SUCCINATE METABOLISM AND TRICARBOXYLIC ACID
CYCLE A C T I V I T Y IN PSEUDOMONAS AERUGINOSA
by
NARAYAN PRASAD TI WAR I
o f J a b a l p u r , 1957
B.V.Sc, University
M.Sc,
Panjab
University,
19&3
A T H E S I S SUBMITTED IN PARTIAL FULFILMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
i n t h e Department
of
Microbiology
We a c c e p t
this
t h e s i s as conforming
required
to the
standard
THE UNIVERSITY OF B R I T I S H COLUMBIA
April,
1969
In p r e s e n t i n g
an
this
thesis
advanced degree at
the
Library
I further
for
shall
the
in p a r t i a l
U n i v e r s i t y of
make i t f r e e l y
agree that
f u l f i l m e n t o f the
permission
British
available
for
for extensive
s c h o l a r l y p u r p o s e s may be g r a n t e d b y t h e
by
his
of
this
written
representatives.
thes.is f o r f i n a n c i a l
gain
permission.
Department o f
Microbiology
The U n i v e r s i t y o f B r i t i s h
V a n c o u v e r 8, Canada
Date
It i s understood
30 A p r i l
1969
Columbia
shall
Columbia,
I agree
r e f e r e n c e and
copying o f this
for
that
Study.
thesis
Head o f my D e p a r t m e n t o r
that
not
requirements
copying o r p u b l i c a t i o n
b e a l l o w e d w i t h o u t my
ii
ABSTRACT
Although
in
the importance o f t r i c a r b o x y l i c a c i d c y c l e
the metabolism o f aerobic
s t u d i e s on t h e u t i l i z a t i o n
to assess
the nature,
bacteria i swell established, detailed
of intermediates
of the cycle
pseudomonads.
l a c k s NAD o r NADP l i n k e d L - m a l ? c d e h y d r o g e n a s e .
ATCC 9 0 2 7
Studies with
acid.
succinate-1,4-
12,
The l a b e l l i n g
patterns
C and s u c c i n a t e - 2 , 3
-
acid to
o f c i t r a t e obtained
other
excluded
possibility.
Phosphofructokinase
extract
from
-|Z|
C have demonstrated t h e
i n v o l v e m e n t o f t h e p a r t i c u l a t e m a l i c dehydrogenase and have
any
cell
h a v e shown t h a t a n NAD a n d NADP i n d e p e n d e n t , p a r t i c u l a t e
dehydrogenase c a t a l y s e s the o x i d a t i o n o f L-malic
oxalacetic
concerned
Results of this
i n v e s t i g a t i o n h a v e shown t h a t Pseudomonas a e r u g i n o s a
L-malic
designed
i m p o r t a n c e a n d c o n t r o l o f t h e enzymes
have n o t been performed w i t h
fractions
activity
preparations
c o u l d n o t be d e t e c t e d
and thus a c c o u n t i n g
Embden-Meyerhof pathway
f o rthe non-functional
i n t h i s organism.
medium e i t h e r do n o t h a v e o r h a v e e x t r e m e l y
metabolizing
enzymes.
The g l u c o s e
c y c l e enzymes was n o t o b s e r v e d .
i n the c e l l - f r e e
C e l l s grown
i n succinate
low l e v e l s o f g l u c o s e
e f f e c t on t r i c a r b o x y l i c
Further,
acid
the a d d i t i o n o f a-keto-
g l u t a r a t e a n d g l u t a m a t e t o t h e medium d i d n o t r e p r e s s
these
enzymes.
These o b s e r v a t i o n s
is of special
The
data
suggest that t r i c a r b o x y l i c a c i d c y c l e
i m p o r t a n c e f o r g r o w t h and
have a l s o
indicated that
medium, p e n t o s e s y n t h e s i s
upon compounds d e r i v e d
I t has
glucose
shift
from s u c c i n a t e
even
presence of
2-deoxyglucose,
the
medium.
i t was
intermediates.
fructose or
glucose
u p t a k e was
and
permease
not
by
GDP.
c l e a r , however, i t i s
maintained
on
growth
in glucose
and
the
the organism t o r e g u l a t e
medium.
obviously
activity.
the
synthesis
These o b s e r v a t i o n s
the
response t o the
particular
environment.
Tricarboxylic
acid cycle
intermediates
" f i n e c o n t r o l " over the
d e h y d r o g e n a s e and
mechanism
s u c c i n a t e media, whereas
in acetate
been found t o e x e r t
adenine
l e v e l s o f m a l i c enzyme w e r e
l e v e l s were low
c a p a c i t y of
was
inhibited
The
i n the c o n t r o l o f t r i c a r b o x y l i c a c i d c y c l e
High
on
a-CH^-glucoside,
inhibited
a c t i v a t e d by GTP
dehydrogenase.
the
mannose.
d e h y d r o g e n a s e was
r e g u l a t i o n i s not
of glutamic
-citrate
transketolase
p e r m e a s e and
A d d i t i o n o f g l u t a m a t e t o t h e medium r e p r e s s e d
in
succinate
a c t i o n of
The
1 0 0 - f o l d . e x c e s s of
galactose,
nucleotides while
in a
induced simultaneously
s p e c i f i c since glucose
Particulate malic
important
growth
i n pseudomonads.
from t r i c a r b o x y l i c a c i d c y c l e
to glucose
very
this
must o c c u r by
enzymes were
f o u n d t o be
of
during
b e e n shown t h a t b o t h t h e g l u c o s e
metabolizing
i n the
metabolism
activity
isocitrate
lyase
synthesis
and
demonstrated
of
enzymes
glyoxylate
activities
the
of
i n s u c h a way
have
iso-
that
flow of
isocitrate
glyoxylate cycle
eel
1.
t h r o u g h the
is precisely
tricarboxylic
regulated
to
acid
suit
c y c l e and
the
needs o f
the
the
V
TABLE OF CONTENTS
Page
INTRODUCTION
LITERATURE
1
REVIEW • .
I. G e n e r a l
3
.
Pattern o f Carbohydrate
Metabolism
3
i n Pseudomonads
II.
Distribution of Tricarboxylic Acid
Enzymes a n d M e t a b o l i s m
Intermediates
of Dicarboxylic Acid
i n Pseudomonads
. . .
I I I . T r i c a r b o x y l i c Acid Cycle A c t i v i t y
of Glucose Oxidation
Containing
Cycle
Cycle
.• .
.
£
.• .
8
and Enzymes
i n C e l l s Grown on M e d i a
Glucose or a T r i c a r b o x y l i c
Intermediate
.
Acid
.
IV. C o n t r o l o f T r i c a r b o x y l i c A c i d C y c l e and
Glyoxylate Cycle A c t i v i t y
V. M o l a r
Growth Y i e l d
Metabolic
VI. Succinate
i n Microorganisms.
as a Tool
Pathways . . .
Utilizations
.
.
.
.
.
as D i s t i n c t f r o m
o f the T r i c a r b o x y l i c
Cycle
.
VI I . M e t a b o l i c
.MATERIALS AND
.
.
.
.
.
11
.
.
16
i n the Study o f
Dicarboxylic Acids
.
.
.• .
.
Other
Acid
.
.
.• .
P a t t e r n and E v o l u t i o n .
.
.
.
.
.
. . .
.
.
17
.
18
METHODS
I . O r g a n i s m s and G r o w t h M e d i a
.
.
.
.
.
.
23
vi
Table
of
Contents
(Continued)
Page
II.
Preparation
Cell-Free
III.
of
Extracts
Determination
IV.
V.
VI .
VII.
VIII.
Resting
of
Substrates
Isolation
of
Manometric
Enzyme A s s a y s .
Thin
Layer
Uptake
of
X.
Analytical
XI .
Chemicals
RESULTS AND
I.
IV.
Growth
.
.
Yield
. •
.
. . . .
24
on
of
P.
25
a e r u g i hosa
27
.
27
.
Reaction
Products
Chromatography,
.
.
.
.
.
.
.
Labelled
31
Autoradiography
31
Measurements.
.
32
.
33
.
.
34
Pseudomonas a e r u g i n o s a
.
35
Substrates
Methods
.
Oxidation
Growth
of
Tricarboxylic
Intermediates
Yields
of
o x y l i c Acid Cycle
III.
.
.
. . .
.
DISCUSSION
Related
I I.
.
S u s p e n s i o n s and
25
Mutants
Methods
Analysis of
.
Molar
Different
and R a d i o a c t i v e
IX.
.
Cell
Studies
with
Lack of
NAD o r
Dehydrogenase
P.
by
Acid Cycle
a e r u g i nosa w i t h
Some
Tricarb-
Intermediates.
Mutants
P.
41
.
NADP D e p e n d e n t
in
and
aeruginOsa
44
L-Malic
44
Table o f Contents
(Continued)
Page
V.
D e t e c t i o n and
VI. Labelling
S i t e o f Ma l a t e O x i d i z i n g A c t i v i t y .
of C i t r a t e
from Succinate-1,4-
14
.
.
45
.
52
C
14
and
Succinate-2,3
-
C
V I I . O x i d a t i o n o f S u c c i n a t e , F u m a r a t e and
by a C e l l - F r e e
V I I I . The
Extract
o f A.
aerogenes.
i n S u c c i n a t e or Glucose Media
1. G l u c o s e d e g r a d i n g enzymes
2. T r i c a r b o x y l i c
acid
of Malic
G 1 u c o s e o r A c e t a t e Med i a
r e l a t e d enzymes.
activity
Enzyme i n C e l l s
.
59
XI. Control
Harvested
.
.
.
.
.
.
61
.
68
.•
.
76
.•
.
.
76
. . . . .
79
.
. . . .
c y c l e and
80
Utilization
activity
of the flow of i s o c i t r a t e
tricarboxylic acid
.
.
Cycle A c t i v i t y .
1. P a r t i c u l a t e m a l i c d e h y d r o g e n a s e
Control
.
f r o m S u c c i n a t e Medium .
of Tricarboxylic Acid
.
Grown i n S u c c i n a t e ,
I n d u c t i o n o f t h e Enzymes o f G l u c o s e
in C e l l s
.
. . . .
cycle activity
b. G l u t a m i c d e h y d r o g e n a s e
|iX. L e v e l
. . . .
c y c l e and
a. T r i c a r b o x y l i c a c i d
2.
. . . . .
Enzymes o f C a r b o h y d r a t e M e t a b o l i s m - i n - P _ .
a e r u g i n o s a Grown
X.
Malate
.
. . .
83
. . .
89
. . .
89
v i a the
glyoxylate cycle
i n P_. a e r u g i h o s a . • . .
.
.• .
.
a. I s o c i t r a t e d e h y d r o g e n a s e a c t i v i t y
,
.
.
.
.
. . . . .
.
94
97
VI I I
Table o f Contents
(Continued)
Page
b.
Isocitrate
lyase a c t i v i t y
c. A c o n i t a s e a c t i v i t y
GENERAL DISCUSSION
BIBLIOGRAPHY
.
. . . .
.- .
101
.• .
.• .
.• .
.
.
105
108
. . .
.
.
.
.
. . .
.
117
ix
L I S T OF
TABLES
Page
Table
Table
I
II.
Table I I I .
Table
Table
Table
IV.
V.
VI.
Accumulation of ketp acids during the
o x i d a t i o n o f s u c c i n a t e , f u m a r a t e and
ma l a t e i n t h e p r e s e n c e o f 1 mM s o d i u m
arsenite
40
G r o w t h y i e l d o f P_. a e r u g i n o s a
v a r i o u s carbon sources
43
from
P a r t i c u l a t e m a l i c d e h y d r o g e n a s e and
m a l i c enzyme i h P_. a e r u g i n o s a
48
E f f e c t o f EDTA and r i b o n u c l e a s e t r e a t m e n t
on t h e d i s t r i b u t i o n o f p a r t i c u l a t e m a l i c
d e h y d r o g e n a s e o f P_. a e r u g i n o s a
51
The t r i c a r b o x y l i c a c i d c y c l e a n d
e n z y m e s o f P_. a e r u g ? n o s a M32
55
related
Specific activity of radioactive citrate
formed from
succinate-1,4-^and
succinate-2,3- ^C
57
A c t i v i t y o f some e n z y m e s o f c a r b o h y d r a t e
m e t a b o l i s m i n P_. a e r u g i n o s a ' grown i n
s u c c i n a t e o r g l u c o s e media
69
Glucose-6-phosphate forming a c t i v i t y from
r i b o s e - 5 - p h o s p h a t e and r i b o s e p h o s p h a t e
isomerase act i v i t y
72
S p e c i f i c a c t i v i t i e s of the t r i c a r b o x y l i c
a c i d c y c l e and r e l a t e d e n z y m e s i n P_.
a e r u g i n o s a grown w i t h d i f f e r e n t c a r b o n
sources
78
E f f e c t o f c a r b o n s o u r c e s on t h e s p e c i f i c
a c t i v i t i e s o f m a l i c enzyme a n d i s o c i t r a t e
l y a s e i ri P_. a e r u g i n o s a
82
I n d u c t i o n o f some enzymes o f g l u c o s e
o x i d a t i o n by g l u c o s e
84
1
Table
Table
Table
Table
Table
VII.
VIII.
IX.
X,
XI
X
L i s t o f Tables
(Continued)
Page
Table
X I I . E f f e c t o f n u c l e o t i d e p h o s p h a t e s on t h e
p a r t i c u l a t e m a l i c dehydrogenase i n
Table X I I I .
Table
Table
Table
XIV.
XV.
XVI.
105,000 x £ p e l l e t
92
Partial p u r i f i c a t i o n of i s o c i t r a t e
d e h y d r o g e n a s e and i s o c i t r a t e l y a s e from
c e l l e x t r a c t s o f P. a e r u g i n o s a
96
I n h i b i t i o n o f i s o c i t r a t e dehydrogenase
a c t i v i t y by v a r i o u s o r g a n i c a c i d s and
r e l a t e d compounds
98
Inhibition
by o r g a n i c
o f i s o c i t r a t e lyase a c t i v i t y
a c i d s and r e l a t e d compounds
Aconitase inhibition i n c e l l - f r e e
e x t r a c t o f P. a e r u g i riosa
102
106
xi
L I S T OF
FIGURES
Page
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
1.
2.
3.
4.
5.
6.
7.
8a.
O x i d a t i o n o f s u c c i n a t e , f u m a r a t e and m a l a t e
e e l 1 s u s p e n s i o n o f P. a e r u g i n o s a
by
O x i d a t i o n o f s u c c i n a t e , f u m a r a t e and m a l a t e
c e l l - f r e e e x t r a c t o f P. a e r u g i n o s a
by
O x i d a t i o n o f a c e t a t e and p y r u v a t e by c e l l s
c e l l - f r e e e x t r a c t o f P. a e r u g i n o s a
49
Autoradiogram o f the t h i n - l a y e r chromatograph
from the r e a c t i o n mixture c o n t a i n i n g c e l l - f r e e
e x t r a c t o f P. a e r u g i n o s a w i I d t y p e a n d r a d i o active succinate
53
Autoradiogram o f the t h i n - l a y e r chromatograph
from the r e a c t i o n m i x t u r e c o n t a i n i n g c e l l - f r e e
e x t r a c t o f P. a e r u g ? h o s a M32 and r a d i o a c t i v e
succinate
54
14
Citrate
f o r m a t i o n from succinate-1,4-
C.
58'
C
58
14
Fig.
9.
O x i d a t i o n o f s u c c i n a t e , f u m a r a t e and m a l a t e
c e l l - f r e e e x t r a c t o f A. a e r o g e n e s
11.
38
O x i d a t i o n o f L - m a l i c a c i d by c e l l - f r e e e x t r a c t
and 100,000 x g_ p e l l e t o f P. a e r u g i n o s a
C i t r a t e f o r m a t i o n from s u c c i n a t e - 2 , 3
Fig.
and
39
8b.
10.
37
O x i d a t i o n o f s u c c i n a t e , f u m a r a t e and m a l a t e by
r e s t i n g c e l l s u s p e n s i o n o f P_. a e r u g i n o s a i n
t h e p r e s e n c e o f 1 mM s o d i u m a r s e n i t e
Fig.
Fig.
36
-
by
60
G r o w t h o f s u c c i n a t e grown i n o c u l u m i n t h e m e d i a
containing succinate, glucose or succinate plus
glucose
63
G r o w t h o f g l u c o s e grown i n o c u l u m i n m e d i a
containing glucose, succinate or glucose plus
succinate
64
XI
List
o f Figures
(Continued)
Page
Fig.
12.
F i g . .13.
Fig.
Fig.
Fig.
Fig.
14.
15.
16.
17.
Oxidation of succinate, glucose o r a mixture of
s u c c i n a t e and g l u c o s e by e e l 1s h a r v e s t e d f r o m
a s u c c i n a t e m i n i m a l medium
65
Oxidation of succinate, glucose o r a mixture
o f s u c c i n a t e and g l u c o s e by c e l l s h a r v e s t e d
f r o m a g l u c o s e medium
66
O x i d a t i o n o f g l u c o s e a n d s u c c i n a t e by t h e c e l l f r e e e x t r a c t s from t h e c e l l s grown i n g l u c o s e
o r s u c c i n a t e media
67
G l u c o s e - 6 - p h o s p h a t e f o r m a t i o n from r i b o s e - 5 p h o s p h a t e by P_. a e r u g i n o s a g r o w n i n s u c c i n a t e
or g l u c o s e media
73
Increase i n the level of 3 phosphoglyceraldehyde d e h y d r o g e n a s e and
glucose-6-phosphate
f o r m i n g a c t i v i t y f r o m r i b o s e - 5 - p h o s p h a t e , on
s h i f t f r o m s u c c i n a t e t o g l u c o s e medium
74
U p t a k e o f r a d i o a c t i v i t y by g l u c o s e grown e e l I s
o f P.. a e r u g i n o s a w i t h 1 0 " 5 M g l u c o s e - U - C and
w i t h 10"i> M a - m e t h y l - g l u c o p y r a n o s i d e
86
I n c o r p o r a t i o n o f g l u c o s e - U - ^ C i n the presence
o r a b s e n c e o f o t h e r s u g a r s by t h e w h o l e c e l l s
o f P. a e r u g i n o s a h a r v e s t e d f r o m t h e g l u c o s e
m i n i m a l medium
87
-
1 4
Fig.
Fig.
Fig.
Fig.
18.
19.
20.
21.
G l u c o s e - U - C i n c o r p o r a t i o n by t h e w h o l e c e l l s
o f P. a e r u g i n o s a w i I d t y p e and i t s m u t a n t
s t r a i n M5, h a r v e s t e d f r o m s u c c i n a t e m i n i m a l
med i um
88
Increase i n the level o f p a r t i c u l a t e malic
d e h y d r o g e n a s e a c t i v i t y on s h i f t t o g l u c o s e
med i um
90
Lineweaver-Burk p l o t f o r p a r t i c u l a t e m a l i c
dehydrogenase i n the absence o r presence o f
1 mM ATP
93
1 / |
XI
List
Fig.
Fig.
Fig.
Fig.
22.
23.
24.
25.
of
Figures
E f f e c t of a mixture of
c y c l e i n t e r m e d i a t e s on
activity
(Continued)
tricarboxylic acid
i s o c i t r a t e dehydrogenase
Page
99
Lineweaver-Burk p l o t f o r i s o c i t r a t e
d e h y d r o g e n a s e o f P_. a e r u g i n o s a
100
E f f e c t of a mixture of
c y c l e i n t e r m e d i a t e s on
activity
103
Lineweaver-Burk p l o t
o f P. a e r u g i n o s a
tricarboxylic acid
i s o c i t r a t e lyase
for
isocitrate
lyase
104
I I
ACKNOWLEDGEMENTS
I would
J.J.R.
like
Campbell
critcism
during
t o e x p r e s s my s i n c e r e g r a t i t u d e
for his interest,
the course o f t h i s
supervision
t o Dr.
and c o n s t r u c t i v e
s t u d y and w r i t i n g
of the
thesis.
I would
helpful
also
like
t o thank
f o r her
s u g g e s t i o n s and c r i t i c i s m .
My s i n c e r e a p p r e c i a t i o n
for her forbearance during
typing
Dr. A.F. Gronlund
a part
i s extended
Kamla
t h e c o u r s e o f t h i s work and f o r
of the thesis.
Rosbergen f o r t h e f i n a l
t o my w i f e
typing
I a l s o w i s h t o thank Mrs.
of this
manuscript.
Rita
INTRODUCTION
Pseudomonas a e r u g i h o s a d o e s n o t p o s s e s s a
Embden-Meyerhof pathway
(Campbel1
and N o r r i s ,
Wang and G i l m o u r , 1960), and when o r g a n i s m s
tricarboxylic
acid
cycle
the o x i d a t i v e p o r t i o n o f
T i g e r s t r o m and
on
pentose
( H a m i l t o n and
C a m p b e l l , 1966b).
t h a t when 6 - p h o s p h o g l u c o n a t e
proposed
Stern,
a r e grown
p a t h w a y a r e a b s e n t o r e x t r e m e l y low
Dawes, I960; Von
1950;
i n t e r m e d i a t e s , t h e enzymes o f t h e
E n t n e r - D o u d o r o f f p a t h w a y and
phosphate
functional
dehydrogenase
I t has
been
activity
i s a b s e n t , t h i s o r g a n i s m s y n t h e s i z e s p e n t o s e by t h e c o n d e n s a t i o n
of
and
and
C^ f r a g m e n t s
(Wang, S t e r n and G i l m o u r , 1959;
N e i d h a r d t , 1967a), w h i c h c o u l d be d e r i v e d
acid cycle
intermediates.
t r i c a r b o x y l i c acid
Thus, under
from
Lessie
tricarboxylic
these c o n d i t i o n s ,
c y c l e m e e t s t h e m a j o r m e t a b o l i c and
biosynthetic
demands i n P. a e r u g i h o s a .
H o w e v e r , o n l y a weak s u c c i n i c
activity
i n t h i s organism
has b e e n r e p o r t e d
S t r a s d i n e , 1962;
cell
Von T i g e r s t r o m and
s u s p e n s i o n s s u c c i n a t e was
compound.
dehydrogenase
( C a m p b e l l , Hogg
and
C a m p b e l l , 1966b), a l t h o u g h i n
utilized
at a rapid'rate
t o o t h e r d i c a r b o x y l i c a c i d s s u g g e s t i n g a u n i q u e mode o f
of this
the
comparable
utilization
S u c c i n a t e has a l s o been f o u n d t o r e p r e s s t h e
i n d u c t i o n o f c e r t a i n d e g r a d a t i v e enzymes
( L e s s i e and N e i d h a r d t ,
2
1967b;
1969).
R o s e n f e l d and F e i g e l s o n ,
The u t i l i z a t i o n
o f succinate
a t t e m p t h a s b e e n made t o c o r r e l a t e
utilization
of cycle
extensive studies
activity
W a r m s l e y , 1968)
and
I t was t h e o b j e c t
acid
ATCC 9 0 2 7 .
special
(Hanson e t a l , 1964;
i s known a b o u t t h e r e g u l a t i o n
i n pseudomonads.
investigation
t o s t u d y t h e mode o f
reference to.succinate
i n P. a e r u g i n o s a
i n succinate
and t h e E n t n e r - D o u d o r o f f pathway i n
and g l u c o s e m e d i a h a s b e e n c o m p a r e d i n
o r d e r t o a s s e s s t h e r e l a t i v e r o l e o f these pathways
metabolism.
cycle
cycle
The l e v e l o f t h e enzymes o f t h e t r i c a r b o x y l i c a c i d
grown
mechanism
been
a c i d members o f t h e t r i c a r b o x y l i c
c y c l e , pentose phosphate c y c l e
cells
of tricarboxylic acid
little
of this
of dicarboxylic
cycle with
W h i l e t h e r e have
and i n B a c i 1 l u s s p e c i e s
the a c t i v i t y o f t h i s cycle
utilization
o f t h e enzymes t o
( G r a y , Wimpenny a n d M o s s m a n , 1 9 6 6 b ; Wimpenny
Hanson a n d Cox, 1 9 6 7 ) , v e r y
of
the a c t i v i t y
intermediates.
on r e g u l a t i o n
in coliforms
cycle
i n d e t a i 1 i i i P. a e r u g i n o s a a n d n o : ,
members h a s n o t b e e n s t u d i e d
the
and o t h e r t r i c a r b o x y l i c a c i d
Further,
a n a t t e m p t h a s b e e n made t o e l u c i d a t e t h e
c o n t r o l 1ing t r i c a r b o x y l i c a c i d
activities
i n succinate
in this
organism.
cycle
and g l y o x y J a t e
LITERATURE REVIEW
I .
General
Pattern of
Although
a f a c t which
of
our
this
Bergey's
is
genus,
only
has
largely
fluorescens
of
the pathways
of
come f r o m s t u d i e s
h a s been e s t a b l i s h e d
i n the
that
the
utilization
respectively
via
the p e n t o s e - p h o s p h a t e
The
Entner-Doudoroff
degradative
and G i l m o u r ,
to
pathway
1959).
capabilities
of
for
particular
this
Pseudomonas a e r u g ? n o s a ,
putida
of glucose
P.
metabolic studies
(Stanier
in
has
1966).
et a l ,
pathway
plays
an
pseudomonads.
glucose exclusively
via
this
r e p t i 1 i v o r a m e t a b o l i z e 7 1 % and
this
(Stern,
i n P.
pathway and t h e
Wang and G i l m o u r ,
fluorescens
(Wang,
remainder
I960).
major
Stern
t h e Pseudomonas s p e c i e s h a v e been
l a c k a complete Embden-Meyerhof
Pseudomonas
f1uorescens
has a l s o b e e n shown t o be t h e
glucose
Most o f
P.
In
metabolism of
S t r a i n A3.12 of
via
pathway
pathway
in d e t a i l .
Entner-Doudoroff
P_. a e r u g ? n o s a and P.
72% o f a d d e d g l u c o s e
range o f m e t a b o l i c
in exhaustive
Pseudomonas s a c c h a r o p h ? 1 a d e g r a d e s
scheme w h i l e
Pseudomonas,
carbohydrate
a n d Pseudomonas p u t i d a .
role
149 s p e c i e s o f
a few h a v e b e e n s t u d i e d
now b e e n c l a s s i f i e d a s a s t r a i n o f
important
1ists
the wide
u s e d by W . A . Wood and c o w o r k e r s
It
M e t a b o l i s m i t i Pseudomonads
(1957)
manual
indicative
knowledge of
genus
Carbohydrate
scheme.
The
first
found
indication
4
of
t h i s was p r o v i d e d by a s t u d y o f t h e d i s t r i b u t i o n
carbohydrates
that
and
this
i n P. a e r u g i h o s a , on t h e b a s i s o f w h i c h
organism
1950).
Norris,
lacked
Wood a n d S c h w e r d t
(1954)
reported
and
DeMoss ( 1 9 6 2 ) .
a l (i960)
i n Pseudomonas
(Campbell
f u r t h e r showed
that
i n P. f l u o r e s c e n s a n d t h u s t h e
E m b d e n - M e y e r h o f p a t h w a y was n o t o p e r a t i o n a l .
was
i t was c o n c l u d e d
t h e Embden-Meyerhof pathway
p h o s p h o f r u c t o k i n a s e was a b s e n t
et
o f phosphorylated
1ihdrier?
A similar
observation
(Zymomonas m b b i l i s ) by Raps
Using radiorespirometric experiments, Stern
showed t h e a b s e n c e
o f t h e Embden-Meyerhof pathway i n
t h r e e s p e c i e s o f pseudomonads.
P r o d u c t i o n o f g l u c o n a t e and 2 - k e t o g l u e o n a t e from g 1 u c o s e has
been o b s e r v e d
i n pseudomonads by many w o r k e r s
the
significance of this
and
Campbell
(1949)
observation
first
(DeLey,.1960).
became c l e a r when N o r r i s
f o u n d t h a t P_. a e r u g i n o s a o x i d i z e d
glucose to
c a r b o n d i o x i d e and w a t e r v i a g l u c o n a t e a n d 2 - k e t o g l u c o n a t e .
discovery
together with
the finding
However,
that t h i s organism
This
lacks the
Embden-Meyerhof pathway e n a b l e d t h e a u t h o r s t o d e s c r i b e t h e o p e r a t i o n
of
a new p a t h w a y , t h e non-phosphory,l;ated p a t h w a y o f g l u c o s e
degradation.
T h i s has s i n c e been found
and Wood, 1 9 5 6 ;
Frampton
i n P. f l u o r e s c e n s
a n d Wood, 1 9 6 1 ) .
G1uconokinase
g l u c o n o k i n a s e h a v e been d e m o n s t r a t e d a n d t h e p r o d u c t s ,
and
2-keto-6-phosphogluconate
converting
have been
2-keto-6-phosphogluconate
demonstrated.
I t h a s been p r o p o s e d
identified.
(Narrod
and 2-keto-
6-phosphogluconate
A reductase
t o 6-phosphogluconate
further
that
has been
6-phosphogluconate
5
and
2-keto-6-phosphogluconate
Entner-Doudoroff
pathway.
are metabolized to pyruvate v i a the
2-keto-
E v i d e n c e has been p r e s e n t e d t h a t
g l u c o n a t e f o l lows a s i m i l a r m e t a b o l i c p a t t e r n i n P. a e r u g i r i o s a .
1965).
(Kay,
The
e a r l i e s t attempts
t r i c a r b o x y l i c acid
cycle
s u s p e n s i o n s were used
apparently
t o demonstrate
i n microorganisms
the presence o f a
met w i t h f a i l u r e .
i n t h e enzyme s t u d i e s and t h e s e
lacked the a b i l i t y
to oxidize c i t r i c
fa?led
tricarboxylic
and
t o o x i d i z e a number o f t h e members o f t h e
dried
o f t h e same p r e p a r a t i o n o x i d i z e d a l l o f t h e i n t e r m e d i a t e s
They c o n c l u d e d
a e r u g i n o s a was i n i t i a l l y
"Apparently the c e l l
t h e s u b s t r a t e s a c r o s s t h e membrane".
and
w i t h r e s t i n g c e l l s was t h e
time necessary f o r the e l a b o r a t i o n o f the system
Kallio
membrane o f
i m p e r m e a b l e t o t h e compounds m e n t i o n e d ,
the period o f adaptation encountered
f o r transferring
Subsequently, B a r r e t t ,
Larson
(1953) u s i n g P, f l u o r e s c e n s showed t h a t d u r i n g p r o l o n g e d
lags before the o x i d a t i o n o f c e r t a i n
protein essential
cell
Campbell
acid cycle without a prior period of induction,
without a lag.
and
suspensions
(1951) o b s e r v e d t h a t a l t h o u g h a e e l 1 s u s p e n s i o n o f P_.
a e r u g i hbsa
cells
Cell
a c i d o r some o f t h e
dicarboxylic acids without a period of induction.
Stokes
complete
t r i c a r b o x y l i c a c i d c y c l e members,
t o t h e t r a n s p o r t o f t h e s e compounds a c r o s s t h e
membrane was b e i n g s y n t h e s i z e d .
F o r m a t i o n o f g l y o x y l a t e and s u c c i n a t e from c i t r a t e o r c i s -
a c o n i t a t e was d e s c r i b e d by C a m p b e l l ,
Smith and E a g l e s
(1953) i n
6
P_. a e r u g i n o s a .
Discovery of this
reaction
together with
s y n t h a s e by Wong a n d A j 1 ( 1 9 5 ° ) l e d t o t h e f o r m u l a t i o n
steps o f glyoxylate
cycle
f o r growth on a c e t a t e
I t has been f o u n d
level
cycle directly
"glycerate
glyoxylate,
give
i n pseudomonads a n d v a r i o u s
cannot enter
The
pathway"
derived
( K o r n b e r g , 1961)
the
from t h e o x i d a t i o n
Dicarboxylic Acid
o f g l y c o l l a t e , condense t o
in bacteria
Enzymes and M e t a b o l i s m
i n Pseudomonads
established.
cycle
i n aerobic
In a n e x c e l l e n t
Using d i f f e r e n t experimental
presented evidence against
as
review
o f d i f f e r e n t steps o f t r i c a r b o x y l i c acid c y c l e i n
mammalian t i s s u e a n d m i c r o o r g a n i s m s h a s b e e n d e s c r i b e d
1961).
semi-
t h e Embden-Meyerhof scheme.
Ihtermedi ates
i swell
Tartronic
i s phosphorylated to
importance o f the t r i c a r b o x y l i c a c i d
formulation
oxidation
i n w h i c h two m o l e c u l e s o f
i s reduced t o g l y c e r i c a c i d which
metabolism
organisms.
and i s m e t a b o l i z e d v i a a n o t h e r scheme named t h e
D i s t r i b u t i o n o f T r i c a r b o x y l i c Ac?d Cycle
Of
i sessential
the t r i c a r b o x y l i c acid or glyoxylate
p h o s p h o g l y c e r i c a c i d and f u r t h e r enters
I I.
other
i s at a higher
t a r t r o n i c s e m i a l d e h y d e and c a r b o n d i o x i d e .
aldehyde
o f malate
of different
( K o r n b e r g a n d K r e b s , 1957) w h i c h
that g l y c o l l a t e , which
than a c e t a t e ,
that
Kogut and P o d o s k i
approaches, Krampitz
the operation
p r o p o s e d e a r l i e r by Thunberg
(1953)
(1920)
studied
(Krampitz,
also
of dicarboxylic acid
a n d by A j 1 ( 1 9 5 0 ;
the oxidation
of
cycle
1951).
succinate
7
by w h o l e c e l l s
On t h e g r o w t h medium r i c h
i n s u c c i n a t e a n d l o w i n ammonia, a c c u m u l a t i o n
o f a - k e t o g l u t a r a t e was o b s e r v e d .
possess
was
the tricarboxylic
proposed
acid
acid
C e l l - f r e e e x t r a c t s w e r e shown t o
a c i d c y c l e enzymes.
and Smith
c y c l e enzymes
(1956)
showed t h e p r e s e n c e o f t r i c a r b o x y l i c
C a m p b e l l , Hogg.and
d i s t r i b u t i o n o f many i m p o r t a n t enzymes
in this
Strasdine
organism
(1962)
in different
P_. a e r u g i n o s a a n d f o u n d t h a t s u c c i n i c d e h y d r o g e n a s e
the cell
membrane w h e r e a s f u m a r a s e ,
isocitrate
and
i s o c i t r a t a s e were i n t h e s o l u b l e c y t o p l a s m .
and
Gunsalus
P.
(1953)
reported that
they d i d not attempt
aba
that
and
(Campbell,
studied the
was a s s o c i a t e d
dehydrogenase
Stanier,
Gunsalus
activity in
fraction.
However,
t o e l u c i d a t e t h e n a t u r e and l o c a t i o n o f t h i s
While s t u d y i n g o x i d a t i o n o f L-malate
i r i Pseudomonas B?
a n d Pseudomonas o v a 1 i s C h e s t e r , F r a n c i s e_t a j _ (1963)
the former organism
i t sc e l l - f r e e extract
possessed
uti1ized
incompletely while extracts
this
substrate
earlier
fractions of
the malate o x i d i z i n g
f l u o r e s c e n s was p r e s e n t i n t h e p a r t i c u l a t e
enzyme.
possibilities.
i n P_. a e r u g i n o s a , t h u s c o n f i r m i n g t h e i r
Stokes, 1951).
with
study i t
v i a the t r i c a r b o x y l i c
not e l i m i n a t e other
observation o f the presence o f t h e c y c l e
and
From t h i s
t h a t s u c c i n a t e was b e i n g o x i d i z e d
c y c l e , although they could
Campbell
o f P_. f l u o r e s c e n s .
and c e l l - f r e e e x t r a c t s o f a s t r a i n
soluble
malate
L-malic
dehydrogenase
v e r y s l o w l y and
from t h e l a t t e r organism
r a p i d l y and c o m p l e t e l y .
found
oxidized
This investigation
t h a t P. o v a l i s C h e s t e r p o s s e s s e s an NAD, NADP
independent
showed
8
p a r t i c u l a t e L-malic
III.
dehydrogenase.
T r i c a r b o x y l i c Acid
Oxi d a t i o n
C y c l e A c t i v i t y and
i h C e l 1 s Grown oh
a T r i c a r b o x y l i c Acid
The
to
inhibit
the
r e c o g n i z e d by
of
the
synthesis
Epp
and
of
Gale
as
the
that
factor
the
glucose effect
as
the
d e a m i n a s e s by
lowering of
is exerted
by
the
pH
1961).
found t h a t
when g r o w n on
g l u c o s e , a c e t a t e and
tricarboxylic acid
nutrient
b r o t h S.
intermediates of
was
t r i c a r b o x y l i c acid
correlated
extracts
Strasters
of
with
the
and
a
low
organism.
Winkler
glucose
cycle
the
inhibition
medium
been
cycle
suggested
formed
from
enzymes
Lascelles
aureus,
has
(1962)
oxidized
tricarboxylic acid
cycle.
organism f a i l e d
i n t e r m e d i a t e s and
activity
first
repression
C o l l i n s and
H o w e v e r , when g r o w n i n a g l u c o s e medium, t h e
oxidize
or
i i i E.' c o l i ;
catabolites
(Magasanik,
in various bacteria.
of
I t has
hence i t i s s i m i l a r t o c a t a b o l i t e
been r e c o g n i z e d
the
gl ucose
the
for this effect,
g l u c o s e e f f e c t on
a b i 1 i t y of
(1942) when t h e y o b s e r v e d
g l u c o s e and
The
G1ucose
c e r t a i n d e g r a d a t i v e enzymes was
the
responsible
Glucose
Intermediate
is defined
f o r m a t i o n o f amino a c i d
These workers e l i m i n a t e d
Med i a C o n t a ? h i ng
Cycle
glucose e f f e c t which
Enzymes o f
o f enzymes o f
this
to
inability
this cycle
in
S i m i l a r o b s e r v a t i o n s were r e p o r t e d
(1963) i n t h e
same o r g a n i s m .
Glucose
by
has
9
a l s o been o b s e r v e d
c y c l e enzymes
Srinivasan
t o repress
i n Baci1lus
the synthesis
subtilis
of t r i c a r b o x y l i c acid
and B a c i 1 l u s
cereus (Hanson,
a n d H a l v o r s o n , 1 9 6 3 a ; 1 9 6 3 b ; Hanson e t a l ,
H a n s o n and C o x , 1 9 6 7 ) . An
t h e s e enzymes o c c u r r e d
increase
1964;
i n the s p e c i f i c a c t i v i t y
after depletion
of
o f g l u c o s e f r o m t h e medium.
In an e x h a u s t i v e s t u d y on t h e r e g u l a t i o n o f m e t a b o l i s m o f
Escherichia
glucose
this
col i ,
repressed
repression
G r a y , Wimpenny and Mossman
t r i c a r b o x y l i c a c i d c y c l e enzymes.
was
partially
used
showed
However,
glucose
i n which s i t u a t i o n the
f o r synthesis.
The g l u c o s e e f f e c t on enzymes o f t r i c a r b o x y l i c a c i d
has
n o t been r e p o r t e d
species,
this
i n pseudomonads w i t h
i . e . Pseudomonas
species
hitriegens
i s so a t y p i c a l that
the exception
i t p o s s e s s e s an a c t i v e
d o e s n o t show
D o u d o r o f f p a t h w a y a c t i v i t y when g r o w n on g l u c o s e
(Eagon,
activity
cycle
o f one
(Cho a n d E a g o n , 1 9 6 7 ) .
M e y e r h o f p a t h w a y , c a n grow a n a e r o b i c a l l y ,
Wang, 1962)
that
r e d u c e d when t h e c e l l s w e r e grown
i n a s y n t h e t i c medium c o n t a i n i n g
c y c l e must be
(1966)
But
EmbdenEntner-
(Eagon and
a n d d o e s n o t p o s s e s s NADPH o x i d a s e o r t r a n s h y d r o g e n a s e
1963). Von T i g e r s t r o m a n d C a m p b e l l
of t r i c a r b o x y l i c acid
grown on g l u c o s e ,
c y c l e enzymes
d-ketoglutarate
l e v e l s o f t h e enzymes
were not s t r i k i n g l y
and a c e t a t e
i n the c e l l s
different.
harvested
(T966b) c o m p a r e d t h e
iriP.
and found
that the
from the three
I t h a s been r e p o r t e d
t r i c a r b o x y l i c a c i d c y c l e enzymes may
aerug?nosa
that
be c o n s t i t u t i v e i n
media
pseudomonads
(Campbell
but permeases f o r the c y c l e
intermediates are
inducible
a n d S t o k e s , 1951 ; B a r r e t t et_ a j _ , 1953).
In c o n t r a s t i t a p p e a r s
i n pseudomonads
t h a t t h e enzymes o f g l u c o s e c a t a b o l i s m
are inducible.
Hamilton
t h a t c u l t u r e s o f P. a e r u g i r i o s a : w h 1 c h
medium p o s s e s s
a n d Dawes (1960)
h a v e b e e n grown on a g l u c o s e
g l u c o s e and g l u c o n i c d e h y d r o g e n a s e s ,
gluconokinase, 2-ketogluconokinase,
found
hexokinase,
glucose-6-phosphate
6-
and
p h o s p h o g 1 u c o n a t e d e h y d r o g e n a s e s a n d t h e enzymes o f t h e E n t n e r Doudorof f pathway.
However, organisms
members o f t h e t r i c a r b o x y l i c
acid
grown on m e d i a c o n t a i n i n g
c y c l e showed e x t r e m e l y
low
l e v e l s o f t h e s e enzymes and t h e y w e r e i n d u c e d on i n c u b a t i o n o f s u c h
c e l l s with glucose.
were induced
shown
that a l l of these
on t r a n s f e r o f c i t r a t e grown c e l l s
containing glucose
Campbell
L a t e r i t was
(Ng a n d Dawes, 1967).
enzymes
t o a medium
Von T i g e r s t r o m a n d
(1966b) a l s o f o u n d t h a t when grown on a - k e t o g l u t a r a t e
and
a c e t a t e P. a e r u g ? r i o s a was e i t h e r d e v o i d o f o r p o s s e s s e d
low
l e v e l s o f t h e enzymes
required f o r glucose oxidation.
grown c e l l s w e r e shown t o b e h a v e s i m i l a r l y
1967a).
resembles
G r o w t h on t r i c a r b o x y l i c a c i d
cycle
( L e s s i e and
Succinate
Neidhardt,
intermediates probably
t h a t on a c o m p l e x medium s i n c e when grown
P. f 1 u o r e s c e n s
very
i n such
a medium
h a s been shown t o l a c k t h e enzymes o f t h e E n t n e r -
Doudorof f pathway
( E i s e n b e r g and D o b r o g o s z , 1967)•
11
IV.
C o n t r o l o f T r i c a r b o x y l i c A c i d C y c l e arid G l y o x y l a t e
Activity
The
in
Microorganisms
topic of regulation of tricarboxylic acid
glyoxylate cycle activity
has
i s a recent s u b j e c t .
c y c l e and
Most o f t h e s t u d y
been w i t h c o l i f o r m b a c t e r i a and B a c i 1 l u s s p e c i e s .
e f f e c t o f a e r o b i o s i s and a n a e r o b i o s i s a p a r t
"fine" control
has been s t u d i e d .
made by A m a r s i n g h a m a n d D a v i s
glutarate
completely
Interesting observations
(1965)
d e h y d r o g e n a s e ?h E. c o l i .
repressed
a substantial
The
from " c o a r s e " and
were
on t h e r e g u l a t i o n o f a - k e t o T h i s enzyme was f o u n d
t o be
i n a n a e r o b i c a l l y grown c e l l s w h i l e i n
a e r o b i c c u l t u r e s grown on g l u c o s e o r l a c t a t e
until
Cycle
accumulation
i t was n o t f o r m e d
of metabolites
(presumably
a-keto-
g l u t a r a t e o r a c e t a t e ) had o c c u r r e d .
They p r o p o s e d t h a t
dehydrogenase serves
l i n k between t h e r e d u c t i v e
branch
l e a d i n g t o s u c c i n a t e and t h e b i o s y n t h e t i c branch
a-ketoglutarate.
not
as a c o n n e c t i n g
Under a n a e r o b i c
r e q u i r e d and i s a b s e n t .
conditions this
leading t o
connection i s
Under a e r o b i c c o n d i t i o n s t h e second
h a l f o f the c y c l e can serve a c a t a b o l i c
dehydrogenase
a-ketoglutarate
r o l e and a - k e t o g l u t a r a t e
i s synthesized to completely
o x i d i z e s u b s t r a t e s by
recycling.
Gray et_a_l_ (1966b)
acid
c y c l e enzymes
have r e p o r t e d t h a t t h e l e v e l
i s g r e a t l y reduced
of tricarboxyl ic
i n a n a e r o b i c a l l y g r o w n E. c o 1 i .
The
coarse
a way
and
control
o f t h e enzymes o f t h i s c y c l e i s e x e r t e d
t h a t the a c t i v i t y
a n a b o l i c need.
is modified
When g l u t a m a t e
a c c o r d i n g t o the
o f g l u c o s e , but
and
can
The
authors
( D a v i s , 1961)
pathway
g l u t a r a t e and
c a n n o t be
regarded
as
glutamate;
constitutive
t h e a and
The
s o t h a t t h e c y c l e may
On
c g r o u p s o f enzymes a r e
needed f o r g l u t a m a t e
presence
repressed
of glutamate,
repression of aconitase
Cox,
glutamic
operate
s y n t h e t i c media w i t h
induced
while
as
glucose
a-ketoglutarate
since a-ketoglutarate
is
now
biosynthesis.
r i s e to glutamate
( H a n s o n and
the
g l u c o s e , a l 1 enzymes e x c e p t
pathway.
dehydrogenase remains
under
c. t h e 4 - c a r b o n d i c a r b o x y l i c a c i d s .
and
dehydrogenase are derepressed
an e n e r g y g e n e r a t i n g
this
the 5-carbon d i c a r b o x y l i c a c i d s , a-keto-
complex media w i t h o u t
giving
t h a t t h e enzymes o f
i . e . t h e enzymes c a t a l y z i n g : a .
t r i c a r b o x y l i c a c i d s ; b.
presence
derepressed
be d i v i d e d i n t o a t l e a s t t h r e e g r o u p s w h i c h a r e
independent c o n t r o l ,
On
suggest
the
i n the
t h e o t h e r enzymes o f t h e c y c l e a r e n o t
proportionately.
amphibolic
i n c r e a s e d even
such
catabolic
must be s y n t h e s i z e d ,
enzymes l e a d i n g t o i t s f o r m a t i o n a r e
in
a-ketoglutarate
i n glucose minimal
i n B.
1967), but
s i i b t i 1 i s and
o r compounds
medium c a u s e d
B.
complete
1 ichen?formis
fumarase, s u c c i n i c
dehydrogenase,
m a l i c d e h y d r o g e n a s e and
i s o c i t r i c dehydrogenase were not a f f e c t e d .
These workers suggested
that
t o r e g u l a t e c a t a b o l i c and
individual
control
mechanisms
a n a b o l i c s e c t i o n s of the
operate
tricarboxylic
acid
is
cycle activity
in the b a c i l l i ,
d i f f e r e n t • from-Ej-'col i s i n c e
both
and t h i s
c o n t r o l mechanism
i n the l a t t e r organism a c t i v i t y o f
t h e a n a b o l i c and t h e c a t a b o l i c s i d e o f t h e c y c l e was
repressed
u n d e r t h e same c o n d i t i o n s o f g r o w t h .
Recently,
observations
S h i i o and Ozaki
on c o n c e r t e d
dehydrogenase a c t i v i t y
observed w i t h
(1968)
inhibition
o f t h i s enzyme w h i l e
not result
that
t h e enzyme when a l r e a d y
t h a n when i t i s f r e e .
oxalacetate
caused s t r o n g
inhibition.
stronger with
(oxalmalate)
inhibition
i n e q u i m o l a r amounts
K i n e t i c a n a l y s i s showed
bound t o one o f t h e s e
The c o n d e n s a t i o n
and p i g h e a r t .
a d d i t i o n o f small
their addition singly
in significant
isocitrate
w h i c h was a l s o
B. s u b t ? l i s
the simultaneous
combines about 60,000 times
and
flavum
t h i s enzyme f r o m E. c o l i ,
amount o f g l y o x y l a t e a n d o x a l a c e t a t e
interesting
o f NADP-specific
i n Brevibacterium
These w o r k e r s found t h a t
did
have r e p o r t e d
the other
product
inhibitors
inhibitor
of glyoxylate
was n o t f o u n d t o be a s t r o n g
o f t h e i s o c i t r a t e dehydrogenase from t h e above s o u r c e s ,
i t was a l s o shown t h a t t h e c o n d e n s a t i o n
physiological
product
by
R u f f b e t _ a l _ (1967)
observation
tissue.
was more i n h i b i t o r y
a s was p r o p o s e d
On t h e b a s i s o f f u r t h e r
to.isocitrate
d e h y d r o g e n a s e o f B. f l a v u m ,
under
i n the
t h a t t h e combined e f f e c t o f t r i c a r b o x y l i c a c i d
intermediates
isocitrate
i n p i g heart
although
i s n o t formed
c o n d i t i o n s and i s t h e r e f o r e n o t i m p o r t a n t
regulation of tricarboxylic acid cycle activity
inhibitor
cycle
lyase than t o t h e
i t was p r o p o s e d
that a
high
the
concentration
o f t r i c a r b o x y l i c a c i d c y c l e members
a c t i v i t y o f t h e g l y o x y l a t e c y c l e by r e p r e s s i n g
(Ozaki
a n d S h i i o , 7968).
On t h e o t h e r
produced as a r e s u l t o f i s o c i t r a t e
i s o c i t r a t e dehydrogenase i n concert
acetate
all
i s p r e s u m e d t o be p r e s e n t
with
lyase
glyoxylate
inhibits
oxalacetate.
i n the c e l l
Oxal-
a t low l e v e l s a t
times.
I t h a s b e e n shown t h a t when E. c o l i
in glucose,
pyruvate o r metabolizable
tricarboxylic
repressed
E.
isocitrate
hand, excess
lyase a c t i v i t y
inhibits
cOli
acid cycle, isocitrate
( K o r n b e r g , 1965; 1966).
induce the c y c l e
been i d e n t i f i e d
isocitrate
lyase synthesis i s
the glyoxylate cycle
i s not
by.removing t h e r e p r e s s e r which has
as p h o s p h o e n o l p y r u v a t e .
The f i n e c o n t r o l o f
t o be e x e r t e d
again
by p h o s p h o -
i h E_. c o l _ i _ a n d t h i s m e c h a n i s m seems t o be d i f f e r e n t
found
unaffected
indirectly
of the
d i r e c t l y , b u t t h a t t h e s e compounds
l y a s e has been p r o p o s e d
enolpyruvate
than that
o r acetyl-CoA
intermediates
I t has been p r o p o s e d t h a t i n
and Achromobacter s p e c i e s ,
i n d u c e d by a c e t a t e
o r pseudomonads a r e grown,
i n B. f l a v u m ,
j n which
by p h o s p h o e n o l p y r u v a t e
isocitrate
(Ozaki
lyase a c t i v i t y i s
a n d S h i i o , 1968).
Pseudomonads a r e known t o u t i 1 i z e a n d grow on a g r e a t
oforganic
developed
control
activity
variety
compounds a n d , t h e r e f o r e , t h e y must p o s s e s s a w e l l
r e g u l a t o r y mechanism.
However, r e f e r e n c e s
on t h e
o f t h e • t r i c a r b o x y l i c -acid c y c l e and g l y o x y l a t e
i n pseudomonads a r e n o t a v a i l a b l e .
cycle
Smith and Gunsalus
(1957)
studied
t h e r e a c t i o n mechanism o f
P. a e r u g i n o s a and
by
i t was
i t s p r o d u c t g l y o x y l a t e and
succinate
and
found t h a t
i s a "mixed"
Howes, 1963)
Shiio,
1968).
nitrate
on
type
as was
There
succinate.
h a v e b e e n a few
acid
grown u n d e r . t h e s e c o n d i t i o n s
enzyme
(McFadden
flavum
(Ozaki
the e f f e c t
cycle activity
i f nitrate
c y c l e was
B.
iridigofera,
r e p o r t s on
deri?trificans,
grow a n a e r o b i c a l l y
The•tricarboxylic acid
In P.
inhibitor of this
the t r i c a r b o x y l i c
lyase i n
non-competitively inhibited
a l s o observed w i t h
C e r t a i n s p e c i e s s u c h as P.
stutzeri
isocitrate
P. a e r u g ? n o s a
shown t o f u n c t i o n
S i m i l a r l y , Wimpenny a n d Warms l e y (1968)
that
and
P.
i n t h e medium.
i n P.
stutzeri
1966).
Gilmour,
found
of
o f pseudomonads.
is included
( S p a n g I e r and
and
fumarase
and
a e o n i t a s e w e r e u n a f f e c t e d i n P_. a e r u g i n o s a , P: s t u t z e r i • and
aerobes
s u c h as B. s u b t i 1 i s and
a l l y with
A.
nitrate.
aerogenes,
undetectable
of
levels
and
under
high l e v e l s of n i t r i t e
suggest
activity
different
B. m e g a t h e r i u m when grown a n a e r o b i c -
However, i n f a c u l t a t i v e organisms
E. c o l i
B. m a r c e r a n s
and
i n t h e medium.
facultative
such
t h e s e enzymes f e l l
t h e s e c o n d i t i o n s due
types of c o n t r o l
i n aerobes
other
as
to
to accumulation
These o b s e r v a t i o n s thus
of t r i c a r b o x y l i c acid
anaerobes.
cycle
-16
V. M o l a r G r o w t h Y i e l d a s a T o o l
M o l a r growth y i e l d
i n t h e Study o f M e t a b o l i c Pathways
(MGY) i s d e f i n e d
as t h e d r y w e i g h t o f c e l l s
i n gms o b t a i n e d p e r m o l e o f c a r b o n s o u r c e u s e d d u r i n g g r o w t h .
aerobic bacteria,
relatively
i n which
e a s y , were f i r s t
assessment
tested
o f energy
Gunsalus
productioni s
f o rdeviations
E m b d e n - M e y e r h o f p a t h w a y u s i n g MGY d a t a .
An-
from t h e
DeMoss, B a r d a n d .
(1951)'• c o m p a r e d t h e MGY o f S t r e p t o c o c c u s f a e c a l i s and
LeuconOstoc
m e s e n t e r o i d e s grown on g l u c o s e a n d c o n c l u d e d
L. m e s e n t e r o i d e s
less energy
f e r m e n t e d g l u c o s e by a pathway w h i c h
that
yielded
t h a n t h e E m b d e n - M e y e r h o f p a t h w a y u s e d by S_. f a e c a l i s .
F u r t h e r work l e d t o t h e d i s c o v e r y o f t h e mechanism o f h e t e r o l a c t i c
fermentation
( G u n s a l u s and G i b b s , 1 9 5 2 ) .
i n L, m e s e n t e r o i d e s
and
E l s d e n (1960)
per
m o l e o f ATP i s s i m i l a r
showed t h a t
v
^
T p
, the y i e l d
great
importance
in correlating
c a r b o n s o u r c e i n t h e medium w i t h
Under a e r o b i c c o n d i t i o n s
the
Moreover,
c o n d i t i o n s , energy
a large part
h a v e no s i g n i f i c a n c e .
limiting
(20 t o 5 0 % ) o f t h e
material
(Hernandez and
balance
i t i s w i d e l y accepted t h a t under
i s never
is of
t h e m e t a b o l i c route used.
V967) w h i c h makes t h e c a l c u l a t i o n o f e n e r g y
impossible.
using the
This observation
o b t a i n e d on a
energy source i s converted t o c e l l u l a r
Johnson,
i n gms o f d r y w e i g h t
f o r d i f f e r e n t microorganisms
same p a t h w a y o f c a r b o h y d r a t e m e t a b o l i s m .
Bauchop
limiting
aerobic
a n d t h e r e f o r e , Y^.p v a l u e s
H e r n a n d e z a n d J o h n s o n Q967)
found
that
under a e r o b i c
the
c o n d i t i o n s ATP i s n o t t h e f a c t o r l i m i t i n g
Y^-p v a l u e
i s not a
constant.
It has been o b s e r v e d
yield
i s proportional
substrates
t h a t under a e r o b i c
and E a g l e s ,
H o w e v e r , i t c a n be a r g u e d t h a t t w o s u b s t r a t e s
metabolized
could
give d i f f e r e n t c e l l
M a y b e r r y , P r o c h a z k a and Payne
(1967)
by t h e s e
1952).
y i e l d s i f they a r e
i n v o l v e s an
i ; e . CC^ f i x a t i o n .
Recently,
made an i n t e r e s t i n g s t u d y
growth y i e l d s o f b a c t e r i a under a e r o b i c
values
t h e growth
of equivalent
by d i f f e r e n t p a t h w a y s , one o f w h i c h
advantageous step o f g a i n i n g carbon,
the
conditions
t o t h e amount o f c a r b o n s u p p l i e d
(Monod, 1942; C a m p b e l 1 , L i n n e s
carbon content
g r o w t h and
c o n d i t i o n s and found
However, t h e y found t h a t
t h e growth y i e l d
of a v a i l a b l e electrons . in the substrate
and
was a p p r o x i m a t e l y
avai1able
VI.
3.14 gm o f c e l l s
utilized
a s Di s t ? n e t
o f t h e T r i c a r b o x y l i c Ac?d
In s e v e r a l
per equivalent
was
constant,
per equivalent o f
f r o m O t h e r Di c a r b o x y l i c
Cycle
s t u d i e s , i t was f o u n d t h a t
in relation
t r i c a r b o x y l i c a c i d c y c l e enzymes, a low l e v e l
d e h y d r o g e n a s e was p r e s e n t
Von
wide
electrons.
Succinate.Ut?1ization
Acids
that
o f y i e l d s p e r m o l e o f s u b s t r a t e ; p e r gm a t o m o f s u b s t r a t e
c a r b o n ; a n d p e r m o l e o f o x y g e n consumed v a r i e d o v e r a v e r y
range.
on
Tigerstrom
i n P. a e r u g i n o s a
t o other
of succinic
(Campbell e t a l , 1962;
and C a m p b e l l , 1 9 6 6 b ) , b u t i n s p i t e o f t h i s t h e
organism oxidized
cycle
succinate at rates
intermediates.
difficult
T h u s , on
i n h i b i t o r of h istidase
Hunter,
1968).
urocanase which
indirectly
It would
histidase
is
a r e two m a j o r
i s ATP
and
the
c o n c l u d e d t o be
a
( L e s s i e and
histidase.
inhibiting
be c a l l e d
metabolites
to
not
the
i n reduced
However, o t h e r
also
by t h e same m e c h a n i s m as
e f f e c t was
succinate
urocanase.
suppress
succinate.
inhibition
by
studied.
forms o f a v a i l a b l e b i o c h e m i c a l e n e r g y .
i n the d i r e c t i o n
more e n e r g y on c o m b u s t i o n .
the m o l e c u l a r hydrogen
form
in primitive
life
( o r NADH) w h i c h
of reduction, forming
This reducing
the " m e t a b o l i c hydrogen" which
replaces
inhibition
Thus,
such as c i t r a t e
(Hug,
E v o l u t 1 on
the reactions
compounds w h i c h y i e l d
form o f l i f e
due
t h e o t h e r t h e r e d u c i n g p o w e r o f NADPH
to drive
p o w e r may
by
i n P. a e r u g i h o s a
have been r e l e v a n t t o have o b s e r v e d
M e t a b o l i c P a t t e r n and
used
by
support r a p i d growth
s y n t h e s i s presumably
There
One
ih vivo
inhibited
f u m a r a t e and m a l a t e , b u t t h e i r
VII.
previously
Urocanate accumulated
in turn
inhibited
compounds w h i c h
histidase
solely
1967b) has b e e n f o u n d t o i nh ?b? t u r o c a n a s e i n P. p u t i d a
Neidhardt,
of
utilized
i t was
dehydrogenase.
R e c e n t l y , s u c c i n a t e w h i c h was
R o t h and
to those o f other
the b a s i s o f these s t u d i e s ,
t o c o n c l u d e t h a t s u c c i n a t e was
action of succinic
feedback
comparable
i n t h e modern
that maintained the
forms
(Horecker,
1965).
Horecker
(1965) h a s d e s c r i b e d t h e r e l a t i o n s h i p o f NADH a n d
NADPH w i t h o x y g e n a n d c e l l u l a r
b i o s y n t h e s i s as f o l l o w s :
Substrate
NADH
Substrate
NADPH
fast
r
I
slow
No~P
Reduction
reactions
T h e r e w o u l d be n o need f o r t w o c o e n z y m e s
i n t h e absence o f m o l e c u l a r
o x y g e n , s i n c e a s i n g l e coenzyme c o u l d a c t b o t h
for
f e r m e n t a t i o n and as a source
what
In
i s seen
these
i n thepresent
species
developed
first
o f metabolic hydrogen.
day s t r i c t anaerobes such
fermentation
the major m e t a b o l i c
as t h e coenzyme
This i s
as
f o r t h e p r o d u c t i o n o f energy
activity.
This also implies that
Clostridia.
is still
anaerobes
i nevolutionary history since i ti sbeiieved that
t h e a t m o s p h e r e o f t h e p r i m i t i v e e a r t h was h i g h l y r e d u c i n g c o n t a i n i n g
h y d r o g e n compounds s u c h
itself.
in
a s m e t h a n e , ammonia a n d m o l e c u l a r
L a t e r , i n t h e presence
o f oxygen, p r e e x i s t i n g
t h e hydrosphere were c o n v e r t e d
NADH was now u t i l i z e d
phosphorylation,
i t was n o t a v a i l a b l e
a s e c o n d c o e n z y m e , NADP was e v o l v e d
appeared.
NADPH p r o v i d e d
a source
subunits
t o more o x i d i z e d f o r m s .
f o r the production o f energy
hydrogen
Since
in oxidative
f o r r e d u c t i v e r e a c t i o n s and
f r o m NAD when
respiration
o f m e t a b o l i c h y d r o g e n w h i c h was
not
i n equilibrium with
The
oxygen.
h e x o s e m o n o p h o s p h a t e p a t h w a y h a s b e e n shown t o be r a t e -
limited
p r i m a r i l y by t h e r a t e o f NADP s u p p l y
tissues
such as l i v e r
(McLean, I 9 6 0 ) .
rate
limiting
( C a h i 1 1 et_ a l _ , 1958) a n d i n mammary
Eagon
( 1 9 6 2 ) showed t h a t
i n the operation
i i i P. l i i t r i e g e n s . T h i s
but
i n a v a r i e t y o f animal
t h e s u p p l y o f NADP was
o f t h e hexose monophosphate pathway
o r g a n i s m p o s s e s s e s enzymes o f t h i s
l a c k s NADH o x i d a s e a n d t r a n s h y d r o g e n a s e .
A strong
b e t w e e n t h e s i m u l t a n e o u s p r e s e n c e o f NADPH o x i d a s e p l u s
hydrogenase w i t h
tissue
the u t i l i z a t i o n
pathway
correlation
trans-
o f t h e hexose monophosphate
p a t h w a y and t h e E n t n e r - D o u d o r o f f p a t h w a y when grown on g l u c o s e h a s
b e e n shown
i n d i f f e r e n t microorganisms
( E a g o n , 1963) -
Although complete t r i c a r b o x y l i c a c i d c y c l e a c t i v i t y i s
associated
with
aerobic
life,
two p o r t i o n s
o f the cycle
serving
biosynthetic
purposes o p e r a t e under a n a e r o b i c c o n d i t i o n s .
now
i n c r e a s i n g l y common t o f i n d
viz.
becoming
the c i t r a t e
activity
leading
synthase, aconitase
t o biosynthesis
It has been r e p o r t e d
the anaerobic
and i s o c i t r a t e
of a-ketoglutarate
It i s
route
dehydrogenase
i n anaerobes.
i n a v a r i e t y o f anaerobes such as C l o s t r i d i u m
k l u y v e r i , M e t h a n o b a c i 1 l u s onie 1 i a i i s k ? i , P e p t o s t r e p t o c o c c u s e l s d e n i 1
and
Ch 1 o r o p s e u d o m o h a s e t h y l i c u m
and
B a r k e r , 1967; S o m e r v i l i e ,
In c y t o c h r o m e c o n t a i n i n g
'succinate
(Stern
and Bambers, 1966;
1 9 6 8 ; C a l l e l y and F u l l e r ,
a n a e r o b e s s u c h as
h a s been shown t o be f o r m e d
Gottschalk
1967).
Bacteroides'ruminicola,
from pyruvate o r i t s
derivative
cycle
(White,
The
some m e t a b o l i c
reductive steps
changes
originally
in fatty
to facultative o r aerobic
i n microorganisms.
evolved
acid
i n anaerobes f o r c a t a l y s i n g
synthesis, but the f l a v o p r o t e i n
t o o x y g e n must h a v e b e e n t h e f i r s t
to a e r o b i o s i s ( D o l i n , 1961).
life
The coenzyme
system t o c a t a l y s e t r a n s f e r o f e l e c t r o n s from
directly
acid
and C a l d w e l l , 1962).
from a n a e r o b i c
was p r o b a b l y
oxidase
the r e d u c t i v e branch o f t h e t r i c a r b o x y l i c
Bryant
shift
involved
FAD
through
substrates
step towards
I t has been o b s e r v e d
adaptation
that l a c t i c
acid
b a c t e r i a w h i c h h a v e no c y t o c h r o m e s , c a r r y o u t c e r t a i n o x i d a t i o n
reactions
that
in this
fashion.
1 ti S. f a e c i um a n L (+)
induced
on a e r o b i c g r o w t h .
coenzyme a t t a c h e d
Recently,
specific
London
(1968)
h a s shown
lactate oxidizing
enzyme i s
T h i s o x i d a s e w h i c h h a s FAD a s t h e
t o t h e apoenzyme e n a b l e s
under a e r o b i c c o n d i t i o n s on l a c t a t e .
t h e o r g a n i s m t o grow
I t was s u g g e s t e d
that
this
enzyme c o n f e r s on t h e o r g a n i s m t h e a b i 1 i t y t o . s u r v i v e ' i n a
carbohydrate
depleted
a e r o b i c environment c o n t a i n i n g L(+)
lactate.
F a c u l t a t i v e organisms are capable
o f developing
and
a e r o b i c m e t a b o l i s m d e p e n d i n g upon t h e c o n d i t i o n s
The
extensive study
b y G r a y e t a l _ ( 1 9 6 6 a ; 1966b)
and
Warms l e y ( 1 9 6 8 )
provide
The
m e t a b o l i c p a t t e r n o f E_. co]_i_, when g r o w n u n d e r
good
•conditions,' resembles'that-of
insight
into this
an a e r o b e w h i l e
both
anaerobic
provided.
a n d by Wimpenny
phenomenon.
aerobic
i t s i m u l a t e s an
a n a e r o b e when g r o w n
Autotrophs,
i n t h e absence o f oxygen.
which a r e considered
a step
beyond
heterotrophs
i n e v o l u t i o n , a r e c o m p r i s e d o f a e r o b i c and a n a e r o b i c
have developed
different metabolic
patterns.
t h a t o b l i g a t e chemoautotrophs have e v o l v e d
ancestors
thiobacilli
conserving
and b l u e - g r e e n
reducing
Several
algae
proposed
from h e t e r o t r o p h i c
i n the loss
species of autotrophic
l a c k NADH o x i d a s e
(thus
power o f NADH) a n d a - k e t o g l u t a r a t e
dehydrogenase a c t i v i t y , which
results
i n thedestruction o f the
o f t h e o r g a n i s m t o u s e o r g a n i c compounds f o r g r o w t h a n d
production
o f energy
l o s s o f NADH o x i d a s e
( S m i t h , London and S t a n i e r ,
defect
However,
i s not
S t u d i e s oh N i t r o c y s t i s
t o show t h a t o b i i g a t e a u t o t r o p h y
metabolic
1967).
and a - k e t o g l u t a r a t e dehydrogenase
always t h e cause o f autotrophy.
failed
I t h a s been
by e l i m i n a t i o n o f c e r t a i n enzymes r e s u l t i n g
of heterotrophic metabolism.
ability
s p e c i e s and
( W i l l i a m s and Watson,
was s o l e l y
1968).
t h a t a n o r g a n i s m was a u t o t r o p h i c b e c a u s e
oceanus
due t o t h i s
The a u t h o r s
i t either
concluded
l a c k e d o r had low
l e v e l s o f o n e o r more enzymes o r i t s membrane was n o t p e r m e a b l e
to o r g a n i c molecules
at a rate s u f f i c i e n t
o f two o r more o f t h e s e
It
of
i s thus
and
from t h i s
i n f o r m a t i o n c o u l d be g a t h e r e d
mechanisms
v
obvious
d e f e c t s may be
and l e v e l
of carbon sources
A
combination
expected.
d i s c u s s i o n that a vast
about t h e behaviour
i n t h e o r g a n i s m by t h e s t u d y
the nature
f o r growth.
amount
and c o n t r o l
o f i t s metabolic
activity
o f e n z y m e s p r e s e n t , when g r o w n on a v a r i e t y
and e n v i r o n m e n t a l
conditions.
MATERIALS AND METHODS
I .
0 rgari ? srfis arid Growth • Med i a
Pseudomonas aeruginosa ATCC 9027 and Aerobacter aerogenes
(departmental stock) were grown in Roux flasks without shaking at
30 C and 37 C respectively.
NH H P0 , 0.3%;
!j
2
Zf
K HP0^,
2
The growth medium c o n s i s t e d o f
0.2%; iron as F e S O ^ H ^ , 0.5 p.p.m;
MgS0^.7H 0, 0.05% and carbon source (minimal medium).
2
The pH was
adjusted to 7.0 and s t e r i l e MgSO^^H^O and the carbon source were
added to the autoclaved medium before inoculation., This medium
without the carbon source w i l l be referred to as the basal salts
medium.
The organisms were grown with 0.2% carbon source for
manometric experiments and with 0.3% when used for enzyme assays.
The carbon sources used and their concentration as w e i r as-.pH of
the medium when d i f f e r e n t than mentioned above wi11 be s p e c i f i e d .
In some growth experiments
the organisms were grown i n 2 5 0 ml
side arm flasks in a shaking water bath and density of the culture
was recorded with a Klett-Summerson colorimeter.
Cultures were routinely checked for purity by'. streak?ng on
suitable media.
P. aeruginosa produces pyocyanine on King's
medium (King, Ward and Raney, 1954) which was used to check the
purity of this
II.
organism.
o f R e s t i hg C e l 1 S u s p e n s i o n s
Preparation
The
o r g a n i s m s w e r e h a r v e s t e d by c e n t r i f u g a t i o n
temperature
the
in early
logarithmic
0.01
M tris
pH 7 - 0 .
buffer,
sodium c h l o r i d e
stationary
Extracts
a t room
phase f o r manometric s t u d i e s
(hydroxymethyl)aminomethane
(Tris)
0.3%
In some e x p e r i m e n t s n e u t r a l i z e d
s o l u t i o n was u s e d f o r w a s h i n g .
Resting
cell
s u s p e n s i o n s w e r e o b t a i n e d by r e s u s p e n d i n g t h e p e l l e t i n 0.1
Tris
For
buffer,
pH J.k,
preparation
of cell-free extracts
0 . 2 M T r i s b u f f e r , pH 1.k,
of
deoxyribonuclease
t h e p e l l e t was
t o a suitable concentration
The c e l l s w e r e d i s r u p t e d
and c e n t r i f u g e d
and d e b r i s .
referred
e x t r a c t , was u s e d
t o as t h e c e l l - f r e e
e x p e r i m e n t s a n d f o r enzyme a s s a y s
c e n t r i fuged
every
i n a French
The s u p e r n a t a n t ,
f o r respirometry
respectively.
f r a c t i o n a t i o n was d e s i r e d
the c e l l - f r e e
extract
f o r 30 m i n a t 2 5 , 0 0 0 x g_ a n d f o r 90 m i n a t 1 0 0 , 0 0 0
x £ i n a S p i n c o Model
was
and 1 drop
a t 8 , 0 0 0 x g_ o r 2 0 , 0 0 0 x £ f o r
15 m i n t o remove w h o l e c e l l s
was
resuspended
(1 mg/ml a q u e o u s s o l u t i o n ) was a d d e d f o r
2 . 0 ml o f t h e s u s p e n s i o n .
When f u r t h e r
M
t o a p p r o x i m a t e l y 5 mg d r y w e i g h t o f c e l l s / m l . ,
in
pressure c e l l
or in
p h a s e o f g r o w t h f o r enzyme a s s a y s . . T h e y w e r e
washed t w i c e w i t h
chloride
and C e l 1 - F r e e
L Ultracentrifuge.
w a s h e d o n c e wrt-th 0.1
M Tris buffer,
The 100,000 x £ p e l l e t
pH 7 . 0 , a n d r e s u s p e n d e d a t
a suitable
c o n c e n t r a t i o n i n 0.2 M T r i s
" P o t t e r " g l a s s homogenizer.
scheme t h e y h a v e b e e n
III.
specified.
a e r u g i n o s a 9 0 2 7 was grown w i t h s h a k i n g
7.4 w i t h 0.2% g l u c o s e
other carbon
equivalent
pH
When m o d i f i c a t i o n s w e r e made i n t h i s
D e t e r r h i h a t i o n o f M o l a r G r o w t h Y i e l d on Di f f e r e h t
P.
pH
b u f f e r , pH 7 . 4 , u s i n g a
o f t h e medium was 6 . 7
above 7 . 7 .
going
t h e c o n c e n t r a t i o n a d d e d was
i n o r d e r t o keep t h e f i n a l
pH
from
C e l l s w e r e h a r v e s t e d a t room t e m p e r a t u r e a n d
b u f f e r , pH 7 . 0 .
T h e p e l l e t was
i n t h e same b u f f e r t o a d e f i n i t e v o l u m e i n a
volumetric flask
volume o f t h i s
weight
When
c o n t e n t o f g l u c o s e and t h e i n i t i a l
washed t w i c e w i t h 0.025 M T r i s
resuspended
i n t h e medium a t
(11.1 u m o l e / m l o f t h e m e d i u m ) .
s o u r c e s were used,
t o t h e carbon
Substrates
and d r y w e i g h t s
suspension
a t 95 t o 9 7 C;
were determined
on a s u i t a b l e
by d r y i n g d u p l i c a t e samples t o a c o n s t a n t
For c a l c u l a t i o n
o f growth y i e l d s
the value
a t t h e p e a k o f t h e g r o w t h c u r v e was t a k e n .
IV.
I s o l a t i o n o f M u t a n t s o f P_. a e r u g i n o s a
The
followed.
^fumarate
m e t h o d recommended by A d e l b e r g , M a n d e l a n d Chen
Wild
minimal
type
(1965) was
l o g a r i t h m i c phase c e l l s were h a r v e s t e d
medium b y c e n t r i f u g a t i o n a t room
from
temperature.
The
cell
p e l l e t was
washed t w i c e w i t h
and
resuspended
and
s h a k e n a t 30
was
added t o g i v e a f i n a l
C for 1 hr.
the
Again,
this
cell
enrich
f o r the mutants.
a
spread
on
This
suspension
s u c c i n a t e or
100
Ug/ml and
was
on
fumarate minimal
was
then
c h e c k e d by
different
patching
on
i n the
and
on
eel Is , were a l s o i s o l a t e d
i z a t i o n was
medium c o n t a i n i n g a
The
small
diluted
for
tricarboxylic
For
I t was
glucose
in these
acid
isolation
was
limiting
c o l o n i e s were p i c k e d
p l a t e c o u n t a g a r and
carbon sources.
a-ketoglutarate
to
i n fumarase a c t i v i t y , the suspension
of glucose.
salts
medium c o n t a i n i n g
(0.0\%)- o f a - k e t o g 1 u t a r a t e
fumarate minimal
concentration
basal
f o r 4 to 6 hr
suspension
i n some s t e p
was
volume i n n u t r i e n t b r o t h .
incubated
cell
it
then c e n t r i f u g e d
l e a d i n g to b i o s y n t h e s i s of a - k e t o g l u t a r a t e .
of mutants blocked
on
was
The
a c q u i r i n g mutants blocked
spread
of
suspension
t o the o r i g i n a l
limiting concentration
cycle
concentration
p e l l e t , a f t e r washing twice with
resuspended
5.5
N-methyl-N'-nitro-N-nitrosoguanidine
concentration
min.
medium, was
and
M c i t r a t e b u f f e r , pH
i n t h e same b u f f e r a t t h e o r i g i n a l
f u r t h e r s h a k e n f o r 45
a t 3 t o 4 C and
0.01
minimal
and
media
containing
found t h a t mutants u n a b l e t o grow
w h i c h grew s l o w e r
experiments.
than w i l d
Further
a t t e m p t e d by m a n o m e t r i c e x p e r i m e n t s and
type
character-
enzyme
assays.
V.
Manometric Methods
Utilization
extracts
of different substrates
and c e l l
30 C u s i n g W a r b u r g
VI.
cell-free
f r a c t i o n s was i n v e s t i g a t e d by c o n v e n t i o n a l
manometric techniques
endogenous
by w h o l e c e l l s ,
(Umbreit,
B u r r i s a n d S t a u f f e r , 1957) a t
respirometers.
C o r r e c t i o n s w e r e made f o r
values.
Enzyme A s s a y s
Soluble
Ochoa
(1955)
coenzymes.
L-malic
d e h y d r o g e n a s e was a s s a y e d a c c o r d i n g t o ,
using T r i s
b u f f e r , pH 7 . 6 a n d NADH o r NADPH as
Ethylenediamine
tetraacetic acid
( E D T A ) , 5 mM was u s e d
in the reaction mixture
t o e l i m i n a t e t h e i n t e r f e r e n c e due t o
m a l i c enzyme a c t i v i t y .
M a l i c enzyme was a s s a y e d a t pH 8 . 5 ,
t h e method o f J a c o b s o n e t a l (1966).
EDTA b u t w i t h o u t
A c o n t r o l c o n t a i n i n g 5 mM
MgC.^ was u s e d t o c h e c k
was
solely
was
measured s p e c t r o p h o t o m e t r i c a l l y using'
indophenol
due t o m a l i c e n z y m e .
according
i f the a c t i v i t y
P a r t i c u l a t e L-malic
measured
dehydrogenase
2,6-dichlorophenol
t o F r a n c i s e t _ a j _ (1963).
a d d i t i o n o f 5 mM EDTA was u s e d t o c o n f i r m
m a l i c enzyme a c t i v i t y
using
Again, the
the elimination of
w h i c h m i g h t show up due t o t h e p r e s e n c e o f
endogenous coenzymes
i n the c e l l - f r e e e x t r a c t s .
For i s o c i t r a t e
7.8,
dehydrogenase
t h e r e a c t i o n m i x t u r e c o n t a i n e d T r i s b u f f e r , pH
50 mM; MgC.l
10. mM; NADP, 0 . 2 5 mM; D L - i s o c i t r a t e , 3 . 0 mM a n d t h e
enzyme.
T h e r e d u c t i o n o f c o e n z y m e was r e c o r d e d a t 3^0
A c o n i t a s e and fumarase
(1950).
were assayed
DL-isocitric acid
my.
by t h e method o f R a c k e r
( p o t a s s i u m s a l t ) was p r e p a r e d f r o m i t s
l a c t o n e a c c o r d i n g t o t h e p r o c e d u r e g i v e n by Hanson e t a l ( 1 9 6 3 b ) .
a - K e t o g l u t a r a t e dehydrogenase
Amarsingham and D a v i s
coenzyme.
(1965)
was a s s a y e d
u s i n g a c e t y l p y r i d i n e - N A D as t h e
For s u c c i n i c dehydrogenase
method d e s c r i b e d by K i n g
Glutamate
by t h e p r o c e d u r e o f
the phenazine
methosulfate
( 1 9 6 7 ) was u s e d .
dehydrogenase
was a s s a y e d
i n a 1.0 ml
reaction
m i x t u r e w i t h NADH o r NADPH as t h e c o e n z y m e a n d e x c e s s NH^Cl and
a-ketog1utarate
(Von T i g e r s t r o m a n d C a m p b e l l ,
1966a).
When t h e
a s s a y was i n t h e d i r e c t i o n o f a - k e t o g l u t a r a t e 1 ml o f t h e r e a c t i o n
mixture c o n t a i n e d potassium phosphate
b u f f e r , pH 8 . 0 ,
50 mM;
L - g l u t a m a t e , 25 mM; NAD o r NADP, 0 . 2 5 mM a n d t h e enzyme.
was
assayed
by t h e d e t e r m i n a t i o n o f g l y o x y l a t e f o r m e d
Isocitratase
f r o m DL-
i s o c i t r a t e by t h e a c t i o n o f t h e enzyme ( O z a k i a n d S h i i o ,
1968).
In some p r e l i m i n a r y e x p e r i m e n t s , h o w e v e r , t h e a c t i v i t y was
by t h e method o f D i x o n and K o r n b e r g
was
assayed
(1959).
Succinyl
sodium
determined
CoA s y n t h e t a s e
by t h e h y d r o x a m a t e a s s a y method a s d e s c r i b e d by G i b s o n ,
Upper and G u n s a l u s
(1967).
the assay o f glucose-6-phosphate
For
b u f f e r , pH 8 . 0 ,
mixture contained Tris
dehydrogenase
50 mM; M g C l ,
the reaction
5 . 0 mM;
2
0 . 2 5 mM; g l u c o s e - 6 - p h o s p h a t e , 2 . 5 mM a n d t h e c e l l - f r e e
For
6-phosphogluconate
was
t h e same e x c e p t t h a t 6 - p h o s p h o g l u c o n a t e
glucose-6-phosphate.
Tris
dehydrogenase
assay, the reaction mixture
was s u b s t i t u t e d f o r
50 mM; M g C l ,
2
10 mM; A T P , 2 . 5 mM; g l u c o s e ,
5 mM; NADP, 0 . 5 mM;
t h e c e l 1 - f r e e e x t r a c t and e x c e s s
glucose-6-phosphate
dehydrogenase.
was d e t e r m i n e d
Glucose
dehydrogenase
by t h e combined
measured
and
Campbell, 1966b).
-
using commercial
l a c t i c dehydrogenase
and
The f o r m a t i o n o f 3 - p h o s p h o g l y c e r a l d e h y d e
using commercial
according
Ling et_aj_
isomerase
(Bock a n d N e i d h a r d t , I 9 6 6 )
i s o m e r a s e and f r u c t o s e d i p h o s p h a t a s e were a s s a y e d
(1967).
t o G a l e and Beck
( 1 9 6 6 ) was f o l l o w e d
kinase a c t i v i t y
(1967)
aldolase
t r i o s e phosphate
3-phosphoglyceraldehyde dehydrogenase
Phosphohexose
aldolase
(Von T i g e r s t r o m
from f r u c t o s e - 1 , 6 - d i p h o s p h a t e by f r u c t o s e d i p h o s p h a t e
was m e a s u r e d
Pyruvate
a c t i o n o f 6-phosph
and 2 - k e t o - 3 d e o x y - 6 - p h o s p h o g l u c o n a t e
was
activity
commercial
u s i n g t h e m e t h o d o f Hauge ( 1 9 6 6 ) .
f o r m a t i o n from 6-phosphogluconate
gluconate dehydrase
extract.
F o r g l u c o k i n a s e t h e r e a c t i o n m i x t u r e had
b u f f e r , pH 8 . 0 ,
activity
NADP,
The m e t h o d d e s c r i b e d by
f o r the assay o f phosphofructo-
and t h a t o f D e V r i e s , Gerbrandy
and
f o rfructose-6-phosphate phosphoketolase.
g l y c e r a l d e h y d e dehydrogenase
b u f f e r , pH 7 . 4 ,
50 mM;
F o r 3-phospho-
the reaction mixture contained Tris
DL-3 phosphoglyceraldehyde,
_
Stouthamer
6 mM;
cysteine
h y d r o c h l o r i d e , 10. mM;
20 mM;
sodium
a r s e n a t e , 17 mM;
NADP o r NAD, 0 . 2 5 mM a n d t h e enzyme.
sodium
The f o l l o w i n g
enzymes w e r e m e a s u r e d by t h e m e t h o d s d e s c r i b e d
given a f t e r
t h e name o f e a c h enzyme: t r i o s e
3-phosphoglyceric acid
and
1966); enolase
Tris
i n the references
phosphate
isomerase
k i n a s e ( C a m p b e l l , H e l l e b u s t and W a t s o n ,
( W e s t h e a d , 1966)
a s f o r t h e y e a s t enzyme b u t w i t h
b u f f e r ; p y r u v a t e k i n a s e ( V a l e n t i n e and T a n a k a , 1 9 6 6 ) ;
aldolase
( T c h o l a and H o r e c k e r , 1966); and r i b o s e
(Axel rod and J a n g , 1 9 5 4 ) .
the a c t i v i t i e s
50 mM;
cysteine hydrochloride,
commercial
triose
dehydrogenase.
1.5
phosphate
The a b i l i t y
action of several
mM;
mM; NADH, 0 . 2 5 mM; enzyme a n d e x c e s s
i s o m e r a s e and a - g l y c e r o p h o s p h a t e
of the cell-free extracts
(i.e.
ribose-5 phosphate) which
-
endogenous pentose phosphate
r i b o s e phosphate
b u f f e r , phi 8 . 0 ,
thiamine pyrophoshate, 1
ribose-5-phosphate t o glucose-6-phosphate
forming a c t i v i t y from
together with
i s o m e r a s e and r i b u l o s e - 5 - p h o s p h a t e
i n a system c o n t a i n i n g T r i s
r i b o s e - 5 - p h o s p h a t e , 1 mM;
trans-
phosphate"isomerase
Transketolase a c t i v i t y ,
o f r i b o s e phosphate
e p i m e r a s e was a s s a y e d
as
fluoride,
to transform
glucose-6-phosphate
involves
the coupled
pathway enzymes
isomerase, r i b u l o s e - 5 p h o s p h a t e epimerase,
_
such
trans-
k e t o l a s e a n d t r a n s a l d o l a s e was a s s a y e d a c c o r d i n g t o t h e p r o c e d u r e
o f G a l e and Beck
(1967).
S p e c t r o p h o t o m e t r i c assays were performed e i t h e r
s p e c t r o p h o t o m e t e r model DB-G
specific; activities
utilized
or
Gilford
i n a Beckman
model 2000 a t 30 C.
a r e e x p r e s s e d a s mymoles o f t h e s u b s t r a t e
p e r m i n p e r mg o f p r o t e i n .
The
31
VII.
Analysis of Reaction
Products
F o r t h e i d e n t i f i c a t i o n o f k e t o a c i d s o n e ml p o r t i o n s o f t h e
spent
reaction mixtures
poured
i n t o o n e ml o f a 1 u m o l e / m l s o l u t i o n o f 2 , 4 - d i n i t r o p h e n y l
hydrazine
i n 2 N HCl.
centrifugation.
min.
from Warburg v e s s e l s o r from c u v e t t e s were
The p r e c i p i t a t e d
The s u p e r n a t a n t
p r o t e i n was removed by
was i n c u b a t e d
The h y d r a z o h e s w e r e e x t r a c t e d i n t o e t h y l
extracted
a t 37 C f o r 30
a c e t a t e and r e -
i n t o 1 M T r i s b u f f e r , pH 11 f r o m e t h y l
acetate.
The
T r i s e x t r a c t was a c i d i f i e d w i t h 5 N HCl and t h e h y d r a z o h e s w e r e
finally
no.
extracted into ethyl
a c e t a t e a n d c h r o m a t o g r a p h e d on Whatman
4 paper using n-butanol-ethanol-0.5
described
by S m i t h
(1960).
Standards
samples were c o n c l u s i v e l y i d e n t i f i e d
case
and
VIM.
N NH^OH (70:10:20) a s
w e r e r u n s i m u l t a n e o u s l y and
by c o - c h r o m a t o g r a p h y .
In t h e
o f r a d i o a c t i v e samples, s t r i p s o f t h e chromatograms were c u t
scanned
i n an a c t i g r a p h
Thin Layer
(Nuclear
Chicago).
Chromatography, Autoradiography
and R a d i o a c t i v e
Measurements
Reaction
0.12 ml o f 5.8
i n t h e W a r b u r g v e s s e l s was s t o p p e d
by t h e a d d i t i o n o f
M HC10. p e r ml o f r e a c t i o n m i x t u r e .
The p r e c i p i t a t e d
p r o t e i n was removed b y c e n t r i f u g a t i o n
neutralized
and
1967).
chromatographed
(TLC)
A s u i t a b l e volume o f t h e s u p e r n a t a n t
t w o - d i m e n s i o n a l l y on t h i n
thin
p u r p l e o r by a n i 1 i n e - r i b o s e ( H i g g i n s a n d Von B r a n d ,
on Kodak m e d i c a l
contents corresponding
p l a t e s were scraped
counted
X-ray
films
1966).
the autoradiograms
f o r 7 t o 10
days.
t o t h e d e s i r e d r a d i o a c t i v e s p o t s on t h e
and p l a c e d
i n a Nuclear Chicago
i n the s c i n t i l l a t i o n
scintillation
o f S n y d e r and S t e p h e n s
v i a l s and
spectrometer
t o l u e n e c o n t a i n i n g PPO, P0P0P a n d C a b - 0 - S i l
procedure
IX.
by s p r a y i n g w i t h brom
l o c a t e t h e r a d i o a c t i v e s p o t s on TLC p l a t e s ,
using
chromatography
I d e n t i f i c a t i o n of standards
l a y e r c h r o m a t o g r a m s was a c h i e v e d
were developed
The
layer
f l u i d was
p l a t e s c o a t e d w i t h e e l l u l o s e CC-41 a c c o r d i n g t o t h e
cresol
To
bicarbonate
p e r c h l o r a t e was removed by c e n t r i f u g a t i o n
method o f M y e r s and Huang . ( 1 9 6 6 ) .
on
f l u i d was
by t h e a d d i t i o n o f 0.24 ml o f 2 . 9 M p o t a s s i u m
the precipitated
(Weiss,
and t h e s u p e r n a t a n t
725,
model
according t o the
(1962).
Uptake o f Label led S u b s t r a t e s
The
incorporation o f the labelled
s u b s t r a t e s , glucose-U-
14
C
14
and
a-methyl-g1ucoside-U-
Millipore
The
C i n t o whole c e l l s
was s t u d i e d b y t h e
f i l t r a t i o n t e c h n i q u e o f B r i t t e n and M c C l u r e . ( 1 9 6 2 ) .
l o g a r i t h m i c phase c e l l s
w e r e h a r v e s t e d a t room
a n d w a s h e d o n c e w i t h b a s a l s a l t s medium.
temperature
The p e l l e t was
resuspended
added
of
jet
to
0.3
in
the
my.
The
addition
by t h e
New Y o r k )
of
addition
of
intervals
mixture
on a T r a c e r l a b
Waltham,
Mass.)
Millipore
The
placed
in v i a l s
by c o l l e c t i n g
of
filters
1iquid
X.
Protein
Stern
Corp.,
(1957).
the
1 ml
minimal
y
on a M a g -
scintillation
for
prior
to
at
the
incubation
(Tracerlab,
medium.
were
s o u r c e and
fluid
counting
(liquifluor,
in a
spectrometer.
Methods
Citrate
determined
was e s t i m a t e d
P y r u v a t e was
by t h e
by t h e
method o f
Lowry
method o u t 1 i n e d by
assayed using l a c t i c
pH 7 . 4 ,
d e h y d r o g e n a s e and
NADH i n a s y s t e m c o n t a i n i n g T r i s
buffer,
NADH, 0 . 2 5
d e h y d r o g e n a s e and t h e
sample.
allowing
to
mM; c o m m e r c i a l
a-Ketoglutarate
the
started
pore s i z e
infra-red
Mass.)
density
terminated
from
and 0 . 4 5
of
Boston,
bath
apparatus
2 ml o f
under
10 ml
containing
optical
Scientific,
and was
cells
s u s p e n s i o n was
were s t i r r e d
C water
precipitation
c o n c e n t r a t i o n was
et_ a_l_ ( 1 9 5 1 ) .
the
25 mm d i a m e t e r
scinti1lation
Analytical
E8B
were d r i e d
New E n g l a n d N u c l e a r
a final
T h e r e a c t i o n was
substrate
and w a s h i n g w i t h
filters
used.
i n a 30
substrate.
labelled
desired
give
(Bronwi11,
stirrer
10 m i n
for
radioactive
to
cel1
This
incubation mixtures
submergeable magnetic
Rochester,
medium.
incubation mixtures
660
at
s u c c i n a t e minimal
lactic
was e s t i m a t e d
by
the
40 mM; M g C l ,
reaction
2
5 mM;
proceed
in
the d i r e c t i o n
dehydrogenase
Tris
o f glutamate synthesis
a n d e x c e s s ammonia.
b u f f e r , pH 8.0,
i n the presence o f g l u t a m i c
The r e a c t i o n m i x t u r e c o n t a i n e d
40 mM; N H ^ C l , 200 mM;.NADH, 0.25
g l u t a m i c dehydrogenase
mM; c o m m e r c i a l
and t h e sample.
S u c c i n a t e was a s s a y e d m a n o m e t r i c a l l y u s i n g c r u d e
oxidase preparation
f r o m b e e f h e a r t ( U m b r e i t £t_ aj_,
succinic
1957).
F u m a r i c a c i d was a l s o a s s a y e d m a n o m e t r i c a l l y i n a c o u p l e d a s s a y
system
using commercial
fumarase;
Lactbbaci1lus arabinosus-culture
as a s o u r c e o f NAD, m a l i c enzyme a n d l a c t i c
S e m i c a r b a z i d e was a l s o
Lusty, T 9 6 3 ) .
included
XI.
(Sanger and
spectrophotometrically
m a l i c dehydrogenase
(Hohorst,
1963).
Chemi c a l s
Succinate-1,4International
was
i n the system
M a l a t e was d e t e r m i n e d
u s i n g NAD a n d c o m m e r c i a l
dehydrogenase.
14
C and s u c c i n a t e - 2 , 3
Chemical
-
14
C were the p r o d u c t s o f
and N u c l e a r C o r p o r a t i o n .
o b t a i n e d from Schwarz B i o r e s e a r c h Inc.
Glucose-U-
14
C
a-Methy1-D-gluco-
14
pyranoside-U-
C was t h e p r o d u c t o f N u c l e a r C h i c a g o C o r p .
c h e m i c a l s a n d enzymes u s e d
obtained
from commercial
throughout t h i s
sources.
study were
Other
similarly
RESULTS AND
I . Oxidation
DISCUSSION
o f T r i c a r b o x y l i c A c i d C y c l e arid R e l a t e d
I ri t e r m e d i a t e s
by Pseudomonas ae r u g i h o s a
Since
and o t h e r
cultures
i t was
proposed
dicarboxylic
were normally
s u s p e n s i o n s and c e l l
oxidized
succinate,
1 a n d 2).
higher
acids
extracts
fumarate
of
a-ketoglutarate
c i t r a t e only
as
this
utilized
a c e t a t e and p y r u v a t e
substrate
oxidation
these
substrates
relatively
of c e l l - f r e e
results
cultures
rate
similar
for
(Figs.
the
were o b t a i n e d
Washed c e l l
three
oxidized
extracts
whole
cells
rapidly, cell-free extracts
(Fig.
3).
These
or c o f a c t o r s ,
did
observations
required
were d e s t r o y e d
e x t r a c t , w h i l e those
was
with
suspensions
Although
the t r i c a r b o x y l i c a c i d cyc|e
undamaged.
cell
the t h e o r e t i c a l oxygen uptake
o f a c e t a t e and p y r u v a t e ,
of
from such
cycle,
Washed
and m a l a t e a t a r a p i d
immediately.
i n d i c a t e d t h a t t h e enzymes
preparation
medium.
a f t e r a l a g o f 60 m i n , w h i l e c e l l - f r e e
utilized
oxidation
prepared
Comparable
substrate.
succinate
the t r i c a r b o x y l i c a c i d
t h e c e l l - f r e e e x t r a c t a n d was
dicarboxylic acids.
not o x i d i z e
of
the u t i l i z a t i o n of
g r o w n on s u c c i n a t e
The p e r c e n t a g e
with
to study
for
the
during
required
intermediates
the
for
the
were
o
'
0
30
'
60
'
90
,
120
•
t
150
MINUTES
F i g . 1. Oxidation of s u c c i n a t e , fumarate. and malate by. c e l 1 suspensions •
of P.-aeruginosa. The Warburg vessels contained T r i s . b u f f e r , .
pH 7.4, 50 ymoles; c e l l suspension (5 mg dry weight); 7.5 ymoles
of substrate in a total.-volume-of 3.0 m l . Center wel 1 contained
0.15 ml of 20% KOH.c: Symbols: •., succinate; A , malate; 0., fumarate
I
0
60
120
180
240
MINUTES
Fig.
2.
O x i d a t i o n o f s u c c i n a t e , f u m a r a t e and m a l a t e by c e l l f r e e
e x t r a c t o f P. a e r u g i n o s a .
The W a r b u r g f l a s k s c o n t a i n e d
T r i s b u f f e r pH 7 . 4 ,
150 y m o l e s ; c e l l f r e e e x t r a c t a p p r o x .
30 mg p r o t e i n ; 7.5 y m o l e s s u b s t r a t e i n a t o t a l v o l u m e o f
3.0 m l .
C e n t e r w e l l c o n t a i n e d 0.15
ml o f 20% K0H.
Symbols:
• , s u c c i n a t e ; A, m a l a t e ; 6 ,
fumarate.-
MINUTES
. O x i d a t i o n o f a c e t a t e and p y r u v a t e by c e l l s arid c e l l - f r e e
e x t r a c t o f P. a e r u g i h o s a . The W a r b u r g v e s s e l s w e r e s e t
up as f o r F i g s . 1 and 2.
Symbols: open, c e l l s ; closed,',
c e l l - f r e e e x t r a c t ; • , a c e t a t e ; O,
pyruvate.
39
200 r
0
30
60
90
120
MINUTES
Fig', k.
•
O x i d a t i o n o f s u c c i n a t e , , f u m a r a t e and m a l a t e ; ; by r e s t i n g c e l l
s u s p e n s i o n o f P. a e r u g ? n o s a i n t h e . p r e s e n c e o f 1 mM s o d i u m .
arsenite.
Experimental c o n d i t i o n s ; w e r e s i m i l a r to those inF i g . 1.
S y m b o l s , • , s u c c i n a t e ; A, m a l a t e ; O,
fumarate.
Table
I.
Accumulation of keto a c i d s during the o x i d a t i o n o f
s u c c i n a t e , f u m a r a t e and m a l a t e i n t h e p r e s e n c e o f
1 mM s o d i u m a r s e n i t e .
Carbon source i n
the Warburg v e s s e l
/., . • , \
(7.5
ymole)
r
Keto acid,, accumulated
Pyruvate
.
... y m o l e s
1
a-ketogl utarate
,
y m o l e s ...
Succi nate
6.9
<0.2
Fumarate
6.5
<0.2
Malate
5.1
<0.2
In a s u b s e q u e n t
experiment
the o x i d a t i o n of the f i r s t
a r s e n i t e was
intermediate keto acid
of o x i d a t i o n of the d i c a r b o x y l i c
acids
( F i g . k).
showed t h a t t h e s e compounds w e r e o x i d i z e d
or pyruvate.
pyruvate
Paper
i n every
o f p y r u v a t e and
that
II.
i n the
prevent
pathway
Calculations
to either oxalacetate
c h r o m a t o g r a p h y showed t h e a c c u m u l a t i o n
case.
Table
I shows t h e r e l a t i v e
a-ketoglutarate.
o f p y r u v a t e as was
fumarate
and
of
accumulation
From t h e s e r e s u l t s
u n d e r t h e c o n d i t i o n s e m p l o y e d , s u c c i n a t e was
by way
i t appeared
oxidized
solely
malate.
G r o w t h Y i e l d s o f P_. a e r u g i n o s a w i t h Some T r i c a r b o x y l i c
Acid
Cycle
Intermediates
In a f u r t h e r a t t e m p t
differently
t o see
i f s u c c i n a t e was
than o t h e r d i c a r b o x y l i c a c i d s , growth
s u c c i n a t e , fumarate,
malate
and
Glucose
comparison
I I ) . To e n s u r e
(Table
and
i n the media were u t i l i z e d ,
were a n a l y z e d .
instance.
t o be
I t was
not attempted
in aerobic metabolism
with
t h a t a l l of the s u b s t r a t e s
supernatants
from
the growth
t h a t no s u b s t r a t e r e m a i n e d
(Campbel1 e t a l , 1956)
i n the present
accepted
yields
p y r u v a t e were i n c l u d e d f o r
Under s i m i l a r c o n d i t i o n s o f growth
c o m p l e t e l y used
I f one
found
degraded
a - k e t o g l u t a r a t e as s u b s t r a t e s
were d e t e r m i n e d .
was
employed t o
the statement
t h e n one
g l u c o s e was
and
media
i n any
found
i t s estimation
experiments.
t h a t energy
would expect
i s never
limiting
that succinate,
f u m a r a t e and
be
the
On
this
malate would g i v e
identical
growth y i e l d s f o r i t would
amount o f c a r b o n t h a t w o u l d d e t e r m i n e t h e
basis
succinate
metabolism that
are
However, i f energy
will
be
This
latter
not
capable of entering
s h a r e d by
is limiting
a f u n c t i o n of
situation
must be
the
f u m a r a t e and
i n an
state of
aerobic
t e s t e d when e x p r e s s e d on
avai l a b l e electrons.
the
The
basis
malate
of
the
operative
of y i e l d
average value
per
f o r gms
3.26
Therefore,
under these c o n d i t i o n s
observations
t h o s e o f M a y b e r r y et_ a l _ (1967) , who
or per
gm
atom o f
the
substrate
cells
per
equivalent
energy a v a i l a b l e from the
constant
the
end
anything
-aerobic
regardless
products are
i n the
present
substrates
per
(Table
are
of
ll).
i n agreement
that
in
the
mole o f
the
substrate
carbon or per
mole o f
the
oxygen
a constant
the y i e l d
and
was
per
close
equivalent
t o 3.14
gm
of a v a i l a b l e e l e c t r o n s .
It would appear that
of
the
growth y i e l d
substrate.
route
C t ^ and
H^O)
yield
per
consumed v a r i e d o v e r a w i d e r a n g e w h i l e
o f a v a i l a b l e e l e c t r o n s was
± 0.1
reported
c a s e o f b a c t e r i a grown a e r o b i c a l l y , y i e l d
II).
substrate.
of c e l l s
o f a v a i l a b l e e l e c t r o n s was
the
of
(Table
equivalent
equivalent
with
pathways
s i m i l a r f o r al1
growth y i e l d s are
yield.
system then growth
reduction
s i t u a t i o n w o u l d a p p e a r t o be
f o r the
growth
is a function of
Since
this
of degradation
, one
i s not
about p o s s i b l e d i f f e r e n c e s
i n routes
b a c t e r i a by m e a s u r i n g g r o w t h
yields.
the
is essentially
(assuming
going
to
that
learn
of metabolism
in
of
Table
II.
Carbon
source
Growth y i e l d
o f P. a e r u g i n o s a f r o m v a r i o u s c a r b o n s o u r c e s .
Equivalent to
available electrons
per mole
M o l a r growth
y i e l d (gm/mole)
Gm/equivalent
of available
electrons
Gm/gm
atom
of carbon
Glucose
24
79.0
3.29
13.2
Succinate
14
44.2
3.16
11.0
Fumarate
12
40.3
3.36
10.1
Malate
12
40.3
3.36
10.1
a-Ketoglutarate
16
51.1
3.19
10.2
Pyruvate
10
32.0
3.20
10.7
III.
Studies
All
with
Mutants
a t t e m p t s t o i s o l a t e m u t a n t s o f P. a e r u g i n o s a
t o g r o w on f u m a r a t e b u t c a p a b l e o f g r o w t h on s u c c i n a t e
failure.
M u t a n t s u n a b l e t o grow o n f u m a r a t e w e r e a l s o
9027
unable
met w i t h
incapable
o f g r o w t h on s u c c i n a t e .
The
r e s u l t s presented
to.support
the suggestion
utilization.
utilization
succinate
above d i d n o t p r o v i d e
t h a t t h e r e was a u n i q u e p a t h w a y o f s u c c i n a t e
Hence, f u r t h e r s t u d i e s were f o c u s s e d
of tricarboxylic acid cycle
pseudomonads t h a n
IV.
o n . t h e mode o f
intermediates
with
as t h e example t o e l u c i d a t e t h e r o l e and c o n t r o l o f t h e
t r i c a r b o x y l i c acid c y c l e which obviously
forming
any e v i d e n c e
bacilli
seems t o be d i f f e r e n t i n
i n f a c u l t a t i v e o r g a n i s m s and a e r o b i c
as o u t l i n e d
spore
earlier.
L a c k o f NAD o r NADP D e p e n d e n t L - M a l i c
Dehydrogenase i n .
P. a e r u g i n o s a
P r e l i m i n a r y s t u d i e s on t h e u t i l i z a t i o n
other
o f s u c c i n a t e and
dicarboxylic acids of the t r i c a r b o x y l i c acid
indicated
that c e l l - f r e e
e x t r a c t s , without
coenzymes, o x i d i z e d s u c c i n a t e ,
cycle
the addition of
f u m a r a t e and m a l a t e a t a r a p i d
rate
( F i g . 2) and a l m o s t
to completion.
However, s e v e r a l
attempts
t o d e t e c t NAD
o r NADP d e p e n d e n t L - m a l i c d e h y d r o g e n a s e i n t h e
free extracts
and
i n t h e 100,000 x g_ s u p e r n a t a n t w e r e u n s u c c e s s f u l .
Since a e r o b i c organisms
possess
a relatively
p a r t i c u l a t e ' o r membrane b o u n d enzymes
25,000 x g_ and
l a r g e number o f
( M a r r , 1960;
Smith,
100,000 x g_ p e l l e t s w e r e a l s o s c r e e n e d
1961),
f o r the
p r e s e n c e o f t h i s enzyme w i t h t h e same n e g a t i v e r e s u l t s .
ensure
that the procedure
of preparation of c e l l - f r e e
and
t h e a s s a y o f t h e enzyme w e r e n o t a t f a u l t ,
was
prepared
from A e r o b a c t e r aerogenes
and
showed a v e r y h i g h a c t i v i t y o f NAD
(specific activity,
V.
assayed
A.
linked
aerogenes
c o n f i r m i n g t h a t the procedure
being
S ? te of Malate Oxidizing A c t i v i ty
the f o r m a t i o n of o x a l a c e t i c a c i d
to oxalacetate.
acid
is essential
c y c l e , malate
a "carrier
type" experiment
washed c e l l
s u s p e n s i o n and
Succinate-2,3-
14
for
must
To show t h a t o x a l a c e t a t e i s i n d e e d
a p r o d u c t d u r i n g o x i d a t i o n o f m a l a t e by t h e c e l l s
.employed.
f o r NAD
extract
dependent L - m a l i c dehydrogenase
the f u n c t i o n i n g of the t r i c a r b o x y l i c
be o x i d i z e d
extracts
reliable.
D e t e c t i b h and
Since
3410)
To
a eel 1-free
L - m a l i c d e h y d r o g e n a s e u n d e r t h e same c o n d i t i o n s .
f o l l o w e d was
cell-
was
carried
out
o f P_. a e r u g i n o s a ,
( K r a m p i t z , 1961).
c o n v e n t i o n a l Warburg t e c h n i q u e were
C was
tipped
in at zero time, while
A
o x a l a c e t a t e was t i p p e d
i n from t h e second s i d e arm a few m i n u t e s
60 u4 o f o x y g e n had b e e n consumed a s a
l a t e r , when a p p r o x i m a t e l y
F i v e t o 10 m i n a f t e r
result of succinate oxidation.
of unlabel led oxalacetate, the reaction mixtures
prepared
and chromatographed.
identified
as
i n a l l the flasks
the i n i t i a l
substrate.
enzyme) was i n c l u d e d
of
receiving radioactive succinate
I f 3 - 3 mM EDTA (an i n h i b i t o r o f m a l i c
i n the reaction mixtures,
from L - m a l i c
to determine
i fthis
a direct
(Table
I I I ) and
Since
pyruvate
sequence o f o x i d a t i v e d e c a r b o x y l a t i o n
species
During
f o roxalacetate
formation
then
as observed
( F u l l e r and K o r n b e r g , I96I) o r whether
s t u d i e s on t h e o x i d a t i o n o f L - m a l i c
o f P s e u d o m o n a s , F r a n c i s ' e t ' a l (1963)
o f Pseudomonas
B^, a b a , p o s s e s s i n g
w h i l e e x t r a c t s o f P. o v a l i s C h e s t e r ,
dehydrogenase, c a t a l y z e d
oxidation of this
substrate.
found
a c i d by two
that extracts
s o l u b l e NAD d e p e n d e n t L - m a l i c
dehydrogenase, o x i d i z e d L-malate very
L-malic
oxalacetate
o x i d a t i o n by a p a r t i c u l a t e m a l i c d e h y d r o g e n a s e was
involved.
species
by w h i c h
a n d R e m b e r g e r , 1 9 6 1 ) , an a t t e m p t was made
r e c a r b o x y l a t i o n was u t i l i z e d
i n a Chroma H u m
accumulation
( F r a n c i s e t a l , 1963)•
acid
pseudomonads p o s s e s s t h e m a l i c enzyme
(Seubert
higher
be d e m o n s t r a t e d .
There a r e several a l t e r n a t i v e routes
carboxylase
were removed, hyd
R a d i o a c t i v e o x a l a c e t a t e was
radioactive oxalacetate could
c o u l d be f o r m e d
the addition
s l o w l y and
incompletely
possessing p a r t i c u l a t e
t h e r a p i d and a l m o s t
This
complete
suggested a s i m i l a r i t y i n
t h e enzyme c o m p l e m e n t o f P. a e r u g i n o s a
9 0 2 7 a n d P. o v a 1 i s
Chester
since
the c e l l - f r e e extract preparations
malate at a rapid
rate.
o f t h e former a l s o
And i n d e e d i t was f o u n d t h a t
does p o s s e s s t h e p a r t i c u l a t e L - m a l i c dehydrogenase
Oxalacetic
a c i d and p y r u v i c
a c i d were i d e n t i f i e d
reaction mixtures f o rp a r t i c u l a t e malic
malic
enzyme r e s p e c t i v e l y . To r e d u c e
e n d o g e n o u s NAD o r NADP w h i c h m i g h t
cell-free extracts
comparable
a c t i v i t y was
In a n o t h e r a t t e m p t
P. a e r u g i n o s a
(Table I I I ) .
i n t h e assay
dehydrogenase
interference
and
due t o
be p r e s e n t i n c r u d e
e x t r a c t s , m a l a t e o x i d i z i n g a c t i v i t y was a l s o a s s a y e d
treated
cell-free
i n charcoal
( H o c h s t e r a n d S t o n e , 1956) a n d
demonstrated.
t o assess the importance o f the
p a r t i c u l a t e malate o x i d i z i n g system, L-malate o x i d a t i o n
cell-free
The
extract
initial
74.6%
a n d t h e 1 0 0 , 0 0 0 x g_ p e l l e t was s t u d i e d
o f t h e t h e o r e t i c a l oxygen
i t slowly
required
f o rcomplete o x i d a t i o n .
oxidation
by t h e p e l l e t .
revealed
C a l c u l a t i o n showed t h a t when
l e v e l l i n g o f f , a p p r o x i m a t e l y 0.5
Analysis
indicated
of the reaction mixture f o r keto
as t h e m a i n p r o d u c t a n d p y r u v a t e as
T h i s amount o f p y r u v a t e c o u l d
the non-enzymatic
This
either to oxalacetic acid or pyruvic
oxalacetate
a minor p r o d u c t .
of
1 7 . 8 % o f t h e amount o f o x y g e n
p e r u m o l e o f m a l a t e was t a k e n up.
t h a t m a l a t e was o x i d i z e d
acids
(Fig. 5).
uptake, whereas t h e resuspended p e l l e t
and consumed o n l y
curve started
umole o f oxygen
acid
i n the
c e l l - f r e e e x t r a c t a c t i v e l y o x i d i z e d m a l a t e a n d consumed
oxidized
the
oxidized
breakdown o f o x a l a c e t a t e .
be t h e r e s u l t
Table
III.
P a r t i c u l a t e m a l i c d e h y d r o g e n a s e and m a l i c
i n P. a e r u g i n o s a
Preparation
Particulate malic
. dehydrogenase
enzyme
NADP m a l i c
enzyme
NAD m a l i c
enzyme
Specific.Activity
Cell-free
extract
100,000 x £ p e l l e t
100,000 x g_ s u p e r n a t a n t
17.1
574.0
24.3
n i l
nil
972.0
133.0
nil
194.0
C e n t r i f u g e d a t 100,000 x £ f o r 90 m i n t w i c e .
If centrifugation
was c a r r i e d o u t o n l y o n c e , a b o u t 10% d e h y d r o g e n a s e a c t i v i t y
remained- i n the supernatant.
400
3OO
<
h-
200
z
LU
O
>-
X
°
100
60
MINUTES
Fig.
5.
•
90
120
' O x i d a t i o n o f L-mal i c a c i d by (A) e e l 1 - f r e e e x t r a c t and .(B)
100,00Q\ x £ p e l l e t o f P. a e r u g i n o s a . / The r e a c t i o n m i x t u r e
c o n t a i n e d 250 y m o l e s o f p o t a s s i u m p h o s p h a t e buffer,\ .pR
7.2;
. 2.0 ml c e l l - f r e e e x t r a c t o r p e l l e t s u s p e n s i o n ; 7.5 y m o l e s
o f m a l a t e i n a t o t a l / v o l u m e o f 3.0 m l .
The c e n t e r wel1 o f
t h e W a r b u r g v e s s e l c o n t a i n e d 0.15
ml o f . 20%
KOH.
:
These o b s e r v a t i o n s
show t h a t
the malic
dehydrogenase i s
p a r t i c u l a t e and i s p r o b a b l y s i m i l a r i n d i s t r i b u t i o n
( F r a n c i s e t _ a j _ , 1963).
P. O v a l i s C h e s t e r
not
exclude the p o s s i b i l i t y
ribosomes.
the
To r e s o l v e
this
that
5 x 10\
then c e n t r i f u g e d
resuspended
much h i g h e r
total
t h a n t h e c o r r e s p o n d i n g p e l l e t , was t r e a t e d
H_ EDTA and a l l o w e d
J
associated
t o stand
with
a 2 5 , 0 0 0 x g_ s u p e r n a t a n t o f
c e l l - f r e e e x t r a c t , which contained
o f t h e enzyme
However, t h e d a t a d i d
t h e enzyme.was
question
to that i n
a t 0 C f o r 15 m i n .
a t 1 0 0 , 0 0 0 x g_ f o r 90 m i n .
i n a s u i t a b l e volume o f 0.25
The p e l l e t
activity
with
I t was
was
M_ T r i s b u f f e r , pH
7.4.
"3
Portions
of this
EDTA a n d 500 ug p a n c r e a t i c
15 C f o r 4 h r .
to avoid
and
t h e p e l l e t was w a s h e d o n c e w i t h
so obtained
IV shows t h a t
this
the ribonucleic acid
A t t h e end o f t h e r e a c t i o n
a t 1 0 0 , 0 0 0 x g_ f o r 90 m i n
0.25
resuspended
M_ T r i s b u f f e r , pH
enzyme
7-4.
i n t h e same b u f f e r .
from the p e l l e t
I t i s seen t h a t
indicating
there
t h e enzyme h a v i n g t h e same d i s t r i b u t i o n
or being
used
Table
t r e a t m e n t was q u i t e e f f e c t i v e i n r e m o v i n g most
ribosome d e g r a d a t i o n .
of
was
M_
( W o r t h i n g t o n ) p e r ml a t
The l o w t e m p e r a t u r e o f i n c u b a t i o n was
t h e m i x t u r e was c e n t r i f u g e d
The p e l l e t
of
ribonuclease
i n a c t i v a t i o n o f t h e enzyme.
period,
5 x 10
pel l e t s u s p e n s i o n were t r e a t e d w i t h
solubilized.
Therefore,
i s not associated
with
i s no i n d i c a t i o n
as r i b o n u c l e i c a c i d
i t i s concluded
ribosomes.
extensive
that
this
Table
IV.
E f f e c t o f EDTA a n d r i b o n u c l e a s e t r e a t m e n t o n t h e
d i s t r i b u t i o n o f p a r t i c u l a t e m a l i c dehydrogenase o f
f_. a e r u g i n o s a
.j.
i
^ ...
Total protein
Enzyme a c t i v i t y
.
7
No.
Fraction
7
I
1
(mg)
I
O.D.
I
sp. a c t . total a c t .
280/260
P31 IO
(1)
1 0 0 , 0 0 0 x g_ p e l l e t
26.6
24.3
646.0
0.67
(2)
(1) a f t e r EDTA and
RNAse t r e a t m e n t
8.1
16.1
T30.5
0.688
100,000 x £ pel l e t
o f (2)
5.8
19.85
115.0
0.966
7-3
0.607
(3)
(4) 1 0 0 , 0 0 0 x £
s u p e r n a t a n t o f (2)
5V
2.5"
2.9
A f t e r c o r r e c t i n g f o r t h e q u a n t i t y o f RNAse a d d e d .
U l t r a v i o l e t s p e c t r a f r o m 340 t o 230 mu o f a l l t h e f r a c t i o n s ;
w e r e r e c o r d e d i n Beckman S p e c t r o p h o t o m e t e r model DB-G.
52
VI .
L a b e l 1ing
The
malic
but
as
f r o m S u c c i nate-1 ,4-
above e x p e r i m e n t s
i n d i c a t e d the
C and
S u c c i nate-2,3~
involvement of p a r t i c u l a t e
dehydrogenase i n the d i r e c t o x i d a t i o n of malate t o
d i d not
exclude oxidative decarboxylation
a sequence of
malate.
To
activity
i s the
reactions
f o r the
vessels with
important
The
system
r e a c t i o n was
NaHCO^ and
substrates.
i n the
The
reaction mixture
( F i g . 6).
r e c y c l i n g of c i t r a t e ,
Oyketoglutarate
the
pH
and
The
other
35%
(see
mutant c e l l s
Warburg
oxygen -
P.
aeruginosa
aeruginosa
oxidized
5%
possess
in w i l d type
the
of
lacking
M32,
was
reaction
a-ketoglutarate.
a-ketog1utaric
t r i c a r b o x y l i c a c i d c y c l e and
than
C
succinate
s u p e r n a t a n t s of the
showed a c c u m u l a t i o n o f
enzymes a r e a t a l o w e r l e v e l
footnote
( U m b r e ? t e t a l , 1957).
t h e m u t a n t o f P.
T a b l e V shows t h a t t h i s m u t a n t d o e s n o t
d e h y d r o g e n a s e and
oxalacetate
e l i m i n a t e the p o s s i b i l i t y
a n a l y s i s o f the
for keto acids
i n the
dehydrogenase
found to accumulate i n
dehydrogenase, designated
( F i g . 7).
incompletely
To
of
from
C or succinate-2,3-
f l a s k s were gassed w i t h
U n d e r t h e s e c o n d i t i o n s , c i t r a t e was
mixtures
of o x a l a c e t a t e
formulated
succinate-1,4-
oxalacetate,
recarboxylation
formation
c a r r i e d out
carbon d i o x i d e mixture to maintain
a l s o used
formation
a c i d a n o t h e r e x p e r i m e n t was
of Table VI).
slight
and
c o n c l u s i v e l y show t h a t p a r t i c u l a t e m a l i c
from L-malic
as
of C?trate
cells
related
C
citrate
I-
F i g . 6.
ETHER-FORMIC ACID-WATER
Autoradiogram o f the t h i n - l a y e r chromatograph from the
r e a c t i o n m i x t u r e c o n t a i n i n g e e l 1-free e x t r a c t o f P_.
'aeruginosa-wild
type and r a d i o a c t i v e s u c c i n a t e .
E x p e r i m e n t a l c o n d i t i o n s w e r e t h e same a s i n T a b l e VI.
54
•C-k-gl
citrate
ETHER-FORMIC ACID-WATER
Fig.
7.
A u t o r a d i o g r a m o f the t h i n - l a y e r chromatograph from t h e
r e a c t i o n m i x t u r e c o n t a i n i n g c e l l - f r e e e x t r a c t o f R.
a e r u g ? n o s a M32 a n d r a d i o a c t i v e s u c c i n a t e .
Experimental
c o n d i t i o n s w e r e t h e same a s i n T a b l e V I . a - k - g l =
a-ketoglutarate.
Table
V.
The t r i c a r b o x y l i c a c i d
P. a e r u g i n o s a
M32
Enzyme
cycle
and r e l a t e d
Specific Activity
Fumarase
380.0
Particulate malic
dehydrogenase
6.9
Soluble malic
dehydrogenase
n i l
Aconitase
3 5 . 5
I s o c i t r a t e dehydrogenase
1 4 6 . 0
a-Ketoglutarate dehydrogenase
n i l
NADP m a l i c enzyme
259.0
NAD m a l i c enzyme
5 7 . 0
mumoles s u b s t r a t e u t i l i z e d
enzymes o f
p e r m i n p e r mg o f
protein.
(Table
IX).
occurred
C i t r a t e accumulation
t o a much l e s s e r e x t e n t
a c o n i t a s e and
was
isocitric
almost completely
and
t h e enzyme f o r 15
Apparently
t h i s was
accumulation
The
14
before
mM
of c i t r a t e ,
i s not
The
the
assay.
accumulation.
The
specific activity
i n much
clear.
a c i d formed from s u c c i n a t e -
c l o s e to double that obtained
(Table V l ) .
type
in contact
i n the mutant s t r a i n , which r e s u l t s
s p e c i f i c a c t i v i t y of c i t r i c
C was
NaHCO^
i n both w i l d
starting
the cause o f c i t r a t e
mechanism o p e r a t i n g
lower
min
of
aconitase
i n t h e p r e s e n c e o f 21
i n the Warburg v e s s e l s )
t o 20
substrate
Determinations
t h e m u t a n t c e l l - f r e e e x t r a c t s , i f b i c a r b o n a t e was
with
2,3"
i n M32.
the
dehydrogenase i n d i c a t e d t h a t
inhibited
(the c o n c e n t r a t i o n used
w i t h s u c c i n a t e as
14
from s u c c i n a t e - 1 , 4 -
of c i t r a t e obtained
C
from
14
s u c c i n a t e - 1 ,4f r o m 84
t o 87
expected
was
C was
percent
close to that of s t a r t i n g
of that value.
i n the presence o f excess b i c a r b o n a t e
as shown i n F i g s . 8a
o x i d a t i v e d e c a r b o x y l a t i o n and
any
These r e s u l t s
d i r e c t l y o x i d i z e d t o o x a l a c e t a t e and
formation
and
8b.
then
These data
specific a c t i v i t y of starting
be
i f malic acid
utilized
r e c a r b o x y l a t i o n as
from s u c c i n a t e - 1 ,4-^C
could
only
importance f o r o x a l a c e t a t e formation, s i n c e
c i t r a t e obtained
substrate, varying
for
citrate
eliminate
a mechanism o f
in this
w o u l d h a v e one
situation
half
the
substrate while c i t r a t e obtained
from
14
succinate-2,3
C w o u l d show d o u b l e t h e s p e c i f i c a c t i v i t y o f
s t a r t i n g s u b s t r a t e w h i c h i s f o u r times t h a t of c i t r a t e formed
-
14
from s u c c i n a t e - 1 , 4 Thus, the data
C.
presented
h e r e have d e m o n s t r a t e d
the
involvement
Table VI.
Specific a c t i v i t y of radioactive c i t r a t e formed from succinate-1,4and succinate-2,3 C
C
-
T
e of C e l l .
.
free extract
£
P_. aeruginosa
9027 W+
c
Citrate formed
per Warburg
^
. •
vessel
(ymoles)
, .
Substrate
3
v
K
...
succinate-1,4-
C
2.91
6.85
84.0
succinate-,2,3-
C
3.18
11.85
145.5
succinate-1,4-
C
0.70
7.07
87.O
c
0.76
13.60
162.0
14
P_. aerug?nosa
9027 M32
Sp. Act.
Sp.Act. as % of Sp.
, ,
,
(cpm/ymole
Act. of succinate
x.10'5)
.
at zero time
14
succinate-2,3
-
The reaction mixture in Warburg vessels contained T r i s buffer, pH 7.5, 50 mM; c e l l free extract equivalent to approximately 30 mg protein; NaHCO,, 21 mM; succinate1,4-^C or s u c c i n a t e - 2 , 3 ^ C , 7.5 ymoles (5 yc) in a total volume of 3.15 ml.
KOH was not put in the center w e l l . Atmosphere^ a mixture of 95% O2 and 5% CO2.
The reaction was stopped a f t e r 120 min. Zero time samples were prepared by
addition of HC10. before adding succinate to the reaction mixture.
-1
OH
COOH
H C-C OOH
2
HC-COOH
HC-COOH
H C-COOH
HC-COOH
H, C-COOH
2
HC-COOH
II
HC-COOH
H.C-COOH
r
H C - COOH
2
y
COOH
0
_ . i .
/ '
C
H
=
0
3
^co„
CH,-CO~S -CoA
• 3 •
0=-C —COOH
0=C-COOH
I
H C-COOH
2
COOH
I
HOOC-HoC-C-OH
I
H C-COOH
2
2
8a.
H C-COOH
H, C —COOH
2
COOH
•
COOH
I
HOOC-H C-C-OH
HOOC-H
•J
2
C-C-OH
H C-COOH
P
2
H C-COOH
2
C i t r a t e f o r m a t i o n f r o m s u c c i n a t e - 1 ,h- C
R a d i o a c t i v e c a r b o n i s i n d i c a t e d by a s o l
c i r c l e (•).
Fig.
8b.
Citrate formation
Radioactive carbon
cTrcle (•).
from s u c c i n a t e - 2 , 3 i s indicated
C.
by a s o l i d
VJI
oo
o f t h e NAD, NADP i n d e p e n d e n t p a r t i c u l a t e m a l i c d e h y d r o g e n a s e
in
t h e o x i d a t i o n o f m a l a t e t o o x a l a c e t a t e and t h e p a t t e r n o f
labelling
of citrate
o b t a i n e d from s u c c i nate-1 ,4-^C
and
14
succinate-2,3"
VII.
C has e l i m i n a t e d
any o t h e r p o s s i b i 1 i t y .
O x i d a t i o n o f S u c c i n a t e , Fumarate
a n d M a l a t e by a C e l 1 - F r e e
E x t r a c t o f A. a e r o g e n e s ,
S i n c e t h e r e a r e d i f f e r e n c e s between t h e m a l a t e
systems
o f P. a e r u g i n o s a and A. a e r o g e n e s , t h e o x i d a t i o n o f
dicarboxylic acids
was a l s o s t u d i e d
oxidized
and
in cell-free
( F i g . 9).
extracts of the latter
A cell-free extract
t h e s e , compounds o n l y v e r y s l o w l y .
fumarate were u t i l i z e d
is j u s t
oxidizing
Initially
These f i n d i n g s
a r e i n agreement w i t h
Jones
(1968) who h a v e r e p o r t e d
i n t h e Warburg v e s s e l s
t h a t a t 60 m i n , 4.1 y m o l e s
reaction mixture.
malate
the observations o f .
such
receiving
differences
Determination
succinate
o f malate accumulated
showed
i n t h e A_. a e r o g e n e s
A l t h o u g h P. a e r u g i n o s a c e l l - f r e e
extract
showed some m a l a t e a c c u m u l a t i o n , t h e amount was o n l y 0.57
per
also
ymoles
vessel.
The
that
which
P. a e r u g i n o s a .
b e t w e e n pseudomonads a n d t h e c o l i f o r m g r o u p .
of malate
aerogenes
a t a slower rate than s u c c i n a t e
the opposite t o the s i t u a t i o n with
and K i n g
o f A.
organism
pattern o f oxidation o f these d i c a r b o x y l i c acids
P. a e r u g i h o s a h a s an a d v a n t a g e
o v e r A. a e r o g e n e s
indicates
because
60
0
60
120
MINUTES
Fig.
v
9.
180
240
,
O x i d a t i o n o f s u c c i n a t e , f u m a r a t e and m a l a t e by
e x t r a c t s . o f A . . a e r o g e n e s . The r e a c t i o n m i x t u r e s
s e t up as i n F i g . 2.
S y m b o l s ; • , s u c c i n a t e ; A,
•'. O, f u m a r a t e .
cell-free
were
malate;
•
t h e l a t t e r o r g a n i s m d e p e n d s on t h e a v a i l a b i l i t y
the o x i d a t i o n o f malate.
aerogenes
obviously
The u t i 1 i z a t i o n
is a limiting
i n pseudomonads and c o l i f o r m s
s u c c i n i c oxidase system.
step.
o f m a l a t e by A_.
Oxidation of succinate
i s catalyzed
This
o f f r e e NAD f o r
by t h e p a r t i c u l a t e
could explain
the rapid
oxidation
o f s u c c i n a t e by t h e c e l l - f r e e e x t r a c t s o f b o t h g r o u p s o f o r g a n i s m s
The m a l a t e o x i d a s e s y s t e m has b e e n shown t o c o n t a i n
p r o s t h e t i c group
1966)
i n P. o v a l i s C h e s t e r ( P h i z a c k e r l e y a n d
and A c e t o b a c t e r x y l i h u m ( B e n z i m a n
initial,
rapid
FAD a s t h e
and G a l a n t e r , 1 9 6 4 ) .
o f A_. a e r o g e n e s
c a n be a s c r i b e d
the p a r t i c u l a t e malate oxidase system.
i n the c e l l
t o the possession o f
Thus, organisms
possessing
particulate
L - m a l i c dehydrogenase
are b e t t e r equipped f o r
utilization
of dicarboxylic acids
since their
compounds
of free
VIII.
The
r a t e o f o x i d a t i o n o f m a l a t e and f u m a r a t e o b s e r v e d
i n t h e c e l l - f r e e e x t r a c t o f P. a e r u g i n o s a and n o t s e e n
free extract
Francis,
i s not rate
limited
activity
on t h e s e
by t h e l a c k o f a c o n s t a n t s u p p l y
NAD.
The Enzymes o f C a r b o h y d r a t e M e t a b o l i s m i h P_. a e r u g i n o s a
Grown
i n S u c c i n a t e o r Glucose Media
The pseudomonads p o s s e s s a g r e a t c a p a c i t y
nutritional
activity
environment.
That the t r i c a r b o x y l i c
i s c o n s t i t u t i v e and i s o f s p e c i a l
metabolism
t o adapt t o t h e i r
i n these organisms
acid
cycle
importance i n the
i s s u p p o r t e d by t h e s t u d y o f t h e
p a t t e r n o f u t i l i z a t i o n o f s u c c i n a t e and
The
organisms
t h e y showed a
g r o w n on s u c c i n a t e d i d s o w i t h o u t a l a g , w h i l e
l o n g l a g i n g l u c o s e medium
medium c o n t a i n i n g b o t h
was
o b t a i n e d and
observed
glucose.
s u c c i n a t e and
initially
( F i g . 10).
In
the
g l u c o s e , the d i a u x i c
the growth
p a t t e r n resembled
growth
that
i n s u c c i n a t e medium.
Figure
11
s u c c i n a t e and
glucose.
shows t h e g r o w t h
pattern in glucose, glucose
s u c c i n a t e media o f organisms
T h e r e was
no d i a u x i c g r o w t h
on
no p r o n o u n c e d
p r e v i o u s l y grown
l a g on s u c c i n a t e medium
plus
on
and
t h e medium c o n t a i n i n g g l u c o s e p l u s
succi nate.
Very
s i m i l a r p a t t e r n s w e r e o b t a i n e d when r e s t i n g
cell
suspensions, harvested
from g l u c o s e o r s u c c i n a t e media,
t h e s e compounds
12 and
(Figs.
i n g l u c o s e medium o x i d i z e d
theoretical
oxygen uptake
13).
The
extracts
a t 240
min,
( F i g . 14).
1 Umole o x y g e n was
of the
i n t h e a b s e n c e o f a d d e d c o f a c t o r s , h o w e v e r , was
level
is oxidized
(Campbel1 e t a l , 1 9 5 6 ) .
g r o w n i n s u c c i n a t e medium o x i d i z e d
the weaker a c t i v i t y
The
but d i d not o x i d i z e
This behaviour
s i n c e under these c o n d i t i o n s g l u c o s e
gluconate
e x t r a c t o f the c e l l s
indicated
The
the
glucose
consumed
cell-free
t o be
o n l y t o the
e x t r a c t of the
expected
2-ketocells
glucose very slowly suggesting
of glucose oxidase
above r e s u l t s
grown
s u c c i n a t e r a p i d l y consuming 65% o f
beyond t h e s t a g e where a p p r o x i m a t e l y
per umole o f g l u c o s e
oxidized
system.
t h a t t h e enzymes n e c e s s a r y
the u t i l i z a t i o n o f s u c c i n a t e were p r e s e n t
in cells
grown on
for
either
63
I
O
H
0
«
120
•
240
360
480
Minutes
• F i g . 10.
•
G r o w t h o f s u c c i n a t e grown i n o c u l u m i n t h e m e d i a c o n t a i n i n g '
succinate, glucose or succinate + glucose.
Log/phase
e e l I s f r o m s u c c i n a t e m i n i m a l medium w e r e h a r v e s t e d , washed
o n c e and i n o c u l a t e d i n t h e m e d i a .
! Incubation'.'was. a t 37 C
w i t h s h a k i n g . . S y m b o l s : O, 0 . 2 % . s u c c i n a t e ; # , 0 . 2 % g l u c o s e ;
• , 0.04% s u c c i n a t e + 0.2% g l u c o s e . .
10
«—
0
120
»
240
Minutes
11.'
.
>
i
360
480
\,
G r o w t h o f g l u c o s e grown i n o c u l u m ' i n m e d i a c o n t a i n i n g g l u c o s e ,
succinate or glucose + succinate.
Experimental conditions
w e r e t h e same as i n F i g . 10.. S y m b o l s : O, 0.2% s u c c i n a t e ;
: •,' 0.2% g l u c o s e ; •, 0.04% g l u c o s e + 0.2% s u c c i n a t e .
60
120
180
240
INUTES
Fig.
12.
Oxidation of succinate, glucose or a mixture of succinate
and g l u c o s e by c e l l s h a r v e s t e d f r o m a s u c c i n a t e m i n i m a l
medium. The W a r b u r g v e s s e l s w e r e s e t up as i n F i g . .1.
S y m b o l s : • , s u c c i n a t e , 7.5 p m o l e s ; A, g l u c o s e , 5.0 y m o l e s ;
O,
s u c c i n a t e , 3.75
y m o l e s + g l u c o s e , 2.5
ymoles.
66
0
20
40
60
80
100
120
MINUTES
Fig.
13'.;V O x i d a t i o n o f s u c c i n a t e , g l u c o s e o r a: m i x t u r e o f . s u c c i n a t e ' +
g l u c o s e by c e l l s h a r v e s t e d f r o m a g l u c o s e medium.
The
e x p e r i m e n t a l c o n d i t i o n s and t h e s y m b o l s u s e d a r e s i m i l a r t o
t h o s e i n F i g . 12.
140
400
r
0
60 .
120
180
240
MINUTES
.14.
O x i d a t i o n o f g l u c o s e ;and s u c c i n a t e b y . t h e e e l 1 - f r e e e x t r a c t s
f r o m t h e . c e l l s grown i n g l u c o s e (o) o r s u c c i n a t e (•) m e d i a .
The W a r b u r g v e s s e l s w e r e s e t up a s i n F i g . 2.
Symbols:
open.,, r e c e i v i ng 7 i 5 . y m o l e s s u c c i n a t e ; c l o s e d , r e c e i v i n g
5.0 y m o l e s g l u c o s e .
g l u c o s e o r s u c c i n a t e medium, w h e r e a s t h e enzymes o f g l u c o s e o x i d a t i o n
were e i t h e r
medium.
low o r n o t d e t e c t a b l e i n c e l l s
The a b s e n c e o r p r e s e n c e
harvested
from
o f o n l y a low l e v e l
the succinate
o f t h e enzymes
o f g l u c o s e o x i d a t i o n i n p s e u d o m o n a d s , when grown on
tricarboxylic
acid
(Hamilton
and
c y c l e i n t e r m e d i a t e s , has b e e n r e p o r t e d e a r l i e r
Dawes,
1960; Von T i g e r s t r o m and C a m p b e l l ,
Dawes, 1967;
metabolism
1967a), b u t t h e d e t a i l e d
L e s s i e and N e i d h a r d t ,
study o f the level
o f t h e enzymes
has n o t been c a r r i e d
regulating
out.
grown
1.
metabolism
i n s u c c i n a t e o r g l u c o s e medium was
Glucose
degrading
o r at a very
these pathways would
attempted.
t h e enzymes o f t h e
p a t h w a y and t h e o x i d a t i v e p o r t i o n o f p e n t o s e
were e i t h e r absent
low l e v e l
(Table V I I ) .
that
I t h a s b e e n r e p o r t e d t h a t t h e Embden-
has n o t been d e f i n e d .
the organism
This
f u n c t i o n i n g o f t h e Embden-Meyerhof pathway.
a s s a y was c h e c k e d
but the
investigation
lacks phosphofructokinase,
phosphofructokinase
Hence,
be u n a v a i l a b l e f o r t h e o x i d a t i o n o f
by r e c y c l i n g .
reason
Entner-Doudoroff
phosphate pathway
M e y e r h o f p a t h w a y i s n o t o p e r a t i v e i n P. a e r u g i n o s a
actual
in
enzymes
In s u c c i n a t e g r o w n c e l l s
substrates
the pattern o f
Therefore, the determination
o f t h e l e v e l s o f t h e enzymes o f c a r b o h y d r a t e
cells
1966b; Ng a n d
thus
has
shown
preventing the
Validity
by p r e p a r i n g a
of
cell-free
Table VI I.
A c t i v i t y of some enzymes of carbohydrate metabolism
in P. aerugi nosa grown in succinate or glucose media.
Speci f ? c Act ? vi ty
Enzyme
Succinate medium
Glucokinase
G1ucose-6-phosphate dehydrogenase
, 6-Phosphog1uconate dehydrogenase
Glucose medium
16.4
98.7
2.4
224.0
nil
26.0
6-Phosphogluconate dehydrase +
2-keto-3 deoxy-6-phosphogluconate aldolase
<1
49.2
Phosphohexose isomerase
35.7
43.1
_
Phosphofructokinase
nil
nil
Fructose diphosphatase
36.0
39.0
Fructose-1,6-diphosphate aldolase
74.6
93.0
1780.0
1145.0
76.0
346.0
15-0
81.0
435.0
375.0
Enolase
408.0
635.0
Pyruvate kinase
.61'.8
71.6
Triosephosphate isomerase
NADP 3-phosphoglyceraldehyde
dehydrogenase
NAD
3 phosphoglyceraldehyde
-
dehydrogenase
3-Phosphoglycerate
kinase
Transketolase
33'.3 '
41.0
Transaldolase
12.0
12.0
S p e c i f i c a c t i v i t y = mymoles substrate u t i l i z e d per min per mg of protei
extract
o f A. a e r o g e n e s and a s s a y i n g
A. a e r o g e n e s showed h i g h
activity
extract
o f 292.0).
under the s i m i l a r
a c t i v i t y o f t h i s enzyme
The a s s a y s
f o r t h i s enzyme
of phosphofructokinase
t h e same n e g a t i v e
i s associated
with
1 9 5 4 ) ; Z_. mobi 1 i s (Raps and DeMoss,
of
Rhodotorula
and
Neidhardt
(Wood a n d
their
fructose diphosphate aldolase
species
However,
strain
o f P.
activity.
i n the s t r a i n
studied
by L e s s i e
and
Lessie
aeruginosa
I t i s seen
that
be s y n t h e s i z e d
r e v e r s a l o f Embden-Meyerhof scheme, but t h i s w i l l
possible
Lack
Schwerdt,
1962) and s e v e r a l
(Brady and Chamb1iss, 1967).
(1967a) found t h a t
results.
P_. a e r u g i n o s a 9 0 2 7 h e x o s e p h o s p h a t e s c o u l d
the
in a cell-free
absence o f f u n c t i o n i n g
E m b d e n - M e y e r h o f p a t h w a y i n P;. f 1 u o r e s c e n s
in
(specific
o f P. a e r u g i n o s a w e r e a l s o c a r r i e d o u t i n t h e p r e s e n c e
of phosphoenolpyruvate with
lacked
conditions.
n o t be
Neidhardt.
T h r e e - p h o s p h o g l y c e r a l d e h y d e d e h y d r o g e n a s e showed h i g h e r
with
NADP as t h e c o e n z y m e .
higher
level
i n g l u c o s e grown c e l l s t h a n
The enzymes whose
level
have been s u g g e s t e d
in
The
o f t h i s enzyme was
i n c e l l s g r o w n on
fluctuates with
t o be r e g u l a t o r y .
nutritional
This
by
enzyme
activity
much
succinate.
conditions
i s involved
t h e a c t i v i t i e s o f t h e E n t n e r - D o u d o r o f f pathway, t h e Embden-Meyerhof
p a t h w a y a n d p e n t o s e p h o s p h a t e c y c l e and may
in
the r e g u l a t i o n o f metabolism.
in
later
This
have s p e c i a l
aspect w i l l
be
importance
discussed
sections.
A consideration
of pentose phosphate c y c l e a c t i v i t y i n
g l u c o s e grown and s u c c i n a t e
grown c e l l s b r i n g s
out a
very
interesting
Fig.
15').
activities
phosphate
pattern of metabolism o f pentoses
When grown
activities
i n a g l u c o s e medium c e l l s
o f a l l t h e enzymes a s s a y e d
indicating
p a t h w a y c o u l d o p e r a t e as a c y c l e
the s u b s t r a t e s .
(Table V I I , VIM
exhibited
that the
H o w e v e r , i n s u c c i n a t e grown c e l l s , w h i l e
identical
to those i n glucose c e l l s ,
glucose-6-phosphate
dehydrogenase
this
cycle
indicated
dehydrogenase
that hexoses
i n these c e l l s .
a c t i v i t y , which
o f hexose
the
o f pentose phosphate
activities
carbohydrates
In an e f f o r t
o f the anaerobic
c y c l e m e a s u r e d by
transformation
low i n t h e
that
importance i n degrading
i f the a b i l i t y
t o form g l u c o s e -
r i b o s e - 5 p h o s p h a t e i s i n d u c e d by g l u c o s e , an
-
e x p e r i m e n t was
l e v e l was
f o u n d t o be
cells.
t o determine
aldehyde dehydrogenase
its
for degradation
t o g l u c o s e - 6 - p h o s p h a t e was
in these
6-phosphate from
activity
c y c l e are not o f
cycle,
phosphoketolase
i n s u c c i n a t e medium, a g a i n i n d i c a t i n g
reactions of this
trans-
c a n n o t be u t i l i z e d v i a
( D e V r i e s e t a l , 1 9 6 7 ) , was
p o r t i o n of the pentose phosphate
additional
the
6-phosphogluconate
Fructose-6-phosphate
A l s o , the o v e r a l l
grown
and
i s i m p o r t a n t i n some b a c t e r i a
phosphates
insignificant.
organisms
pentose
f o r the breakdown o f
o f t h e enzymes o f o x i d a t i v e p o r t i o n o f p e n t o s e p h o s p h a t e
i.e.
good
o f r i b o s e p h o s p h a t e i s o m e r a s e , t r a n s k e t o l a s e and
a l d o l a s e were
and
assayed
was
undertaken.
also
Since
3-phosphoglycer-
f o u n d t o be a r e g u l a t o r y
i n t h i s experiment
( F i g . 16).
a p p a r e n t t h a t on t h e t r a n s f e r o f s u c c i n a t e c e l l s
enzyme,
It i s
t o the glucose
Table V I M .
G1 u c o s e - 6 - p h o s p h a t e
f o r m i n g a c t i v i t y from
r i bose-5-phosphate and r i bosephosphate
isomerase a c t i v i t y .
Growth
Glucose
Ribosephosphate
isomerase
G1ucose-6-phosphate forming
a c t i v i t y from r i b o s e - 5
phosphate "
medium
Succinate
0.33
0.30
28.0
2.5
-
1
T h e a c t i v i t y o f r i b o s e p h o s p h a t e i s o m e r a s e i s e x p r e s s e d as
A O.D. 520 my p e r m i n p e r mg o f p r o t e i n .
The r e a c t i o n
was a l l o w e d t o p r o c e e d f o r 20 m i n .
The a c t i v i t y i s e x p r e s s e d as mymoles NADP r e d u c e d p e r
m i n p e r mg o f p r o t e i n .
0
5
10
15
MINUTES
15.
;
G 1 u c o s e ^ 6 - p h o s p h a t e f o r m a t i o n f r o m r i b o s e - 5 - p h o s p h a t e by
P. a e r u g i n o s a g r o w n i n s u c c i n a t e (•, 1.2 mg p r o t e i n ; • ,
0.6 mg p r o t e i n ) o r g l u c o s e ( o , 0.315 mg p r o t e i n ) m e d i a .
One ml r e a c t i o n m i x t u r e c o n t a i n e d T r i s b u f f e r , pH 8.0,
50 mM; t h i a m i n e - p y r o p h o s p h a t e , . 0.5 mM; r i b o s e - S ^ p h o s p h a t e ,
2.0 mM; NADP, 1 mM; c o m m e r c i a l
glucose-6-phosphate
d e h y d r o g e n a s e and c e l l - f r e e e x t r a c t .
In A, NADP t o s t a r t
t h e r e a c t i o n was a d d e d a f t e r 40 m i n i n c u b a t i o n o f t h e . a s s a y
r e a c t i o n m i x t u r e a t room t e m p e r a t u r e .
20
74
HOURS
Fig.
16.
Increase i n the level of 3-phosphoglyceraldehyde
dehydrogenase
and g I u c o s e - 6 - p h o s p h a t e f o r m i n g a c t i v i t y f r o m
ribose-5 phosphate,
oh - s h i f t : f r o m s u c c i n a t e t o g l u c o s e medium.
Log p h a s e c e l l s
f r o m . s u c e i n a t e medium w e r e h a r v e s t e d , washed, i n b a s a l . s a l t s
medium and i n o c u l a t e d i n t o 0 . 3 % g l u c o s e medium i n ' E r l e n m e y e r
f l a s k s . . I n c u b a t i o n was a t 30 C w i t h s h a k i n g .
Samples were
t a k e n o u t a t d e s i r e d i n t e r v a l s a n d u s e d f o r enzyme a s s a y s .
S y m b o l s : o, o p t i c a l d e n s i t y ; •,
3-phosphoglyceraldehyde
d e h y d r o g e n a s e ; A, G-6-P f o r m a t i o n f r o m R-5-P ( m y m o l e s /
min/mg o f p r o t e i n ) .
-
medium these a c t i v i t i e s
are
greatly
increased.
3-phosphoglyceraldehyde dehydrogenase
easily
The i n c r e a s e
i n g l u c o s e medium can be
r a t i o n a l i z e d f o r 3-phosphoglyceraldehyde i s a product
o f E n t n e r - D o u d o r o f f pathway a c t i v i t y on g l u c o s e .
increase
the c a p a c i t y
In o r d e r
to
t o u t i l i z e t h i s s u b s t r a t e and d i r e c t
it
the t r i c a r b o x y l i c a c i d c y c l e , h i g h a c t i v i t y o f t h i s enzyme
necessary.
This
i s not so
in a s u c c i n a t e
phosphoglyceraldehyde dehydrogenase
reversal
and hexoses f o r c e l l
From the above o b s e r v a t i o n s
i n o r d e r to grow on s u c c i n a t e
intermediate,
medium where
3
is u t i l i z e d o n l y f o r
o f Embden-Meyerhof r e a c t i o n s
s i z i n g pentoses
cycle
in
to
is
-
limited
f o r the purpose of s y n t h e biosynthesis.
i t c o u l d be concluded t h a t
or any o t h e r t r i c a r b o x y l i c a c i d
t h i s organism must u t i l i z e the
transketolase
r e a c t i o n , u s i n g compounds which c o u l d be d e r i v e d from t r i c a r b o x y l i c
acid cycle
intermediates,
of the s e v e r a l
alternative
t o s y n t h e s i z e pentoses
reactions
(Racker,
by one o r more
1954; Sable,
1966).
These mechanisms c o u l d be summarized as f o l l o w s : -
1) F r u c t o s e - 6 - p h o s p h a t e
Pentose; phosphate
+
+
2)
Triose-phosphate
Erythrose-4-phosphate
Triose-phosphate
Pentose
+
An " a c t i v e g l y c o l a l d e h y d e " donor
( e . g . Hydroxypyruvate)
phosphate
Evidence o f such
laboratories.
DeLey
were
an
reactions
organism
confirmed
transketolase-transaldolase
i n pentose phosphate s y n t h e s i s
by t h e w o r k o f F o s s i t t
and B e r n s t e i n
A similar
(1963).
faeca1is
involvement o f such
Wang e t
phosphate
p e n t o s e s by a l t e r n a t e
c o n c l u s i o n was drawn by L e s s i e a n d N e i d h a r d t
(1967a) i n d i s c u s s i n g s u c c i n a t e
Streptococcus
T h i s was
grown P. a e r u g i h o s a l a c k e d t h e
p a t h w a y a n d t h e r e f o r e , must s y n t h e s i z e
oh
i n P_. s a c c h a r o p h ? l a ,
o f the o x i d a t i v e p o r t i o n o f t h e pentose
schemes.
reactions
l a c k i n g 6-phosphogluconate dehydrogenase.
a l _ (1959) p r o p o s e d t h a t g l u c o n a t e
activity
h a s come f r o m many
The w o r k o f DeLey and D o u d o r o f f a s q u o t e d by
(1960) showed t h a t
involved
i n pseudomonads
grown P. a e r u g i n o s a .
and A l c a l i g e n e s
reactions
Studies
faecal is also
i nbiosynthesis
showed
o f pentoses
( S a b l e , 1966).
2. T r i c a r b o x y l i c a c i d c y c l e a n d r e l a t e d enzymes
a. T r i c a r b o x y l i c a c i d c y c l e
The
activity
r e s u l t s o f s t u d i e s on t h e enzymes o f g l u c o s e
o x i d a t i o n d i d n o t a c c o u n t f o r t h e growth and o x i d a t i o n
of glucose
and s u c c i n a t e
grown c e l l s
or a mixture o f these substrates.
glucose
medium u t i l i z e d
i n media w i t h
The c e l l s
succinate without
one o f these
harvested
a lag.
pattern
from
Therefore,
a study
of the
levels
t y p e s o f c e l l s was
study
of t r i c a r b o x y l i c
considered
acid
essential.
i s t h e o n l y pathway
left
o f the Entner-Doudoroff
not
Table
IX shows t h e a c t i v i t y
important
i n t h i s medium, t h e
the pentose phosphate
i n the c e l l s
a-ketoglutarate
and
of these
and
s u c c i n a t e + glutamate
media.
t h a t t h e s e compounds w e r e u t i l i z e d
succinate
f o r growth.
The
activities
c y c l e e n z y m e s w e r e c o m p a r a b l e and
The
v a r i a t i o n s observed
and
a-ketoglutarate
The
level
as g r e a t
Again,
cells
as
observed
i n the
in cells
i n spore
forming
bacilli
(Gray
present
i n t h e m e d i a do
leading
to a-ketoglutarate
not
i s not
i n d i c a t e that the a c t i v i t y
with
aconitase
to be'reproducible.
about
deserved
twice
f u r t h e r study.
m e c h a n i s m on
the
s i m i l a r t o the
(Hanson and
Cox,
shut o f f the a c t i v i t y
formation.
glutamate
tricarboxylic
levels of
et a l , 1966b), s i n c e a-ketoglutarate
E. c o l ?
rates
g r o w n on s u c c i n a t e .
r e g u l a t o r y and
cycle activity
or
growth
no s t r i k i n g d i f f e r e n c e s
a l s o show t h a t t h e c o n t r o l
tricarboxylic acid
recycling.
Higher
o f most o f t h e
dehydrogenase were found
t h i s enzyme c o u l d be
These r e s u l t s
pathway
simultaneously
o f p a r t i c u l a t e m a l i c d e h y d r o g e n a s e was
in glucose
activities
g r o w n on g l u c o s e , s u c c i n a t e , s u c c i n a t e +
indicated
were seen.
aerug1nosa
r e l a t e d enzymes f o r
s u c c i n a t e media supplemented w i t h a - k e t o g l u t a r a t e
acid
two
detailed
a d e q u a t e f o r t h e o x i d a t i o n o f t h e c o m p o u n d s , by
comparison
on
p a t h w a y and
becomes
i n the
a t t h e d i s p o s a l o f P.
grown on s u c c i n a t e s i n c e , d u r i n g g r o w t h
are
Moreover, the
of t r i c a r b o x y l i c acid cycle a c t i v i t y
because t h i s
c y c l e enzymes
These
pattern
1967)
and
and
glutamate
o f enzymes
observations
of the t r i c a r b o x y l i c a c i d
cycle in
Table
IX.
Specific a c t i v i t i e s o f the t r i c a r b o x y l i c acid cycle
and r e l a t e d enzymes, i n P. a e r u g i n o s a g r o w n w i t h
d i f f e r e n t carbon sources
Growth
Enzyme
Glucose
44.0
Aconi tase
NADP i s o c i t r a t e
NAD i s o c i t r a t e
dehydrogenase
dehydrogenase
a-Ketoglutarate
Succinic
dehydrogenase
dehydrogenase
Particulate malic
dehydrogenase
Succinate +
a-ketoSucci nate
glutarate
31.6
Succinate
+
G1utamate
44.8
30. 0
438
464
600
483
nil
nil
-
-
50.4
128
91.7
102
93.3
80. 8
-
-
1230
1185
1260
Fumarase
Medium
1165
16.5
14. 2
-
-
28.2
270
15.0
294
343
75.0
76.0
300
nil
nil
nil
nil
NADP M a l i c enzyme
438
524
481
471
NAD M a l i c enzyme
117
141
153
125
Succinyl
CoA s y n t h e t a s e
NADPH G l u t a m i c
dehydrogenase
NADH G l u t a m i c
dehydrogenase
NADP G l u t a m i c
dehydrogenase
NAD G l u t a m i c
dehydrogenase
Specific activity
62.0
273
57.0
70.0
53.0
= mymoles o f s u b s t r a t e u t i l i z e d
of protein.
163
48. 0
28. 0
p e r m i n p e r mg
a - K e t o g l u t a r a t e a n d g l u t a m a t e a d d i t i o n s , when s p e c i f i e d , w e r e a t ;
0.3%
level.
pseudomonads
occurs
i s not only e s s e n t i a l
f o r e n e r g y p r o d u c t i o n when g r o w t h
i n g l u c o s e medium, b u t i s o f s p e c i a l
degradation of intermediates of this
are growing
on such
compounds.
importance
for the
c y c l e when t h e o r g a n i s m s
Under any c i r c u m s t a n c e s t h e
enzymes o f t h e t r i c a r b o x y l i c a c i d
c y c l e c a n be c o n s i d e r e d
constitutive.
Good a c t i v i t y
and
o f s u c c i n i c dehydrogenase i n both
s u c c i n a t e g r o w n c e l l s was o b t a i n e d
method used
i n the present
u s e d by p r e v i o u s w o r k e r s
enzyme i r i P. a e r u g i n o s a
and
Campbell,
1966b).
indicating
who f o u n d
very
low a c t i v i t y
of this
(Campbel 1 e_t a l _ , 1962; Von T i g e r s t r o m
The i n f l u e n c e o f t h e k i n d o f a s s a y
p o i n t e d o u t by S i n g e r a n d K e a r n y
synthetase,which
that the
i n v e s t i g a t i o n was s u p e r i o r t o t h e o n e
u s e d on d e t e c t i n g a d i f f e r e n c e i n a c t i v i t y
was
glucose
has been found
of succinic
(1963).
method
dehydrogenase
S u c c i n y l CoA
t o increase about 8-fold i n
E_. c o l j _ g r o w n o n s u c c i n a t e a s c o m p a r e d t o t h e c e l l s grown i n
g l u c o s e medium
difference
( G i b s o n , Upper and G u n s a l u s ,
inactivity
i n P. a e r u g i n o s a
s u c c i n a t e o r g l u c o s e media.
1967), showed
little
d u r i n g growth i n
T h u s , t h i s enzyme d i d n o t seem t o b e
regulatory.
The
b. G l u t a m i c
dehydrogenase
variation
i ntheactivity
in d i f f e r e n t
growth media
activity
o f glutamic
i s interesting
dehydrogenase
t o note.
T h e enzyme
activity
i n t h e d i r e c t i o n o f g l u t a m a t e s y n t h e s i s was
as g r e a t as
the
i n the d i r e c t i o n o f o x i d a t i o n .
activity
was
in the d i r e c t i o n
with
NAD
Such
1961;
the
Leech
and
plants
and
Kirk,
1968)
and
i s ammonia
1968).
high a c t i v i t y
formation indicates
i n c o r p o r a t i o n which
o b s e r v a t i o n s made i n o t h e r s y s t e m s
Leech
found
that the presence o f glutamate
activity
o f t h e enzyme and
both d i r e c t i o n s .
genase
1968).
Kirk,
and
activity
indicate
Sanwal
and
that
Lata,
i t s primary
i s i n agreement w i t h
the
Rickenberg,
s u g g e s t i o n i t was
i n t h e medium r e p r e s s e d t h e
fell
to less
Repression of the a c t i v i t y
i n g l u t a m a t e medium ha:s b e e n f o u n d
a l , 1965)
activity
o f t h e enzyme i n
In s u p p o r t o f t h i s
the l e v e l s
(H)
b e e n shown i n
( V e n d e r , J a y a r a m a n and
1965;
and
as has
S c h n e i d e r , 1957;
d i r e c t i o n of glutamate
function
probably
( H o l z e r and
The
no
i n Rhizobium japonicum
than h a l f
in
of g l u t a m i c dehydro-
i n E_. c o l ?
(Mooney and
(Vender
Fottrell,
1968).
IX.
Level
or
of Malic
Enzyme i n C e l l s Grown
i n Succinate, Glucose
A c e t a t e Med ? a
T h i s enzyme was
NADP t h a n w i t h
NAD
found t o e x h i b i t
as t h e c o e n z y m e
times
r e s u l t s were a l s o r e p o r t e d
p r e s e n c e o f two g l u t a m i c d e h y d r o g e n a s e s
some f u n g i
et
NADH g a v e some
o f g l u t a m a t e s y n t h e s i s , b u t t h e r e was
i n the o p p o s i t e d i r e c t i o n .
five
A l s o , w i t h NADP
high i n both d i r e c t i o n s .
i n Ch1oropseudomonas e t h y l i c u m ( S t e r n ,
the
about
much h i g h e r a c t i v i t y
(Table IX).
The
levels
with
were
high
i n both
activity
dropped
w e r e grown
reported
"coarse
is
t h e s u c c i n a t e a n d t h e g l u c o s e grown c e l l s b u t t h e
t o a b o u t k0% o f t h i s
i n a c e t a t e medium
i n P. p u t i d a
acid
cycle
has
by
acid
t o be c a t a l y s e d m a i n l y
earlier.
indicates
o f both
that i t s a c t i v i t y
When o r g a n i s m s a r e
Hence, a c e t y l
CoA i n a d d i t i o n
succinate.
acid
In pseudomonads
cycle
i salso
d u r i n g growth
t h e s e o r g a n i s m s a r e grown
o b t a i n e d by t h e d i r e c t
level
important
level
of acetate.
similar
the further
of malic
CoA i s
T h i s c o u l d be
t o t h a t i n E_. c o l i
a c t i v i t y has been d e m o n s t r a t e d
a e r u g i n o s a g r o w n on v a l i n e
t h i o k i n a s e which
(Sokacth, 1967);
has been r e p o r t e d
enzyme
H o w e v e r , when
by a c e t a t e k i n a s e a n d p h o s p h o t r a n s a c e t y l a s e ,
phosphotransacetylase
enzyme
f o r the complete
i n a g l u c o s e medium.
activation
by t h e
of this
i n a c e t a t e medium, a c e t y l
a c h i e v e d e i t h e r by a s y s t e m
catalysed
i s achieved
t h e a c t i v i t y of the.
o x i d a t i o n o f g l u c o s e and hence a g a i n a h i g h
is maintained
This
CoA c a n t h e n be d e r i v e d f r o m
oxidation o f pyruvate.
tricarboxylic
t o oxalacetate
v i a malate
o f m a l i c enzyme a n d t h u s , t h e h i g h
Acetyl
i t
by t h e r e a c t i o n s o f t h i s c y c l e a s
the o x i d a t i o n o f succinate t o pyruvate
is maintained.
This type o f
c y c l e member,, e . g . s u c c i n a t e ,
t o be c o n s t a n t l y d e r i v e d from
activity
also
t h e g l u c o s e and t h e
intermediates.
grown on a t r i c a r b o x y l i c
discussed
Such r e s u l t s w e r e
e t a l, 1966).
(Jacobson
i n the metabolism
tricarboxylic
has
(Table X ) .
c o n t r o l " on m a l i c enzyme
important
v a l u e when t h e o r g a n i s m s
since
i n f_.
o r by a c e t i c
i n P. f 1 u o r e s c e n s
(Kornberg
Table X.
Effect of carbon sources on the s p e c i f i c a c t i v i t i e s of
malic enzyme and i s o c i t r a t e lyase in P_. aeruginosa.
Growth Medium
Glucose
Enzyme
NADP Malic enzyme
Isocitrate lyase
Succinate
Acetate
speci f i c act i vi ty'
435
24.3
407
11.3
mymoles substrate u t i l i z e d per min per mg of protein
174
450
and
Madsen, 1958).
necessary
Thus, high
f o rthe generation of acetyl
for maintaining the a c t i v i t y
tricarboxylic acid
It
l e v e l s o f m a l i c enzyme a r e no
i s thus
variation
seen t h a t t h e organisms possess
X.
to their
enzymes.
i n the level
g r o w t h medium
either
cycle.
according
the necessary
acids
of glyoxylate cycle or of the
regulate the flow o f metabolites along
metabolism
CoA f r o m
longer
a capacity to
s e l e c t e d pathways o f
n e e d by a l t e r i n g
the synthesis o f
The commonly made o b s e r v a t i o n o f t h e g r e a t
of isocitrate
lyase according to the
( T a b l e X) i s y e t a n o t h e r
example t o support
this.
I t i d u c t i o n o f t h e Enzymes o f G l u c o s e U t i 1 i z a t i o n ' i t i ' C e l l s
Harvested
To
f r o m S u c c i h a t e Med ium
determine
i n P. a e r u g i n o s a
i fthe i n i t i a l
a r e induced
s u c c i n a t e grown c e l l s ,
Warburg e x p e r i m e n t s
enzymes o f g l u c o s e
f o rglucose
cell
(5 mg d r y w e i g h t / m l ) ,
suspensions
glucose
flasks
identical
M_Tris
Each
b u f f e r , pH l.k, 10 m l ; s u c c i n a t e g r o w n
contained
10 m l ; a n d H^O
15 mg c h l o r a m p h e n i c o l .
w e r e s h a k e n a t 30 C f o r 30 m i n a n d t h e n
solution
t o those f o r
w e r e s e t up i n t w o E r l e n m e y e r f l a s k s .
0.1
One o f t h e s e
utilization in
incubation.mixtures
flask contained
suspension
metabolism
(75 u m o l e ) was added t o e a c h f l a s k .
f u r t h e r shaken f o r 2 h r a t which time
t h e c e l l s were
t o 27 ml .
These
3.0 ml o f
They w e r e
harvested,
Table X I .
I n d u c t i o n o f some enzymes o f g l u c o s e o x i d a t i o n by
glucose.
Glucose
dehydrogenase
Source
of cells
Specific
Glucose
Glucokinase
medium
Glucose-6-phosphate
dehydrogenase
•
activity"
32.0
98.7
224.0
S u c c i n a t e medium
1.5
16.4
2.5
C e l I s t r a n s f e r r e d from
s u c c i n a t e medium t o g l u c o s e
suspension containing c h l o r amphenicol
3.0
13.8
1.0
10.0
23.0
9.0
C e l l s t r a n s f e r r e d from
s u c c i n a t e medium t o g l u c o s e
s u s p e n s i o n c o n t a i n i n g no
chloramphenicol
mumoles s u b s t r a t e u t i l i z e d
p e r m i n p e r mg o f p r o t e i n .
cell
e x t r a c t s prepared
and
assayed
f o r glucose-6-phosphate
h y d r o g e n a s e , g l u c o s e d e h y d r o g e n a s e and
glucokinase.
The
results
( T a b l e X l ) showed t h a t a l 1 o f t h e s e enzymes w e r e i n d u c e d
cells
was
not
suppressed
Although
when a s h i f t
be s e e n
by
chloramphenicol.
are
included for
The
values f o r glucose
t h e enzymes o f g l u c o s e m e t a b o l i s m
from
succinate to glucose
i f i t t o o has
g l u c o s e has
t o be
t o be
and
comparison.
i f s u c c i n a t e grown c e l l s
glucose or
for
in the
incubated with chloramphenicol, while t h e i r synthesis
succinate cells
to
de-
induced
i s made, i t s t i l l
possess
induced.
i n d u c e d , how
are
remained
the permease f o r
I f the t r a n s p o r t
system
l o n g does i t t a k e f o r t h e
14
induction?
To a n s w e r t h e s e q u e s t i o n s g l u c o s e U -
was
studied.
Firstly, cells
and
s u c c i n a t e s e r v e d as
g r o w n i n g l u c o s e medium w e r e u s e d
the energy source
Under t h e s e c o n d i t i o n s g l u c o s e uptake
about 5 min
( F i g . 17).
of
i . e . a-CH^-glucoside
glucose,
the glucose
permease system,
tried
I t was
curve
interesting
and
i n these
levelled
to find
C.
with
that
analogues
To
f u r t h e r see
the s p e c i f i c i t y
None.of t h e s e sugars
competed w i t h
I t was
a l s o found
a-CH^-glucoside
E'. c o l i s y s t e m
and
which
retard
concentr-
of
c o m p e t i t i o n w i t h g a l a c t o s e , f r u c t o s e and
( F i g . 18).
aerug?hbsa.
accumulate
o f f at
2-deoxyglucose d i d not
i n d i c a t i n g a h i g h l y s p e c i f i c mechanism o f g l u c o s e
P.
experiments.
i n c o r p o r a t i o n , e v e n when a d d e d a t 1 0 0 - f o l d t h e
14
ation of glucose-U-
was
C transport
glucose
mannose
glucose
transport in
t h a t g l u c o s e grown c e l l s
did
not
i s i n c o n t r a s t t o the o b s e r v a t i o n
i s i n agreement w i t h
the f i n d i n g of
Hamilton
86
6-Or
MINUTES
Fig.
17. Uptake of r a d i o a c t i v i t y by glucose grown c e l l s of P . . aerugi nosa
with 10'- M glucose-•U- C (0.5yc/ymole) and with 10 M
a-methyl-D-glucopyranoside (0.5yc/ymole). Symbols:
0-0 , glucose-U- C; • - • ,_glucose-U- C + 250 mM chloramphenicol;
o - o , g l u c p s e - U - ^ C with 10'"* M, 2-deoxygl ucose; • - •,
g1ucose-U- ^C with 10"3 M a-methyl-D-glucopyroside, A- A,
a-methyl-D-glucopyranoside-UC.
14
1
5
MINUTES
Fig.
18.
I n c o r p o r a t i o n o f g l u c o s e - U - C i n the presence o r absence
o f o t h e r s u g a r s by t h e w h o l e c e l l s o f P. a e r u g i n o s a
harvested
f r o m t h e g l u c o s e m i n i m a l medium. T h e e x t e r n a l g l u c o s e
c o n c e n t r a t i o n was 1 0 ~ M; s p e c i f i c a c t i v i t y , 0 . 5 uc/umole.
S y m b o l s : • - • , no a d d i t i o n ; o - o , w i t h 10"- M f r u c t o s e ;
• - •, w i t h ,10"3 M g a l a c t o s e ; • - • , w i t h 10~3 M mannose.
1 4
5
5
•8.8.:
MINUTES
fig.
19.
Glucose-UC i n c o r p o r a t i o n by t h e w h o l e c e l I s o f PI a e r u g i h o s a
w i l d t y p e and i t s m u t a n t s t r a i n M5, h a r v e s t e d f r o m s u c c i n a t e
m i n i m a l medium. E x t e r n a l g l u c o s e c o n c e n t r a t i o n was 1 0 " ^
M;
s p e c i f i c a c t i v i t y 0.5 u c / y m o l e .
Symbols:A-A, w i l d type c e l l s ;
o-o, w i l d t y p e c e l l s w i t h 250 mM c h l o r a m p h e n i c o l ;
w i 1 d t y p e c e l 1 s . w i t h 30 mM s o d i u m a z i d e + 1
mM
iodoacetamide;
• - • , M5 c e l l s .
:.
'•'•^r::[
••
'' • V
' *• •
Dawes ( 1 9 6 0 ) , who made s i m i l a r
and
P. a e r u g i n o s a .
The a b i l i t y
observations
t o accumulate
in their
strain of
2-deoxyglucose
was
not t e s t e d .
S i n c e t h e c e l l s grown
i n g l u c o s e medium a c t i v e l y
g l u c o s e a c r o s s t h e membrane i n t h e s y s t e m
the t r a n s p o r t o f glucose
i n t h e c e l l s grown
was f u r t h e r s t u d i e d w i t h t h i s
i t s mutant s t r a i n M 5 .
and
system
T h i s mutant
cycle intermediates.
presented
i n F i g u r e 19.
permease i s induced
The r e s u l t s
The s t u d y
study,
t y p e P. aerug?nosa»
i s unable
t o g r o w on
b u t does grow on
tricarboxylic
o f these experiments
clearly
revealed that
are
glucose
s l o w l y over a long p e r i o d o f time.
I t was c o n c l u d e d
from
t h e a b o v e d a t a t h a t on s h i f t
s u c c i n a t e t o g l u c o s e medium b o t h
as w e l l
in this
i n s u c c i n a t e medium
in wild
glucose, gluconate o r 2-ketogluconate
acid
used
transported
the s p e c i f i c
glucose
from
permease
a s g l u c o s e o x i d a t i o n enzymes h a v e t o be s y n t h e s i z e d
simultaneously
i n o r d e r t h a t t h e o r g a n i s m s c a n a d a p t t o g r o w t h on
g1ucose.
XI.
C o n t r o l o f Tr? c a r b o x y l ? c A c i d C y c l e Act? v i t y
1. P a r t i c u l a t e
The
level
of particulate
. i n t h e c e l l s grown
to
determine
m a l i c dehydrogenase a c t i v i t y
m a l i c d e h y d r o g e n a s e was much
i n g l u c o s e medium ( T a b l e
the increase i n the level
IX).
higher
The e x p e r i m e n t
o f t h i s enzyme on s h i f t t o
90
HOURS
F i g . 20.
Increase in the level of p a r t i c u l a t e malic dehydrogenase
a c t i v i t y on s h i f t to glucose medium. The experimental
conditions were the same as in F i g . 16, except f o r the
higher rate of shaking at 37 C. Symbols, O , o p t i c a l
d e n s i t y ; • , p a r t i c u l a t e malic dehydrogenase a c t i v i t y .
glucose
medium showed
the expected
results
(Fig. 20).
I t was
t h e r e f o r e thought d e s i r a b l e t o observe t h e c o n t r o l o f malate
oxidase
and
activity.
Substrates
like acetate, c i t r a t e ,
succinate at a concentration
activity.
o f 3 mM d i d n o t i n f l u e n c e t h e
When ATP was a d d e d t o t h e r e a c t i o n m i x t u r e
c o n c e n t r a t i o n , a b o u t h a l f o f t h e a c t i v i t y was l o s t
This
i n h i b i t i o n was n o t c o m p e t i t i v e w i t h
addition of higher
inhibition.
and
concentrations
Essentially
AMP ( T a b l e X I I ) .
with
similar
Cumulative
t w o o r more o f t h e s e
(Table X I l ) .
o f ATP d i d n o t c a u s e a n y f u r t h e r
r e s u l t s were o b t a i n e d
inhibition
o f GTP.
o r concerted
However,
activation
The k i n e t i c a n a l y s i s showed
Km f o r m a l a t e o f 3.77 x 10
partially
W h i l e GDP was a s s t i m u l a t o r y a s
Adenosine,
i s common t o t h e s t r u c t u r e o f A T P , ADP a n d AMP, was
effect.
increased
reaction mixture
GTP; GMP, CTP a n d ITP d i d n o t i n f l u e n c e t h e a c t i v i t y .
any
inhibition
A d d i t i o n o f an e q u i v a l e n t
o f ATP a n d GTP i n t h e a s s a y
o v e r c a m e t h e ATP i n h i b i t i o n .
which
w i t h ADP
n u c l e o t i d e s was n o t o b s e r v e d .
increasing concentrations
concentration
i n 1 mM
the substrate,as the
GTP was f o u n d t o a c t i v a t e t h e enzyme a n d t h i s
with
pyruvate
t h a t t h i s enzyme
m o l e / l i t e r and t h i s
without
has a
i s increased t o
8.33 x 10 ^ m o l e / l i t e r i n t h e p r e s e n c e o f 1 mM ATP.
From t h e
L i n e w e a v e r B u r k p l o t i t i s s e e n t h a t ATP i s a " m i x e d " t y p e o f
inhibitor,
i . e . i ti s neither a competitive
i n h i b i t o r o f t h e enzyme a c t i v i t y
present
these
nor a
non-competitive
( F i g . 2 1 ) . The r e s u l t s o f t h e
i n v e s t i g a t i o n do n o t a l l o w t h e p r e c i s e i n t e r p r e t a t i o n o f
observations
but such
results
have been r e p o r t e d w i t h
NAD-
Table X I I .
E f f e c t o f n u c l e o t i d e p h o s p h a t e s on t h e p a r t i c u l a t e
d e h y d r o g e n a s e i n 1 0 5 , 0 0 0 x g_ p e l l e t " .
. Inn i b i t o r
Concent r a t ion
(mM)
-
Specifi c activi ty
(mumoles/min/mg
-
29?0
0.5
18.4
1.0
15.6
2.0
15.6
3.0
15.6
0.5
15.6
1.0
14.2
2.0
14.2
0.5
17.0
1.0
17.0
2.0
15.6
0.5
34.0
1.0
41.0
2.0
53.0
GDP
1 .0
39.6
GMP
1.0
29.7
ATP+AMP
ATP
ADP
AMP
GTP
1.0
a
15.6
ATP+ADP
1.0
8
17.0
ATP+ADP+AMP
1.0
a
ATP+GTP
i.o
CTP
1 .0
29.7
ITP
1.0
28.9
a
malic
protein)
15.6
22.4
2 x 10
M EDTA was added t o 2 0 , 0 0 0 x g _ . s u p e r n a t a n t b e f o r e
c e n t r i f u g a t i o n a t 1 0 5 , 0 0 0 . x g_ f o r 90 min.. The p e l l e t was
w a s h e d ;;with 0 . 2 M T r i s b u f f e r , pH 7 . 2 c o n t a i n i n g 1 0 " 3 M
EDTA a n d r e s u s p e n d e d i n 0 . 2 M T r i s , pH 7 . 2 .
J
'Each n u c l e o t i d e p h o s p h a t e was added a t a c o n c e n t r a t i o n
o f 1 mM.
-40
0
20
40
[Sl ( mM
F i g . 21.
60
80
, 100
MALATE )
Lineweaver-Burk plot for p a r t i c u l a t e malic dehydrogenase
in the absence (A) or presence (B) of 1 mM ATP.
linked
heart
particulate
i n which
assayed
( m i t o c h o n d r i a l ) m a l i c dehydrogenase from p i g
A T P , ADP a n d AMP w e r e f o u n d
i n the direction
from peas
i n which
o f NADH o x i d a t i o n ; a n d w i t h t h e enzyme
t h e i n h i b i t i o n was o b s e r v e d
( K u r a m i t s u , 1966; K u r a m i t s u , 1 9 6 8 ) .
by a d e n i n e
t o be i n h i b i t o r y when
i n both
directions
This pattern o f i n h i b i t i o n
n u c l e o t i d e s was p r e s u m e d t o m i m i c t h e c o n t r o l
by y e t a n o t h e r compound
regulating
t h e enzyme a c t i v i t y .
However,
i n t h e absence o f a r e g u l a t o r y mechanism f o r i s o c i t r a t e
by a d e n i n e
dehydrogenase
n u c l e o t i d e s i n P. a e r u g i n o s a t h e s i g n i f i c a n c e o f s u c h an
observation
acid
exerted
i s very great.
i s necessary t o ensure
cycle, a control
at this
Since a constant supply o f o x a l a c e t i c
the a c t i v i t y
s t e p on m a l a t e
of the tricarboxylic
oxidase system
acid
can thus
regulate the a c t i v i t y o f the cycle.
2.
Control
acid
The
cycle
o f the flow of isocitrate
c y c l e and g l y o x y l a t e c y c l e
inhibitory
v i a the t r i c a r b o x y l i c
i n P_. a e r u g ? h o s a
and r e p r e s s i v e e f f e c t s
of tricarboxylic
i n t e r m e d i a t e s and p h o s p h o e n o l p y r u v a t e
on i s o c i t r a t e
of
E. c o l i h a v e b e e n r e p o r t e d ( K o r n b e r g , 1 9 6 6 ) .
on
the control
of this
R u f f o and c o w o r k e r s
acid
lyase
This type o f study
enzyme f r o m pseudomonads h a s n o t b e e n made.
(1959,
1962,
1963,
1967)
have r e p o r t e d t h a t
o x a l m a l a t e , a c o n d e n s a t i o n p r o d u c t o f g l y o x y l a t e and o x a l a c e t a t e ,
inhibits
has
mammalian a e o n i t a s e and i s o c i t r a t e
been proposed
that this
dehydrogenase and i t
c o n d e n s a t i o n p r o d u c t may be i m p o r t a n t
in
the r e g u l a t i o n
S h i i o and Ozaki
(1968)
not formed
from g l y o x y l a t e
and o x a l a c e t a t e
conditions.
two
They
reported
flavum
enzyme when o b t a i n e d
It
was o f
dehydrogenase
and r e l a t i v e l y
from
interest
look
oxalmalate
under p h y s i o l o g i c a l
inhibition
by t h e s e
dehydrogenase
lower
f o r such
i n P. a e r u g i n o s a , s i n c e
(Von T i g e r s t r o m a n d C a m p b e l l ,
also
found
inhibition
study
it
1966;
that
control
of
Table
unlike
from
of
this
heart.
isocitrate
shows a c t i v i t y
IX).
only
with
It was
the N A D - s p e c i f i c
dehydrogenase o f Neurospora and y e a s t
(Atkinson,
1966)
N A D P - s p e c i f i c enzyme o f P . a e r u g i n o s a was n o t i n f l u e n c e d by
AMP, ADP o r A T P .
showed
that
addition
study,
t h e enzyme
isocitrate
sulfate
Preliminary
of glyoxylate
grown c e l l s
is strongly
procedure
Malic
d e h y d r o g e n a s e a s s a y when
isocitrate
and r e l a t e d
isocitrate
Xlll)
is
lyase a c t i v i t y
lyase
likely
to
of different
respectively.
acetate
by ammonium
by O z a k i
interfere with
o f malate
T h e P4 f r a c t i o n
detailed
from
as o u t l i n e d
the e f f e c t
compounds on i s o c i t r a t e
for
purified
d e h y d r o g e n a s e and PS f o r
T a b l e X I V a n d XV show t h e e f f e c t
cycle
(Table
extracts
by s i m u l t a n e o u s
Therefore,
partially
enzyme w h i c h
low i n Pk a n d P5 f r a c t i o n s .
assay of
the e e l 1-free
inhibited
and o x a l a c e t a t e .
o f P. a e r u g i n o s a were
(1968).
isocitrate
results with
d e h y d r o g e n a s e and i s o c i t r a t e
fractionation
and S h i i o
was
However,
E_. c o l i , B_. s u b t i 1 i s o r p i g
to
in the present
found t h a t
isocitrate
NADP
isocitrate
have
strong concerted
compounds o f NADP s p e c i f i c
Brevibacterium
the
acid cycle activity.
recently
more
is
of tricarboxylic
is studied,
was u s e d f o r t h e
isocitrate
the
lyase.
tricarboxylic
d e h y d r o g e n a s e and
acid
'Table X I I I .
P a r t i a l p u r i f i c a t i o n o f i s o c i t r a t e d e h y d r o g e n a s e and i s o c i t r a t e
f r o m t h e c e l l e x t r a c t s o f P_. a e r u g i n o s a ;
lyase
Enzyme
(NH^)2^^ZJ
Fractionation
procedure
Fraction
Isocitrate
Isocitrate
T o t a l .dehydrogenase
lyase
protein
—:— —:
(mg)
sp. a c t . t o t . a c t . s p . a c t . t o t . a c t .
;
105,000 x £
supernatant
(A)
P r e c i p i t a t e b e t w e e n 0.5
and 0.8 s a t u r a t i o n o f (A)
P r e c i p i t a t e f r o m P2 b e t w e e n
0 . 5 5 a n d 0.65 s a t u r a t i o n
P5
sp.act.
tot.act.
148,300
1090.
184,300
877
68.
1200
82,000
672
45,900 .
31. 2
2140
66,700
880
27,450
37.5
1,170
5.68
3290
18,700
1265
7,180
14.8
.84
6 . 16
3480
21,450
1695
10,450
18.9
116
P1
P4
:
169. 2
P r e c i p i t a t e from.PI between
0.5 a n d 0.6 s a t u r a t i o n
P2
P r e c i p i t a t e f r o m P2 a t
0.55 s a t u r a t i o n
NADP m a l i c
enzyme
.
210
14,350
a.
Isocitrate
dehydrogenase
activity
The a n a l y s i s o f t h e r e s u l t s showed t h a t
dehydrogenase, although
extent,
i s very
concentrations
inhibited
by a l l t h e compounds t o some
s e n s i t i v e t o concerted
of glyoxylate plus
isocitrate
inhibition
oxalacetate
by l o w
(Table X I V ) .
When a d d e d s i n g l y , o x a l a c e t a t e c a u s e d some i n h i b i t i o n o f t h e
enzyme, but g l y o x y l a t e a c t u a l l y
condensation
product
as o x a l a c e t a t e
a c t i v a t e d t h e enzyme.
was n o t f o u n d t o be as s t r o n g an
and g l y o x y l a t e t o g e t h e r .
the assay
oxalacetate.
i n the reaction mixture
This
found
a t t h e end
receiving glyoxylate plus
indicated that a condensation
f o r m e d a n d t h u s was n o t i m p o r t a n t
inhibitor
M o r e o v e r , i t was
t h a t a l m o s t a l l o f t h e g l y o x y l a t e c o u l d be r e c o v e r e d
of
The
product
was n o t
i n t h e r e g u l a t i o n o f enzyme
activity.
The m i x t u r e
reaction mixture
striking
o f t r i c a r b o x y l i c a c i d c y c l e a c i d s added t o t h e
i n equimolar concentration
inhibition
reaction mixture,
d i d not cause a
b u t i f g l y o x y l a t e (1 mM) was i n c l u d e d
the a c t i v i t y
i n the
dropped s h a r p l y t o almost z e r o a t
1.5 mM c o n c e n t r a t i o n o f t h e m i x t u r e
of acids
(Fig. 22).
Striking
i n h i b i t i o n was a l s o o b s e r v e d e v e n when 0.1 mM g l y o x y l a t e was
added t o t h e a s s a y
mixtures.
Lineweaver-Burk p l o t
( F i g . 23)
showed t h a t t h e Km.of t h e
Table XIV.
I n h i b i t i o n o f i s o c i t r a t e d e h y d r o g e n a s e a c t i v i t y by
v a r i o u s o r g a n i c a c i d s and r e l a t e d compounds.
0.2 mM
D L - i s o c i t r a t e was u s e d i n t h e a s s a y r e a c t i o n m i x t u r e .
Additions
Concentration
% Inhibition
* Glyoxylate
recovered
(mM)
Citrate
10
57.7
Cis-aconitate
10
66.7
a-Ketoglutarate
10
60.0
Succinate
10
46.7
Fumarate
10
33.3
Malate
10
35.6
Oxalacetate
10
88.5
Condensation
42.5
"»0.1
11.4
-33.5
10
Glyoxylate
Oxalacetate
glyoxylate
1.0
1 .0
-16.7
0.1
nil
+
10 e a c h
product
The c o n d e n s a t i o n
S h i i o and Ozaki
100
1.0
each
0.1
each
10
100
94.0
91.3
105.0
100
1 .0
77.8
nil
0.1
35.7
nil
product
(1968).
was
prepared
by t h e m e t h o d o u t l i n e d
by
99
O-O
0
n
r
,
2«0
g
4*0
6*0
CONCENTRATION( mM)
Fig.
22.
E f f e c t o f a mixture o f t r i c a r b o x y l i c acid c y c l e intermediates
on i s o c i t r a t e d e h y d r o g e n a s e a c t i v i t y .
Assays were c a r r i e d
o u t i n 1.0 ml r e a c t i o n m i x t u r e c o n t a i n i n g 6.8 y g enzyme
p r o t e i n and i n d i c a t e d c o n c e n t r a t i o n o f D - i s o c i t r a t e ( o - o ) ;
an e q u i m o l a r m i x t u r e o f D - i s o c i t r a t e , c i t r a t e , c i s - a c o n 4 t a t e ,
a - k e t o g l u t a r a t e , s u c c i n a t e , f u m a r a t e , L-malate and o x a l acetate ( • - • ) .
G l y o x y l a t e , 1 mM (• - • ) o r 0.1 mM ( • - • )
was a d d e d t o t h e a s s a y m i x t u r e s c o n t a i n i n g t h e m i x t u r e o f
organic acids.
100
IS] ( m M DL-ISOCITRATE )
F i g . 23. Lineweaver-Burk p l o t f o r i s o c i t r a t e dehydrogenase of
P. aerug?hosa.
4-'
enzyme f o r D - i s o c i t r a t e i s 2.3
b.
Isocitrate
Succinate
competitive
Gunsalus, 1957).
product
which
did
activity
of this
The p r e s e n t
enzyme i n P. a e r u g i n o s a
results
i n h i b i t o r n o r was
i s known t o be a s t r o n g
important
included
enzyme.
inhibitory
This
7 acids o f the t r i c a r b o x y l i c
a c t i o n o f these
the a c t i v i t y
with
remained
the mixture
further
condensation
enzyme f r o m E.
pattern
of acids
mM
glyoxylate
increasing
c o n c e n t r a t i o n , but
a c i d c y c l e were a l s o
i n equimolar c o n c e n t r a t i o n , the
a c i d s was v e r y
low.
1.5
coli.
i s a l s o seen
The a c t i v i t y o f t h e enzyme i n c r e a s e d w i t h
i n the r e a c t i o n mixture
and
phosphoenolpyruvate,
inhibitor of this
c o n c e n t r a t i o n o f D - i s o c i t r a t e u n t i l about
when o t h e r
The
o b s e r v a t i o n was t h a t o x a l a c e t a t e p l u s
not s t r o n g l y i n h i b i t t h i s
i n F i g . 2k.
(Smith
show t h a t s u c c i n a t e was t h e
i n h i b i t o r o f a l l t h e compounds t r i e d .
was n o t a s t r o n g
Another
mole/liter,
and g l y o x y l a t e p r e v i o u s l y w e r e shown t o be n o n -
inhibitors
most p o t e n t
lyase
x 10
I f 0.1
mM
s t r o n g o n . t h e enzyme and
g l y o x y l a t e was a d d e d
i n the reaction mixture,
along
i t d i d not
i n h i b i t the a c t i v i t y .
The Km o f t h e enzyme f o r D - i s o c i t r i e . a c i d was f o u n d t o be
2.5
x 10 ^ m o l e s / l i t e r ( F i g . 2 5 ) .
lyase f o r D - i s o c i t r i c
dehydrogenase.
acid
Thus, the a f f i n i t y o f
i s a b o u t one t e n t h
that of
isocitrate
isocitrate
102
Table
XV.
I n h i b i t i o n o f i s o c i t r a t e l y a s e a c t i v i t y by o r g a n i c a c i d s
and r e l a t e d compounds. - 3.0 mM D L - i s o c i t r a t e was u s e d i n
the assay r e a c t i o n mixtures.
Additions
Concentration
.%
Inhibition
(mM)
Citrate
10
13.5
Cis-aconitate
10
42.4
a-Ketoglutarate
10
57.0
Succinate
10
81.0
Fumarate
10
16.0
Malate
10
39.0
Oxalacetate
10
55.0
Glyoxylate
Oxalacetate
glyoxylate
0.5
40.0
0.1
31.0
plus
0.5
each
32.0
Condensation
product
o f g l y o x y l a t e and
oxalacetate
1.0
16.0
Phosphoenolpyruvate
1.0
5.0
103
l-5p
6
w
O
E
w 0-5
u
<
o.
1-0
2-0
3-0
CONCENTRATION (mM)
F i g . "24.
Effect of a mixture of t r i c a r b o x y l i c a c i d c y c l e intermediates
on i s o c i t r a t e l y a s e a c t i v i t y .
Assays were c a r r i e d o u t i n
2 . 0 ml r e a c t i o n m i x t u r e s c o n t a i n i n g 48 y g o f enzyme p r o t e i n
a n d i n d i c a t e d c o n c e n t r a t i o n o f D - i s o c i t r a t e (O) o r a n
equimolar mixture of D - i s o c i t r a t e , c i t r a t e , c i s - a c o n i t a t e ,
a - k e t o g l u t a r a t e , s u c c i n a t e , f u m a r a t e , m a l a t e and o x a l a c e t a t e
(•).
0 . 1 mM g l y o x y l a t e was a d d e d t o t h e a s s a y m i x t u r e
c o n t a i n i n g t h e m i x t u r e o f o r g a n i c a c i d s , when i n d i c a t e d (•).
A f t e r s t o p p i n g t h e r e a c t i o n , t h e m i x t u r e was d i l u t e d t e n
times before c o l o r development t o m i n i m i z e t h e i n t e r f e r e n c e
due t o t h e a d d e d o r g a n i c a c i d s .
.104
c. Aeon i t a s e a c t i v i t y
Aconitase
with
inhibition with
condensation
be i m p o r t a n t
product
glyoxylate plus
was a l s o t e s t e d t o s e e i f t h i s
i n the regulation of t r i c a r b o x y l i c
activity.
This
inhibition
by t h e c o n d e n s a t i o n
enzyme was
f o u n d t o be v e r y
product
physiological
and
XVI).
The
p l u s g l y o x y l a t e was
i t was c o n c l u d e d
that this
could
acid cycle
i s not formed
c o n d i t i o n s and i s u n i m p o r t a n t .
with oxalacetate
and
sensitive to
(Table
shown p r e v i o u s l y t h e c o n d e n s e d p r o d u c t
inhibition
oxalacetate
However, as
under
concerted
not so pronounced
on
aconitase
enzyme
in
the r e g u l a t i o n o f the a c t i v i t y of t r i c a r b o x y l i c
i s not
important
a c i d and
glyoxylate cycle.
Interpretation of this
isocitrate
study
l y a s e and a c o n i t a s e
on i s o c i t r a t e
activity
a pattern of control of tricarboxylic
cycle which
At
is similar
low l e v e l s
isocitrate
order
formed
lyase
i s induced
isocitrate
the
carbon source.
i n a c e t a t e medium
of i s o c i t r a t e
dehydrogenase a c t i v i t y
flavum.
the glyoxylate c y c l e in
g r o w on t h i s
The g l y o x y l a t e f o r m e d a s a r e s u l t
inhibits
i n Brevibacterium
acid cycle intermediates
by g r o w t h
brings out
a c i d c y c l e and g l y o x y l a t e
f r o m a c e t a t e must e n t e r
t h a t the organisms could
isocitrate
i n P. a e r u g i n o s a
to that described
of tricarboxylic
dehydrogenase,
lyase
The
(Table X ) .
activity,
concertedly with
oxal-
106
Table XVI.
Aconitase inhibition
P_. a e r u g i n o s a .
Add! t i o n s
Concentration;
Oxalacetic acid
Glyoxylic
in cell-free extract
acid
Oxalacetate
glyoxylate
Condensat ion
product
%
Inhibi t ion
0.1
17-5
1.0
30.0
0.1
14.0
1.0
30.0
p i us
0.1
of
each
14.0
1 .0 e a c h
41.8
0.1
100
1 .0
100
acetic acid.
present
i n the c e l l .
activity
cycle,
Small amounts o f o x a l a c e t a t e are presumably always
However, even when i s o c i t r a t e
i s weak, some i s o c i t r a t e
e n t e r s the t r i c a r b o x y l i c a c i d
because o f the high a f f i n i t y o f
this substrate,
dehydrogenase
isocitrate
dehydrogenase f o r
thus e n s u r i n g a supply of a - k e t o g l u t a r a t e .
When
l a r g e amounts of o r g a n i c a c i d s have accumulated due to the high
a c t i v i t y o f the g l y o x y l a t e c y c l e o r when these t r i c a r b o x y l i c a c i d
c y c l e members serve as the carbon source
suppress the a c t i v i t y o f
they do the
This
isocitrate
results
isocitrate
isocitrate
i n the medium,
they
l y a s e more e f f e c t i v e l y than
dehydrogenase a c t i v i t y
(Figs.
22 and
2k).
i n a lowering of g l y o x y l ' a t e p r o d u c t i o n and thus
dehydrogenase is r e l e a s e d
from the
inhibition.
complete t r i c a r b o x y l i c a c i d c y c l e now f u n c t i o n s and the
glyoxylate cycle a c t i v i t y
remains s u p p r e s s e d .
The
GENERAL DISCUSSION
Pseudomonas
utilizes
a e r u g i nosa
like
o t h e r members
a w i d e v a r i e t y o f compounds
disadvantage
to the c e l l
degradation of
this
f o r growth.
to maintain at a l l
large
for
the synthesis
towards
Thus,
the
of e s s e n t i a l
the synthesis
are o t h e r enzymes,
cell
at
of c e r t a i n enzymes,
advantage
its
Sharp,
neighbours
enzymes
diverted
However,
little
levels
o f enzyme
has
gives
there
are
independent
induction
a t t e n t i o n has
It
necessary
been
been proposed
inducible
(Pardee and B e c k w i t h , 1963;
reports
of this
that
i n v e s t i g a t i o n are
i n P. a e r u g i n o s a
that
it a selectional
which possess
counter-
Moses
and
i n agreement
t h e enzymes
of
with
glucose
has
given
i.e.
1968).
The r e s u l t s
other
synthesize
to c o n t r o l the s y n t h e s i s ,
constitutivity
o f such
enzymes.
o f c o n s t i t u t i v e enzymes.
i n a b i l i t y o f an o r g a n i s m
parts
m a t e r i a l are not
r e l a t i v e l y constant
been e x t e n s i v e l y s t u d i e d but v e r y
over
inductively
The phenomenon
for
and h e n c e b a c t e r i a
i . e . the c o n s t i t u t i v e enzymes, which
of n u t r i t i o n a l c o n d i t i o n s .
the
t h e enzymes
i n t e r m e d i a t e compounds
of unnecessary
p r o d u c e d by t h e o r g a n i s m
to the study
I t w o u l d be a
times
number o f compounds
h a v e d e v e l o p e d a s y s t e m by w h i c h t h e y c a n
enzymes when r e q u i r e d .
o f t h e genus Pseudomonas .
the
oxidation are inducible while those of the t r i c a r b o x y l i c a c i d
cycle are c o n s t i t u t i v e (Von Tigerstrom and Campbell, 1 9 6 6 ;
Ng and Dawes, 1 9 6 7 ) .
Lack of phosphofructokinase a c t i v i t y has
been found to be the reason f o r a non-functional Embden-Meyerhof
pathway in t h i s organism.
When the c e l l s were grown in succinate
medium,.the enzymes of glucose oxidation were e i t h e r at a low
level or were absent.
Repression of these enzymes, i . e .
the
enzymes of the Entner-Doudoroff pathway and the o x i d a t i v e portion
of the pentose phosphate pathway is a very important
regulatory
mechanism a v a i l a b l e to the c e l l when the organisms are grown
on any t r i c a r b o x y l i c acid cycle intermediate.
Under these
conditions s u f f i c i e n t energy is a v a i l a b l e from the t r i c a r b o x y l i c
acid cycle and t h e r e f o r e , the breakdown of glucose and related
compounds which are synthesized in l i m i t e d q u a n t i t i e s by the
reversal of Embden-Meyerhof pathway reactions and are required
for biosynthesis of s t r u c t u r a l components, is stopped.
P. aeruginosa
does not accumulate any s p e c i a l storage product which could serve
as a readily a v a i l a b l e source of hexose (MacKelvie, Campbell and
Gronlund, 1968) and hence i t w i l l be a disadvantage for the organism
to c a t a b o l i z e a l 1 of the a v a i l a b l e hexoses in the c e l l .
It has
been reported that at low concentrations of g1ucose-6-phosphate,
ATP strongly
i n h i b i t e d the glucose-6-phosphate dehydrogenase of
P. aeruginosa, while at high concentrations of t h i s substrate
the i n h i b i t i o n was i n s i g n i f i c a n t (Lessie and Neidhardt, 1 9 6 7 a ) .
These o b s e r v a t i o n s
context
rationalized
in.this
o f t h i s enzyme,
present
g r o w n i n s u c c i n a t e medium, may d e p l e t e
t h e low
o f g l u c o s e - 6 - p h o s p h a t e w h i c h may be d e r i v e d
Thus, strong
from
succinate.
i n h i b i t i o n o f g 1 u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e by
a t low l e v e l s o f g 1 u c o s e - 6 - p h o s p h a t e e n s u r e s t h e s u p p l y o f
ATP
this
compound f o r c e l l u l a r b i o s y n t h e s i s .
is present
ATP
be m e a n i n g f u l l y
s i n c e even a minor a c t i v i t y
in the c e l l s
levels
could
i n excess
inhibition
f o r example d u r i n g growth
i s released
o x i d i z e t h e compound w i t h
The
species.
medium,
to
facility.
repression o f the synthesis o f glucose
intermediates
Baci1lus
i n glucose
and t h e enzyme i s p e r m i t t e d
i n P_. a e r u g ? h o s a upon g r o w t h
cycle
glucose-6-phosphate
When
i n medium w i t h
o x i d a t i o n enzymes
tricarboxylic
i s i n contrast t o the observations
In one r e p o r t w h e r e t h e s t u d y
acid
made w i t h
on t h e r e g u l a t i o n
of g1ucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrog e n a s e a n d h e x o k i n a s e was c a r r i e d o u t w i t h
found t h a t t h e l e v e l s o f these
B. s u b t i 1 i s ,
t h r e e enzymes i n c e l l s
s u c c i n a t e o r g l u t a m a t e medium d i d n o t d e c r e a s e .
c e l l s were capable
to those o f c e l l s
As
of metabolizing
glucose
grown i n a g l u c o s e
a matter o f fact
t h e o r g a n i s m s grown
showed more t h a n t h r e e t i m e s
t h a n o r g a n i s m s grown
medium
i t was
g r o w n on
Therefore,
at rates
these
comparable
(Moses a n d S h a r p ,
i n a succinate
1968).
medium
the rate o f synthesis o f hexokinase
i n a glucose
medium.
vto w h a t w o u l d be p r e d i c t e d s i n c e g l u c o s e
This
i s contrary
w o u l d be e x p e c t e d t o be
the
inducer
o f t h e enzyme.
No e x p l a n a t i o n
was o f f e r e d
b e h a v i o u r o f a s e e m i n g l y c o n s t i t u t i v e enzyme.
f o r this
Particulate malic
d e h y d r o g e n a s e o f P. a e r u g i n o s a h a s b e e n f o u n d t o b e h a v e
in
that
i t showed
rather than
(Table
higher
i n the presence o f a l i k e l y
IX, F i g . 20).
were n o t c o n c l u s i v e ,
enzymes
rates o f synthesis
Although
inducer,
that
glucose
a r e c o n s t i t u t i v e i n B. s u b t i 1 i s .
aerogenes c e l l s
glucose without/a
grown
namely
in this
succinate
and Sharp
metabolizing
A l t h o u g h no s u c h
h a s b e e n made on g l u c o s e o x i d a t i o n enzymes
appear t o resemble b a c i l l i
i n g l u c o s e medium
t h e r e s u l t s o f Moses
i t appeared
similarly,
in coliforms,
c o n t r o l mechanism
study
they
s i n c e A_.
i i i c i t r a t e medium h a v e b e e n shown t o o x i d i z e
l a g ( D a g l e y and Dawes, 1 9 5 3 ) .
Indeed,
glucokinase
has b e e n c o n c l u d e d t o be a c o n s t i t u t i v e enzyme i i i A. a e r o g e n e s
(Kamel, A l l i s o n
The
Horecker
and A n d e r s o n ,
1966).
r e s u l t s presented here a l s o support the suggestion
(1965)
that
i n most
instances
of
t h e p e n t o s e phosphate pathway
does n o t f u n c t i o n as a c y c l e , b u t r a t h e r as two mechanisms f o r
cellular biosynthesis.
The o x i d a t i v e p o r t i o n o f t h e p a t h w a y
o p e r a t e s when NADPH i s r e q u i r e d
transformations
It
v
while
non-oxidative
a r e u t i l i z e d when p e n t o s e p h o s p h a t e s a r e r e q u i r e d .
i s o n l y when t h e r e q u i r e m e n t o f t h e c e l l
requirement
to
by t h e c e l l
f o rthe pentose phosphates that
f o r NADPH e x c e e d s i t s
i t o p e r a t e s as a c y c l e
return excess pentose phosphates t o the metabolic
carbohydrates.
I t would appear, t h e r e f o r e ,
that
pool o f
t h e demand f o r
NADPH o f t h e c e l l s
by
the reactions
o f the pentose phosphate c y c l e
does n o t o p e r a t e .
pentose synthesis
low
during
growth
F o r t h e same r e a s o n s ,
i n the c e l l s
i n pseudomonads.
that
i n succinate
grown
This
finding
medium t h e demand f o r
reaction
( D e L e y , 1960;
indicates
pentose
b e t w e e n compounds
from t r i c a r b o x y l i c a c i d c y c l e
i s i n agreement w i t h
other workers
its contribution to
The a n a l y s i s o f t h e r e s u l t s a l s o
i n succinate
be d e r i v e d
i s unnecessary
i n g l u c o s e medium may a l s o be
p h o s p h a t e must be met by t h e t r a n s k e t o l a s e
which could
medium i s met
o f t h e t r i c a r b o x y l i c a c i d c y c l e and hence, t h e
oxidative portions
and
o f P. a e r u g i n o s a grown
intermediates.
t h e p r o p o s a l s made by s e v e r a l
S a b l e , 1966;
Lessie
and N e i d h a r d t ,
1967a).
F a c u l t a t i v e b a c t e r i a a n d some s p e c i e s
forming b a c i l l i
the
of aerobic
w h i c h c a n l i v e a n d grow w i t h o u t
t r i c a r b o x y l i c acid cycle
found
t o show a s t r i k i n g
cycle
(Col
spore
the a c t i v i t y of
under c e r t a i n c o n d i t i o n s
h a v e been
g l u c o s e e f f e c t on t h e enzymes o f t h i s
1 i n s a n d L a s c e l l e s , 1962; G r a y e _ t a l _ , 1966b; Hanson e t
al_,
1963a; 1963b; 1964; Hanson a n d C o x , 1967).
the
g l u c o s e e f f e c t on t h e t r i c a r b o x y l i c a c i d c y c l e e n z y m e s i s
not
seen
cycle
i n P. a e r u g i n o s a ,
i n d i c a t i n g that
i s e s s e n t i a l f o r g r o w t h on g l u c o s e .
On t h e o t h e r
the a c t i v i t y
hand,
of this
The a d d i t i o n o f
g l u t a m a t e a n d a - k e t o g l u t a r a t e t o t h e g r o w t h medium d i d n o t r e p r e s s
the
synthesis
leading
o f t h e enzymes o f t h e t r i c a r b o x y l i c a c i d
to a-ketoglutarate synthesis.
<
cycle,
In a d d i t i o n , g r o w t h
in a
succinate
medium
oxidation
as
acid
and
cycle
repressed
already
fulfils
therefore,
Succinate
effectively
i s of
than
1967b).
been
to
shown
(Rosenfeld
possess
a
coli
and
and
A.
two
in
has
various
again
that
the
groups
of
oxidation
both
cycle
as
the
and
importance
shown
the
the
biosynthetic
to
these
aeruginosa
acidovorans
induction
1969).
of
of
to
to
which
bacteria.
they
are
possess
sole
complete
lead
to
pathways
difference
cycle
recently
oxygenase
rise
to
succinate,
repression
more o f
required.
of
which
activity,
catabolite
active
source
and
i n pseudomonads,
exert
not
more
has
tryptophan
give
needs
(Lessie
succinate
Thus,
appears
absence of
degradative
suggest
-glucose
P.
since
the
been
The
and
metabolic
glucose
the t r i c a r b o x y l i c
i n P.
activities
glucose
even
glucose
Thus,
glucose
aerogenes which
utilize
enzymes o f
hist idine degradation
Feigelson,
this
of
repress
In
intermediates
growth
on
of
enzymes,the
cycle
above.
constitutive tricarboxylic acid
component
on
to
prevent
and
major
special
does
Neidhardt,
synthesis
discussed
the
i s known
the
the
Whereas
in
Embden-Meyerhof
carbon
and
effective catabolite
(Magasanik,
in metabolic
1961).
control
pathways
energy
tricarboxylic acid
E_.
for
cycle,
repression
These
facts
pattern
in
the
organisms.
study
i n P.
of
the
induction
aeruginosa
induction
permease were
of
of
grown
glucose
responsible
the
systems
in succinate
metabolizing
for
the
long
for
medium
enzymes
lag
glucose
transport
suggested
and
periods
the
required
before
utilization.
cells
The f i n d i n g t h a t g j u c o s e
in t h i s organism
permease which
d e v e l o p e d an a c t i v e s y s t e m f o r g l u c o s e
i s unique s i n c e
permease i s i n d u c i b l e
i n E. c O l i
i s also responsible
f o r glucose
shown t o be c o n s t i t u t i v e (Cohen and Monod,
Many o r g a n i s m s ,
a-methyl-glucoside
i n c l u d i n g animals
u p t a k e h a s been
1957).
and p l a n t s , p o s s e s s b o t h a
particulate mitochondrial
and a s o l u b l e c y t o p l a s m i c
dehydrogenase.
i t h a s b e e n shown t h a t t h e s o l u b l e
malic
dehydrogenase, which
acetate,
i s repressed
necessary
by g r o w t h
the m i t o c h o n d r i a l
i s required
by g l u c o s e
f o rcatabolism
influenced
has
In y e a s t ,
f o rgluconeogenesis
the mitochondrial
from
enzyme
v i a the tricarboxylic acid cycle
i n a glucose
medium
(Hoizer, 1966).
enzyme o f p i g h e a r t , p e a s and o t h e r
i s not
It is
plants
that
b e e n shown t o be i n f l u e n c e d b y a d e n i n e n u c l e o t i d e s w h i l e t h e
s o l u b l e enzyme i s u n a f f e c t e d
the
while
malic
particulate malic
the m i t o c h o n d r i a l
(Kuramitsu,
1966; 1968).
d e h y d r o g e n a s e o f P. a e r u g i n o s a
enzyme o f h i g h e r
organisms
Therefore,
resembles
in i t s control pattern.
I t h a s b e e n f o u n d t h a t t h e i s o c i t r a t e d e h y d r o g e n a s e o f P_.
aeruginosa
Glutamic
formation
i s NADP s p e c i f i c a n d shows no a c t i v i t y w i t h
dehydrogenase
i n t h e d i r e c t i o n o f a-ketog 1 u t a r a t e
shows a c t i v i t y
coenzyme u t i l i z e d
NAD.
only with
NADP a n d NADPH i s t h e m a j o r
i n the d i r e c t i o n o f glutamate synthesis.
M a l i c d e h y d r o g e n a s e i s p a r t i c u l a t e and h a s e l i m i n a t e d t h e need
f o r f r e e NAD, r e q u i r e d by o t h e r
bacteria at this step.
Therefore,
although
not
this
organism
relies
on NADPH f o r a n a b o l i c r e a c t i o n s , i t does
r e l y on f r e e NAD f o r many c a t a b o l i c r e a c t i o n s .
w h e r e t h e o r g a n i s m o b v i o u s l y n e e d s NAD a r e p y r u v a t e
and
steps
dehydrogenase
a - k e t o g l u t a r a t e dehydrogenase c a t a l y z e d r e a c t i o n s where
this
coenzyme i s c a r r i e d
possesses
and
The o n l y
a s apoenzyme i n a c o m p l e x . P_. a e r u g i n o s a
( E a g o n , 1 9 6 3 ; Von T i g e r s t r o m
a c t i v e transhydrogeriase
Campbell, 1966b).
Other a e r o b i c organisms possessing
trans-
h y d r o g e n a s e , e . g . P. f 1 u o r e s c e n s a n d A. v i he1 a r i d i i a r e known t o
p o s s e s s o n l y NADP l i n k e d
isocitrate
T h u s , t h e r e seems t o be a g e n e r a l
possessing
on
energy generation
transhydrogenase
roles
oxidized
(Horecker,
Although
proposal
NADPH t h r o u g h t h e
agrees w i t h
the postulated
1965; Kaplan,
1963).
i s not r e a d i l y
available for biosynthetic
Pseudomonads a r e known
c a p a c i t y o f b i o s y n t h e s i s a n d i t i s an a d v a n t a g e t o
o f NADPH r a t h e r t h a n
rapidly oxidizable
purpose.
P_. a e r u g i n o s a
carboxyl ic acid
-exerted
This
from excess
oxygen and i s thus
t o produce a pool
NADH f o r t h i s
dependent
r e a c t i o n s a n d NADH f o r t h e p u r p o s e o f
o f h i g h e n e r g y p h o s p h a t e a n d o f NADPH w h i c h
t h e i r great
the c e l l
are less
organisms
i s r a p i d l y o x i d i z e d by t h e o x y g e n w i t h t h e
by m o l e c u l a r
reactions
for
reaction.
( K a p l a n , T'963)'.
i n the aerobic
i n that they
c o u l d be d e r i v e d
o f NADH w h i c h
formation
pattern
a c t i v e transhydrogenase
f r e e NAD f o r t h e m e t a b o l i c
dehydrogenase
a p p e a r s t o have c o n s t i t u t i v e
cycle activity,
by m e t a b o l i t e s
tri-
t h e c o n t r o l on k e y s t e p s i s
t o r e g u l a t e t h e f l o w o f compounds
through
the
by
cycle.
P a r t i c u l a t e malic
adenine nucleotides
m e c h a n i s m by w h i c h t h e
and
dehydrogenase which
activated
cell
could
by
GTP
control
and
the
is inhibited
GDP
provides
t r i c a r b o x y l i c acid
cycle
activity.
S i m i l a r l y , i s o c i t r a t e d e h y d r o g e n a s e and
lyase
activities
are
acid
c y c l e and
the
c o n t r o l l e d by
glyoxylate
cycle
compounds t h r o u g h t h e s e c y c l e s
manner, but
needs o f
The
cycle
is regulated
the
does not
a way
proceed
fashion
that
in a
the
flow
of
haphazard
to best s u i t
the
organism.
activity
bacteria
isocitrate
members o f t h e t r i c a r b o x y l i c
in;such
in a precise
lack'of .similarities
b e t w e e n P.
the
a
and
the
activity
a e r u g i n o s a and
suggests that
control of
t r i c a r b o x y l i c acid
of glucose oxidation
e n t e r i c and
during
o r g a n i s m s have d e v e l o p e d
mechanisms s u i t i n g the
i n the
the
aerobic
spore
enzymes
forming
process of evolution
c h a r a c t e r i s t i c m e t a b o l i c and
environment which they
the
control
found themselves
in.
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