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ADENINE DINUCL‘EOTIDE OXIDASES FROM VEGETATWE
CELLS AND SPORES 0F QLOSTRIDIQM OIQL HEM 6:2 A

I Thesis {or {he Defife‘e éfi Rh D. "
MICHIGAN STATE UNIVERSITY
‘ John Henry Green
1963 ’

THESIS

This is to certify that the

thesis entitled

Comparison of Soluble Reduced Nicotinamide
Adenine Dinucleotide Oxidases from vegetative

Cells and Spores of Clostridium botulinum
62-A

presented by

John Henry Green

has been accepted towards fulfillment
of the requirements for

Ph.D. degreem Microbiology and

PfiBlic Health

L)’ ’40:sz 0/] 44/6”

Major professoyfl

Date 14 November 1963

0-169

 

LIBRARY

Michigan State
University

 

ABSTRACT

COMPARISON OF SOLUBLE REDUCED NICOTINAMIDE ADENINE
DINUCLEOTIDE OXIDASES FROM VEGETATIVE CELLS
AND SPORES OF CLOSTRIDIUM BOTULINUM 62-h

by John Henry Green

A comparison has been made of the properties of a
vegetative cell and a heat stable spore reduced nicotinamide
adenine dinucleotide (NADH) oxidase from a strain of
Clostridium botulinum 62-A. Both NADH oxidases were soluble
and could be separated from their respective particulate
diaphorases by centrifugation at 32,000 g for 6-8 hours.

Forty and eighty-fold purifications respectively were obtained
for vegetative cell and spore NADH oxidases.

Comparisons were based on heat inactivations,
solubility in ammonium sulfate solutions, substrate (NADH)
and inhibitor (atabrine) kinetics, relative activity in
buffers between pH 5.2 to 9.4I cofactor requirements,
elution from an anion exchange column, estimations of
molecular weights derived from gel filtrations, immunological
responses, and serological cross reactions between heterologous
antigens and antibodies. Differences were found in all of

the above properties and are given below.

John Henry Green

The spore NADH oxidase was extremely heat stable and
survived 65 C for 2 hours with little or no loss while the
vegetative oxidase lost 90% activity in 3 minutes at 65 C.
The spore oxidase precipitated in a narrow band at 55%
ammonium sulfate saturation while the vegetative oxidase
precipitated over a wide band showing a peak at 70%
saturation.

The spore oxidase exhibited less affinity for the
substrate than the corresponding vegetative cell enzyme.
Its Michaelis constant (Km) wasB times greater than the
vegetative oxidase Km of 7.9 x 10’6M The kinetics also
showed that atabrine is‘a non-competitive inhibitor for
both enzymes and was less inhibitory to the spore oxidase.
The pH optimum of both enzymes was approximately 7.5 but
the spore oxidase displayed more activity in the lower pHs
while the vegetative oxidase was more active in the pH range
above optimum.

A difference was shown in their ionic charge when
both NADH oxidases were mixed together and eluted from a
DEAE cellulose column by a linear NaCl gradient. They
appeared separately in the effluent. the spore enzyme being
eluted first.

Several attempts to remove the flavin by acid—

ammonium sulfate treatment revealed that this cofactor was

John Henry Green

tightly bound to the spore oxidase. The flavin was readily
removed from the vegetative oxidase and flavin adenine
dinucleotide (FAD). not flavin mononucleotide (FMN),
reactivated the enzyme.

The spore oxidase was excluded from the imbibed
volumes of both Sephadex G-100 and G-200 gel particles and
was estimated to have a molecular weight of 200,000 or greater.
The vegetative oxidase was included in both and was therefore
100.000 molecular weight or less. The spore oxidase elicited
higher levels of antibodies in rabbits than did the
vegetative cell oxidase. Neither NADH oxidase would cross
react with its heterologous antibody in a precipitation
reaction.

The above differences in characteristics studied
showed a distinct difference between the vegetative cell and
spore NADH oxidases.

The evidence excluded the possibility of the
conversion of a single vegetative cell enzyme molecule into
a heat stable spore NADH oxidase. Stable spore proteins may
originate by the polymerization of vegetative proteins or

g§_novo protein synthesis during sporulation.

COMPARISON OF SOLUBLE REDUCED NICOTINAMIDE ADENINE
DINUCLEOTIDE OXIDASES FROM VEGETATIVE CELLS

AND SPORES OF CLOSTRIDIUM BOTULINUM 62-A

 

BY

John Henry Green

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

DOCTOR OF PHILOSOPHY

'

Department of Microbiology and Public Health

1963

(4;)
O 9x»
( N1
~9

$3

ACKNOWLEDGMENTS

I wish at this time to express my deepest thanks
to Dr. H. L. Sadoff for his interest and generous counsel
toward me during my course of studies at this institution.
I would like to extend my thanks to Dr. R. N. Costilow for

his helpful and wise counsel.

ii

TABLE OF CONTENTS

Page
INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . - . . ... . . . , . . . . . . 5
Sporogenesis: Biochemical and Morphological

Considerations 5

Spore Protein Synthesis ll
Antigenic Studies of Spores 15
MATERIALS AND METHODS . . . . . . . . . . . . . . . . . 19
Production of Cells and Spores l9
Assay Procedures ‘ 21
Purification Procedures 23

Gel Filtration 26
Procedures for Enzyme Characteristics 28
Immunization and Serological Procedures 29
EXPERIMENTAL RESULTS . . . . . . . . . . . . . . . 32
Purification of Vegetative and Spore NADH Oxidase 32

Gel Filtrations 39
Separation of Mixed Vegetative and Spore Enzymes 45
Determination of Enzyme Characteristics 47
Serological Studies 58
DISCUSSION . . . . . . . . . . . . . . . . . . . . . 62
CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . 68
REFERENCES . . . . . . . . . . . . . . . . . . . . . . 71

iii

LIST OF TABLES

 

 

 

 

Table Page

1. Diaphorase activity in spore and vegetative

cell extracts before and after

centrifugation . . . . . . . . . . . . . . 33
2. Attempt to reduce NAD with spore enzyme . . . 35
3. Purification procedures and typical results

for vegetative cell NAD oxidase from

Q, botulinum . . . . . . . . . . . . . . . 40
4. Purification procedures and typical results.

for spore NADH oxidase from g. botulinum . 41
5. RD values determined from gel filtrations for

vegetative cell and spore NADH oxidase

from Q. botulinum . . . . . . . . . . . . . 44
6. Enzyme kinetic constants determined for

vegetative cell and spore NADH oxidase

from g, botulinum . . . . . . . . . . . . . 53
7. Flavin activation of vegetative cell

NADH-oxidase . . . . . . . . . . . . . . . 57

iv

Figure

LIST OF FIGURES
Page

Heat inactivation determinations of vegetative
cell and spore NADH oxidase . . . . . . . . 34

Vegetative and spore precipitation profiles of
NADH oxidase activity and protein in various
saturations of ammonium sulfate at 0 C . . 37

Vegetative and spore precipitation profiles of
NADH oxidase activity and protein in various
saturations of ammonium sulfate at
room temperature and a constant pH of
6.5 I O I O .. O O O O O O n. O O O O O O O O 38

Vegetative and spore elution profiles of NADH
oxidase activity and protein from
respective Sephadex G—100 columns . . . . . 42

Vegetative and spore elution profiles of
NADH oxidase activity and protein from
respective Sephadex G-200 columns . . . . . 43

Normal and heat resistant elution profiles
of NADH oxidase activity and protein.
also serological reaction profile. from
region of peak A, Figure 4 . . . . . . . . 46

Profiles for NADH oxidase activity and
protein eluted from a Sephadex G-100
column loaded with a mixture of
vegetative and spore enzyme extracts . . . 48

Profiles for NADH oxidase activity and
protein eluted from a Sephadex G-200
column loaded with a mixture of
vegetative and spore enzyme extracts . . . 49

Profiles for NADH oxidase activity eluted
from a DEAE cellulose column loaded with
a mixture of vegetative and spore enzyme
extracts . . . . . . . . . . . . . . . . . 50

V

Figure Page

10. Effect of various concentrations of substrate
and inhibitor on reaction velocities of
both vegetative cell and spore NADH

oxidases . . . . . . . . . . . . . . . 52

ll. Plot of relative velocity Kg. pH of
. medium for both vegetative cell and

spore NADH oxidase . . , . . . . . . . . . 54
12. Flavin reactivation of both vegetative cell

and spore NADH oxidases . . . . . . . . . . 55
13. Effect of vegetative cell NADH oxidase and

FAD incubation on maximal activity . . . . 56
14. Serological reactions with vegetative cell

NADH oxidase as antigen . . . . . , . . . . 59
15. Serological reactions with spore NADH

oxidase as antigen . . . . . . . . . . . . 60

vi

INTRODUCTION

In the family Bacillaceae, both aerobic and anaerobic
species are capable of forming endospores which are meta-
bolically dormant and usually heat resistant. The thermal
stability of endospores is of economic interest in the food
and drug industry. Certain of the sporeforming bacteria are
pathogenic and represent a hazard to livestock and human
lives.

Sporogenesis in itself is of academic interest.
for here can be observed an unusual case of morphogenesis
in which a normally labile vegetative cell differentiates
into a biological entity able to withstand extremely adverse
conditions. This transformation entails morphological,
biochemical and physical changes, and it has received
attention from many scientific disciplines.

The attempts at resolving the phenomenon of spore
durability have been multifold but no clear explanation for
the resistance of spores exists today. Studies on the
nature of spores have been concerned with the factors in—
fluencing sporulation and germination, fine structural
changes, mineral and water content, formation and function

of dipicolonic acid (DPA), the development of heat

1

resistant proteins, and interrelationships of other coa—
stituents associated with bacterial spores. One of the

above topics, heat resistant proteins, is the area with

which this present research is concerned.

The elucidation of bacterial spore heat resistance
depends in part upon resolving the mode of transformation
from labile proteins to stable proteins. When biological
systems are subjected to elevated temperatures, their
proteins are among their most vulnerable entities By
comparison, nucleic acids are relatively heat stable
and consequently have received little attention when
factors involved in thermal destruction are being con—
sidered. Spores have a mechanism or mechanisms by which
they are able to preserve proteins and other constituents
necessary for germination and outgrowth 0f the many spore
enzymes which have been identified in extracts, all are
heat stable in the intact spore and some are heat stable
in the spore extracts. These extractable, heat resistant
proteins provide reasonable systems for studying mechanisms
ofspore stability. Of the many enzymes which have been
identified in spore extracts, some are heat stable and have
been shown to develop during Sporogenesis.

Simmons (1961) reported a reduced nicotinamide

adenine dinucleotide oxidase (NADH oxidase, formerly called

DPNH oxidase) found in both vegetative cells and spores of
Clostridium botulinum. The Spore NADH oxidase is heat stable in
extracts while the vegetative enzyme is a normally labile
flavoprotein.

The discovery of analogous enzymes in both vegetative
cells and spores suggested a possible means of studying the
formation and properties of a heat stable protein. A wide
variety of possible mechanisms may exist for the transfor—
mation from the vegetative protein state to the spore
protein state. In general, these could be stated briefly as
consisting of four alternatives. First, the spore protein
would essentially have the same primary, secondary, and
tertiarystructureas the analogous vegetative enzyme, heat
resistance being accomplished by the addition or the modifi-
cation by non-protein factors such as DPA, metal ions or pH,
The second alternative might involve a structural modifi-
cation of the original vegetative protein to form a heat
resistant spore enzyme, the primary protein structure of the
two analogous enzymes remaining essentially the same. The
third possibility is polymerization or depolymerization of
vegetative proteins to form stable spore structures. Fourthly.
the spore enzyme may be synthesized ggmngyg during sporulation
and has little or no primary structural relationship to its

analogous vegetative enzyme. Elimination of one or more of

4‘.

these possible alternatives would help to elucidate the
origin of one known stable protein.

The research presented in this thesis is a study of
the characteristics of the spore and vegetative NADH

oxidases of Q. botulinum. Differences in heat stability,

 

molecular size, enzyme kinetics, ionic charges, solubility,
serological reactions and other characteristics will be
presented and evaluated. The possible significance of the
difference between these two analogous enzymes will be
discussed in relationship to the formation of resistant

spore protein.

LITERATURE REVIEW

Sporogenesis: Biochemical and
Morphological Considerations

Ever since Alfred Koch (1888) first reported his
observations on the Sporogenesis of several sporeforming
bacteria, scientists have been interested in this unique
process of development. In the sixty years following this
report, most studies were confined to routine microscopic
observations, requirements for growth and sporulation, some
biochemical studies, and antigenic characteristics. Good
reviews of these early works have been written by Cook
(1932) and Knaysi (1948).

In recent years, new biochemical and morphological
findings have put a different emphasis on the study of
bacterial spores. Several of the important initial findings
that stimulated the new biochemical and morphological
approaches to Sporogenesis will be mentioned.

The first major biochemical approach to studying
spores was made by Hills (1949, 1950) who observed that
specific factors from yeast extract were necessary for the
germination of Bacillus anthracis. He determined from

fractionations of yeast extract that several different

amino acids, glucose, and certain nucleotides were stimulative
for rapid germination of Spores of the genus Bacillus. The
amino acids, L-alanine, and tyrosine and the nucleoside
adenosine were found to be the only nutrients necessary for
the germination of a few species of these Bacillus spores.
The stereoisomer, D—alanine, was occasionally found to
inhibit germination, especially in spores of Bacillus
subtilis. Hills concluded that a biochemical action on the
part of the spores was probably responsible. This research
stimulated subsequent biochemical studies of bacterial
spores and thus led to further important findings.

Stewart and Halvorson (1953) were studying the
effects of L- and D-alanine on the germination of Bacillus
cereus spores and found an active alanine racemase in the
intact spores. Later (1954) they studied this enzyme in
spore extracts and showed that it was heat stable when
associated with particulate matter. Since this first report
of alanine racemase, a variety of oxidative and non-
oxidative enzymes, all heat resistant in the intact spore,
have been reported (Lawrence, 1957: Murty, 1957: and
Levinson, 1957). Church and Halvorson (1955, 1956) reported
that the oxidative enzymes of aerobic spores were activated
by heat shocking the spores. Most spore enzymes appear to

be dormant until activated by heat shocking, mechanical

rupture or germination.

Powell and Strange (1953) observed that a number of
organic compounds were secreted during the initial stages
of germination and that excretion was one of the first changes
associated with the germination process. An unusual organic
acid with an absorption band between 250 and 300 mu was
detected in these exudates. Powell (1953) later identified
this compound as pyridine 2:4 dicarboxylic acid, dipicolinic
acid, by spectrophotometric comparison with known organic
compounds. This compound is found uniquely in spores of
Bacillaceae in the approximate range from 5 to 15% of their
dry weight. It is released during germination (Powell,
1957 and Rode and Foster, 1960). DPA has been shown to
develop during Sporogenesis, before the formation of heat
resistant Clostridium roseum spores (Halvorson, 1957 and
Collier, 1957). A similar relationship was found for B.
cereus (Hashimoto, Black and Gerhardt, 1960). Church and
Halvorson (1959) were able to vary the amount of DPA in
B. cereus spores by changing the phenylalanine content of
the media. They found that spores containing less DPA per
spore were less heat resistant. The above and similar
findings show that DPA is associated with spore heat
resistance. As yet, no direct evidence or mechanism by

which DPA functions in spore heat resistance has been shown.

Our initial knowledge of Spore enzymes and, to a
limited extent, spore physiology came from studies of
germination. Hardwick and Foster (1952) developed a useful
technique for studying spore formation, which they termed
endotrophic sporulation. Actively growing vegetative cells
are removed from their nutrient medium and replaced in
distilled water. They were able to show that at a certain
stage of growth, the vegetative cells became irreversibly
committed to sporulation. This technique has since been
used by many authors to study sporulation, primarily in the
aerobic sporeforming bacilli. Lund (1957) and Day (1960)
reported unsuccessful attempts at endotrophic sporulation in

anaerobic sporeforming bacilli (PA 3679 and 91 botulinum,

 

respectively); however, Collier (1957) was successful with

Clostridium roseum.

 

Hardwick and Foster (1953), using endotrophically
produced spores, studied seventeen different enzyme systems
in vegetative cell and spore extrhcts of several Bacillus
species. Their spore extracts were devoid of enzyme
activity. The conclusion they drew from these experiments
was that Sporogenesis occurs at the expense of proteins
pre-existing in the vegetative cell. Foster and Perry (1954)
further substantiated this claim in their studies of the

free amino acid, purine and pyrimidine pool found in

Bacillus mycoides cells. Isotopic tracer techniques

 

revealed an appreciable reduction in this pool of free
materials as the cells underwent endotrophic sporulation.
They concluded that spore synthesis during sporulation drew
from the vegetative cell's pool of free materials, perhaps
at the expense of degraded vegetative constituents.

The higher levels of divalent metal ions in spores
than in cells, especially calcium, was observed by Curran,
Brunsteller and Myers (1943). Their spectrochemical analyses
showed a definite increase in calcium and a slight increase
in copper and manganese. Spores were lower in potassium
and phosphorous content. Grelet's studies (1952a, b, c)
focused attention on the fact that specific chemical
species such as calcium, magnesium and manganese were
necessary for sporulation. In his studies, Grelet (1952c)
indicated an association between the amount of calcium in the
medium and the amount and heat resistance of spores produced
Curran (1957) showed that magnesium ions could replace
calcium ions in synthetic media with the result that the
same number of spores were produced, but they were less
heat resistant. Knaysi (1961a, b), using a spodcgram
technique, found most of the mineral content of spores was
concentrated in or near the spore cortex. In vegetative

cells, the mineral content was evenly distributed.

10
The development of plastic embedding and ultrathin
sectioning, coupled with electron microscopy, has provided
useful techniques for studying bacteria and spores.
Robinow (1953) was among the first to use these tebhniques
to study spore structures. His observations revealed the
complexity of the spore coat and membrane layers in resting

spores of B. cereus and g. megaterium.

 

Fine structural changes occurring during Sporogenesis
have been observed and reported by other workers. Hashimoto
and Naylor (1958) were among the first to apply these above

techniques to studies of synchronized cells of Clostridium

 

sporogenes that were undergoing sporulation. Hashimoto,
Black and Gerhardt (1960) made similar observations on
sporulating cells of g. cereus and they coupled their
studies with other observable spore characteristics such as
the synthesis of DPA, radioactive phOSphorus uptake, and the
appearance of heat stable spores. Young and Fitz-James
(1959a, b, c) studied the synchronized sporulating cells
of_§. cereus and B. cereus var. Alesti using an electron
microscope and various chemical techniques Their obser—
vations reveal the development of a spore septum which forms
and encloses future spore desoxynucleic acid (DNA) They
also observed an active ribonucleic acid (RNA) turnover

during sporulation. Fitz—James (1960, 1962) has described

11

the possible role that mesosomes and cytoplasmic membranes
may play in the early development and formation of the spore
septum in the genus Bacillus and in Clostridium patinovorum.
These and similar observations show that sporulation is an
ordered process with definite spore structures being formed
in conjunction with or followed by the appearance of other
spore characteristics.

Recent interest has been directed to observations
on the formation and properties of the spore cortex. Young
and Fitz-James (1962) have described the cortex formation
in B. cereus. Warth, Ohye and Murrell (1963a, b) have
studied the mucopeptide, containing 1, c -diaminopime1ic
acid (DAP), that is closely associated with spore cortex.
This recent interest in spore cortex is due to its close
association with heat resistance and will be discussed

later.

Spore Protein Synthesis

Some enzymes found in spores are heat stable
analogues of vegetative enzymes while others have no
counterpart in the vegetative cell. Many enzymes show
a decrease or increase in activity during sporulation
or undergo conversion from a soluble system to a particulate
system, or from particulate to soluble systems. In

many studies, periodic sampling of synchronized cultures

12

have shown that some spore enzymes are synthesized during
sporogenesis.

A system for this synthesis would require an active
turnover of RNA. Studies of sporulating cultures show that
an active turnover of RNA does occur and that there is little
or no synthesis of DNA (Young and Fitz-James, 1959b, c:

Rosas de Valle and Aronson, 1962; and Day, 1963).

Working with asporogenic Strains (SP‘) Of Bacillus
subtilis. Schaeffer and Ionesco (1960) observed that the
addition of purified DNA from sporogenic strains (SpT) to
Sp“ strains caused the latter to sporulate. It seems
evident that the vegetative cells of sporeforming bacteria
have DNA containing the information for sporogenesis. These
genes for synthesis of spore matter are apparently inactive
until some mechanism, as yet unknown, "instructs” the cells
to initiate sporulation whereby they form the RNA necessary
for spore synthesis.

The synthesis of spore protein during Sporogenesis
has been demonstrated by several workers. Heat stable
glucose dehydrogenase from B. cereus has been shown to
develop after exponential growth of vegetative cells, but
prior to the appearance of forespores (Bach and Sadoff, 1962).
A similar finding was made for heat resistant spore catalase

of_§. cereus (Sadoff, 1961). Szulmajster and Schaeffer

13

(1961a, b) studied several NADH oxidizing systems (NADH
oxidase, —dehydrogenase, -cytochrome c reductase, and
diaphorase) in Sp‘ and Sp+ strains of B. subtilis. They
observed a definite increase in the various NADH oxidizing
enzymes during the sporulation of Sp+ strains For the
corresponding interval, there was not a similar increase in
the 8p” strains. In the same study, the authors found a
decrease in succinic cytochrome c reductase, cytochrome c
oxidase and cytochrome c peroxidase during sporulation of
Sp+ strains; no corresponding decrease in Sp' strains
Hanson, Srinivasan and Halvorson (1963) have
studied changes in enzymatic activities, during sporulation
of B. cereus, by periodically sampling synchronized cultures
between early vegetative stages through maximum spore
production. Vegetative cells, apparently lacking a
functional tricarboxylic acid (TCA) cycle enzyme system,
rapidly synthesize aconitase, malic dehydrogenase and other
enzymes during the cell's transition into a spore. Anti—
sporogenic agents, such as a—Picolinic acid, not only
prevented sporulation when added to the medium but inhibited
formation of these active TCA cycle enzymes. The authors
feel that the formation of certain TCA cycle enzymes may be
necessary prior to the beginning of sporulation They

suggest that the antisporogenic agents may function by

merely inhibiting this enzyme formation

14

Doi and Halvorson (1961) have made comparisons
between vegetative and spore electron transport systems in
B, cereus and they observed a variety of differences. The
primary difference was the change from a predominantly
cytochrome-rich, particulate system of vegetative cells to
a soluble, flavoprotein oxidase system found in spores.

They also showed that the soluble oxidases of spores
function in electron transport during dormancy and germi-
nation.

Aubert and mullet (1963), using Sp+ and Sp‘ strains
of Bacillus megaterium, were able to induce B-galactosidase
during sporulation of the Sp+ strains. Just prior to
sporulation, the cells were washed and resuspended in fresh
media. At any time, up to the appearance of 100% refractile
spores (about six hours), they were able to induce
B-galactosidase in the Sp+ strains. After this time, the
enzyme could no longer be induced in the Sp+ strains.

There was no time limit for the induction of this enzyme
in the Sp’ strains.

Monro (1961) has studied the formation of a parasporal
inclusion body (Fcrystalfi) during the sporulation of Bacillus
thuringiensis,an insect larva pathogen. The "crystal” can
be readily isolated in pure form and it is composed

entirely of protein. Tracer studies using carbon14 labeled

15
amino acids, added at various times prior to and during
sporogenesis, show that the "crystals" are synthesized
during sporulation. This same study with labeled amino
acids showed that many of the spore proteins were also
synthesized during sporulation.

The various reports cited reveal that protein synthesis
is active during sporogenesis and ceases abruptly when the
mature, refractile spore is formed In some cases, a loss
or gain of a certain enzyme activity has been noted during
sporogenesis Increased RNA synthesis has been detected
and this also indicates an active protein synthesizing

mechanism.

Antigenic Studies of Spores

Kabat (1958) points out the following limitation in
the use of serological techniques to study and compare
protein structures. An antigenic structure is a site or
series of sites on the protein molecule and not the whole
molecule. Alterations of part of the protein structure can
occur without changes in the antigenic structure and more
than one antibody can react with a given protein.

For nearly 60 years microbiologists have attempted
serological comparisons between spores and vegetative cells

of Bacillaceae. They have encountered some limitations

1.

which in some ways are peculiar to sporeforming bacteria.
The question of possible vegetative contamination of spore
preparations can always be asked. Another consideration is
the effect on spore antigens of heat or chemicals used to
destroy vegetative material- Thirdly, when viable spores
are injected into animals, there is the possibility of spore
germination, in yiyg, producing vegetative antigens.

In his classical serological studies on spores,
Defalle (1902), was aware of these problems. He used vegetative
cells, viable and heat treated spores as antigens.
Observations were made by agglutination or sensitizing
reactions with whole cells or spores and he was able to
distinguish distinct vegetative and spore antigens. When
cross reactions between vegetative cells and spore anti—
bodies were observed, they were generally attributed to
either vegetative impurities in the antigenic preparation
or possible spore germination in the animal. In many
experiments, vegetative antibodies did not react with
spores.

Howie and Cruickshank (1940), using several Bacillus
species were able to produce crops containing 100% spores.
In their experiments, they did not observe a cross reaction
between vegetative cells and spore antibodies. They

concluded that vegetative cells were distinctly different

17

from spores in their antigenic structure. The same authors
did observe some common H (flagelar) and 0 (somatic) antigen

between vegetative cells and old cultures of Clostridium

 

sporogenes. ‘They noted, however, that the old cultures of
.Q. sporogenes they used as antigens were not pure spores.
Norris and Wolf (1961) did find one common H antigen between
cells and spores of Bacillus alvei, but found no other common
antigens between spores and cells of eleven other Bacillus
species-

Bekker (1944) heated spores of various Bacillus
species for 30 minutes at 120°C and injected these into
rabbits. When the resulting serum was tested with viable
spores, it was noted that the autoclaving had little or no
effect on changing the antigenic properties of spores.
Control sera were produced by injecting viable spores.
Antisera from autoclaved spores did not react with
vegetative cells and antigens from various Bacillus species
were mutually distinct, Similar observations had been made
by Defalle and others.

Vennes and Gerhardt (1957, 1959) have produced
specific antibodies against isolated vegetative cell
structures of B. megaterium. (The isolated structures were

found to be, for the most part, antigenically distinct

Antibodies produced from isolated spore wall did not show

18

any cross reaction with cell walls or any other cell
component.

Norris (1961) in his review of spore antigens,
sums up the six decades of research since Defalle. He
concludes that spore antigens are distinct from vegetative
cell antigens. When apparent cross reactions have been
shown between cells and Spore antibodies, there is always

the question of germination or vegetative contamination.

MATERIALS AND METHODS

Production of Cells and Spgres

Cells and spores were produced from Clostridium

 

botulinum 62~A, American Type Culture Collection (ATCC 7948).
The strain that was used by Simmons (1961) was employed for
most of these experiments. The stock cultures were stored

at -20(:as 0.5 m1 suspensions of spores.

The medium used for spore cultures was a modification
of the 4% Trypticase (Baltimore Biological Laboratories,
Baltimore, Md.) medium developed by Day (1960). It con-
tained 1 ppm thiamine and following mineral salts: 200 ppm

each of MgCl -6H 0, MnCl ~4H 0 and CaCl 7 20 ppm each of

2 2 2 2 2
FeSO , ZnSO and CuSO ; and 50 ppm of K HPO .

4 4 4 2 4

Vegetative cultures were grown in a medium
containing 2% Trypticase, 1 ppm thiamine and 0.2% dextrose.
In both vegetative and Spore cultures, 0.2% sodium thiogly-
coloate was used only in the inoculating series to be
described below.

The procedure for growing these cultures was
modified from Simmons (1961) and was adapted to 18 liters

of media in carboys incubated at 37 C in water baths.

To establish actively growing cultures, the following

19

2O

inoculating sequence was employed. A tube containing 0 5

ml of frozen spore suspension was thawed, poured into 10 ml
of media, heat-shocked at 100 C for 1-2 minutes and incubated
at 37 C for 12 to 18 hours until turbid growth was apparent.
This initial inoculation culture was then transferred to a
flask, containing 120 ml of medium and incubated at 37 C

for 4 hours. The entire contents of this flask was added

to 480 ml of medium and this was incubated for 4 hours.
Finally, this culture was transferred into 2400 ml of

medium and incubated 4 hours yielding a 3 liter inoculum of
actively growing cells. This last flask was poured into

the carboy containing 15 liters of medium. In spore cultures,
sterilized mineral salts were also added at this time The
age of the culture (in hours) was measured from the time

of this final inoculation until the culture was removed

from the 37 C water bath and cooled.

The cultures were routinely sampled and examined
microscopically. In spore cultures, swelling appeared at
approximately 14 to 16 hours and the maximum percentage of
refractile spores in sporangia were observed at about 24 hours
These cultures were then allowed to incubate another 24 to
36 hours followed by 5 to 10 days storage at 4 C. Harvesting
of spore cultures was done when microscopic examination

showed that the spores were free of their sporangia.

21

Vegetative cell cultures were harvested after 7 to 11 hours
of growth at which time they were rapidly chilled in ice
baths to arrest growth and then harvested immediately.
Harvesting of the spores and cells was accomplished
either batchwise in Servall centrifuges (model G or SS-4,
Ivan Sorvall Inc., Norwalk, Conn ), or in a closed model
Sharples Super Centrifuge (type 313D—49A-24BY, Sharples
Corp., Philadelphia, Penn ). The collected cells and spores
were washed 3 to 5 times in cold 0.85% NaCl solutions and

were then stored in the freezer at —20 C.

Assay Procedures

The NADH oxidase activity was routinely assayed
by following the reduction in absorption at 340 mu in a
Beckman DU spectrophotometer (Beckman Instruments Inc.,
Fullerton, Calif.). To a diluted portion of enzyme in a
3 ml cuvette (1 cm light path), the following final
concentrations of materials were added: 10 uM flavin
adenine dinucleotide (FAD): 0.067 M phosphate buffer,
pH 7 7; approximately 125 uM NADH: and distilled water to
a total volume of 3.0 ml. It was found necessary to
mix the enzyme and FAD and incubate them at room temperature
for several minutes in order to measure maximal activity.

A unit of enzyme activity is defined as a 0.01 optical

22

absorption change at 340 mu using an initial concentration
of 125 uM NADH. Units are reported as 0.01 A0D340/minute/ml
of sample. Specific activity is the number of units per m1
of sample divided by the mg/ml of protein for that sample.

Protein levels were determined by turbidimetric
procedures (Stadtmann, Novelli and Lipmann, 1951), the
Lowry technique (1951), or the spectrophotometric deter-
mination of Warburg and Christian (1942). Choice of the
method employed depended upon the experimental situation.
Protein concentrations are reported as mg of protein per
ml of extract or suspension.

The assay procedure for diaphorase was identical to
NADH oxidase assay except 10 to 100 ppm of methylene blue
was added to the mixture. The total activity was measured
by recording the decrease in absorption at 340 mu and
diaphorase was estimated as the activity above the known NADH
oxidase activity for the same sample. Units are reported
as 0.01 AOD34O/minute/ml of sample.

An assay for the spore enzyme was necessary to
distinguish between vegetative and spore NADH oxidase
activity. This was accomplished by testing the enzymatic
activity before and after heating a portion of the sample
to 70 C for 10 minutes. Spore enzyme was little affected

by this heat treatment while vegetative enzyme was completely

23

denatured. Spore enzyme can also be identified serologically
by reacting rabbit antispore serum with the sample, removing
the precipitate by centrifugation, and assaying for remaining
NADH oxidase activity.0nly spore NADH oxidase is removed

by antibodies against the spore enzyme.

The toxin of g. botulinum was qualitatively assayed
by the following procedure. A half milliliter of the
material in question was injected intraperitonealily into
a mouse weighing approximately 25 9. Death of the mouse
within 3 to 4 days was interpreted as indicating the presence
of toxin. Preparations used for rabbit immunization were
negative by this toxin assay. All preparations, following
either vegetative or spore enzyme purification heating

procedure, had little or no detectable toxin.

Purification Procedures

Purification of the spore and vegetative cell NADH
oxidases was accomplished by the following procedures.
All extracts, unless otherwise specified, were suspended
in 0.067 M 2—amino-2-(hydroxy methyl)—l,3—propanediol (tris)
buffer at pH 7.7 and maintained at 0 to 4 C. Routine assays
for activity and protein concentration were performed at
the various procedures to determine specific activity.

Hemogenates were prepared in a high speed Servall

Omnimixer at full speed, using a medium sized cup (50 ml

capacity) which was cooled by immersion in an ice bath.
Cells or spores, 10 to 12 grams, were mixed with 45 grams
of Superbrite #110 beads (Minnesota Mining and Manufacturing
Co., St. Paul, Minn.) and tris buffer. The duration of the
run was usually 10 minutes. Cell debris and glass beads
were removed by centrifugation, washed and often rehomogenized
for recovery of more enzyme. The supernatant fluids were
then pooled and referred to as initial extracts.

The extracts were subjected to centrifugation for
6-8 hours at 32,000 g. This procedure removed the diaphorase
and other particulate matter which interfered with enzyme
assay and other subsequent procedures.

The nucleic acids which would interfere with
protein fractionation procedures (below) were removed by
the following sequence of steps. The extracts were treated
with ribonuclease and desoxyribonuclease (Calibiochem,
Los Angeles, Calif.), 1 mg each per 100 ml of extract,
and incubated 37 C for two hours in the presence of
0.005 M Mg++ (Hatch, gt..aI., 1961). The nucleic acid
degradation products formed were separated from the enzyme
during subsequent’ammonium sulfate and gel filtration
procedures.

Extracts containing a heat stable enzyme can be

subjected to controlled heating procedures to remove other

25

proteins which are less stable. Both vegetative and spore
NADH oxidaseS'exhibit some degree of thermal stability.
The extracts were placed in a boiling water bath and when
the treatment temperature was approached, transferred to a
water bath of the proper temperature. Vegetative extracts
were treated for 3 minutes at 50 C. Spore extracts were
treated for 30 minutes at 75 C. Denatured material was
removed by centrifugation.

Proteins can be precipitated from an aqueous
solution by a procedure known as "salting out" in which
ammonium sulfate is often used as the salt. The increased
concentrations of highly ionic salts force out of solution
the organic solutes (proteins) which have low affinities
for the solvent. A protein with a specific affinity for
water can usually be "salted out" by a definite concentration
of salt and thus separated from other proteins with different
affinities. Variation in protein behavior can be achieved
by altering the type of salt used, the solution temperature
and the pH (Dixon and Webb, 1958, 1961).

Initially the proteins were precipitated by slowly
adding ammonium sulfate until 90% saturation was achieved
The precipitated proteins were separated from their super-
natant solutions by centrifugation. The proteins were

resuspended in a small volume of tris buffer and either

26

of two routine procedures was followed. In the first method,
solid ammonium sulfate was slowly stirred into the protein
suspension maintained at 0 C. The pH varied with the
addition of ammonium sulfate and was approximately 5.2 to
5.4 as 90% saturation was approached. The second method
employed a saturated solution of ammonium sulfate adjusted
to a pH of 6.5 with NH40H. The fractionation was carried
out at room temperature by slowly adding the calculated
amount of ammonium sulfate solution to give the desired
concentration.

Active enzyme fractions were further purified by
gel filtration on Sephadex G-100 or G-200 columns
(Pharmacia, Uppsala, Sweden). Fractions were collected
and assayed for activity and protein concentration. Most
of the purified vegetative and spore enzymes used for
rabbit immunization, serological reactions, and kinetic

studies were from these columns.

Gel Filtration

 

Sephadex gel filtration columns were prepared
according to the manufacturer's instructions and consisted
of two sizes: 2 Sephadex G—100 of approximately 3.5 cm x 100 cm
each and several Sephadex G—100 or G—200 of approximately

2.5 cm x 60 cm. The total volume (Vt) was determined from

27

the geometry of the column and the void volume (Vb), by
measuring the volume between start until the first protein

material appeared in the effluent. The imbibed volume

(Vi) was determined from the formula: V,

a W , where a
l r

equals dry weight of Sephadex used and Wr water regain
for a given gel.

The solute (protein) distribution coefficient (KD)
can be determined from experimental results using the

formula
= K (Flodin, 1962)

where Ve is the effluent volume in which the solute (NADH
oxidase) appears. In the event that Ve = V0, the solute
has been completely excluded from the imbibed volume and K
is considered to be 0. The molecular size of a solute

having a K of 0 would be equal to or greater than the

D
exclusion size which is approximately 100,000 and 200,000
mol. wt. for Sephadex 6-100 and G-200 respectively.

The eluant used in gel filtration columns was 0.01 M
tris buffer at pH 7.7. A constant flow rate was achieved

by use of a Sigmamotor Pump (Sigmamotor, Inc., Middleport,

N.Y.) and 2 to 5 ml fractions were collected.

Procedures for Enzyme Characteristics

The heat inactivation studies were performed on
NADH oxidases derived from either vegetative cells or
spores of g, botulinum. The source of the vegetative NADH
oxidase was active Sephadex G-100 fractions from routine
purification procedures. For this particular investigation
the spore NADH oxidase preparation was modified. The spore
extracts were heated at 60 C for 5 minutes instead of 75 C
for 30 minutes. Aliquots of enzyme were heated at 55 to
100 C in individual tubes (13 x 100 mm) placed in a regulated
water bath. At various times, tubes were removed, cooled
in an ice bath, supernatant fluid collected by centrifugation
and assayed for enzyme activity. Loss of activity was
determined by comparison to unheated controls.

Enzyme kinetics were studied by noting the effect
of substrate concentration on the oxidase activity. The
NADH concentration was varied over the range of 20 uM to
240 uM while maintaining a constant concentration of enzyme.
The effect of atabrine as a noncompetitive inhibitor of
flavin enzymes was ascertained by repeating the above
experiments in the presence of 0.12, 0.6 and 1.2 mM
atabrine. Michaelis constants for substrate (Km) and
inhibitor (Ki) were determined from the slopes of Lineweaver

and Burk (1934) plots of data and the maximum velocity,

29

Vmax' determined from the line intercept on the ordinate
axis.

The effect of pH on enzyme activity was determined
in 0.2 M'buffers over the pH range of 5.2 to 9.4 using a
constant amount of enzyme and NADH. The activity was
reported in terms of percent of maximum activity.

The flavin cofactor requirements for vegetative
cell and spore NADH oxidase were studied by attempts at
removal of the cofactor component and then activation of the
resulting enzyme with the addition of either FAD or flavin
mononucleotide (FMN). A vegetative cell NADH oxidase
suitable for these studies was obtained from ammonium
sulfate fractionation or gel filtration procedures. The
acid-ammonium sulfate technique for flavin removal (Warburg

and Christian, 1938) was attempted several times in an

endeavor to free the spore NADH oxidase of its cofactor.

Immunization and Serological Procedures

The vegetative cell and spore NADH oxidases (antigens)
employed in either immunization or serological reaction
procedures were obtained from respective Sephadex G—100
columns as described. Two different immunization methods
were tried with different sets of Dutch Belt rabbits. A11

suspensions used in immunization procedures described below

30

were maintained from 0 to 4 C until just prior to rabbit
injection. All glassware used for serological reactions
was thoroughly cleaned with non-ionic detergent.

The first method employed either vegetative cell
or spore antigens suspended in Freund's adjuvants, as
directed by the manufacturer (Difco, Detroit, Michigan),
and injected by subcutaneous and intramuscular routes. The
first injection consisted of 0.5 to 1 ml of antigenic
material containing 1 to 2 mg of protein and suspended in
an equal volume of Freund': complete adjuvant. In subsequent
injections, similar suspensions were prepared with Freund's
incomplete adjuvant and administered once a week for 4 to 6
weeks. Over a period of a year a total of ten rabbits were
injected with vegetative protein and four with spore
protein by this above method. An alternative immunization
method using only vegetative suspensions was also attempted.
Foot pads (toe pads) of a rabbit were injected with 0.2
to 0.5 m1 of vegetative protein suspension (no adjuvants)
followed 3 to 4 weeks later by one subcutaneous injection
of 0.5 ml protein suspension (Leskowitz and Waksman, 1960).

Blood was collected by cardiac puncture withdrawing
15 to 20 cc from each rabbit. The fresh blood was placed
in clean 25 x 150 mm tubes and slanted, incubated at room

temperature for 2-4 hours, then stored overnight at 4 C.

31

Serum was decanted from these tubes and clarified by centri-
fugation. Further treatment of the serum was necessary in
order to remove serum enzymes that oxidized NADH. An
ammonium sulfate fraction of 0—40% saturation was prepared
from the serum proteins. This fraction was resuspended in
0.85% NaCl solution and used in the serological reactions
described below. A control serum was prepared from blood
drawn from non-immunized rabbits and treated in a similar
manner.

The method of Cohn and Torriani (1952) was used to
determine cross reactions between spore or vegetative cell
NADH oxidases. Vegetative and spore antigens, obtained
from Sephadex G-100 columns, were serially diluted in 0.85%
NaCl solution. The dilutions were then distributed equally
into several sets of 13 x 100 mm clean test tubes. To
each identical set of dilutions, an equal volume of anti—
serum, control serum, or 0.85% NaCl solution was added. All
tubes were incubated at room temperature 1 to 2 hours,
stored at 4 C overnight and any precipitate was removed by
centrifugation. The supernatant fluid from each tube was
collected and assayed for NADH oxidase activity. The
dilution sets receiving the various serums were compared
to the controls. Spore antisera usually had to be diluted

10 to 100 fold prior to reaction with spore antigen.

EXPERIMENTAL RESULTS

Purification of vegetative and
Spore NADH Oxidase

 

Several procedures in sequence were utilized to
purify the spore and vegetative NADH oxidases. The highest
purification ratios obtained varied from 25 to 40 fold for
vegetative enzyme and from 60 to 80 fold for spore edzyme.

The initial extracts were subjected to centrifugation
for 6 to 8 hours at 32,000 g to remove particulate matter.
There was an initial loss of 10 to 15% of total activity
following this centrifugation

Table 1 shows a distribution of enzymatic activities
found in initial extracts, supernatant fluid and resuspended
pellets. It is apparent that methylene blue (MB) stimulates
the oxidation of NADH by the particles and has little or no
effect on the activity of the supernatant fluid. This
result indicates that the initial loss was due to removal

of a diaphorase.

The centrifugation procedure also removes the
particulate protein from the initial extracts which amounts
to 50% or more of the total protein. Both the spore and

vegetative cell NADH oxidase remain in the supernatant fluid

32

33

and thus are considered soluble enzymes.

I

Table 1. Diaphorase activity in spore and.vegetative cell
extracts before and after centrifugation.

 

 

Source (fraction) Vegetative Spore
in 10 uM FAD (units) (units)

 

Initial Extract
no MB 96 75
100 ppm MB 180 126

Supernatant Fluid
no MB 92 76
100 ppm MB 99 81

Resuspended Pellets

no MB 9 6
10 ppm MB 30 15
100 ppm MB 66 36

 

Results of heat inactivation studies are presented
in Figure l. The information obtained from these heat
inactivation studies was applied to the heating step of
the purification procedure. The selection of 75 C treatment
for the spore extract was based on the convenience of
adjusting and controlling the temperature of a laboratory
water bath.

The purified spore and vegetative cell NADH oxidases
exhibit notable differences in their heat stability.

Simmons (1961) also noted similar differences based on
experiments with crude preparations. He reported a heat

activation of the spore enzyme at 70 C but the present

LOG ’Io MAXIMAL ACTIVITY

 

 

34

 

 

 

 

 

VEGETATIVE
2.0 .
\v
\V 500
~~.~....~~\~v~
I A
I .0 “- LO”
150’
Q
I I
O
IIOO'
65‘
'W0'
L O I l l l L 3 l l e I L A J
O 5 l0 0 20 4O 60 90 I20
MINUTES

Figure 1. Heat inactivation determinations of vegetative
cell and spore NADH oxidases.

35

data show heat activation at 55 C with a slight loss at 65 C.
The activation of the spore enzyme could be explained by

the presence of a NAD reducing system that was labile at

55 C. Data in Table 2 indicate that the same spore

extract used in thermal inactivation studies does not reduce
NAD under the conditions of the NADH oxidase assay. This

apparent activation may be a property of the spore NADH

oxidase.

Table 2. Attempt to reduce NAD ‘with spore enzyme.

 

 

 

Spore Enzyme Activity
in 10 uM.FAD (units)
Control
125 uM NADH 195
250 uM NAD 0
500 uM.NAD 0

 

A detailed study was performed to determine the
range of ammonium sulfate concentration necessary to pre—
cipitate the vegetative or spore NADH oxidases. vegetative
or spore proteins which had been initially precipitated by
90% saturated ammonium sulfate were resuspended in tris
buffer and dialyzed 4 to 6 hours to remove excess salts.

Calculated amounts of ammonium sulfate were added to these

suspensions to give 5 or 10% increases in saturation in

36

the range of 33 to 90% saturation. Assays were performed
to determine NADH oxidase activity and protein concentration
of precipitates which formed within these increments.

The results of NADH oxidase precipitation by either
method described in Methods and Materials were very similar.
Figures 2 and 3 represent the enzyme and protein profiles
resulting from increments of 5 to 10% over the ammonium
sulfate saturation range studies. The spore enzyme preci-
pitates within a narrow range at an ammonium sulfate con-
centration of 55% saturation. The vegetative preparations
precipitated over a wide band of ammonium sulfate con-
centrations with a peak at 70% saturation.

The vegetative cell NADH oxidase profiles reveal
a small initial peak coinciding with the spore enzyme profiles.
In Figure 3, this was labeled peak A, and is heat resistant.
This spore-like enzyme appearing in vegetative extracts
will be referred to again in this thesis.

The vegetative NADH oxidase that was obtained
from the ammonium sulfate fractionation of these procedures
was partially inactive and FAD was needed for maximal
activity. This vegetative enzyme was used for two flavin
cofactor studies that will be described in detail later.

The nucleic acids constituted more than 20% dry

weight in crude extracts as detected by the Warburg and

37

mg/mL

 

o—O PROTEIN

 

 

 

O
,0], VEGETATIVE
I
\
O I \
300 900" I \
\
I I
I \
I \
20-- 6000 I \
U \
0 \
II 0’0 \
I o~~ 300» .so' \ I
'0 / O \
t z’ \\
g ./ Ne
. O" “I O'A‘. I 1 I I I U I I I l
E 4h 50 so 70 so so
>
Z SPORE
O
aw “ 300-"-
I
5.. ' 1‘
l / ‘
n I
4» 200-" I, o\
31> I \O
I I
I \
20 '00 d- O
I o ‘\
'4: y \‘ O—-O\O ’0
o" 0N.— .." . . . I
40 so 60 70 so 90

AMMONIA SULFATE: °/o SATURATION

Figure 2. vegetative and spore precipitation profiles of NADH
oxidase activity and protein in various
saturations of ammonium sulfate at 0 C.

 

38

 

 

 

VEGE TA TIVE B I"
O O I \t
4." . 2000 O ,’ \
I ‘\
’ \
3» I50» 0 ’ \
o )\ \
\
II \
0 O O \
2.1L I00 ’ \
I, ‘
.. A I .
E 'oT :2 50" _ ...-- .
\o E ’v.’ X. . / Heated
E 3 IX 'g”...u)t' . 'x.... y
E O'I >- O‘I-D v" I I A 'v 4
— t- 30 40 50 60 7O 80
u, _
t- 2
o» F'
c' (a
a 3... 4 4004, SPORE .
It 0
o I l \
I l i
300% I
2d I o l I O
C) . II ‘3‘7-C>‘
200.. \ / I *0
t I
b O l \
'0‘ O/ ' ‘
I000 I I
I I
.”. ‘~‘s
-- ‘
oi out,_1_p , , 4__.q
3O 4O 50 60 70 80
AMMONIA SULFATE: °lo SUTURATION
Figure 3. vegetative and spore precipitation profiles of NADH

oxidase activity and protein in various saturations
of ammonium sulfate at room temperature and a
constant pH of 6.5. In the vegetative profiles,

a spore-like enzyme, peak A, is identified by

the heating method (X ..... X). Peak B is
vegetative enzyme.

39

Christian (1942) spectrophotometric method. After the
initial 0-90% ammonium sulfate treatment, they were reduced
to about 3% of the resuspended precipitate and after further
ammonium sulfate fractionation were less than 1% by dry
weight of all succeeding suspensions.

Further purification of vegetative cell and spore
NADH oxidases was accomplished by gel filtration procedures
Two large Sephadex G-100 columns of approximately 700 cc
each. were used for these enzyme purifications.

Tables 3 and 4 show results from purification of
vegetative cell and spore NADH oxidases. These procedures,
previously described in detail, are briefly indicated in
the first vertical column. The gel filtrations reported
represent single fractions showing the highest purification
ratios. There is no loss of either NADH oxidase from these

gel filtrations.

Gel Filtrations

Gel filtration elution profiles were determined by
protein and NADH oxidase assays. The vegetative cell and
spore NADH oxidase profiles from both Sephadex G-100 and
G-200 are shown in Figures 4 and 5 respectively.

Table 5 lists the K values calculated from the

D

Flodin (1962) equation The effluent volume (Vé) was

4O

 

 

m mm 0.0mm mm.a mm.H oam m ooauw xmpmcmwm
. - . . . e N v
m we H h 0 am m me 0 me come OH om A mzv Koblmm
. . . . . e N w
m mm mm N m mm 0 5H 0 mm omm ooa om A mzv Romlo
m.ao mm.m o.vm m.h m.mm mom NNN mucmEummuu
sea m o om +
0 mm wmdznlmmfizm
m.nm mm.s m.mH m.ma mm com mmm m n
mooo.~m .ua “\auh
coaummsmwnucmo
ooa o.a m.ma 5.0m ,hm 0mm mew uomuuxm HmfluflcH
Ago AmE\muHssv AHE\mEv Amoa xv AHE\muHssv AHEV wusmmooum
pamflh coflumoflMflusm .uo¢ camaommm cflmuoum muflcs Hmuos >ua>fluo¢ mESHD>

 

 

Eonm wmmcflxo

 

.EDCHHSuOQ am
nflz Hamo w>flumummm> Mom muaammu amoammu Dam mousvmuoum soaumoflmflusm

.m wanna

l

 

m.m ms mam mm.o omm.o omm m ooauo xmcmnmmm

fig mm o.mma N.HH H.ma OVBH h wommfifimzv Xmmlmv
:. . . . . v N w
u 0v 5 ea 0 mm o N m ma mma OOH Om A mzv Romlo
mm o.¢a m.o© m.o b.0H 00 0mm mucmfiumwuu

CHE CM 0 mm +
0 PM wmflznlmmmzm

 

mm ©.m m.na o.m N.©N hm com 0 u
mooo.mm .H: o
soaummSMHHuch

ooa o.H n.¢ m.om m.mm om oam uomuuxm HMHUfisH

E 35323 2.35 i: 5 . 35333 :5 3880:

came» soflumoHMHHsm .uu¢ UHMflowmm cfimuonm mufiss Hmuoa >ua>fluo¢ wEsHo>

 

 

 

.Essfiasuon aw
Eoum mmmpflxo mndz mnomm How muasmwu HMUflmhu paw mmugpwooum GOHuMUHMflHDm .w manna

42

SEPHADEX G-IOO

   
  
   

 

 

 

 

s.-- 400" a VEGETAT/VE
,"\ V" 720 cc
4 300‘ V0 3 2| 5
’ Vi . 445
200'
‘2':-
E wI00<
\ L"
o .z
E 3
H 00.. O
S C {/0 250 300 350
P E: h
o I— , \
I U l ‘
n. 3... 4. 400 ' | SPORE
I
i n ‘ Vt- 675 cc
300 ,3 \ Vo= I90
2.‘ .
‘ ,' \ v:-435
l \
200‘ n \
n \
I 4: ' ‘
' l \
IOO- I \
I \
,' \
\
0‘ I l - I I
V0 200 250 300 - 350
mLEFFLUENT—§
Figure 4. vegetative and spore elution profiles of NADH

oxidase activity and protein from respective
Sephadex G-100 columns. In the vegetative
NADH oxidase profile, purified enzyme was
routinely collected from peak B fractions.
Profiles in region of peak A were studied in
detail (to be described, Figure 6).

Y

l.6%

|.i! ‘P

.4»

PROTEIN: mg [ml

 

Figure 5.

43

SEPHADEX 6-200
vznmsxarnns

IZOW

90+

 

 

 

 

 

601
a) 30*
t
2
D
$3 0‘
E:
g I
~ :
3I2m~ : ‘ SPORE
l |
I ' Vo-50
n ' ‘
9°" : Vt =I70
I
a |
60-1» ' ‘
u l
: I
30" . |
I
y .
0 LK‘W \ I r l I
Va 75 IOO l20 I50

EFFLUENTiml

vegetative and spore elution profiles of NADH
oxidase activity and protein from respective
Sephadex G—200 columns.

Table 5. RD values determined from gel filtrations for
vegetative cell and spore NADH oxidase from
'9. botulinum.

 

 

 

 

 

 

NADH
oxidase Column
activity

peak Sephadex G-100 Sephadex G-200
Spore 0 0.01
Vegetative 0.110 0.280
peak A 0.035 ----
determined from the various elution profiles. It is evident

that the spore NADH oxidase is excluded, or nearly excluded,
from the imbibed volumes of the gel particles of Sephadex
G—100 and G—200. The vegetative NADH oxidase "enters" the
imbibed volume of the gel particles of either Sephadex G-100
and G—200 This implies that the spore enzyme is equal to
or greater than the greatest exclusion size of the columns,
in this case approximately 200,000 molecular weight for
Sephadex G—200. The vegetative NADH oxidase is included
in the imbibed volumes of both Sephadex G-100 and G-200.
This implies that it is equal to or less than the smallest
exclusion size, approximately 100,000 molecular weight for
Sephadex G-100.

The vegetative cell NADH oxidase elution profiles

from Sephadex G-100 columns reveal an initial peak, labeled A

45

in Figure 4, whose position nearly coincides with that of the
spore enzyme. A detailed study of this region was performed
as follows. Pooled fractions were routinely assayed for
NADH oxidase activity and protein concentration. They were
then retested for spore enzyme by heating and serological
methods. Protein concentrations were also redetermined on
the heated samples. The results, seen in Figure 6, show that
peak A contains a heat resistant NADH oxidase that precipitates
with antispore serum. The majority of the other proteins

in this region are also heat stable. Similar evidence from
ammonium sulfate fractionation studies, previously

mentioned (Figure 3), also show that a heat resistant,
spore-like NADH appears early in cultural development.

Separation of Mixed vegetative and
Spore Enzymes

Equal units of vegetative cell and spore NADH oxidase
were mixed and placed on Sephadex G—100 and G-200 columns
and a diethyl-amino ethyl (DEAE) cellulose column.

A 1 cm x 20 cm ion exchange (DEAE) column was
prepared according to Peterson and Sober (1962) and adjusted
to a pH of 7.7 with 0.01 M tris buffer. Proteins were eluted
from this column by using a linear NaCl gradient between
0.07 to 0.2 M (Bock and Ling,l954). A Sigmamotor Pump was

used to control the flow rate at 50 ml per hour and 5 ml

\

46

     
   
   

 

 

 

a}.
I"' ‘8
5<r 50» r
UNHEATED
\
I
_4."w4°"
E I-
\ E
E .‘3
.. >-
5 t
“513-02300
c: 5
IL 4
I <3
\
lea-I20» i“
\
II 0 s
O O s
I <|3 / '
' l
I In», no» ANTI SPORE
- o SERA y/
\\
\
Os
x \O‘~~O
0‘ 0 fl # I I a I # I :‘L?
56 5a 60 62 64 66 so 70 72 74
TUBENO
225 250 275
EFFLUENTIInL

Figure 6. Normal and heat resistant elution profiles of
NADH oxidase activity and protein, also serological
reaction profile, from region of peak A, Figure 4.
Spore-like enzyme is identified by both heating
and serological (X ----- X) methods of assay.

47

fractions were collected. The effluent fractions from these
columns were assayed for proteins, total NADH oxidase activity.
and for heat resistant NADH oxidase activity. These
elution profiles are shown in Figures 7, 8, and 9.
The elution profiles show that the vegetative cell
and spore NADH oxidases have been separated on Sephade:
G-lOO and G—200. A complete separation is obtained on
Sephadex G-200 and this establishes a useful means to dis-
tinguish or separate spore from vegetative NADH oxidases.
These results could have been predicted from the KD values.
However, the actual mixing of these two NADH oxidases,
eluting them from gel filtration columns and being able
to demonstrate separate peaks definitely establishes that the
vegetative and spore NADH oxidases differ in molecular size.
The elution profile for spore enzyme on DEAE
cellulose is quite narrow compared to the vegetative enzyme
which has a tendency to trail. These results indicate
probable differences in ionic charge between the spore and

vegetative NADH oxidase.

Determination of Enzyme Characteristics

Studies of enzyme kinetics, pH optima, and cofactor
requirements were performed on the vegetative cell and spore

NADH oxidases The purpose was to show the differences that

48

SEPHADEX G- IOO
VEGETAT/VE- SPORE

  

 

 

 

 

4.0» eo~I A
’1‘ B Vos 50
I I ,‘I VI -I90
I, ‘I "
I ' |
I ‘ l I
-' 3.00 60" I |' |
E l ‘ I
\ m «I I
a t " |
.5. g .‘z‘ . I
E .. ': o I
III >‘ " ' I
s t '3 I I
a: 2.00 2 40-» '; 1 I
a P . I I
u 'I I
( I: I
l l I
l l
l I
I . II) '
l.0 * )5 20 | ‘
s ,I .
3k , I
A . ‘
3 ’ '- I
I; 1‘. '. I‘
O
0.. 5 o x'x x‘x
“ l r T I I
V0 75 IOO l25 |50
EFFLUENTC ml
Figure 7. Profiles for NADH oxidase activity and protein

eluted from a Sephadex G—100 column loaded with a
mixture of vegetative and spore enzyme extracts.
Spore enzyme. peak A, is identified by the
heating method (X ..... X) which also identifies

peak B as vegetative enzyme.

4.0+»

2.0T

— PROTEINS mg /ml

 

Figure 8 .

(Hound) X----

49

   
 

 

 

SEPHADEX G- 200
30.. VEGETAT/VE - SPORE
Vo= 50
A VI=|72
m 60-» f,
L' I ‘ B
I
g l I ,‘I
-- ' .".I II
>' ‘ '.I |
t '5‘ '.I I, I|
> - '.| I
— 40“ I- .
I- .' a " I
2 a I ‘
I
: I I
I I '.
' |
8 20¢.
I
I
I
I
I
I I
\‘( I
0 R? x x x x x L I
V0 75 I00 '25
EFFLUENTIml

Profiles for NADH oxidase activity and protein
eluted from a Sephadex G-200 column loaded with

a mixture of vegetative and spore enzyme extracts.
Spore enzyme, peak A. is identified by the
heating method (X ..... X) which also identifies
peak B as vegetative enzyme.

SO

 

 

 

 

 

 

l25¢
A DEAE Cellulose
.lOv I000 I;
.s :3
E ; .
\. ,1
.090 g ; 1
q 75‘} : 2
3; I 1 ,
P I 1 ,-v’
.081' E : .
P . '
o l
.1 50» k-
.07" "fl” :
.. x" E
0 : n '
. ; 25 2!
z x .0
a ‘6 5
I 3 :
O I .
' ' Xe. V
I 3'; l T “x .
IO 20 30
TUBE No.
Figure 9. Profiles for NADH oxidase activity eluted from

a DEAE cellulose column loaded with a mixture
of vegetative and spore enzyme extracts. Spore
enzyme, peak A, is identified by the heating
method (x ..... X) which also identifies peak

B as vegetative enzyme.

51

might exist between these two enzymes. Figure 10 represents
typical Lineweaver and Burk plots of the relationship between
substrate concentration and the reaction rates. From these
plots the kinetic values were determined. Table 6 summarizes
the Michaelis constants. Km and Vmax' for spore and vegetative
enzymes and the Ki values for various concentrations of
atabrine, a non—competitive inhibitor. The Km of the spore
enzyme is approximately 8 times that of the vegetative enzyme
implying a greater substrate binding by the vegetative enzyme.
Inhibitor kinetic values indicate that the spore enzyme is
less inhibited by atabrine.

The optimum pH for activity was determined for
vegetative and spore NADH oxidases and the resulting
curves are seen in Figure 11. The pH optimum for the two
enzymes appears to lie between 7.3 to 7.6. It is apparent
the spore enzyme is more active in the lower pH regions
than the vegetative enzyme. Conversely, the vegetative
enzyme shows more activity in the pH regions above the optimum
pH.

Differences in affinity for FAD were noted between
vegetative and spore NADH oxidases. The vegetative enzyme
readily loses half or more of its activity after ammonium
sulfate fractionation but the activity can be restored by the

addition of FAD. Following elution from Sephadex columns,

52

 

 

 

.I o... VEGETATIVE J OT I spans
1.2 mM I
.09"K\/ db Iv
‘h / l.2 m M
66‘/ /
.08" o. / v
I /

. / Hy

.07 I /
[fl
.. A It ..
A, r , ’
.05" ’8’ .05“
I
. ’/ ‘3
[(0.3 «/
049/6) '0' 09/ $
0 1’
/ 0329 O
030 «II- >/ / /
/ ,/
024» «I- ’l I
e / l
I

.0l0

0 .6 ' 36 5‘0 0 ' Io so 510

I
’S x 10’3

Figure 10. Effect of various concentrations of substrate
and inhibitor on reaction velocities of both
vegetative cell and spore NADH oxidases.

Results of substrate (solid lines) and substrate
in different atabrine concentrations (dashed
lines) are shown above.

53

Table 6. Enzyme kinetic constants determined for vegetative
cell and spore NADH oxidase from Q. botulinum.

 

 

 

 

Constant Inhibitor
20 to 240 uM.NADH concentration Vegetative Spore
(atabrine) NADH oxidase NADH oxidase
—6 —5
Km ——-- 7.9 x 10 M 5.9 x 10 M
Vmax ---- 130 280
-5 -5
Ki 0.12 mM 1.2 x 10 5. x 10
-5 -5
0.60 mM 2.3 x 10 9 X 10
1.2 * 4.5 x 10"4

 

*Completely inhibited.

the vegetative NADH oxidase is nearly free of FAD Ceveral
unsuccessful attempts were made to resolve the partially
purified spore NADH oxidase by the warburg and Christian
acid-ammonium sulfate treatment. The cofactor remained
strongly bound to the spore NADH oxidase and the enzyme was
rapidly destroyed below pH of 3. The best experimental
results for removal of flavin from spore enzyme are shown

in Figure 12. Comparison is made to a vegetative enzyme from
an ammonium sulfate fractionation. In neither case does
FMN restore activity. Simmons (1961) also treated his cell-
.free preparations to remove flavin groups and reported about
55%.reactivation with FMN.

The effect of incubation time on activity was noted

when FAD and enzyme were mixed prior to assaying. Figure 13

54

 

 

 

I00--
800
).
t
Z
l-
0
9 so»
2
D
2
X
<
2
u. 40-»
O
.-
2
MI
0
K
E 20‘» / I—I vaosnmve
I’-
' 0---0 SPORE
° : l : : :
5 6 7 a 0
pH

Figure 11. Plot of relative velocity _I_I_s_. pH of medium for
both vegetative cell and spore NADH oxidase.

iiililil
IIIII' .

55

 

 

 

0"‘\\\ VEGETATIVE °l"\ SPORE
\
0
I00 '\
. ‘I
.ZOqu 0 \\\z
(D \ 0
e ~-
'0
O .3004- ..
o
‘3 \-
.400“ ._. NO FAD v ' 6 0—0 NO FAD
u—u lpM FAD \‘ I--I 3.3,pIII FAD
V
.500» e—‘ 5pm ..
v—v IOpM
we : : : t : A: ; : a : :
I5 30 45 50 75 90 IS 30 45 so 75 90
SECONDS

Figure 12. Flavin reactivation of both vegetative cell and
spore NADH oxidases. In either enzyme assay.
the presence of 10 uM.FMN has no effect and
simulates the no FAD curve.

fl! IIIII. I I I’ll 3! I ll {:1 l I

 

56

 

 

 

 

 

 

 

 

.OOOT
.IOO‘P
.200‘I
.. \ e
o .300 \-
e A
IO
C) o:::::::.
(D D
.4 .
<1 GOP \‘
v
0 O No FAD O
D
.5004} I I I5 sec. V
o
A A 50 sec. 0
.600" v v 60 sec.
0 O l20 sec.
.700" U 0 I hour
we: I : I I I —:
I5 50 45 60 75 90 I05
SECONDS
Figure 13. Effect of vegetative cell NADH oxidase and FAD

incubation on maximal activity.

The above

enzyme-FAD incubation times include the 15
seconds between initiating the assay reaction
and the first observation of reduction in
absorption at 340 mu.

57

represents the data from experiments using a constant amount
of enzyme and FAD (10 HM) and varying only the time of incu-
bation. In assaying procedures, the FAD was always
pre—incubated with the enzyme for 3 minutes.

A vegetative cell NADH oxidase obtained from gel
filtration column was used in another study and results
are seen in Table 7. Various concentrations of FAD,
ranging from 1.67 uM to 30 uM and 10 HM FMN, were added
to this inactive vegetative cell NADH oxidase. FMN
reactivated the enzyme about 10% of the maximal activity
obtained by FAD. For the enzyme concentration in this exper-
iment, the amount of FAD used in routine assays (10 uM)

achieved maximal activity.

Table 7. Flavin activation of vegetative cell NADH oxidase.

 

 

 

(::n::::::tion) ACtiVitY‘ units
0.0 6
10 FMN 21
1.67 FAD 84
3.30 114
10 132
20 135

30 135

 

58

Serological Studies

 

Serological studies were performed in an endeavor to
find antigenic relationships between vegetative cell and
spore NADH oxidases. If these two antigens had one or more
common antigenic sites, then a cross reaction would be expected
between either protein and antibodies formed against the
other protein. If they were identical or very closely
related, either protein would react fully with its heterologous
antibody.

The Cohn and Torriani technique used is described in
Methods and Materials. It was selected for the following
reason. It is an ideal method for showing a Specific
serological reaction when a wide variety of antigens and
their homologous antibodies are present in the same mixture.
If the antigen is an enzyme, then the specific antibody-
antigen reaction is detected by removal of the enzyme from
the mixture.

The results of serological studies are presented in
Figures 14 and 15 and are typical for vegetative and Spore
antigen-antibody reactions. The striking result of these
investigations is that there is no cross reaction between
Vegetative cell or spore NADH oxidase and heterologous

antisera.

 

59

    
 
  
 

 

O
6
VEGETAT/VE
IOOT
o—o Control I
o—-o Anti Spore Sero
I--l Anti Vegetative Sara
0’.
.5
E
\
O
'.
u)
c:
O
.‘i 504»
).
l:
2
...
c)
Id
/8’ '
0’ .-
’0’ ,I"
./
Mrs . . .
“15 85 I70 340
ENZYME ADDEDZUnHS
Figure 14. Serological reactions with vegetative cell

NADH oxidase as antigen. Both saline and serum
controls (0 0) and the antispore sera
curve are identical and indicate no cross
reactions. The anti vegetative sera curve is
typical for all anti vegetative rabbit sera
tested.

 

ACTWVFTY2150|D34KL/m|n

IOO‘

In
9

60

   
   
  
  

SPORE

e—e Control

 

 

’ I
0--O AMI v.9.to'ivo so”
AMI spar. s.r0
.000... I/‘o
0"” '/I00
......"0
eeeesseseee.eeseeeee
sessessessss.
LPQFO—k 4;.‘
I80 360

' ENZYME ADDED! Units

Figure 15. Serological reactions with spore NADH oxidase as

antigen. Both saline and serum controls

(0 0) and the anti vegetative sera are
identical and indicate no cross reactions. The
diluted (1/10 and 1/100) anti spore sera curves
are typical for all anti spore rabbit sera
tested. undiluted anti spore sera completely
remove the spore NADH oxidase (antigen) in these
concentrations.

 

61

Rabbits receiving spore antigen produced high levels
of antibody. Undiluted antispore sera completely removes
spore NADH oxidase for the enzyme levels shown in Figure 15.
All rabbits receiving antigen derived from vegetative cells
by either Freund's method or by the foot pad technique, had
low titers of antibodies against the vegetative NADH oxidase.
These observed differences in titers indicate an apparent

distinction in antigenic properties of these enzymes.

DISCUSSION

The purpose of this research, as outlined in the
Introduction, was to compare the properties of vegetative

cell and heat resistant spore NADH oxidases of g. botulinum.

 

It was hoped that the comparison of these analogous enzymes
would elucidate some possible mode by which a heat resistant
spore protein could be derived from vegetative cells.

A comparison of the properties of these two enzymes
shows the following differences. Besides the extreme dif-
ferences in their heat stability, there appear to be dif-
ferences in their solubility at various levels of ammonium
sulfate saturations. There are differences in their ionic
surface charge as observed by characteristic differences in
their elution from DEAE cellulose anion exchange chromato-
graphic columns. The optimum pH activities of the two enzymes
show a slight but consistent difference. The important
kinetic differences are their reaction with substrate and
inhibitor and their binding capacities for the flavin cofactor.

The above cited differences could be interpreted
as resulting from some modification of the vegetative NADH
oxidase molecule. However, there are two characteristics

which show distinct protein structural and mohacular size

62

63

differences between these analogous enzymes.

In the first case,they do not cross react serologically.
Current interpretation of this information is that the two
antigens (enzymes) are different. ‘Kabat (1958) points out
that antigenic sites on a protein molecule are not necessarily
an expression 0f the whole molecule. Therefore, differences
in one or all expressed antigenic sites cannot be absolutely
interpreted as complete differences in protein structure.

The possibility that the basic vegetative protein structure.
has been changed to form a spore protein should not be
completely disregarded. \

The information obtained from gel filtration studies
shows a definite molecular size difference between the two
proteins The spore enzyme has an estimated molecular weight
of 200,000 or more and is therefore at least twice the size
of the vegetative enzyme which is estimated at 100,000 or
less The fact that gel filtration separates solutes by
molecular size is well documented (Flodin, 1962; Granath and
Flodin, 1961; Killander and Flodin, 1962). Molecular size
differences quite definitely exclude the possibility that a
single vegetative enzyme molecule has been modified into a
single spore enzyme molecule. However, the interpretation
that a vegetative NADH oxidase molecule has polymerized with

one or more other proteins molecules, including other

64

vegetative NADH oxidases, is not excluded. Such a polymeri-
zation could lead to the formation of a protein which need
not react serologically with its monomer.

In the Introduction, four general alternatives were
give) to embrace all conceivable ways a spore protein might
be derived from vegetative cells. The first two alternatives
were concerned with possible modificatiOn of a single vegetatiVe
protein, either by extrinsic protein factors such as DPA and
calcium or intrinsic protein structural changes.' Possible
non-protein modifications and protein structural modifications
of vegetative cell NADH oxidase molecules can be eliminated
as the sole means of forming heat resistant spore NADH
oxidase.

The second two alternatives were briefly stated as
either polymerization or depolymerization of Vegetative
proteins to form stable structures, or the g_ gqgg synthesis
of a spore protein structurally unrelated to pre-existing
vegetative proteins. As mentioned, our current findings
cannot completely exclude either of these two as operational

for spore NADH oxidase from g. botulinum.

 

In reviewing the literature, some authors have indi-
cated evidexce for gg_novo synthesis of spore proteins.'
Isotopic tracer studies show the incorporation of labeled~

amino acids into the spore protein (Hardwick and Foster, 1952

'65

and 1953; Foster and Perry, 1954, and Monro, 196l). The
existence of spore enzymes not found in the vegetativecell
certainly support this View (Bach and Sadoff,ll962).
Other authors, working with spores as antigens, generally
indicate spores and their vegetative cells are antigenically
distinct (Norris, 1962).

There is no.a priori reason for all spore proteins
to arise g§_ngyg and the possibility does exist that cells
utilize some protein conserving mechanism. Although the
enzymes observed in the present work are distinctly different,
it is not possible to exclude polymerization as a means
of formation. If this is truly the case, the flavin cofactor
may be involved in some way in the mechanism of heat resistance.

Some current theories explaining spore heat
resistance can also be considered in the light of the
results and the above interpretation. The main stress has
been to find a direct connection between calcium ions,
DPA and a mechanism for protecting spore constituents,
especially proteins. Young (1959) demonstrated that DPA
could form a DPA-amino acid complex which was calcium
dependent and she speculated that a similar complex might
exist with spore proteins.g Doi and Halvorson (in Halvorson

and Howitt, 1961) demonstrated that DPA stabilized a soluble

NADH oxidase from g. cereus, but Powell (1957) was unable to

66

show any increase in thermal stability of her soluble adenosine
deaminase. Halvorson and Howitt suggest that if DPA protects
proteins directly, such stabilization might be selective for
those enzymes requiring divalent ions for tertiary structure.

If a DPA—Ca-NADH oxidase complex was responsible for
the extreme heat resistance of the spore enzyme,it was

unique to the particular strain of Q. botulinum 62A used.

 

There exists in our laboratory a culture of Q. botulinum

 

62A which does not possess the stable spore NADH oxidase
(Day, 1963).

Lewis, Snell and Burr (1960) have proposed a mechano-
chemical hypothesis for spore heat resistance and dormancy.
Their concept involves an elastic layer, such as the spore
cortex, that would swell and exert pressure upon the vital
core and thus force water out. The water content within the
interior would be greatly reduced and this semi-dehydrated
state could explain both dormancy and heat resistance.
Support for their hypothesis comes from several sources:
concentration of metal ions in the periphery of spores,
Knaysi (1961a, b); apparent location of DPA in spore
peripher‘, Hashimoto and Gerhardt (1960); the effect of
Ca:DPA ratio in heat resistance, Levinson, Hyatt and Moore
(1961); and base (ion) exchange studies of spores, Alderton

and Snell (1963).

67

The Lewis hypothesis may explain a possible mechanism
for the intact spore, but it does not explain the development
of certain heat stable proteins. Bach and Sadoff (1962) have
shown the appearance of a heat stable protein prior to the
development of definite Spore structures such as spore cortex.

This research performed on NADH oxidases of_g. botulinum

 

shows the early development of a heat stable, Spore-like
protein in vegetative cells as shown in Figures 3 and 6.
At this stage, the heat stability of spore NADH oxidase
cannot be explained by a contractible spore cortex.

Any theory which attempts to explain the unique

heat resistance of Bacillaceae spores must incorporate

 

into it a mechanism whereby the configuration of the spore
proteins is maintained. A viable spore must have enzyme
systems capable of initiating germination, carrying on the
synthesis of outgrowth and finally capable of producing a
viable vegetative cell. Failure of any proteins in this
reaction sequence result in what the microbiologist would

normally classify as a non—viable organism.

CONCLUSIONS

A study of the enzymatic characteristics of vegetative

cell and spore NADH oxidases from a strain of Clostridium

 

botulinum 62-A has been presented. The heat stability of
the isolated spore enzyme was tested and compared to that
of the vegetative enzyme. At 60 C, the protein derived
from vegetative cells was readily denatured while the
spore enzyme was unaffected by this heat treatment. These
results would tend to validate this as a test system for
studies of mechanisms of heat resistance.

Certain similarities were found for vegetative cell
and spore NADH oxidases. Both are soluble in their extracted
state and can be separated from their respective particulate
diaphorases by centrifugation. The pH optima (approximately
7.5) are not significantly different. However, a slight
difference is observed in the spore enzyme's higher
relative activity in pHs below optimum and the vegetative
enzyme is conversely more active above optimum pH.

This study has revealed the following significant
differences between the two analogous NADH oxidases.

There are the above mentioned differences in the thermal

stability of the purified enzymes as well as their respective

68

69

solubilities in solutions of ammonium sulfate. The spore
NADH oxidase precipitates at 55% saturation while the
vegetative enzyme precipitates at 70% saturation.

The enzymes differ in their reactions with substrate
and the inhibitor atabrine. The Km value for the spore
enzyme is about 8 times greater than that for the vegetative
NADH oxidase. This can be interpreted as showing that the
spore enzyme has a lesser affinity for substrate. On
the other hand, spore enzyme is less inhibited by atabrine
than the vegetative enzyme. This may be related to the
relative abilities of the two proteins to bind cofactor.

Important differences in their ability to bind
flavin were observed. The flavin can be completely
removed from the vegetative cell NADH oxidase by relatively
mild treatments and FAD, not FMN, restores activity.
Stronger acid-ammonium sulfate treatments fail to remove
an appreciable amount of flavin from the spore enzyme.

Thus, there are only indications that FAD may be the primary
cofactor.

The results of gel filtration studies show that
there are at least two fold differences in the molecular
size of the two oxidases. Estimations of 100,000 or less
molecular weight for the vegetative NADH oxidase and 200,000

or more for the spore enzyme have been made. Differences in

70

the charge on the two proteins is indicated by their
elution characteristics on DEAE cellulose anion.exchange
columns.

Neither oxidase interacts with the heterologous
antibodies elicited by its analogous enzyme. This can be
interpreted as indicating that there exist no structural
similarities in their antigenic sites. Apparent differences
in their antigenic properties are observed by quantitative
differences in antibody formation in rabbits receiving

similar immunization treatments.

REFERENCES

Alderton, G. and N. Snell. 1963. Base exchange and heat
resistance in bacterial spores. Biochem. Biophy.
Res. Commun. 10:139-143.

Aubert, J. P. and J. Millet. 1963. L' induction de la
B-galactosidase au cours de la sporulation chez
Bacillus megaterium. Compt.-Rend. 256:1866-1868.

Bach, J A. and H L. Sadoff. 1962. Aerobic sporulating
bacteria. I. Glucose dehydrogenase of Bacillus
cereus. J. Bacteriol..§;:699-707.

 

Bekker, J. H. 1944. The antigenic properties of bacterial
spores. Antonie van Leeuwenhoek. J. Microbiol.
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Bock, R. M. and N. S. Ling. 1954. Devices for Gradient
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Church. B. D. and H. O. Halvorson. 1955. Glucose metabolism
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Church, B. D. and H. 0. Halvorson. 1956. Effect of heating
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Church, B. D. and H. Halvorson. 1959. Dependence of the
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Cohn, M. and A M. Torriani. 1952. Immunological studies
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spore production in Clostridium roseum. ‘23
H. O. Halvorson (ed.), Spores. American Institute
of Biological Sciences, Washington, D. C

71

72

Cook, R. P. 1932. Bacterial spores. Biol. Rev. Cambridge
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Curran, H. R., B. C. Brunstetter, and A. T. Myers. 1943.
Spectrochemical analysis of vegetative cells and
spores of Bacteria. J. Bacteriol. 45:485-494.

Curran, H. R. 1957. The mineral requirements for sporulation.
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Institute of Biological Sciences, Washington, D. C.

Day, L. E. 1960. Studies on the sporulation of Clostridium
botulinum Type A. M.S. Thesis, Michigan State
University.

Day, L- E. 1963. some physiological changes occurring during
the sporulation of Clostridium botulinum Type A.
Ph.D. Thesis, Michigan State University.

Defalle, W. 1902. Resherches sur les anticorps des spores.
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Dixon, M. and E. C. Webb. 1958. Enzymes. Academic Press,
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Dixon, M and E. C. Webb. 1961. Enzyme fractionation by
' salting-out: a theoretical note. Advances in
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Doi, R. H. and H. Halvorson. 1961. Comparison of electron
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of B. cereus. J. Bacteriol._§l:51-58.

Doi, R. H. and H. Halvorson. 1961. The role of DPA in
bacterial spores. ‘In H. O. Halvorson (ed.),
Spores II. Burgess Publishing Co., Minneapolis
15, Minn.

Fitz-James, P. C. 1960. Participation of the cytoplasmic
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Bacilli. J. Biophy. Biochem. Cyto. §z507~528.

Fitz-James, P. C. 1962. Morphology of spore development
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Flodin, P. 1962. Dextran gels and their applications in
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Uppsala, Sweden.

73

Foster, J. w. and J. J. Perry. 1954. Intracellular events
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bacillus mygoidcs. J. Bacteriol._§l:295-302.

Granath, K. A. and P. Flodin. 1961. Fractionation of
dextran by the gel filtration method.
Makromolekulare Chemie 4§:160-171.

Grelat, N. 1952a. Le déterminisme de la sporulation de
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