University of Southern California Dissertations and Theses

Studies on the aerobic oxidation of fatty acids by bacteria

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Studies on the aerobic oxidation of fatty acids by bacteria

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Content STUDIES ON THE AEROBIC OXIDATION OP PATTY ACIDS BY BACTERIA
A Dissertation
Presented to
the Faculty of the Graduate School
The University of Southern California
In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy
by
John Harold Sllllker
September 1950
'^1
This dissertation^ w rliie u by
.......CrQHN..JHAJRQLD..SJ:LLIKER........
under the guidance of F a cu lty Com m ittee
on Studies, and approved by a ll its members, has
been presented to and accepted by the C o u n c il
on Graduate Study and Research, in p a rtia l f u l
fillm e n t of requirements fo r the degree of
D O C T O R O F P H IL O S O P F IY
Dean
Date.
Committee on Studies
Chairmà:
ACKNOWLEDGMENTS
The author wishes to express his sincere apprecia
tion of the personal interest shown by so many persons in
his work—
His thanks to Drs. M. D. Appleman, James W. Barth
olomew, Walter Marx, Charles Pait, Robert P. Williams, and
to Dean Harry J. Deuel, the members of his committee on
studies, for their constructive criticism, for their gener
ous advice.
His thanks to fellow students Mr. Daniel Ivler and
Mr. Eugene P. Hess for drafting and photographing the
figures and to Mr. Tod Mittwer whose helpful suggestions
clarified difficulties both in research and in the draft
ing of this manuscript.
Especial and deepest appreciation to Dr. S. C.
Rittenberg for his unstinted interest and encouragement,
but most of all, for the quality of his guidance.
TABLE OP CONTENTS
CHAPTER PAGE
I. INTRODUCTION ................................ 1
Stimulatory effects produced by fatty
acids............................... 2
Fatty acids as oxidizable substrates for
microorganisms ....................... 5
Mechanisms of fatty acid oxidation In
animals............................. l4
Simultaneous adaptation— a possible means
of studying the mechanism of fatty acid
oxidation of microorganisms .......... 20
II. THE NATURE OF THE ENZYMES, CONSTITUTIVE OR
ADAPTIVE, IN PATTY ACID OXIDATION BY VARIOUS
BACTERIA: A SURVEY..................... 26
Experimental........... 27
Discussion............................... 37
Summary................................. 38
III. APPLICATION OF THE TECHNIQUE OF SIMULTANEOUS
ADAPTATION TO THE STUDY OP FATTY ACID OXIDA
TION IN SERRATIA MARCESCENS.............. 39
Experimental ..................... . 39
Oxidative patterns in relation to growth
on capric acid medium................ 39
Ill
CHAPTER • PAGE
Oxidative patterns in relation to
specific adaptation to various acids . . A3
The response of Escherichia coll #2 to
growth on and exposure to fatty acids . A9
Discussion...................... 55
Permeability as a factor in the lag periods 57
The possibility of a single enzyme .... 59
The possibility of the accumulation of an
intermediate . ..................... 63
Distinct reaction chains merging in a com
mon intermediate ..................... 64
Summary........................ 67
IV. THE EFFECT OF CELL POISONS ON FATTY ACID
OXIDATION BY SERRATIA.......... 68
Experimental.................... 70
Effect of DNP and sodium azide on oxida
tion at pH 7 . 0 .............. 72
The oxidation of fatty acids at pH 8.0 in
the presence and absence of DNP .... 85
Evidence that pelargonic and capric acids
are completely oxidized at pH 7*0 in the
presence of D N P ............ 89
Discussion...................... 90
Summary........................ 97
iv
CHAPTER PAGE
V. STUDIES ON THE RELATIONSHIP OP VARIOUS PATTY
ACID DERIVATIVES TO THE PRIMARY OXIDATIVE
REACTIONS IN FATTY ACID OXIDATION........ 99
Experimental . ........................... 100
The oxidation of caprate derivatives by
glucose and caprate-grown cells .... 101
The relationship between adaptation to
caprate derivatives and the oxidation of
other derivatives...................... 105
The effect of adaptation to caprate deriva
tives on the oxidation of capric, capryl-
ic, and undecylic acids.............. 107
Discussion........................... IO8
Summary................................... II8
VI. THE RELATIONSHIP BETWEEN THE CITRIC ACID CYCLE
AND THE OXIDATION OF FATTY ACIDS BY SERRATIA
MARCESCENS......................... 120
Experimental......... 121
The oxidation of citric acid cycle com
pounds by Serratia...................... 122
The effect of prior oxidation of succinate
and malate on the oxidation of capric
acid................................... 122
CHAPTER PAGE
The effect of cooxidation of citric
acid cycle compounds..................... 123
Discussion................................. 129
Summary................................... 130
VII. ATTEMPTS TO OBTAIN ENZYMATICALLY ACTIVE PRE
PARATIONS PROM SERRATIA..................... 132
Experimental............................... 134
Dry cell preparations.................... 134
Acetone dried preparations ................ 137
Freezing and thawing...................... 138
Autolysis............................... 139
Discussion................................. 139
Summary............................ 142
VIII. RESUME.................................. 143
BIBLIOGRAPHY ....................................... 131
LIST OP TABLES
TABLE PAGE
I. Oxidation of Capric and Pelargonic Acids
by Various Bacteria .............. 30
II. Lag Periods in the Oxidation of Patty Acids
in Relation to Specific Adaptation of
Serratia (Alphin) Cells to Various Acids . 44
III. Effect of 2:4-Dinitrophenol on the Oxidation
of Fatty Acids by Serratia Marcescens
(Alphin).......................... 74
IV. The Effect of pH on the Oxidation of Fatty
Acids by Serratia (Alphin) Cells in the
Presence and Absence of 2:4-Dinitrophenol . 8l
V. Lag Periods in the Oxidation of Capric, Un
decylic, and Caprylic Acids in Relation to
Adaptation to Capric Acid Derivatives . . . 109
VI. Capric Acid Oxidation in Relation to the
Simultaneous Oxidation of Malate ........ 125
VII. Capric Acid Oxidation in Relation to the
Simultaneous Oxidation of Oxalacetate,
Succinate, and Citrate by Unadapted Cells . 126
LIST OF FIGURES
FIGURE PAGE
1. Oxidation of Various Fatty Acids by Glucose-
Grown Serratia (Alphin) ................... 33
2. Oxidation of Various Patty Acids by Glucose-
Grown Serratia (Baker)........... 35
3. Oxidation of Capric Acid by Four Strains of
Pseudomonas SP. ......................... 36
4. Oxidation of Various Fatty Acids by Caprate-
Grown Serratia (Alphin) .............. 4l
5» Oxidation of Various Fatty Acids by Caprate-
Grown Serratia (Baker) .................... 42
6. Oxidation of Capric Acid by Serratia (Alphin)
Cells Specifically Adapted to Other Acids . . 47
7* Oxidation of Caprylic and Caproic Acids by
E. Coli # 2 ............................... 50
8. Oxidation of Patty Acids by Adapted and Unadapt
ed Cells in the Presence and Absence of DNP . 73
9. Oxidation of Alpha-Beta Unsaturated Capric
Acid by Serratia (Alphin).................. 102
10. Oxidation of Beta Hydroxy Capric Acid by
Serratia (Alphin) ......................... 103
11. Oxidation of Beta keto Capric Acid by Serratia
(Alphin) ........................... 104
12. The Oxidation of Caprate in the Presence and
Absence of Malate......................... 124
CHAPTER I
INTRODUCTION
The effects of fatty acids on microorganisms are
many and diverse, but the influence exerted usually takes
one of three forms:
1. The acids may be toxic, causing inhibition of
growth, cessation of respiration, or death of the cells.
2. They may be stimulatory, allowing rapid initia
tion of growth, increased cell yields, and increased res
piration.
3- They may serve as oxidizable substrates yield
ing energy to the cell.
A given compound may exert one or more of these
influences on a given organism depending upon the experi
mental conditions imposed. Thus Bergstrom, Thorell, and
DaVide (1946) report inhibition of respiration in the
tubercle bacillus by oleic acid in 1:10,000 dilution; Dubos
(1 9 4 9) has repeatedly shown that the same acid stimulates
growth of Mycobacterium tuberculosis; Gray (19^9) has re
cently shown that many species of mycobacteria, including
the tuberculosis organism, not only oxidize oleic acid but
in fact depend upon it as well as upon certain other fatty
acids as a source of energy in the absence of exogenous
oxidizable substrates.
The toxic action of fatty acids has received
considerable attention since such activity suggests possible
application of these compounds as therapeutic agents and as
a means of killing or inhibiting microorganisms in foods and
other commercial products. Toxicity may be due to hydrogen
ions, to the undissociated molecule, or to the lowered sur
face tension produced by these surface active materials. A
detailed discussion of toxicity will not be attempted since
two theses from this department will treat this subject ex
tensively (Simon, 1950; Ward, 1950).
Stimulatory effects produced by fatty acids. The
most striking study of the stimulatory effects produced by
fatty acids is that of Dubos and his co-workers on Myco
bacterium tuberculosis. This work has recently been re
viewed in detail (Dubos, 19^9)- The tubercle bacillus, in
the common aqueous media used by the bacteriologist, grows
in the form of heavy pellicles or large clumps of organisms
which are but little wetted by the water phase of the. medium.
This hydrophobic character can be overcome by adding wetting
agents to the culture medium. One class of compounds use
ful in this respect includes polyoxyethylene derivatives of
sorbitan esters of long chain fatty acids, commercially
known as Tween compounds. As a result of adding these sub
stances to culture media, dispersed and homogeneous cultures
of tubercle bacilli can be obtained after one week of incuba
tion. Likewise, with proper care being taken to overcome
toxic effects, palmitic, stearic, oleic, linoleic, arachi-
donic, and lignoceric acids (as sodium soaps or water
soluble esters) increase yields of avian and h^an tubercle
bacilli from serum albumin medium, and the yields increase
in direct proportion to the concentration of fatty acid in
the medium.
With the use of bacteria as a tool in vitamin assay
came the necessity for growing organisms in chemically de
fined media. Quite frequently it was found that fatty
acids were a limiting factor for growth in such media.
Bauemfeind, Sotier, and Bo ruff (1942) found that oleic,
stearic, and palmitic acids stimulated the production of
lactic acid by Lactobacillus casei in the presence of sub-
optimal amounts of riboflavin or pantothenic acid, while
linolenic acid either stimulated or inhibited, depending
upon the amount used. Strong and Carpenter (1942) noted
that oleic and stearic acids exerted strong stimulatory
action in riboflavin assay with L. casei, while palmitic
and linoleic acids were strong inhibitors. Neal and Strong
(1 9 4 3) observed both stimulation and inhibition of bacterial
response in pantothenate assay, depending upon the level of
both the vitamin and the acid used. Krehl, Strong, and El-
vehjem (19^3) obtained increased acid production with added
fatty acids when Lactobacillus arabinosus was used for the
assay of pantothenic acid. Kodicek and Worden (1945) re
ported that there was an increase in acid production with
stearic and palmitic acids and an inhibition with un
saturated acids in the same assay. Williams and Fleger
(19^5) found that fatty acids stimulated bacterial response
when L. casei and L. arabinosus were used in biotin assay.
In a later study (Williams and Fleger, 19^7) it was postulat
ed that biotin functions as a cell permeability factor and
that it could be replaced by the proper lipids. Guirard,
Snell, and Williams (1946) have shown that sodium acetate
greatly stimulates the growth rate of Lactobacillus easel
and permits rapid Initiation of growth. This property was
found to be shared by the longer chain length saturated
acids beginning with caprylic, myrlstic displaying highest
activity.
Hutner (1942) obtained data which indicated that
oleic acid is a necessary factor for the growth of Listerel-
la organisms. Collins, Nelson, and Parmelee (1949) found
that certain strains of lactic acid streptococci failed to
grow in a basal medium unless oleic acid was added as an
accessory factor. Quite recently, Lein and Lein (1949) have
obtained a mutant strain of Neurospora which requires oleic,
linoleic, or linolenic acid for growth in a minimal medium
supporting growth of the parent strain.
It is clear therefore that fatty acids may play an
essential role in the metabolism of many microorganisms.
The media usually used for the cultivation of heterotrophic
organisms probably contain sufficient amounts of these
compounds to meet the requirements for growth. Were
it necessary to grow many types of bacteria in purely
synthetic media, further requirements for fatty acids
might become apparent. It is evident that in some cases
the role of fatty acids is to modify the physical condi
tions of the environment (in the case of the tubercle
bacillus) while in others fatty acids may actually serve
functions analogous to bacterial vitamins (in the case
in Neurospora). It is quite likely that these compounds
are much more important in bacterial nutrition than
previously supposed and that future investigations on
nutritional requirements will reveal that these sub
stances are important constituents of the chemical environ
ment.
Fatty acids as oxidizable substrates for micro
organisms . The study of fatty acid metabolism in micro
organisms has lagged far behind that of carbohydrate and
protein metabolism. Breusch (1948) has pointed out that
a similar situation exists in animal physiology and at
tributes it to the following:
"l. Fatty acids and their salts are difficultly
soluble in water; this fact, together with low diffus
ibility through the cell walls, has made it difficult ex
perimentally to bring substrate and enzymes together.
Therefore incubation of macerated tissue or tissue sections
6
with fatty acids has produced few results.
2. The poisonous action on tissue respiration of
saponified and therefore water-soluble fatty acids permits
only low substrate concentrations in vitro incubation
experiments and may bring about reactions of no consequence
in vivo.
3* The quantitatively relatively small conversion
capacity of tissue enzymes with respect to fatty acids, as
compared to their capacity to act on carbohydrates and
proteins.
4. The lack of suitable microanalytical methods
for proper differentiation, isolation, and identification
of small quantities of fatty acids and their metabolites.”
For the most part these difficulties are to be ex
pected in the study of microbial metabolism of fatty acids
and explain in part the dearth of information on the
mechanisms of attack by microorganisms.
Anaerobic transformations of fatty acids by micro
organisms have been subjected to more detailed investiga
tion than aerobic conversions. Probably the most important
of these is the well known reduction of butyric acid to
butyl alcohol which occurs in the acetone-butyl alcohol
fermentation (Prescott and Dunn, 1940). Similarly,
propionic acid is reduced to propyl alcohol by Clostridium
acetobutylicum (Blanchard and MacDonald, 1935) and by
Aerobacter Indologenes (Mlckelson and Werkman, 1939).
Schonbrunner (1940) has demonstrated the saturation of
oleic acid by pure cultures of bacteria, and Rosenfeld
(1948) noted that saturation of fatty acids buried in
sedimentary materials increased with depth and traced this
to the activity of anaerobic bacteria.
Interest in anaerobic transformations of fatty acids
was stimulated by the postulate that fatty acids might under
go decarboxylation and yield hydrocarbons with one less
carbon atom than the original acid. Such a process might
point to the origin of hydrocarbons as a result of micro
biological processes. Sohngen (1 9 0 6) working with mixed
cultures of bacteria, showed that in an otherwise mineral
medium formates, acetates, and normal butyrates were
quantitatively decomposed to methane and carbon dioxide.
Strong indications were obtained that the decomposition of
capronates, caprylates, and caprinates followed the same
pathway. Coolhaas (1 9 2 7), working with thermophilic
bacteria, established fermentation of formates, acetates,
and isobutyrates with the formation of methane. All other
acids tried, including butyrate, gave inconclusive results.
Taylor (1928) mixed fats and oils with sand amd covered
the mixture with clay, incubating at room temperature.
The cultures thus obtained soon produced gas in sufficient
quantity to lift the clay from the sand. The gas consisted
mainly of methane, but Taylor concluded that the fat was
8
hydrolyzed, and the resulting glycerol converted to methane
and the fatty acids to the corresponding paraffins. Thayer
(1 9 3 1) reinvestigated the problem, adding marine mud samples
to a mineral salts medium containing salts of various fatty
acids. Methane escaped from the fermenting mixture, but
this was the only hydrocarbon detected in the breakdown of
fatty acids under these conditions. Barker (1936) was the
first to isolate methane producing bacteria in pure culture.
Barker, Ruben, and Kamen (l94o) later showed that methane
production arose from the reduction of carbon dioxide, the
hydrogen required arising from organic substrates, includ
ing fatty acids. Buswell and Sollo (1948) present evidence
which suggests that in the methane fermentation in mixed
culture the gas produced arises through some mechanism
other than reduction of carbon dioxide, a simple decarboxyla
tion of acetic acid appearing most likely. They suggest that
the mechanism found by Barker et al. in Methanosarcina may
be limited to pure cultures of that organism. The evidence
seems to lead to the conclusion that bacteria do not produce
hydrocarbons, other than methane, when acting on fatty acids
under anaerobic conditions.
In the work of Sohngen (1 9 0 6) it was shown that hydro
gen sulfide was one of the first compounds produced in mud
samples containing fatty acids. Later investigation showed
that sulfide arose as a result of the action of sulfate
reducing bacteria which used fatty acids as hydrogen donors
for the reduction of sulfate. The reaction involves con
version of fatty acids to carbon dioxide and hydrogen,
with the hydrogen being used to convert a corresponding
amount of sulfate to sulfide (Baars, 1930).
A unique mode of fatty acid metabolism has been
discovered and thoroughly studied by Barker and his co
workers. It was shown that Clostridium kluyveri obtains
energy from the synthesis of fatty acids (Barker, 1937).
This anaerobic organism converts two-carbon compounds
(ethanol and acetate) to fatty acids of four and six car
bons (Bomstein and Barker, 1948a, 1948b). Tracer experi
ments revealed that this conversion is an oxidation-
reduction process in which ethanol is oxidized to a two-
carbon compound ("active” acetate) that is in approximate
equilibrium with acetate. This active acetate is condensed
with acetate to a four-carbon compound that serves as an
oxidant for the reaction and is reduced to butyrate; in a
similar manner, the "active" acetate derived from another
molecule of ethanol may condense with butyrate to form
caproate (Barker, Kamen, and Bornstein, 1945)- Cell-free
preparations of the organism (Stadtman and Barker, 1949a)
under anaerobic conditions quantitatively convert ethanol
and acetate to butyrate. Aerobically, with oxygen as an
electron acceptor, ethanol was oxidized to acetyl phosphate
10
and acetate. A condensation of acetyl phosphate and
acetate was catalyzed and the product reduced to butyrate.
Acetaldehyde proved to be an intermediate in the conver
sion of ethanol to acetyl phosphate (Stadtman and Barker,
19^9b). The conversion of acetyl phosphate plus acetate
to butyrate involves the uptake of two moles of hydrogen.
Where the acetate concentration is low, caproic acid, as
well as butyric, is formed. In the presence of excess
acetate, the reaction goes almost quantitatively to buty
rate (stadtman and Barker, 1949c). Evidence obtained from
experiments with cell-free preparations indicated that
acetoacetic acid is not the condensation product formed
I in the interaction between acetyl phosphate and acetate,
since acetoacetate could be reduced by enzyme prepara
tions only as far as beta hydroxy butyric acid (Stadtman
and Barker, 1949c). The conversion of the condensation
product (whatever it might be) to butyrate should involve
the formation of two intermediate products. The first
should be in a state of oxidation corresponding to aceto
acetate (but is not this compound). The second should
correspond to crotonic acid, beta hydroxy butyric acid,
or vinyl acetate. Both crotonic acid and beta hydroxy
butyric acid were ruled out as possible intermediates.
While vinyl acetate was shown not to be an obligatory
intermediate in butyrate synthesis, this compound may
11
play a role in the conversion of acetyl phosphate and
acetate to butyrate. In the anaerobic oxidation of vinyl
acetate hydrogen is evolved. This reaction leads to a net
production of phosphate bond energy in the form of acetyl
phosphate. It is probable that most of the energy for
synthesis of cell materials is derived from this or an
analagous reaction. The following represents the general
ized mechanism for butyrate synthesis by Clostridium
kluyverit
ethanol + > acetyl phosphate + 4h
acetyl phosphate + acetate > compound + H^PO^
C i | . compound + 4h » butyrate
Our most detailed knowledge of aerobic dissimila
tion of fatty acids by microorganisms comes from studies
on the nature of rancidity in natural fats and oils. The
rancidity is due to oxidative breakdown of fatty acids
liberated after hydrolysis of lipids by various fungi. In
many cases the oxidation is incomplete, and the products
which accumulate, notably ketones, give characteristic
odors and flavors associated with rancidity. Starkle (1924)
was the first to discover the role of fungi in rancidity.
He was able to isolate Pénicillium glaucum from rancid oil
and showed that this mold, as well as Aspergillus niger
and Aspergillus fumagatus, could form methyl ketones with
one less carbon atom than was present in the fatty acids
12
on which the organisms were grown. These early observa
tions have since been confirmed by other workers (Stokoe,
1 9 2 8; Coppock et al., I9 2 8; Acklin, 1939; Thaler and Geist,
1939b; faufel et al., 1939)• The ability of fungi to pro
duce methyl ketones from fatty acids appears to be limited
to the members of the series from C i | . ( butyric ) to 0^4
(myristic). Palmitic, stearic, and oleic acids are not
converted to ketones (Taufel et al., 1936).
Considerable speculation has surrounded the possible
mechanisms involved. Beta hydroxy acids give rise to
methyl ketones when attacked by molds which form ketones
from fatty acids (Thaler and Geist, 1939b). Thaler and
Eisenlohr (1941) have shown that the reaction goes through
alpha-beta unsaturated acids since these derivatives of
butyric, caproic, capric, and myristic acids all yield
methyl ketones. More recently Thaler and Stahlin (1950)
have shown that Pénicillium glaucum cannot form methyl
ketones from beta methyl— beta hydroxy acids, but with
alpha methyl acids ketone formation occurs. The following
sequence was suggested:
saturated fatty acid— 2h-» alpha beta unsaturated
acid— +HgO— * > beta hydroxy acid—- -2H»beta keto
acid » methyl ketone + carbon dioxide
Methyl ketones are not invariably mold products, since acids
may be oxidized without a trace of ketone formation, and
13
yield of ketone is never quantitative. Apparently two
mechanisms compete for beta keto acids— ketone formation
and complete oxidation but that the long chain acids may
be poisonous to the extent that they inhibit oxidative
processes and ketone formation results (Stokoe, 1 9 2 8).
Rahn (1905a, 1905b) was one of the first to study
fat oxidation by bacteria. In the course of other work
he showed that bacteria could grow on a medium with inor
ganic nitrogen and stearic or palmitic acid as a carbon
source. Pozerski (1937a) showed that Escherichia coli could
use sodium stearate as a sole source of carbon for growth
but that oleic acid could not serve a similar function
(Pozerski 1937b). Loebel, Shorr, and Richardson (1933)
studied metabolism of the tubercle bacillus on oleate,
palmitate, and stearate and concluded that the acids could
furnish energy but questioned whether they could be used
to form protoplasm. Bernheim (1941) found that sodium salts
of fatty acids increased oxygen uptake of tubercle bacilli
but that the effect was slight with the acids above valeric.
At the same time Cutinelli (1940) reported that mono-
carboxylic acids from 0% to were oxidized fairly rapid
ly, Cj to 0^2 most rapidly and 0^5 to C20 intermediate
speeds by the tuberculosis organism. Studying Pseudomonas
aeruginosa. Peppier (194l) reported that the rate of oxygen
uptake was greater on fatty acid substrates than on glycerol.
14
Attemonelll (1942) showed that Brucella melltensls, and
several other species of the same genus, oxidized to
fatty acids. Acetic and Cg to 0^0 acids were most
readily attacked. Yamaguchi (1946) found that sodium
salts of to Gjg acids (except untested C1 3, and
C^y) were readily oxidized by Pseudomonas aeruginosa and
Micrococcus ochraceus. Since the rate of oxidation was
generally greater with the fatty acids than with glucose,
lactic and succinic acids, it was concluded that there is
a close relationship between fatty acids and the normal
metabolijsm of the cell. Gray (1949), using Mycobacterium
tuberculosis, found that endogenous metabolism took place
at the expense of stored lipid material. Oginsky, Smith,
and Solotorovsky (1950) reported that the oxidation of
Cg to Cio acids by Mycobacterium tuberculosis var. avis
was not affected by streptomycin but that of laurlc,
palmitic and stearic was partially inhibited. Myristic
acid had an inhibitory effect on respiration. It was sug
gested that there may be two alternate pathways for fatty
acid oxidation: One for Cg to C^o &nd possibly to un
affected by streptomycin, and one for Cx2 to sensitive
to streptomycin.
Mechanisms of fatty acid oxidation in animals. It
is apparent that our knowledge of fatty acid oxidation in
15
microorganisms is meager and that far more is known of
anaerobic dissimilation processes than of aerobic
metabolism, little or nothing is known of the actual
mechanisms involved. The problem of lipid metabolism
in animals has long concerned the biochemist. While many
fundamental questions await answer, it would seem of
value to review the various theories which have been pro
posed for the mechanism of fatty acid oxidation in animal
tissue. A detailed review of the literature will not be
attempted, since excellent review articles on the subject
have recently appeared (Breusch, 1948; Stadie, 1946).
Beta oxidation, the earliest theory of fatty acid
catabolism, was proposed by Knoop (1904) who fed phenyl
derivatives of fatty acids to dogs and analyzed the ex
cretion products in the urine. He noted that when even
chain acids were fed hippuric acid was invariably excreted
in the urine; odd chain acids yielded phenaceturic acid.
Knoop made no assumptions concerning the possible splitting
off of two-carbon fragments or whether the oxidations were
successive or simultaneous. The hypothesis came to mean
that each fatty acid molecule is oxidized through a
series of fatty acids, each shorter by two carbon atoms
than its immediate precursor. Finally one molecule of
butyric acid remains, and this in turn is oxidized to
acetoacetic acid. The balance of the molecule is converted
i6
presumably to acetic acid molecules.
The original hypothesis of Knoop did not explain,
however, the great capacity of the liver to form acetone
bodies, since it allowed for the formation of only a
single acetone body per fatty acid molecule. Further, the
hypothetical acetic acid molecules were never isolated nor
were fatty acids of intermediate chain length found in the
tissues and fat deposits of mammals. On this basis, a
second theory of oxidation was formulated (Jowett and
Quastel, 1935; Butts, et al., 1935). According to their
theory of multiple alternate oxidation, oxidation begins
at the beta carbon atom and simultaneously at each alternate
carbon atom along the whole length of the carbon chain. Ac
cordingly, fatty acids are oxidized first to polyketo com
pounds, and following this preliminary oxidative step, the
molecule splits into a number of acetoacetic acid molecules,
the number depending upon the length of the chain. This
then accounted for the observed increase in acetone body
production with increased chain length. Breusch (1948)
believes that this mechanism is the major pathway of
fatty acid oxidation in the liver but not in the muscle or
kidney tissue.
In the mechanism of multiple alternate oxidation it
was implied that long chain polyketo acids were formed and
that these then split into four-carbon units forming ketone
17
bodies. The question arose as to the origin of ketone
bodies from fatty acids with carbon atoms not an even
multiple of four, since these also are converted into
ketone bodies. Likewise, odd chain acids are to some
extent ketogenic. The sole postulate offered was that
ketone bodies might arise from C2 condensations. The
theory of successive beta oxidation— condensation was
formulated by McKay (1943): the fatty acid molecule, re
gardless of its nature, is split into C2 units which are
then re-assembled into ketone bodies. The sole difference
between this theory and that of multiple alternate oxida
tion is that the fatty acids are split on the one hand in
to four-carbon units and on the other into two-carbon
fragments which are re-assembled into ketones (four-carbon
units). There is experimental evidence to support both
of these theories, and the possibility exists that both
mechanisms occur in the body (Stadie, 1946).
Verkade and van der Lee (1932) noted that trigly
cerides of saturated fatty acids of medium chain length
(C3 to C^i) are partially excreted in the urine as di-
carboxylic acids. The evidence indicated that the fatty
acid molecule was oxidized first at the omega carbon atom
and then simultaneously beta oxidation occurred at both
ends of the chain, since capric acid (C^q) yielded di-
carboxylic acids with ten, eight, and six carbon atoms.
18
Experimental evidence indicated that with fatty acids of
intermediate chain length omega oxidation competes with
beta oxidation, with about 90 per cent of the acid follow
ing the latter pathway. The mechanism of omega oxidation
is of little importance in animal nutrition, since most
of the catabolism of fatty acids apparently occurs in
the liver by the multiple alternate route, and the inter
mediate acids do not accumulate (Breusch, 1948).
Whatever the mechanism of primary attack, fragments
are formed which must undergo further oxidation. The pres
ent evidence indicates that products of the primary oxida
tion undergo terminal degradation in a manner similar to
carbohydrates. Breusch (1948) has summarized as follows:
"Acetoacetic acid, the primary metabolite of fatty acids
in the liver, and to a smaller extent, higher beta keto
fatty acids eventually formed in muscle tissue, undergo
condensation with oxalacetic acid in muscle and kidney
tissue, resulting in the formation of Cg-tricarbosylic
acids due to the action of an enzyme system (citrogenase).
The acids, citric acid;,===^^ cis-aconitic acid iso-
citric acid, are in enzymatic equilibrium with one another,
of which citric acid makes up 80 per cent. These acids
lose two carbon atoms through oxidation and then are readi
ly oxidized to oxalacetic acid (Ci^), which in turn under
goes new condensations and thus bums fatty acids at the
19
rate of two-carbon fragments; this is called the C^-
tricarboxylic acid cycle." The reactions involved may be
represented as follows:
R
c=o
C H j >
COOH
Fa.t'ty A cid
4-
O^C C H g .
COOH COOH
Oxa.1 a c e tic Acid
R
C
C--------C ----------C
I I I
C O O H COOH C O O H
H y p o t h e t ic a l I n t crH «d ia.te
R
COOH
Fatty Acid
1 ess tw o
carbons
+
H.C
OH
C - CHe
COOH COOH COOH
C itri C V ^ I S o c i t r i c
^ C1 s -a c o n itic
While there is general agreement that fatty acid
derivatives undergo terminal oxidation through the tri
carboxylic acid cycle, the above scheme remains only an
outline. Neither the reactants nor the products in the
primary condensation reaction have been definitely estab
lished. Lehninger (19^5) and Green (1945) present evidence
which indicates that a two-carbon fragment (not acetate)
derived from beta keto acids, rather than the beta keto
acids themselves, participates in the condensation reaction.
20
The chemical nature of the two-carbon fragment has not
been definitely established.
Likewise, the mechanism involved in primary attack
on saturated fatty acids in animal tissues has still not
been discovered. It is generally assumed that the saturat
ed acid is converted first to the alpha-beta unsaturated
derivative, then to the beta hydroxy> and finally to the
beta keto derivative, but definite proof for this sequence
is still lacking. Breusch (1948) has stated that alpha-
beta dehydrogenation may be quite unrelated to beta oxida
tion of fatty acids. It is of interest that he suggests
no alternate mechanism for primary attack on fatty acids.
It is difficult to conceive of a mechanism whereby saturat
ed acids are converted to beta keto acids without a pre
liminary dehydrogenation occurring. Since the intermediates
in primary oxidation have not been established, it follows
that little is known of the number and specificity of the
enzymes which catalyze the breakdown of various fatty
acids in animal tissue.
Simultaneous adaptation— a possible means of studying
the mechanism of fatty acid oxidation of microorganisms.
The difficulties encountered in the study of fatty acid
metabolism have previously been pointed out; similar prob
lems are to be anticipated in studying lipid catabolism in
21
bacteria. In considering possible methods of approach to
the problem, the technique of simultaneous adaptation sug
gested itself (Stanier, 1947). The method has as its
basis the Kluyverian axiom (Kluyver, 1931) that every bio
logical transformation is the result of a series of simple,
chemically intelligible step-reactions. Thus, the oxida
tion of even a relatively simple organic molecule will
involve the formation of numerous intermediate compounds.
If one accepts the existence of several discrete steps in
the dissimilatory process, it follows that for each step
a specific enzyme is involved. It has been claimed that
two types of enzymes exist in microorganisms, i.e. con
stitutive enzymes which are present in the cell regardless
of its previous history and adaptive enzymes which are form
ed in response to contact between the cell and the specific
substrate (Kamstrom, 1930). If a given compound is at
tacked through adaptive enzymes, the possibility exists
that some of the intermediates in the catabolic process are
also attacked adaptively. It follows, then, that if cells
are adapted to a particular compound, these organisms
should become simultaneously adapted to all intermediates
in the dissimilatory process, even though they have not
been previously exposed to the intermediates. Thus, by
growing cells on a primary substrate or on intermediates
in the oxidation of this substrate and then testing for
22
adaptation to a variety of related substances, it should
be possible to obtain evidence as to whether or not as
sumed intermediates actually occur. Stanier (194?) has
summarized the technique as follows:
1. "If the dissimilation of a given substance A
proceeds through a series of intermediates B, C, D, E, F,
G . . . , and if the steps in this chain of reactions are
under adaptive enzymic control, then growth on a medium
which contains A will produce cells that are simultaneous
ly adapted to B, C, D, E, F, G . . . .
2. "If growth on A fails to adapt cells to a
postulated intermediate X, then X cannot be a member of
the reaction chain.
3. "Growth on E will adapt cells for F, G . . .
but not necessarily for A, B, C, and D. The probability
that growth on E will adapt cells to precursors decreases
with the number of intervening steps; i.e., adaptation to
D is more probable than adaptation to A."
The technique of simultaneous adaptation is superior
to the customary approach to the problem of detecting
metabolic pathways by determination of utilizability of
potential intermediates. The inability of an organism
to utilize a suggested intermediate can of course be used
as positive evidence that the proposed intermediate does
not fall in the pathway of oxidation of the compound in
23
question, provided that the intermediate is permeable.
Stanier (1948) has pointed out, however, that all too
frequently the utilizability of a compound has been used
as positive evidence for the occurrence of that compound as
an intermediate. He points out that such a finding may be
coincidental and not sequential. The simultaneous adapta
tion technique, however, makes it possible to determine
whether the relationship between two given compounds
utilized by the cell is sequential or coincidental, for
if both substances are attacked adaptively, then adapta
tion to the higher compound should not cause adaptation to
the proposed intermediate unless the substance is a normal
product in the breakdown.
The assumptions on which Stanier*s technique is
based have recently been subject to attack (Campbell,
Norris, and Norris, 1949). The authors present data which
indicate that acetate is an intermediate in the oxidation
of glucose by Pseudomonas aeruginosa. Yet, growth on
glucose failed to adapt the organisms to acetate. These
authors recall the theory of Yudkin (1 9 3 8) who holds that
all examples of enzyme production are cases of increase
in enzyme and none are instances of the formation of new
enzyme. They point out that Stanier*s theory contradicts
this idea, for it assumes that when a substrate is attacked
by an adaptive enzyme the same amount of enzyme is produced
24
regardless of the amount of substrate present In the
growth medium so when cells are transferred from condi
tions where the compound in question is a metabolic inter
mediate, and therefore present in only small aimounts, to
conditions where the same compound is present as the
parent substrate and in much larger quantity, no increase
in enzyme will occur.
Despite the objections raised, the technique of
simultaneous adaptation has been applied with success to
study the mechanism of nitrogen fixation (Burris and Wil
son, 1946); to study the oxidation of aromatic compounds
(stanier, 194?> 1948; Sleeper and Stanier, 1950; Stanier,
Sleeper, Tsuchida, and MacDonald, 1950; Sleeper, Tsuchida,
and Stanier, 1950); to establish the nonparticipation of the
tricarboxylic acid cycle in the oxidation of acetate by
Azotobacter agilis (Karlsson and Barker, 1948); to establish
the mode of utilization of uronic acids by Escherichia coli
(Cohen, 1949); to elucidate the initial steps in trypto
phane oxidation by Pseudomonas sp. (Stanier and Tsuchida,
1 9 4 9); Sind to establish the non participation of the
tricarboxylic acid cycle in the oxidation of acetate by
E. coli and Aerobacter aerogenes (AJl, 1950).
It was felt that the technique of simultaneous
adaptation offered a logical means of approach to the study
of fatty acid oxidation in bacteria, since fatty acids are
25
relatively long molecules and thus should involve a long
series of enzymecontrolled reactions for complete oxida
tion. This was predicated on the assumption that organisms
showing adaptive attack on fatty acids could be found.
CHAPTER II
THE NATURE OP THE ENZYMES, CONSTITUTIVE OR ADAPTIVE, IN
FATTY ACID OXIDATION BY VARIOUS BACTERIA: A SURVEY
The role of fatty acids in the metabolism of
aerobic bacteria has been but little investigated, al
though certain workers have described organisms with
strong affinities for these substrates. Giesberger
(1 9 3 6) found that fresh water spirilla oxidize many of
the lower fatty acids and, in fact, this property char
acterizes one species in that group. Peppier (l94l) re
ported that certain strains of Pseudomonas aeruginosa
oxidize fatty acids more rapidly than they do glycerol.
Gray (1949) found that the tubercle bacillus, as well as
other mycobacteria, metabolize at the expense of stored
fatty acids when an exogenous source of carbon is un
available. Rittenberg and Williams (1950) have recently
found that many strains of marine and fresh water spirilla
can grow on media containing fatty acids as sole sources
of carbon. Yamaguchi (1946) found that certain micrococci
and pseudomonads showed a more rapid rate of oxygen up
take on fatty acids than on glucose or Krebs cycle com
pounds. He has suggested that there may be a close rela
tionship between fatty acid metabolism and the normal life
of the cell. It is well known that many bacteria.
27
particularly the aerobic spore formers, store fat globules
as reserve food material. It follows that these organisms
must have active fatty acid oxidases, since this lipid
material serves as a source of energy for the cell when
other food supplies are depleted. It is apparent that
the ability to oxidize fatty acids may be a common pro
perty of aerobic bacteria, but the available literature
gives no clue as to the nature of the enzymes involved,
i.e., whether they are constitutive or adaptive. In the
present survey the fatty acid oxidizing ability of a
relatively large group of organisms was examined with the
hope that one or more strains might show an adaptive pat
tern. Such organisms would be valuable tools for the
study of the mechanism of fatty acid oxidation using the
technique of simultaneous adaptation.
EXPERIMENTAL
Bacteria utilizing stearic acid as a sole source
of carbon were isolated from soil samples by the enrich
ment culture technique. A medium of the following composi
tion was used: K2HPO4, 5*0 g; KH2PO1 1. , 2.0 g; NH1 1.NO 3, 1.0 g;
MgSO]^, 0 .5 g; stearic acid 1,8 g; distilled H2O, 1000 ml.
The pH of the medium was adjusted to 7*0 with potassium
hydroxide. For initial isolation the medium was not
28
sterilized. After three or four serial transfers in the
unsterilized medium, isolation was accomplished on a solid
medium of the same composition but containing 1 .5 per cent
agar. Even though the medium contained considerable amounts
of insoluble soap, no difficulty was encountered in obtain
ing bacteria from many different soil samples by this tech
nique. Without exception, the isolated organisms proved
to be Gram negative rods. They were not further identified,
although the indications were that most of them belonged
to the genus Pseudomonas.
In addition to the organisms obtained by the en-
richment technique, pure cultures of various organisms
^ recently isolated from natural habitats were tested for
fatty acid oxidizing ability. These organisms had never
been exposed to fatty acids under experimental conditions
prior to the time when substrate was poured from the side-
arm of the Warburg vessel.
Before tests were conducted to determine fatty acid
oxidizing ability, both groups of organisms (stearic acid
enrichment culture group and stock cultures) were trans
ferred three or four times on a mineral salts medium of
the above composition but with glucose in place of stearic
acid. In a few cases, the stock cultures failed to grow
on the glucose medium; these cultures were transferred on
nutrient agar.
29
The organisms were tested for ability to oxidize
capric and pelargonic acids, using conventional mano-
metric techniques (Umbreit et al., 19^9)» Cultures were
grown on glucose-mlneral salts medium and harvested after
16 to 24 hours at 37®G. The cells were washed from the
slants with M/20 phosphate buffer at pH 7.0. Subsequent
ly, the cells were centrifuged and re-suspended in fresh
buffer. The process was repeated twice, the centrifugate
being suspended in fresh buffer each time. The twice-
washed cells were finally suspended in buffer for use in
the Warburg vessels. Since the primary purpose of the
study was to determine oxidative patterns, no attempt was
made to adjust all suspensions to a standard turbidity;
in most cases, however, the suspension used gave a reading
of about 300 on the Klett-Summerson apparatus (using the
540 millimicron filter). Each Warburg vessel received
1.0 ml of cell suspension. The side-arms of the flasks
contained 1.0 ml of O.OOO6 M fatty acid in M/20 buffer
and the center-wells 0.1 ml of 10 per cent KOH for the
absorption of carbon dioxide. The flasks were equilibrat
ed for 15 minutes before substrate was poured from the
side-arms. Oxygen uptake was measured for two or three
hours, or until all substrate had been utilized.
The results shown in Table I indicate that all the
organisms tested utilized capric and pelargonic acids. In
30
TABLE I
OXIDATION OF CAPRIC AND PELARGONIC ACIDS BY VARIOUS BACTERIA
Organism
Capric acid (Cio)
total oxygen uptake
Pelargonic acid (C9)
total oxygen uptake
in
20
uL after*
minutes
4o 60
in
20
uL after
minutes
4o 60
Pseudo, sp. - 19 4l
95
128
Pseudo, sp. - 6 30 62 92 20
49
78
Pseudo, sp. - 13 11
37
— —
15
40
67
Pseudo, sp. - 21 58 116
169
20 44
69
Pseudo, sp. - 24 54 111 170
27 57 83
Pseudo, sp. - 20
25
61 94 13
36 61
Pseudo, sp. - 9 48
103 140
— —' — — — —
Pseudo, sp. - 7 48
95
154
— —
Pseudo, sp. -110
55 115
176
— — — — — —
Pseudo, sp. - 8
49
100 200
— — — — • — —
Pseudo, sp. - 11 80
165
— — — » — — —
Enrich. #3 ** 22 44
65 13 35 53
Enrich. #43b 40 90
137
— — — — — —
Enrich. #83b 40 100
175
— — — * —
Enrich. #43a 82 156 274 21
51
81
Enrich. #63
95
200 332 29 71 95
Enrich. #83a 40 104
175
24 48
71
E. coli #2 21 40
53
— — — —' — —
B. brevis
13 45
80
15
46 84
B. firmus 8
17 25 5
12
19
B. subtilis
13 29 49 19
44
71
B. megatherium -1 6
13
20 8 16 24
B. megatherium -2 14 32
51
26
45
64
N. catarrhalis 70
— — — —
90
— — —
Serrâtia (Baker) 2
9
21 2 11 17
Serratia (Alphin) 2
7 19 3
10 22
Experiments conducted in air atmosphere with KOH (10$) to
absorb COg; pH 7-0; 30°C.
* Figures not corrected for autorespiration-
♦♦ Enrichment isolates from soil, originally grown in
stearic acid— mineral salts medium.
31
general, the rates were somewhat higher on capric than on
pelargonic, although with some organisms the reverse was
true (see Neisseria catarrhalis). There was considerable
variation in activity, the Gram positive spore formers
showing the lowest oxidation rates. There was no con
sistent difference between the rates observed for enrich
ment isolates and those for other Gram negative bacteria.
Bacillus megatherium - 1 and Bacillus firmus showed very
low rates, only slightly above that of the cells in the
absence of substrate; this may be indicative of a slight
stimulation of endogenous metabolism rather than actual
utilization of the fatty acids.
With the exception of the two Serratia strains and
Bacillus brevis, all strains tested consumed oxygen im
mediately at nearly maximum rate when exposed to fatty
acid. In certain cases the initial uptake was slightly
less in the first twenty minutes than during subsequent
twenty minute periods, but this was likely due to a per
meability effect. This immediate oxygen uptake at nearly
maximum rate suggests that in these organisms the enzymes
involved are constitutive. The fact that most of the
cells had never previously been exposed to fatty acids
under experimental conditions materially strengthens this
conclusion.
The initial uptake by the Serratia strains and
32
Bacillus brevis was low. The rate steadily increased
until a maximum was reached at the end of twenty to forty
minutes. In later work, cells were washed three times
instead of twice. With this added washing, the cells
showed,even lower initial uptake. This lag in the oxida
tion of the fatty acids is presumptive evidence for at
tack through adaptive enzymes. To investigate this pos
sibility, the Serratia strains were tested on several
different fatty acids. The results are shown in Figures 1
and 2. Figure 3 shows comparable curves for some Pseudo
monas strains on capric acid.
Figure 1 shows that Serratia marcescens (Alphin)
attacked the fatty acids only after an initial lag period.
Propionic, butyric, and valeric acids were not oxidized,
and the oxidation curves for flasks containing these com
pounds coincided with the autorespiratory curve. The lag
periods varied somewhat with the different acids. The
length of the lag period was estimated by extension of
the steepest part of the curve to the time axis. Such an
operation shows the following lag periods: acetic (Cg)f
20 minutes; caproic (C5), 25 minutes; heptylic (C^)^ 20
minutes; caprylic (Cg), 22 minutes; pelargonic (Cg), 22
minutes; capric (C^q)^ 36 minutes; lauric (C^g)^ 32
minutes; tridecylic (C^g) ^ 22 minutes; myristic (Cjii.),
26 minutes. Similar effects were noted with the Baker
33
9Qr
BO -
7 0 -
€0-
Z
u
>
X
o
5 0 -
50-
20-
ISO 1 2 0 l + Q 160 1 0 0 80 60 40 20
M/20 phosphate buffer, pH 7*0, 30°C., air atmosphere with
0.1 ml 10 per cent KOH in center-well. Curves for propionic,
butyric, and valeric acids coincide with autorespiratory.
Flask concentrations: acetic acid (Cg) 0.001 M; caproic
acid (C6) 0.0005 M; heptylic (Cj) and tridecylic (Cto) 0.0003
M.
FIGURE 1
OXIDATION OF VARIOUS FATTY ACIDS BY
GLUCOSE-GROWN SERRATIA (ALPHIN)
34
IfO
a
>
X
o 80-
J
i.
70- -
50'
4 0 -
5 0 -
2 0 40 SO 8 0 100 160 140
T1 M E IN M I N U T E S *
M/20 phosphate buffer, pH 7*0, 30^G., air atmosphere with
0.1 ml 10 per cent KOH in center well. Flask concentra
tions: caprylic (Cg), pelargonic (Co), capric (C^n)* lauric
(C^g) f tridecyelic , and myristic (Ciij.)— all 0.0003 M.
FIGURE 1 (Continued)
OXIDATION OF VARIOUS FATTY ACIDS BY
GLUCOSE-GROWN SERRATIA (ALPHIN)
35
z
o
z o 80
T I M E I N M I N U T E S
M/20 phosphate buffer, pH 7*0, 30^0., air atmosphere with
0.1 ml 10 per cent KOH in center well. Propionic, butyric,
and valeric acids not oxidized by same cell suspension.
Flask concentrations: caprylic (Co), capric (Cig), and
lauric (Cig)--all 0.0003 M.
FIGURE 2
OXIDATION OF VARIOUS FATTY ACIDS BY
GLUCOSE-GROWN SERRATIA (BAKER)
36
M/20 phosphate buffer, pH 7*0, 30®C., air atmosphere with
0.1 ml 10 per cent KOH In center well. P5, Pgo, Pp%, and
P2 4: four different strains of Pseudomonas sp. Glucose-
grown cells, never previously experimentally exposed to
fatty acids. Flask concentrations: 0.0003 M capric acid
(Cio)-
PIGÜBE 3
OXIDATION OP CAPRIC ACID BY POUR STRAINS OF PSEUDOMONAS SP
37
strain, although the lag periods were somewhat shorter and
less definite (Figure 2). In contrast, the four strains of
Pseudomonas (Figure 3) showed essentially no lag period in
the oxidation of capric acid. The data suggest that the
fatty acids are attacked through adaptive enzymes in the
two strains of Serratia marcescens. It has not been de
termined whether Bacillus brevis shows a similar pattern
on other fatty acids.
The Alphin strain was also tested for ability to
oxidize the ten, seven, six, and four-carbon dicarboxylic
acids. Of these acids, only succinic acid was oxidized.
DISCUSSION
The data indicate that the ability to oxidize fatty
acids is not an uncommon property of aerobic bacteria. All
strains tested showed some ability to oxidize the nine and
ten-carbon fatty acids. All organisms, with the exception
of the spore formers and Neisseria catarrhalis which were
not tested, grew well on a medium with capric acid as the
sole carbon source.
The enzymes concerned with fatty acid oxidation are
apparently constitutive in most eases since cells never
previously exposed to these substrates under experimental
conditions show a high level of metabolism when placed
38
in contact with fatty acids. Oxygen uptake begins almost
immediately and at a constant rate. This would seem to
strengthen the conclusions of Yamaguchi that these enzymes
have an intimate relationship to the metabolism of the
cell.
Although the lag periods observed with the two
strains of Serratia marcescens and with Bacillus brevis
are suggestive of attack through adaptive enzymes, such
a conclusion can only be tentative. The lag phase in
these instances may be a special case in which the cells
are permeated by the acids at a slow rate. In any case,
it would seem that these three strains of bacteria would
bear further investigation and that they might be used in
the study of fatty acid oxidation, utilizing Stanier*s
technique of simultaneous adaptation.
SUMMARY
The ability to oxidize fatty acids is a wide-spread
property of bacteria. In most cases studied the attack
was through constitutive enzymes, although two strains of
Serratia marcescens as well as one strain of Bacillus brevis
gave evidence of attack through adaptive enzymes. Both odd
and even chain acids were oxidized, the rates being some
what lower with the odd chain acids.
CHAPTER III
APPLICATION OP THE TECHNIQUE OP SIMULTANEOUS ADAPTATION TO
THE STUDY OF FATTY ACID OXIDATION IN SERRATIA MARCESCENS
The previous results suggested that bacterial attack
on fatty acids under aerobic conditions is usually catalyzed
by constitutive enzymes. It was noted, however, that two
strains of Serratia marcescens oxidize fatty acids only
after a lag period of from twenty to forty minutes. This
suggested that adaptive enzymes might be involved. If the
lag period were indeed due to adaptive enzyme formation,
then growth on a medium with a fatty acid as the sole source
of carbon should adapt organisms to that acid, and subse
quent exposure to the acid should result in the immediate
uptake of oxygen at maximum rate, i.e. the lag should be
eliminated. In addition, simultaneous adaptation to all
intermediate compounds in the oxidation of the fatty acid
should take place.
EXPERIMENTAL
Oxidative patterns in relation to growth on capric
acid medium. The two strains of Serratia marcescens
(Alphin and Baker) were grown on a medium containing 0.01
M capric acid as the sole source of carbon. The mineral
40
composition of the medium was the same as previously used
(Chapter II). Cells were harvested after forty hours
growth at 37^C. by washing down the growth on slants with
M/20 phosphate buffer. Subsequently the cells were washed
twice with buffer, using the technique described in Chapter
II. The washed cells were suspended in M/20 phosphate
buffer and the turbidity adjusted to a 325 reading on the
Klett-Summerson apparatus, using the 540 millimicron filter.
Oxygen uptake was measured, using the technique previously
described.
The results are shown in Figures 4 and 5* It is
obvious that buffer suspensions of cells grown on capric
acid medium show immediate oxygen uptake when exposed to
caprate. The lag period noted with glucose-grown cells
is eliminated, and the oxidation curves are similar to those
for glucose-grown pseudomonads (Figure 3)* This response
would be expected if capric acid were attacked through
adaptive enzymes.
It will be noted that caprate-grown cells also
attack caprylic, caproic, and acetic acids without a lag
period, while butyric acid is not oxidized. In the pre
vious study it was found that neither Serratia strain is
capable of butyrate oxidation. If this is a case of
adaptive enzyme attack, then the adaptation of caprate-
grown cells to the two, six, and eight-carbon acids indicates
2
igr 120 ■
>
O 1(0-
So..
8 0 -
70
30-
30
20-
IO--I
80 40 SO
2 ».
i»o..
160 -
160-
I50--
140-
° n o -'
SO-
6 0 "
5 0 "
40-
2 0 6 0 80 4 0
4l
T IM E IN M IN U T E S T IM E IN M IN U TES
M/20 phosphate buffer, pH 7*0, 30°C., air atmosphere with
0.1 ml 10 per cent KOH in the center-well. Propionic,
butyric, and valeric acids not oxidized by same cell sus
pension. Flask concentrations: acetic acid (Cg), 0.002M;
caprolc acid (0 5) and caprylic acid (Cg), O.OOO5 M; pelar-
gonic acid (Co), capric acid (Cin), and lauric acid (Ctq),
0 .0 0 0 3 M.
FIGURE 4
OXIDATION OF VARIOUS FATTY ACIDS BY
CAPRATE-GROWN SERRATIA (ALPHIN)
42
||0 -
1 0 0
z
\S
>
X
o
8 0-
C .
50
30--
3 0 -
2 0 -
B O
eo f e O 20
m/20 phosphate buffer, pH 7 * 0, 30 C., air atmosphere with
0.1 ml 10 per cent KOH in the center-well. Propionic,
butyric, and valeric acids not oxidized by same cell sus
pension. Flask concentrations: caprolc and heptylic
[Cj) and caprylic (Cg) acids, 0.0005 M; pelargonlc (Cq),
capric (Cin), lauric (C12)> tridecylic (Cno), and myrlstic
(C1 4) acids, 0 .0 0 0 3 M.
FIGURE 5
OXIDATION OF VARIOUS FATTY ACIDS BY
CAPRATE-GROWN SERRATIA (BAKER)
43
that these substances are intermediates in the oxidation
of the ten-carbon acid. The failure of the organisms to
utilize butyric acid eliminates this substance as an
intermediate, unless this failure to oxidize butyrate is
due to impermeability.
Quite unexpectedly, the caprate grown cells were
also adapted to the oxidation of lauric and myristic acids,
twelve and fourteen-carbon fatty acids (see Figures 4 and 5
and Table II). Still more curious was the adaptation of
caprate-grown cells to heptylate, pelargonate, and tridecyl-
ate (seven, nine, and thirteen-carbon compounds). Propionic
and valeric acids, substrates not attacked by glucose-grown
cells, were not oxidized by caprate-grown organisms.
Oxidative patterns in relation to specific adapta
tion to various acids. A series of experiments were con
ducted in which Serratia marcescens (Alphin) cells were
adapted to each of the fatty acids utilized by glucose-
grown cells, and then the specifically adapted cells were
tested for oxidation of all the other acids.
All the fatty acids tested, except heptylic and
acetic, could serve as sole sources of carbon for the growth
of the Alphin strain of Serratia marcescens. Repeated at
tempts to grow cells on acetate and heptylate failed even
though both compounds were readily oxidized by buffer
44
TABLE II
LAG PERIODS IN THE OXIDATION OF FATTY ACIDS IN
RELATION TO SPECIFIC ADAPTATION OP SERRATIA
(ALPHIN) CELLS TO VARIOUS ACIDS
Oxidation Lag period in minutes after specific adaptation
of of Alphin cells to
glucose Cg
C6
C7
OQ
C9 Cio Cii
C12
Ci3 Cl.
acetic 20
5
2 8 6 0 8
7
6 8 l4
caproic 28
5 7
18 2 18 2 XX
5
10 6
heptylic
19
8
9 7
0
5 9
0 8 XX 14
caprylic
39
0 4 11 1 12
3 7
6 4
5
pelargonic
29
0
9
XX 0 1
3
XX 4 XX 4
capric
27
8 8 16 0 16 4
7 7 5
0
undecylie 32
3
XX XX XX XX 0 0 XX
9
XX
lauric 40 20 8 XX 0 28 6 XX 0 XX XX
tridecylic 14
7
0
9
6
9
0 8 0 1 10
myristic 34 9
18
17
0 20
3
6 0
7 17
^ Lag periods estimated by extension of steepest part of the
curve to time axis. Most values represent averages from
several experiments.
XX— no data available.
Air atmosphere with 10 per cent KOH to absorb CO2; flask
concentration of acetic acid, 0.001 M; other acids,
0 .0 0 0 3 M; M/2 0 phosphate buffer, pH 7-0; temperature
300c.
45
suspensions. No attempts were made to obtain growth on
these compounds with media supplemented with growth factors.
It was found that adaptation to the oxidation of a
particular acid could be accomplished either by growth on
a medium containing the acid as the sole source of carbon
or by allowing buffer suspensions of glueose-grown cells to
oxidize a small amount of acid in the Warburg flask. In
preliminary work it was found that there was no essential
difference in subsequent oxidative patterns between cells
adapted by exposure to fatty acids and those adapted by
growth on fatty acid medium. Since maximum growth on
fatty acid medium was not reached until after 40-48 hours
of incubation at 37^G., most of the experiments were con
ducted with cells grown on glucose-mineral salts medium.
Under these conditions, cells could be harvested after
16 to 24 hours growth. Adaptation was accomplished by
suspending the glucose-grown cells in buffer containing
a small amount of fatty acid (l.O micromole per ml for
acetate adaptation, 0.6 micromole per ml for the other
acids). The cells were then allowed to oxidize the added
acid to completion. When the autorespiratory level was
reached, the test substrate was poured from the side-
arm into the main well of the Warburg vessel.
Table II summarizes the effects noted when specifical
ly adapted cells were allowed to oxidize various fatty acids.
46
Figure 6 shows the oxidation of capric acid by cells
specifically adapted to other acids.
The exposure of Serratia (Alphin) cells to any of
the oxidizable fatty acids had a profound effect on the
pattern of oxidation of all the other fatty acids tested.
In general, this procedure reduced the length of the lag
period that was noted when glucose-grown (unadapted) cells
oxidized the same acids. Quantitatively, it will be noted
that in certain cases (acetic, caprolc, heptylic and
myristic) exposure to a particular acid did not completely
eliminate the lag period when the cells were subsequently
exposed to the same acid. Likewise, considerable varia
tion exists in the degree to which adaptation to a par
ticular acid shortens the lag period in the oxidation of
other acids. In some cases oxygen uptake is immediate,
leaving little doubt that adaptation has occurred. In
other instances the length of the lag period is inter
mediate between that for unadapted cells and that for
completely adapted organisms. Without exception, however,
the exposure of glucose-grown cells to any of the oxidiz
able fatty acids resulted in organisms which oxidized all
the other oxidizable fatty acids with shorter lag periods
than did the original cells.
Throughout the course of this study and in the
investigations to be reported in subsequent chapters, the
47^
Cg
Z
o
30 -
2 0 "
T IM E IN M INUTES
M/20 phosphate buffer, pH 7-0, 30^0., air atmosphere with
0.1 ml 10 per cent KOH in the center-well. Exposed cells
adapted by allowing glucose-grown cells to oxidize in
dicated acid. Flask concentration: capric acid {Cjq),
0.0003 M.
FIGURE 6
OXIDATION OF CAPRIC ACID BY SERRATIA (ALPHIN)
CELLS SPECIFICALLY ADAPTED TO OTHER ACIDS
48
occurrence of short lag periods has caused great difficulty
in the interpretation of experimental results. At best> a
considerable experimental error is to be exptec,t,ed in the
calculation of these periods. Technical difficulty is
also encountered in establishing the first point on the
experimental curves. At "time zero" substrate was poured
from the side-arm of each Warburg vessel and the initial
reading on the manometer recorded. This operation always
required a longer time than the process of reading the
manometers at subsequent twenty minute intervals. Even
when precautions were taken to make allowances for this
technical difficulty in the drawing of experimental curves,
short lag periods were noted in the oxidation of fatty
acids by cells which might be expected to be completely
adapted. Stanier (1950) has encountered similar dif
ficulties and has suggested that such lag periods repre
sent a permeability effect, a time necessary for substrate
to contact preformed enzymes in the cell. This explana
tion may account for the shorter lag periods noted (up to
six or seven minutes, perhaps), but in some cases the lag
periods noted may be too long to be explained on the basis
of slow permeability (see heptylic-adapted cells vs.
myristic, capric, and caproic acids— Table II).
Failure to eliminate the lag period completely may
be a manifestation of partial adaptation; i.e., as a
49
result of exposure to a given acid enzymes are formed
which are active in the oxidation of other acids. For
example, if a lower acid were an intermediate in the
oxidation of a higher acid, then adaptation to the lower
acid would result in activation of enzymes involved in
the oxidation of the higher acid and hence shorten the lag
period in the oxidation of the higher acid. If such were
the case one would expect a definite pattern in complete
adaptation and conversely a definite pattern in partial
adaptation. Such does not appear to be the case— (l)
lauric-adapted cells show no lag in the oxidation of
myristic and tridecylic acids, compounds which could not
logically be considered as intermediates in the oxidation
of laurate; (2) lauric-adapted cells show a seven minute
lag period in the oxidation of capric acid, a substance
which is a possible intermediate in the oxidation of
lauric acid. It is apparent that a more critical inter
pretation must await work with cell-free preparations.
The response of Escherichia coli #2 to growth on and
exposure to fatty acids. A considerable amount of work was
done with E. coli #2, an organism which in preliminary
experiments showed slow oxidation of fatty acids. Curve A,
Figure 7 shows the oxidation of caprylic acid by glucose-
grown E. coli #2. Similar curves were obtained for other
fatty acids, both odd and even chain.
160
150
KO--
I30--
120-
1 1 0
8 0 -
60-
<50--
20 4 0 60 80 100 120
50
TIM E IN M IN U TES
J
M/20 phosphate buffer, pH 7-0, 30°C., air atmosphere with
0.1 ml 10 per cent KOH in the center well. Flask concen
trations: 0.01 M. Curve A: Oxidation of caprylate by
glucose-grown cells never previously exposed to fatty
acids. Curve B: Oxidation of caprylic acid by caprylate-
grown cells. Curve C: Oxidation of caproate by caprylate
grown cells. Curve D: Oxidation of caprylate by cells
back-transferred through four cultures on glucose-mineral
salts medium.
FIGURE 7
OXIDATION OF CAPRYLIC AND CAPROIC ACIDS BY E. COLI #2
51
Experiments were conducted in which glucose-grown
cells were allowed to oxidize fatty acids in buffer
suspension. After complete utilization of substrate,
more acid was poured from the side-arm of the Warburg
vessel and the oxygen uptake measured. Under these con
ditions no increase in the rate of fatty acid oxidation
was noted, the curves for the exposed cells showing the
same low rates as those for cells never previously in
contact with fatty acids under experimental conditions.
These are qualitatively different from the results obtain
ed with Serratia (Alphin). Exposure of this organism to
fatty acids had a marked effect on subsequent oxidative
patterns. When E. coli #2 was grown on a medium with
fatty acid as the sole source of carbon, the cells harvest
ed from this medium showed a tremendous increase in the
rate of oxidation of that acid. Curve B, Figure shows
the oxidation of caprylic acid by cells grown on a mineral
salts medium with caprylic acid as the sole source of
carbon. The rate of oxygen uptake is many times greater
than that observed in caprylate oxidation by glucose-grown
cells. Caprylic-grown cells showed a similar increase
in the rate of caproic acid oxidation (Curve C). Glucose-
grown cells oxidized caproic acid at a somewhat lower
rate than caprylate (Curve A). Similar effects were noted
when cells were grown on acetic, caproic, capric, lauric and
52
myristic acids. In each case the fatty acid-grown cells
showed a tremendous increase in the rate of oxidation of
the acid which had served as a carbon source and a corres
ponding increase in oxidation rate on the next lower even
chain acid. In the case of acetate-grown cells, there
was no increase in the rate of butyrate oxidation; no
other attempt was made to study oxidation of the next high
er even chain acid after growth on a particular acid.
While E. coli #2 appears to attack fatty acids
through constitutive enzymes and the Serratia strains at
tack the same compounds only after a definite lag period,
it is apparent that other fundamental differences exist.
With the Serratia strains, either exposure to the fatty
acids or growth on media containing the acids as sole
sources of carbon results in cells with essentially no
lag period when allowed to oxidize the same acids. The
maximum rate of oxidation is not increased, the rate in
the steepest part of the unadapted curves being about the
same as that in the initial stages of oxidation by adapted
cells. With E. coli #2, on the other hand, exposure of the
cells to fatty acids does not affect the rate of oxidation
of fatty acids, while growth on fatty acid media greatly
increases the rate of oxidation.
In attempting to grow the Serratia strains and E.
coli #2 on fatty acid media, a further difference was
53
noted. The collform organism grew only sparsely on trans
fer to fatty acid media, only a few scattered colonies
developing on initial sub-culture. On transfer of isolat
ed colonies to fresh media, the growth became more luxuriant
and appeared sooner with each sub-culture until good growth
could be obtained in eighteen to twenty-four hours. Four
to five days were required before colonies appeared on
first transfer. The cells thus obtained through successive
transfer on fatty acid media showed a tremendous increase
in metabolism on the acid serving as a carbon source for
growth. When such cells were transferred on glucose medium
in the absence of fatty acids, they did not revert to cells
showing the low rate of metabolism characterizing cells
never exposed to fatty acids. Curve D, Figure 7» shows the
oxidation of caprylic acid by cells transferred on glucose
medium through four sub-cultures, after growth on caprylic
acid. The rate is much higher than in Curve A and only
slightly lower than that of the caprylic-grown cells. It
was found that after long storage (several months) on
glucose medium, the cells of the type shown in Curve D did
lose the characteristic of rapid metabolism on caprylic
acid. Both Serratia strains, on the other hand, grew
luxuriantly on first transfer to fatty acid medium, al
though maximum growth was not attained until after forty
to forty-eight hours. Further transfer did not increase
54
growth rate.
The data indicate that the increased metabolism of
E. coli #2 as a result of growth on fatty acid medium is
a selection phenomenon, the method of serial transfer of
isolated colonies serving to select from the population
those cells best suited to fatty acid oxidation. The re
sults with Serratia strains suggest that growth on fatty
acid medium does not involve selection, although no experi
ments were conducted in which fatty acid-grown cells were
tested after transfer in the absence of fatty acids.
Ajl (1 9 5 0) has investigated the mechanism of acetate
oxidation by E. coli and Aerobacter aerogenes. These organ
isms oxidized acetate, as well as Krebs cycle compounds, at
a low rate but without a lag period. As a result of growth
on a medium containing acetic acid as a carbon source, the
rate of oxidation of acetate was greatly increased. These
cells showed corresponding increases in the metabolism of
certain Krebs cycle compounds but not of others. Ajl has
used this evidence to rule out the tricarboxylic acid
cycle in acetate metabolism and has suggested an alternate
pathway. He has suggested that this technique may be a
broader application of Stanier*s method of simultaneous
adaptation, since it allows one to study metabolic path
ways in organisms attacking compounds through constitutive
enzymes. It would appear that Ajl was dealing with a
55
phenomenon similar to that observed in E. coli #2. In this
instance, cells grown in media containing fatty acids show
ed increased rates of fatty acid oxidation. Even though
the process may involve selection, it would appear that
the results permit the conclusion that the enzymes catalyz
ing the oxidation of even chain fatty acids are closely
related. The data are insufficient to allow one to pos
tulate the mechanism involved.
DISCUSSION
The technique of simultaneous adaptation failed
to give the clear-cut results that were expected. Although
adaptation to a particular acid did simultaneously adapt
cells to compounds which were logical intermediates in its
oxidation, the procedure also adapted cells to substances
which could not possibly have been intermediates.
For example, adaptation to capric acid simultaneous
ly adapted cells to caprylic, caproic and acetic acids.
If oxidation were to occur by simple beta oxidation or by
beta oxidation followed by acetic acid condensation, then
the Cg, OS9 and Cg fatty acids would be intermediates in
caprate oxidation. In the process of adaptation to capric
acid, however, cells also became adapted to heptylic and
pelargonic acids. The seven and nine-carbon acids could
56
not be intermediates in caprate oxidation unless the mech
anism involved the splitting off of a single carbon atom
at a time. No mechanism of this type is known to occur in
animal tissue, and it appears unlikely that bacteria oxidize
fatty acids in this manner. This is emphasized by the fact
that the C5, C4 and C3 acids are not oxidized by caprate-
adapted cells; these substances would be intermediates if
degradation of the molecule were to involve the loss of a
single carbon atom at a time. Furthermore, adaptation to
capric acid also adapted Serratia cells to undecylic, lauric,
tridecylic and myristic acids; these acids are higher than
capric and thus could not be considered intermediates in
its oxidation. Still more curious is the fact that adapta
tion to acetic acid simultaneously adapts cells (partially
or completely) to all acids higher than acetic (up to
myristic), except for the three acids not utilized by the
cells (C^, C4 and G^). Likewise, adaptation to the seven,
nine, eleven, and thirteen-carbon acids adapts cells to the
oxidation of other acids, both odd and even, higher and
lower.
The question arises as to the significance of the
lag period that is noted when glucose-grown cells oxidize
fatty acids. Three possibilities suggest themselves: (l)
The enzymes are constitutive and the lag represents a time
necessary for fatty acids to enter the cell. (2) The
57
enzymes are truly adaptive but a single enzyme is involved
in the initiation of oxidation of any of the oxidizable
acids and thus adaptation to one acid means adaptation to
all the others. (3) The lag represents a time during which
some intermediate is accumulating in the cell, a product of
the metabolism of fatty acids and a substance necessary for
metabolism to take place at maximum rate.
Permeability as a factor in the lag periods. Breusch
(1948) has pointed out that fatty acids and their salts
are difficultly soluble in water, and this fact, together
with slow diffusibility of fatty acids through cell walls,
has made it difficult experimentally to bring substrate and
enzymes together. The acids from acetic (Cg) through capric
(Ciq) were soluble in M/20 buffer at all concentrations used
in the manometric studies. The acids from undecylic (C],^)
through myristic (Cx4) were only partially soluble at the
0.0003 M concentration used in most of the manometric studies;
insoluble particles were visible to the naked eye. Despite
these differences in solubility (and therefore in substrate
concentration), the lag periods with the lower acids were
almost as long as those with the less soluble substrates.
Little is known with regard to the permeability of the
bacterial cell membrane to various substances. If a compound
is oxidized by an organism, it is logical to conclude that
the cell is permeable to that compound, but no general method
58
Is available which allows one to measure the rates at which
they contact endoenzymes. Hober (1945) asserts that the
ability of fatty acids to permeate lipoidal membranes of
many types of cells is low for the short chain acids but
increases sharply with chain length, starting with valeric
acid. He suggests that polar substances have difficulty
in entering the cell. If the observed lag periods were
due to slow permeability of the acids, and if Hdber's
remarks may be applied to bacterial cells, then one would
expect longer lag periods with acetic acid than with the
longer chain compounds. But the data show that the lag
period and the rate of oxidation of acetic acid is not un
like that for the higher acids. It is of course possible
that the property of slow diffusibility just offsets the
property of infinite solubility so that acetic acid gives
the same pattern as the higher acids. This appears unlike
ly. If, however, the observed lag periods are due to per
meability effects, then by appropriate techniques it should
be possible to demonstrate the presence of fatty acid oxidiz
ing enzymes in glucose-grown cells. This would involve ex
traction of enzymes from Serratia cells which had never
been experimentally exposed to fatty acids. Further, if
constitutive enzymes are involved, then dry cell prepara
tions of Serratia cells should oxidize fatty acids without
lag periods, since permeability effects should play no role.
59
Further, certain cell poisons, such as 2:4-dinitrophenol
and sodium azide are known to prevent the fomation of
adaptive enzymes. Oxidation of fatty acids should be
inhibited by these poisons if adaptive enzyme formation
is involved; conversely the poisons should have no over
all inhibiting effect if oxidation is through constitu
tive enzymes. The problem has been attacked from all
these angles and the data will be presented in subsequent
chapters.
The possibility of a single enzyme. Since expos
ing Serratia marcescens (Alphin) cells to any one of the
oxidizable fatty acids essentially eliminates the lag
period in the oxidation of most of the other acids tested,
it may be that a single enzyme, or group of enzymes, is
responsible for the initiation of the oxidation of any pf
the oxidizable fatty acids. Breusch (1948) has postulated
that attack on fatty acids involves attachment of the
enzyme to the carboxyl group and that the beta carbon atom
of the molecule is spatially close to the enzyme surface.
The remaining portion, the paraffin end of the molecule,
is flexible and possibly arranges itself in a ring struc
ture, with the carboxyl group being at the open end of the
ring. The essential feature of Breusch*s theory bearing
on the problem at hand is that it allows one to visualize
60
a single enzyme initiating attack on a wide variety of
fatty acids. If this were the case, then formation of
adaptive enzymes for one acid should allow the cell to
attack all other acids without a lag period.
The question arises as to why butyric, valeric and
propionic acids are not utilized by the Serratia organisms.
Propionic acid is oxidized in animal tissues by a mechan
ism unlike that for any of the other acids; i.e., it under
goes alpha oxidation to pyruvic acid, and this acid is
oxidized through the tricarboxylic acid cycle. Presumably
all odd chain acids are finally transformed to a propionic
acid fragment which is then oxidized by this different
mechanism (Breusch, 1948). Conceivably an organism might
have enzymes for the production of propionic acid but not
for the oxidation of that substance. This then might ex
plain the non-utilization of propionic acid. The failure
of the organisms to utilize butyric and valeric acids is
not easily explained. It may be that with these short-
chain acids the paraffin end of the fatty acid chain is
sufficiently short to exert steric hindrance to the at
tachment of the enzyme to the carboxyl group. Another
explanation, albeit a weak one, is that the cell is not
permeable by these acids. If such is the case, then cell-
free preparations oxidizing higher acids should as readily
oxidize butyric and valeric acids.
6l
If one were to accept the hypothesis of a single
enzyme being involved in the primary attack on the six to
fourteen carbon acids, it is nevertheless difficult to
visualize acetic acid being metabolized in the same manner.
Yet, exposure of cells to the two-carbon acid does to a
large extent eliminate the lag period in the oxidation of
the other acids by Serratia cells. The details of acetate
metabolism in animal tissue remain to be discovered, but
there is general agreement that oxidation is through the
tricarboxylic acid cycle (Green, 1948). Saz and Krampitz
(1 9 5 0) have recently given evidence for the same mechanism
in Micrococcus lysodeikticus. In lysed preparations citric
1 i i
acid was synthesized from oxalacetic and acetic acids. C
carboxyl labeled magnesium acetate, when incubated aerobical
ly with unlabeled oxalacetate, gave rise to citric acid
with radioactivity centered in the primary carboxyl groups.
The results are interpreted as meaning that at least a por
tion of the complete oxidation of acetate occurs through
the tricarboxylic acid cycle. Karlsson and Barker (1948),
using isotope studies in conjunction with the technique
of simultaneous adaptation, present evidence against the
occurrence of the tricarboxylic acid cycle in Azotobacter
agilis, an organism which oxidizes acetate. Ajl (1950)
has shown that the cycle does not occur in E. coli and A.
aerogenes. He suggests that in these organisms acetate
62
metabolism is via a condensation with the formation of
succinic acid from two molecules of acetate. The succinate
is then metabolized through fumarate, malate, oxalacetate
and pyruvate. Pyruvate then undergoes decarboxylation and
oxidation to form one molecule of acetate which is then
free to condense and go through the cycle again. Nord and
Vitucci (1 9 4 7) have suggested that oxalic acid formation
by wood destroying fungi may involve a condensation between
two molecules of acetate, with the formation of succinate.
Slade and Workman (1948), using labeled acetate, demon
strated the formation of succinate from acetate in the
presence of glucose under anaerobic conditions. Randles and
Birkeland (1947) reported that when E. coli was grown under
aerobic conditions in the presence of acetate, the rate of
methylene blue reduction with succinate was greatly in
creased. There thus appears to be at least two mechanisms
in microorganisms for acetate oxidation, i.e., coupling
with oxalacetate with oxidation through the tricarboxylic
acid cycle and succinic acid formation with dissimilation
through an entirely different cycle.
If acetate is metabolized through the tricarboxylic
acid cycle in Serratia marcescens, then it is possible
that the reactions involved in coupling acetate to oxal
acetate are adaptively controlled. Since higher acids
probably can give rise to acetate, or a closely related
63
two-carbon fragment, adaptation to acetate would simultan
eously adapt to higher acids, at least as far as the con
densation is concerned. If, on the other hand, acetate
metabolism involves condensation to succinate, a remark
able similarity between succinate metabolism and beta
oxidation of fatty acids is evident, since succinic acid
is transformed to fumaric by an alpha-beta desaturation
and subsequently to malate by addition of water. Malate
then forms oxalacetate, a beta keto acid. These steps are
presumably involved in primary attack on fatty acids of
longer chain length than acetic acid. It may be that adapta
tion of the Serratia strains to any of the acids, including
ascetic acid, means activation of the enzymes involved in
the formation of beta keto acids. Alternatively, the meta
bolism of all the fatty acids may require the formation
of succinic acid from two-carbon fragments. If one can
visualize the formation of acetate as a constitutive
process and the formation of succinate from acetate an
adaptively controlled process, then the peculiar simultan
eous adaptation to higher acids becomes comprehensible.
The possibility of the accumulation of an inter
mediate . There remains the possibility that the observed
lag periods represent a time during which some compound
necessary for metabolism at a maximum rate is accumulating
64
in the cell. This substance would likely be a normal
intermediate in metabolism of all fatty acids or at least
a compound readily produced from fatty acids. If one
were to assume oxidation of fatty acids through the tri
carboxylic acid cycle, then the rate of metabolism of
fatty acids would be a function of the concentration of
tricarboxylic acid intermediates in the cell since meta
bolism of the acids would involve coupling of fragments
to oxalacetate. If acetate is formed in the metabolism of
fatty acids, then the known formation of succinate from
acetate suggests a mechanism whereby increasing concentra
tions of tricarboxylic acid intermediates might build up
in the cell as a result of normal metabolism of fatty
acids. If the lag periods were due to this or a similar
factor, it should be possible to increase the rate of fatty
acid oxidation by addition of members of the tricarboxylic
acid cycle. Such a mechanism would mean that the enzymes
involved in fatty acid oxidation are truly constitutive.
The relationship between tricarboxylic acid cycle compounds
and fatty acid oxidation is discussed in Chapter VI.
Distinct reaction chains merging in a common inter
mediate . Stanier et al. (1950) have recently considered
the possibility of two or more distinct oxidative reaction
chains, one of which is freely reversible, merging in a
65
common Intermediate:
A--->B------C
A *---» B *----» C *---- ^ D >E »etc.
B* »^=îB* ‘ * *
They point out that adaptation to any compound on the
chains originating in A and A* would cause complete adapt
ation to the common intermediate D, and consequently the
possibility of back adaptation from D into the freely re
versible chain of reactions leading from A*’ would exist.
In the light of this logic, it seems possible that there
are distinct reaction chains involved in the oxidation of
the various fatty acids but that these chains merge at
some common intermediate, perhaps acetate. If one accepts
the possibility that the reactions involved are freely
reversible, then adaptation to any one of the acids could
simultaneously adapt the cells to the other acids. It
seems quite likely that the oxidation of all fatty acids
does merge in a common intermediate, but at the moment
there appears to be no logical means of determining the
reversibility of the reactions involved. If the curious
results from simultaneous adaptation experiments prove
to be explained only on the above basis, then determina
tion of metabolic pathways in fatty acid oxidation by
this technique would prove exceedingly difficult, if not
66
impossible.
On the basis of the data given it is evident that no
definite conclusions can be drawn as to the mechanisms in
volved in the oxidation of fatty acids. Certain mechanisms
can be ruled out, however. The inability of the organisms
to utilize butyric acid would indicate that this compound
is not an intermediate in the oxidation of even chain acids,
while the inability to utilize valeric acid rules this com
pound out in the oxidation of odd chain acids. These con
clusions must necessarily be tentative, pending proof that
inability to utilize these compounds is not due to lack of
permeability. If such proves to be the case then the
mechanism of beta oxidation and that of beta oxidation with
acetic acid condensation to give acetoacetic acid are ruled
out, since even chain oxidation must go through butyric acid
and odd chain oxidation through valeric. Further, omega
oxidation appears unlikely since ten, six, and seven carbon
dicarboxylic acids are not used by the Alphin strain. Suc
cinic is utilized, but inability to oxidize the higher
dicarboxylic acids seems to indicate that succinic is not
formed through omega oxidation. Of the known mechanisms
of oxidation occurring in animals, the only remaining scheme
is that of multiple alternate oxidation. None of the data
67
rule out this mechanism, although there is no positive
evidence for its occurrence.
SUMMARY
Patterns of fatty acid oxidation by Serratia
marcescens (Alphin) were studied in relation to growth
on or exposure to various fatty acids. It was found
that adaptation of cells to any of the oxidizable fatty
acids from Cg to Cii|. simultaneously adapts cells to the
oxidation of all the other acids; i.e., the lag periods
are shortened or completely eliminated. The question of
the significance of the lag period in unadapted cells was
discussed.
CHAPTER IV
THE EFFECT OF CELL POISONS ON FATTY ACID
OXIDATION BY SERRATIA
The Investigations reported in Chapters II and III
were qualitative in nature, the primary object being to
study oxidative patterns. As a consequence, no quantita
tive data were obtained showing the relationship between
substrate utilization and gas exchange. Knowledge of the
extent to which the various acids are utilized would be
of value since it would indicate the probable end products
of oxidation and thus possibly throw light on the mechan
isms involved.
Quantitative studies of this type are complicated
by the fact that microorganisms, either in the process of
growth or in the resting state, convert part of the avail
able substrate to new cellular material while another
part is subjected to an oxidation process involving the
consumption of oxygen. Barker (1936), studying the metabol
ism of the colorless alga, Prototheca zopfii, first de
scribed this phenomenon, now termed oxidative assimilation.
He observed that the amounts of oxygen consumed during the
dissimilation of various organic substrates varied with the
nature of the compound. Glycerol oxidation resulted in a
net oxygen consumption of only twenty-nine per cent of the
69
theoretical amount for breakdown to carbon dioxide and
water* Similarly, ethyl alcohol gave forty-six per cent
aid acetic acid fifty per cent of the calculated values
for complete oxidation. That this represented actual
assimilation rather than incomplete utilization was shown
by the complete disappearance of the substrates from the
medium. Giesberger (1936) noted similar differences
between theoretical and experimental uptake in his studies
on the oxidation of organic compounds by Spirillum species.
Clifton (1 9 3 7) observed oxidative assimilation in Pseudo
monas calco-acetica and Escherichia coli but found that
in the presence of suitable concentrations of mono-
iodoacetate, sodium azide, 2:4-dinitrophenol, and methyl
urethane the oxidation of organic compounds resulted in
theoretical oxygen consumption. He concluded that at
critical concentrations these agents completely prevent
synthesis and force the reaction in the direction of com
plete oxidation to carbon dioxide and water. Clifton’s
discovery led to the wide use of cell poisons in quantita
tive studies on oxygen consumption by microorganisms.
The inhibition of oxidative assimilation by cell
poisons in closely related to a second property of these
substances, i.e., inhibition of adaptive enzyme formation.
Monod (1 9 4 4) noted that 2:4-dinitrophenol (DNP) did not
prevent fermentation of glucose by E. coll but that xylose
70
fermentation was completely inhibited. Since the oxidative
pattern for xylose in the absence of DNP indicated that it
was attacked through adaptive enzymes, Monod concluded that
in the presence of DNP assimilation is blocked and conse
quently the formation of adaptive enzymes is inhibited.
Reiner (1946) and Spiegleman (19^7) showed that DNP and
sodium azide prevent adaptation of Saccharomyces carls-
bergensis to maltose and galactose fermentation. The
use of cell poisons thus suggests a valuable approach to
the problem of determining the reason for the lag periods
observed when fatty acids are attacked by glucose-grown
Serrâtia cells.
EXPERIMENTAL
Methods employed were similar to those previously
described. Unadapted cells were harvested from glucose-
mineral salts medium, while adapted cells were obtained
either by exposure of glueose-grown cells to specific
acids in the Warburg vessels or by growing cells on media
containing a fatty acid as the sole carbon substrate.
Stock solutions of DNP and sodium azide were prepared in
M/20 phosphate buffer, pH 7.0, and for experimental work
appropriate concentrations were prepared from these
solutions. Preliminary experiments showed that it made no
71
difference whether cell poison was poured from the side-
arm at the time of substrate addition or was added to
the cell suspension prior to the addition of substrate
from the side-arm. In the experiments to be reported,
cell poison and fatty acid were added simultaneously after
a fifteen minute equilibration period.
Osygen uptake was measured in the usual manner and
was followed until it was apparent that all substrate had
been utilized and the rate for autorespiration had been
reached. In determination of net oxygen uptake it was
necessary to consider the effect of endogenous respiration.
Barker (1936), Clifton (1937) and Doudoroff (19^0) have
found that when oxidation proceeds at a rapid rate, auto
respiration is negligible. In those cases where substrate
is used at a slow rate, oxygen uptake due to endogenous
respiration becomes a significant factor, and appropriate
corrections must be made; this usually involves subtracting
the endogenous uptake from the uptake observed in the
presence of substrate. Norris, Campbell and Ney (19^9)
showed that in Pseudomonas aeruginosa endogenous respira
tion continues, even when oxidation takes place at a rapid
rate, and in quantitative studies with this organism
it was necessary to subtract autorespiratory uptake from
total uptake, under all experimental conditions. In the
present study, net uptake due to substrate utilization was
72
determined by projection of the steepest part of the curve
to meet the projection of the portion of the curve after
autorespiration had been reached (see Figure 8). With
this procedure, the values obtained in different experi
ments were comparable, and oxygen uptake per unit amount .
of substrate was nearly constant over the range of con
centrations tested. When oxygen uptake was calculated
by subtraction of autorespiratory, the values were ob
viously erroneous; in some cases, however, the rate of
oxidation was very slow, and in these instances net uptake
was determined by subtracting the autorespiratory uptake
from the total uptake observed in the presence of sub
strate .
Effect of IXfP and sodium azide on oxidation at
pH %.0. Table III summarizes experimental data obtained
when buffer suspensions of Serrâtia marcescens (Alphin)
were exposed to fatty acids in the presence and absence
of DNP. The values given represent averages of the results
of several different determinations. In the absence of
cell poisons the oxygen uptake was always lower than the
theoretical value for complete oxidation of substrate to
carbon dioxide and water. With acetic acid as the sub
strate the oxygen consumption most nearly approached the
theoretical value. The amounts of oxygen consumed relative
73
QlwtC C„ «O.ClOO*r4DN»
9f^Vfc.C»>0 0 Q 0 1 5 0 N f >
i
z
8
,4kK«W vy Cy * O 0007J' ONf
gfowm V» C* + o . o o o r j ONP
2
o
m/20 phosphate buffer, pH 7-0, 30°C., 0.1 ml 10 per cent
KOH in center well. Flask concentrations: acetic (Cg)^
0.001 M; caproic {C5), heptylic (C7), caprylic (C3),
pelargonic (Cg), and capric (C^q)— 0.0002 M.
FIGURE 8
THE OXIDATION OF FATTY ACIDS BY ADAPTED AND UNADAPTED
CELLS IN THE PRESENCE AND ABSENCE OF DNP
74
TABLE III
effect OF 22 4-DINITROPHENOL ON THE OXIDATION OF FATTY
ACIDS BY SERRATIA MARCESCENS (ALPHIN)
Substrate
Theory )xl
Experimental conditions Og per
pM acid
Observed*
% of
theo
retical
acetate Unadapted 44.8
33 7 3 .5
Cg exposed
39
8 3 .8
Unadapted, 0.0005 M DNP
3
5 .6
Cg exposed, 0.0005 M DNP 8
1 6 .7
Cio grown, 0.00075 DNP 0 0
caproate Unadapted
179
130 7 2 .6
Unadapted, 0.00075 DNP 0 0
Cio grown, 0.00075 DNP 0 0
heptylate Unadapted 2 1 2 .6 142 6 6 .6
Cj exposed 112
5 2 .7
Unadapted, 0.0005 M DNP 0 0
Unadapted, 0.00075 M DNP 0 0
Cj exposed, 0.0005 M DNP 23**
1 1. 0**
Cio grown
150
7 0 .5
Cio grown, 0.00075 M DNP 75**
3 5. 5**
caprylate Unadapted 246 142
5 7 .7
Cg exposed 130
53
Unadapted, 0.00075 M DNP
50**
2 0. 3**
Gio grown, 0.0005 M DNP
212 86
Cio grown, 0.00075 M DNP
210 8 5 .6
pelargonate Unadapted 280 147 5 2 .3
Co exposed 175 6 2 .5
Unadapted, 0.00075 M DNP 23**
8.1**
Cg exposed, 0.0005 M DNP 173 6 1 .7
Cio grown, 0.00075 M DNP 237 84.5
caprate Unadapted 314
175 5 5 .7
Cio exposed 173
5 5 .0
Unadapted, 0.00075 M DNP
261
C%o exposed, 0.0005 M DNP
264 84.2
* Values corrected for autorespiration.
** Rate of oxidation very slow; accurate values difficult to
determine.
75
TABLE III (Continued)
Theory jil ^ of
Substrate Experimental conditions Og per Observed* theo-
)M acid retical
Cio exposed, 0.00075 M DNP 256 8I .5
Cio grown, 0.0 0 0 5 M DNP 257 82.0
Cno grown, 0.00075 M DNP 272 8 7.0
Cio grown, 0.001 M DNP 252 8O .3
Substrate concentrations: acetic acid, 2.0 micromole per
2 .0 ml fluid in flask; all other acids, 0.4 or 0 .6 micro
mole per 2 .0 ml fluid in flask, depending on particular
experiment.
Experiments conducted in air atmosphere, with 10 per cent
KOH to absorb COg; pH 7.0; 30^0.
76
to theoretical amounts for complete oxidation decreased
with increasing chain length, the percentage of theoretical
uptake being about the same for caprylic, pelargonic, and
capric acids. Cells adapted to acetic and pelargonic acids
showed greater uptake on the homologous acids than did the
unadapted cells; with cells adapted to heptylic, caprylic,
and capric acids the reverse effect was noted. It is ap
parent that oxidative assimilation occurs with all the acids
tested and that the amount of assimilation increases with
molecular weight of the fatty acids.
In preliminary experiments with capric acid oxida
tion, it was found that DNP increased oxygen uptake by
both adapted and unadapted cells. Caprate oxidation was
most nearly complete in the presence of 0.00075 M DNP;
0 .0 0 0 5 M and 0.001 M concentrations gave slightly lower
values. In most cases tests were conducted with 0.0005
and 0.0 0 0 75 M DNP.
DNP completely inhibited the oxidation of caproic
acid by both adapted and unadapted cells. Metabolism of
acetic acid by C^o-g^own cells (and thus cells simultan
eously adapted to acetate) was completely inhibited;
unadapted cells and cells exposed to acetic acid showed
a very low level of oxygen uptake in the presence of DNP.
It seems probable that no actual utilization of acetate
occurred under these conditions, the values in excess of
77
autorespiratory uptake perhaps representing a slight stim
ulation of endogenous metabolism. As a working hypothesis
it may be assumed that DNP at pH 7*0 blocks acetate and
caproate metabolism in Serrâtia marcescens (Alphin).
Reference to Figure 8 and Table III shows that DNP
brings about both quantitative and qualitative changes in
the oxidation of heptylate. Unadapted cells show no
oxygen uptake in excess of the autorespiratory level,
while cells grown on capric acid-mineral salts medium
(and thus adapted to heptylic acid) show an oxygen uptake
significantly in excess of endogenous but at a slow rate
as compared with adapted cells in the absence of cell
poison. It will be noted that CjiQ-grown cells showed
a greater uptake (75 }il perjim) than did the Cj-exposed
cells (23jiil per Jim). In the experiment with Gig-grown
cells 0.00075 M DNP was used; while 0.0005 M DNP was used
with the exposed organisms. Whether this difference in
concentration of poison accounts for the observed differ
ence in uptake is not apparent. In both cases, the rate
was so slow it was necessary to subtract autorespiration
directly from total uptake; this may be an important
factor.
DNP brought about a marked inhibition of caprylate
and pelargonate oxidation by unadapted cells. Uptake in
both cases was low as compared with that for unadapted
78
cells in the absence of DNP (see Figure 8). With pelargon
ic acid it is questionable whether the uptake above the
endogenous level represents utilization of substrate. It
must be emphasized that in repeated experiments with oxida
tion of pelargonate by unadapted cells the rate was always
slightly in excess of autorespiratory uptake, even with
low concentrations of TOP. Unadapted cells show a higher
level of oxygen uptake on caprylate than pelargonate in the
presence of DNP. It is, nevertheless, at a slower rate and
the net uptake is less than that with the same cells in the
absence of poison. DNP had no inhibitory effect on caprylic
acid oxidation when Serratia (Alphin) cells were grown on
capric acid medium and then tested for caprylate oxidation
in the presence of DNP. The net oxygen uptake was in excess
of that in the absence of poison, being approximately 86 per
cent of the theoretical value for complete oxidation. No
data are presented for capiylate-grown or exposed cells; in
preliminary studies it was found that DNP did not inhibit
caprylic acid oxidation in these cells, the rate and quan
titative uptake being comparable to that for capric-grown
cells. Similarly, DNP had no inhibitory effect on pelar
gonate oxidation by cells exposed to pelargonic acid or
grown on capric acid-mineral salts medium. With Cg-exposed
cells the uptake was essentially the same as that for the
exposed cells in the absence of poison, but with the G^q-
79
grown cells the total uptake was Increased, approximately
85 per cent of the theoretical amount of oxygen being
consumed. It will be noted that O.OOO5 M DNP was used
with the exposed cells, 0.00075 M DNP with the caprate-
grown cells. In many experiments with pelargonic acid
oxidation it was noted that cells adapted to capric acid
showed greater uptake on pelargonic acid than did cells
specifically adapted to pelargonic acid. One might argue
that in the absence of cell poisons the cells specifically
adapted to the odd-chain acid can more efficiently as
similate that acid than can the caprate-adapted cells.
In the presence of cell poisons this assimilation is
presumably inhibited, and the divergence between results
obtained with the two types of adapted cells seems to have
no logical explanation.
The reactions of Serratia marcescens cells to capric
acid were unlike the reactions for the lower acids. DNP
did not inhibit oxidation by either adapted or unadapted
cells. Total oxygen uptake with unadapted cells, with
Ciorgrown cells, and with C^g-exposed cells was always in
excess of the value obtained in the absence of DNP. C^g-
grown cells showed 87 per cent of the theoretical uptake
for complete oxidation, unadapted cells 99 per cent, and
C^g-exposed cells 8 1 .5 per cent. The unadapted cells
utilized the acid only after an initial lag period, just
80
as did the unadapted cells in the absence of DNP. Further,
if unadapted cells were allowed to oxidize capric acid to
completion and then were exposed to fresh substrate (poured
from the side-arm) the oxidation once again proceeded with
an initial lag period. Such cells also showed a lag period
in the oxidation of other fatty acids. It will be recalled
that cells allowed to oxidize capric acid in the absence of
DNP subsequently oxidized caprate and all the other acids
tested with no lag period.
Neither undecylie nor trldecylic acids were oxidized
by unadapted cells in the presence of DNP (Table IV); under
these conditions there was a slow rate of oxygen uptake
slightly above the autorespiratory uptake, similar to that
observed in the oxidation of pelargonic acid under the
same conditions. Laurie and myristic acids were oxidized
by the unadapted cells in the presence of DNP, but the rate
of oxygen uptake was only about a fourth of that observed
in the absence of the cell poison; the amount of oxygen
consumed was much less than that taken up by unadapted
cells without DNP. When C^g-grown cells were used, the
Oil to Gi4 acids were all oxidized in the presence of DNP.
No quantitative data on the extent of oxidation of these
acids were obtained.
Sodium azide in O .06 M concentration had effects
similar to 0.00075 M DNP, but the over-all oxygen uptake
81
TABLE IV
THE EFFECT OF pH ON THE OXIDATION OF FATTY ACIDS
BY SERRATIA (ALPHIN) CELLS IN THE PRESENCE
AND ABSENCE OF 2:4-DINITROPHENOL
Sub- pH 7.0^ pH 8.0
strate^ Unadapted Adapted Unadapted Adapted Unadapted Adapted
DNP DNP DNP DNP
Gg
_*
/ / / (93$g)
/ (62$g)
06
- - - - - -
°7
- -
/
— / (60^)
0& / / /
/ (74^)
Cg / / /
/ (64$)
ClO
/ / / /
/ o m / (74$)
Gil
/ / /
_*
/
Gi2
/ / / /
Ci3
_*
/ / / /
Gi4
/ / / /
^ All substrates oxidized by Serratia (Alphin) at pH 7*0 in
absence of DNP.
2 Acetic acid flask concentration— 0.001 M; all other acids—
0.0002 M.
* Oxygen uptake slightly above endogenous.
** Rate of oxygen uptake lower than that in the absence of
DNP; total oxygen uptake less than when DNP absent.
Figures in parenthesis represent percentage of theoretical
oxygen uptake for complete oxidation to carbon dioxide and
water.
All experiments at 30°C., air atmosphere with 10 per cent
KOH to absorb carbon dioxide.
Unadapted— glucose-grown Serratia (Alphin) cells.
Adapted— caprate-grown Serratia (Alphin) cells.
82
was not as great as In the presence of the phenolic com
pound. Azide Inhibited acetate oxidation to the same
extent as did DNP. Caproate and heptylate oxidation
were not studied in the presence of azide. The azide
increased oxygen uptake on caprylic, pelargonic, and
capric acids when adapted cells were used but not to the
same extent as did DNP. With unadapted cells, azide had
no inhibitory effect on caprate oxidation; the oxidation
of pelargonate was inhibited to the same extent as was
noted with DNP. No data were obtained for unadapted cells
in the presence of azide and caprylate.
The data indicate that the oxidation of certain
acids is chemically blocked by DNP. With acetic and
caproic acids, for instance, cells completely adapted to
these compounds fail to metabolize them in the presence
of DNP. It appears likely that adaptation is also in
hibited, but the data do not conclusively prove this, i.e.
adaptation (whatever it may involve) might occur in the
presence of DNP, but the presence of DNP also might pre
vent its observation. No attempts have yet been made to
determine whether adaptation can occur without fatty acid
oxidation.
The oxidation of certain other acids, caprylic and
pelargonic for instance, by unadapted cells is prevented
by DNP, but such oxidation by adapted cells is unaffected
83
by DNP. This indicates that DNP prevents an adaptation pro
cess which normally occurs when glucose-grown cells are
exposed to fatty acids. Whether this adaptation process
involves the formation of adaptive enzymes has not been
determined.
Only in the presence of capric acid was the amount
of oxygen consumption indicative of complete oxidation.
This occurred only when glucose-grown cells were used;
caprate-grown cells consumed only 8j per cent of the amount
of oxygen required for dissimilation of cparic acid to
carbon dioxide and water. Since it is improbable that the
oxidative pathways are different in the two types of cells,
it appears likely that the lower uptake with adapted cells
is due to oxidative assimilation. This suggests that a
higher concentration of DNP is necessary to inhibit com
pletely oxidative assimilation in adapted cells. A similar
effect was noted in the study of acetic and capric acid
oxidation at pH 8.0 (see Table IV).
Pelargonate oxidation by caprate-grown cells result
ed in the uptake of 237 jul of oxygen per jam of substrate—
85 per cent of theoretical. This value is quite close to
that observed in the oxidation of caprate by the same cells
(8 7 per cent). Since pelargonate oxidation was inhibited
by DNP in unadapted cells, these data do not permit the
conclusion that the failure to show theoretical oxygen
84
uptake is due to assimilation rather than incomplete oxida
tion. If an unoxidized fragment were to accumulate during
the oxidation of pelargonate in the presence of DNP, this
substance should be in a state of oxidation corresponding
to acetic acid. The oxidation of pelargonic acid with the
accumulation of one mole of acetic acid per mole of pelargon
ic acid would require the consumption of 235 )il of oxygen
per jAm of pelargonate. Since acetate is not oxidized at
pH 7 .0 in the presence of DNP, the results suggest that
acetate may accumulate during the oxidation of the nine-
carbon acid. Data to be presented later in this chapter
indicate that this is not the case and that it is probable
that the apparent incomplete oxidation is due to oxidative
assimilation.
Oxidation of caprylic acid resulted in the uptake
of 210 yil of oxygen per jm. of acid when caprate-grown cells
were used— 8 5 -6 per cent of the theoretical amount for
complete oxidation. As with pelargonate, oxidation by un
adapted cells was inhibited by DNP. The percentage of
theoretical uptake with caprate-grown cells is sufficiently
close to that observed in pelargonate and caprate oxidation
by the same organisms to allow the assumption that DNP is
only about 85 per cent effective as an inhibitor of oxida
tive assimilation in caprate-grown cells.
Since neither heptylate nor caproate were appreciably
85
oxidized in the presence of DNP at pH 7.0, indications as
to the extent of oxidation of these acids can be obtained
only from the data obtained in the absence of DNP. Under
these conditions, caprate-grown cells consumed 150 yl of
oxygen per ym of heptylic acid. Without considering the
effect of assimilation on the observed uptake, this in
dicates oxidation beyond the propionic acid stage. Similar
ly, unadapted cells showed an uptake of 130 jil of oxygen
per jam of caproate, indicating disimilation at least to a
stage of oxidation corresponding to acetate. Since work
with the higher acids indicated that in the absence of DNP
30-40 per cent of the substrate is assimilated, it is prob
able that caproate is oxidized beyond the acetate stage,
possibly to carbon dioxide and water.
The oxidation of fatty acids at pH 8.^ ^ the pres
ence and absence of DNP. Unlike sodium azide, the effective
ness of DNP as an inhibitor of oxidative assimilation is in
direct relationship to the hydrogen ion concentration
(Doudoroff, 194 0). The preceding work indicated that
0.00075 M DNP at pH 7 .0 might exert at least two distinct
inhibitory effects on fatty acid oxidation by Serratia
(Alphin), i.e. direct blockage of the enzymes concerned
with oxidation and inhibition of adaptation to fatty acid
oxidation. It seemed possible that two different concentra
tion of DNP might be responsible for the inhibitory actions.
86
one for Inhibition of oxidation and one for inhibition of
adaptation. Experiments were conducted in which the oxida
tion of fatty acids was studied at pH 8.0, both in the
presence and in the absence of DNP.
The results summarized in Table IV show that with the
exception of caproic acid, all acids oxidized at pH 7.0 were
also oxidized at pH 8.0. Heptylic acid is oxidized by cells
previously adapted to heptylate oxidation by growth on capric
acid medium but is not oxidized by unadapted cells. Failure
to oxidize caproic acid under any of the conditions imposed
at pH 8.0 indicates that at this pH the acid may become
toxic to the cell; at the higher pH the ionic equilibrium
shifts to a higher concentration of the salt of caproic
acid and away from the free acid. Alternatively, there may
be something unique in the metabolism of caproic acid (as
compared to the other acids tested) in that its oxidation
is blocked at a pH at which the other acids are metabolized.
At pH 8.0 DNP prevents oxidation of heptylate,
caprylate, pelargonate, undecylate, and tridecylate by un
adapted cells. C2f ^1 2^ and C^ii. acids are oxidized
by unadapted cells in the presence of DNP. Most notable
is the fact that acetic acid is nearly completely oxidized
in the presence of DNP at pH 8.0. It will be recalled that
acetate oxidation was blocked by DNP in both adapted and
unadapted cells at pH 7-0.
87
With the exception of caproic acid, all the acids
are oxidized by adapted cells (Cig-grown) in the presence
of DNP at pH 8.0. Quantitatively, the amount of oxygen
consumed under these conditions is less than at pH 7?0.
Oxygen uptake on acetic and heptylic acids is actually
less than that observed when adapted cells oxidize these
acids at pH 7*0 in the absence of DNP. With the other
acids, uptake is greater than that for adapted cells at
pH 7.0 in the absence of DNP but is less than that by or
ganisms at the lower pH in the presence of the phenolic
compound. Further, heptylic acid oxidation is completely
inhibited by DNP at the lower pH, but the acid is oxidized
by adapted cells at pH 8.0. Thus DNP is not as effective
as an inhibitor of oxidative assimilation at pH 8.0. Thus
DNP is not as effective as an inhibitor of oxidative as
similation at pH 8.0 as at pH 7.0.
Difficult to explain is the fact that neither capric
nor acetic acids are oxidized to completion by adapted
cells at pH 8.0 in the presence of DNP; unadapted cells
show oxygen uptake indicating complete oxidation to carbon
dioxide and water under the same conditions. This suggests
that a higher concentration of DNP may be required to in
hibit oxidative assimilation in adapted cells. A similar
finding was reported in the previous section--at pH 7*0
in the presence of DNP capric acid was completely oxidized
88
by unadapted cells (9 9 per cent) but showed only 87 per
cent of theoretical oxygen consumption with Cig-grown
organisms. The age of the cells may account for these
differences, since the C^Q-grown cells were harvested
from 4o hour cultures and the glucose-grown cells from
l8 hour cultures. It is often stated that poisonous sub
stances are most effective against young cells. The fact
that Gio-exposed cells (l8 hour glucose-grown cells) show
ed less uptake than cells at pH 7*0 would in
dicate that perhaps the differences in uptake between adap
ted cells and unadapted cells is due to something other than
the age of the organisms.
If one studies the patterns of oxidation at the two
pH levels, it will be noted that the odd chain acids-'C^^,
and Cg--gave identical patterns; the even chain acid,
caprylic, shows the same pattern. and C^g acids
have similar patterns; here, however, DNP had some inhibit
ory effect on the oxidation of lauric and myristic acids
by unadapted cells. The rate was significantly decreased
and also the total oxygen uptake. Capric acid oxidation
is unaffected by DNP. Acetic, caproic, and heptylic acids
gave patterns unlike the higher acids and also differed
from one another. The significance of these findings will
be discussed later.
89
Evidence that pelargonic and capric acids are com
pletely oxidized at pH 7.0 lu the presence of DNP. It will
be recalled that in the presence of DNP (0.00075 M) un
adapted cells showed 99 per cent of the theoretical oxygen
uptake for complete oxidation of capric acid. Gig-exposed
cells showed 87 per cent of the calculated amount. Similar
ly, Cj^Q-grown cells consumed only 85 per cent of the amount
of oxygen required for complete oxidation of pelargonic
acid. Analysis of the data indicated that if the uptakes
below theoretical were due to incomplete oxidation, the un
oxidized fragments should correspond closely to acetic
acid. Since acetic acid is not oxidized under these condi
tions, it seemed possible that it might accumulate. It was
found, however, that acetate was metabolized at pH 8.0 in
the presence of DNP. Experiments were conducted in which
capric acid was oxidized by adapted cells at pH 7-0 in the
presence of DNP. When the autorespiratory level was reach
ed, alkaline buffer was poured from the side-arm and the
pH in the flask was thus raised to 8.0. Similarly, pelargon
ic acid was oxidized by C^o'S^own cells at pH 7-0 and then
the pH raised to 8.0 in the same manner. In both cases,
oxygen uptake was followed to the point at which endogenous
respiration started at the lower pH, and then the uptake
was measured after addition of the buffer solution. In
neither case was there any further oxygen uptake above the
90
autorespiratory level after the pH was raised. If acetic
acid had accumulated during the oxidation at pH 7.0, one
would have expected further uptake of oxygen after the pH
had been raised, since acetate is metabolized at the high
er pH. Since there was no further uptake above the endo
genous level, the results indicate that acetate does not
accumulate in either pelargonate or caprate oxidation at
pH 7-0 in the presence of DNP. Since unadapted cells had
previously been shown to oxidize caprate to completion at
pH 7.0, it appears reasonable to assume that the apparent
incomplete oxidation of pelargonate, caprate, and caprylate
is due to incomplete inhibition of oxidative assimilation.
DISCUSSION
Work with cell poisons gives evidence, in some cases
indirect, that the Cg, G5, Cy, Cg, Cg, and C^^g ^*atty acids
are completely oxidized to carbon dioxide and water by
cell suspensions of Serratia (Alphin). With capric acid
99 per cent of theoretical oxygen uptake was obtained with
unadapted cells in the presence of 0.00075 M DNP at pH 7.0.
This was the only case in which unequivocal proof for
complete oxidation was obtained. With acetic acid 93 per
cent of the theoretical uptake was observed in the presence
of DNP with unadapted cells at pH 8.0; it is assumed that
91
failure to observe the consumption of theoretical amount
oxygen under these conditions is due in part to experi
mental error and in part to the failure of DNP to inhibit
completely oxidative assimilation at this pH. Caprate-
grown cells showed approximately 85 per cent of the oxygen
uptake necessary for complete oxidation of caprylic and
pelargonic acids in the presence of DNP; indirect evidence
indicated that this value represents the effectiveness of
DNP as an inhibitor of oxidative assimilation rather than
incomplete oxidation of the eight and nine-carbon acids.
Since neither heptylic nor caproic acid was oxidized in
the presence of DNP at pH 7 . 0, it was possible to obtain
evidence as to the extent of oxidation of these acids only
from quantitative results obtained in the absence of DNP.
Such data indicated that in the absence of DNP oxidation
of heptylate proceeds beyond the propionic acid stage and
the oxidation of caproate beyond acetic acid. When one
takes into account the fact that 3 0 -^ 0 per cent of the
available substrate is assimilated, it appears likely that
both these acids are completely oxidized. No quantitative
data were obtained on the extent to which the higher acids
are oxidized.
In previous studies it was found that Serratia
(Alphin) cells do not oxidize butyric, valeric, or pro
pionic acids. The quantitative data indicate that the
92
oxidation of the C6> Cg, and G^q acids proceeds beyond the
butyric acid stage and that the Gy and Cg acids are oxidiz
ed beyond the propionic acid stage. This indicates that
butyric acid is probably not an intermediate in the oxida
tion of higher even chain acids and that neither propionic
nor valeric acids are intermediates in the oxidation of
higher odd chain acids. Since the data show that oxida
tion goes beyond the propionic and butyric acid stage, it
is not likely that the G3, G i | . , or G^ acids accumulate in
the oxidation of higher acids. According to the classical
theory of beta oxidation, butyrate should be an intermediate
in the oxidation of higher even chain acids and propionate
in the oxidation of higher odd chain acids (Breusch, 1948).
If one assumes that the repeated failures to demonstrate
oxidation of butyric, propionic, and valeric acids is due
to lack of appropriate enzymes in the cell for the metabol
ism of these compounds, then beta oxidation is not the
mechanism for attack on C5 to Ciq fatty acids. If, on the
other hand, this failure to observe oxidation is due to
impermeability of the cells to these compounds, and if the
appropriate enzymes for the oxidation of these acids are
present in the cells, then it is possible that the three,
four, and five-carbon acids are intermediates and that
beta oxidation occurs. The only feasible manner in which
this problem can be solved is through studies with cell-free
93
enzyme preparations. Under these conditions, permeability
effects should be eliminated, and if the enzymes for the
oxidation of Cg-C^ fatty acids are present in the cells,
they should be as readily demonstrated as those for the
higher acids. Should cell-free preparations capable of
catalyzing the oxidation of the higher acids fail to
activate oxidation of C^-C^ acids, then it would have to
be concluded that some mechanism other than beta oxida
tion occurs. This problem will be more completely dis
cussed in the following chapter.
The quantitative data on oxygen uptake by unadapted
cells in the presence of DNP at pH 7*0 indicated that
capric acid is completely oxidized to carbon dioxide and
water. Under the same conditions, unadapted cells fail
to oxidize pelargonic, caprylic, heptylic, caproic, and
acetic acids. Thus, the nine, eight, seven, six and two-
carbon fatty acids cannot be direct intermediates in the
oxidation of caprate by Serratia cells. Indirect evidence
suggested that adapted cells completely oxidize capric,
pelargonic, and caprylic acids at pH 7-0 in the presence
of DNP; but the same cells fail to oxidize heptylic,
caproic, and acetic acids in the presence of DNP. Hence,
neither heptylate, caproate, nor acetate can be direct
intermediates in the oxidation of caprylic and pelargonic
acids. Since the technique of simultaneous adaptation
94
Indicated a close relationship between the enzymes
catalyzing the primary steps in the oxidation of all
these acids, the results suggest that DNP interferes with
a reaction necessary for activation of heptylate, caproate,
and acetate oxidation. The nature of this reaction is not
apparent. . Cross et al. (1949) have shown that DNP inhibits
a reaction involving the transformation of phosphate; this
phosphate transformation is necessary for the activation
of the oxidation of certain lower fatty acids by the cyclo-
phorase system. It may be that a similar reaction is neces
sary for the activation of oxidation of acetate, caproate,
and heptylate. If such is the case, then "activated" acet
ate and caproate may be intermediates in the oxidation of
caprate and caprylate, and "activated" heptylate may be an
intermediate in the oxidation of pelargonate.
DNP inhibited oxidation of acetate, caproate,
heptylate, caprylate, pelargonate, undecylate, and tri-
decylate in unadapted cells at pH 7.0. The same concen
tration of DNP did not interfere with the oxidation of
caprylate, pelargonate, undecylate, and tridecylate in
adapted cells. At pH 8.0 DNP failed to inhibit acetate
oxidation in either adapted or unadapted cells; heptylate
was oxidized by adapted cells. This inhibition is ap
parently a blocking of the adaptation process (whatever
it involves), since cells adapted to the oxidation of
95
these acids (by growth on caprate) were able to oxidize
the fatty acids in the presence of DNP. Caproate appears
to be the only exception, since it was not oxidized under
any of the conditions imposed when DNP was present.
Monod (194 4), Reiner (1946), and Spiegleman (1947)
have indicated that DNP inhibits the formation of adaptive
enzymes in microorganisms. This fact would lead one to the
conclusion that the Cy, Cg, C9, and C^g fatty acids
are attacked through adaptive enzymes, while the Cg, C^o^
C^gf and C^ii. acids are catalyzed by reactions involving
only constitutive enzymes. The type of attack on caproic
acid might be either adaptive or constitutive, since DNP
apparently interferes directly with some enzymatic step
in its oxidation. On the basis of experiments with simul
taneous adaptation (Chapter III) it appears unlikely that
some of the acids are attacked adaptively and others con-
stitutively. Exposure of cells to any of the fatty acids
known to be oxidized by the Serratia produces organisms
which have no significant lag period in the oxidation of
the other acids. If certain acids were oxidized by adap
tive enzymes and others by constitutive enzymes, one could
not expect this reciprocal adaptation to occur. It is
probable that the enzymes involved in the oxidation of
these acids are either all adaptive or all constitutive.
It will be recalled that despite the fact that caprate was
96
oxidized by unadapted cells in the presence of DNP, these
cells did not become adapted to any of the acids, including
capric acid. When such organisms were subsequently exposed
to capric acid, oxidation proceeded after an initial lag
period characteristic of unadapted cells* While the un
adapted cells are capable of caprate oxidation in the
presence of DNP, the process of adaptation by such cells
to other acids is blocked; in fact even adaptation to
capric acid is prevented (if elimination of the lag period
is to be considered the criterion for adaptation). Since
DNP inhibits oxidative assimilation, it is probable that
the adaptation process involves utilization of exogenously
supplied substrate. In the presence of DNP, this process
of assimilation is blocked, and although certain acids may
be oxidized by unadapted cells in the presence of DNP, und
er these conditions there is no adaptation. It is not pos
sible to say on the basis of the present information whether
adaptation is a process involving the formation of adaptive
enzymes. The only logical approach to this problem appears
to be through preparation of cell-free enzyme systems from
both adapted and unadapted cells.
The failure of both adapted and unadapted cells to
oxidize caproic acid in the presence of DNP suggests that
some reaction essential for the oxidation of the six-carbon
acid is blocked. While it appears unlikely that this acid
97
is oxidized in a different manner than all the other acids,
the results with DNP, together with the failure of cells
to oxidize caproate at pH 8.0, suggest this possibility.
The unique results from experiments with caproic acid
bear further investigation.
SUMMARY
The effects of 2:4-dinitrophenol and sodium azide on
fatty acid oxidation by Serratia (Alphin) were studied.
Both substances inhibited oxidative assimilation at pH 7*0,
the former compound proving most effective in this respect.
On the basis of direct and indirect evidence from the experi
ments with DNP it was concluded that capric, pelargonic,
caprylic, and acetic acids are completely oxidized by the
Serratia cells. There was good evidence to indicate that
heptylic and caproic acids are also completely metabolized
without the accumulation of intermediate substances. At pH
7.0 appropriate concentrations of DNP prevented the oxida
tion of acetic, caproic, heptylic, caprylic, pelargonic,
undecylic, and tridecylic acids by glucose-grown cells.
Under the same conditions, capric, 1auric, and myristic
acids were oxidized after lag periods. Caprate-grown cells
oxidized the eight, nine, and thirteen-carbon acids in the
presence of DNP at the same pH. At a pH of 8.0 acetic acid
98
was oxidized by both types of cells and heptylic acid
by caprate-grown cells only. The reactions of the other
acids were the same as that at the lower pH. Under no
circumstances was caproate oxidized in the presence of
DNP in the concentrations used.
CHAPTER V
STUDIES ON THE RELATIONSHIP OF VARIOUS FATTY ACID
DERIVATIVES TO THE PRIMARY OXIDATIVE
REACTIONS IN FATTY ACID OXIDATION
All proposed mechanisms for fatty acid catabolism
involve the formation of beta keto derivatives of fatty
acids. If it is assumed that beta keto acids are key com
pounds in fat metabolism, then the question arises as to
the steps involved in the conversion of a saturated acid
to its beta keto derivative. The oxidation of a saturated
acid to the corresponding keto acid should result in the
net removal of four hydrogen atoms. According to current
concepts of biological oxidations, such a chain of reac
tions should involve at least two intermediate steps, two
hydrogen atoms being removed in each step. Various theories
have been proposed for the mechanism of ketone formation
(Breusch, 1948; Foster, 1949)« While absolute proof is
lacking, evidence indicates that the following sequence
occurs both in animal tissues and in molds:
-PH H H +H0O OH H . oLi
R-CHg^ CHaCOOH -----------^ R-C=C-COOH — > R -C -C -C O O H
Fatty acid ^ ^
II H
R - C - C ' COOH
H
From the standpoint of comparative biochemistry, the sequence
is analagous to the Thunberg-¥ieland mechanism for the
100
oxidation of succinic to oxalacetic acid and to the primary
steps in the oxidative deamination of amino acids.
The present study concerned itself with the rela
tionship of the alpha-beta unsaturated, beta hydroxy, and
beta keto derivatives of capric acid to the metabolism of
capric acid and other fatty acids.
EXPERIMENTAL
Solutions of the potassium salts of beta hydroxyl
and beta keto^ capric acid were prepared in M/20 phosphate
buffer. Alpha-beta unsaturated capric acid^ was added to
M/20 phosphate buffer to give a 0.01 M solution; the pH was
adjusted to 7 .0 by the addition of potassium hydroxide-
For manometric studies, 0.0006 M solutions of the three
caprate derivatives were prepared. Oxygen uptake in the
presence of these compounds was measured in the manner
described in previous chapters, using 1 .0 ml of diluted
stock solution in the side-arm and 1 .0 ml of cells in the
main well. Cell suspensions were prepared by washing l6
to 20 hour glucose-grown or 40 hour caprate-grown Serratia
marcescens (Alphin) cells three times in M/20 phosphate
^ Prepared according to the method of Thaler and
Geist (1939b).
^ Prepared according to the method of Stenhagen (19^5)
3 Prepared according to the method of Tulus (1944).
101
buffer. All suspensions were adjusted to a standard
turbidity as measured on the Klett-Summerson apparatus.
In most cases, oxygen uptake was measured until sub
strate had been completely utilized, but in certain in
stances experiments were stopped as soon as it was pos
sible to establish the shape of the oxidation curve.
The oxidation of caprate derivatives by glucose and
caprate-grown cells. Curves for glucose-grown cells in
Figures 10, 11, and 12 show that the alpha-beta unsaturat
ed, the beta hydroxy, and the beta keto derivatives of
capric acid were oxidized after lag periods of 2 6, 40, and
19 minutes, respectively. The same cells showed a 30
minute lag in the oxidation of capric acid. The lag periods
were estimated by extension of the steepest part of the
curve to the time axis.
Cells harvested from capric acid medium showed no
lag in the oxidation of beta keto capric acid. The lag
periods in the oxidation of the unsaturated and hydroxy
derivatives were shortened but not eliminated, being ap
proximately 8 minutes in each case. The caprate-grown
cells showed a 4 minute lag in the oxidation of capric
acid.
If one assumes that the lag periods represent a
time during which adaptive enzymes are being formed, then
102
80-
TO-
60-
z
w
< o
X
o
120 14 0 160 160 2 0 0 80 20 6 0 4 0
T IM E IN M IN U T E S
M/20 phosphate buffer, pH 7.0, 30^0.
Experiments conducted in air atmosphere with 0.1 ml KOH
in center well. Flaskconcentration of alpha-beta un
saturated capric acid— 0.0003 M. Glÿcose-grown cells
used for "exposure."
FIGURE 9
OXIDATION OF ALPHA-BETA UNSATURATED CAPRIC ACID
BY SERRATIA (ALPHIN)
103
no- -
IOO--
9 0 -
80--
w w a t u r & t * j c x p o t c d
Z
e r
>
X
o
60-
40 -
3 0 -
2 0-
20 6 0 8 0 100 120 WO 160 180 2 0 0
T IM E IN M IN U T E S
M/20 phosphate buffer, pH 7.0, 30^0.
Experiments conducted in air atmosphere with 0.1 ml KOH
in center well. Flask concentration of beta hydroxy capric
acid— 0.0003 M. Glucose-grown cells used for exposure.
FIGURE 10
OXIDATION OP BETA HYDROXY CAPRIC ACID
BY SERRATIA (ALPHIN)
104
HOT
IOO--
9 0 --
7D--
Z
VÎT 6 0 •
X
J
3 0 -
10-
160 ISO SOO 80 2 0 40 6 0 1 0 0
T IM E IN M IN U T E S
M/20 phosphate buffer, pH 7*0, 30^0.
Experiments conducted In air atmosphere with 0.1 ml KOH
in center well. Flask concentration beta keto capric
acid— 0.0003 M. Glucose-grown cells used for "exposure."
FIGURE 11
OXIDATION OF BETA KETO CAPRIC ACID
BY SERRATIA (ALPHIN)
105
according to the theory developed by Stanier (194?) it
must be concluded that beta keto capric acid is an inter
mediate in the oxidation of capric acid. While the lag
periods In the oxidation of the unsaturated and hydroxy
derivatives of capric acid were not completely eliminated
as a result of growth on capric acid medium, the same
cells showed a 4 minute lag period in the oxidation of
capric acid. The difficulties encountered in the calcula
tion of lag periods have previously been discussed; the
question of the significance of short lag periods is still
unanswered. The periods in the oxidation of the alpha-
beta unsaturated and the beta hydroxy acids were 26 and
40 minutes respectively; as a result of growth on caprate
these periods were reduced to 8 minutes. It would seem
permissible to assume that this represents adaptation to
these acids and that the observed lag periods are due
to technical difficulties both in the conduct of the ex
periments and in the mechanics of calculating the lag.
This leads to the conclusion that all three derivatives
of capric acid are intermediates in its oxidation by Alphin
cells.
The relationship between adaptation to caprate
derivatives and the oxidation of other derivatives. Glu
cose-grown cells were specifically adapted to the three
caprate derivatives by adding 0 .6 ym of substrate to each
106
ml of cell suspension and shaking in Warburg Vessels.
After the added substrate was utilized (as indicated by
return to autorespiratory oxygen uptake) the test sub
strates were poured from the side-arms.
Figures 9> 10, and 11 show that exposure of glucose-
grown cells to any of the three caprate derivatives caused
a shortening of the lag periods in the oxidation of the
other two compounds. Organisms specifically adapted to
alpha-beta unsaturated capric acid show no lag in the ox
idation of the beta hydroxy or the keto acids. Beta hydroxy
caprate exposed cells showed no lag in metabolizing the keto
acids. Beta hydroxy caprate exposed cells showed no lag in
metabolizing the keto and the unsaturated compounds. Cells
adapted to the keto acid had a 9 minute lag in the oxida
tion of the hydroxy acid and an 8 minute lag in the oxida
tion of the unsaturated derivative of caprate.
Adaptation of cells to the oxidation of the hydroxy
and keto derivatives as a result of exposure to the un
saturated acid Indicates that the keto and hydroxy acids
are intermediates in the oxidation of the alpha-beta un
saturated compound. The simultaneous adaptation of beta
hydroxy caprate-exposed cells to the keto derivative
supports this conclusion. Although it is inconceivable
that alpha-beta unsaturated caprate is an intermediate in
the oxidation of the beta hydroxy acid, adaptation to the
107
hydroxy derivative simultaneously adapted organisms to
the unsaturated acid. This suggests a close relationship
between the two compounds. Chemically the hydroxy acid
differs from the unsaturated derivative in that the former
is fully saturated with a molecule of water. It may be
that in the cell the unsaturated acid is in equilibrium
with and rapidly converted to the hydroxy acid through
the action of a reversibly acting enzyme; similar enzymes
are known to be involved in the reversible conversion of
fumaric acid to malic acid and in the conversion of cis
aconitic acid to citric acid. If an enzyme exists in the
cell for the conversion of alpha-beta unsaturated fatty
acid to beta hydroxy fatty acid, then one would expect
that adaptation to one of the compounds would result in
simultaneous adaptation to the other.
The relatively short lag periods noted when beta
keto capric acid exposed cells were allowed to oxidize the
hydroxy and the unsaturated acids are probably indicative
of simultaneous adaptation to the oxidation of these deriva
tives as a result of exposure to the keto acid. The results
reported in the next section support this conclusion.
The effect of adaptation to caprate derivatives on the
oxidation of capric, caprylic, and undecylic acids. In
order to determine the effect of adaptation to caprate
derivatives on the oxidation of the normal saturated acids.
108
glucose-grown cells were specifically adapted to the three
caprate derivatives and then allowed to oxidize caprate,
caprylate, and undecylate. The results are shown in
Table V. Adaptation of cells to any one of the three com
pounds produced organisms which were adapted to oxidation
of the C0, CiQ, and acids. As in the previous sec
tions, there were instances where the lag periods were not
completely eliminated, but the great differences between the
calculated lag periods for exposed and unexposed cells allows
the conclusion that exposure to any of these three compounds
adapts the cells to the three normal fatty acids tested.
DISCUSSION
Although aspects of the data already discussed (i.e.,
the oxidation of caprate in the presence of DNP by glucose-
grown cells) makes it impossible to conclude definitely
that Serratia marcescens (Alphin) attacks fatty acids by
means of the "classical" type of adaptive enzymes, it is
certain that exposure to or growth on fatty acids or their
derivatives profoundly affects the metabolism of this organ
ism with respect to this group of compounds. The basic
theory behind the concept of simultaneous adaptation
(stanier, 19^7) or "simultaneous activation" (AJl, 1950)
will still apply, even though qualitative differences in
the properties of the enzymes exist.
109
TABLE V
LAG PERIODS IN THE OXIDATION OP CAPRIC, UNDECYLIC,
AND CAPRYLIC ACIDS IN RELATION TO ADAPTATION
TO CAPRIC ACID DERIVATIVES
Oxidation
^ Lag period
ol
alpha-beta
unsaturated
capric acid
in minutes after specific
beta hySroxy beta keto
capric acid capric acid
adaptation
Unadapted
caprylic
5 3
6
25
capric 0 0
7
30
undecylic 2 6 4 32
Specific adaptation accomplished by exposing Serratia
(Alphin) cells to 0.6 micromole of derivative per ml of
cell suspension. Unadapted cells were unexposed. All
cells were glucose-grown.
Flask concentrations of fatty acids: 0.0003 M.
Experiments conducted in air atmosphere with 10 per cent
KOH to absorb carbon dioxide.
Temperature--30°C.
Lag periods estimated by extension of steepest part of
curve to time axis.
110
The data indicate that the alpha-beta unsaturat-
ed, beta hydroxy, and beta keto derivatives of capric
acid are intermediates in the oxidation of caprate.
The evidence for this is conclusive, since adaptation
of cells to capric acid simultaneously adapts the organ
isms to the oxidation of the three caprate derivatives.
Chemical logic would dictate that the oxidative path
way should involve a preliminary oxidation of capric acid
to its unsaturated derivative and that this should be
followed by formation of the hydroxy and then the keto
acid. An examination of the formulae of these compounds
indicates that an alpha-beta dehydrogenation should be
the primary attack on capric acid and also on its hydroxy
derivative. Neither the keto acid nor the unsaturated
acid can undergo an alpha-beta dehydrogenation, yet,
adaptation to either of these compounds simultaneously
adapts Serratia cells to the saturated and hydroxy acids.
The adaptation to the hydroxy acid is expected, since this
compound should be an intermediate in the oxidation of
alpha-beta unsaturated capric acid; but neither the un
saturated acid, the hydroxy acid, nor capric acid can be
intermediates in the oxidation of beta keto capric acid.
To complicate the picture, adaptation of cells to any of
these four compounds simultaneously adapts the organisms
to the oxidation of undecylic acid, an eleven carbon com
pound. Furthermore, previous studies showed that
Ill
adaptation to any of the acids oxidized by the Alphin
strain simultaneously adapted the cells to the oxida
tion of all the other acids. These facts suggest that
a single enzyme system catalyzes the oxidation of all
the acids oxidized by the Serratia cells. Two mechanisms
might be postulated: Scheme I— Beta Oxidation and Scheme
II— Multiple Alternate Oxidation as shown on the follow
ing page.
112
§
M
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s
i
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M
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C O
O
( d
iH O C V I
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I 0 I
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§^43
c 5 Ü
I 0 3
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I x:
O OiO
I I — I
Ü 0 3
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o o
0 3 O
o
O I
*H O
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Q.O
0 3 O
O I
X O
P I
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( W
I
OH
c
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C V J
O C V J « 3
0 3 W O U
O « M
O O ^
•H O ^ C V J
^ I izj o G O
0 .0 *0 0 ^ +>
0 3 I *H O C
O o o O *H «HO)
I 0 3 I O 3 3 Ü 6
O O O 0 3 O 0 3 b O
-P I O I O 0 3
0)O*HOOOOJ^
M 1 rH I n H I n H < M
O ^ O p O ^
I ^ I ^ I C V I I 0 3
O -P
10) . _
o x a o o o oox>cvi
O 0 .0 0 .0 -P o
I 0 3 I 0 3 I 0
s
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s
M
1 — 1 C V I oo
3! 3 3
O 3 3 o
O 3 3 O o
3 3 3 3 O O O o
O O I O O
0 * 0 0 o O 1 o
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I o 1 o o I o
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I I 1 o o 1
o o o o o 1 o
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1 0 3 I I 3 3 o o 1 0
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I I 1 I o 1
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o o o O o I o b O
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o .o o o o o o m
EH
<
=t s t = 3 - K
M
EH
OO OO OO « 0 3 OO oo OO
o O O
33 C V J C V I C V J 3, C V J 33 C V J
33 C V J C V J
33 C V J 33 M C V I 33 C V I
1 \
A
1 1
1 —1 rH rH pH rH rH
G O G O GO
1
1 GO G Q GO
o C O M 0 c C
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+s P M P P P
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< D < 1 > < 1 > TO Ü ) < ! > 0>
-
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C O 3 3 3 3
3 3 O 3 3 o
o O 3 5 o o
o 3 3 3 3 3 3 O o o o
3 3 3 3 o O 3 3 O O I o o 1
O O I o O o O O o 1 o
O O o o O o O I 1 o I
O O I I
O 1 I o o
I
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1 1 3 3 o 3 3 I o O o
I o o
O O Q 1 O O I I I o o 1
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o o O o o o o o I I o I
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3 3
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o o o o o Ü O O o o o o o o
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1 0 3 I I 1 1 1 O I 0 3 I I 1 t o o
o o o o o o O o O o o o o 1
I O 1 I 1 1 1 1 I O I I I 1 1 o
O *H o o o o o o o •H O o o o o 1
1 k I I 1 1 1 1 1 1 1 I 1 1 o
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o o o o o o o o o O O o o o o o
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C V J
33
113
The Initial steps are the same in both schemes.
They involve (l) the conversion of saturated acid to alpha-
beta unsaturated acid by removal of two hydrogen atoms,
(2) the conversion of unsaturated acid to beta hydroxy acid
by addition of water, and (3) the conversion of hydroxy
acid to keto acid by removal of two more hydrogen atoms.
Since reactions 1 and 3 involve dehydrogenation at the
alpha-beta position, the possibility exists that a single
enzyme may catalyze both reactions. Since both the saturat
ed acid and its hydroxy derivative are oxidized by glucose-
grown Serratia cells only after a lag period, it follows
that reactions 1 and 3 are catalyzed by adaptive enzymes.
Reaction 2 involves addition of water at the alpha-beta
position, and hence the enzyme active in this step must
be different from those involved in steps 1 and 3» This
reaction is analogous to the reversible conversion of fumar
ic to malic acid which occurs in the tricarboxylic acid
cycle and is catalyzed by the enzyme fumarase. The data
do not indicate whether the enzyme Involved in reaction 2
is constitutive or adaptive, since conversion of unsaturat
ed acid to hydroxy acid produces a compound which itself
is attacked by adaptive enzymes. These three reactions
and the enzymes catalyzing them must be involved in the
oxidation of all the fatty acids oxidized by the Serratia
cells, since adaptation to any of these acids simultaneously
114
adapts the cells to the oxidation of all the other acids.
The peculiar position of acetic acid will be discussed
later.
The two schemes differ in the mechanism of keto
acid oxidation. The first mechanism visualizes classical
beta oxidation occurring. Accordingly, the keto acid
undergoes hydrolytic cleavage with the formation of a two-
carbon fragment and a fatty acid with two less carbon atoms.
Since the lower acid should be oxidized through the same
pathway as the higher homologue, reactions 1, 2, and 3
repeat themselves and are catalyzed by the same enzymes
that were involved in the oxidation of the higher acid.
Reaction 4, the cleavage of beta keto acid, may be catalyzed
by either an adaptive or a constitutive enzyme. The data do
not indicate which type is involved, since the cleavage of
keto acid produces a compound which is attacked through
adaptative enzymes. This mechanism would explain the adap
tation of cells to the oxidation of capric acid and its
hydroxy and unsaturated derivatives after exposure to beta
keto acid, since adaptation to the beta keto compound simul
taneously adapts the cells to reactions 1, 2, and 3 in the
oxidation of caprylate. The enzymes catalyzing these re
actions are the same as those catalyzing the first three
steps in caprate oxidation.
According to Scheme II, the keto acid formed as a
115
result of reactions 1, 2, and 3 does not cleave, but a
second oxidation occurs at the delta carbon atom. As a
result a second keto group is formed; subsequently, simi
lar oxidations occur at alternate carbon atoms on the
fatty acid with the eventual formation of a polyketo
acid. According to this scheme, reactions 1, 2, and 3
repeat themselves, i.e. the enzymes involved in the forma
tion of the delta keto group are the same as those catalyz
ing the formation of beta keto acid. The results from ex
periments with simultaneous adaptation would indicate that
these enzymes are involved in the formation of polyketo
acids from all the oxidizable acids (except acetic). Re
action 4a, the cleavage of polyketo acids to give two-
carbon fragments, may or may not be catalyzed by an adap
tive enzyme.
The question arises as to the significance of acetic
acid in the oxidation of higher acids by Serratia cells.
When glucose-grown Serratia cells are exposed to acetate,
they become adapted not only to acetate oxidation but also
to the oxidation of higher acids. It has been postulated
that higher acids are oxidized to beta keto acids; acetic
acid, being a two-carbon compound, cannot be metabolized
in this manner. This suggests that acetate is an inter
mediate in the oxidation of both odd and even-chain acids
and indicates that the reactions involved in acetate
116
formation are reversible. Rlttenberg and Bloch (1945)
have shown that In animal tissue higher even chain acids
are synthesized from acetate. The results would Indicate
that a similar mechanism occurs in Serrâtla. The synthet
ic process must be the reverse of the oxidation reaction.
Both schemes suggested Indicate that two-carbon
fragments arise from the cleavage of keto acids. The re
sults from experiments with DNP Indicated that acetate Is
not a direct Intermediate in the oxidation of higher acids.
It was suggested, however, that an "active" form of acetate
may be an Intermediate. It follows that this two-carbon
fragment, related to acetate, must also be active In the
reverse reaction. I.e. the synthesis of higher acids from
acetate.
The experimental data does not allow an unequivocal
choice between the two suggested mechanisms. The failure
to demonstrate oxidation of propionic, butyric, and valeric
acids by Serrâtla cells would appear to rule out beta oxi
dation In Its classical conception, since butyric acid
should be an Intermediate In the oxidation of higher even
chain acids. Since the higher acids are completely oxidiz
ed to carbon dioxide and water, these substances do not
accumulate. Three possibilities suggest themselves:
1. The Serratla cells are Impermeable to butyric,
propionic, and valeric;acids, but these substances can be
117
oxidized when formed in the cell as the result of the oxi
dation of higher acids.
2. These lower acids cannot be oxidized as such,
but during the oxidation of higher acids, "active" three,
four, and five-carbon compounds are formed, and these "ac
tive" substrates undergo oxidation.
3. Propionic, butyric, and valeric acids are per
meable to the cells but are not oxidized.
The first postulate. If correct, would mean that
beta oxidation In its classical conception Is the mode of
fatty acid oxidation in Serratla. In this case, cell free
enzyme preparations should as readily oxidize the C3, Ci|^,
and C5 acids as they do the higher acids. If "activated"
forms of C3, C4, and C5 compounds are Intermediates, then
the essential features of beta oxidation are retained, but
the chemical nature of the Intermediate compounds Is some
what different than generally supposed. The work with
DNP Indicated, It will be recalled, that neither caprylic
nor caprolc acids are direct Intermediates In the oxida
tion of caprate and similarly that heptylate Is not a
direct Intermediate In the oxidation of pelargonlc acid.
There Is good evidence, then, that If beta oxidation oc
curs the Intermediates are not the "normal" form of the
saturated acids, but direct evidence Is still lacking for
the occurrence of "active" forms of the C^, and acids
118
as Intermediates In the oxidation of higher acids. If
oxidation were to occur by the second scheme. I.e. mul
tiple alternate oxidation, then It would possible for high
er acids to be oxidized without the formation of propionic,
butyric, or valeric acid. Such a mechanism would lead to
the formation of odd chain dlcarboxyllc acids during the
oxidation of odd chain acids. It appears that these sub
stances might be oxidized through a terminal decarboxyla
tion of one of the acid groups with the formation of an
even chain monocarboxyllc acid.
It would seem that work with cell-free enzyme systems
would be the most promising approach to the problem of
determining the relationship of the Gg, Cj^, and acids
to the oxidation of higher acids. An understanding of
this relationship would lead to clarification of the basic
mechanism whereby saturated acids are oxidized by the
Serratla cells.
SUMMARY
The alpha-beta unsaturated, beta hydroxy, and beta
keto derivatives of caprlc acid were oxidized by glucose-
grown Serratla (Alphln) cells after lag periods. Cells
harvested from caprlc acld-mlneral salts medium showed no
lag periods In the oxidation of the three derivatives of
caprlc acid. Cells adapted to the oxidation of any one of
119
the three derivatives were also adapted to the oxidation
of the other two compounds and also to the oxidation of
caprlc, caprylic, and undecyllc acids.
The results suggested that a single enzyme system
catalyzes the oxidation of all the fatty acids known to
be oxidized by the Serratla cells. The preliminary steps
appear to Involve (1) alpha-beta dehydrogenation of saturat
ed acid with the formation of an alpha-beta unsaturated
fatty acid, (2) addition of water at the double bonds with
the production of a beta hydroxy fatty acid, and (3) alpha-
beta dehydrogenation of the hydroxy acid with the formation
of beta keto fatty acid. Two mechanisms for beta keto acid
oxidation were proposed. I.e. classical beta oxidation and
multiple alternate oxidation. The data are Insufficient to
allow an unequivocal choice between the two pathways.
The role of acetate In the metabolism of higher acids
was discussed. Although the data Indicate that acetate Is
not a direct Intermediate In the oxidation of higher acids, a
two-carbon fragment readily formed from acetate must be an
Intermediate In the oxidation of both odd and even chain
acids.
CHAPTER V I
THE RELATIONSHIP BETWEEN THE CITRIC ACID CYCLE AND THE
OXIDATION OF FATTY ACIDS BY SERRATIA MARCESCENS
Numerous investigators have shown a relationship
between the citric acid cycle and the oxidation of fatty
acids In animal tissues. Compounds In this cycle appar
ently have two distinct functions with respect to the oxida
tion of fatty acids: (1) Grafflln and Green (1948) and
Knox et al. (1948) have shown that the oxidation of a small
amount of a citric acid cycle compound Is an obligatory
"sparking" reaction In the oxidation of fatty acids by the
cyclophorase system prepared from rabbit kidney. All at
tempts to demonstrate fatty acid oxidation by the cyclo
phorase system without cooxldatlon of tricarboxylic acid
cycle compounds have failed. (2) When fatty acids are
oxidized to completion, the terminal oxidation of these
substances Involves a coupling reaction with oxalacetate
to form citric acid or a closely related compound. The con
densation Involves acetate or acetoacetate, substances pro
duced In the primary oxidation of fatty acids (Green, 1948),
To a lesser extent, higher beta-keto fatty acids may con
dense directly with oxalacetate to form a labile compound
which yields citric acid and a saturated fatty acid with
two less carbon atoms than were contained In the keto acid
121
participating In the condensation (Breusch, 1948). The
sparking reaction Initiating fatty acid oxidation and the
condensation reaction Involved In the terminal oxidation
of fatty acid derivatives are Independant processes, even
though tricarboxylic acid cycle compounds are active In
both reactions.
It seemed Important to determine the relationship
between the citric acid cycle and the oxidation of fatty
acids by Serratla:
1. Evidence from the previous studies Indicated that
the two, eight, nine, and ten-carbon acids are oxidized to
completion by these bacteria, and there were Indications
that the six and seven-carbon acids are also completely
oxidized. The question thus arises as to the mode of ter
minal oxidation of these compounds.
2. Previous work suggested the possibility that the
lag periods In the oxidation of fatty acids by glucose-
grown cells might be due to a time necessary for the accumu
lation of some compound necessary for the oxidation of fat
ty acids. It was postualted that such a compound might be
a member of the citric acid cycle.
EXPERIMENTAL
The methods employed were the same as described In
previous chapters. All suspensions used were adjusted to a
122
standard turbidity measured on the Klett-Summerson ap
paratus. Cells were harvested from glucose or caprlc
acid medium and treated In the manner described In prev
ious sections. One experiment was conducted In which cells
grown on a succinate medium were used. In this case the
mineral salts medium contained 0.01 M succinic acid as
the sole source of carbon; these cells were grown at room
temperature and harvested at 40 hours. Growth at 37®C.
was very slow when succinate was the sole source of carbon.
The oxidation of citric acid cycle compounds by
Serratla. Glucose-grown cells were tested for ability to
oxidize succinic, malic, citric, and oxalacetic acids. Ci
tric acid was not metabolized but had no Inhibitory effect
on endogenous respiration. Oxygen uptake In the presence
of the other three compounds was about 50 per cent of the
amount required for complete oxidation. No attempt was
made to block assimilation with DNP, but If one assumes
that oxidative assimilation Is as great as that found when
fatty acids are oxidized, then It follows that the oxida
tion of malate, succinate, and oxalacetate Is complete.
The effect of prior oxidation of succinate and
malate on the oxidation of caprlc acid. Experiments were
conducted In which glucose-grown cells were exposed to
malate and succinate In Warburg vessels. After added sub
strate had been oxidized, caprlc acid was poured from the
123
side-arm, and the oxygen uptake by the exposed cells was
measured. This prior oxidation of citric acid cycle
compounds had no effect on the pattern of fatty acid oxida
tion by glucose-grown cells; caprlc acid was oxidized after
a lag period of the ssune length observed with unexposed
cells. Cells grown on succinic acid medium also oxidized
caprate with an Initial lag period characteristic of
glucose-grown cells.
The effect of cooxldatlon of citric acid cycle com
pounds . Experiments were conducted In which Serratla
cells were tested for caprate oxidation In the presence
of citric acid cycle compounds. Caprlc acid was used In
0.0003 M concentration; In most Instances, the tricarboxyl
ic acid cycle compounds were added In a concentration
equlmolar with the fatty acid, but In certain experiments
higher concentrations were used. Oxygen uptake was also
measured In flasks containing caprlc acid and citric acid
cycle compounds separately. The results for malate are
plotted In Figure 12 and tabulated In Table VI. The data
for citrate, succinate, and oxalacetate are shown In
Table VII. The figures presented In the two tables repre
sent averages obtained from duplicate (and sometimes tri
plicate) flasks.
Figure 12 shows that caprlc acid was oxidized by
glucose-grown cells after a lag period of approximately
124
IÎO-.
no-
70 .
4 0 -
30 -
20-
10-
lOO 120 140 160 ISO to o *20 t40 8 0 6 0
TIME IN MINUTES
M/20 phosphate buffer, pH 7*0, 30*^0., 0.1 ml 10 per cent
KOH in center well. Flask concentrations: malate as
indicated in figure; caprate, 0.0003 M. Where both sub
strates were present, they were added simultaneously from
the side-arm.
FIGURE 12
THE OXIDATION OF CAPRATE IN THE PRESENCE
AND ABSENCE OF MALATE
125
TA B LE V I
CAPRIC ACID OXIDATION IN RELATION TO THE
SIMULTANEOUS OXIDATION OF MALATE
Substrate Cells
Total Oxygen uptake In mlcro
uters at the end of *
20
minutes
40
minutes
0.0003 M. malate (l) glucose
0.0003 M. caprate (2) grown
Total (1) (2)
malate /.caprate
0.0015 M. malate (l) glucose
0.0003 M. caprate (2) grown
Total (1) (2)
malate / caprate
0.0015 M. malate (l) caprate
0.0003 M. caprate (2) grown
Total (1) (2)
malate / caprate
- 60
minutes
6 11 l6
2
19 37
8 30
53
15
28 44
10 34
59
2
19 37
12
53
96
18 48 74
16
51 77
24 61
83
40 112 i6o
29 73
114
Experiments conducted in air atmosphere with 0.1 ml 10 per
cent KOH in center well.
Temperature 30^C.
In flasks containing malate / caprate, both substrates were
added simultaneously from the side-arm afjbei^the usual 15
minute equilibration period. '
* Values represent averages from duplicate, and in some
cases triplicate, flasks.
126
TABLE V I I
CAPRIC ACID OXIDATION IN RELATION TO THE SIMULTANEOUS
OXIDATION OF OXALACETATE, SUCCINATE, AND
CITRATE BY UNADAPTED CELLS
Substrate
Total oxygen uptake In microliters
at the end of *
20 minutes 40 minutes 60 minutes
0.0003 M. oxalacetate
0.0003 M. caprate (2)
Total (1) (2)
oxalacetate / caprate
(1)
0 .0 0 0 3 M.
0.0003 M.
Total (1)
succinate
0 .0 0 0 3 M.
0 .0 0 0 3 M.
Total (1)
citrate /
succinate (1)
caprate (2)
(2)
/ caprate
citrate
caprate (
(2)
caprate
8 16 21
1 16 42
16
32
63
33
52
0 11 12
0 24 52
0
35
64
10
31
56
0 0
3
0 24 52
0 24
55
8 22 47
Experiments conducted in air atmosphere with 10 per cent KOH
to absorb carbon dioxide.
Temperature 30®C.
In flasks containing two substrates, both were added simul
taneously from the side-arm after the usual 15 minute
equilibration period.
* Values represent averages from duplicate, and in some
cases triplicate flasks.
127
30 minutes. When malate and caprate were oxidized simul
taneously, the lag was eliminated; oxygen uptake commenced
immediately at maximum rate. The data in Table VI shows
that this effect was not due entirely to the oxidation of
malate. During the first 20 minutes, the uptake in flasks
containing both malate and caprate was greater than the sum
of the uptakes when malate and caprate were oxidized separate
ly. At the end of 40 minutes, the sum of the separate up
takes was about the same as the total uptake in flasks con
taining both compounds. At 60 minutes, the effect noted
in the first 20 minutes of the experiment was reversed,
i.e. the amount of oxygen uptake in flasks containing both
malate and succinate was less than the sum of the uptakes
when the two compounds were oxidized separately. Refer
ence to Figure 12 shows that the oxidation of 0.0003 M
malate is complete before the curve for 0.0003 M caprate
has reached the autorespiratory level. An experiment was
conducted in which 0.0015 M malate was used. Under these
conditions malate oxidation was not complete until caprate
was nearly completely oxidized. Table VI shows that in
creasing the malate concentration had no effect on the
initial stimulation of fatty acid oxidation. As with the
lower malic acid concentration, initial uptake (first 20
minutes) in flasks where both malate and caprate were being
oxidized was greater than the sum of the separate oxygen
128
uptakes. At 4o and 60 minutes the reverse effect was
noted.
When similar experiments were conducted with
caprate-grown cells, no initial stimulatory effect due
to simultaneous oxidation of malate in the presence of
caprate was noted. Table VII shows that malate had a
depressing effect on the oxidation of*fatty acid; this
effect was apparent throughout the course of the experi
ment. Quantitatively the inhibitory effect was much great
er with caprate-grown cells than with unadapted organisms.
During the first 20 minutes the oxygen uptake in flasks
containing both substrates was approximately three-fourths
of the sum of the separate uptakes; thereafter the simul
taneous uptake was only about two-thirds of the sum of the
separate oxygen uptakes. With glucose-grown cells there
was a definite depressing effect on caprate oxidation after
20 minutes, but the magnitude of this effect was not nearly
as great as that noted with adapted cells.
Table VII shows that succinate, oxalacetate, and
citrate produced effects on unadapted cells similar to
those noted with malate. The presence of these compounds
caused an initial stimulation of caprate oxidation, but
this effect was reversed by the end of 60 minutes. Of
particular interest is the fact that citrate, a substance
not oxidized by Serratia cells, reacted in a manner
129
similar to malate, succinate, and oxalacetate. Whether
citric acid is actually utilized in the presence of
caprate has not been determined.
DISCUSSION
The initial stimulation of caprate oxidation by
citric acid cycle compounds appears to be a general ef
fect with glucose-grown cells. It was not observed in
cells harvested from capric acid-mineral salts medium.
Since glucose-grown cells show a negligible scnount of
oxygen uptake in the presence of caprate during the first
twenty minutes, it appears unlikely that the stimulatory
effect noted involves any condensation reaction between
caprate derivatives and citric acid cycle compounds. The
effect appears to be analagous to the sparking effect of
citric acid cycle compounds on fatty acid oxidation by
animal tissues. As previously indicated, a cooxidation
of a citric acid cycle compound is an obligatory priming
reaction in fatty acid oxidation by the cyclophorase
system. If a similar reaction occurs in Serratia cells,
it is probable that the glucose-grown cells have an endo
genous supply of citric acid cycle compounds, since aerobic
oxidation of carbohydrates involves the tricarboxylic acid
cycle. If the supply were small, however, then an exogen
ous supply of these compounds might be expected to stimulate
130
this preliminary oxidation. Caprate-grown cells may al
ready be primed with respect to fatty acid oxidation,
and hence the initial cooxidation of citric acid cycle
substrate may be unnecessary. This would account for the
failure of these compounds to stimulate oxidation of
caprate by adapted cells.
The inhibitory effect warrants further investigation.
The complete oxidation of fatty acids involves two distinct
reaction systems, i.e. the production of fatty acid inter
mediates and the oxidation of these intermediates, perhaps
through the tricarboxylic acid cycle. Citric acid cycle
compounds should not decrease the rate of terminal oxida
tion if the citric acid cycle is involved. But it is
possible that during the early stages of fatty acid oxida
tion the enzymes catalyzing the oxidation of citric acid
cycle compounds can compete with those involved in the
primary stages of fatty acid oxidation. For example the
competition may be for a limited supply of hydrogen
carriers.
SUMMARY
Previous exposure of glucose-grown Serratia (Alphin)
cells to tricarboxylic acid cycle compounds failed to
eliminate the lag period in the oxidation of caprlc acid.
When citric acid cycle compounds and capric acid were.
131
oxidized simultaneously, there was a stimulatory effect
during the first 20 minutes of caprate oxidation and a
depressing effect thereafter. The citric acid cycle com
pounds has a depressing effect on caprate oxidation through
out the course of experiments with caprate-grown cells.
The stimulatory effect may be analagous to the sparking
effect of tricarboxylic acid cycle compounds on fatty
acid oxidation in animal tissue. The mechanism of the
inhibitory action is in doubt.
CHAPTER V I I
ATTEMPTS TO OBTAIN ENZYMATICALLY ACTIVE
PREPARATIONS PROM SERRATIA
At best the technique of simultaneous adaptation is
an indirect approach to the study of metabolic pathways.
It depends, of course, upon the use of organisms showing
adaptive attack on the substrates oxidized. In the pre
vious investigations the question arose as to what con
stitutes proof that attack on a particular compound is
through the agency of adaptive enzymes. Previously no
rigid criteria have been established, but it has usually
been assumed that if a substance is oxidized by micro
organisms only after a lag period, then this lag period
represents a time during which the adaptive enzymes are
being formed in the cell. Further, if growth on a medium
containing the compound as a sole source of carbon produces
cells which show no lag period in oxidation, then the con
clusion that attack is adaptive is strengthened. As an
indirect approach, inhibition of oxidation by cell poisons,
such as sodium azide and 2:4-dinitrophenol, gives further
evidence that adaptive enzymes are involved, provided the
same concentrations of poisons produce no over-all inhibi
tion when appropriately adapted cells are exposed to the
carbon substrate.
133
In the present studies it was found that Serratia
strains, grown on glucose-mineral salts medium, attack
odd and even chain acids only after a lag period of fifteen
to forty minutes. Exposure of buffer suspensions to small
amounts of these acids or growth on media containing them
as the sole source of carbon produced cells which oxidiz
ed the substrates without an appreciable lag period. Quite
unexpectedly, however, cells which were adapted to any of
the fatty acids from Cg to (with the exception of C^,
C4, and which were not oxidized under any conditions)
became simultaneously adapted to all the other acids.
Several explanations for this non-specificity were suggest
ed, the most likely being that a single enzyme system is
responsible for primary attack on all the fatty acids test
ed. Work with cell poisons indicated that attack on the
fatty acids from Cg to did not involve identical enzy
matic reactions in each case. The oxidation of acetic and
caproic acids was completely blocked by DNP at pH 7*0;
adaptation to the oxidation of heptylate, caprylate,
pelargonate, undecylate, and tridecylate was inhibited.
Capric, 1auric, and myristic acids, on the other hand,
were oxidized by both adapted and unadapted cells under
the same conditions which produced the aforementioned in
hibitory effects on the oxidation of other acids.
As other possible explanations it was suggested that
134
the lag period might be due to a slow permeability of the
cells to the acids or that perhaps the lag period repre
sents a time during which some product necessary for oxida
tion at maximum rate was accumulating in the cell.
It became apparent that the most direct approach to
the problem would be to attempt isolation of the enzyme
systems involved in fatty acid oxidation and to determine
the conditions under which these enzymes occur in the
cell. If attack on the fatty acids were to involve adap
tive enzymes, then it would be expected that only cells
appropriately adapted to the fatty acids would contain the
enzymes. If, on the other hand, some permeability effect
were involved (and oxidation were through constitutive
attack) then with isolated enzyme systems the permeability
effect should be eliminated; Serratia cells should show
the enzymes regardless of their previous history.
Several general methods are available for prepara
tion of bacterial enzyme systems. The most widely used
are grinding, autolysis, lysis with added agents, and
freezing and thawing. Attempts were made to obtain enzyme
preparations by each of these methods.
EXPERIMENTAL
Dry cell preparations. Sleeper, Tsuchida, and
Stanier (1950) have recently perfected a method for
135
preparation of enzymatically active dried cells. In these
preparations the vast majority of the organisms are non-vi
able, and permeability effects play no role. Working with
organisms attacking substrates through adaptive enzymes,
they found that dried cells prepared by their methods oxid
ized the adaptively attacked substrates only when the liv
ing cells from which they were prepared had been adapted
to these substrates. By appropriate procedures, cell free
enzyme systems were isolated from the enzymatically active
dried cells.
Cells of Serratia marcescens (Alphin) were grown in
quantity in large prescription bottles containing capric
acid-mineral salts medium. After forty hours incubation
at 37^C., the cells were harvested by washing down the
agar slants with phosphate buffer at pH 7*0. After wash
ing twice with buffer, the cells were treated according
to the method of Sleeper et al.: The paste of packed
cells was spread out in a beaker which was placed in a
vacuum dessicator. The dessicator was evacuated with an
oil seal pump. The inlet to the dessicator was clamped
off, and the cells were allowed to dry at room tempera
ture over calcium chloride. Drying was complete in seven
hours. A glassy residue was obtained and was ground to a
fine powder with mortar and pestle. The powder was sus
pended in phosphate buffer, each Warburg vessel receiving
1 .0 ml of buffer containing 10 mg. of dried preparation.
136
After 15 minutes of equilibration, 0.6 jim of fatty acid in
1.0 ml of buffer was poured from the side-arm. In most
cases capric acid was used, but with each preparation tests
were made with acetic and pelargonlc acids also. Repeated
attempts to obtain fatty acid oxidation with such prepara
tions failed. A high autorespiration was noted, but this
decreased after storage four or five days at ice box
temperature. The activity of the preparations did not im
prove with age. Attempts were made to "spark" oxidation
of fatty acids by addition of succinate and malate. Suc
cinate was oxidized by the preparations at a slow rate
which did not increase in the presence of fatty acids;
malate was not attacked. The preparations attacked glucose
at a high rate; no data were obtained on the extent of
glucose oxidation. Considering the possibility that hydrogen
acceptors might be destroyed in the drying process, methy
lene blue was added as an auxiliary acceptor; this had no
effect. The methylene blue did not inhibit glucose oxida
tion, even in high concentrations.
With the hope of preventing enzyme inactivation,
cells were frozen in an amyl alcohol-dry ice bath before
the drying process was begun. Cells obtained after dry
ing by this method showed the same reactions as described
above.
A final attempt to obtain active dried cells was
137
made by freezing the cell paste in an acetone-dry ice bath,
.thus giving a thin shell in the bottom of a 500 ml flask.
This flask was then immersed in an acetone-dry ice bath
and evacuated with an oil seal pump. The flask was kept
under constant vacuum in the acetone bath until the cells
were completely dry. The process took approximately six
hours. A fluffy powder was obtained, not a glassy residue
as in the previous preparations. This powder gave the
same reactions as the preparations obtained by drying at
room temperature.
Acetone dried preparations. Washed cells prepared
in the manner described above were dried with acetone ac
cording to the methods given by Umbreit, Burris, and
Stauffer (1949)* The bacterial paste was added dropwise
to about ten times its volume of ice cold acetone, and
the mixture was stirred rapidly. The cells flocculated
and were allowed to settle from solution (about ten min
utes). The supernatant was decanted and the sediment
filtered with gentle suction. The residue on the filter
paper was spread out and dried under a slight vacuum for
about two hours. The dried preparation was then ground to
a fine powder, and tests were conducted in the manner
described above, using 10 mg. of dried cells for each
flask. The preparation thus obtained was devoid of
activity on glucose, fatty acids, or succinate. Addition
1 3 8
of methylene blue as a hydrogen acceptor was without ef
fect. The cells had no autorespiration.
Following the suggestion of Stanier (1950), a batch
of cells was treated in the manner described above, but
a final wash with ether was made in order to aid in re
moval of last traces of acetone. The preparation showed
no activity.
Freezing and thawing. Avery and Neill (1924) and
Koepsell and Johnson (1942) have successfully obtained
cell free preparations of bacterial enzymes by rupturing
the cell with alternate freezing and thawing.
Cells were grown in quantity on capric acid medium
and washed by centrifugation from buffer solution. The
washed cells were suspended in an equal volume of buffer
at pH 7.0 and immersed in an acetone-dry ice bath. The
suspension froze in a minute or two and was removed from
the bath and thawed at room temperature. As soon as the
suspension became fluid, it was once again placed in the
dry ice bath. This procedure was repeated eight times.
The cell debris was then sedimented three times by centri
fugation, the supernatant fluid being retained after each
sedimentation. The clear fluid thus obtained was used
for activity tests. Ten ml. of fluid were obtained from
one gram of cells (dry weight).
For activity tests, one ml. of fluid was used in
139
each Warburg vessel. The side-arms contained one ml. of
carbon substrate, as described previously. The prepara
tion obtained showed no activity against fatty acids, glucose,
or succinate. Methylene blue, added as a hydrogen acceptor,
did not activate oxidation.
Autolysis. Stephenson (1928) prepared cell free
dehydrogenases from Escherichia colin by suspending washed
cells in phosphate buffer and incubating at 37°C. Under
these conditions, the cells autolyzed and the enzymes were
set free into the solution. Maximum activity of the super
natant fluid was attained after five or six days.
The technique of Stephenson was followed using washed
capric-grown Serratia. A heavy suspension was stored in a
stoppered centrifuge tube at 37^C. At one, three, and
seven days the suspension was centrifuged and the super
natant tested for ability to oxidize fatty acids. No
activity against fatty acids or against glucose was detect
ed in any of the three preparations tested.
DISCUSSION
All attempts to obtain fatty acid oxidation by dry
cell and cell free preparations of Serratia marcescens
(Alphin) were without success. While several different
approaches were applied, it must be admitted that no one
l4o
technique was investigated sufficiently to rule it out
as a possible method of obtaining active enzyme prepara
tions .
Monoz and Leloir (1943) and later Lehninger (1945)
succeeded in separating fatty acid oxidase activity from
tissue homogenates by centrifugation. Grafflln and Green
(1948) studied fatty acid oxidation by cyclophorase pre
parations obtained from liver and kidney. The system of
Leloir and Munoz required magnesium ions, phosphate ions,
adenylic acid or adenosine triphosphate, and cytochrome c.
Lehninger*s system required the same components and in
addition, catalytic amounts of malic or oxalacetate. The
preparations of Grafflln and Green had the same require
ments, but it was found that oxidation could occur only
if a small amount of cyclophorase substrate was first
oxidized to initiate the reaction. There have been no
reports of successful isolation of cell free preparations
from bacteria capable of fatty acid oxidation. Grey
(1 9 4 9) has reported unsuccessful attempts to obtain fatty
acid oxidation in cell free preparations obtained from
the tubercle bacillus. The addition of adenosine tri
phosphate, cytochrome c, and tricarboxylic acid cycle
intermediates (separately and in combination) failed to
activate oxidation.
In the present studies all attempts to "spark"
I4l
fatty acid oxidation by the enzyme preparations with
tricarboxylic acid intermediates failed. Any require
ments of the oxidase system for phosphate ions were sup
plied by the phosphate buffer. Neither adenosine tri
phosphate nor magnesium ions were added to the prepara
tions. With the preparations obtained by freezing and
thawing and by lysis, it is conceivable that failure to
add these components accounts for the inactivity of these
systems. Both components should have been present in
adequate amounts in dry cell preparations.
While the attempts to obtain active enzyme pre
parations were far from exhaustive, the results indicate
that the enzymes systems involved are extremely labile.
The technique of Stanier and his co-workers was most
completely investigated. Using this method of prepara
tion, enzymes involved in glucose dissimilation were still
active after the drying process. Even when the drying was
carried out from the frozen state (actually lyophilization),
the fatty acid oxidation system was inactivated. This sug
gested that the system is sensitive to drying per se, and
perhaps no technique involving drying of cells can be ex
pected to yield fruitful results. The negative results
obtained with acetone drying techniques can probably be
attributed to a general inadequacy of the technique em
ployed, since the preparations thus prepared failed to
142
show activity against any of the compounds tested. The
same is probably true of the autolyzed and the frozen and
thawed preparations. It may be mentioned that it was not
possible to carry out these procedures entirely in the cold.
It is possible that had all procedures been carried out
at a near freezing temperature, then the freeze-thaw and
acetone techniques might have yielded more positive re
sults. The work of Singer and Barron (1945) suggests that
inactivation of -SH groups may be involved. They have
shown that the oxidation of stearate, oleate, acetate, and
beta hydroxy acids by Corynebacterium creatinovorans are
all blocked by -SH inhibitors. Furthermore, the addition
of glutathione and cysteine restored lost oxidative ability.
As a possible means of attack in future work one might ex
pect that addition of glutathione and cysteine might prevent
inactivation of fatty acid oxidase.
SUMMARY
Attempts were made to isolate active enzyme prepara
tions from Serrâtia marcescens (Alphin) by application of
dry cell techniques, freezing qnd thawing, and autolysis.
All attempts failed to produce positive results, even in
the presence of tricarboxylic acid intermediates and
methylene blue as a hydrogen acceptor.
CHAPTER VIII
RESUME
An attempt was made to determine the mechanism of
fatty acid oxidation in bacteria, using the technique of
simultaneous adaptation. Since this method depends upon
analysis of adaptive behavior, use of the technique
necessitated the isolation of bacteria attacking fatty
acids through adaptive enzymes. Twenty-five different
strains of bacteria were examined for the ability to oxi
dize pelargonic and capric acids, using manometric methods.
Some of the organisms were isolated from the soil, using
the enrichment culture technique with stearic acid-mineral
salts medium; others were stock strains of bacteria, re
cently isolated from natural habitats. All organisms test
ed oxidized the nine and ten-carbon fatty acids. All or
ganisms except three showed immediate oxygen uptake at
nearly maximum rate when exposed to these acid substrates.
This pattern was suggestive of attack through constitutive
enzymes and hence eliminated these strains from studies in
volving the use of the technique of simultaneous adaptation.
Two strains of Serratia marcescens and one of Bacillus brevis
oxidized capric and pelargonic acids only after lag periods
of from twenty to forty minutes. Since this type of oxida
tive pattern has previously been the major criterion for
the conclusion that attack is catalyzed by adaptive enzymes.
144
these three organisms appeared to be promising tools for
the study of the mechanism of fatty acid oxidation by
simultaneous adaptation. The two Serratia strains were
selected for further study.
The Serratia strains oxidized all the fatty acids
from Cg to with the exception of and
acids. In each case oxidation was preceded by a lag
period similar to that noted in caprate oxidation. With
the exception of acetate and heptylate, all oxidizable
compounds supported growth of the Serratia organisms in
a mineral medium with fatty acid as the sole source of
carbon. Growth on a medium containing a fatty acid as
the sole source of carbon resulted in organisms which
showed no lag period in the oxidation of that acid. Allow
ing buffer suspensions of Serratia (Alphin) cells to oxidize
a small amount of fatty acid gave the same effect. Cells
adapted to any of the oxidizable acids showed either no
lag period or a shortened lag period in the oxidation of
all the other acids utilized by the Alphin strain. Hence,
adaptation to the oxidation of a particular acid simultan
eously adapts cells to compounds which are logical inter
mediates in its oxidation. The process also causes adapta
tion to substances which could not possibly be intermediates.
Three possible explanations were given for the lag period
noted in unadapted cells (glucose-grown):
145
1. The lag is due to slow permeability of the
cells to the fatty acids, and exposure to these compounds
in some way alters permeability of the cell membrane to
fatty acids.
2. The lag represents a time during which some
intermediate is accumulating in the cell, the intermedi
ate being a product of the oxidation of fatty acids and
also a substance necessary for metabolism at maximum rate.
3. A single enzyme system is responsible for attack
on all oxidizable fatty acids, and adaptation to the oxida
tion of any acid results in simultaneous adaptation to all
other acids.
Later work indicated that the third explanation
was most nearly correct, although definite proof must
await work with cell-free enzyme preparations.
Dinitrophenol inhibited oxidative assimilation in
Serratia marcescens (Alphin). At pH 7*0 dinitrophenol
inhibited adaptation to the oxidation of heptylate,
caprylate, pelargonate, undecylate, and tridecylate; ap
propriately adapted cells oxidized these substrates under
the same conditions. Neither acetate, caproate, nor
heptylate were oxidized at pH 7*0 in the presence of DNP,
but at pH 8.0 acetate was oxidized by both adapted and
unadapted cells. Under none of the experimental conditions
imposed was caproic acid oxidized in the presence of DNP.
146
The ten, twelve, and fourteen-carbon acids were oxidized
by both adapted and unadapted cells in the presence of
this phenolic compound, but work with capric acid indicat
ed that even though the acid was metabolized by unadapted
cells, DNP prevented these organisms from becoming adapted
to the oxidation of other acids. After oxidation of capric
acid in the presence of DNP, the glucose-grown cells still
showed a lag period in the oxidation of capric acid and
in the metabolism of all the other oxidizable acids. This
suggested that adaptation (the elimination of the lag
period) involves assimilation of substrate. It was con
cluded that the enzymes involved in fatty acid oxidation
by Serratia cells are adaptive, even though qualitative
differences exist between the properties of these catalysts
and the "classical" type of adaptive enzyme.
Quantitative data indicated that the acids are
completely oxidized to carbon dioxide and water. Only
unadapted cells oxidizing capric acid in the presence of
DNP showed the theoretical oxygen consumption for complete
oxidation. Auxiliary data indicated, however, that failure
to show the theoretical oxygen consumption was due to
incomplete oxidation of fatty acid substrates. The data
indicated that higher DNP concentrations are necessary to
inhibit oxidative assimilation in adapted cells than are
required in unadapted organisms.
147
The alpha-beta unsaturated, beta keto, and beta
hydroxy derivatives of capric acid were attacked by
glucose-grown cells only after a lag period. Exposure
to any one of these derivatives resulted in the adapta
tion of cells to the oxidation of the other two compounds
and also to the oxidation of capric, caprylic, and un-
decylic acids. It was concluded that the alpha-beta un
saturated, beta hydroxy, and beta keto derivatives of
caprate are intermediates in the oxidation of capric acid.
These findings, together with the fact that adaptation of
Serratia cells to any of the oxidizable acids results in
simultaneous adaptation to the oxidation of all the other
acids, suggested that a single enzyme system catalyzes
the oxidation of all the fatty acids (except acetic) which
are oxidized by Serratia (Alphin) cells. It was proposed
(1) that fatty acids are converted to alpha-beta unsaturat
ed acids with the removal of two hydrogen atoms, (2) that
the unsaturated acids add water and are changed to beta
hydroxy acids, and (3) that the hydroxy acids are oxidized
to beta keto acids with the removal of two hydrogen atoms.
Two possible mechanisms for keto acid oxidation
were proposed: (1) cleavage of the molecule at the beta
position with the formation of a two-carbon fragment and
a fatty acid with two less carbon atoms; (2) secondary
oxidation at the delta carbon atom and on each alternate
148
carbon atom with the formation of a polyketo acid which
undergoes cleavage to form a series of two carbon frag
ments. In the first instance, oxidation of the lower
acid is catalyzed by the ssime enzyme system that was
responsible for oxidation of the higher acid. Alternative
ly, if polyketo acids are formed, reactions leading to the
formation of ketone groups are catalyzed by the same enzymes
as were involved in the formation of the beta keto acid.
The experimental data do not allow a choice between
the two mechanisms. The first scheme, cleavage of the
beta keto acid with the formation of an acid with two less
carbon atoms, is analogous to classical beta oxidation.
The failure of Serratia cells to oxidize propionic, butyric,
and valeric acids indicated that these substances are not
intermediates in the oxidation of higher acids. If classic
al beta oxidation were to occur, the and acids should
be formed in the oxidation of higher odd-chain acids and
the C4 acid should be formed during oxidation of higher
even-chain acids. It was suggested that failure to oxidize
these compounds might be due to impermeability of the cells
to the acids; alternatively, "active" three, four, and five
carbon compounds related to the C3, C4, and acids might
be intermediates. Work with DNP indicated that the "normal"
forms of caprylic and caproic acids are not direct inter
mediates in the oxidation of caprate and similarly that
149
the "normal" form of heptylic acid is not an intermediate
in the oxidation of pelargonic acid. Hence, if beta oxida
tion occurs, the intermediates are chemically different than
is usually supposed. If, however, three, four, and five
carbon compounds are not intermediates, then some mechanism
other than beta oxidation must occur. If polyketo acids were
to be formed during oxidation, then it would be possible for
higher fatty acids to be oxidized without the formation of
C^, C4, and compounds. Work with cell-free enzyme systems
should clarify the position of propionic, butyric, and valer
ic acids with respect to the oxidation of higher acids.
Work with DNP indicated that acetic acid is not a
direct intermediate in the oxidation of higher acids. Yet,
adaptation to acetate results in simultaneous adaptation of
cells to the oxidation of higher acids. It was suggested
that an "active" form of acetate may be an intermediate
in the oxidation of higher acids. The simultaneous adapta
tion of acetate-exposed cells to the oxidation of higher
acids suggested that the formation of two-carbon fragments
from higher acids may be a reversible process and hence
the,oxidation process may be the reverse of the mechanism
for synthesis of higher acids.
Prior oxidation of citric acid cycle compounds by
glucose-grown Serratia cells failed to eliminate the lag
period when these organisms were exposed to capric acid.
150
When glucose-grown cells were allowed to oxidize malic,
succinic, oxalacetic, or citric acids simultaneously with
caprate, the oxygen uptake was greater than the sum of the
uptake on caprate plus the uptake on the citric acid cycle
compound when determined separately. The stimulatory ef
fect of citric acid cycle compounds was evident only dur
ing the first twenty minutes. Thereafter the presence of
these substances depressed caprate oxidation. In caprate-
grown cells, citric acid cycle compounds had a depressing
effect throughout the course of the experiment. It was
concluded that the stimulatory effect is analogous to the
"sparking effect" of citric acid cycle compounds in fatty
acid oxidation by animal tissues. It is probable that in
caprate-grown cells fatty acid oxidation is already primed,
and hence only the depressing effect is noted. Insuffi
cient experimental data is available to warrant interpreta
tion of the nature of the inhibitory action of citric
acid cycle compounds.
All attempts to obtain enzymatically active cell-
free preparations from Serratia (Alphin) cells failed.
The techniques of drying vacuo, acetone drying, lysis
with distilled water, and lysis by freezing and thawing
were investigated.
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Asset Metadata

Creator Silliker, John Harold (author)

Core Title Studies on the aerobic oxidation of fatty acids by bacteria

School Graduate School

Degree Doctor of Philosophy

Degree Program Bacteriology

Degree Conferral Date 1950-09

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biological sciences,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c30-261686

Unique identifier UC11228151

Identifier DP23891.pdf (filename),usctheses-c30-261686 (legacy record id)

Legacy Identifier DP23891.pdf

Dmrecord 261686

Document Type Dissertation

Rights Silliker, John Harold

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA