r---------
--··
·-- ----------
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:
!i
CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
ATEMPERATURE
\I
SENSITIVE MUTANT OF BACILLUS SUBTILIS:
ANALYSIS OF PHYSIOLOGICAL SPORE DEFECTS
A THeSIS
SUBMITTED IN PARTIAL SATISFACTION OF THE
• REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN
BIOLOGY
BY
JoEL ERIC RoTHMAN
JUNE,
1974
i
I!
·~--·--~-----~----- ------~-------~----~----·----------·--
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---»·-----------···---------~----------------·--------------------~--------------~~J
THE THESIS a~ JoEL ERIC RoTH~ N IS APPROVED:
. zloMfvliTTEE [HAl RMAN
CALIFORNIA STATE UNIVERSITY. NoRTHRIDGE
1
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JUNE,
1974
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ii
DEDICATIONS AND ACKNOWLEDGEMENTS
I WANT TO DEDICATE THIS THESIS TO MY WIFE, VALINDA,
IFOR HER NEVER ENDING FAITH AND INSPIRATION, AN~ TO MY
JPARENTS, FOR THEIR ENDURING ENCOURAGEMENT.
SPECIAL ACKNOWLEDGEMENT AND THANKS TO DR. CHARLES
R.
SPOTTS, FOR WITHOUT WHOM, THIS THESIS WOULD NOT HAVE BEEN ·
'possiBLE.
To DRs. MARY CANTOR AND DoNALD BIANCHI, MY
APPRECIATION FOR THEIR ASSISTANCE IN THE PREPARATION OF
THIS T.~Esrs.
lI
To MR. ALBERT BLIFELD,OF DAMON MEDICAL LABS.,
MY GRATITUDE AND. APPRECIATION
FOR HIS HELP DURING THE
..
COURSE OF THIS PROJECT.
THIS THESIS PROJECT WAS PARTIALLY SUPPORTED BY A
CALIFORNIA STATE UNIVERSITY, NORTHRIDGE FoUNDATION GRANT,
NUMBER
3234.30013.
II
L---------·-·--------- --------·--------------·--------·-·----·--------------------_1
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TABLE OF CONTENTS
DEDICATIONS AND AcKNOWLEDGEMENTs •••••••
Ill II
ABSTRACT
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I NJRODUCTI ON.
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METHODS AND MATERIALS •••••
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Bl 1 1 1 1 1 1 1 1 1 1 1 1 I
RESuLTS ;
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DIscuss I ON
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LITERATURE cITED
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FIGURE
1.
FIGURE
2
FIGURE
3111111101111111111111111111111111111
FIGURE
41.
FIGuRE
5
FIGURE
TABLE
61
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TABLE
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ABSTRACT
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A TEMPERATURE SENSITIVE MUTANT OF BACILLUS SUBTILIS:
ANALYSIS OF PHYSIOLOGICAL SPORE DEFECTS
I
BY
JOEL ERIC ROTHMAN
MASTER oF SciENCE IN BIOLOGY
JUNE,
1974
A temperature sensitive sporulation mutant of
1 Bacillus
subtilis was isolated and its chemical and
physical characteristics were investigated.
This mutant is'
able to grow equally well at 30° and 42°C, but is unable to
!produce a
normal~
healthy spore a.t the restrictive
!temperature of 42°C.
Properties of the spore such as heat
resistance, refractility, and resistance to osmotic shock
were normal in the parent strain but were altered in the
mutant at the restrictive temperature.
Calcium levels
were found to be lower in the mutant than in the normal
I
crosslinked and thus exhibit the altered phenotypic
I characteristics
I
of the mutant spore.
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v
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_ _ _ _ _ _J
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INTRODUCTION
CHAPTER
The bacterial endospore is a metabolically dormant,
heat resistant resting cell formed by bacteria of the
family Bacj.llaceae.
It can be readily recognized
microscopically by its characteristic intracellular
location, extreme refractility, and resistance to staining
by basic analine dyes which stain the vegetative cells.
Only one spore is formed by each cell and each endospore
gives rise to only one cell.
Thus, the endospore should
be considered a resting form, rather than a reproductive
structure.
Since the dormant endospore completely lacks
any biosynthetic activity, it preserves the species until
favorable growth conditions are once again encountered
I (Murrell
and
Irepresents
Warth~
1969).
Therefore, the endospore
a stage in the life cycle which allows certain
, bacteria to bypass unfavorable conditions without dying.
Previous work with this mutant (Goldman, 1970) has
shown it to be a temperature sensitive asporgenous mutant.
This study will attempt to define the physical basis for
l
its failure to attain the characteristics of a normal,
I healthy
spore, such as heat resistance, refractility, and
I resistance
j
to osmotic shock.
An examination of its c
chemical composition and physical characteristics was
I performed
and is reported here.
This information will be
I
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L----------------------··------·-----~------~-------·----------__j
j compared vd th known data on the normal wild type spore to
1
~2
. attempt to identify the physiological defect.
l
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iI
I
Considerable information has been obtained on the
1 cytology
l
and chemical composition of the normal spore in
I
!I recent years through electron microscopy, and chemical and i
!physical analysis of fractionated spores.
To better
understand the various aspects of spore structures and its
functions, it is essential to have a working knowledge of
the background of spore development and organization.
When the level of critical nutrients has decreased
and growth ceases, changes are induced in sporogenic
bacte=ia which cause a rearrangement of nuclear material
which could be interpreted as a unique type of cell
division.
This particular type of division requires an
extremely long period as compared to a normal cell
generation time of 49 minutes, and it marks the beginning
of the formation 0£ the forespore septum.
separ~tes
This septum
the sporal area from the parasporal part of the
sporangium.
These changes are part of the series of
sequential processes leading to sporogenesis and
I
I
eventuall~
I
result in the production of mature spores.
Sporulation is a process of cellular differentiation
1.vhich appears to be quite simple 1.'Jhen compared to the
complex intercellular interaction characteristic of a
multicellular organism.
However, it is nevertheless a
very complex biochemical and morphogenetic process.
Information obtained through research over the past
L!ifteen years (for
re~~~-~-=-~--=-~-~--~~ tz -Jame.s
I
and Young,
J
3
~(\0~9.
!
..L ::::1
,
~~
Tipper and Gauthier, 1971) indicates that
isporulation consists of an ordered sequence of biochemical
I
land morphological changes.
I
By means of electron microscopy, it has been found
I
!convenient to identify eight stages of spore formation
(Schaeffer, Ionesco, Ryter, and Balassa, 1965) (see
Figure I):
Stage 1) formation of the axial chromatin
filament, 2) beginning of septation, 3) completion of the
forespore, 4) formation of the cortex layer, 5) completion
of the spore coat development, 6) spore maturation, giving
an in·tac.t, included spore, 7) lysis of the vegetative cell
liberating the mature spore, and 8) complete refractility
of the spore.
Up to stage 3, sporulation is reversible
and the cell can return to the vegetative form after
regaining favorable medium and/or environmental conditions.
At stage 4, the cell becomes committed to proceed with the
spore formation through stage 8 (Fitz-James and Young,
1969) and will proceed through the process regardless of
any changes in the medium.
Stage 4 is represented as the
period of development when the underlying spore
layer is formed.
~ortex
Although the stages represented are
arbitrary frames in a continuous process, it is possible
to isolate morphological mutants in which sporulation is
blocked at any of the stages.
The development and biosynthesis occurring during
sporulation involves a series of several integrated
_ sequential biochemical pathways (Murrell, 1969), which can ·
t______
····· - - - - - - - -
_::_j
4
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FIGURE 1
STAGE 5
STAGE i
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STAGE 6
STAGE 2
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STAGE 3
STAGE Z
STAGE 4
STAGE 8
!
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1
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DIAGRAM OF EIGHT MoRPHOLOGICAL STAGES DuRING
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SPORULATION IN BACILLUS SUBTILIS (SEE TEXT
1
FOR DESCRIPTION)
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·5
!be correlated with the morphological stages previously
I
I described:
stage 1) formation and release of antibiotics,
i
lproteases, ribonuclease, and amylase, protein turnover and
synthesis of alanine dehydrogenase, 2) initiation of
synthesis of glucose dehydrogenase, 3) production of
aconitase, alkaline phosphatase, and heat-resistant
catalase, 4) production of DPA and the uptake of calcium,
5) incorporation of large amounts of cysteine into the
spore coat, 6) production of an enzyme, alanine racemase,
involved in both heat-resistance and germination.
The
mature spore, as diagrammed in Figure 2, is
composed of three cytological components:
coat, and the spore cell.
cortex, spore
The chemical composition of the
the spore's dry weight (Tipper and Gauthier, 1972).
The
proteins of the coat have a high concentration of glycine,
lysine, aspartic acid and non-polar amino acids.
The
cysteine content of the spore coat is especially high,
! being
four to five t.imes ·that of vegetative cell proteins.
I The high
I
-~
concentration of non-polar amino acids suggests
I
I
that the polypeptides are very capable of forming
I extensive
intra- and intermolecular bonds of the
1
1
j "hydrophobic" type as lvell as the production of
j
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I
.
L~~tramole~ul_~~-dis_ulfi~~--~2_id_ge: and io~ pairs . __._!h~_:__: ___j
6
r
· FIGURE 2
SPORE CELL
CoRTEX
DIAGRAMATIC REPRESENTATION OF THE
I
STRUCTURE OF BACILLUS SUBTILIS
l
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I special
I
chemical properties of spore coats have been
I
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!postulated to explain the resistance of spores shown to 8M
!
!
!urea, 80% phenol, 98% formic acid, and detergents
(Warth,-~
!1963).
I
The cortex layer contains a mucopeptide polymer,
similar to that found in the vegetative cell walls of gram
positive bacteria.
A hypothetical structure of this
polymer has been formulated.
The evidence suggests the
presence of glucosamine, alanine, o< -
I
E -diaminopimelic
acid (DPA), glycine, aspartic acid, and glutamic·acid in B.
subtili.s (Warth, 1963).
Similar polymers have been found
in four other species of Bacillus.
In electron micrograph1
the cortex appears as a layered matrix (Mayall and Robinow,l
1957).
It is, therefore, likely to be cross-linked,
perhaps with the chains twisted and coiled or interwoven
as they are laid down during cortical formation.
More
recently, (Murrell, Ohye, and Gordon, 1969) the existence
of the peptidoglycan polymer was postulated as a three
dimensional array of polymer stands, forming a layer about
0.1 micrometers in thickness, whose function is laying
down the Ca:DPA matrix.
It has been found (Curran, Brunstetter, and Myers,
1943) that spores contain more calcium than do vegetative
II
1
cells and recent findings have shown that calcium plays an •
I
important but unknown role in the development of heat-
I
! resistant
l
1
spores (Sugiyama, 1951; Grelet, 1952; Amaha and
~:.~~-1_,_1_9_5_7_~_an_d_ B1 ac k_~ -~a~ h im_e>_~~__!-~~Ge r h_C:_::d t ,
I
_j
19 6 0) . _
-8
in the mature heat-resistant spore, calcium and dipicolinicj
liacid
(DPA) occur in nearly equimolar amounts (Lund, 1959;
j
IWalker
I
I
et al. ' 1961; El Bis i' et al. ' 1963; Pelcher'
r
jFleming, and Ordal, 1963) and are released together with
!the spore peptide during germination (Powell and Strange,
11953).
Cells grown in calcium deficient media, or under
conditions where DPA is not incorporated do not develop
heat-resistance.
This has been correlated with the Ca:DPA
ratio with two species of Bacillus (Levinson, et al., 1961;
Lechowick and Ordal, 1962).
The relationship between the function of each of the
spore components and the behavior of the spore and the
sequence of morphogenetic events may be analyzed by a study
of morphogenetic mutants.
From a morphological point of
view, two types of mutants may be described.
First, there !
are some mutants which are completely blocked at a definite,1
step of sporulation and unable to proceed beyond it.
Such
I
lesions result in the accumulation of incomplete spores
blocked at the formation of a recognizable normal
intermediate.
Secondly, mutants have been isolated which form whole
spore bodies which lack one specific spore structure.
1
!These mutants are apparantly capable of synthesis of
Jstructures formed before and after the expected time of thel
I deleted structural change.
Il this
A well described mutant of
second type is one which completely lacks the ability
!
II
I to form a cortex.
Complete cortexless mutants have been
L
___________________
----------------------__l!
9
!fowid i:: 3. cereus, and B. megaterium (ritz-James, 1965).,
'The defects on these mutants have been attributed to
!'
jdefects
l:l
calcium and DPA incorporation, ultimately
I
I leading to an ·imperfectly formed cortex.
I
I
The utilization of temperature sensitive mutants
I
!creates a further refinement in the study of sporulation
I
.
when cells are grown _at low temperatures, the sporulation
process proceeds normally, but when the temperature is
raisec above some critical level, still within the normal
physiclogical temperature range, the cells behave as
mutants.
In this type of mutant, a minor alteration in
amino acid sequence causes a critical polypeptide to
become non-functional at a relatively low temperature
while other functions remain unaffected (Fitz-James and
Young, 1969).
It is thus possible to turn sporulation off
and/or on when desired, by raising or lowering the
temperature, a decided advantage 1vhen attempting to
I
I
I
determine the precise role of biochemical reactions in the
I
::::::::;:: :::::::·pe:::t:s:h:fr:::::::::r:os:::::~:e
~
many aspects of the process not accessible with regular
!mutants, since they would normally be lethal.
I
A great deal of research (reviewed by Murrell, 1969)
I
~points toward a relationship between the structure of the
!cortex and development of dormancy and heat-resistance.
!
~Since the defect in the mutant manifests itself as a
·
I
II
1
Il
~--~~ ~-~~ ~~~--~~--!eve~ o ~---~ e a~ ~-:-~_:~~_:~_c ~--!-~~_:__~-=-~~~c-~-~~i t y , -=-~~=--j
1-0
~tudy
will be directed toward a comparison of cortex
l
!structure with respect to murein chemistry and Ca:DPA
!
i
jratios.
The primary problem is to attempt to identify
l'
I the specific spore component which has the defect causing
I
I
I the aberrant behavior.
II
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!'
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'--~---·---------~~---------------~------- --··-------·---- ·-------~--~·-----·-·----~~-----------------·----'
CHAPTER II
Ii
I
· METHODS AND MATERIALS
I
/ORGANISM
I
I
I
Bacillus subtilis 168, a stable tryptophan auxotroph,
!was used as the wild type organism and source of the
!mutants.
This organism demonstrates normal sporulation.
!Temperature sensitive sporulation mutants of Bacillus
subtilis
168~were
obtained from Mr. Robert Goldman (1970).
These mutants were induced by heating dried pellets of
\1
spores~ at 100°C in a vacuum oven for 9 to 12 hours.
The
treated spores were then suspended in sterile distilled
water, diluted, and replica-plated on Nutrient Broth
0
Sporulating Medium (NBSM, described below), at 45 C and
I 30°C.
Those colonies which did not turn the characteristic,·
!dark brown color of the wild type sporulants after four
days of incubation at 42oc, but which did show increased
sporulation at 30°C were retained as the temperature
sensitive mutants.
fv]ED I A
The standard sporulating medium used, Nutrient Broth
Sporulating Medium (NBSM), consisted of (per liter): 8
Igrams Nutrient Broth
!Mgso 4 • 7Hz0.
(Difco) , 1 gTam KCl, and 0. 2 5 grams
After autoclaving, this medium was
i
I supplemented with 0.1% glucose, 10- 3 M MgClz, and 10-6M
I
I FeS04.
i
The glucose and calcium salt were autoclaved
l s~parately and then ad~~-~-'---~hil_~-~~e --~-a-nga~ese_:-n~ iron _j
i
11
12
!salts were filter sterilized prior to their addition to~
I
!
I
!the
medium.
i
Two percent agar (Difco) was added for solid
i
I
I
d"1a.
1me
jGROWTH AND SPORE PRODUCTION
i
1
Cultures were started from a frozen suspension
!(approximately 107 spores/ml in 75% glycerol) in the case
of the mutant, t-6, and from a refrigerated slant in the
case of the wild type 168.
The strains were inoculated
into 100 ml of NBSM and were allowed to grow at 30°C until
Ia conce=tration of 10 7 to 10 8 cells per ml was obtained.
!The cultures grown at 30°C were incubated in a New
Brunswick Psychrotherm Controlled Environmental Incubator
Shaker, at 175 rpm.
Cultures grown at 42°C were incubated
in a New Brunswick Gyrotory Shaker at about 175 rpm.
!Growth was determined by monitoring the optical density
I
at 600 mu in a Spectronic 20 colorimeter.
Growth rate
!experiments were performed in nephaloflasks, flasks
I
equipped with side-arms for the measurement of the optical
density.
This procedure was a repeat of work done by
jGoldman (1970), to insure that the spores were grown under
I identical
conditions to that of Goldman's research.
i
I
1
Large amounts of spores required for amino acid and
/ chemical assays were obtained by growirig cultures in four
!
! one-liter
flasks, each with 500 ml of NBSM.
These
'
I cultures
l
i culture.
i
were inoculated with 10 ml of a 12 hour old batch
Spore suspensions were prepared in this manner atl
I
l----·---·--···--------------·--------··· ---------·-----------·-·--------~--·--------·-----------------i
13
~-----------
'both the Testrictive and permissive temperatures for both
the muta=.t and 'dld type organisms.
The amount of
sporulation and whether phase dark or bright was monitored
via direct spore counts, in triplicate, on a PetroffHauser counting chamber
:us~il.g
a phase contrast microscope.
The frequency of sporulation was expressed as percent
sporulation:
spores/.Ccells+spores) times 100.
were harvested 48 hours after inoculation.
data
to
~ad
The spores
Although such
been obtained by Goldman (1970), it was necessary
re~eat
these measurements to insure that data from
all S?ore groups were made under comparable conditions.
Normal spores were observed to be phase bright, while
phase dark spores were taken to be abnormal.
Phase bright
spores appear as very bright spots because of their high
refractive index.
Mutant or defective spores appear
darker than the bright spores since they never achieve
the high refractivity of normal spores.
Determination of
size of normal and abnormal spores was achieved by
measurements from photographs taken with a Zeiss phase
contrast microscope at 1250 X magnification, using a
Pentax 35 mm camera (Figure 3).
PREPARATION OF SPORES FOR ANALYSIS
Both mutant and wild type spores were prepared by the
i
same procedures.
I usually
I
When sporulation was judged complete,
after 48 hours, the spores were collected by
Ii centrifuging
in the Sorval
'-------------·--·---
------·-----------------------------------·---- - - -
RC-2
centrifuge at 5000 rpm at
1
_j
/5000 rpm at 3°C for 30 minutes.
The spores were suspended
!
I
iin 150 ml 0.5 M phosphate buffer, pH 7.0, and lysozyme was
'
jadded to give a final concentration of 0.2 mg/ml.
This
I
!suspension was incubated at 30°C for 24 hours to allow
llysis of any remaining vegetative cells.
It was then
jcentrifuged again at 5000 rpm for 1 hour, and the pellet
jwas washed three times with a 0.05 M phosphate buffer,
pH 7.0.
The resulting pellet was layered upon a 66%
sucrose solution (w/v) and centrifuged at 3°C for 1 hour.
Very gcod separation was obtained between the spores and
twice in distilled water, and spore counts were performed
to determine the number of spores per ml of original spore
suspension.
The counts were done in triplicate, on the
Petroff-Hauser counting chamber.
Dry weights of the spore
!pellets obtained for each of the four conditions were
jdetermined after drying at 90°C in vacuo to a constant
iproteins and other compounds, 1 gram (dry) of spores was
I
I
I
I
,I
I
I
..
,
1 we1gnt.
I
To obtain broken spores for the solubilization of
I suspended
J
in 20 ml distilled water and placed in the
j
l_!::_~_:tc~--~r~!-~u~-~-~~-~-~--- T!:~--su_~-e.~:_ i~~ wa.~---.Y-~.~~-ed ~hro~gh _j
15
the
six tines at 25,000 psi resulting in about 80%
,breakage.
This was performed on each of the four
!different spore groups.
I
I
The broken spore extracts were
tre~ted
with pepsin in
!preparation for the amino acid determination via thin
!layer chromatography.
I
Fifteen ml of the broken spore
suspension were conce?trated to 0.5 ml using
Lyphogel~
(Gelman Instrument Company), a water absorbant, and
centrifuged at 5000 rpm for 30 minutes.
resi~~e
The resulting
of broken spores was digested in a 1 ml solution
of 1% pepsin (Difco) in 0.1 N HCl and incubated for 48
hours at 45°C.
After the incubation period, the solution
was neutralized by the addition of 0.12 ml of 1 N NaOH.
An equal volume of ethanol (100%) was then added.
Finally,
11 drop of 0.5 M phosphate buffer, pH 7.0 was added to
l
I stabilize the
pH of the solution.
The solution was then
centrifuged at 5000 rpm for 30 minutes and-the supernatant
was analyzed by thin layer chromatography.
Temperature sensitivity determinations were performed
on aliquots of each spore suspension prior to drying.
I
One
iml of Nutrient Broth spore suspensions (approx. 3.5 X 1010
Ispores/ml)
were added to 9 ml Nutrient Broth blanks,
/maintained at 45°C and increasing by 5° intervals.
!
I
After
:five minutes at each temperature level, 0.5 ml of the
!
!
I particular
1
suspension was withdrawn, serially diluted in
Nutrient Broth at 45°C, and plated on solid Nutrient Broth
I
I
I media.
I
j
I
I
I!
After 48 hours growth at 30°C, the number of ---·------_1!
L--- --------------- - - - - - - - - - - - - - - - - · - - - - - - - - · -- -------- - - · - - - - - - - - - · - - - - - - · - - - - - - - · - - - - - - - - - - - - - - - -
16
jviable colonies was counted on a Quebec Colony Counter and
I;the
I
percent viability was determined for each spore group.
. !
!Each suspension was assayed in triplicate, with duplicate
I
l
.
!spore suspensions prepared for each determination.
I
Further studies to determine the sensitivity to
osmotic shock were performed on similar aliquots of spore
suspension.
The
different media:
Broth, and water.
spo~e
suspensions were diluted in fouf
0.1 M NaCl, 0.1 M Sucrose, Nutrient
All the operations were performed at
room te=perature (24°C), except for an additional dilution
in wate::- at 10°C which was included as a temperature
control.
After the dilution, 0.5 ml of each suspension
was withdrawn at five minute intervals, serially diluted
in the same media from which it came, at room temperature,
and plated on solid Nutrient Broth media.
After 48 hours,
at 30°C, the viable colonies were counted and the percent
viability determined for each temperature interval and
in each of the four media.
THIN LAYER CHROMATOGRAPHY
Thin layer chromatography was performed on 20 em X
1
20 em Eastman Kodak Cellulose TLC sheets without
flourescent indicator.
The solvent system consisted of
ethanol:butanol:propionic acid:waterr (10:10:6:5).
The
TLC sheets were heated for 15 minutes at 90°C prior to
spotting with the spore extracts and controls.
I were
The sheets
then developed through 50 ml solvent three times to
;f'
I
'----··-----·-------------------·--·-·-----··---·------·-------------·----------·---_j
17
obtain optimal separation of the amino acids.
On the
run~ 6 ml of ninhydrin (3.1 g/ml 50 ml solvent) was
l
!added to the solvent system to stain the amino acids.
1 third
!Following the development of the sheets, they were heated
lance again for 15 minutes at 90°C to fully develop the
color of the amino acid spots.
The controls for the amino
acid TLC consisted of aliquots from a solution of 5 mg
alanine, 10 mg aspartic acid, 5 mg glutamic acid, and
5 mg glycine in 10 ml of 7 5% ethanol.
A separate control
of DAP alone was also used at a concentration of 10 mg in
10 ml water.
A control TLC run was performed to determine
the Rf values of the individual amino acids.
A separate TLC sheet was used for each spore group.
Each TLC sheet was divided into 8 equal strips, 4 containing replicate amounts of
being tested.
the~particular
spore extract
For both the controls and the extracts, 10
microliters of solution were spotted upon the TLC sheet
with microcapillary pipets, and dried with a hot air gun.
j
When the chromatography was finished, the strips were
Icut
apart and each individual spot which represented a
particular amino acid was eluted in 5 ml of distilled water1
II for 10 minutes while being vigorously agitated.
I
ICentrifuging the solution cleared the TLC binding powder I
! from the water,
I
leaving only the colored amino acid
l complex
since the ninhydrin stain is completely soluble in
i water.
The optical density of these solutions were
l
i determined with the Beckman DB- G spectrophotometer at the
1
L--------~---------o-----_:._-~------------ --------------------·------~-----------~-------------- ---~---------- -- -·--------·-----_J
!wavelength of 525 urn.
!jall
The data from all eight strips of
four TLC sheets was then averaged for each individual
!
!control amino acid and each individual unknown amino acid.
lA standard curve was then established plotting the amount
!
lof known amino in
mg~
against the colorimetric
concentration hi optical density units.
The amountoof
unknown amino acid was then determined from the curve for
each particular amino acid.
Then their individual molar
quantity was determined and finally their molar ratios to
glutamic acid.
DEIERM!NATIONS OF CALCIUM, MAGNESIUM, AND DPA
For the
de~ermination
of calcium, 50 mg of dried
spores were suspended in 100 ml distilled water, and
autoclaved for two hours.
Icentrifuged for
the suspension was then
11 hour at 7500 rpm.
The resulting
I supernatant solution contained the calcium in solution.
The suspension for the determination of magnesium
. contained 50 ml dried spores in 10 ml distilled water.
I was
similarly autoclaved and centrifuged.
It
The assays were
performed in triplicate on a Perkin-Elmer Atomic
IAbsorbtion Spectrophotometer.
A combination of controls
I
I were
I the
1
utilized to standardize the equipment and maintain
integrity of accuracy of the calcium and magnesium
concentrations in the spore extracts.
I
II
These controls
l
i consisted of commercially prepared samples which had both
I
I, ions
I
in the free state, and individual samples which were
-------------------'--------~---------------------·--·---·-----------------··--·-'
I
19
• speci:f:.cs..lly prep-ared using- salts ofootfi calcium anCf
•magne s i 1..:.:-::..
I
The DPA determination was performed in triplicate
lJon the
i
!assay.
1 (1958).
sane spore suspension as used for the magnesium
The procedure utilized was that of Lund, et al.,
Pure DPA (Sigma) was used as the control for these
!determinations.
The assays were read on the Beckman DB-G,
at a wavelength of 525 mu.
For the remainder of this text, the spore groups will
be
re~erred
to as t-6 42 or mutant 42 for those mutants
prod1::cec at 42°C, and 168 30 or wild type 30, for those
wild type spores produced at 30°C.
comparisons will be made
be~ween
The significant
these two groups.
I
I
:
I
'
I
l
L-----------~---- __________________________________________________j
r
I
II
CHAPTER III
RESULTS
The average weight of a spore of each of the four
!spore groups is shown in Table I.
I
The values given are the I
I
average of duplicate measurements of two separate spore
,pellets.
I
I
The value of the t-6 42 group is 33% less than
that of the other spo"re.groups.
This indicates a marked
difference between the t-6 42 group, and the other three
groups.
Measurements taken from photographs of spore
prepa::.:-at:ions demonstrated that there are two completely
separated populations of spores.
The t-6 42 spores were
!smaller (an average 1.3 microns) than those of the 168-30
spores (an average 2.0 microns)
(see Table I and Figure 3).
From growth curves of the four groups, generation times
were determined to average 49 minutes in each case"
The amount of sporulation (Figure 4) was quite
extensive in the wild type cultures.
The levels were 83%
and 91% at 30°C and 42°C, respectively.
All of the spores
observed appeared normal and phase bright.
culture sporulated to a lesser extent.
The mutant
At the permissive
temperature (30°C) only 18% sporulation occurred, and this
I
I fell
to 3% at the restrictive temperature (42°C).
I
j Approximately one-half of the mutant spores (44%) at 30°C
appeared abnormal and phase :ark, while almost all (96%)
I
I
of the spores produced at 42 C appeared similarly abnormal.!
These results are summarized in Figure 4.
i
All further
I
I
L------·----------~~------··---~··---~----- ----------·-·-----~---~------~-----------'
20
21
,.--.
:compar1sons will be made between the mutant, t-6 42 and the!
1
!wild type 168-30. Mutant spores formed at 30°C
I
ltimes be included for control purposes.
m~y
some-
i
l
;
I
. I
Data obtained from the wild type spore suspensions are
1
1
j
!similar to that obtained by Goldman (1970).
However~
in
the case of the mutant cultures, the extent of sporulation
obtained here slightly exceeds (by about 6%) that which
was obtained by Goldman.
Temperature sensitivity determinations clearly
demonstrate the mutant spores to be much more sensitive to
heat
th~~
the wild type spores (Figure 5).
The mutant 42
group was almost completely unable to sporulate and grow
vegetatively after 5 minutes at 70°C.
The wild type 30
spores were decreased by 22% under these conditions.
Results of studies of sensitivity to osmotic shock show
that the mutant 42 group did not survive exposure to \vater
for more than 20 minutes at either 10°C or 24°C, while the
wild type was not affected at all at either temperature.
Control studies using saline, sucrose, or Nutrient Broth
as the suspending fluid, demonstrated complete protection
in all cases, indicating that the mutant was osmotically
sensitive and did not require a specific stabilizing
I1 component.
I
Table
!
II summarizes the final levels cif the calcium
!magnesium and DPA in the prepared spores.
Chemical
i
I determinations
showed the magnesium and DPA concentrations
'
;
I to be normal and comparable between the mutant and wild
1
!
·
L-----·---·-------·-----~----·----~---······---------- ------------·--·--·-----------~-·-
I
11
22
!demonstrate little if any difference between the four spore
types in regard to their respective molar ratios of amino
acids.
The amino acids are compared to glutamic acid for
their molar ratios, hence its value is 1;0 for all the
spore groups.
I
I
l
l---~-------------------------~---··
II
~----··-------. --J
!
i
TABLE I
AVERAGE LENGTHS AND WEIGHTS
OF THE FOUR SPORE GROUPS
SPORE GROUP
~-·
'
I
-.
LENGTH*
(u)
WEIGHT** 8
(mg x 10 )
r-6 30°C
1.7
1.47
r-6 42°C
1.3
0.98
168 30°C
2.0
1.49
168 42°C
1.9
1.43
* AVERAGE LENGTH PER SPORE
** AVERAGE WEIGHT PER SPORE
I
!
L.~.--------------·- -~-------------·-- ----------- ·----- ----- ...------------------·------·---------------------~----- ________ }
--·----------1
~---··---------··
TABLE I I
I
i
f1AGNES I UM, CALC I Uf L AND DP/\ CONCLNT 1{1\TION
1
;
l
.!
DPAa
CA:DPA-b
0.24
0.26
0.92
0.066
0.20
0,26
0.77
168 30°C
0.068
0.26
Oa21
1.24
168 42°C
0.066
0.29
0.24
1.21
SPORE GROUP
-
r~AGNES IUf~~
CALCIUMa
T-6 30°C
0.066
T-6 42°C
-
.-
~
VALUES INDICATE AMOUNT OF fi]G++, CA·\+, AND DPA IN MI CROMOLES/MG SPORES
b
!
- VALUES REPRESENT MOLAR RATIO OF CA++ TO DPA
I
I
I
L~~--·-~~
. . ····--·---.. ·-·--.. . . - . -·-~~--·~-. . _,__________________________
-------
··-···--·-j
N
'
.j:::..
~
..-......
---···----·~-~~-
.. ···---···-- ·-·--
·~··--
..... --.
~
I
TABLE III
!
AMINO ACID MOLAR RATIOS*
T-6 30°C
i
T-6 42°C
168 30°C
168 42°C
-
ALANINE
3.6
4.3
3.7
3.8
GLUT AI~ IC ACID
1.0
1.0
1.0
1.0
GLYCINE
0.34
0.40
0.35
0.37
ASPARTIC ACID
0.30
0.29
0.31
0.37
DAP
0.86
0.88
0.75
0.87
* VALUES INDICATE AVERAGE OF DUPLICATE SPORE PELLETS
I
I
lL-.·-·-·------·--··--,--------·
I
_________j
.'N
ln.
--···-·-···--····-···----,
,-·~·-··-·-~-·~-·· ··-· ~-·--···----------
!
TABLE IV
SENS IT IV ITY TO OSMOTIC
SIIOCI<
.f:ERC.E.HI VIABILITY AI RooM TEMPERATURE
~
MUTANT 42°C
TIME
0.1 M
NACL
0.1
M
NuT.
SucRosE BROTH
5 MIN, 100%
10
98
15
98
20
96
25
97
100%
98
.97
98
98
100%
100
98
99
98
WILD TYPE 30°C
1o 0 c 0.1
VIATER
WATER
91%
80
47
19
0*
90%
68
43
18
0*
11
M 0.1 M
NACL SucRosE
100%
98
100
98
100
100%
100
98
100
100
10°C
NuT.
BROTH. WATER
WATER
100%
100
98
98
100
100%
100
98
100
100
100%
98
100
98
98
* INDICATES VALUES BELOW THE LIMITS OF DETERMINATION WITH THIS PROCEDURE
iL--··----------·----------
--------------·-
-··--··-·-N,
0\
27
FIGURE 3
P:-tCTOGRAPHS OF MUTANT SPORES (UPPER)
AND WILD TYPE SPORES ( L.OvlER)
MAGNIFICATION:
5000 X
NoTE THE DIFFERENCE IN SIZE AND THE
AMOUNT OF REFRACTILITY BETWEEN THE
MUTANT AND WILD TYPE SPORES.
'
;
:
i
j
l
L---------------------···-------~-------------------------------. --~-J
2"8
lI
I
FIGURE 4
!
I
!
SPORULATION OF r-6 AND 168 IN NBSM
I
100
I
I
I
15
75
10
50
25
1<C
._I
:::>
0::
0
CL
;(/)
!~
II
I
i
!
I
i
I
D
r-6
ToTAL
SPORULATION
L-~-~------.-------~------------------···
~NoRMAL
~SPORES
168
BQJ ABNORMAL
~
SPORES
.
-··----------------.---. -------------------------------··-- -------~-------~-------~-------__]
29
----------·------·--·--·---
FIGURE 5
100
I
t
80 -1
40
~
fv]UTANT
20
-o
Ll
45
I
50
1
55
I
60
J
65
I
70
I
75
I
80
I
I
85 90
DEGREES CENTIGRADE
I
TEMPERATURE SENSITIVITY
L__, _____________ ,_, - · · · - - - - . - - .. - ..........- - . - - - · · · -..- - - · - - · - - - - - - - - · - - - - - -..
i
-·------·--------·-·---1
30
·--------·--------··-·----------,
FIGURE 6
PHOTOGRAPH OF A TYPICAL THIN LAYER
CHROMATOGRAPHY SHEET
STRIPS
1-4:
STRIPS
5-8: AN EXTRACT OF ONE GROUP OF
CONTROLS RUN IN QUADRUPLICATE
SPORES, RUN IN QUADRUPLICATE
;
l
I
'---~-----------··----------·--------·-------------·---~-J
1
2
'-
''
DAP
i~·
i'l
l
J
IV
DISCUSSION
CHAPTER
t
I
I
The bacterial
I!complex structure,
1
I'Compounds
endospore has been shm.m to be a
both chemically and morphologically.
.
are found in the spore which are not found in
vegetative cells.
These compounds are produced during
spore membrane synthe.sis.
In some cases, a genetic lesion
may occur and a spore will not develop properly or
compl-etely.
I
Spores such as these are mutants and are of
great interest, for the study of them allows the researcher
to gain greater knowledge and insight into the presently
unknown
~echanism
of spore
formatio~.
Mutant t-6 is temperature sensitive with regard to
sporulation and produces spores with altered properties at
the restrictive temperature.
Sporulation frequency at the
!permissive temperature of 30°C was found to be sixfold
greater than sporulation to the restrictive temperature at
I
II 42°C. (Figure 4)~
The wild type strain sporulated normally 1
I
with about 87% sporulation at both temperatures.
It is
evident from the data presented in Figure 4 that the mutant
I
culture at 30°C contains only 56% normal spores, the
I remaining 44% being similar to
I mutant cultures grown at 42°C.
the abnormal spores in the
For purposes of discussion,
!
i
1
all the t-6 42 spores are considered to be abnormal, and
!
ll the fraction of 4% normal is ignored.
Previous research
I
I
j performed on this mutant (Goldman, 1970) has shown very
L
---'-
I
------·----·- · ·- - - - - ·. ----·----"--'_j
31
31
'similar results with regard to the extent of sporulation.
j
Growth rates determined via optical density showed
I
I
that vegative growth_ proceeded equally for all four of
;the spore groups.
Also, all the vegetative cells appeared
!equal in size and shape.
The spore weight and
size~
were
!different for the t-6 42 kroup which had a markedly smaller
I
mass and smaller size. than the wild type group of spores
(Table I).
I
Studies determining temperature sensitivity and
sensitivity to osmotic shock which were performed showed
differences between the mutant and wild type groups.
Osmotic shock determinations showed that saline, sucrose,
and Nutrient Broth had no osmotic effect upon the mutant.
1
Hmvever, water had an extreme effect upon the mutant spores
l
Comparable osmotic shock values existed at both 10°C and
I 24°C, thereby discounting a metabolic change in the spore,
/and supporting a structural change when compared to the
!wild type group (Table IV).
. Physical properties of the abnormal spore suggest a
I
I!possible
l have
1
deficiency in the cortex of the spore.
The spores 1
been shown to be smaller than normal size, in both
I length
and mass, lysozyme resistant, temperature sensitive,
1
~partially or non-refractile, and unstable to osmotic shock.!
!,.
1•
These properties are very similar to those found by Murrell
and 11/arth {196 5) in spores with an abnormal cortex.
II .
Murrell and Warth found normal spores to have a basic
Icortex structure
L_______________
composed of muropeptide polymer
l
.... ------------------· ·-------.--~----·-"··---------------------·---~
33
consisting of aianinecz:--87), diaminopimelic acid (1. 24),
glutamic acid (1.0), glycine (0.25), and aspartic acid
(0.26).
These values are molar ratios to glutamic acid.
Comparison of the mutant and wild type spores showed the
chemical composition of murein was found not to be affected
since the molar
r~lations~ip
on the amino acids via TLC
were found to be identical for all four types (Table III).
The t-6
mutatio~
therefore, does not affect the final ratio
of amino acids necessary for inclusion in the cortex of the
spore.
Since these results are calculated as a ratio
I
jagair3t glutamic acid, one can not determine the total
jamount of amino acids present in each spore group.
j
Normal amino acid ratios in murein do not necessarily
imply a-normal cortex structure.
I formed
The murein is first
during early sporulation and is not exuded in
jgermination but rather remains to become the cell wall of
lthe new vegetative cell (Vinter, 1965).
Thus, this
j (Levinson, et al., 1961).
The results of calcium
ldeterminations-;resented in Table
II
'
show essentially the
!normal values for the wild type cultures (0.26, 0.29).
I
Thej
!mutant spores showing a value some 27% less (0.2 pmoles/
l
!spore), is comparable to that found by Levinson et al.,
I
(1961)
in calcium deficient spores
(0.18 - 0.22
pmoles/
jspore).
Spores usually contain equimolar
concentrations of
I
calcium and DPA (Tipper and Gauthier, 1972) and several
studies (Murrell, 1969; Murrell, et al., 1969) indicate
that heat-resistance· and siability are relaied to the
Ca:DPA ratio though the relationship is not a simple one.
Additional research (Levinson, et al., 1969) comparing the
Ca:DPA ratios against the osmotic stability and heatl resistance of the spore have demonstrated that the highest
Ca:DPA ratio of 1.37 is associated with the most stable
Iand
I
resistant and the lowest ratio of 0.85 is associated
!with the lowest stability and lowest resistance(0.77 in the
!mutant
I
group) .
Hence, the mutant spore having the lowest
I Ca:DPA
that it also has the lowest record of stability to osmotic
1
shock and heat-resistance"
expla::et::w::::i::::i:: :::e:u::n:h:p:::t:: :::::nc::::le I
I
!
fduring storage and suffer osmotic shock.
I to
!
This might be duel
a weakness of the cortex murein which could result in
I
J
: osmotic sensi ti vi ty (Lewis, et al., 1960). This implies j
l--------------------------------------------------·-----------------------J
3-5
·!'_that these mutants have a defect which at the same time
i
I
jcauses a weakness in the murein complex and an inability toi
!
I
!bind calcium.
II
I
Cortexless mutants, cortex-deficient spores and spores!
formed in the presence of penicillin (a specific inhibitor j
of muropeptide polymer formation as found in stage 4 of
spore development) indicate that a properly formed cortex
is necessary for a stable resting spore with full heatresistance (Murrell, 1969).
This may require
murein~
but
may also require a subsequent reaction involved with
calcium and DPA incorporation.
When the process of calcium
uptake and DPA formation are interferred with during the
sporulation process (Murrell, 1969) the resulting spores
do not acquire full refractility or heat-resistance.
indicates that there may be a sequence of reactions
This
~--
involved in cortex formation, possibly in the laying down
of the crosslinkage which incorporates the Ca:DPA with the
murein, or perhaps the direct incorporation o£ the calcium
into the Ca:DPA complex.
Therefore, it-could be
postulated that the amount of calcium and DPA bound in the
spore is quantitatively determined at the time and by the
I degree
of the peptidoglycan
~olymerization
and that DPA is
1 requried to bind a large part of the calcium (Murrell,
I
let al., 1969).
!
•
1
It has been shown that the mutant spore under
I investigation
l! and
1,•
l
has a markedly lowered calcium concentration,!
a visibly lowered Ca:DPA ratio.
'--·---
!
-----
Thus, a probable
iI
·-----------····-------------------·-----------__j
lcause of the mutant's lowered or lack of stability and heat!
!resistance is based upon the decreased Ca:DPA ratio and a
I
!subsequently weakened polymer chain itself.
The
I
!temperature sensitivity of the spore is thus manifested in
lsome probable genetic lesion which in some way disrupts the
Ispore's
ability to fix the· uptake of calcium and its
binding properties with DPA.
The inability for calcium
binding may be due to the lack of a regulatory of
transport protein, utilized in the transport and binding ofl
the calcium ion into the murein-Ca:DPA complex.
It is
possible that the functional absence or suppression of this
protein, brought about by growing the.mutant at the
restrictive temperature, results in a genetic lesion and
the present phenotype.
One of the
characteristic.si:of~_such
a phenotype is the inability to incorporate calcium into
!the murein complex required for spore stability and
!rigidity.
In so doing, the spore's cortex may not be
!properly formed, giving the decreased heat-resistance and
!decreased stability to osmotic shock.
I component.
Biochemical and genetic analysis of other
!
I conditional
Imore
sporulation mutants will certainly provide
information on the molecular basis of the regulatory
,
IL----·-----------------------------------~----·--·
program involved bacterial sporogenesis.
l
-------·-------.. __________ ..______________,
37
LITERATURE CITED
~
ii
i
Amaha, M. and Z. Ordal (1957), "Effect of divalent cations j
in the sporulation medium on the thermal death rate ofi
Bacillus coagulans var. thermoacidurans."
J. Bacterial., Z!:596-604.
I
Black, S., T. Hashimoto, and P. Gerhardt (1960), "Calcium
reversal of the susceptibility and dipicolinate
deficiency of spores formed endotrophically in water." 1
Can. J. Microbial., 16:2!3-224.
Curran, H., B. Brunstitter, and A. Myers (1943),
"Spectrochemical analysis of vegetative cells and
soores of bacteria." J. Bacterial., -45:485-494.
~
El Bisi, H., R. Lechowich, M. Amaha, and Z. Ordal (1962),
;'Chemical events during death of bacterial endospores
"by moist heat."
J. Food Sci., 27:219-231.·
I
I
Fi tz_-::_James, P.. and E. Young, "Morphology of Sporulation"
in The Bacterial Spore, Ed. Gould and Hurst, Academic
Press, New York, 1969, p. 39.
1 Fitz-James,
1
I
P.(l965), "Regulations chez les Micro-organisms." Centre Nat._Recher. Sci., Paris, p. 529.
R. (1971), A Temperature Sensitive TCA Cycle
Mutant of Bacillus subtilis:
Physiological Effects
Sporulation." Master's Thesis, CSU Northridge.
!Goldman~
1
1
Gould, G. and A. Hurst, The Bacterial Spore, Academic
Press, N~w York, 1969.
Grelet, N. (1952), "Le determinisme de la sporulation de
Bacillus megaterium.
IV. Constituants mineraux du
· milieu synthetique necessaires a la sporulation."
Ann. Inst. Pasteur, ~:71-79.
jLechowich, R. and Z. Ordal (1962), "The influence of the
j
sporulation temperature on heat-resistance and
chemical <;=ompo~ition ~f bacterial spores."
Can. J. M1crob1ol., 8.287-295.
I!
-- -
-
I
I
I
onj
I
I
I
I
I
!Levinson, H., M. Hyatt, and F. Moore (1961), "Dependence of!
1
the heat resistance of bacterial spores on the
i
calcium-dipicolinic acid ratio." Biochem. Biophys
1
Res. Commun., -5:417-431.
I
--
I
I
! Lewis,
!
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