WEE GiUCOSE WYDROGENASE OF BACRLUS CEREUS:
A MODEL FOR THE STUDY OF SPORE HEAT
R315? ANCE

Thai: for f!» Dear» of M. S.
MBCMGAN “ATE UEIVERSITY
John Aifrod Bach

1960 '

 

 

 

‘LIBRARY_

Michigan State

University

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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MSU Is An Affirmative Action/Equal Opponunity Institution

 

 

 

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This research w(o u1lsrtnben ii an effrrt to
siloiify the Stuly rf sgzre teat r distance by

heat resastance from viability. ihe
assumoticn was aide that the heat resistance of store
entymes contributes to the teat reelstance of whole
egores. ?rom this it followed thut a heat stable
spore enZVLe end its honolcgoue heat labile counter-
tart from germinated stores or vevetcti"e cells could
Le purified end use; as a model system in the studv of
spore heat resisterce. Tor this reason, various er-

tracts of Zecillus cereus were examined for a satis-

 

factory enzyme system.

in extr cts of

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Glucose dehydrogeneses vere foun
'egetetive cells, spores and germinated spores, which
had different levels of heat resistance but egpeured
to be very similer by other criteria. The vegetative
cell enzyme,‘which subsequently was shown to be score
protein, had a half-life of 15 minutes at gg C and
retained this level of heat resistance throughout
purification. The enzyue from scores was slightly less

heat resistant than the vegetative cell enzyme. The

glucose dehvdrogenase from germinated spores‘was heat

labile, having a half—life of about one minute at §_ C.
Various procedures were employed to test the

similarity of the enzymes in an attempt to show that
they are homologous proteins. A study of the time
course of biosynthesis of glucose dehydrosenase in a

growing culture of Bacillus cereus revealed that neither

 

a heat labile nor heat stable enzyme is present in the

culture during the exponential growth plase. A heat

v r.‘
staole glu

C

ose denydrogenase appears after the ex-
ponential growth phase has ended, and prior to the
detection of dipicolinic acid and cellular heat resist-
ance. This enzyme is present in sigh concentration in
spores and gerainated spores, but, as stated before,
the enzyue from germinated spores is markedly heat
labile.

The glucose dehydrogenases from vegetative cells

D

and spores were purified extensively and characterized
according to their catalytic aid serological preperties.
Purification was accomplished by the following steps:
extraction by mechanical disruption of vegetative cells
or spores, heating to 65 C, protamine precipitation of
nucleic acids, ammonium sulfate fractionation, and DEJ<
cellulose column chromatography. A purification as high

as 560 fold was obtained by this procedure. The hichaelis

constants of the vecetative cell enzyme were determined
and compare ed to th hichaelis cons t ants of the spore
enzyme reported in the literature. The constants for
both enzy.xnesxmere very sin ilar. Purified en ymes
froni veg etative cells and spores were injected into
rabbits to produce specific antisera. Th 1e two enzymes
were compared by a modified inmunodiffusion technique
and found to be serologically identical

On the basis of the results obtain ed by sero-

logical and enzymatic comparisons, the coincidence of

the )roduction of this enzime with the :roduction of
.t-

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spores, and the coincidence of the change in he t
resistance of this enzyme with the change in heat
resistance of whole spores, it was concluded that the
glucose dehydrogenase from vegetative cells, spores and
germinated spores is essentially the same enzyme with
different levels of heat resistance. The resistant
glucose dehydrogenase is easily purified, is stable
during storage, and is readily assayed. All of these
considerations favor the use of this enzyme ystem as

a model for studying spore heat resistance at the

molecular level.

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by

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A TingQ

Submitted to
Tichigan State Vniversity
n wartial fulfillment of the requirements
for the degree of

H-

“-0 «‘m - T0, jfiTw—“UI‘ 7
MJI- 3.1. AB C: usz. :L-".‘UL
,.

Department of Xicroticlogy and Public Eealth

1960

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I wish to exhress .y gratitude to Dr. H. L. badof ,
Department of licrotiolo;y aid Pttlic Heal h, for his
patient guidance and continued interest. His original
thought provided the hygothesis on thici this work is
based.

1.

I would like to thank also Dr. R. .. Costilow,
Sebartment of Iicrobiology and Eubli -ealth, for his

0 F
helpful suggestions on experinental techniques.

-
..

Finally, I wish to thank my wife, Virginia, whose
c1611 . F1 L 01 at? 1.1511 hasten d all [31"]. ' 1
‘at' rce a d enc Lr He t * re + e co f etior of
this'work.

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RESULTS

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A.

l . “N
Iii-1.43 :11. J.'-' -firnl FOL S o o o o o o o o o o o

Organism and Iedium . . . . . . . . .

Growth of Vegetative Cell and
Spore Crops . . . . . . . . . . . . .

Q

Frenaration of Cell-free Extracts . .

1-.

Glucose ue ydroa enase Assay . . . . .

DPI‘TH CXidase $1888.), I o o o o o o o o

YT ' .'

Assay for heat has 1stance . . . . . .

Purification of Glucose Dehydrogenase
Irotein and Iucleic Acid Assay . . .

Chloride Ion Assay .

Acetone Extra tion of whole Cells . .
Diticolinic acid Assay . . . . . . .
Production of nn.une Serums . . . . .
Serological Comparison of Enzymes . .
Germination of Spores . . . . . . . .

. . . . . . . . . . .
Screening for Heat Resistant Enzrmes
"Activation" of Glucose Dehydrogenase

Time Course of Glucose 7ehydrogenase
L1093_r.theSis O O O O O O O C O O O O

Durification of Vegetative Cell and
Spore Glucose Denydrogenase . . . . .

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11-7

11
12
13
13
15
18
19
20

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1“ ‘ fin“!
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E. Characterization of Yegrtative Cell
and Store Glucose Dehydrogenase . . . . . . 54
1. Tea Resistance . . . . . . . . . . . . . 34

'fi 0

2. Lichaelis Constants . . . . . . . . . . . 37

3. Serology . . . . . . . . . . . . . . . . 37

F. Heat Labile Glucose Dehydrogenase . . . . . 43
DISGtialCN . . . . . . . . . . . . . . . . . . . . 44
SCILARY . . . . . . . . . . . . . . . . . . . . . . -1.

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.LLQl JQJL‘u‘NJLb o o o o o o o o o o o o o o o o o o o 0 M5

TABLE EnGE
l. tarification of the glucose dehydro-

genase from vegetati'e cells of Bacillus

0938118 0 O O O O O O O O O O O O O O O O O O 32

 

onstants for th

el es from vegeta
a a - -0 ‘ro .--.-'"!--—. so” 7“
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LIST CF FIGURE"

TRE

The heat inactivation at 69 C of the
glucose dehydrogenas in an extract of

vegetative cells of scilli cereus . . .

A cougarison of the effect of heating
at o0 ‘ on t lucose lehydrogenase
- s in an x ract of

 

 

A Line Course of the bi synthesis or
heat resistant glUCODB dehydrogenase

and dioicclinic acid by a urowing
culture of hacillus cereus . . . . . . .
The heat inactivation at 69 C of the
purified glucose oeiyo13enases from
vegetative cells and SpO re: of cillus

 

083'“??? S O O O O O O O O O O O 0 O O O O O
The effect of substrate concentration

on tne activity of the glucose dehydro-
genase from vegetative cells of Bacillus
cereUE . Q o o O 0 O O O O O O o 0 O o O

A serological comparison of the glucose
ehydrogenases from ve3etative cells and
stores of Bacillus cereus by the agar
gel method . . . . . . . . . . . . . . .

A serological con parison of the glucose
dehydrogena see from ve3etative cells

and spores of baClllLIS cereus by the
cellulose acetate filn method . . . . . .

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40

INTRODUCTION

Bacterial endosgores are produced, by mehbers of

L “ I“ -21 ‘1‘ A 3,—5- .3 . 1‘
the 3enera_3gg_13ds and Clost i§1dm, as a res1stan ,

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dormant stage in the life cvcle of these cr3arisms.
Endospores are formed within ve3etative cells duIin3 the
stationary 3rowth phase of the culture. After endospore
formation is completed, the su rroundin3 ve3etative cell
material (Sporan31um) is usually digested, and the spores
are released. In qualitative chemical wmpo:;ition, these

rom v33ete tiv ve cells only in their content

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of dipicolinic acid (pyridine 2,6 dicarboxylic acid).

Tigicolinic acid has not been found in exgonentia1l1y 3rowin3

eristics, how-

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ve cells, being

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ever, spores differ 3rCSS1y 1
almost inactive metabolically, and resistart to many of
the physical and chehical a3cn s capable of k llin3 ve3e-
tative cells. Durin3 a process called 3er1ination, the
typical resistant properties of spores are lost, and

certain latent metabc lic ezzyues once a3 ain become active.

At the same time, lar3e amounts

released from the spores, includin3 peptides and the ca1-

cium salt of dipicolinic acid. If environmental conditions

are favorable, the 3er minated spores 3row into true ve3e-

tative cells which.repeat the life cycle by reprouuc1ng

l

and sporulatin3 exactly as their predece‘scrs did.
Cne of the most unique properties of bacterial
spores is their extremely high resistance to heat. The

spores of some bacterial species are capable of with-
standin3 autoclavin3 at 115 C for as lcn3 as three hours,
while, in contrast, the ve3etative cells of this species,
and most other sporeformers, are killed in a few minutes
at 55 C to 65 C (Curran, 1952). AlthOU3h this is an
extreme example, it is, nevertheless, true that most
bacterial spores are much more resistant to heat than the

ve3 tative cells from which they were formed.

(1)

To study the effect of any factor on the heat re-
sistance of bacterial spores, a suitable means of measur—

1at heat resistance is needed. Up to the present

7““

ing t
time, this has been done by enumerating the survivors of
a given heat treatment. HOwever, the detection of these
survivors depends on the proper functioning of the
multitude of reactions involved in 3erninaticn, outgrowth,
and reproduction. Although these reactions have nothing
to do with spore heat resistance, they may be affected

by the factor under study. Thus, the study of spore

heat resistance with such a complex, variable system can
not be expected to yield reliable information. A more
suitable method cf studying heat resistance wculd be one

which does not depend on spore viability; capperhaps one

O]

which does not even depend on whole spores. In fact,

L )J

the simples , most .irect method of studying heat resist—
ance would be at the molecular level, providin3 of
course that spore heat resistance is eXpressed at the
nolecular level.

Because many of the essential components of cells,
such as proteins and nuclecproteins, are extremely labile
to hea , it is reasonable to suspect that these compounds
are protected from heat denaturation in spores, and, con-
versely, unprotected in ve3etative cells and germinated
spores. If this protective mechanism involves molecular
structure, ather than 3 st a change in cell environment,
it should be possible to isolate and purify heat resist-
ant spore material and homolorous heat sensitive vege-

tative cell or germinated Spore material. Together, these

/

‘/

two types of cellular material would constitute a simple,
cell-free model system for studying spore heat resistance
without the problems inherent in Spore viability methods.
In fact, if the two types of material were actually homo-
logous and could be sufficiently purified, a simple
physical-chemical comparison mi3ht indicate at least one
mechanism of spore heat resistance. The most reasonable
approach to this problem would be to select, for examin-

ation, one cellular component which is heat stable in

spores and heat labile in ve3etative cells and germinated

spores. In addition, this component should be easily
purified and must possess some measurable index of
denaturation. The obvious choice for such a component
is some type of enzyme, since enzyme denaturation is
easily detected by loss of enzymatic activity.

The enzyme catalase, from vegetative cells and
spores of Eacillus cereus, appeared to fulfill the
requirements for such a model system, but extensive
attempts by Sadoff, Kools, and Ragheb (1959) to
crystallize the heat stable enzyme failed because of
the low cellular concentration of this enzyme, the poor
yields obtained during purification, and the instability
of the enzyme during stora3e at low temperatures. It
became apparent that, to obtain the amount of pure pro-
tein needed for a physical-cheiical comparison, a new
model was needed. For this reason, the glucose dehydro-
genase from Bacillus cereus was examined as a possible
model for the study of spore heat resistance. The
isolation, partial purification, and partial character-
ization of this enzyme system is the topic of this

thesis.

HISTORICAL REVIEW

Many heat resistant enzymes have been found in

resting spores of Bacillus cereus and in cell free

 

extracts of these spores. Host of these enzymes
differ greatly in their catalytic specificities and
almost as greatly in their level of heat resistance.
Although the heat resistance of many of these enzymes
has been reported, to the author's knowledge, only
catalase has been purified and characterized expressly
for the purpose of formulating a mechanism of spore
heat resistance.

In connection with their studies on the germina-
tion requirements of Bacillus cereus spores, Stewart
and Halvorson (1955) described an alanine racemase
which converted L-alanine to D-alanine. The enzyme
was completely resistant to heating at 80 C for two
hourswvhile it was in the intact spore.

Lawrence and Halvorson (1954) found a heat re-
sistant catalase in spores of Bacillus cereus which
was completely resistant to heating at 80 C. When
this enzyme was extracted from the Spore, its heat
resistance was somewhat diminished. In addition to
the stable catalase, a heat labile catalase was also

present in spore extracts, but the labile enzyme was

not active in intact spores.
A remarkably heat stable adenosine ribosidase
which cleaved adenosine into adenine and ribose was

found in spores of Eacillus cereus by Lawrence (1955).

 

This enzyme retained 50 per cent of its activity after
heating at 100 C for four hours. Powell and Hunter
(1956) reported the same adenosine ribosidase to be
stable both in intact and disintegrated spores. These
authors also described an adenosine deaminase in

Bacillus cereus spores. The deaninase in the intact

 

Spores was stable to heating at 60 C for several hours.
However, in spore homogenates or germinated spores, the
enzyme was destroyed in 15 minutes at 60 C.

Sadoff, Kools and Ragheb (1959) found, in vege-

tative cells of Bacillus cereus, a heat stable catalase

 

which had heat resistance similar to that of spore
catalase. Recent immunochemical data (unpublished)
indicate that the heat stable vegetative catalase is
identical to the heat stable catalase found in spores
by Lawrence and Halvorson (1954).

Church and Halvorson (1955) discovered a DPN-
specific glucose dehydrogenase in extracts 0f.§é£lll£§
cereus spores. This enzyme was partially purified and
characterized by 301, Church and Halvorson (1959) in

connection with their study of the intermediate metabolism

of aerobic spores. The effect of heat on this enzyme

was not reported.

'4
’v(
61
C?
01

MATERIALS ANDZW‘

and Hedium

 

A. Organigp
Bacillus pageus T. was the organism used through-

out this study. Stock cultures were maintained by

allowing the organism to sporulate on nutrient agar

slants, and refrigerating the slants at 4 C until needed.
All vegetative cell crops, and most of the spore

crops, were grown at 50 C, in G medium, as described by
‘tewart and Halvorson (1955). This medium contains the
following components per liter:

0.00001 g.

ZnSO4
CuSO4 0.00001 g.
CaClg 0.00001 g.
Fe504.7Eé0 0.000001 g.
KQHP04 1.0 g.
(KH4)ZSC4 4.0 g.
Yeast Extract 8.0 g.
MnSO4.H20 0.1 g.
MgSO4 0.8 g.
Dextrose 4.0 g.
0.7 ml.

Antifoam B

Some of the score crors were grown at 28 C with the
1 :1

dextrose concentration reduced to three grams per liter.

This modification prevented premature lysis of the
sporulating cells by lowering their oxygen require-
ment, thereby increasing spore yield.
3. Growth of Vegetative_§ell_and gpore Cropg

Large amounts of vegetative cells and spores were
needed to obtain enou3h glucose dehydrogenase for
purification and characterization of the enzyme. All
of the vege Ha ive cell crops and some of the Spore

crops were produced in three liter fernanters (New

;—h

Erunswick Sci ntif 0 Company). The medium in,these
fermenters was oxygenated by forced aeration and me-
chanical a5it ation. Some of the spore crOps were
produced in 18 liter pyrex carboys in which the medium
was oxygenated hv forced aeration through six, radially
arran 3e d sparger tubes. The force of the air flowing
through the s a.rge s rotated and agitated the medium
thereby increasinr the efficiency of oxvzenation. The

temperature of be 'th the New Prunswick ferme nter and the
carboy was maintained by a thernostatically controlled
water bath.

The large volume of medium use d in either fermenter
required a large, actively growing inoculum. This was
prepared i11 dign; le d leeks, each contain in3 50 milliliters

of G medium. The di pled flasks are 530 milliliter

.rleniey r flasks with four depressions, equally spaced

around the lower side of the flash. these .epressions

, 1 1 -n'v“ 5,
cause turtule ence .xren tne linens are blidxul, thereby
‘. " A ‘ . ‘r’ " 4- I‘ ‘ R."'* " " '1‘ " 1“ : r““‘, Lin ‘. --‘ J '3 -‘"\‘.' fl * ,5 . h J‘- ar fin '1
1-;JF-3aSluQ mm:- UA_H.DU'1151L;.L'VL; 01 one A..L§DL;_LQ1110 alt-.11 la]. LIKJDQI -,‘l 9

throng h an increasin; number of dimpled flasks synchrcrizes
t? physiological age of he cells and increases the
volume of the inoculum.

The following specif:c prclcedure was used to grow
venetative cell and spore crops. A nutrient agar slant

was inoculated from a s,ock culture and incuba ed at

n m \ 4-..- .AF A u“ «'1’ fi Y .3

50 e approximat—ly L113 hours. n dllleu ildbm or medium
_.., :5A ‘1 .1. ,q ‘. _| 5' L, ,‘l, n. 1-

‘N30 ino elated ire. tie nine nour are: and siaxen for

iour hours. gen mill ilit ers of culture from this flasi
Jere put into each of two more flasks, and these flasks
were shaken for two hours. Ten milliliters of culture
from he two flasks were put into each of seven more

01f "1‘- 4 1 $1, w-‘r , ~. 1A.'1" '61 ‘-‘ I.- ~1 .\ r-I‘A
ilrsms, and these "13045 were snanen ior two nears. lme

ent its con tents of the seven flasks wer2 transferred into

,J -..

I.

a Lew Brunswick fermenter tank containing ‘,ECC milli-

‘L' A r‘ *N A 5‘ ~‘ ' " I“ u“ I "‘ ’ '."- ‘ A“ . n
ters of isd_un. The tonr: contents were agitated at

L

F-J
H0

450 revolutions per ninute, and aerated 3t 3 rate 0f five

liter: per minute. If the 18 liter carb oy ferirnt‘” we
used, abort 1.5 liters of a two hour culture from the

New Brunswick ferienter were used for inoculation.

"egetative cell crops were harvested at about seven

11

and one quarter hours “ft r inocuM ng the fermentor.
For spore crops, the cultures were allowed to grow until
microscopic examination of stained smears indic ted a

large proportion of free spores. The vegetative cell

an

d

(L

and spore crops were harvest n a Sher lee Super
Centrifuge, type T-41-23 l-l-H (The Sharples Specialty

om‘any). Spores .ere cleaned by differential centri-

C.)

*C:

*1

ugation in water or dilute saline until practically

7’

free of vegetative debris and germinated spores. vege-

tative cells were washed once or twice 1n 0.1 molar,
pH 7.6, tris (trishydroxynethylaninome hane) buffer.
The cells and spores were cleaned in the cold in a
Servall centrifuge, model SS-l (Ivan So 11, Incorpor-
ated). Unless specifically stated otherwise, all

further mention of centrifugation implies the use of

this centrifuge. The cells and spores were stored at

C. Preparation of Cell-free Extracts

All cell-free extracts were obtained by mechanical
disruption of cells in a Servall Omnimixer (Ivan Sorvall,
Incorporated). In most cases, three parts, by weight,
of number 110 super ri te beads ( innesota Ti ining and
Lanufacturing Con 1pany), one part, wet weight, of cell

or spore paste, and enough 0.1 molar, pH 7. 6, tris

buffer to fill the 80 milliliter Cmnimixer cup were mixed

12

at top speed for 10 to 30 minutes. The cup was cooled
in crushed ice. After breakage, the brei was centri-
fuged at 52,700 times gravity for 15 to 60 minutes,
and the supernatant liquid was retained for enzyme
studies.

D. Glucose Dehydrogenase Assay

 

Glucose dehydrogenase was assayed spectrophoto-
metrically at 340 millimicrons using DPH and glucose
as substrates. This assay is based on the difference
in light absorption of DEN and DPXH. Both compounds
absorb at 230 millimicrons, but only DPNH absorbs at
540 millimicrons. Since glucose dehydrogenase catalyses
the oxidation of glucose and reduction of DPN, the rate
of increase in absorbance at 340 millimicrons can be
used to express enzyme activity. One unit of glucose
dehydrogenase activity is defined as an increase of
0.001 optical density units per one hundred seconds at
540 millimicrons.

The assay procedure was the same for almost all
experiments. In addition to enzyme, the assay cuvettes
contained the following reagents: 500 micromoles of
pH 7.6, tris buffer; 100 micromoles of glucose; 2 micro-
moles of DPN; and water up to three milliliters. Endo-

genous cuvettes contained no glucose but otherwise were

the same. Cno exception to this procedure was that used

13

for assaying glucose dehydrogenase in the heat inactiv-
ation of vegetative cell extract. The assay cuvettes

in this experiment contained the following reagents:

50 micromoles of pH 7.3, tris buffer; 100 micronoles

of glucose; 1 micromole of DPT; and water up to three
milliliters. All assays were done at approximately 32 C
in a Beckman spectrophotometer, model DU (Beckman
Instruments, Incorporated). The temperature'was main-
tained by heat from the hydrogen lamp.

E. DPNH Oxidase Assay

 

DPNH oxidase was also assayed spectrophotometrically,
using DENH as the only substrate. The oxidation of DPNH
decreased the absorption at 340 millimicrons. One unit
of DPNH oxidase activity is defined as a decrease of
0.001 Optical density units per 100 seconds at 540 milli-
microns. In addition to enzyme, the assay cuvettes con-
tained the following reagents: 500 micronoles of pH 7.6,
tris buffer; 0.5 micromole of DPNH; and water up to three
milliliters. Endogenous cuvettes contained no DPNH.

F. Assay for Heat Resistance

 

Heat inactivation rates were used to screen cell
extracts for heat resistant enzymes and to determine the
inactivation Kinetics of purified enzymes. In general,
these rates*were obtained by heating samples at a fixed

temperature for various times and detersining the enzyme

14

activity of each heated sahple. Since the heating

times vary widely among samples, the time required for
temperature equilibration of the samples constitutes a
different percentage of the heating time for each sample.
This introduces an error in the tine-temperature value
reported for each sample. When screening for heat re-
sistant enzymes, this error is unimportant but in kinetic
studies it can not be ignored. For this reason, two
methods of heating samples were used, one for crude ex-
tracts and another for purified enzyme solutions.

Crude extractsxwere heated in rubber stoppered, 15
millimeter, pyrex test tubes. Before adding extract,
each tube was pre-heated to the inactivation temperature
in an unstirred water bath. After adding the extract,
the tubes were stoppered and agitated for one.minute to
increase the rate of heat transfer. The tubes were with-
drawn and iced at various intervals after the addition
of the extract and the heated extracts were centrifuged
to remove precipitated protein before activity measure-
ments.

Purified enzyme solutions were heated in thin-walled
capillary tubing. The tubes were made by drawing 23
millimeter pyrex test tubes to a diameter of two milli-
meters in a gas-oxygen flame. These capillary tubes had

xtremely thin walls to facilitate heat transfer and re-

F)
()1

duce the temperature la~

suffi01ent volume

.I. V
U

needed for an accurate assay.

was drawn into the tubes and
melting then in a flame. it
were placed inul nebusly in

Zach tube
and cooled in a 10 C water

I. r.,-.‘1 .
35101; Salli/1.8 1.43.3

time.

,1 1,: -
'v iCuLu the

'I'ec’iS Wit hdl" Sh-‘l’l after

r . -°-L-, .
measured W1bh"

both ends were .sealed by
zero tide, all of the tubes
a stirred water bath.

the specified time interval

 

G. ~uri ,ation of filucose Behydrg;engse

To deteriine the progertier of the :luc ose dehydro-
genases frcn ve3ztati 1e cells and Spores, both crude
extracts had to be purified. The pur1ifu01tion procedure
for both extracts Was sinilar to thzt used by Doi, Church
and Ialvorson (1959} in their purification of spore glucose

ehydrogenase.

35 C, cooled

to remove precipitated material. I e

tlfiae-

w1tn stirring, until the solut

precipitated protein was collec

separated
one tenth the riei

buffer. The pH of the ex

The crude extract

im1eu iately and rapidly in ice,

and centrifuged
act was
ammonium sulfate slowly,
d.

was saturate The

ted by centrifugation,

from the supernatant liquid, and resu spended in
nal volume of 0.1 molar, pH 7.6, tris

tract was not controlled during

the anmoniun sulfate saturation.

Nucleic acids were removed from the concentrated
ex trac t becaus they inter are with annoniun sulfate
fre ctiona tion. They were precioitated by adjustirg
the pH 0 the concent ated extract to 5.1 with acet
acid and addin5 snall anounts of 2 per cent protanine
ate in a fine strean awhile stirring t: e extract.
The precipitate was removed by centrif-ga tion, and the
280/260 ratio (see section H. of HAT53IALS n. HST ICE 3)
of the supernatant liquid was determined. This pro-

cedure was re pe-1ted until a ratio of 0.7 or greater was

obtained. The supernatant liquid was then dialysed

a
m
o
O
1:
d
F4
m
rr
0
a
H
01

against 0.1 nolar, pH 7.6, tris buffer fo

cipitation occurred during dialvsis, but, since the

preciuitate contained no alucose dehydro: enase, it was

”5 in t1e dialysa d extract was

fractionated with ammonium sulfate. The desired satur-

(.0

tion pe wcenta5 e was obtained by measuring the volume

of the extract before each addition of annonium sulfate,

and addin5 a calculated amount of solid Sclt, slowly and
vvith constant stirrin5. No attenpt was made to control

the pH of the solution during addi tio on of the a:unonium

sulfate. Each precipitated fraction was collected by

entrifugation and resuspended in 0.1 mole

(L)

r, pH 7.6,

17

tris buffer.

The almonium sulfa fractions which com1 ained
glucose dehydro5enase were further fractionated on an
anion exch an-e column. Phe material used in the column

was KK-diethylamino ethyl cellulose (DEAE cellulose).

"fl ,. ‘fW

olumn was atproxiaately 20'nilliliters.

d—
t
(D
O

The almonium sulfate Fractiors containing glucose

dehydrOQenase were comoined and dial'sed against 0.05 molar,

r4
.)

pH 7.6, tris buffer to prepare t en for adsorption on

the coluhn. Dial3; is at this buffer concentration pre-

bv

ci‘itate d a la 'e anount of material, out, since this

1at rial Was enzyaatically inactive, it was discarded.

H

(1‘

d-

The supernatant solution was added to he colunn Eld
washed throu-h with bufifer. The effluent from washing
contained no lucose dehydroger ase , indicatin5 that
the enzyme was adsorbed on the DLAE cellulose.
Adsorbed protein was removed from tr a column by

‘

31a dient elation. The gradient was obtainea by neing

o ‘ a l “' ’ 3 'Y‘Vh. “-‘9
two reservoirs in a serles arranpemont. -ne lcm,r

reservoir was an aspirator bottle conta1n1aa 1c mill1-

liters of bu,Fer, and the ugger res r:

,1 _ .—
."O ‘-I

A

‘1 JUL- J.

LA.

'4

9’7]

343

L
V”

.4
\‘J

_.' L171; ] 0

.--.—~

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flyik

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34-“:(1

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.50

a
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51

a V *3

2/}

C1
./

m‘

19

In samples containing less than 10 per cent nucleic

6*

CD

acid, protein was determined spectrophotom trically a
280 and 260 millimicrons, according to the method of
Warburg and Christian (1942). This method depends on
light absorption at 280 and 250 millimicrons by protein
and nucleic acid. Both protein and nucleic acid absorb
at these wavelenqths, but, since their absorption co-
efficients differ, the ratio of absorbance at 280 milli-
nicrons to absorbance at 230 millinicrons (280/260 ratio)
can be used to determine the relative amounts of each in
a given sample. Information on the relative amounts of
protein and nucleic acid, plus the optical density at

280 millimicrons, is used to calculate the actual concen-
tration of each in the samples. A Becknan DU Spectro-
photometer was used for these readings.

I. Chloride Ion A§§§X

 

The sodium chloride concentration required to elute
each fraction from the cellulose colunn was estimated
from the chloride ion concentration of the fractions.
This information was used to compare the exchange charac-
teristics of both glucose denydrogenases. The chloride
ion concentration in each fraction was determined by
titration with silver nitrate using potassium chromate
as an adsorption indicator. Since protein affects the

adsorption endpoint, a correction had to be made for the

20

protein in each fraction. The correction factor was
obtained empirically by titrating known concentrations
of sodium chloride and protein, with silver nitrate.

J. Acetone Extraction of Whole Cells

 

A quantitative method for assaying glucose dehydro-
genase in small cell samples was needed to study the
rate of formation of this enzyme in growing cultures.
Several assay procedures were tried, but most of these
were not successful. Whole cells could not be assayed
directly because they were impermeable to substrates
used in the assay. Heating the cells to 69 C for two
minutes did not increase their permeability. Breaking
the cells in the Omnimixer diluted the activity excess-
ively because of the large cup volume and the large
volume cf buffer required to thoroughly wash the glass
beads.

Acetone extraction of the cell samples increased
their permeability to assay substrates and also permitted
the assay of small cell samples. Glucose dehydrogenase
assay by acetone extraction was done in the following
manner. Cell samples were taken from a New Brunswick
fermentor at various times during growth, washed by
alternate centrifugation and resuspension in 0.5 per cent
saline, and suspended in just enough saline to disperse

the cells. One part of each suspension was pipetted into

‘7

sl

at -lS C, and cent:

etone,

."t
\J

a

ten

cpany).

0"
L1

("1

‘J
for about

7

dry

,3

oui'nent

A

.1
4

tional
in

Cal

LVL

V1

.L

ion,

Inter

L;

(

uga

L
was crerated

"1
‘J

.11

——

3.9
v1}

‘1‘.”3 re

ls

1L

Du

drie

#118

rd:

Z81".

.p
lI‘i-ECE

L.-

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A

l

ide.

alor'

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ssayod for

C

4—4
wk 5

C

.1
x 1.

cent
anv

cts.

- J.
20.ng

n1

1
-4.

cells

e
to

i Anderso
th
iron

’5‘ ll \

d from

- a

D ‘54

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divalent

1" 1 C

L.
L

GIL;
ti

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Lomb "Spectronic 20"

z; .1. D
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ltOC"

 

cal

tri
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produce

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w

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j

(“q *"
: -II‘LLIL-

V‘

 

" ”Lin 3

ill-KL

ve lengt

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CJ.

Ianune

cl

n3

serums,

e
S

111

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«.0
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~RL

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31317.8 I’ o

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22

centrated enzyme and one part of Freund's complete
adjuvant (1944) were emulsified by forcing in and out
of a one cubic centimeter tuberculin syringe and in-
jected subdermally in the necks of rabbits. These
rabbits had been heart bled prior to injection to
obtain normal serum for serological controls. Twenty-
four days after injection, the rabbits were heart bled
to obtain immune serums. The blood was incubated at
room temperature for three hours to start clot re—
traction and refrigerated for about three days to finish
the retraction. The serum was poured off, centrifuged
to remove blood cells, and stored at -°O C. Forty-six
days after the first injection, a second, smaller in-
jection of enzyme plus Freund's incomplete adjuvant (1944)
was given to keep the antibody level high. At fifty—six
days, the rabbits were bled again to obtain more immune
serum.
M. Serolggical Comparison of Enzymes

The serological comparison of spore and vegetative
cell glucose dehydrogenase was accomplished by the
Cuchterlony agar gel diffusion method (1949a, 1949b) and
by Consden and Kohn's (1959) modification of this method.
In the agar gel diffusion method, several wells are cut
in a layer of solid agar, sealed with melted agar, and

filled with various antigens and antisera. The antigens

23

and antisera diffuse through the agar, until, in a region
of optimal proportions, they combine and precipitate,
forming a visible line. Since various antigens and anti-
sera differ in diffusion rates and optimal proportion
values, this method is capable of separating the com-
ponents of a compound antigen-antibody reaction. Consden
and Kohn's modification of the Ouchterlony technique is
the substitution of cellulose acetate film for the agar
gel. In this method, moist cellulose acetate film is
spotted with very small amounts of antigens and antisera,
and incubated under mineral oil to isolate the aqueous
phase. During incubation, the antigens and antisera
diffuse through the aqueous phase, and precipitate in

the cellulose aCetate matrix. After ~’ncubation, the

film is washed and stained with a suitable dye to reveal
the precipitin lines.

A slight modification of the cellulose acetate
technique, especially useful in this study, is the en-
zymatic identification of precipitin lines. This method
depends on the enzymatic reduction of DPN (in the presence
of glucose) by serologically precipitated glucose dehydro-
genase, and the fluorescence of DPNH under a Black-ray
ultraviolet lamp (Ultraviolet Products, Inc.). The pro-
cedure consists of spreading a solution of glucose and

DPN on the completed cellulose acetate film, and examining

the film for fluorescent precipitin lines.

N. Spore germination

 

 

The heat labile glucose dehydrogenase was present
only in germinated spores. Germination was accomplished
by the following procedure. Five grams, wet weight, of
partially germinated spores, which had been stored at
-20 C for about five months, were suspended in 50 milli-
liters of germinating solution, and heat shocked at about

hating solution contained

'0
F'-

70 C for ten minutes. The germ

enosine in 0.07

d-
C0
L1:

0.2 per cent alanine and 0.02 per cen
molar, pH 7.0 potassium phosphate buffer. After heat
shock, the suspension was incubated at 50 3 for five

hours, and then refrigerated at 4 C for 12 hours. At the
end of this time, almost all of the spores were germinated,
as indicated by their permeability to crystal violet stain.

In addition, a small number of germinated spores had grown

into vegetative cells.

A. Screening for Heat Pa esi stant dQZXLjfi

The purpose of this study was to ide tify and purify
an enzyme sys teanhich could be used as a model in the
study of spore heat resistance. my enzyne would be
useful, as long as it had a heat stable and heat labile
form. Results of experiments on the catalase of Bacillus
cereus suggested that heat labile enzymes are present
during all stages of vegetative growth, and that heat
stable enzymes appear near the beginning of the station-
ary phase. For this reason, stationary phase cell ex-
tracts were screened for heat resistant enzymes by the
method given in Section F. of MATERIALS iND l3 THODS.

The heat inactivation curve for glucose dehydro-
genase, the first enzyme examined, is given in figure 1.
This graph of enzyme activity versus time of heating
indicates that the glucose dehydrogenase loses half of
its activity in about 15 minutes at 69 C. A half-life
of this magnitude means that the enzyme has a greater
than average resistance to heat.

B. "Activation" of Glucose Dehydrggenase

An interesting feature of the curve in fig ure 1 is
the large increase in activity during the first minute
of heating. This increase was reminiscent of the activ-

ation of glucose oxidation in spores of Bacillus cereus,

UNITS ENZYME/ ML

OJ

 

300

 

200

 

IOO

 

l l J

 

 

 

0 IO 20 3O 40
TIME OF HEATING- MINUTES

h=‘+ inactivatio

.13

 

_;:;u_; ~. Th. head n at 09 C of the

ease dehyireguazae in an extract cf ve etat‘ve cells
vaillus cef“ns. This curve also shows the a arent
t activatiti of the enzyme.

M
\1

reported by Church and Halvorson (1957). This increase
could be the result of true activation or merely an

expression of the difference in heat stability of two

p.

coupled enzymes - n this case, glucose dehydrog enase
and DPNH oxidase.
To test the latter possibility, s mples of extracts
were heated to 60 C for various times, and assayed for
both glucose dehydrogenase and DENH oxidase. The lower
temperature was used to slow the apparent activation to
a measurable rate. The results are plotted in figure 2,
which shows the activity for each enzyme versus heating
time at 60 C. As DPNH oxidase activity decreases due to
thermal inactivation, the activity of glucose de hydro-

genase increases. Apparently the loss of the oxide se

permits DEER to accumulate at a faster rate.

J

C. Time Course of Glucose Dehydrog “a e Bio vnthesLs

L

Although glucose dehydrogenase had been found in
spores of Bacillus cereus (Church and Halvorson 1955)

and in stationary phase cells or forespores of the same

[—10

organ am, either a heat labile nor heat stable glucose
dehydrogenase could be found in exponentially growing

at

{31

cells. Therefore, biosynthesis had to be initiate
a time between the exponential and stationary growth
phase of this organism.

In order to observe the appearance of the enzyme

 

800

600

   
 
 
 

GLUCOSE DEHYDROGENASE .

400L 0

UNITS ENZYME / ML

DPNH OXIDASE

 
 

ZOO

 

 

 

1 1 l a
O 30 60 9'0 IZO
HEATING TIME -- SECONDS

‘ - - n . -._-. ’_ an 1.1.. -00 - .9...
Q’UIE é° A-conrarison o ane allaot cf Leatin at
. -‘ Tm, ‘ x~ ‘
I
a .‘ a
w

03 (‘2! _j

\ ‘ 1" \ ~ ‘ 5 .~. " " I ‘ i "‘ IA > T 7“ V I“ "

(11 tie glinnose de,vdrrfgenlse and the handic. 1
4- +- ' t - - ' . ,

n eitracc of verétati e ce s of :qoll.lfi cetets.

{U
(0

and to follow its concentration throughout the sporulation
process, cell samples were harvested from the New Brunswick
fermentor at various times during growth. The cells were
acetone dried, heated, and assayed for glucose dehydro-
genase activity. The results are shown in figure 3 as

a graph of glucose dehydrogenase activity and dipico-
linic acid concentration versus culture time. The curve
for dipicolinic acid is included as a parameter of
sporulation. Glucose dehydrogeLase increased rapidly
after six hours, but then appeared to decline after eight
hours. However, the decline in glucose dehydrogenase
corresponds to an increase in the number of Spores, as

indicated by increased di icolinic acid concentration.

I’C)

This suggested that spores are more refractory to
acetone extraction than vegetative cells and thus remain
impermeable to the assay substrates in spite of the
acetone treatment.

To check the validity of the acetone extraction pro-
cedure, an alternate extraction method was employed.
Two large samples were taken from a fermentor at nine
and thirteen hours after inoculation, ruptured in the
omnimixer, heated at 69 C for two minutes and assayed
for glucose dehydrogenase. The enzyme concentration of
the culture, obtained by this procedure, was consider—

ably higher than the concentration obtained by acetone

I.)
C)

1w1vaa°n

8 o

4 ‘HD

 

 

 

I

d

MECHANICAL
DISRUPTION

   
  
     
 

EXTRACTION

‘---o

ACETONE

Q

GLUCOSE
DEHYDROGENASE

 

IO I2 l4

s ROWTH TIME - Hou'Rs

 

 

GOOr

1
O 0
O

v N
WWI HWAZNB Slan

OO»-

. .
. 1 N 1 1 i '3 .

culture

Cf heat

-, . _ .9
5.3. 13wa 1 51-3

. . .
3 sell by a

31

extraction. These data point out the fact that the
apparent enzyme concentration can be grossly altered
by changing the extraction method.

D. Purification of Vegetative Cell and Spore Glucose
Dehydrogenase

The results for the purification of vegetat ve cell
and spore glucose dehydrogenase are summarized in tables
1 and 2. The meaning of the various columns is as
follows: Specific activity is defined as units of en-
zyme per milligram of protein; purification number is
the ratio of specific activity in a particular step to
the specific activity in the heated extract; and yield
is the percentage of total enzyme activity remaining
at a particular step, relative to the total activity
in the cell extract.

The total activity of the crude extracts cannot
be determined directly, because they contain DPNH
oxidase. Loss of the oxidase during purification results
in an apparent increase in the total amount of glucose
dehydrogenase, while, at the same time, glucose dehydro-
genase is lost by inactivation. To obtain an estimate
of the total amount of glucose dehydrogenase in the
extracts, all increases in activity, throughout the

purification, were added to the apparent total activity

of the crude extracts.

TABLE 1

 

Purification of the glucgse dehydrogenase from

vegetative cells of Bacillus cereus

 

 

 

 

 

 

 

 

 

 

 

 

» . Specific Purifi— Yield
krocedure activity cation %
Heated Extract . 44 l 62
Protamine and Dialysis
(0.1 n. tris) 195 4 72
Amflonium 54-75% 155 4
Sulphate
fractionation 75-86% 1,720 59 44
55-96% 554 a
Combined, Reheated,
Dialysed (0.05 M. tris) 3,420 75 46
DEAE Fract. 9 29,400 660 17
Cellulose
Chromatography Fract. 10 12,600 290 13
Fract. 11 2,240 51 7

 

 

 

 

 

 

 

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SO 7 0.~
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...... E... «I. flu C
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U C; I.\ L n x. W . A I,"

 

 

 

 

 

 

 

 

16 8:)

a _1

T cific activity and purification number of
the heated spore extract are not prese ed because the
protein concentration of the extract was not deter-
mined. Instead, the specific activity of the protar ins
and dialysis step is considered a purification of one.
The heated spore extract probably iid not have a specific
activity much lo'er than tha.t cf the uialys 3d ex tract
because very little precipitation occurred during the
protamine and dialysis step.

Al hough the purif ication obtained in the 75-85
per cent saturated ammonium sulphate fraction was far

sreater than that in both the 54-75 and 85-96 per cent

_7

p

saturated fractions, all three fractions were combined
before cellulose chromatography in order to conserve
total activity.

The similarity between vegetative cell and spore
glucose dehydrogenase was indicated by their solubility
and charge properties. Both enzymes were prec cipitated
at an ammonium sulfate conce entration cf approximately
80 per cent of saturation (see Doi, Church and Kalvorson,
1959, for data on spore enzyme), and both enzymes were
eluted from the cellulose column at a sodium chloride

concentration cf abcut 0.1 molar.

E. Charac éterization of Vegetative Cell and Score Glucose
Dehydro crenase

 

 

 

T‘) stability of some proteins is increased

L
.L .LLV

when they are conjugated with nucleic acids. Since
the crude vegetative cell and spore extracts contained
a large amount of nucleic acid, the heat resistance of

-1 H- .- :1-
the glucose c .3tr

‘,_'_‘IV\Q
’.J~¢K

U)

e in these extracts might have

C)
If‘

been due to binding with this material. To elizhlnate
his possibility and to compare the heat resistance of

L .. — .,.-,..-, ..., - 39:. - -_,._. ,Lg- ... ., ,1 .
the two enzymes, pur111ed VCSGtQU1V8 cell and spa:

(D

enzymes were tested for heat resistance by the capil-
lary tube method. The vegetative cell enzyme used in
this ex xperi dent had been purified 290 times and con-
tained less than 1 per cent nucleic acid. The Spore
enzyme had been purified 20 fold and contained about
6 per cent nucleic acid.

Inactivation data for both enzymes are presented

in fioure 4 as the logarithm of activity versus time

I}(

(3

of he atinl" at 59 C. The purified vegetative cell

:1

enzyme has a half-life of 17 minutes compared to a

q

half-life of 15 minutes for the crtae mgetative cell

1.).

extract. The inactivation of the puril ed vegetative
cell enzyme follows first order reaction kinetics very
closely. 0n the other hand, the inactivation of purified
spore slucose dehy dro.enase does not appear to follow
first order kinetics and this enzyme has a half-life of

only one minute at 69 G.

Since the removal of practically a l nucleic acid

LOG UNITS ENZYME / ML

 

4.0 -

 

 
 
 

VEGETATIVE CELL
ENZYME

    

3.0

 
 

SPORE ENZYME
2.0

 

i J _A

 

 

O 20 4O 6O
HEATING TIME -- MINUTES

 

57

from the vegetative cell extract does not lower the heat
stability of glucose dehydrogenase from this source,

it is unlikely that nucleic acids contribute to the heat
stability of this enzyme.

2. Liohaelis constants

 

..3

To prove the enzytatic sim larity of vegetative

cell and spore lucose dehydrogenase, the Lichaelis

constants of the two enzymes were compared. The Iichaelis
constants of the vegetative cell glucose dehydrogenase
were deternined from the data in figure 5. These are
Lineweaver-Eurke plots (1954) of enzyme activity versus
substrate concentration. The enzyme preparation used
for this experiment was an 86 per cent saturated amnonium
sulfate fraction, having a specific activity of 11,700.
The Spore glucose dehydrogenase Iichaelis constants were
reported by Doi, Church, and Halvorson (1959). Table 3
is a compilation of results obtained for both enzymes
which shows that the enzymes have similar substrate
affinities.
3. Serology

The vegetative cell and Spore glucose dehydro-
genases were compared serologically to test the structural
similarity of the two enzymes. The results obtained by
the agar gel diffusion technique are shown in the photo~

graphs in figure 6. These plates demonstrate clearly that

3

 

 

 

 

 

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1.1123? '18 JULIO-173 ‘3 :

 

‘ ‘~ n‘! ,,.,-..-.. ‘, .
--‘ , ILL .. L» .7 I73 7 o :1

 

 

 

 

Spore Enzyme DLN 9.1 X l

 

 

 

 

 

 

 

 

1%

Figure 6. A serological comparison of th glucose .
dehydrogenases from vegetative cells and spores of bac1llus
cereus by the agar gel method. See page 42 for key to
well contents.

41

at least two heat resistant proteins are present both
in vegetative cells and spores. To determine if one

Q

eenase, a mixture

p
‘-

of the proteins was glucose dehydro
of DPH and glucose was Spread over the agar, and the
precipitin lines were examined for fluorescence under
an ultraviolet lamp. although fluorescence was ob-
served, it could not definitely be attributed to the
precipitin lines, because of the large anount of un-
precipitated glucose dehydrogenase in the agar.

The serological identity of the two enzymes was
proved by using cellulose acetate film in place of
agar and developing the precipitin lines with a DPH-

nd

s

glucose mixture. The arrangement of antigens
antisera on the film was essentially the same as the
arrangenent used for the agar di.fusion experiment,
but, when a DPN-glucose mixture was used to develcp

the washed film, the only visible precipitin lines were
those produced by the reaction of glucose dehydrogenase
with its homologous antibody. After spreading the
glucose-DEN mixture on the washed films, distinct

fluorescent lines appeared between the ant1gens and each

antiserum, but not between the antigens and the normal

serum controls. These results are presented diagrana-

tically in figure 7, where the inked lines represent

fluorescence.

 

 

 

ABV ' ABV

CV VE ABS CV SE ABS

 

 

(38 (38

 

 

 

'- rrv

mi

d'e azainst vevetative

m
G

ABV - Serum containing antibo
cell enzyme
abs - serum containing antibodies against spore enzyme
VB - Vegetative cell enzyme
SE - Spore enzyme
CV - Control serum from rabbits receiving vegetative
cell enzyme

0° Control serum from rabbits receiving spore enzyme

Figure 2. A serological comparison of the glucose
.1.

dehydrogenases from vegetative cells and spores o
Bacillus cereus by the cellulose acetate film method.

45

F. Heat labile Glucose Dehydrogenase

Because veg

(D

tative cells contained no heat labile
glucose dehydrogenase, a germinated Spore extract was

exanined for this enzyme. A :luccse d hydrogenase

is s was
found, in this extract, which was completely inactivated
in less than one ndnute at both 70 and 60 C. At go C,
this enzyme had a half-life of less than one minute,

compared to a half-life of seventeen minutes at 69 C for

the vegetative cell enzyme.

DISCUSSICE

Following the exponential growth of Bacillus
cereus cultures, a complex series of events occurs
resulting in the production of heat resistant spores.
Heat resistant glucose dehydrogenase is synthesized

by these cells early in the sporulation process and

(D

1‘ tr—

0‘)
L U

is eventually incorporated into spores. Aft
mination, when the spores have completely lost their
heat resistance, the glucose dehydrogenase is still
present in high concentration but it too has lost its
heat resistance. The glucose dehydrogenase can not
be detected after vegetative growth commences because
it is diluted by the multiplication process.

The results of serological and enzymatic com-
parisons, the coincidence of the production of this
enzyme with the production of spores, and the coincidence
of the change in heat resistance of this enzyme with the
change in heat resistance of spores, all indicate that
the glucose dehydrogenase in vegetative cells, spores,
and germinated spores is the sage enzyme with different
levels of heat resistance. For this reason, if both
the heat labile and heat stable forms can be completely
purified, this enzyme system should provide an excellent

model for studying spore heat resistance. However, as

stated in the introduction, the purpose of this study
was not merely to obtain any model system, 'but one in
which the enzymes are plentiful, easily purified, and

we sonably stable during store as EnzyneS'with these

U

.2

characteristics can be accumulated in sufficient
quantity to permit physical-chemical characterization.

"1

ins ve‘e tative cell glucose dehydrogenase appears
to meet these requirements since it is very stable, and
can be purified easily and in high yields. Although
its cellular concentration is not especially high, its
stability during storage will permit the accumulation
of the large amount of pure enzyme needed for its
complete characterization. In fact, a partially puri-
fied preparation of this enzyme has been stored for
eight months, at -20 C, without losing appreciable
activity.

On the other hand, the accumulation of germinated
spore glucose dehydrogenase may not be as easy because
this enzyme is heat labile. Enwever, since the spore
enzyme is much more stable, large amounts of unger-
minated spores, rather than germinated Spores, could be
accumulated. Germination of the spores and purification
of the heat labile enzyme could then be accomplished in
a short time, with minimum loss of enzyme activity.

The aberrant inactivation kinetics of the spore

Q

“t to

.J.‘

«.4

° 1a)."
Yul ‘1‘

.4‘” V

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TL

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i

'is

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UAL

Tr

true,

10
l C

said.

a?

at will by conditions sinilar to those required for
germination of whole Spores. Such a syste a would

era 1t one to study loss of heat resistance by a

*(i

"dynamic" method rather than by a "static“ comparison

of heat stable and he at labile e11zyme 8. Much more lg.
in Mornation about the Inec1anism of heat resistance

could be obtai: ed from a dynamic system than from a a

static one because loss in heat resistance may involve

(D

more tr an one simpl step. In fact, the loss of heat

resistance by a series of events is suggested by the
Spore enzyme inactive mi n etrve shown in fi5ure 4. If
only two levels of heat resistance were possible for
each glucose dehydrogenase molecule - those of the
Vegetative cell and germinated spore enzyme - the bend
in this curve would be extremely sharp at 69 C, because
of the large difference in inactivation rates of the
two enzymes. Instead, the rather broad bend observed
indicates the pose sibi li 1ty of glucose dehydrogenase with
heat resistance intermediate to that of the vegetative
cell and germinated spore enzymes. Thus, tr 1e loss of
heat resistance by this enzyme may be a sequential
phenomenon, requiring more than one unit event for the

change from maximum to minimum heat resistance. This

statement, however, should be considered as no more 1

than an hypothesis, since the meager inact 1v vation data

48

Ho

in gure 4 can not support any real conclusions. The

(D

v rtheles

(L‘
(0

heat inactivation results in figure 4 do, n ,
contain l? portant inplications for the purification of
this model system. If phm ical-c hemical comparisons of
the heat stable and heat labile enzymes are to be made, W
the stable enzyme must be obtain1ed from vegetative cells
because the enzyme at this stage of Spore developALnt
retains its heat stability during storage. On the other
Lani, since loss of heat resistance by purifled enzyme
appears to depend on the raturity of the spores from

which the enzyt1e is isolated, the heat ste ble en: :yute
from 1 ature spores would be useful in "cell-free
germination" studies

Compared to other heat resist ant spore enz 1nes and

to the whole spores th mselves, the stability of the
zlucose dehydrogenase1rom.vegetativ e cells and spores

of Bacillus cereus is rather low. For example, the

 

adenosine ribosidase found in Eacillus cereus spore

 

extracts by Powell and Hunter (1956) had a half-life of
four hours at 100 C, compared to a half-life of fifteen
minutes at 69 C for the gl lucose dehydrogenase. However,
the glucose dehydrogenase has both a heat stable and
heat labile form, and the difference in heat stability
of the two forms is quite large. Thus, the glucose

dehydrogenase appears to be a reasonable model for the

study of heat resistance. The fact that t1mchange in
heat resistance of the glucose dehydrogenase parallels

the Change in heat resistance of whole Spores during

germination 13 additional proof of its usefulness as a
model system. Sorrelation with the heat res 1s stance of

whole spores is, in fact, a requirement for any enzyme

5 stem to be used for the study f spore heat resistance,

O

stant enzyme 1n

t3”

\

cf
(I)
H.

since the mere presence of a re re

1

spores does not necessar11y m

(D

an it contributes to Spore

L
11

(I)

at resistance. For exa1tple, a very heat stable in-
organic pyrophosphatase was found by Johnson and Johnson

(1959) in azotoba a2 lis, a nonsporeforming meso-

 

phile. This enzyme Ob'v iously bears no relation to

"-v-J
:1.
O

:7
P.
C’-

cellular heat resistance because the cell from w

is derived is not heat resistant.
In spite of the foregoing considerations, a certain

amount of caution must be exercised in predictions of
the usefulness of this model system for determining the
mechanism of spore heat resistance. Since the purified
heat stable glucose dehydrogenase is rapidly inactivated

at temperatures used to heat shock spores prior to ger-

nination, and the heat labile glucose dehyer enase is
abundantly present in these germinated Spores, it is

obvious that differences in structure of these two puri-

fied enzymes cannot completely account for the heat

(51
C)

resistance of the enzyme in intact Spores. It is possible
that the structural differences between these enzymes are
diminished when they are extracted, or that intraspore
environment also plays a role in the pro action of this
enzyme‘while it is in the spore. In any event, the
author feels that the application of the model systei,
described in this thesis, to the study of spore heat
resistance is almost certain to yield infornation of a
fundamental nature, and to provide at least the beginning

of a theory for the mechanism of spore heat resistance.

(“YT '7" ' " ' T“, T
JUliliu”h—L\1

A model system for the study of the heat resistance

of spores of Bacillus cereus has been described. T113

 

system consists of a heat stable glucose dehydrogenase
from vegetative cells and Spores, and a heat labile
glucose dehydrogenase from germinated spores. In crude
extracts, the vegetative cell enzyme has a half—life
of about fifteen minutes at g3 C, while tre
spore enzyme has a ha f-life of about one minute at

An increase in the g1ucose dehydrogenase activity
of vegetative cell extracts was obser ed during the

A

first minute of heating at 69 o. This app

d"

heat

on

I‘

(D

I1

0.1

activation was investigate because of its possible
connection with the heat activation of glucose oxidation
in whole spores. It was concluded that the heat in—
activation of DPNH oxidase, without grossly affecting
the glucose dehydrogenase activity, was reSponsible for
the apparent heat activation of the extracts.
Biosynthesis of the heat stable glucose dehydro-
genase is initiated shortly after the exponential growth
of cultures ceases and before any dipicolinic acid can
be detected in the cells. The heat stable enzyme is
present in the cells during most of the sporulation pro-

0

cess but a true measure of its concentration during this

period was impossible with the extraction procedures
available.
The enzyme from vegetative cells was purified about

30 t

C}
Pa

mes, and the enzyme from spores was purified about

...4
Q
(11

mes by the following steps: extraction by mechan-

H0

t

H.

cal disruption of cells or spores, heating to 65 C,
protamine precipitation of nucleic acids, anmonium

“fig-3,1

nation, and nzns cellulose colunn chro-

U)
:1
pa
*0
:3”
$13
C,-
(D
F9
‘fi
CD
(D
d.
F“
O

1he purified enzymes from vegetative cells and spores
had almost identical substrate affinity and serological
characteristics, but differed in heat stability and
kinetics of heat inactivation. The difference in heat
stability and heat inactivation kinetics was attributed
to a partial conversion of the heat stable enzyme to the
heat labile enzyme.

The possibility of completely purifying the glucose
dehydrogenase system and using it as a model for studying

spore heat resistance was discussed.

D

 

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1

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10.

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13.

14.

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17.

18.

 

Lawrence, F. L. 1955a The cleavexe of adenosine

by spores of Bacillus cereus. J. of

Bacteriol., 73, 577-582.
L138.€&V8T, ?. and Burk, D. 1954 The deter—

mination of enzym dissociation constants.

T A 'r‘. ‘ ,«~‘ A (”A

f) O .1124. Cfldlll. .DCC. , 55’ 658-6CO. '
Iowrv, C. 3., Posebrougu I J. *Ure, A. L.

.. t ' : 9*

and iandall, R. I. 1951 Protein measure-
ment with the Folin p a r gent. J. Biol.
Chem., 193, 265-27

 

Cuchterlory, 0. 19491 antigen-antibody reactions
in gels. Arkiv. Yemi, Kin. Cch Tecl. 2%3, l-v

Cuchterlony, O. 1949b eatiren-antibod' reactions
in gels. Acta gath. e: microbiol. 3.9:fli:av.,

Iowell, J. F. anl 7untsr, J. F. 1956 uierosine
dea.inase and ribosiiase in spores of Eacillgs
Careus. Eiocheg. 3. (London), 62, 381-387.

 

’T

Eadcff, F. L. Fools J. 3. ani Rasheb, a. S.
e 6 ant catalase of Bacillus

Stewart, E. T. and Halvorson, H. C. 1953
Studies on the spores of aerobic bacteria.
I The occurance of alan ae racemase. J. of
6

1. . 1?!‘
_h\

 

Warburg, C. and Christian, K. 1942 Isolierung
and kristallisatian des garungsferments
enolase. Biocren. 2., 310, 384-421.

 

k

 

AZT' ":r’j1
‘Md'fl!

USE emLY

 

 

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9

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