SOME PEWSEG-LOGECAL CHANGE OCCURRING
D‘URENG THE SPGRULATION (DP
CL’JSTRIDM W
TYFE A

Thesis {or the Degree cf Ph‘ D.
MECHEGAN STATE UNWERSITY

Lawrence E. bay
19653

 

This is to certify that the

thesis entitled

SOME PHYSIOLOGICAL CHANGES OCCURRING
DURING THE SPORULATION OF
CLOSTRIDIUM BOTULINUM.

TYPE A

presented by

 

Lawrence E. Day

has been accepted towards fulfillment
of the requirements for

_Eh.._D_._ degree in _Mi.CI_Qb.iOl ogy
and Public Health

gag/g 72 @7me

V Major professor

 

Date January 7, I963

 

0-169

   
     

  

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. Unchrsil)

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'tr» : ~ v :

 

 

ABSTRACT

SOME PHYSIOLOGICAL CHANGES OCCURRING
DURING THE SPORULATION OF
CLOSTRIDIUM BOTULINUM,
TYPE A

by Lawrence E. Day

An investigation of the sporogenesis of Clostridium

 

botulinum. type A, was conducted to determine some of the
fundamental physiological events taking place during this
process. The final step in sporulation, from the immature
forespore to the mature spore, was studied by placing the
immature forespores in a simplified environment. It was
observed that the only exogenous nutrient requirement for
this step was fulfilled by L-alanine and L-proline and

that these amino acids were metabolised via the Stickland
Reaction. It appeared that these amino acids were required
only as an energy source. L-arginine and L-alanine, and
L-isoleucine and L-proline would also serve in this
capacity. but L—ornithine and L-alanine would not. Neither
L-arginine alone nor L-citrulline alone would furnish the
energy requirements for this final step. With the use of

chloramphenicol it was found that no protein synthesis was

occurrin:
phosphor
acid syr
conclusi
complete
growth 1
Iibonu:
formati
maturat

the en:

Lawrence E. Day

occurring during this developmental stage. Radioactive
phosphorus studies coupled with known inhibitors of nucleic
acid synthesis, mitomycin C and 8-azaguanine, led to the
conclusion that deoxyribonucleic acid (DNA) synthesis is
completed almost simultaneously with the end of the log
growth phase and the initiation of sporulation, while
ribonucleic acid (RNA) synthesis is required through the
formation of the immature forespore but not during the
maturation process. A study of the synthesis of some of
the end products of growth and sporulation in a medium of
4% Trypticase revealed that valeric, propionic. and acetic
acids were being formed. Using acetic acid-l-C14, it was
found that acetic acid was being utilized during vegetative
growth at a slower rate than that at which it was being
formed. However, during the early phase of sporulation acetic
acid was being utilized at the same rate but the production
of this material had slowed, resulting in a net decrease in
the level of acetic acid in the culture. Fractionation of
cells indicated that the acetate was being used for the synthesis
of cell wall material, although this was not verified con-
clusively. The synthesis of dipicolinic acid was studied.
and it was found that this material was formed prior to the

appearance of heat resistant spores but after the appearance

of re
diphc

to de

Spor;
initi
Eluci
tap
at th
SPOru

COUtr

Lawrence E. Day

of refractile spores. Two enzymes. acetokinase and reduced
diphosphopyridine nucleotide (DPNH) oxidase, were studied

to determine the time of formation of heat resistance.
Acetokinase was observed to have no resistance to heat in
either the vegetative cell, intact spore, or an intermediate
stage, or in cell-free extracts of these forms. The DPNH
oxidase was found to develop stability towards heat during
sporulation. the development of this resistance being
initiated at the onset of sporulation. The study did not
elucidate the mechanism(s) which initiate sporulation. If
the pH and concentration of end products known to be present
at the time of sporulation were preset in a fresh culture,
sporulation was observed to occur no sooner than in a

control culture.

 

in

Do

M

pa;

SOME PHYSIOLOGICAL CHANGES OCCURRING

DURING THE SPORULATION OF

CLOSTRIDIUM BOTULINUM:

 

TYPE A

BY

Lawrence E. Day

A THESIS

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

durir

critj

exte:

 

(, Zs’VW/

sflyas

ACKNOWLEDGEMENTS

For his generous assistance and technical guidance
during the period of this investigation and for his kind
critical evaluation of this dissertation, I would like to
extend my deepest appreciation to Dr. Ralph N. Costilow.

I am also indebted to Dr. H. L. Sadoff for his helpful

and wise counsel.

ii

._._a_ 1 :-

INTROI

HISTO

rn

5—1 In r—t

TABLE OF CONTENTS

Page
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . l
HISTORICAL REVIEW . . . . . . . . . . . . . . . . . . 3
Nutritional and Environmental Requirements
for Sporulation 3
Replacement and Endotrophic Sporulation 7
The Role of Dipicolinic Acid in Sporulation 9
Nucleic Acids and Sporulation 12
Heat Resistant Enzymes of Spores 15
MATERIALS AND METHODS . . . . . . . . . . . . . . . . 1?
“SETS 0 O O O O O O O O O O O O O O O O O O O O O O 30
Sporulation in Replacement Media 32
Acetate as a replacement nutrient 32
Chemical groups of amino acids as
replacement nutrients 33
L-alanine and L-proline as replacement
nutrients 36
The L—arginine to L-ornithine cycle and
spore maturation 52
The Role of End Products of Cell Metabolism
in Sporulation 55
Nucleic Acid Synthesis and Sporulation 70
Synthesis of DPA and Poly-B-hydroxybutyric Acid 79
The Formation of Heat Resistant Enzymes During
Sporulation 82
DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 87
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . 98

BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . 101

APPENDIX . . . . . . . . . . . . . . . . . . . . . . . 109

iii

Tabb

4;

U1

Table

LIST OF TABLES

Tests for sporulation and acetic acid
utilization following replacement
of Q. botulinum in potassium acetate
and sodium acetate solutions . . . . . . .

Effect of 0.5% sodium acetate on sporulation
of g. botulinum following replacement in
a synthetic medium containing the amino
acids of Trypticase . . . . . . . . . . .

 

Tests for sporulation of g. botulinum following
replacement in solutions of various groups
of amino acids . . . . . . . . . . . . . .

Chromatography of L-alanine, L-proline
replacement medium before and after
sporulation of g. botulinum . . . . . . .

 

The distribution of C14 in a replacement
culture of Q. botulinum in L-alanine,
L-proline. and dl-alanine-l—C14 . . . . .

Replacement of Q. botulinum in solutions
containing L-alanine and L—proline,
and in L-isoleucine and L-proline . . . .

 

Replacement of Q. botulinum in solutions of
L—arginine and L—citrulline . m . . . . .

 

Replacement of Q. botulinum in a solution
containing L-arginine and L-alanine.
and in a solution containing L-ornithine
and L-alanine . . . . . . . . . . . . . .

The identification of acids isolated from
a sporulation medium following
sporulation of g, botulinum . . . . . . .

 

iv

Page

34

35

37

47

51

53

54

56

6O

Table

10

ll.

12.

13.

Table

10.

11.

12.

13.

14.

15.

Distribution of C14 in fractions from
spores of Q. botulinum produced in
sporulation medium containing 10 he of
acetic acid-l-Cl4 . . . . . . . . . . . .

Distribution of C14 in free spores and in
debris of a culture of Q. botulinum
in a sporulation medium containing
4 no of acetic acid-l-Cl4 . . . . . . . .

 

Utilization and distribution of C14 in spores
and in debris of a culture of Q. botulinum
in a sporulation medium with 10 uc of
acetic acid-l-Cl4 added at the onset of
sporulation . . . . . . . . . . . . . . .

Sporulation of Q. botulinum in a sporulation
medium containing valeric, propionic
and acetic acid . . . . . . . . . . . . .

 

Specific activities of the DPNH oxidase and
acetokinase extracted from vegetative
cells, swollen vegetative cells, and
mature spores of Q. botulinum . . . . . .

 

DPNH oxidase activity of extracts of
vegetative cells, swollen vegetative
cells. and mature spores of Q. botulinum
before and after heating at 80 C for
l min . . . . . . . . . . . . . . . . . .

 

Page

65

68

69

71

84

85

Sporulation cycle of Q. botulinum

The

LIST OF FIGURES

 

formation of refractile spores of 9.

Page

31

botulinum in a replacement solution
containing L-alanine and L-proline . . . . 39

 

The formation of refractile spores of g.
botulinum in a replacement solution
containing L-alanine and L-proline and
in L-proline solution and in sodium
acetate solution . . . . . . . . . . . . . 40

The formation of heat resistant spores
of Q. botulinum in a replacement
solution containing L-alanine and
L-proline . . . . . . . . . . . . . . . . 42

 

The effect of chloramphenicol on the
sporulation of Q. botulinum following
replacement in a solution containing
L-alanine and L-proline . . . . . . . . . 44

The formation of refractile spores of Q.
botulinum and the production of acetic
acid in a replacement solution containing
L-alanine and L-proline . . . . . . . . . 46

The formation of refractile spores of Q,
botulinum in a replacement solution
containing L-alanine, L-proline. and
acetic acid-l-Cl4 . . . . . . . . . . . . 49

 

The metabolic activity of cells of Q.
botulinum at various stages of growth
and sporulation . . . . . . . . . . . . . 57

vi

Figure

10.

ll.

12.

l3.

14.

15.

16.

Figure Page

 

 

 

9. The relationship of culture pH and ammonia

production during the sporulation of

Q, botulinum in sporulation medium . . . . 59
10. Acid production and sporulation of g,

botulinum . . . . . . . . . . . . . . . . 61
ll. Acetic acid concentration and acetic

acid-l-Cl4 utilization during growth

and sporulation of Q, botulinum . . . . . 63
12. Nucleic acid synthesis during the sporulation

of Q. botulinum in sporulation medium . . 73

 

13. The effect of 8-azaguanine on the growth and
on the sporulation of Q. botulinum . . . . 76

 

14. The effect of mitomycin C on the growth and
on the sporulation of_g. botulinum . . . . 78

 

15. The synthesis of nucleic acids by g. botulinum
during sporulation in a replacement

solution containing L-alanine, L-proline and
P32

 

O O O O O O I C O C O O O C O O O O O 80

16. The time of appearance of DPA, refractile
spores, and heat resistant spores of
IQ. botulinum in a sporulation medium . . . 81

vii

attribui
biologi:
of resi
a great
the nat
during

Sporula
a Probl
haVE be

USUallE

INTRODUCTION

The current interest in bacterial spores can be
attributed to the remarkable properties of these unique
biological entities. Spores possess an extremely high level
of resistance to various chemical and physical agents, and
a great amount of research has been carried out to determine
the nature of this resistance. Also, the events taking place
during sporulation are of interest to the biologist because
sporulation is fundamentally a process of cell differentiation.
a problem currently under much study. Sporulation studies
have been approached from many different viewpoints, and
usually each investigation has been concerned with only
one facet of the problem. These detailed studies are certain—
1y desirable and give insight into some of the problems
under investigation, but the results are not always related
to other events occurring in the cell during the same
period. This study, therefore, was undertaken to View the
process of sporulation in its entirety. The ultimate goals
were to relate some of the major events occurring during
sporulation, and to determine the event which initiates
sporulation if possible. With this in mind, many aspects

of sporulation were investigated. This involved using

the technique of replacement (Hardwick and Foster, 1952)

in an attempt to simplify environmental conditions during
sporulation. Studies were made of the nutritional require-
ments for sporulation, the production and utilization of
metabolic end products, the synthesis of nucleic acids
during sporulation, and the effects of inhibitors of these
synthetic processes. An investigation concerning the heat
resistance of certain enzymes of the vegetative cell, fore-
spore, and mature spore, and the time of appearance of this
resistance was also carried out.

From these data certain conclusions were made, and
some of the major steps occurring during sporogenesis were
clarified. Although the more subtle changes were not
studied, it is hoped that this investigation might give
further impetus to the elucidation of the processes taking
place during the transition from the vegetative cell to the

spore.

HISTORICAL REVIEW

Nutritional and Environmental Requirements
for Sporulation

 

Ordal (1956) stated three requirements necessary for
sporulation to take place; yi§., (a) the cells must be of
the sporogenous type, (b) the cells must be in the proper
physiological condition, and (c) the cells must be in the
proper environment. Schaeffer et a1. (1959) demonstrated
that the first of these requirements is under genetic
control by observing that a non spore-forming strain of

Bacillus subtilis could be transformed to the sporogenic

 

type by the deoxyribonucleic acid (DNA) extracted from a
spore-forming strain of the same organism. Not much is
known of the second requirement. In a well—synchronized
culture of a spore-forming bacterium, all cells will
sporulate simultaneously over a relatively short period

of time, but it is not known what event or mechanism triggers
the initial process leading to the formation of spores.
Nakata and Halvorson (1960) have shown that large amounts

of acetic acid and lesser amounts of pyruvic acid were
produced and accumulated in the medium in a culture of

Bacillus cereus growing in a glucose—yeast extract-salts

 

medium
sporul
from t
was ob
cell t
induce
leadir
(1960)

by 3—;

medium. After the period of logarithmic growth when
sporulation was initiated, the acids rapidly disappeared
from the medium, and the pH of the medium increased. It

was observed that during the transition from the vegetative
cell to the spore, a system which could oxidize acetate was
induced; this induction being the first recognizable step
leading to the formation of spores. Gollakota and Halvorson
(1960) demonstrated that this induction could be inhibited
by a-picolinic acid with the result that the formation of
spores was also inhibited.

A great deal of investigation has been carried out
concerning the proper environment and the necessary mineral
and nutritional requirements for sporulation of the aerobic
spore-formers, and these have been reviewed extensively
by Cook (1932), Knaysi (1948), Williams (1952), Stedman (1956),
and Halvorson(l957a).

Less is known of the nutritional and environmental
requirements for sporulation of the anaerobes. A medium of

4%Trypticasel was found to support sporulation of Clostridium

 

botulinum, type A, of about 95%»of the cells if the medium

was fortified with 1 ng/ml of thiamine (Day, 1960). Lund

 

lBaltimore Biological Laboratories, Baltimore, Maryland.

(1956)
Tryptic

lation

(1956) also observed the necessity of added thiamine in a
Trypticase medium in order to obtain a high degree of sporu-
lation with the Putrefactive Anaerobe, PA 3679.

wynne (1948) demonstrated that as the concentration
of salts or the ionic strength of the medium increased, the
degree of sporulation decreased with a culture of Q.
botulinum using Na2S04, NaCl, and KCl. At a concentration
of 2% NaCl in brain-heart-infusion medium, sporulation was
completely inhibited. The inhibitory effect of NaCl was

also noted by Leifson (1931), who reported that Q. botulinum

 

would sporulate in a basal medium of 1%.peptone only when
NHI4+ and P04. were added. The addition of 304‘ stimulated
sporulation somewhat.

The inhibition of sporulation by glucose has been
observed many times (Leifson, 1931; Kaplan and Williams,
1941; Blair, 1950), but the reasons for this inhibition are
not clear. Leifson (1931) has stated that the inhibitory
effect is due to the formation of acid, but Costilow (1962)
has shown that sporulation will take place after all of the
glucose has been utilized. Wynne (1948) reported that 0.8%

glucose in brain-heart-infusion broth did not adversely

affect sporulation of g. botulinum.

 

Blair (1950) working with the synthetic medium of

PM»

or

ac

am.

ar

m0]

Roessler and Brewer (1956) demonstrated that the deletion of
methionine and phenylalanine from this medium suppressed
the sporulation of g, botulinum. Sporulation was enhanced
by the addition of L-alanine, L-glutamic acid, glycine,
L-ornithine, sodium butyrate, sodium valerate, L—arginine
and L-proline. Perkins and Tsuji (1962) could not demon-
strate spores in the synthetic medium of Williams and Blair
(1950), but obtained good growth in a more complex synthetic
medium. This medium would support sporulation when the
level of L-arginine was raised to about 70 umoles/ml of
medium. Further study by these investigators revealed that
the L-arginine was metabolized through citrulline to
ornithine, and that the greater requirement for this amino
acid for sporulation was not due to incorporation of the
amino acid into spore material. Rather, it was thought that
a mole of adenosine triphosphate (ATP) was formed for each
mole of citrulline broken down to ornithine, which furnished
additional energy for the syntheses leading to sporulation.
Vinter (1962) found that the cystine requirement for
spores of g. cereus was about five times higher than that
for the vegetative cells. This demand for cystine occurred
in the period prior to the appearance of young refractile

forespores. At this time the organisms showed increased

d

resis*
methi:

(Vint'

was e
7.0-7
SporL

but I

the

anae
incr
note

more

resistance to X-rays. The requirement for alanine or
methionine during the sporulation process did not increase
(Vinter, 1960).

The pH optimum for the sporulation of g. botulinum
was established by Mohrke (1926) and Collier (1956) to be
7.0-7.2. Leifson (1931) stated that the pH at the time of
sporulation is of greater importance than the initial pH,
but he did not state what this value should be.

The effect of oxygen tension on.the sporulation of
the anaerobes is not clear. It was observed that various
anaerobic organisms exhibit different responses under
increasing oxygen tensions (Leifson, 1931). Sommer (1930)

noted that a broth culture of Q. botulinum will sporulate

 

more quickly when exposed to the air than when the culture
is sealed with vaseline, and Esty and Meyer (1922) reported
that small amounts of oxygen in the culture medium were
beneficial to sporogenesis. However, WYnne (1948) observed
that the level of sporulation in broth cultures of Q.
botulinum was identical in either oxygen or in a natural

gas atmosphere.

Replacement and Endotrophic Sporulation

Although it had been observed for many years that

the vegetative cells of the aerobic spore—forming bacteria

r11

V?

w‘

If)

(.0

(I)

will sporulate when placed in distilled water (Buchner, 1890;
Schreiber, 1896; and Knaysi, 1945), it was not until 1952
that this phenomenon was studied in detail (Hardwick and
Foster, 1952). They designated the technique of transferring
vegetative cells to another solution as replacement, and the
sporulation of an organism in the absence of exogenous
nutrients as endotrophic sporulation.

Collier (1956) was the first to observe endotrophic
sporulation in an anaerobic spore-former (Clostridium roseum).
Lund (1956) reported that the Putrefactive Anaerobe (PAr3679-h)
would not sporulate in distilled water and Day (1960)

reported that Q. botulinum would not sporulate when trans-

 

ferred to distilled water or buffer at various intervals of
growth. Neither was sporulation observed when the vegetative
cells of this organism were replaced in other solutions
containing various combinations of amino acids and vitamins,
solutions of enzymatically hydrolyzed casein, and solutions
of various anions and divalent cations known to be of
importance to sporulation in other and related species.
However, Day (1960) showed that 4% Trypticase broth used in
replacement supported sporulation to a high degree, and when
the individual amino acids known to be present in Trypticase

were combined as a synthetic medium and used as a replacement

'1)

’(J

'U

kl.

(D

r!-

solution, a significant degree of sporulation occurred.

The Role 9f Dipicolinic Acid ig_Sporulation

 

Powell (1953) was the first to isolate and identify
dipicolinic acid (DPA), pyridine-2,6-dicarboxylic acid,
from extracts of spores of Bacillus megaterium. Martin
and Foster (1957) attempted to elucidate the biochemical
pathway leading to the synthesis of DPA by testing various
amino acids and carboxylic acids as precursors of this
compound. They found L—glutamic acid, L-aspartic acid,
L-alanine, L-serine and L-proline to be the most efficient
precursors; and that CO2 could contribute 6.5% of the carbon
in DPA. Significantly, L-tryptophane did not contribute
to the synthesis. These workers felt that the formation of
DPA may be the result of a C4 and a C3 condensation, involving
either L—aspartic acid and pyruvic acid or L-alanine and
oxalacetic acid. Powell and Strange (1959) have postulated
the formation of DPA from a, e- diketopimelic acid.

Dipicolinic acid is a strong chelating agent and
occurs only in bacterial endospores in high concentrations
(4-15%.dry weight). It is synthesized just prior to the

formation of resistant spores and is completely lost during

germination (Halvorson and Hewitt, 1961). Its function in

10

sporogenesis is not known although several have been
suggested. The first of these is that DPA has some relation-
ship to the thermostability of the spore. It was noted»
early that the thermoresistance of the spore became apparent
immediately after the synthesis of DPA (Halvorson, 1957b;
Black et al., 1960). Lechowich (1958) pointed out that
sporulation at elevated temperatures resulted in spores which
were more thermostable and which contained a higher level

of DPA. It has also been known for some time that the heat
resistance of the spore is related to the calcium content

of the spore. Curran et a1. (1943) have noted the relation-
ship of the presence of Ca++ and heat stability in several
organisms. There may be an interrelationship between the
role of calcium and DPA in the heat resistance of the spore,
and the several mechanisms proposed for this have been
discussed by Halvorson and Howitt (1961). Young and Fitz—
James (1959a) found that the cortex of the spores of B.
cereus was formed between double layers of the forespore
membrane. When the cortex was about one-third formed, the
spore coat and the exosporium were laid down on the outer
side of the membrane; and at this time the forespore became
refractile and the uptake of calcium commenced. The

synthesis of dipicolinic acid followed. In a medium which

was deficient in calcium, DPA was synthesized only in amounts

'r1

In

11

proportional to the calcium present with the subsequent
lessening of refractility. waever, Halvorson (1957)
reported that the synthesis of DPA preceded or accompanied
the accumulation of Ca++. Shinagawa (1960) suspended the

vegetative cells of B. cereus in CaCl and observed complete

2
sporulation after 18 hours at 37 C. In this technique, the
calcium was essential and irreplaceable. It was found

that the spores thus produced contained a reduced amount

of DPA, were less heat resistant, and showed increased

sensitivity to HgCl and phenol. Analyses of various aerobic

2
spore—formers for nitrogen, carbohydrate, DPA, and phosphate
showed little correlation between the concentrations of
these materials and the heat resistance of the spores
(walker, 1961). It was observed that as the molar concen-
tration of Mg++ increased with respect to the concentrations
of DPA and Ca++ the heat resistance decreased. Portellada
(1961) has made the interesting observation that 60 ug/ml of
DPA inhibited endotrophic sporulation, but in a chemically
defined medium concentrations of DPA up to 80 ug/ml did

not interfere with sporulation. In fact, it was reported
that 20-25% of the DPA was consumed (Portellanda, 1960).

DPA.has also been shown to stimulate the electron

transport system in_§. cereus (Halvorson et al., 1958),

and that this involved a soluble DPNH oxidase (Doi and

12

Halvorson, 1961). However, Simmons (1961) observed that

the DPNH oxidase of Q. botulinum was no more active in the

 

presence of DPA than in its absence.

Other roles that have been assigned to DPA include
stimulation of germination (Rieman and Ordal, 1960; O'Conner
and Halvorson, 1960); the masking of activity of dormant
enzymes in spores (Riemann, 1961); and the rendering of
stability to enzymes through calcium DPA complexes (Young,
1959).

It is apparent that the function of DPA in the
spore is not yet clear and that it may have more than one
role. The fact that heat resistance develops after the
synthesis of DPA (Halvorson, 1957; Black et al., 1960;
Hashimoto et al., 1960) indicates that DPA may play some

other primary role which affects thermoresistance secondarily.

Nucleic Acids and Sporulation

Only a minimum amount of work has been reported which
describes the nucleic acid metabolism of the cell undergoing
sporogenesis. The investigations of Delaporte (1950),
Robinow (1956), and Fitz-James and YCung (1959) indicate
that the nuclear material of the spore is preformed in the

vegetative cell and is enclosed in the spore during

sporulation. The latter workers demonstrated that during

13

the sporulation of B. cereus two nuclear bodies of DNA

were present but only one of these was incorporated into the
mature spore. They found some variants of the same organism
which did not synthesize DNA during sporulation and some

that did, however the spore DNA always arose from that of

the vegetative cell and was not a g§_ggyg synthesis.
Fitz—James (1954) reported that ribonucleic acid (RNA)
comprises 50%.of the total phosphorus of the spores of

Ig. cereus and 3—4% of the dry weight. Hewever in B.
megaterium the RNA accounted for only 25%.of the phosphorus.
The DNA of the spores of both species was about the same:

1% of the dry weight. In endotrophically produced spores of
the same two organisms, Hedson and Beck (1960) found the

DNA content to be less than one-half of that found in the
vegetative cells, and their results indicate that one discrete
nuclear body preformed in the vegetative cell is enclosed

in each spore. They also found that about 99%»of the nucleic
acids of the spores which were formed during endotrophic
sporulation were synthesized from the pre-existing nucleic
acids of the cell, allowing the degree of new syntheses to
be about 1%. Other results indicate, however, that synthesis
of some nucleic acid is essential to sporulation. Foster

and Perry (1954) have shown that the purine analogue,

14

2,6-diaminopurine, will inhibit endotrophic sporulation of
Bacillus mycoides. YOung and Fitz—James (1959b) working

with B. cereus var alesti demonstrated that there was no
synthesis of RNA once sporulation was initiated, although
there may have been some RNA turnover. The purine analogue,
8-azaguanine, inhibited sporulation at a concentration of

100 ug/ml at any time during the vegetative growth of the
organism, but if the antimetabolite was added to the culture
after it had become committed to sporulation no inhibition
occurred. Day (1960) noted that the addition of vancomycin
to a sporulating culture of g. botulinum resulted in the
inhibition of sporulation after a lag period of about 10
hours. Vancomycin at that time was believed to act specifically
through the inhibition of net RNA synthesis (Jordan and Innis,
1959). Thus, it was theorized that RNA synthesis was neces—
sary through some intermediate stage of sporuIation and those
cells sporulating in the presence of the RNA inhibitor had
already completed this step in sporogenesis. More recent
data on the mode of action of this compound casts some doubt
on the validity of this conclusion. This will be discussed

later.

15
Heat—resistant Enzymes g: Spores

Most of the reports concerned with the enzymes of
spores have considered only the aerobic spore—formers; few
of these have reported observations on the thermostability
of the individual enzymes or enzymic systems involved.
Stewart and Halvorson (1953) first demonstrated the presence
of alanine racemase in the spores of Bacillus terminalis
which retained 97% of its original activity after heating
for 120 min at 80 C. Lawrence and Halvorson (1954)
isolated two species of catalase from the spores of the
same organism, B. terminalis later shown to be_§. cereus,
which differed in heat resistance but could not be separated
by centrifugation. Sadoff (1961) reported that the heat
resistant catalase first appeared at the end of the
exponential growth phase at a rate equal to the rate of
synthesis of DPA; however, the maximum in catalase activity
had been achieved by the time that only 2% of the DPA
had been formed. Using the Ouchterlony immuno-diffusion
technique, it was shown that the heat labile and the heat
resistant enzymes were the same proteins immunologically.
Bach and Sadoff (1962) isolatedetglucose dehydrogenase
from cells of B. cereus in the initial stages of sporogenesis,

but the enzyme was not present in cells during the logarithmic

16

phase of growth. The enzyme extracted from cells in an early
stage of sporulation was identical to that extracted from
mature spores in kinetic characteristics, immunologic
characteristics, and in chromatographic and electrophoretic
experiments. However, the enzyme from mature spores was
less heat stable than that extracted from an early stage in
sporulation, and the same enzyme from germinated spores was
quite labile and possessed a higher pH optimum for enzymatic
activity. Simmons (1961) detected a number of the enzymes
of the Embden-Meyerhof—Parnas (EMP) pathway in the extracts
of the spores of Q. botulinum. These enzymes along with
diaphorase, acetokinase, phosphotransacetylase and coenzyme
A transphorase were present in the spores at lower levels

of activity than that found in the vegetative cells; but
DPNH’oxidase in the spore extracts had higher activities

and much more thermostability than a DPNH oxidase of

vegetative cells.

from t
invest
which

When 5
about

harves
Water,
The su
inOcul

which

MATERIALS AND METHODS

Clostridium botulinum, 62 A, originally obtained
from the American Type Culture Collection was used in all
investigations. The culture was maintained in spore suspensions
which were produced in 4% Trypticase broth (Day, 1960).

When spores were present in the growth medium at levels of
about 90—95%.of the final population, the spores were
harvested by centrifugation. washed twice in sterile distilled
water, and resuspended in 0.067 M.phosphate buffer, pH 7.0.
The suspensions were stored at 5 C until needed. Subsequent
inoculations were made from portions of the suspension

which had been heated to 80 C for 10 min.

A high degree of synchrony in the culture was desired
in order to study the various aspects of sporogenesis. This
was achieved by first inoculating 50 m1 of 4% Trypticase broth
containing 1 ug/ml of thiamine hydrochloride and 1 mg/ml of
sodium thioglycollate with 5 ml of a heat—shocked spore
suspension. This culture was incubated at 37 C for 10 hr.
after which it was added to 150 ml of the same medium without
the sodium thioglycollate. Anaerobiosis was then maintained
in this 200 ml culture by fitting the flask with appropriate

gas inlet and exhaust tubes, and allowing natural gas to

17

18

flow slowly through the culture. The entire apparatus was
placed in a water bath at 37 C. Samples of the culture were
then removed at various times for further study as required.
Depending on the type of study being carried out, variations
in the volumes of cultures used were sometimes required.

It was shown by Day (1960) that replacement of the
vegetative cells of g. botulinum could be carried out only
in an atmosphere devoid of oxygen. The replacement was
achieved by manipulation of the cultures in a plexiglass
chamber described by Day (1960) which was filled with an
atmosphere of nitrogen. The cells were removed from the
growth medium by centrifugation in a type G Servall Angle
Centrifuge,2 and the cells resuspended in an equal volume
of the appropriate replacement medium. Anaerobiosis was
maintained either by the addition of 0.1% sodium thioglycollate
to the replacement solution or by the use of the Brewer
anaerobic jar.

The numbers of cells and spores were counted by two
methods. Refractile spores could be counted in a highly
reproducible manner by use of the Petroff-Hausser Bacteria

Counter.3 When the cells present in a suspension numbered

 

Ivan Sorvall, Inc., Norwalk, Conn.

3C. A. Hausser & Son, Philadelphia, Pa.

COUI‘.

ml.

19

above 108/m1, a 1:10 dilution of the suspension was made

and the resulting suspension utilized for enumeration of
cells. Twenty squares of the chamber were counted for

both total counts and for spores and the average of this
count multiplied by 2 x 107 gave the number of cells per

ml. The bodies counted as mature spores were those appearing
to be highly refractile with a well-defined edge. Free
spores were those which were free of the sporangia. Immature
forespores were those bodies which showed a slight degree

of refractility and a diffuse edge and were still within the
sporangium.

Since the Petroff—Hausser Bacteria Counter does not
provide the means of differentiating between viable and non-
viable cells, the determination of heat resistant spores
required the use of a method which counts viable cells.

The yeast-extract-starch—bicarbonate agar (YESB agar) of
wynne et al. (1955) and oval tubes were utilized for this
purpose. In order to enumerate the number of spores,
samples of the suspension to be counted were first subjected
to 80 C for 10 min. Appropriate ten-fold dilutions were
made, and four tubes of YESB agar were poured per dilution.

The method of Kinnory et a1. (1958) was used to

assay Cl4 activity in various cell fractions. Spores which

20

had been grown in 4% Trypticase broth containing acetic
acid-l—Cl4 were collected by centrifugation and fractionated
as follows:

Spores

washed twice, resuspended in(1067 M
phosphate buffer, pH 7.1

heated 80 C, 10 min, centrifuged

 

 

I
dipicolinic acid heat-shocked spores
(DPA)

broken in 5% trichlor-
acetic acid (TCA) in
Omnimixer, centrifuged

 

 

TCA soluble fraction, protein, lipid, nucleic acids,
including residual DPA mucocomplexes

75% ethyl alcohol
50 C, 1 hr, centrifuged

 

 

 

 

 

[
lipids proteins, nucleic acids, muco-
complexes
5%.TCA, 100 C, 30 min,
centrifuged
l
nucleic acids proteins, mucocomplexes
8N H SO , 120 C, 5 hr,
filtere on membrane
filter
(

 

amino acids humins

A 1 ml
20‘ml

lined

anot
acid
L‘Pr

Zuly

21

A 1 m1 sample of each of the cell fractions was placed in a

20 m1 radioactivity counting bottle provided with a foil-

lined cap. The fraction was evaporated at 60 C, and the
residue redissolved in a 1 m1 of formamide. To this was

added 8.6 m1 of absolute alcohol and 10.4 ml of 0.58%
2,5-diphenyloxazole in toluene. All samples were prepared

in duplicate and were counted in a Tricarb Scintillation
Spectrometer with Control Model 314-DC4. The high voltage
setting was 880 volts.

The insoluble humin which was collected on a type

G-5 membrane filter5 in the last step of the cell fractionation
was assayed for C14 activity using a NUclear-Chicago thin—
window gas-flow counter,6 Model 30373, and the counts
registered on a Nuclear-Chicago Model 182 scaling unit. The
voltage setting was 1300 volts.

When the C14 was present as dl—alanine-l-Cl4
another method of assay was used. The radioactive amino

acid was added to a replacement solution of L-alanine and

L-proline containing a sporulating culture of Q. botulinum.

 

Any CO evolved was flushed from the culture by passing a

2

 

4Packard Instrument Company, Inc., La Grange, Illinois.
5Gelman Instrument Company, Chelsea, Michigan.

6Nuclear Instrument & Chemical Corporation, Chicago,
Illinois.

22

stream of natural gas through the culture and then through a
column containing 20% KOH. The culture was acidified at the

. To

end of sporulation in order to release dissolved CO2

assay for C14 activity in CO a 1 m1 sample of the KOH

2:
solution was placed in a 20 ml counting bottle with 15 ml
of a scintillation solution. This scintillation solution
allows for the counting of alkaline aqueous samples. The
composition of the solution is given in the Appendix, Table
A-III. The counts were made in the Tricarb Scintillation
Spectrometer as described above.

In those experiments in which radioactive phosphorus
(P32) was used to assess the synthesis of nucleic acids, the
activity of the various fractions ‘was measured by use of.
the Geiger-Mueller Counter7 with an end-window tube, model 3,
1.6 mg/cmz. The counts were recorded by a Berkeley Decimal
Scaler,8 Model 100, with a high voltage setting of 1250
volts. Counting was carried out for varying lengths of time
depending on the level of activity of the samples. The
method of Hanawalt (1959) was used in assaying the uptake

of P32 by various cell fractions. This method allows the

 

7Radiation Counter Laboratories, Inc., Skokie,

Illinois.

8Berkeley Scientific Corporation, Richmond,
California.

23

assay of small amounts of the cell fractions on membrane
filters directly. A 0.2 ml sample of cell suspension was
filtered by vacuum through a type G-5 filter, 1 inch in
diameter, immediately after being added to 5.0 ml of ice

cold 5% TCA. The residue was then rinsed with 5.0 m1 of
distilled water. The filter was air-dried and counted.

This count represented step 1 and contained the TCA insoluble
fraction. The filter was then placed in a polyethylene cup
containing 75% ethyl alcohol and allowed to remain for 2

hr at room temperature. The filter was then placed on the
filter apparatus and rinsed with 5.0 m1 of 50% ethyl alcohol.
The filter was again dried and counted. This step removed
the lipid portion and was designated step 2. The filter was‘
placed in the polyethylene cup, 3.0 ml of 2N KOH were added,
and the mixture incubated at 37 C for 18 hr. This treatment
dissolves the membrane filter. The mixture was chilled to

5 C in an ice bath, 3.0 m1 of 10% TCA added and filtered
through a new membrane filter. The residue was rinsed with
5.0 m1 of 5% TCA, and the filter was dried and counted.

This was designated as step 3. The radioactivity incorporated
into RNA was considered to be the counts/minute of step 2
minus the counts/minute of step 3. The radioactivity due

32 . . .
to the P in DNA.was the counts/minute of step 3 minus

2% of the counts/minute of step 1 (Hanawalt, 1959).

24

The metabolic activity of cells in the various stages
of growth and sporulation was determined using the warburg
respirometer and manometric techniques as outlined by Umbreit,
Burris and Stauffer (1959).

The organic acids resulting from the metabolism of
IQ. botulinum in 4% Trypticase broth were isolated on a
column of Celite9 containing an internal indicator of 3-
(4-anilino,1-naphthylazo) 2,7-naphthylene disulfonic acid
monoammonium salt. The column was prepared as described by
Wiseman and Irvin (1957), the only modification being the
substitution of the above mentioned indicator for alphamine
red-R indicator due to the lack of availability of the latter.
The developing solvents consisted of various percentages by
volume of acetone in Skellysolve B.10 These were also
prepared as described by Wiseman and Irvin (1957). The lower
concentrations of acetone elute the higher molecular weight
organic acids. The resulting eluates were titrated with
0.005N Ba(OH)2. Standards of known amounts of acetic and
butyric acids placed on the column and eluted resulted in
95-105% recovery.

The method for the assay of the DPA of bacterial

 

9Johns—Manville Products, Detroit, Michigan.

1 . . . .
0Skelly Oil Company, St. Louis, Missouri.

25

cells and spores was that of Janssen, Lund and Anderson
(1958). The cells harvested from 200 m1 of medium were
resuspended in 5 ml of distilled water and autoclaved for
15 min at 15 psi. This suspension was acidified with 0.1 ml
of 1.0 N acetic acid and allowed to stand at room temperature
for 1 hr. The suspension was clarified by centrifugation.
One ml of the color reagent was added to 4 m1 of the super—
natant. This consisted of a 1% solution of Fe(NHZ4)2
(804)2-6H20 and 1%.ascorbic acid in 0.5 M acetate buffer,
pH 5.5. The optical density was read at 440 mu. A
standard curve was prepared using known amounts of DPA.
Poly-B-hydroxybutyric acid was assayed for by the
method of Law and Slepecky (1961). The cells were harvested
by centrifugation in polypropylene tubes which had been
washed in ethanol and hot chloroform. The cells were then
placed in a volume of sodium hypochlorite equal to the volume
of the original growth medium and allowed to stand for 1
hr at 37 C. The suspension was again centrifuged, washed
with water and with acetone and alcohol. Boiling chloroform
was then added to extract the polymer. This step was
repeated three times. The chloroform was then evaporated
and 10 ml of concentrated HIZSO4 added. The tube was stoppered

and heated for 10 min at 100 C in a water bath. After

cooling, the sample was placed in a silica cuvette and the

26

absorbancy at 235 mu measured in a Beckman, model DU,ll

spectrophotometer.

Acetyl posphate was assayed by the method described
by Lipmann and Tuttle (1945) and is based on the reaction
of acyl phosphates with hydroxylamine to form hydroxamic
acids. These complex in the presence of ferric chloride to
form violet compounds. The absorbancy was measured at
540 mu in a colorimeter, and succinic anhydride was used as
a standard.

Paper chromatography was used for the isolation and
identification of several volatile organic acids and several
amino acids. The method of Kennedy and Barker (1951) was
used for the identification of unknown volatile acids.

Due to the volatility of these acids, they were first con-
verted to the non-volatile ammonium salts. After the acids
had been eluted from the Celite column, 1.0 m1 of con-
centrated NHfiOH was added to each sample. The solvent was
evaporated on a steam bath and the aqueous portion used to
spot the chromatogram. A spot of 0.01 ml was applied at the
origin on a 45 x 25 cm piece of Whatman No. 1, chromato-

graphic grade filter paper. The paper was fixed into a

cylinder, stapled and placed in a battery jar containing

 

1Beckman Instruments, Inc., Fullerton, California.

27

200 ml of a solution of ethanol and concentrated NH4OH in
the ratio of 100:1. This was allowed to develop at room
temperature for 6—8 hr. The paper was then dried at 100 C
for 5 min, and sprayed with a solution of 50 mg of bromphenol
blue and 211 mg of citric acid in 100 m1 of distilled water.
Samples of known acids were run in the same manner.
Two-dimensional ascending paper chromatography was
also used in the identification of amino acids, the solvent
system being a solution of isopropanol-acetic acid-water
in a ratio of 3:1:1 by volume. The papers in this case
, were dried in the hood at room temperature for 3 hr. The
ninhydrin spray was prepared by dissolving 0.2 g of ninhydrin
in 100 m1 of water which was saturated with n-butanol.
After spraying the chromatograms, the papers were placed in
an oven at 100 C for 5 min. Standards of known amino acids
were also prepared in the same manner.
Cells and spores were brdken by use of either the
VirTis 945? Homogenizer12 or the Servall Omni-Mixer13

fitted with a Micro-Attachment. With the former, about 3 g

of size no. 100 Superbrite glass beads14 and 3 m1 of cell

 

12The VirTis Company, Inc., Gardiner, New Yerk.

13Ivan Sorvall, Inc., Norwalk, Connecticut.

4Minnesota Mining and Manufacturing Company, St. Paul,
Minnesota.

28

suspension were placed in the glass cup. The cup was placed
in the plexiglas cylinder of the homogenizer, and ethanol
from a cold bath at -20 F was circulated around the cup.

The homogenizer was run at a setting of 100 for about 3 min
for cells and for about 10 min for spores. With the Omni-
Mixer, the ratio of beads to cells by weight was about 6:1.
In this case, the cup was chilled in an ice bath prior to
and during breakage of the cells.

Cell-free extracts were prepared by breakage of the
cells in 0.05 tris buffer, pH 7.1. After breakage the
suspension was clarified by centrifugation at 30,000 rcf
for 1 hr, and the supernatant dialyzed against distilled
water at 5 C for 15-18 hr.

DPNH oxidase activity was assayed by measuring the
decrease in optical density at 340 mu with DPNH as substrate.
To determine the thermostability of the enzyme, it was
assayed immediately after breakage of the cells. The
extract was then heated at 80 C for l min and again assayed.
The activities were compared.

The assay for acetokinase activity was that given
by Rose et a1. (1954) and is based on the principal described
previously for the assay of acetyl phosphate (Lipmann and
Tuttle, 1945). The thermostability of acetokinase was

determined as f'orlPNH oxidase.

29

Protein concentrations were measured by the method
of Stadtman et a1. (1951) and involves the precipitation of
protein by trichloroacetic acid with the resulting turbidity
being measured in the colorimeter at 540 mu. A standard

curve was prepared using known quantities of lysozyme.

RESULTS

Sporulation of Q. botulinum is first indicated

 

morphologically by theswelling of the vegetative cell at one
extremity. This occurs at the conclusion of the logarithmic
growth phase when the maximum number of cells are present in
a culture of 4% Trypticase. In a well synchronized culture
about 90% of the cells initiate the sporulation sequence

at the same time. Two to three hours after swelling has
begun, the larger end of the cell begins to show differences
in refractility. This particular stage of development has
been termed the immature forespore (Vinter, 1962a), and is
characterized by a refractile area with a diffuse edge within
the swollen end of the cell. This refractility continues

to increase, and the edge becomes clearly delineated until

the highly refractile spore is observed within the sporangium.
During the next 12-36 hr the spore is freed from the sporangium
by autolysis. This is the sequence of sporulation from a
morphological viewpoint and is illustrated in Fig. 1. These
are the phases occurring during the transition from the
vegetative cell to the free spore that were considered in

this investigation.

30

31

IMMATURE
FORESPORE

///,/£" «Innis» "\\\\Sk
flswouzu MATURE SPORE

EGETATIVE .3
CELL IN SPORANGIUM
veeamnve ""55 0
CELL SPORE
vase TATIVE
GROWTH GERMINATION
V

Figure l. Sporulation cycle of g. botulinum.

32
Sporulation_ip Replacement Media

Previous work (Day, 1960) demonstrated that when
cells of g. botulinum were replaced in fresh 4% Trypticase
broth, sporulation occurred at the same time as in an un—
replaced control culture. The amino acids known to be
present in Trypticase could be combined into a synthetic
medium which would support some sporulation in replacement.
It was noted during the course of the present investigation
that it was the immature forespore that could complete the
final stages of sporulation to the mature spore in this amino
acid replacement medium. Those cells which had not yet
reached the forespore stage could progress no further in the
sporulation cycle in this particular replacement medium.

The first phase of this investigation was to determine the
minimum nutrient requirements for this spore maturation
process.

Acetate_a§ a replacement nutrient: Previous work
indicated that acetate might play some important role in
sporulation. In order to establish if acetate would support
sporulation in replacement, a 15 hr culture in which about
4% of the cells had completed sporulation was harvested and
resuspended in a solution of potassium acetate containing

6 uc of acetic acid—l—Cl4. The system was agitated with

33

natural gas which was passed through 20% KOH in order to

trap any evolved CO A similar experiment was run utilizing

2.
sodium acetate in phosphate buffer, and in this experiment

the acetic acid was isolated by column chromatography

(Wiseman and Irvin, 1957) and titrated to determine the

extent of utilization. The replacement cultures were acidified
at the end of the incubation period to release any dissolved
C02. The results of these two experiments are given in

Table 1. There were no spores formed in either replacement
solution and no utilization of acetate.

It was known from data obtained from the Baltimore
Biological Laboratories that Trypticase contains a high level
of acetate, and analysis of various samples of this product
revealed that acetate was present in amounts from 3.5—5.4%
by weight. Since the acetate was not observed to support
sporulation when used alone in a replacement system, it was
added to a synthetic medium containing the amino acids of
Trypticase, and the cells replaced in this medium. The
replacement medium of amino acids and acetate resulted in
no significantly higher level of sporulation than did the

control medium containing the amino acids alone (Table 2).

Chemical groups 9; amino acids as replacement nutrients:

 

In an attempt to determine which amino acids contribute to

the excellence of Trypticase as a sporulation medium, the

34

Table 1. Tests for sporulation and acetic acid utilization
following replacement of Q. botulinum cells in
potassium acetate and sodium acetate solutions.

 

 

 

 

 

Total Spores* Cts/min mg/ml
Popula-
tion* acetate CO2 acetate
Experiment 1**
. . 9 7 6
Initial (15 hr) 1.4X10 5.0X1O 5.4X10 185 ---
Final (39 hr) 1.0X109- (1.0X107 5.5x106 247 ---
Experiment 2***
. . 8 7 5
Initial (15 hr) 7.2X10 4.3X10 4.2X10 101 2.898
Final (37 hr) 1.3x1o8 <1.ox107 4.2x105 229 2.876

 

*Counts were made with the Petroff-Hausser Bacteria
Counter.

*fThe replacement solution contained 2.0 mg/ml
potassium acetate and 6.0 no acetic acid-l-Cl4 in aqueous
solutions at pH 7.3, cultures were incubated at 37 C in a
water bath.

***The replacement solution contained 3.0 mg/ml sodium
acetate, 5.0 no acetic acid-l-Cl4 in 0.067 M phosphate
buffer, pH 7.4; incubated at 37 C in water bath.

35

Table 2. Effect of 0.5%»sodium acetate on sporulation of
Q, botulinum following replacement in a synthetic
medium containing the amino acids of Trypticase.

 

 

Amino acids* and

Amino acids*

 

acetate alone
Culture age at replacement 24 hr 24 hr
Time of incubation 23 hr 23 hr
Initial population** 3.0 X 108 4.2 X 108
Final population** 8.0 X 107 9.8 X 107
Initial Spores** 1.0 X 106 2.0 X 106
Final spores** 1.1 X 107 1.3 X 107

 

*Composition of the amino acid medium is given in
the Appendix, Table I—A. Media were in screw-cap vials
with 0.1% sodium thioglycollate and 0.067 M phosphate buffer,

pH 7.2, final volume 10 m1. Incubated at 37 C.

**Counts were made with the Petroff—Hausser Bacteria

Counter.

36

amino acids known to be present in Trypticase were grouped
according to their distinctive chemical characteristics,
i.e., the monoaminomonocarboxylic amino acids, monoaminodi—
carboxylic amino acids, sulfur-containing amino acids,
basic amino acids, aromatic amino acids, and the heterocyclic
amino acids. The acids in each group were combined in the
same concentrations as found in Trypticase to prepare media
to be used in replacement. Table 3 gives the results of
replacement in these groups of amino acids. None of these
would support further sporulation. The control in this
instance was the increase in the number of spores observed
in a medium of 4% Trypticase.

L-alanine and L:proline as replacement nutrients:
The Stickland Reaction is one of the principal means by which
.g. botulinum can obtain energy when growing on an amino acid
medium as the sole source of carbon and nitrogen (Clifton,
1940). This is a coupled reaction between two amino acids,
one acting as a hydrogen acceptor and the other as a hydrogen
donor. One of the most common pairs of amino acids used
by the Clostridia is L-alanine and L—proline (Stickland,

1935a,b), and it is known that g. botulinum rapidly ferments

 

this pair of amino acids (Costilow, 1962). Thus, if an

energy source was the only limitation, these two amino acids

would be expected to support sporulation to the same extent

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37

 

 

 

 

 

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«Edfivmz

mmm ousuaso

 

 

 

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msoausaon GA usofioomHmon mQH3OHHom Edcflasuon .w.mo coaumasuomn How muons .m magma

38

as did the synthetic amino acid medium previously described.

Fig. 2 shows the results of replacement of the cells
of a 12-hr culture of g. botulinum in a solution of L-alanine
and L—proline. At the time of replacement about 40%»of the
cells were in the immature forespore stage and about 1%
of the total population had completed sporulation. During
the next 25 hr most of those cells which had reached the
forespore stage developed to maturity in the L-alanine,
L—proline solution, while those which had not yet reached
the forespore stage showed no further development. The
level of sporulation in the control reached about 93% of
the final population .

If a culture was allowed to proceed in development
until practically all of the cells had reached the fore-
spore phase before replacement in an L-alanine and L-proline
replacement solution, sporulation in replacement proceeded
as in the control culture (Fig. 3). At the time of
replacement there were about 10%.of the final mature spore
population already present, and practically all cells had
advanced beyond the swollen state. In this particular
experiment, cells were also replaced in a sodium acetate
solution and in an L-proline solution. There was no

increase in spore numbers in either of these replacement

solutions.

 

39

 

 

 

 

CONTROL
. ——.O
on o/
O .
ALANINE-PROLINE
A
A/A
Q; A
i ./
K.
<3 7‘"
(a A
C)
~J
XWTIME OF REPLACEMENT
6 1 r 1
IO 20 30 4O
TIME-HOURS
Figure 2. The formation of refractile spores of Q. botulinum

 

in a replacement solution containing L-alanine
and L-proline. The concentration of amino acids
was 2 mg/ml in 0.067 M phosphate buffer, pH 7.2.
The final volume of 10 ml was in screw-cap vials
with 0.1% sodium thioglycollate. Incubation
temperature was 37 C. Replacement was at 11 hr.
The control was replaced in the same sporulation
medium.

L06 OF NO.

 

7
IO

Figure 3.

4O

 

\

 

 

X0
x0

TIME OF REPLACEMENT

CONTROL

ALANINE - PROLINE
SODIUM ACETATE
PROLINE

OXD.

l l l L

20 30 4O 50
TIME -HOURS

The formation of refractile spores of Q. botulinum
in a replacement solution containing L—alanine and
L-proline, and in L-proline solution and in

sodium acetate solution. The concentration in

all solutions was 2 mg/ml in 0.067 M phosphate
buffer, pH 7.2. The final volume of 10 ml was

in screw—cap vials with 0.1% sodium thioglycollate.
Incubation temperature was 37 C. Replacement was
at 13 hr. The control was replaced in the same
sporulation medium.

 

41

The spores that were observed in these experiments
were spores that were counted in the Petroff—Hausser
counting chamber; and, thus, there was no assurance that they
were heat resistant spores. In order to determine if these
were fully mature, heat resistant spores and not just
refractile spore forms, cells were again replaced in a
solution of L—alanine and L—proline, and the numbers of
heat resistant spores determined. This was achieved by heat—
shocking samples of the replacement culture at 80 C for 10
min and plating appropriate dilutions of the samples in oval
tubes with the YESB agar of wynne (1948). Fig. 4 illustrates
that the spores thus formed in a replacement medium of L-
alanine and L-proline were heat resistant. The numbers of
spores formed in the replacement environment were not as
great as those formed in the Trypticase control due to the
fact that a large fraction of the cells had not yet attained
the proper stage of development.

I The results to this point indicated that the energy-
yielding Stickland Reaction involving L—alanine as reductant
and L-proline as oxidant could support that step in the
sporulation cycle of g. botulinum from the immature forespore
to the mature spore. The overall reaction is illustrated

as follows:

 

42

 

 

 

- ——: O
8 (3”,..C>
O/ CONTROL
. O .
C) O"'
2? ,l’TT,
t5 .,' ALANINF-PROLINE
Q 7‘:- /
<3
q 0/0
J.
5 L 1 1 I 1 1 1 4
I5 20 25 3O 35 4O 45 50
TWME-HOURS
Figure 4. The formation of heat resistant spores of Q.

botulinum in a replacement solution containing
L—alanine and L—proline. The concentration of
amino acids was 2 mg/ml in .067 M phosphate
buffer, pH 7.2. The final volume was 50 ml

in a flask stirred with natural gas. Incubation
was at 37 C in a water bath. Replacement was

at 16 hr.

43

 

 

CH3CHNH2COOH + 2 Héc CH2 + 2H20 ~;
I
HZC CHCOOH
\ /
N
H
L-alanine L—proline

CH COOH + 2NH2(CH2)4COOH + NH3 + CO2

acetic acid O-aminovaleric acid

This did not, however, preclude the possibility that some
synthetic reactions requiring either the amino acids or the
products of fermentation were taking place at this time.

In order to determine if proteins were being synthesized
during the developmental step in sporulation which could

be supported solely by L-alanine and L-proline, chloramphenicol
was added to a culture replaced in L-alanine and L-proline
and to a culture replacedin the same sporulation medium.

In this experiment, one culture was replaced early in the
sporulation cycle when about 50% of the cells were in the
forespore stage and another culture when almost the entire
population was at this stage of development. Figure 5a
represents sporulation in replacement of those cultures
which were replaced before all cells had reached the

forespore stage. The presence of chloramphenicol had no

9T

e--

 

LOG OF NO.

 

IO

Figure 5.

44

 

 

/2

‘mTIME or REPLACEMENT

l l l l l 1 l I

I4 IO 22 26 3O 34 38 42

 

/.— g.
M: 3

[ATK— TIME or REPLACEMENT

l

’I o 4%» TRYPTICASE
0 4°/o TRYPTICASE+CAPC
A ALANINE-PROLINE

A AL ANIN E-PROLINE + CAPC

J 1 1 1 1 1 1 I

I4 I8 22 26 3O 34 38 42
TIME-HOURS

The effect of chloramphenicol on sporulation of g,
botulinum, following replacement in a solution
containing L-alanine and L-proline. The amino acid
concentration was 2 mg/ml in 0.067 M phosphate
buffer, pH 7.3. The final volume of 10 ml was in
screw-cap vials with 0.1%.sodium thioglycollate.
The concentration of chloramphenicol (CAPC) was
100 ug/ml. Replacement was 13.5 hr (a) and 16

hr (b). The incubation temperature was 37 C.

Cell counts were made with the Petroff—Hausser
Bacteria Counter.

 

45

effect on the maturation of spores in solutions containing
L-alanine and L-proline. Hewever, the culture in 4%
Trypticase with added chloramphenicol reached only the level
of sporulation observed in the cultures in L—alanine and
L-proline, indicating that protein synthesis was necessary
for that step in sporulation from the swollen vegetative
cell to the immature forespore. When almost all cells were
in the forespore stage at the time of replacement, sporulation
reached the same final level in all cultures even in the
presence of chloramphenicol (Fig. 5b). These data indicate
that no protein synthesis occurs during sporulation in
a solution of L—alanine and L—proline.

.Assays were run for Stickland reaction products of
L-alanine, L-proline fermentation to insure that the
reaction was in operation during the spore maturation process.
Samples were removed from a culture replaced in a solution of
L-alanine and L-proline at various times, the cells removed
by centrifugation, and the supernatant fractionated on a
Celite column (Wiseman and Irvin, 1957). Only one band
appeared on the column during elution with solvents con-
taining concentrations of acetone at 5% or higher. This
band was identified as acetic acid by the method of Kennedy
and Barker (1951). The accumulation of this acid during

sporulation is evident in Fig. 6.

46

 

 

 

 

 

 

 

9"o SPORES
A ACETATE
()--C> O
o/
2 / r‘" ‘ 3””
E a» o /
Q: ‘ \’
O E
q dbe \
TIME OF REPLACEMENT °‘
u,
l
I
#4
71L 1 1 1 1
I5 20 25 3O 35
TIME-HOURS
Figure 6. The formation of refractile spores of g, botulinum

 

and the production of acetic acid in a replacement
solution containing L—alanine and L-proline.

Amino acid concentration was 2 mg/ml in 0.067 M
phosphate buffer, pH 7.3. Final volume was 10 ml
in screw—cap vials. Incubation was at 37 C

in Brewer jar containing nitrogen.

47

Utilizing known samples of L-alanine, L-proline,
and A-aminovaleric acid, it was shown that these acids
could not be eluted from the column with the solvents used.
In order to show the production of d-aminovaleric acid as
another reaction product of the Stickland Reaction, samples
of the replacement medium before the cells were replaced and
after maximum sporulation had been attained were chromato-
graphed. Table 4 summarizes the results of these chromato—
graphs. Only L—alanine and L-proline were present before
sporulation, while d-aminovaleric acid was also present in
the reaction mixture after sporulation.

Table 4. Chromatography* of L—alanine, L-proline replacement
medium before and after sporulation of g, botulinum.

 

 

 

 

 

 

Sample No of spots Color Rf
Medium before 1 purple .34
sporulation 1 yellow .67
Medium after 1 purple .34
sporulation 1 yellow .67

l purple .44
L-alanine l purple .34
L—proline 1 yellow .68
6-aminova1eric acid 1 purple .43

 

*Solvent system used was isopropanol-acetic acid-
water (3:1:1 v/v) and the chromatograph was allowed to
develop for 6 hr at room temp. Ninhydrin, 1 g/500 ml
n-butanol, was used as developer.

48

Samples removed from a replacement culture were also
analyzed for acetyl phosphate since this is an intermediate
in the Stickland Reaction (Nisman, 1954): however, the assay
used did not reveal the presence of this compound.

Even though Fig. 6 indicates the production of acetic
acid during sporulation in alanine and proline, this does
not preclude the possibility that acetate was being utilized
at a rate less than the rate of production. Therefore, cells
were replaced in L-alanine and L-proline containing acetic
acid—1-Cl4, and the medium assayed at various times for the
radioactive acetate. That acetate was not used in sporo-
genesis in the replacement medium is evident in Fig. 7.

An increase in the numbers of spores was observed, but the
activity of the Cl4 acetate remained constant. This was
determined by isolating the acetic acid by the method of
Wiseman and Irvin (1957) and counting the radioactivity by
the method of Kinnory et a1. (1958).

The next step was to determine if the L—alanine in
the medium was being utilized directly by the cells in some
synthetic process in addition to its utilization in the
Stickland Reaction. Cells were replaced in an L-alanine
and L—proline solution containing 5 no of dl-alanine—l—Cl4.
Since the carboxy carbon of L—alanine is released as CO

2
in the Stickland Reaction, it would be expected that all of

49

 

 

 

 

 

9 -
o SPOREs
A ACETIC ACID-I-C"
«3.1
‘
I
9 9
2 ><
IL 0 .. g
c) ’,/” 0 3‘) E
‘9 O ‘\
o S
q a» / 3
0 A
/ - 2.9 g
MTIME OF REPLACEMENT
O
I
u-A—A—1 A
1 - 2.8
7 1 1 1 1
IO I4 I8 22 26

TIME '- HOURS

Figure 7. The formation of refractile spores of g.
botulinum in a replacement solution containing
L-alanine, L-proline and acetic acid—l-C14.
The amino acid concentration 2 mg/ml in 0.067 M
phosphate buffer, pH 7.3. The solution contained
5 uc acetic acid—l-C14. The final volume was
50 ml in a flask stirred with natural gas.
Incubation was at 37 C in a water bath.

50

the Cl4 label utilized would be recovered as C1402 if no
alanine per se were being used for synthesis. The replace—
ment culture was agitated with natural gas to remove CO2
and to provide anaerobiosis. The effluent gases were passed
through a column containing 20%.KOH to trap the CO2 released.
After sporulation had reached the maximum, the culture was
acidified to release all C02. This experiment indicated

that all of the label utilized was found in CO2 (Table 5),
adding to the evidence that L-alanine per se was not used
for synthesis.

The above data did not exclude the possibility that
the acetyl-coenzyme A (acetyl—Co A) or acetyl phosphate
produced as intermediates in the oxidation of L-alanine were
used in synthetic processes. While exogenous acetate was
not used, these high energy intermediates formed intra-
cellularly might be utilized preferentially over exogenous
acetate in synthetic processes. If this were true, another
amino acid which can act as the reductant but not yield
acetic acid as a product would not be able to support the
sporulation when used in replacement with L—proline. .g.
botulinum is known to be active in the presence of

L-isoleucine and L—proline (Costilow, 1962), and the products

are valeric and A-aminovaleric acids respectively (Nisman,

1954). Therefore, these two amino acids were used in

51

Table 5. The distribution of C14 in a replacement culture
of C. botulinum in L-alanine, L-proline, and d1-
alanine- 1- C14.

;—

 

 

 

 

 

 

 

 

 

 

 

Total population, initia1* 1.1 X 109
Total population, final* 4.4 X 108
Spores, initia1* 7.4 X 107

Spores, final* 2.7 X 108
Cts/min, alanine, initial 3.57 X 106
Cts/min, C02, initial ~ 2520

Cts/min, alanine, final 2.40 X 106
Cts/min, C02, final 1.11 X 106
Total cts/min, initial 3.57 X 106
Total cts/min, final 3.51 X 106
Percent Cl4 recovery 98.6%

 

The replacement medium contained 2 mq/m of L-alanine,
2 mg/ml of L-proline, 5 uc of dl-alanine—l -C in 0. 01 M
phosphate buffer, pH 7. 4. The culture was incubated at
37 C for 13 hours.

*Counts were made by the Petroff-Hausser Bacteria
Counter.

52

replacement solutions and the resulting level of sporulation
was compared to the level of sporulation observed in a
solution of L-alanine and L-proline. The results show that
sporulation proceeded to the same level in both cultures
(Table 6). This indicates that an active form of acetate is
not essential to this step of sporulation.

The L—arginine.E9 L-ornithine cycle and spore

 

maturation: The report of Perkins and Tsuji (1962) that

the L-arginine to L-ornithine cycle is an important energy-

 

yielding reaction in the sporulation of_g. botulinum

prompted the study of this cycle as a means of supporting
sporulation in replacement. Cells undergoing sporulation
were replaced in a solution containing L-arginine and

also in a solution of L-citrulline. Since the energy-
yielding step is from L-citrulline to L—ornithine, L—ornithine
was not used in replacement. The results of this study

(Table 7) indicate that neither of these amino acids alone

can support the process of spore maturation.

Both L-arginine and L—ornithine can serve as acceptor
amino acids in the Stickland Reaction, although it is believed
that L-arginine is degraded to L-ornithine which acts as the
final acceptor. When these two were used in conjunction
with L-alanine in replacement systems, it was found that the

L-arginine, L-alanine solution supported sporulation but the

53

.ucsoo Hwfluflzfl mo ofiflu “coumamon no: mm3 Honucoo ceases

.0 um um Hon Museum M GA comOMUHc Hopes poumnsocfl

.e.n mm .ummmsn mumnmmoem_s nmo.o no He OH
CH cmumoficcfl Ufiom ocean zoom «0 HE\mE N cmcfimucoo nOHDSHOm unmaoowamou gnomes

cam mamaP mmonBoHom ca mnm3 acoausHom

.moucsou mwumuomm Mmmmsmmlmmouuom ecu ha coca muo3 nucsoos

 

 

 

 

medxh.v hodnoN moaxa.m moaxm.m n: ma «sewn OH Honusoo
moaxm.a N.odnoN moaxh.¢ moaXm.h Mn ma H: OH eeocaaoumlq
.msaosmaomfllq
mOme.H Roaxo.a moaxm.m moaxe.n be ma an oH «emanaoumuq
.ocficmamlq

«menomm emonoam ecoaumasmom «cofiumasmom soaumnsusw ucofioomammn finance
Hanan Hmaufiea Hanan Hmfiuaea no mafia no mass newswomammm

 

 

 

.oGHHOMmIA Cam mcflosoaomAIn ca cam
.oCHHonmlq cam msflcmHMIA maficamucoo oceansaon Ca Essaasuon .m.mo ucofioosammm

.w mHQmB

54

mCOHusHom

.ucdoo HMHuHcH mo oEHu “noUMHmwM no: mm? Honucou ocaeee

.0 um um woumnsocH cam mHMH> mmol3oHUm CH onm3

.wumHHoosamoch EaHeom AH.o new

.¢.> mm

.ummusn mumcmmoem s Roo.o no HE 0H

CH meMUHccH pHom oaHEm may no HE\mE m poaHmucou COHudHom ucmeUMHmmH summer

.Moucsou MHHouomm Honmsmmlmmouuom man an wcmfi oHoB mucsoue

 

 

 

 

 

 

mOHNm.v h0HXO.H mOHxH.m mOHN¢.H M: NH crew: mH Honusoo
N.OHonoH N.OHo.no.H m0386 NOme.m H: NH H: MH reosHHHsanoIH
hOHNoN bOonH m033.5. mOHNNH H: NH Mn MH eemGHchMMIH
«monomm economm scoHHMHsmom ecoHUMHsmom coHumnsonH ucoEoUMHmmH EanmE
Hcch HMHUHGH HmcHh HMHDHSH mo oEHB no mEHH usmfioumHmmm
.mQOHusHom mcHHHSHuHUIH CH can ocHsHmHMIH CH ESGHHDDOQ .0 m0 unmEmUMHmmm .b GHQMB

L-orni

 

 

 

 

a med:

due t<

 

rathei
There
Sporu
the p

broth

 

 

55

L-ornithine, L-alanine solution did not (Table 8).

The Role 9; End Products 2: Cell
Metabolism i3 Sporulation

 

The possibility exists that the qualities inherent in
a medium that render it suitable for sporulation may not be
due to the presence of any nutrient added initially, but
rather to the products of catabolism by the organism.
Therefore, the metabolic picture of a culture undergoing
sporulation was investigated to determine what role, if any,
the products of metabolism of a culture in 4% Trypticase
broth play in the sporulation of_g. botulinum..

The metabolic activity of cells during vegetative
growth and sporulation was determined by manometric techniques.
Cells removed from culture at various stages of growth were
placed in Warburg vessels containing an L—alanine, L—proline
solution and the evolution of CO2 and H2 determined. The
QHe

CO2

(ul of CO evolved in an atmosphere of helium per

2
mg dry wt of cells per hr) and the Qge (ul H2 evolved in an

2
atmosphere of helium per mg dry of cells per hr) were cal-
culated and plotted against the time of culture age as were
the numbers of total cells and the numbers of spores (Fig. 8).

The cells show maximum metabolic activity after 16 hr

incubation, and at this time 90%.of the cells were in the

56

QMUIBoHUm cH oum3 mGOHusHom

.ucsoo HMHDHGH mo wEHu “coomHmou nos mm? Honuaoo ocasee

.0 5m um COUMQSOGH mvfim mHMH>
.muMHHoosHmoHnu EsHeom xa.o new .¢.R mm .ummmsn mumnnmoem

2 hmo.o HE mH .mCHom osHEm on“ mo HE\mE H coaHmusoo COHusHom pamfimUMHme nommeR

.Hmucsoo MHnwuomm Hommsmmlmmonumm can an meme 0Ho3 mucsooe

 

 

 

 

 

 

 

mOHNm.m N.OHonoN NOHN>.m mOHNo.m Ma NH «ssh: HH Honusoo
N.OHono.m N.0334” m0on.w mOHNN.m H: NH an HH «socHnuHCHOIH
.mGHCMHMIH
mOme.H ROon.N mOme.m mOHxH.m a: «H Hr HH semaHeHmumuq
.ocHCMHMIH

emonomm emonomm RsOHHMHdmom ecoHumHsmom soHumnsocH unoEoUMHmoH SSHcoE
HMCHm HmHuHsH Hmch HMHDHGH mo oEHB mo oEHB usoEoUMHmmm

.ochmHMIH can mchuHcHOIH mcHnHmpcou GOHusHOm m CH can

.ocHGMHmlH cam ochHmHMIH msHaHmucoo COHusHom m CH EscHHsuon .w.mo ucofimumHmmm .m oHQme

Figure 8.

L06 OF NO.

57

 

 

 

 

 

 

f .’.—.\ '1
'\
TOTAL COUNTS” . H I
a .. ' SPORES
/0 - 40
O
I
°Coz—->
130
7 ’ OI
0—0
120
O
I
I 0 4 IO
0H O
gf‘fifilr- ‘~n‘\~
O
0\
O
6 I 1 \ I;
I0 20 so 40
TIME-HOURS

The metabolic activity of cells of g. botulinum at
various stages of growth and sporulation. The CO2
and H2 evolution rates were determined manometri-
cally, the reaction mixtures containing 100 umoles
of L—alanine, 200 umoles L-proline, and 0.067 M
phosphate buffer, pH 7.2. The total volume was 3 m1
and the incubation temperature was 37 C. The
atmosphere was helium. Cell counts were made with
the Petroff-Hausser Bacteria Counter.

 

58

swollen state. As the cells progress in development toward
the mature spore, their metabolic capacity decreases rapidly
to a constant level.

Fig. 9 illustrates the relationship between the pH
of the culture medium, the production of ammonia, and the
formation of spores. The production of ammonia was observed
to increase throughout growth and sporulation. Since one of
the primary energy sources in this type of medium is from
the Stickland Reaction, a great deal of ammonia production
would be expected. The pH of the culture also increased
during this time. Samples from the same and similar cultures
were assayed for various acids. When the samples were placed
on a Celite column (Wiseman and Irvin, 1957), three bands
were observed on elution with varying concentrations of
acetone. Using the chromatographic method of Kennedy and
Barker (1951), the acids were identified as valeric, propionic
and acetic acids. No butyric acid was observed (Table 9).

Samples taken from a culture at various times during
growth and sporulation were then assayed quantitatively by
fractionating the samples into the component acids and titrating
the resultant fractions. There was a steady increase in
acetic and valeric acids until the time sporulation began

as indicated by the appearance of refractile spores (Fig. 10).

At this time there was a decrease in the rate of production

8.2

8.0

7.8

7.4

7.2

7.0

6.8

Figure 9.

 

59

 

 

 

O

I6 '-
\: I4"
E
% ,2 _ NH3PROD'N ,
‘5
E: IO'- I
L‘ ’0’. o o lo
k /. I ‘o 0'
I 8" . O 2:
'5 C’ [K
;: O phi g;
2 _, /
Q 6 Q ‘ 9 K5

4 b

. //
DJ 0 A
2 " 0’ -I 8
.. /0 SPORES
O D/0 A
l
1 1 1
IO 20 3O
TIME-HOURS

The relationship of culture pH and ammonia
production during the sporulation of g. botulinum
in 4% Trypticase broth. Incubation was at 37 C
in a water bath. Cell counts were made with the
Petroff-Hausser Bacteria Counter. NH was

, 3
analyzed by Nessler s reagent.

60

Table 9. The identification of acids isolated from a
sporulation medium following sporulation of
g, botulinum.

 

 

 

Corresponding
Standard acids Rf unknown acids Rf
Acetic acid .32 Fraction 3 .32
Propionic acid .43 Fraction 2 .43
Butyric acid .50

Valeric acid .55 Fraction 1 .56

 

2 m1 of concd NH OH were added to each fraction of
solvent (acetone and SkeIlysolve) from a Celite column and
the sample evaporated on a steam bath. .01 ml of each
fraction was then chromatographed on Whatman #1 chromato—
graphic grade filter paper. Solvent was NH OHeethyl alcohol
(1:100); and the chromatograph was allowed to develop for
3.5 hr at room temp. It was then dried at 100 C for 5 min
and Sprayed with bromphenol blue, 50 mg/100 ml H20)and 200
mg citric acid. ’

61

SPORES -
jo"--O
/ -so
. /

I"
\\

40

I
ACETIC‘ . )A"/
A I

L06 OF NO
\
O
l
01
C)
4450 /ml

7- 4'

- 20
VALERIC —>/

/
I’. I
/ PROPIONIC

u’

1’

I".

I 1 1 1 1

8 I6 24 32
TIME-HOURS

 

 

 

Figure 10. Acid production and sporulation of Q. botulinum.
Incubation was at 37 C in a water bath. Cell
counts were made with the Petroff—Hausser
Bacteria Counter. Organic acids were isolated
by column chromatography.

62

of valeric acid, while there appeared to be a slight but
detectable utilization of the acetate present in the culture.
Propionic acid was produced at a slow rate during growth

and sporulation.

Even though Fig. 10 shows the production of excess
acetate during the growth of the vegetative phase of the
culture, this does not preclude the utilization of acetate
at a slower rate. In order to determine if acetate were
being used for either growth or sporulation, acetic acid-l—Cl4
was added to a culture and samples collected and assayed as
described above. The C14 activity of the acetate was also
determined. Fig. 11 shows that acetate was utilized during
vegetative growth. This utilization continued until about
the time sporulation was initiated, when there appeared to
be a cessation in the demand for acetate. At this time,
the maximum level of acetate in the medium had been reached.
As sporulation continued, more acetate was utilized.

Spores which were produced in 4%.Trypticase containing
acetic acid—l-Cl4 were collected and fractionated in order
to determine into which fraction or fractions the radio-
active carbon had been incorporated. Initial results indi-
cated that only insignificant amounts of the activity could

be recovered in any of the spore fractions; i.e., dipicolininc

acid, protein, nucleic acid, or lipid. The only fraction

T

 

“ I.64
I
9
R
d
3 L48
\~
3
2
\.
to l.32
L.
(3
MC
Figure 11.

63

 

 

 

 

9..
l
I
I
IRSPORES'
I
O
’I
I «$50
0
I ,
8 r I
‘O' ' I
E r '8\ ACETATE i’40
O / I J I E
3 .' ..
. ' 4130 A
\ o
I o I
..0 I4
I\. ACETATE-l-C
“ I \ UTILIZATION “
7 ' 01."; I . 20
I
I
O
l g 1
IO 20 3O 40

TIME 'HOURS

. . . 1
Acetic acid concentration and acetic aCid—l—C 4

utilization during growth and sporulation of g.
botulinum. The 200 ml culture contained 10 ac
acetic acid-l-Cl4; with natural gas passed
through the culture: incubation temperature was
37 C. Cell counts made with the Petroff-Hausser
Bacteria Counter. Acetic acid was isolated by
column chromatography.

64

not assayed was the insoluble portion which remained after
acid hydrolysis of the spore protein. This portion was in
very fine suspension, and could not be removed by centri—
fugation at 30,000 rcf, but it could be collected on a
Millepore filter by vacuum filtration.

Another experiment was run using 10 uc acetic
acid—l—Cl4 in 180 m1 of sporulation medium. The activated
culture technique was used, with the evolved gases being
collected in 20%.KOH. After sporulation was complete, the
spores were collected by centrifugation, and fractionated.
The various fractions were assayed for C14 activity by the
method of Kinnory et al. (1958). The results of this
experiment are given in Table 10. The only fraction con-
taining any significant activity compared to the amount of
activity which was utilized appeared to be the insoluble
fraction which remained after hydrolysis of the protein.

The activity assayed in the insoluble fraction was
low due to greater inefficiency of the gas—flow counter as
compared to the liquid spectrometer scintillation counter,
and due to self-absOrption by the solid sample. A standard
C14 source was counted on both the liquid scintillation
spectrometer and the,gas-flow counter, and it was observed
that the gas flow-counter had about one—half of the

efficiency of the scintillation counter. The standard,

65

Table 10. Distribution of C14 in fractions from spores
of Q. botulinum produced in sporulation medium
containing 10 no of acetic acid-l-Cl4.

 

 

 

 

Fraction Activity, cts/min
Acetate, initial in medium 8.078 X 106
Acetate, final in medium 7.267 X 106
C02, initial .001 x 106
C02, final .001 X 106
lst spore wash .013 X 106
2nd spore wash .004 X 106
Supernatant from spores heated at 80 C 6

for 10 min .009 X 10
TCA soluble (Supernatant from breakage 6

in 5% TCA) .008 x 10
Lipids (70% ethyl alcohol extraction) .021 X 106
Nucleic acids (5% TCA for 30 min at 100 C) .009 X 106
Protein (8N H2804 for 5 hr at 120 C) .007 X 106
Insoluble residue .112 X 106
Activity of Cl4 utilized .811 X 106
Activity of C14 recovered .185 X 106

Percent of Cl4 recovered

22.7%

 

66

benzoic-Cl4-acid, was dissolved in toluene, and 10 ul
counted with each counter. With the gas flow-counter, the
sample was evaporated on a membrane filter; and with the
liquid scintillation counter, the sample was placed in a

20 m1 counting bottle with 8.6 ml of absolute alcohol and
10.4 ml of 0.58%.2,5—diphenyloxazole in toluene. The
standard was calculated to emit 2,000 cts/min. The liquid
scintillation counter registered 1,082 cts/min for an efficiency
of 54%, while the gas-flow counter registered 487 counts/min,
giving an efficiency of 24%. It should be noted also, that
the evaporation of the standard on the membrane filter re-
sulted in a very thin sample, allowing for a minimum of
self—absorption. With the residue from the cell fraction-
ation, a much heavier layer was collected on the filter,
resulting in even greater loss in efficiency with the gas-
flow counter. Thus, it is likely that practically all of
the C14 from the acetate utilized was in the insoluble
fraction remaining after protein hydrolysis.

(The chemical nature of the residue left after
hydrolysis of protein with strong acid is not known with
certainty, but it is probably a protein-polysaccharide
complex. The material after hydrolysis is black to dark
brown in color, and further studies revealed that this

residue contained from 10—15%.of the original dry weight.

67

It is believed that the material consisted of mucopeptide
complexes which are common to bacterial cell walls, and that
it was synthesized during vegetative growth of the cells.

If this were the case, the mucopeptide complex could be an
artifact of the spores.

Upon examination of the previous results, it was
realized that the spores were collected before the sporangia
had been allowed to lyse. Therefore, the spores would be
highly contaminated with the cell walls of the vegetative
phase of growth. In view of this, another study was made
in which the culture was allowed to proceed until the culture
contained 95%.free spores. When this culture was centrifuged
at high speeds, the spores were layered in the lower level
of the pellet while a black layer was observed on the upper
strata. These two fractions were partially separated by
washing off the black layer with water. This procedure was
repeated twice with the spore layer, after which the pellet
appeared fairly homogeneous. Microscopic examination of
the spore fraction revealed that about 90% of the
population was free spores, the rest being comprised of
spores in sporangia or vegetative cells. The fraction which
had been removed by washing showed mostly debris of no
structural organization and barely visible cells or ghost

cells. This fraction was probably the remains of cells and

68

sporangia after lysis. These two fractions were dried and
weighed, and then placed in 10 m1 of 8N H2804 and auto-
claved for 5 hr. The residues after hydrolysis were
collected on Millepore filters and counted in the gas-flow
counter. The results of this study are given in Table 11.
Table 11. Distribution of C14 in free spores and in

debris of a culture of Q. botulinum grown in
sporulation medium containing 4 Ac of acetic

 

 

 

 

 

 

acid-l-Cl4.
Fraction Description weight Activity
Spore 90% free spores, 6.05 mg 2.09 X 104
10% vegetative
cells and spores
in sporangia
Debris Ghost cells, 5.95 mg 3.10 X 104
structures of
little or no
organization
Activity of C14 utilized 31.21 x 104
. . 14 4
ActiVlty of C recovered 5.19 X 10
14
Percent of C recovered 16%

 

The data of Table 11 are not conclusive in determining

whether the acetate utilized was used in synthesis of spore

material, although it does indicate that most of it was

probably used in the synthesis of cell wall material, since

60% of the recovered C14 was found in the debris fraction

69

and the spore fraction was not free of debris.

Figure 11 indicated that acetate was being used to
some degree during sporogenesis. This led to an experiment
in which the labeled acetate was added to the culture at the
conclusion of vegetative growth, at a time when the cells
were undergoing the initial steps of sporulation. At the
time of addition of acetate, there was about 1%»spores in
the population, and the rest of the cells were swollen.

The spores and debris were partially separated and the

fractions digested as described in the above experiment.

The results of this investigation are given in Table 12.

Table 12. Utilization and distribution of C14 in spores and
in the debris of a culture of Q. botulinum grown

in a sporulation medium with 10 uc of acetic
acid-l-Cl4 added at the onset of sporulation.

 

 

 

 

 

 

 

Fraction Activity
4
Spore .838 X 10
Debris 1.699 X 104
Percent C14 utilized during sporulation 4.8%
. . l4 . 5
ActiVity of C utilized 1.580 X 10
. l4 5
Activity of C recovered .254 X 10

Percent Cl4 recovered 17%

 

70

About 5%~of the available acetate was used by the
sporulating culture; however, the activity found in the
hydrolysate residue of the lysed cells and sporangia was
about twice that observed in the acid insoluble portion of
the hydrolysate of free spores. The activity of the latter
portion may represent contamination by vegetative cells and
debris since these could not be completely separated from
the free spores by centrifugation. At the time the culture
was harvested there were about 10%.vegetative cells present.

It appeared possible that the mechanism initiating
sporulation might be a function of the pH and the concen—
tration of end products in the environment. To test this
hypothesis a sporulation medium was prepared with the pH and
the concentrations of acetic, propionic, and valeric acids
adjusted to that observed in a culture at the time of
sporulation. If these conditions are instrumental in initiating
sporulation, a medium thus prepared should support sporu-
lation at a significantly earlier time than a control
culture. HOwever, sporulation occurred no sooner in the

adjusted medium than in the control culture (Table 13).

Nucleic Acid Snythesis and Sporulation

The synthesis of both RNA and DNA by Q. botulinum

prior to and during sporulation was followed by the method

71

Table 13. Sporulation of g. botulinum in a sporulation
medium containing valeric, propionic, and acetic
acids.

 

 

 

 

 

Obgigjazfon Adjusted Medium* Control Medium

PopfiI::ion** Spores** PopEI::ion** Spores**

8 hr 6.4X108 1.0X107 9.4X108 2.0X107

10 hr 8.3X108 3.0X107 1.0X109 3.0X107

12 hr 8.8X108 6.0X'107 6.4X108 7.0X107

15 hr 4.3X108 1.1X108 5.7X108 1.4X108
‘20 hr 5.5X108 3.2X108 4.5X108 3.2X108
32 hr 4.2X'108 4.0X108 3.6X108 3.5X108

 

*The adjusted medium contained 5.0 meq of valeric

acid, 2.5 meq of propionic acid, 6.4 meq of acetic acid,

and l ug/ml of thiamine, and the pH was 7.5. The control
medium was 4% Trypticase containing 1 ug/ml of thiamine with
a pH of 7.1.

**All counts were made by the Petroff-Hausser Bacteria
Counter.

72

of Hanawalt (1959). To a 250 m1 activated culture of
this organism, 140 no of P32 was added at 8 hr of growth.
Fig. 12 illustrates the results of this study. RNA was
synthesized at a more rapid rate than DNA, and at 12 hr of
incubation, when the first swollen cells were observed,
both nucleic acids were being formed at an undiminished rate.
DNA synthesis was complete by the time the first mature
spores were observed. RNA synthesis continued at a de-
creasing rate for the next 5 hr but was complete well before
the maximum number of spores were observed.

Earlier work carried out by the author (Day, 1960)
had indicated that RNA synthesis was essential during
the final sporulation process, but this was based on the
assumption that vancomycin was a specific inhibitor of RNA
synthesis. HOwever, Jordan (1961) reported that the
primary effect of this antibiotic is the inhibition of cell
wall mucopeptide, and that the inhibition of RNA synthesis
is secondary. Experiments in this study revealed that
vancomycin inhibited vegetative growth of g, botulinum and
concomitantly the synthesis of RNA, but no study was made
concerning the effect on cell wall. vancomycin added to
cultures during sporogenesis only partially inhibited
sporulation and RNA synthesis. Since it could not be

positively stated that the primary point of action of

73

 

 

 

 

 

 

9 I-
! ‘-\.TOTAL COUNTS 1
I \. ~ .
C) ,a"—-"‘O
SPORES/
O -I

RNA 90.»
o‘ I ‘I 48 9
z I ><
'6 "7 s
e a P 46 R
° a
N‘ d5 k
I t)

I -3

I
I DNA ‘2
I /D'—D—D O , .
7 1 1 1
IO 20 30 4O
TIME-HOURS

Figure 12. Nucleic acid synthesis during the sporulation
of Q. botulinum. Medium utilized was 4%
Trypticase broth containing 1 ug/ml of
thiamine; incubation temperature was 37 C.
Cell counts were made by the Petroff—Hausser
Bacteria Counter.

74

vancomycin in the metabolism of Q. botulinum was in the

 

inhibition of RNA synthesis, this line of investigation
was not pursued further.

Since no specific inhibitor of RNA synthesis could be
found, 8-azaguanine, a purine analogue was utilized as a
nucleic acid antimetabolite. Figure 13a shows that this

compound did inhibit vegetative growth of Q. botulinum

 

immediately upon addition to the culture. Hewever, when the
purine analogue was added to the culture at the time
sporulation was just beginning, it had only a partial effect
on further sporulation (Fig. 13b). This would indicate

that some nucleic acid synthesis is essential during the
early phase of sporogenesis.

According to Shiba et a1. (1959) mitomycin C will
inhibit the formation of DNA.with no effect on RNA synthesis.
This antibiotic also inhibited the vegetative growth of
g. botulinum (Fig. 14a). Hewever, when a culture under-
going sporulation was similarly treated, no effect was
observed (Fig. 14b). The culture with added mitomycin C
showed the same level of spores as did a control culture.

Finally, the synthesis of both RNA and DNA during
sporulation was followed in a culture which had been

replaced in an L-alanine, L-proline solution when the

predominance of the cells were in the forespore phase of

 

Figure 13.

75

The effect of 8—azaguanine on the growth and on
the sporulation of Q. botulinum. Concentration
of 8-azaguanine was 100 ug/ml. Medium was 4%
Trypticase with l ug/ml thiamine; incubation
temperature was 37 C. Cell counts were made
with the Petroff—Hausser Bacteria Counter.

 

76

ON 0. N. o

mmaox I NSC.

 

 

HI - u H

mz.z§o<~<-o
to 2.34 .._o w:_»I\.Tv

O

.\.
l\o\.
. \

mz.z<:@<N<Io

Jomkzoo

20:. <4320Qm
a

F HI H

mz_z<:0<N<Im
to 2.004 to m2;

 

 

OI mz_z<:o<N<Im /I/
I o

O

.
nomezoo I\\\
I\.

t.» .326 ms: 3 moms
U

l

v.1

 

L

'ON :10 907

 

Figure 14.

77

The effect of mitomycin C on the growth and

on the sporulation of g, botulinum. Concentra-
tion of mitomycin C was 40 ug/ml. Medium

was 4% Trypticase with l ug/ml of thiamine.
Incubation temperature was 37 C. Cell counts
were made with the Petroff—Hausser Bacteria
Counter.

 

 

78

mKDOIIuETr

 

 

 

v~ mm ON 8. m. e. I N. O. o O c m
I . . a a . h 1 a . _ H .
O 28:20::
to 2.34 O u:_e.\1\1
O 26:29::
O O O .O
0
w\ .o o 2826::

to 2.34 to 2:»)

.0

\

Jomhzoo I

0\

293.33% . In .3 2.5 us I E mom:

a o

 

 

'ON J0 907

 

79

development by use of radioactive phosphorus by the method

of Hanawalt (1959). The control was a culture removed from
the growth medium by centrifugation and resuspended in the
original medium with 36.1 “C P32 added. In neither culture
could any synthesis of DNA be observed, and only in the
control was RNA synthesis found (Fig. 15) . However,
sporulation was observed in the L-alanine, L—proline replace-

ment solution in the absence of nucleic acid synthesis,

but not to the level of sporulation as seen in the control.

The Synthesis 2; DPA and Poly-fi-hydroxybutyric Acid

The synthesis of DPA was also investigated to
determine the relationship of its time of formation to the
appearance of refractile and heat resistant spores. Previous
results had indicated that DPA.was formed simultaneously
with the refractile spores (Day, 1960) but Fig. 16 demon-
strates that these data were in error. This evidence indi-
cates that refractile spores were present before the
synthesis of DPA was initiated, and that the heat resistant
entities were not formed until after the synthesis of DPA
had begun. The level of DPA reached about 28 ug/ml of
culture which is in very close agreement with the level
attained in cultures of other spore-formers (Halvorson, 1957).

Law and Slepecky reported (1961) that the accumulation

 

 

 

 

 

80

 

 

 

 

 

 

 

 

9..
CONTROL- SPORES
o ,——-'o
0'
2
IL
9 e-- REPLACEMENT SPORES" 2-5
Q 0 . —_—__. .
<3
CONTROL-RNA ..
.1 ./D D [12.0
.. I.5
/ D - LO
7"? " 0.5
1° REPLACEMENT-RNA
Ill—I I—- H 1 1 1
IS 20 24 28 32
TIME-HOURS
Figure 15. The synthesis of nucleic acids by Q. botulinum

 

during sporulation in a replacement solution
containing L-alanine, L-proline and P32.
Solution contained 36.1 uc P32 and 2 mg/ml
of amino acids in 0.067 M phosphate buffer,
pH 7.4, final volume 20 m1. Solutions in
screw-cap vials. Incubated at 37 C. Cell
counts were made with the Petroff—Hausser
Bacteria Counter.

CTS/Mm/Io-3

'6

L06 % OF TOTAL
.°
0

0.0

Figure 16.

81

 

 

 

II- a. A
.I‘w.1é :3
DPA °
REFRACTILE at
'SPOREs—>
P . /O
./
/
O
h I
HEAT RESISTANT
K SPORES
/.
L
0
. I . . 1 .
0 IO 20 30 4O
TIME-HOURS

The time of appearance of DPA, refractile spores,
and heat resistant spores of Q. botulinum in

a sporulation medium. Refractile counts were
made by the PetrofffiHausser Bacteria Counter:
heat resistant counts were heat shocked samples
plated with YESB agar in oval tubes. The medium
was 4% Trypticase with 1 ug/ml of thiamine,
incubated at 37 C.

82

of fi-hydroxybutyric acid in polymerized form in the cells
of the aerobic bacilli just prior to sporulation. It was
suggested that these granules serve as a reserve energy
source, and an assay for the quantitative determinatiOn of
this compound was described. When this assay was applied *1

to the cells of g. botulinum which were approaching sporu- -H

 

lation, the resulting spectrum was identical to that described

 

by Law and Slepecky (1961) for spores containing no poly-P- *9

hydroxybutryic acid.

The Formation 2: Heat Resistant Enzymes
During Sporulation

 

 

The report of Simmons (1961) that a heat stable
DPNH oxidase could be demonstrated in the spores of Q.

botulinum prompted an investigation to determine when this

 

enzyme was being stabilized. Another enzyme, acetokinase,
reported by Simmons to possess no stability towards heat
when heated in spore extracts, was chosen for assay in order
to compare a labile and a stable system.

Activated cultures in a 4% Trypticase medium were
prepared and allowed to progress until the desired stage of
cell development had been reached. The cells were then
harvested by centrifugation, washed twice in sterile distilled

water, and divided into two equal portions. One portion

was placed in an 80 C water bath for 10 min, and the other

83

portion was left unheated. The cells were then washed once
more, broken in the micro-cup of the Omni-mixer, the extract
centrifuged, and dialyzed for 15—18 hr against distilled
water at 5 C. The extracts thus prepared were assayed for
protein, acetokinase activity, and DPNH oxidase activity.
This procedure was carried out for vegetative cells (8 hr
culture), swollen vegetative cells (cells showing swelling
but little or no refractility), and mature spores (spores
allowed to stand until free of the sporangia). Table 14
summarizes the results of these experiments, and the activi-
ties therein represent the average of the values obtained
for two different extracts of each of the three cultural
stages.

Several aspects of the results given above are at
variance with the results of Simmons (1961). The DPNH
oxidase studied in these experiments was found to have a
higher activity in vegetative cells than in spores.

Also, the actokinase in the spore appears to be completely
heat labile, although Simmons (1961) reported this enzyme to
be active in extracts of heat shocked spores. It is noted
in Table 14 that acetokinase was inactivated in the spore

by heating for 10 min at 80 C, while the DPNH oxidase was

not completely inactivated by this heat treatment either in

the vegetative or the spore state. However, the DPNH

 

 

84

Table 14. Specific activities of the DPNH oxidase and
acetokinase extracted from vegetative cells,
swollen vegetative cells, and mature spores of
'Q. botulinum.

 

 

 

 

 

Cell Sta e Heated at 80 C for 10 Unheated before
9 min before breakage breakage
Acetokinase* PPNH Acetokinase DINH
ox1dase** ox1dase
vegetative 0.0 .011 9.68 .121
Swollen vege- .
tative cells 0.0 .014 6.10 .105
Mature spores 0.0 .022 4.67 .074

 

*Acetokinase activity; umoles of acetyl phosphate
formed per min per mg protein. Reaction mixtures contained:
800 umoles of potassium acetate, .05 m1 of 1.0 M tris
buffer, .25 m1 extract, 10 umoles of MgClz, 10 umoles of
ATP, 700 umoles of hydroxylamine, and 720 umoles of KOH.
Incubation temperature was 30 C; reactions were stopped with
1 ml of 10% TCA.

**DPNH oxidase activity: Change in O.D. per min per
mg protein. Reaction mixtures contained: 2.3 m1 of 0.067

M phosphate buffer, pH 7.4; 0.5 umoles of DPNH; and 0.2 m1 of

extract. Change in O.D. at 340 mu was followed; reaction
temperature was 25 C.

oxidase in spores did have greater resistance to heat than

(did that in the vegetative cells or in the swollen vegetative

cells. The latter appeared to be somewhat more stable to

heat than the enzyme in vegetative cells.

The enzymes in extracts of the three cell forms were

then assayed further for thermostability by heating the cell-free

 

85

extracts for 80 C for l min.’ The acetdkinase activity in
extracts of spores unheated before breakage showed no heat
stability as expected. Thus only the data for DPNH oxidase
is presented (Table 15). The values given in the table are
the averages obtained with two different extracts for each

of the cell forms.

Table 15. DPNH oxidase activity of extracts of vegetative

cells, swollen vegetative cells, and mature spores

of g. botulinum before and after heating at 80 C
for 1 min. -

 

 

 

 

Cell Sta e Heated at 80 C for 10 Unheated before
9 min before breakage breakage
Unheated 80 C for Unheated 80 C for
l min 1 min
Vegetative .011 .009 .121 .025
Swollen vege-
tative .015 .009 .105 .035
Mature spore .022 .010 .074 .041

 

DPNH oxidase activity: Change in O.D. per min per mg
protein. Protein determinations were not made after heating
the extracts at 80 C for l min. Reaction mixtures contained:
2.3 m1 of 0.067 M.phosphate buffer, pH 7.4; 0.5 umoles of
DPNH; and 0.2 ml of extract. Change in O.D. at 340 mu was
followed; reaction temperature was 25 C.

It appears that low levels of heat resistant DPNH
oxidase occurs both in cells and spores. It is obviously

more stable in spores, and the enzyme extracted from spores

appeared more stable to heat than that from cells. However,

 

86

the high stability of thhs enzyme from spores observed by

Simmons (1961) was not reflected in these data.

DISCUSSION

The purpose of this investigation was to define some
of the major physiological events occurring during the

sporulation of g, botulinum. Sporulation, like other

 

developmental biological processes is gradual and cannot be
observed to proceed in discrete, well defined steps. Many
different systems are functioning during the course of
sporulation, and it is probably only at the molecular level
where the individual steps can be Observed. The morphological
stages of spore development present problems in determining
the stage of cell development. The swollen vegetative cell,
the first observable stage in sporulation, varies in its
appearance from the slightly swollen cell with no re—
fractility to the ”club-shaped” body containing a poorly
defined area with some refractility. The assessment of the
number of immature forespores in a suspension is very
difficult; The definition calls this stage the swollen

cell containing an area of refractility with a diffuse

edge. Many cells with varying degrees of refractility are
observed in this stage, and undoubtedly each form represents
a different stage in the development of the mature spore.

Even the mature spore cannot always be recognized by visual

87

 

88

observation. Heat resistance, one of the requirements of
spore maturity, cannot be determined by visual observation
since refractility precedes heat resistance.

In addition to the difficulty in determining the
stage of spore development, another problem is encountered
in the synchrony of the culture. The ideal situation is to
have all cells in exactly the same stage of development at
the same time. This was not possible to achieve in this
investigation. About the best that could be obtained was
to have 50-60%.of the cells in approximately the same stage
of development at the same time. The rest of the culture
would then be slightly ahead or behind of this portion of the
population in spore development. A small portion of the
population would never develop beyond the vegetative stage.
Therefore, it can be seen that these problems present diffi-
culties in defining discrete steps that take place during

the sporulation of g, botulinum. At best the results indi—

 

cate what is occurring in the majority of the cells in a
specific stage of spore development.

The initial event which takes place in sporogenesis
was not determined. The data first indicated that acetate
might play a role in initiating sporulation as was observed
by Nakata and Halvorson (1960) in B. cereus. In a culture

of_§. cereus, maximum growth and maximum acetate concentration

 

89

is reached at the same time, after which an inducible enzyme
system is activated and sporulation takes place. The enzyme
system is responsible for the oxidation of acetate. With

.Q. botulinum, a maximum of acetate concentration was reached

 

concomitantly with maximum population and the initiation of
sporulation, and the level of acetate in the culture decreased
thereafter. However, the use of acetic acid-l-Cl4 indi—

cated that acetate was utilized throughout the growth

 

period and through the initial stages of sporulation: and
the Cl4 activity could only be recovered in the residues
remaining after protein hydrolysis. On the basis of this
evidence, it appears that acetate does not play the same or
a similar role in the sporulation of g, botulinum as it does
in the sporulation of B. cereus.

Srinivasan and Halvorson (1962) isolated a factor
from the cells of B. cereus in the initial stages of
sporulation which, when added to cells not yet committed
'to sporulation in a non—growth medium, caused these cells
'to sporulate. This material is a low molecular weight
<:ompound, but it has not yet been identified. This
:indicates that a compound was formed within the cell which
<2ou1d trigger the sporulation mechanism in other cells which

‘Nould not normally sporulate at that time. Since a non—

Sirowth medium was required, evidently this compound could

 

I III .II III: .

90

not exert its control over the sporulation mechanism during
growth. This may indicate that only after conditions become
unsuitable for growth, either by the exhaustion of nutrients
or through the accumulation of end products, that the
control mechanism for sporulation can function. With E.
botulinum the concentratiOn of end products and the culture
pH alone would not initiate sporulation. When these
conditions were preset in a culture, sporulation occurred no
sooner than in a control culture. Previous work (Day, 1960)

demonstrated that when the cells of Q. botulinum were

 

removed from 4% Trypticase broth and replaced in a fresh
4% Trypticase medium, sporulation proceeded as in the control
culture. This indicates that neither the accumulation of
end products nor the exhaustion of nutrients is a necessary
factor in the sporulation of Q. botulinum. It would appear
that the initial mechanism involves the interplay of several
events, and that more detailed studies are required for the
elucidation of this process.

The next stage of development of the spore, from
the swollen vegetative cell to the immature forespore,
requires no exogenous nutrients in Bacillus species (Hardwick
and Foster, 1952), This was shown by replacing these cells

in distilled water and observing complete sporulation. It

was the theory of Hardwick and Foster that the proteins

 

 

 

91

of the cells were degraded and renobilized for spore protein.

However, g. botulinum will not sporulate in such a simple

 

system (Day, 1960). The present data indicate that both
protein and RNA synthesis occur during this period. An
energy source alone would not support the development of
cells from the swollen vegetative cell to the immature
forespore, chloramphenicol inhibited this stage of develop-
ment, and a purine analogue (8—azaguanine) also inhibited this
development. Thus these data agree with present concepts
that RNA must accompany protein synthesis. Although these
results do not preclude the possibility that some protein
of the vegetative cells is being degraded and reformed into
spore protein, an exogenous amino acid source is required.
The synthesis of DNA does not appear to be essential to this
stage of development of the spore, and this data is in
agreement with the theory of Delaporte (1950), Robinow (1956),
and Fitz-James and YOung (1959) that the DNA of the spore is
preformed in the vegetative cell and is enclosed in the
spore during sporogenesis. The metabolic activity of the
cells was at a maximum value during this phase of sporulation,
so abundant energy was available for synthetic processes.
After the formation of the immature forespore, the
requirements for sporulation were less complex. While no

spore maturation occurred in buffer or water, the addition

 

 

 

92

of an energy source (L-alanine and L—proline, L-isoleucine
and L-proline, or L-arginine and L-alanine) did result

in the formation of mature spores. This energy could not
be stored since it had to be furnished to the cells exo-
genously. This may be another fundamental difference be-
tween the aerobic spore-forming organisms and the anaerobic
spore—formers. Evidence indicates that the former group
can sporulate in the absence of all nutrients, indicating
an ability to store energy. This may be furnished by
B-hydroxybutyrate as suggested by Slepecky and Law (1961),
although the presence of this material is not a prerequisite
for sporulation and energy can be stored in other materials.

No B-hydroxybutyrate was found in g. botulinum cells.

 

Anaerobes, of course, require much more substrate to obtain
the same amount of energy than is required by aerobes. It
is difficult to speculate as to the reason L—arginine alone
would not serve as an energy source for this stage of spore
maturation. Other workers (Perkins and Tsuji, 1962) have

observed that Q. botulinum, type A, actively utilize this

 

amino acid, and it was observed during this investigation

that the cells of g. botulinum are motile in a replacement

 

solution of L-arginine. Although the energy requirements
for motility are undoubtedly small, this observation does

indicate that the cells at this stage of sporulation of

 

 

93

.g. botulinum are able to use L—arginine as an energy source.
The step in sporulation from the immature forespore
to the mature spore was marked by the lack of net protein
and RNA synthesis, and a decreased metabolic activity.
Hewever, during this stage of development the refractility
of the spore became intensified, and the spore became well EH
delineated within the sporangium. DPA was being formed

during this stage, and it was evidently being synthesized

 

entirely from the pre-existing materials of the cell. The B,
cell became resistant to heat shortly after the formation
of DPA, and this would indicate a role for DPA in heat
resistance as postulated by Halvorson (1957) and Black et
a1. (1960); however, the development of the heat resistance
of individual enzymes, as discussed later, complicates this
picture. It is believed that the exogenous energy source
was required to support these processes of final spore
maturation, which appears to be the development of heat
resistance and the organization of previously synthesized
materials into the dense, highly refractile spore body.

One other aspect of sporulation in replacement
culture must be considered. It is felt by some investi-
gators (Powell and Hunter, 1953; Black et al., 1960) that
the replacement solution is actually a dilute nutrient medium

due to the lysis of vegetative cells after the transfer to

94

 

the replacement solution. Lysis of g. botulinum did occur

 

in replacement cultures; and, in the presence of an energy
source, such products may be utilized by sporulating cells.
However, previous work (Day, 1960), showed that the products
of cell lysis alone would not support replacement sporu-
lation. They must not be used in protein or nucleic acid FE
synthesis, since these processes were not active at this

stage. If they serve as an energy source, they are much

 

too dilute for this anaerobe. The possibility does exist éj
that DPA precursors are made available through lysis of
cells which do not sporulate, and that these precursors are
utilized by immature ferespores in the synthesis of DPA.
The concentration of such compounds in a replacement medium
would be extremely low since the maximum lysis observed
amounted to about 5 X 108 cells/m1. This would result in
a great variety of compounds, the total dry weight of which
could not exceed about 0.5 mg/ml. When an L-alanine and
L—proline replacement medium was chromatographed after
sporulation, no other simple amino compounds could be de—
tected other than these two amino acids and A—aminovaleric
acid.

The thermostability of the enzymes studied revealed

some interesting aspects. The acetokinase, although present

in the mature spore at fairly high levels of activity, had

95

no appreciable resistance to thermal inactivation even when
the heat was applied to the intact spore; Thus, this enzyme
apparently has no role in the germination of the spore.
These data indicate that the enzyme was either present in
the spore in an unstabilized form or was only associated
with the sporangial and cellular debris contaminating the
spore suspensions. The DPNH oxidase of the spore develops

a resistance to thermal inactivation during the entire
sporulation process, and this resistance appears to be at
least partially inherent in the enzyme itself. This enzyme
may be one truly significant enzyme of the spore. A
definite role in spore germination has been demonstrated for
a similar enzyme found in aerobic spore—formers (Doi and
Halvorson, 1961). These investigators showed that the

DPNH oxidase of the vegetative cells of B. cereus was
different from that of the spores in that the vegetative
enzyme was particulate while the spore DPNH oxidase was
soluble. Thus, it appears that there is a transition in

enzymes during sporulation. The DPNH oxidase of g. botulinum

 

may exist in different forms in the vegetative cell and in
the spore, and the development of heat resistance of the
enzyme may be due to the synthesis of a new enzyme rather
than the stabilization of the vegetative enzyme. The

present data indicates that heat resistance of DPNH oxidase

 

 

96

is initiated at the time sporulation first begins. This

is well before the synthesis of DPA has begun, and during a
period of protein synthesis. It would appear that heat
resistance involves more than the presence of DPA.

Simmons (1961) demonstrated the presence of both
acetokinase and DPNH oxidase in the extracts of heat shocked
spores of Q. botulinum, and observed no inactivation of the
DPNH oxidase from heat shocked spores on heating of the
extracts at 80 C for l min. The data presented here are
in obvious disagreement with these findings. Two possible
explanations of these variations are (a) the method of
extraction, and (b) the culture used. The extraction pro-
cedures used were similar but differed in volumes. Simmons
(1961) used the 50 m1 cup with the Omnimixer, while results
presented here were obtained with extracts prepared in the
5 m1 Micro-attachment of the Omnimixer. The culture of
.9. botulinum used for both investigations was from the same
original source, but the culture used in this study had
been cultivated in the laboratory for an additional year.
These variables need more investigation.

This is by no means a complete picture of the processes
taking place during sporogenesis, and there are undoubtedly

many subtle control mechanisms in operation which will remain

 

EKELI

97

obscure until more refined experimental techniques are
available. It is believed that this work does give a more
general view of the over-all sporulation process in an
anaerobe, and the manner in which some of the events involved
are related. A great amount of experimental work could have
been carried out in many directions to further clarify the I Equ
physiological events occurring in the sporulating bacterial I

cell, but the solution to one problem invariably leads to ” u

 

I
another problem. It is hoped that this work will lead to E5
more detailed and more fruitful studies in this fundamental

question of cell differentiation.

 

SUMMARY

This investigation was carried out in order to gain
insight into the over-all processes taking place during
sporulation of Q. botulinum, type A. The processes were
viewed from several different aspects including studies of
sporulation in replacement cultures; the role of end products
in sporulation; the syntheses of proteins, RNA and DNA
during sporulation; and the development of heat resistance
of certain enzymes during the transition from the vegetative
cell to the mature spore.

The replacement studies revealed that an exogenous
source of energy as provided by L-alanine and L—proline, or
L—isoleucine and L-proline in coupled deamination reactions
(Stickland Reaction) supported the development of spores
from the immature forespore stage to the final free spore
stage. Using C14 labeled L-alanine, it was shown that no
L-alanine per se was being utilized for synthetic processes
during this time since all the label was recovered in C02.
Acetic acid—l—Cl4 studies showed that this compound was not
used further by the cells in a replacement culture of L-alanine

and L-proline. Acetate was not utilized in replacement,

but was used by cells during growth and the initial stage

98

 

 

99

of sporulation in 4%.Trypticase broth plus 1 ug/ml of
thiamine, and the fractionation techniques used indicated
that acetate was involved in mucocomplex synthesis. It
was observed that L-alanine and L-arginine would support
forespore maturation also, but that L—alanine and L-ornithine
would not, nor would L—arginine or L-citrulline alone.
Studies with chloramphenicol indicated that protein
synthesis was not required during the final step of sporu—
lation but that protein was being formed up to the time of
the appearance of immature forespores. Dipicolininc acid
was synthesized after the appearance of refractile spores
but before the resistance to heat of the spores was developed.
The synthesis of RNA was required through the time
of formation of the immature forespore, this conclusion
being arrived at by the use of radioactive phosphorus and
certain inhibitors of nucleic acid synthesis. Mitomycin C,
a specific DNA inhibitor (Shiba, 1959) would inhibit
vegetative growth, but had no effect on sporulation., Using
P32 and the assay of Hanawalt (1959), it was concluded that
DNA synthesis was completed during the initial sporulation
stage and was not required for the formation of the immature
forespores. Since the purine analogue, 8—azaguanine, in-

hibited sporulation to a certain level at this time, it was

concluded that RNA synthesis was essential to sporulation

 

 

100

during the transition from the swollen vegetative cell to
the immature forespore.

Two enzymes, acetokinase and DPNH oxidase, were
studied in the vegetative cell, spore, and the transition
state between these two forms. Acetokinase was found to be
readily inactivated by heat either in the cell or in
cell—free extracts of any of the stages of development.

DPNH oxidase had a higher specific activity in the vegetative
cells than in the spore, but became more stable to heat
during sporogenesis. The resistance to heat developed during
the initial stages of sporulation and reached a maximum in
the free spores. This resistance appeared to develop before

the formation of DPA by the spores.

LT. -.

 

" may .4 :-
R C

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Young,

5.1128491-

APPENDIX

TABLE I-A

The Amino Acid Composition of Trypticase*

 

Amino Acids Percent
Arginine 2.6
Aspartic acid 5.1
Cystine 0.3
Glycine 1.8
Glutamic acid 17.0
Histidine 2.4
Isoleucine 5.0
Leucine 7.1
Methionine 2.4
Phenylalanine 3.8
Proline 11.5
Threonine 3.5
Tryptophan 0.9
Tyrosine 2.3
Valine 5.6

*Reproduced from the table VApproximate Composition

of B—B-L Peptones? furnished by the Baltimore Biological
Laboratory, Inc., of Baltimore, Maryland.

110

111

TABLE II-A

The Composition of Amino Acid Replacement Solutions.

Monoaminomonocarboxylic amino acids:

glycine .007 g
L-alanine .010 g
L-valine .014 g
L-leucine .028 g
L-isoleucine .020 g
L-serine .010 g
L-threonine .014 9

H20 10 ml

Monoaminodicarboxylic amino acids:

L—aspartic acid .020 g
L-glutamic acid .068 9
H20 10 ml

Sulfur-containing amino acids

L-cystine .001 g
L-methionine .010 9
H20 10 ml

Basic amino acids

L-lysine .021 g
L—arginine .010 g
L-histidine .010 9

H20 10 m1

Aromatic amino acids

L-phenylalanine .015 g
L-tyrosine .010 g
H20 10 ml

Heterocyclic amino acids

L-tryptophan .004 g
L-proline .046 g
H 0 10 ml

2

112

TABLE III-A

The Composition of Scintillation Liquid Used

for C14 Assay.

xylene 385 ml
dioxane 385 ml
ethyl alcohol, absolute 230 ml
2,5-diphenyloxazole 5.9
ANPO 50 mg
naphthalene 80 g
thixotropic gel powder 40 g

All materials were placed in a Waring Blendor and mixed
for five minutes. The material was then stored in glass
bottles at 5 C until used.

ROG-n". L555; 0;.”

   

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