity can be repressed by adding exogenous amino acids
83
while cellular growth is increased.
In addition, growth may occur
without the excretion of NH 4+ and cultures may produce ammonia in the
absence of apparent growth, as they frequently do near the end of
the growth cycle. (figure 11).
In batch cultures nitrogenase activity
and ammonia excretion was observed to continue for days after reaching
the stationary phase with no apparent increase in turbidity.
Andersen, et
~.,
(1977) have found that protein synthesis was
required for continued ammonia production because the addition of
protein synthesis inhibitors resulted in unstable conditiDns which
lead to reduction of nitrogenase activity and cell death.
The final quantity of NH 4+ is not necessarily influenced by the
presence of amino acids~ both nitrogenase specific activity and
turbidity of the culture are influenced.
The cultures with very
little or no added casein hydrolysate show the highest nitrogenase
activities (based on nM NH + produced/hour/mg protein). The cultures
4
with the highest amounts of amino acids, although beginning with
moderate nitrogenase activity, decreased continuously throughout the
experiment.
The cultures showed complete repression of nitrogenase
activity before the stationary phase was reached.
These data indicate
that amino acids repress nitrogenase activity in this mutant.
These nitrogenase activities were based on the quantity of
ammonia excreted into the medium.
To be certain that this ammonia
accumulation was actually the result of nitrogen fixation rather
than other physiological processes, batch cultures with a nitrogen
free, inert atmosphere were employed.
The batch culture experiments
84
with an argon atmosphere showed that the ammonia which is excreted
into the medium is the product of nitrogen fixation (figure 10).
Only
small amounts of ammonia were found in argon sparged cultures in
comparison to N2 sparged cultures.
Enzymes such as glutaminase A
and B may be responsible for the small amounts of NH 4+ produced.
Shanmugam and Valentine (1974) utilizing another derepressed mutant
strain, SK-24, showed that no ammonia was excreted into the medium
when cells were cultured under an argon atmosphere, indicating all
of the NH 4+ excreted into the medium when cultures were grown under
N2 was the result of nitrogen fixation.
The nitrogenase specific activity value based upon molar
quantities of C2H2 reduction are much higher (max >10,000) than those
based upon molar NH + production (max 4,500) in the earlier experi4
ments. This is consistent with the fact that the C2H2 reduction to
C2H4 is a 2 electron transfer whereas 6 electrons (minimum) are
required to fix one N2 molecule.
This gives a ratio of 3:1 and in
theory the C2H2 reduction activities should be 3 times higher than
NH4+ values.
According to Burris (1974), experimental values of
nitrogenase rarely if ever follow this ratio.
There is a negative correlation between the concentration of
glutamine and nitrogenase biosynthesis.
It was found that in the
presence of high levels of L-glutamine alone, the nitrogenase biosynthesis was repressed.
Although both the 100 mg/1 control and the
500 mg/1 glutamine culture initially developed nitrogenase in parallel,
the culture containing high levels of glutamine soon ceased the biosynthesis of nitrogenase, while the enzyme activity of the control
85
culture continued to increase (figure 15). This suggests that Lglutamine when present as the only fixed nitrogen source, is capable
of causing the repression of nitrogenase biosynthesis by an alternate
regulatory mechanism which is shown in the mutant regulatory model
(figure 20).
Batch cultures revealed that L-glutamine as well as mixtures of
amino acids can affect nitrogenase specific activity, ammonia production and turbidity.
However, because cultural conditions such as
nutrient concentration and growth rates change with time over the
growth cycle, it was desirable to examine the effects of L-glutamine
under more closely controlled conditions such as those found in continuous cultures.
Chemostat cultures offer unique advantages over batch cultures.
The primary advantage is the ability to maintain constant concentrations of substrates.
This eliminates the transient nature of sub-
strate concentrations and the sigmoid growth cycle in batch cultures.
Continuous culture provides greater control of growth conditions.
Growth rates, population densities and nutrient supplies can be
controlled and kept at steady state conditions.
Continuous cultures
provided a method for the examination of the effect of an amino acid
on nitrogenase activity while other variables are kept constant.
The
chemostat provided a means by which growth rate and cell population
could be controlled by the sulfate ion concentration while the effect
of various concentrations of L-glutamine could be examined.
Chemostat studies revealed that the steady state turbidity of the
culture was directly proportional to the concentration of L-glutamine
86
supplied at a concentration from 0 to greater than 100 mg/1 (figure 16).
This indicates that L-glutamine was the limiting nutrient in the
concentration range examined.
The continuous culture experiments that used L-glutamine as the
limiting mutrient developed high nitrogenase activities.
The highest
value observed in the chemostat was 7,400 nM CzH4 produced/mg protein/
hour (figure 18).
The nitrogenase activity of continuous cultures was
often variable and under glutamine limitation was often maintained at
only partially derepressed levels, while external controllable parameters remained constant.
The fluctuations of enzyme activity indicate
that the regulation of nitrogenase is complex, being influenced by
unknown uncontrolled factors.
The turbidity of the culture was also
observed to fluctuate, presumably due to a complex interaction between nutrient availability, energy metabolism and nitrogenase.
When the dilution rate was suddenly increased the nitrogenase
activity temporarily decreased.
This can be explained by an excess
of glutamine in the culture which may temporarily occur, resulting
in the repression of nitrogenase.
On several occasions (but not always) increasing nitrogenase
activity trends corresponded with decreasing turbid.ity and vice versa.
Tubb & Postgate (1973) have observed the same phenomenon under
sulfate limited conditions in which a fully repressed population had
a 50% greater bacterial density than a fully derepressed population.
In chemostat cultures, the limiting substrate under steady state
conditions is present in concentrations which are approaching zero.
Thus, it is not possible to provide glutamine in excess when it is
87
also the limiting substrate, but by changing to so4= limitation,
glutamine could be provided in quantities greater than could be
utilized.
Nitrogenase activity was found to be completely repressed in
so 4= limited chemostat cultures when glutamine was in excess.
In
one entire chemostat experiment in which so 4= was limiting or colimiting, extremely low nitrogenase activities were observed (Table
II).
At the end of the experiment when a population of reverse
mutant bacteria developed, excess glutamine was added and the nitrogenase activity dropped to zero.
This was not unexpected because
the reverse mutants have restored NH 4+ assimilating enzymes and
pathways. Nitrogenase was shown to be repressed by the presen~e
of excess L-glutamine in another continuous culture.
This culture
was free from revertants and the chemostat was set up in a manner
that the culture could be freely switched from glutamine to sulfate
ion limitation.
When this chemostat culture was switched from
glutamine to sulfate limitation (figure 19) the nitrogenase activity
dropped to zero.
Under the conditions used 100 mg/1 glutamine did
not cause complete repression but 250 mg/1 did.
The concentration
of sulfate ion (0.0025 mM) should have been sufficient to permit
the biomass to reach the turbidity equivalent to that of 100 mg/1
glutamine (if glutamine was limiting).
In this case glutamine would
not have been in great excess at 100 mg/1, but would have been in
excess at concentrations of 250 mg/1.
Upon switching back to glutamine
limitation, nitrogenase activity was derepressed.
The repression and
derepress.ion of nitrogenase could be controlled at will by switching
88
from glutamine limitation to sulfate limitation with glutamine in
excess.
Tubb & Postgate (1973) have shown that aspartate can completely
repress
f.
pneumoniae M5al chemostat cultures under carbon or sulfate
limitation but not under nitrogen limitation.
This is analogous to
the incomplete repression observed under N-limited (glutamine-limited)
cultures but full repression when another nutrient (i.e.,
so 4=)
becomes
limiting.
The chemostat experiments showed that nitrogenase activity can be
completely repressed in continuously growing
of L-glutamine alone.
c~lls,
in the presence
Because the chemostat experiments were con-
ducted under argon, no nitrogen could be fixed and thus no NH 4+ could
accumulate in the medium. f. pneumoniae SK-25, having no GS protein,
GOGAT, or GDH activities and not sensitive to the repression of
nitrogenase biosynthesis by ammonia, yet has some regulation mechanism
whereby nitrogenase activity is controlled and can be influenced by
the presence of L-glutamine.
The results from both batch and chemo-
stat cultures show that nitrogenase was repressed in the presence of
glutamine alone as well as by mixtures of amino acids.
Another regula-
tory mechanism exists and is shown in the mutant regulatory model
·(figure 20).
This regulation seems to be a crude control, and not nearly as
sensitive as the normal regulatory. pathway.
the presence of a 11 regulator molecule 11 •
The model hypothesizes
Glutamine and amino acids
acting as repressors of nitrogenase most probably exert their influence
through a regulator molecule or molecules which are not yet identified,
89
rather than acting on the DNA themselves.
Likely candidates for such
regulators are cyclic AMP or other cyclic nucleotides.
et
~.,
Prusiner
(1972) showed that AMP can affect the activities of the key
ammonia uptake enzymes in E. coli.
Cyclic AMP added to the culture
medium increased the activities of GDH and GS and decreased GOGAT
and glutaminase A.
Cyclic AMP has also been reported to accelerate
the derepression of nitrogenase in Azotobacter vinelandii (Lepo &
Wiss (1974).
Glutamine is a participant in the synthesis of AMP by
the transfer of an amino group.
In addition AMP is known to be an
allosteric inhibitor of glutamine synthetase.
The picture of how nitrogenase is regulated is not yet complete.
Streicher et
~.,
(1974) proposed that GS and its adenylylation states
were the primary regulators.
Ausub~l
mutants of GS in which the nitrogenase
et
~.,
(1977) have found
~osynthesis
is repressible
in the presence of NH +. They proposed that a NH 4+ specific receptor
4
exists that is capable of binding to a promoter region of the nif
operon and blocking transcription.
A unified concept of the regula-
tion must also include a mechanism that prevents the synthesis of
nitrogenase when o2 is present (St. John et ~·, 1974). Active
uptake of molybdenum can influence nitrogenase activity (Brill et
~.,
1974). Other methods of regulation such as the NADPH/NADP+ ratio and
the ATP/ADP ratio have been suggested but their validity has not been
firmly established,
(Ching 1976, Haaker et
~.,
1974).
It is certain
that the nitrogenase requires vast amounts of ATP and low potential
electrons and any interruption in their supply would decrease the
enzymes activity.
Regulator and promotor regions of the nif genes
90
must also be involved.
This study has implicated L-glutamine alone and amino acids in
the regulation of nitrogenase biosynthesis which is independent of
the adenylylation states of glutamine synthetase and repression by
ammonia.
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