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thesis entitled
THERMOSENSITIVE CELL DIVISION SEPTATION AND
SPORULATION SEPTATION IN A MUTANT 0F

BAC I LLUS MEGATER I UM
presented by

Anthony Dare Hitchins

has been accepted towards fulfillment
of the requirements for

 

 

Ph.D. Jegeem Microbiology stgbléc

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’ ABSTRACT

THERMOSENSITIVE CELL DIVISION SEPTATION AND
SPORULATION SEPTATION IN A MUTANT OF
BACILLUS MEGATERIUM

 

BY

Anthony Dare Hitchins

The objectives of this study were to isolate

conditional cell division mutants of Bacillus megaterium

 

ATCC 19213 and to use them to test the hypothesis that the
early stages of bacterial spore formation are analogous

to stages of normal cell division. Isolation of a thermo-
sensitive mutant, TH 14, defective in cell division sep-
tation made it possible to test a specific corollary of
the hypothesis: that sporulation septation and cell
division septation are analogous processes.

Mutant TH 14 was isolated by replica plating
colonies formed by survivors of N—methyl-N'-nitro-N—
nitrosoguanidine mutagenesis and of a subsequent D-
cycloserine selection step at the restrictive temperature
(37 C or higher). Premissively grown colonies of survivors
were replicated to the restrictive temperature and poorly

grown replicate colonies were picked for subculture. TH 14

Anthony Dare Hitchins

was chosen from such isolates because it formed filaments
at the restrictive temperature. The filaments were shown
to be aseptate and multinucleate by electron microscopy of
ultrathin sections and Giemsa staining. Filaments were
formed in nutrient broth or in simple defined medium. In
the latter, filamentation was independent of the carbon-
source used for growth.

The sporulation frequencies of the mutant at the
permissive temperature (30 C) and at the restrictive
temperature (39 C) were 0.6 and 10-5, respectively, where-
as the corresponding values for the parent strain were
0.8 and 0.1. Revertants of TH 14, selected for ability
to divide normally at restrictive temperature, concomitantly
recovered the ability to sporulate. These facts plus the
fact that the asporogenic property was not directly
selected for in the original isolation of TH 14 suggest
that the filamentation and asporogenic properties are
due to the same mutation.

Asporogeny was due to a block in sporulation prior
to septation (stage II) since no septation or later stages
were detected in ultra-thin sections of filaments by
electron microscopy. The deoxyribonucleic acid (DNA)
yields of permissive and restrictive mutant cultures were
equal. This suggests that the last round of DNA repli-
cation necessary for sporulation is not blocked. Stationary
growth phase filaments shifted from restrictive to permis-

sive conditions sporulated with a frequency of about 10.2.

Anthony Dare Hitchins

Spores were confined to the shorter filaments. It follows
that short filaments at least are not inherently unable to
Sporulate due to some factor unrelated to their aseptate-
ness. Sporulation occurred at higher frequency at the
restrictive temperature if permissively-grown mutant cells
were shifted-up before the last l‘to 2 rounds of DNA
replication in the culture. Similarly, when mutant cells
were shifted to the restrictive temperature about two cell
divisions occurred before the decreased rate of growth and
filamentation started. Together these results suggest that
after shift-up two division septations or one division septa-
tion followed by one sporulation septation can occur before
the effect of the mutation is detectable. £2_E9Eg, the
sporogenesis studies are consistent with the view that the
TH 14 mutation affects both cell division septation and
sporulation septation. This supports the hypothesis that
the early stages (I and II) of sporulation represent a modi-
fied cell division.

The nature of the TH 14 gene product and its
relationship to the cell division and sporulation septation
processes is not yet known. However, the mutant TH 14 was
affected in several noteable properties. It had a doubled
mean generation time, as measured turbidimetrically or by
DNA synthesis, in restrictive culture compared with that
of the parent strain. The restrictively-grown mutant did
not show any qualitative compositional changes in membrane

proteins which are extractable in hot sodium dodecyl

Anthony Dare Hitchins

sulfate plus 2-mercaptoethanol. However, the mutant had a
reduced content of a small molecular weight protein(s),
equivalent in size to cytochrome g, at the restrictive
temperature. Also a membrane protein (molecular weight
about 80,000) was partially derepressed in the restrictively-
grown mutant. Additionally, unidentified cytOplasmic
inclusions (diameter about 100 nm) of a nonmembranous
nature, were found in the mutant at the restrictive
temperature.

Filamentous growth of the mutant was not so sensi-
tive to penicillin as was growth in the rod form. Growth
in either form was equally sensitive to D-cycloserine or

rifampicin at the concentrations tested.

THERMOSENSITIVE CELL DIVISION SEPTATION AND
SPORULATION SEPTATION IN A MUTANT OF

BACILLUS MEGATERIUM

 

BY

Anthony Dare Hitchins

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health

1972

DEDICATION

To my wife and to my mother

ACKNOWLEDGMENT S

My sincere thanks for the guidance of my academic
advisor Dr. Harold L. Sadoff throughout all aspects of my
graduate program. Without his encouragement and financial‘
support I would not have been able to carry out the program.

Appreciation is also due to the other members of
my Ph.D. guidance committee, Dr. Ralph N. Costilow,

Dr. David H. Bing and Dr. John C. Speck, Jr. I thank

the faculty and students of the Microbiology and Public
Health Department with whom I have had helpful discussions.
I am indebted to Prudence J. Hall and H. Stuart Pankratz

for help with the electron microscopy.

iii

TABLE OF CONTENTS

GENERAL INTRODUCTION . . . . . . . . o . . .
LITERATURE SURVEY O O O O O O O O O O O O O

Morphology of Sporulation (Stages I-VII) . . . .

Sporulation Biochemistry . . . . . .
Early Sporulation (Stages I- -II) as a Modified
Cell Division . . . . . . .

Reinterpretation of the Axial Filament (Stage I) .
Septation (Stage II) as a Modified Division
Septation . . . . . . . . . . . . .
Relevance of Division Modifications for Stages
III-VII . . . . . . . . . . . . . . .
Rod to Filament Morphopoiesis . . . . . . .
References . . . . . . . . . . . . . .

ARTICLE 1

Cell Division Septation and Sporulation Septation
in Bacillus megaterium, A. D. Hitchins and H. L.
Sadoff, In H. O. Halvorson, R. Hanson and L. L.
Campbell—TFds.), S ores V, American Society for
Microbiology, Wash1ngton, D. C., p. 148-153 (1962)

ARTICLE 2

Further Properties of a Thermosensitive
Asporogenous Filamentation Mutant of Bacillus
megaterium, A. D. Hitchins and H. L. Sadoff
(manuscript in preparation) . . . . . . . .

 

CONCLUSION O O O O O O O O O O O O O O 0
APPENDIX
Bacterial Sporulation as a Modified Procaryotic

Cell Division, A. D. Hitchins and R. A. Slepecky,
Nature (Lond.), 223: 804-807 (1969) . . . . .

iv

Page

DU!

ll
l4

17

26
27
33

44

51

106

107

GENERAL INTRODUCTION

Morphological differentiationis a common character-
istic of living organisms. In multicellular organisms it
is seen as the development of form (embryogenesis) or as
a change of form (metamorphosis). The reverse process,
dedifferentiation, may occur, sometimes with disastrous
effect as in cancer. In unicellular organisms, morphologi-
cal differentiation involves changes in shape and structure
of single cells.

In the last 15 years, studies of differentiation
in the simplest unicellular organisms, the procaryotes,
and their viruses, have led to an increased understanding
of the genetic and biochemical basis of differentiation.
Aside from the intrinsic interest of procaryotic differ-
entiation, the motivating force of these investigations
has been the hope that they would provide realistic models
for understanding differentiation in the more complex
eucaryotic cells, especially those of multicellular
organisms. In recent years, the relative success of this
approach has prompted its extension to the unicellular
eucaryotic organisms such as yeast. Despite the increase
in knowledge about procaryotic differentiation, under-

standing of its initiation and control is still primitive.

Thus research into procaryotic differentiation remains of
fundamental importance.

Bacterial spore formation (sporogenesis) is the
most studied procaryotic differentiation process. It has
been resolved into seven morphological stages and many of
the chemical changes involved have been analyzed in detail.
However, even such a simple differentiation system contains
about two thousand different proteins as well as other
complex polymers. The immense problem of auditing the
compositional differences and similarities between the
vegetative and spore forms has understandably led to
focusing of attention on the differences rather than on
the similarities. This seems to have led to the implication
that understanding control of the synthesis of the differ—
entiating molecules would be sufficient for understanding
sporogenesis. Recently the validity of this vieWpoint has
been questioned so that interest has shifted to the way in
which vegetative functions and components are adapted to
the process of sporulation.

The characteristic function of the vegetative cell
is the ability to replicate itself by the processes of
growth and cell division. Interestingly, the early stages
of sporulation resemble the process of cell division.
Historically, this has been recognized ever since the
advent of the use of the electron microscope in the late

nineteen-fifties in studying sporulation. Many researchers

have commented on this similarity but without: (1) evalu-
ating the evidence for the idea, (2) pointing out the
importance of the differences from normal division to the
later stages of sporulation, and (3) considering that the
induction of sporulation may involve the way in which

normal division is modified. In contrast, other researchers,
by emphasis of the differences between normal division and
sporulation, seem to imply that cell division and sporula-
tion are unrelated.

As a result of this situation it was decided to
test the hypothesis that the early stages of Sporulation
represent a modified cell division.. This hypothesis is
worth_testing because it may clarify the nature of the
transition from cell division to cell differentiation and
vice versa that occurs in all differentiating or dediffer-
entiating cells.

The test involved isolating and studying the
sporulation prOperties of a mutant defective in one of
the stages of cell division: initiation of deoxyribonucleic
acid (DNA) synthesis, DNA replication, segregation of the
replicated DNA molecules, and cell division septation.
Obviously, such mutations must be conditional since they
affect the process that determines viability.

In fact, a thermosensitive filamentation mutant
defective in cell division septation was used to test

whether division septation and sporulation septation are

related. The results are described in this thesis. The
thesis is divided into four parts: (1) a survey of the
literature pertinent to the modified cell division
hypothesis, (2) a description of the isolation, growth

and Sporulation prOperties of the thermosensitive mutant
defective in cell division septation, presented in the
form of a reprint of a symposium article, (3) a manuscript
of a prOposed publication, describing the membrane proteins
and other prOperties of the mutant, and (4) a short

summary.

LITERATURE SURVEY

Morphologyyof Sporulation (Stages I-VII)

 

Bacterial Spores are special cells that are resistant
and dormant. More correctly, the spores are called endo—
spores because they are formed inside a cell. A spore-
containing cell is called a sporangium. Spore formation
is confined mainly to rod-shaped cells of the family Bacil-
laceae, though a spheroidal-shaped organism, the coccus

Sporosarcina ureae also forms spores. Recently certain

 

filamentous actinomyces have also been shown to form endo-
spores (12, 50). Sporulation occurs With high frequency
at the end of growth in batch cultures although it does
occur at lower frequency also during growth (85). However,
in continuous cultures sporulation can occur with high
frequency at low growth rates (15). Thus total starvation
is unnecessary for sporulation to occur.

Seven morphological steps occur during spore
formation (3, 26, 27, 64). These are diagrammatized in
Figure 1. In stage I, the deoxyribonucleic acid (DNA)
completes replication and becomes aligned on the long axis
of the future sporangium; this is the axial filament or
preseptation stage. Next, in stage II, the septation

stage, a septum or partition is formed near one pole of

 

FIGURE 1.--Division cycle and stages of sporulation
of a typical Spore forming bacillus. The rod shaped cells
are illustrated diagrammatically as they would appear in
longitudinal and/or equatorial thin sections. Symbols:
thick lines, cell walls; thin lines, membranes; dashed
line or medium thickness line, spore coat; hatched areas,
deoxyribonucleic acid (nucleoids). Cytoplasmic areas
and the spore cortex zone have been left blank for
clarity. Mesosomes and the exosporium have been omitted
for simplicity. Scale: the width of a typical bacillus
is about 1000 nm.

 

 

 

 

 

 

 

 

 

 

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the rod-shaped sporangium. This divides the sporangium
into a large compartment and small compartment. These
compartments can be regarded as protoplasts. Each
compartment probably contains one genomic equivalent

of DNA though the evidence for this is not conclusive
(64, 84).

By means of cytoplasmic membrane growth the large
protoplast engulfs or envelopes the small protoplast
(Stage II, engulfment). As a result the small protoplast,
ends up inside the large protoplast and also has an extra
membrane around it called the outer forespore membrane.
The ensemble is called the forespore. The outer membrane
is derived from the larger protOplast's membrane. It has
the novel feature of being inside out relative to the
other membranes in the sporangium.

During stage IV, cell wall material is deposited
between the outer and inner forespore membranes in the
form of two concentric spheroidal shells. The outer
thicker shell is called the cortex and the thinner inner
one is called the germ cell wall or cortical membrane.
The small protoplast and its primordial.cell wall form
the germ cell of the spore.

A coat made of several layers is deposited around
the forespore in stage V (coat formation). Some of the
sporangial cytOplasm is incorporated into the Spore at

this stage (64). Stage VI or maturation is an ill-defined

stage covering the period between coat formation (stage V)
and stage VII, when the mature spore is released into the
environment due to lysis of the sporangium. Biophysical
changes such as increase in resistance to heat and
radiation and increase in refractility are associated
with stages IV and V.

The typical spore thus consists of the germ cell
encased in cortical and coat layers. The final fate of
the outer-forespore membrane is not clear. When conditions
become suitable, the free mature spore germinates and the
germ cell grows out to form a new vegetative cell.

A variety of morphological mutants have been isolated.
These are characterized by blocks in development at specific
stages of sporulation. Freese (27) has proposed the name

cacogenic for developmental mutations causing abnormal

 

forms. The normal genome would then be called protogenic.

 

Sporulation Biochemistry

 

Associated with the 6-8 hour long sequence of
morphological changes occurring during sporogenesis are
biochemical events involving metabolic shifts, the appear-
ance of new metabolic products and modification of some
vegetative components (45). Metabolic shifts occur early
in sporulation because of carbon or nitrogen limitation due
to exhaustion of the supply during growth. Derepression of

enzymes occurs. Reserve material such as poly-B-hydroxybutyric

10

acid and by-products of growth such as acetate and
pyruvate, are utilized for energy and carbon sources.
This involves increased respiration, increased activity
of the tricarboxylic acid (TCA) cycle with concurrent
changes in enzyme patterns and in pH if the environment
is only weakly buffered. Limiting energy, carbon or
nitrogen sources are replenished by turnover of proteins
and ribonucleic acid (RNA). DNA synthesis stops during
stage I, the axial filament stage.

Many enzymes and biochemical products appear at
specific times during sporulation. Exoprotease(s),
antibiotic(s) and toxin(s) are excreted early in sporu-
lation (84). Dipicolinic acid (DPA) accumulates in spores
about stage IV. Associated with DPA Synthesis is cortical
peptidoglycan synthesis and calcium accumulation. Disulfide-
rich coat protein(s) accumulate during stage V. In some
species, there are spore associated structures such as
exosporia and parasporal bodies which appear around mid-
sporulation.

Some enzymes are modified post-translationally by
proteolytic action, e.g. aldolase (79) and RNA polymerase
(53). With RNA polymerase such structural modification
(54) alters its transcriptional specificity with regard
to the genome of phage ¢e (55). The possible relationship
of protease, antibiotic(s) and enzyme modification to the

control of sporulation has been discussed (78). At the

11

translational level changes in transfer—RNAs (107) occur
which could conceivably have important consequences for
sporulation morphogenesis. Changes in other nucleic acids
appear to be relatively minor and have been reviewed else-
where (17).

The sporulation specificity of the biochemical
events is not always clear (30). Many of the metabolic
changes could be typical of stationary phase metabolism
in asporogenic bacteria. Other changes appear to be
sporulation specific such as coat protein or DPA production.
However, the fact that it is possible to obtain cacogenic
forms such as DPA-less spores, coat~less spores or 'spore-
less' coat protein suggests that production of even these
substances need not be obligately coordinated with the
whole process of sporogenesis. The situation resembles that
observed with production of bacteriophage parts (22).

Early Sporulation (Stages I-II) as a
Modified Cell DiVi3ion

 

 

In this section, bacterial cell division (binary
fission) is briefly outlined, and then the relationship
of sporulation to cell division is introduced. Reviews
covering the various aspects of cell division were con-
sulted as indicated: DNA replication (5, 28), wall growth
and wall septation (32, 101) and cytoplasmic membrane

growth and septation (80).

12

Like sporulation, cell division has been resolved
into a sequence of cytological stages at which mutational
blocks may occur. These stages in order of occurrence are:
initiation of DNA synthesis, DNA synthesis, segregation of
duplicate genomes, septum formation by wall and membrane,
and separation of the two resultant cells. Segregation
of the DNA is semi-conservative.

The cell division septum is a partition formed
across the middle of the cell at right angles to the
direction of growth. It consists of cell wall material
sandwiched between two cytoplasmic membranes. The wall
layer of the septum is called the cross wall to distinguish
it from side or lateral wall. The cross wall separates
into two halves thus allowing cell separation. Presumably,
the two halves were formed by the neighboring-cytOplasmic
membranes. After cell separation, the mature septum forms
the new pole of the cell. The old pole is derived from
the septum of the preceding cell division.

.Separation is the last stage of cell division.
Delay of separation results in chains of cells being
formed. Due to this, completion of a septum is a more
convenient way of defining the end of cell division.
Thus physiological separation as judged by survival from
phage infection or ultrasonication provides an endpoint

before separation (11).

13

A major control of normal cell division in
Escherichia coli involves the fact that septation can occur
after completion of a round of DNA replication. However,
initiation of new rounds can occur before an old one is
completed. This control mechanism ensures proper segre-
gation of genomes while allowing increased ratesof DNA
synthesis in rich medium. However, such a control is not

always operative in wild type Bacillus subtilis (20) or

 

in certain mutants of E, coli (35).

In contrast to sporulation, very little is known
about the biochemical changes that occur throughout the
cell cycle. Changes in phospholipid composition during
septation (4) and a transient increase in glycerol
incorporation at septation (13) have been reported.

Robinow (71) was the first investigator to point
out the resemblance between the early stages of sporu-
lation and cell division. The similarity was noted by
successive authors in later years. Hitchins and Slepecky
(34) reexamined the idea in 1969 and used it as a frame-
work on which to arrange much of what was known about
sporulation at that time. They concluded the idea was
a viable one and proposed the following hypothesis: that
the early stages (I and II) of sporulation are equivalent
to a modified cell division and that this modification
results from shift-down growth conditions. A copy of
this review is appended to this thesis (see Appendix)

because it is very pertinent to this literature survey.

14

In the following three sections, the reinterpre-
tation of the first two stages of sporulation as modified
stages of cell division and the consequences of the
modification for later stages are discussed with special
reference to relevant experimental findings obtained
since 1969.

Reinterpretation of the Axial Filament
(Stage I)

 

Although the axial filament configuration of the
DNA in stage I has been considered a peculiar structure
unique to sporulating cells there are reports of its
occurrence in normal cell division of sporeformers (75,
84) and also in a non-sporeformer, E, ggli_(43). Thus,
it has been suggested that axial filaments may be slowly
segregating newly replicated DNA molecules. Consistent
with this viewpoint is the continued synthesis of DNA
during stage I (98) which presumably is equivalent to
the last round of DNA replication since the Spore germ
cell contains half of the DNA originally present in the
sporangium (45, 64). According to the modified cell
division hypothesis, stage I can be regarded as completion
of the DNA replication stage and the beginning of the
segregation stage of cell division.

Recently Mandelstam et al. (14, 57) have reported
interesting results obtained with the temperature-sensitive

(ts)- 134 B, subtilis mutant of Mendelson and Gross (61).

15

Initiation of DNA synthesis is ts in this mutant. Cells
transferred to 45 C complete a round of replication and
can sporulate when transferred to sporulation medium at
35 C. However, the nature of the medium used at 45 C to
complete the round is important. If the medium at 45 C is
rich in amino acids, no sporulation occurs in sporulation
medium at 35 C. If the 45 C medium was a poor one then
sporulation was possible. The nature of the medium
affected the disposition of the DNA molecules formed.
In poor medium an axial filament was formed. In rich
medium there were discrete nucleoids. This result seems
to support the role of shift-down conditions in modifying
cell division for sporulation suggested by Slepecky (91)
and by Hitchins and Slepecky (34). Mandelstam et a1. (57)
concluded that sporulation is induceable only during a
round of replication and not at the end of a round. Other
experiments demonstrated that DNA synthesis was essential
for sporulation to occur. Thus, as in normal cell division,
a sporulation septum is not formed unless the preceding
round of DNA replication is both initiated and completed.
Until recently it has been believed that the
functioning of the TCA cycle is essential for sporulation
of Bacillus species since certain asporogenous mutants
have defective TCA cycles. Yousten and Hanson (116)
studied an aconitase-less mutant that is blocked at

stage 0 or I of sporulation with little or no axial

16

filament formation. They suggested two alternative explana-
tions. The first was that the modified cell division
hypothesis is not correct since the mutation did not affect
normal cell division, though of course it would if the
carbon source was acetate. Alternatively, they suggested
that the round of DNA replication preceding sporulation is
more sensitive than normal replication to adverse conditions
since few or no axial filaments were produced. The second
explanation would appear to be very likely in view of the
results of Mandelstam, Sterlini and Kay (57). Presumably
the TCA cycle block prevents a shift-down of metabolism
thus precluding formation of the axial filament configuration
of DNA and hence sporulation. It is not clear from the
results of Yousten and Hanson (116) whether or not the
last round of DNA replication did occur in the TCA cycle
mutant. In any case, recently Carls and Hanson have found
that certain TCA cycle mutants exhibit a sporulation
frequence of l to 10% (6). Thus the idea that a functional
TCA cycle is essential for Sporulation is less clear than
it used to be.

Studies with a conditional serine protease mutant
(53) have led Santo et a1. (82) to the conclusion " . . .
that forespore formation may not be the result of an
essentially vegetative asymmetric cell division. . . ."
They reason that since sporulation genes are only expressed

in sporulation due to the proteolytic modification of RNA

17

polymerase such genes cannot be involved in normal cell
division. Thus the early stages of sporulation are not
analogous to cell division. This does not prove that the
early stages are not a modified cell division.

Since the completion of DNA synthesis and axial
filament formation that occur during stage I are consistent
with the modified cell division hypothesis, what about
the segregation of the resu1ting daughter DNA molecules?
Kogoma and Yanagita (44) showed that the segregation of
the "old" and the "new" daughter DNA molecules into the
spore germ cell is randomly determined in B. cereus. This
agrees with the random segregation of daughter DNA molecules
in B, subtilis cell division (77). However, the latter
report conflicts with a previous one (21). Furthermore,
determination of DNA segregation patterns during sporu-
lation may be complicated by the demonstration of DNA
excretion (about 40%) during B. subtilis sporulation (2).
Balassa has recently reviewed the subjects of DNA synthesis
during sporulation and the origin of spore DNA in greater
detail than is possible here (3).

Septation (Stage II) as a Modified
Division Septation

 

 

Septation, stage II of sporulation, involves
partitioning of the future sporangium into two compartments
by a process of membrane invagination due to its growth.

Dividing a cell into two compartments is also the function

18

of the cell division septum. The common compartmentali-
zation function of the two kinds of septa is the main

reason for suggesting that division and sporulation septation
are analogous processes. An additional reason is that the
two kinds of septation have in common the property of
completely segregating the daughter DNA molecules formed

by the previous round of replication.

However, there are two very important differences
between the two kinds of septa that have to be accounted
for if their analogous nature is to be accepted. The
first difference is the asymmetric location of the
sporulation septum as opposed to the symmetric location
of the cell division septum. The sporulation septum is
much nearer to one of the poles of the rod-shaped future
Sporangium. This results in two unequal sized compartments.
In contrast, the cell division septum is centrally located
and septation results in two equal sized compartments (58).
The second difference is that sporulation septa contain
no detectable cell wall materials such as peptidoglycan
whereas cell division septa do contain such material.

The importance of these differences for subsequent events
in sporulation will be discussed in the next section.

One reason for the asymmetry of the location of
the sporulation septum could be that it is a novel process
entirely unrelated to cell division septation. Indeed,

Kretschmer (46) has prOposed that the sporulation septum

19

may be the product of a reinitiated, additional membrane
synthesis at an annular zone. This zone occurring at the
newly formed poles of daughter cells would be normally
responsible for cell elongation. The activity of this zone
is usually repressed by the next division and remains
repressed under starvation conditions. This alternative
model for sporulation septation seems to be more applicable
to stage III (engulfment) which involves post—septation
membrane growth. Interestingly, even Kretschmer suggests
that Sporulation septation timing is related to initiation
of cell division.

However, there is no a_priori reason to assume that
an asymmetrically located septum could not be related to a
cell division septum. For instance, asymmetric septation
is not specific to sporulation but occurs in a mutant of
E. 9211 that forms DNA-less minicells (1), and in redivision
of filaments (19). Thus asymmetry seems to be a cell
division septation related phenomena but its cause is
unknown.

In view of the postulated effect of shift-down
growth conditions (34, 91) during stage I of sporulation
(7, 105) and the implied support for this view from the
work of Mandelstam andcoworkers (14, 57) it seems likely
that the unbalanced growth effect could continue into
Stage II and result in asymmetric septation. The relative
independence of sporulation septation and cell elongation

is also sometimes seen with cell division and growth (16).

20

This effect of the medium in determining the choice
between division septation or sporulation septation is well
exemplified by the phenomenon of microcycle sporulation
(106). Microcycle sporulation occurs when a cell, growing
out from a germinated spore, forms a spore instead of
dividing. This effect is caused by low levels of nutrients
often resembling shift-down growth conditions. Similarly
log-phase sporulation (85), the production of spores at
low frequency under growth conditions emphasizes the
alternative types of septation. The frequency of log
phase Sporulation varies with the nature of the carbon
source. The choice between cell division septation and
sporulation septation, i.e. between division and spore
formation, can be regarded as a probability event that
depends on environmental conditions (85).

The sporulation septa are not always peptidoglycan-
less. Under certain conditions they contain cell wall
material as when growth of stage II cells is rejeuvenated
by addition of fresh nutrients (25). Also certain
sporulation mutants have septa that contain cell wall
material (69, 75, 76, 115). Similarly spontaneous
aberrant forms which look like minicells, appear during

sporulation of Clostridium EB! and these septa contain

 

cell wall material (81). Spikes of wall material are
associated with Sporulation septa in B, subtilis (27, 114).

Such observations suggest that under certain conditions

21

sporulation septa can be converted into division-like
septa by addition of wall material.

Even though sporulation septa of B, cereus do
not contain detectable cell wall material such material

has been detected in 3 Clostridium species (109). Also

 

there is evidence of changes in peptidoglycan being
associated with septation. The situation is complex
because the evidence is conflicting. Vinter (104) first
reported a peak of new peptidoglycan synthesis associated
with stage II in B, cereus and this has been confirmed in

B, megaterium (R. Greene and R. A. Slepecky, Unpublished

 

results; 34). However, Pitel and Gilvarg (66, 67) could
not detect net synthesis of peptidoglycan in a diamino-

pimelic acid (DAP) requiring mutant cf B, megaterium.

 

Neither could the latter authors deteCt turnover at stage II.
Even data for peptidoglycan turnover in vegetative cells is
conflicting (32). Thus turnover is reported in B, subtilis

(60) and in B, megaterium (8), but with their DAP-requiring

 

B. megaterium mutant, Pitel and Gilvarg (66) found no turn-

 

over..

Chow and Takshashi (10) have recently studied the
acid-soluble nucleotides of asporogenous mutants of B,
subtilis. In the wild type strain the nucleotides, uridine
diphosphate (UDP) galactose and UDP-N-acetyl-glucosamine
increase at stage II. In a mutant blocked at stage II just

after septum formation, these nucleotides also increased.

22

However, in a mutant blocked at stage 0, i.e. prior to
axial filament formation there was no increase in these
two nucleotides but a third one, UDP-glucose accumulated.
These observations suggest that some changes in wall
metabolism occur between stage 0 and stage II in B.
subtilis. Consistent with this vieWpoint is the obser-
vation by Hitchins and Slepecky that penicillin added at
stage I prevents subsequent septation (33). Furthermore,

binding studies with 14

C-benzylpenicillin (51) show a peak
of binding presumably to glycopeptide transpeptidase
associated with stage II. Binding studies with sproula-
tion mutants confirmed this conclusion (73). However,
penicillin also binds to the D-alanine carboxypeptidase
of B, subtilis though it is postulated this enzyme may
be an uncoupled transpeptidase (52). If net wall synthesis
or wall turnover cannot explain these results then it is
still possible that there is some rearrangement of wall
structure that is necessary for sporulation septation or
more probably its initiation. Alternatively, an effect
of penicillin at the control level of septation has been
postulated by Pitel and Gilvarg (67). It is interesting
to note that spores, in contrast to vegetative cells, do
not contain teichoic acids (9).

Understanding wall metabolism at stages I and II

is important for understanding septation. One theory of

septation is that wall synthesis stops and membrane

23

synthesis occurs resulting in "buckling" (67). That a

rigid wall envelope is necessary is suggested by obser-
vations of sporangial protoplasts (24). A stage II
protoplast consists of a large protoplast (future sporangium
protoplast) linked with a tiny one (germ cell protOplast).
Envelopment cannot occur under these conditions. Whether

a stage I protoplast can undergo septation however is not
clear. Division of vegetative cell protoplasts of B.

megaterium has been reported (49) suggesting that a rigid

 

wall may not be necessary for septation though the fact
that such protOplasts still contained some wall components
may imply that some cell wall is necessary for septation.
It is difficult to compare the mechanisms of cell
division septation and sporulation septation with regard
to cell wall growth. In rod-shaped cells wall extension
and cross wall (septum) formation seem to be relatively
independent processes (32). The concept of the unit cell
developed by Donachie and Begg (18) is probably relevant
to sporulation septation. With small cells of B, 921;, at
slow growth rates, elongation of the cell is in one
direction only and is towards the youngest pole. In
larger, faster growing cells, elongation occurs in two
directions towards both poles. Presumably in sporulation,-
cell wall extension in at least one direction is repressed
so that septation occurs without the normal preceding wall

elongation. In this simplest model, septation (stage II)

24

occurs at what would have been the middle of the cell had
elongation occurred. Freese (27) has considered 3 other
models of asymmetric septation. Also cross wall formation
in the septum is repressed though the preceding discussion
does not rule out small but important rearrangements of
the wall necessary for septation. In a sense, a sporulating
rod represents a reversion of a dividing rod to the more
primitive state of the dividing coccus (32) but without
cross wall formation in the septum. As a result, the germ
cell of the spore resembles a coccus.

An unanswered question with respect to stage I and
II sporulation mutants is how they can exist without
affecting normal cell division if they are indeed equi-
valent to modified stages of cell division. One answer
is that such mutations are in a sense, conditional being
expressed only under the metabolic conditions prevailing
during the stationary phase.‘ Such conditional sporulation
mutants might be expected to be more frequently isolated
than non-conditional mutants according to the hypothesis,
for routine methods for selecting sporulation mutants
depend on the mutant being able to form a pOpulation (84)
by normal cell division. Thus the same genotype will be
expressed as one of two different phenotypes according to
the environmental conditions. For example, since the
occurrence of the sporulation division process seems to

depend on a shift-down in growth conditions, impairment

25

of the TCA cycle will affect sporulation division without
necessarily affecting normal division. Hopefully such
reasoning as this will help to explain how a mutant of
B. subtilis isolated by Ito and Spizizen (40) and blocked
early in sporulation (no extracellular proteases or anti-
biotic) lacked one membrane protein, had one modified
membrane protein and yet could divide normally. They did
not present these data as evidence against the cell division
hypothesis which they seem to accept since they refer to
"symmetric cell division" and "asymmetric cell division."
Phenethyl alcohol (PEA) is believed to affect the
site(s) controlling DNA replication and cell division (117)
though PEA also affects permeability and so may also affect
DNA synthesis this way. Cell division septation and
sporulation septation are both inhibited by PEA (70, 90).
Sporulation septation is, however, more sensitive than
cell division septation. Interestingly, Kretschmer (47)
who confirmed this observation also found that stabilization
of potential transformants of the preseptation stage in
sporulation in B. subtilis is affected by the same con-
centration of PEA (0.3%). She suggests that PEA may be
stabilizing some stage common to the two processes. The
differential effect of PEA on division and sporulation is
not unusual since sporulation is generally more sensitive

to environmental influences than division (63).

26

Relevance of Division Modifications
for Stages III—VII

 

 

Events subsequent to septation can be regarded as
being facilitated by the modifications of the last cell
division postulated to occur during stage I and II.
Envelopment (stage III) of the small germ cell protoplast
by the larger future sporangial protoplast can be regarded
as tn: consequence of membrane synthesis being out of step
with net cell wall synthesis which is delayed until stage
IV (cortex formation). Since there is no wall elongation
envelopment seems to be inevitable. The sporangium cell
wall is necessary for envelopment (24). Envelopment is
helped by the size differences between the two protoplasts
and by the lack of a rigid cross wall in the sporulation
septum.

Cortex formation (stage IV) involves peptidoglycan
formation and it has been suggested that this is equivalent
to cell wall formation by the larger protOplast (23). The
cortical membrane is also peptidoglycan and can be regarded
as germ cell primordial wall formation. In B. subtilgs
this primordial wall is formed before the cortex (75). The
spore coat (stage V) is believed to be synthesized in the
sporangium protoplast (64, 84, 92) since it is located
there.

The end result of the modified cell division process

is a dormant resistant germ cell encysted in products of

f

27

the sporangium, i.e. the coat and cortex. Most of the
sporangium is destroyed by lysis (stage VII) thus freeing
the encysted germ cell. Thus the imbalance and asymmetry
that first becomes apparent during stage I and II is
progressively amplified and in the end a major part of
one of the cells resulting from the division is lyzed.
The morphological changes that occur during sporulation
seem to be principally the result of changes in the rela-
tive timing and quantity of synthesis of membrane and cell
wall materials rather than changes in quality of the
structural materials. This and other aspects of the
modified cell division are reviewed in the Appendix.

The modified cell division hypothesis of sporulation
provides a convenient framework on which to arrange consist-
ently our knowledge of sporulation. Such an inductive
approach, as the previous literature survey shows, cannot
as yet explain every observation about sporulation. Many
of the apparent contradictions will probably be settled

as our understanding of this complex process increases.

Rod to Filament Morphopoiesis

 

As mentioned in the General Introduction, the

availability of a thermosensitive mutant of B, megaterium

 

which forms filaments at restrictive temperatures due to
a defect in cell division septation made it possible to
test a corollary of the hypothesis. The corollary is that

mutants defective in cell division septation should be

28

asporogenic due to the defect also impairing the ability
to make sporulation septa. In fact, there was already a
suggestive precedent supporting this idea. Shaforostova

(86) observed a dissociation of B, megaterium in continuous

 

culture into normal rods and filaments with no cross walls
visible by light microscopy. The dissociation occurred at
intermediate mass doubling rates. The filamentous dis-
sociant was stable and asporogenous. However, the stage
at which sporulation was blocked and the possible presence
of crosswall-less septa in the filaments were not checked
by electron microscopy. ‘In this thesis, these points have
been checked with a mutant that has the added advantage of
being temperature conditional. The results that were
obtained are consistent with the postulated relationship
between the two kinds of septation.

Bacterial filaments (synonyms "snakes" or "long
forms") are elongated rod-shaped cells that contain no
cross walls or septa. This definition of filaments excludes
chains of cells but such a distinction is not always made
in the filament literature. Filament formation can occur
in Gram-positive and -negative rod-shaped cells. Rod-
shaped cells are endowed with a cell lengthening mechanism,
and filamentation is an exaggeration of this function due
to inability to control it by means of septation. Spheroidal
shaped cells or cocci, which do not have a well developed

extension mechanisms, do not form filaments as easily.

29

Although long forms of cocci have been reported (32) they
never reach the exaggerated lengths of filaments derived
from rods. When rods lose their elongation capacity, as
in certain mutants, coccoidal forms result (72).

Filaments sometimes occur naturally in growing
cultures but with a low frequency. High-frequency fila-
mentation can be induced by a variety of environmental
and genetic effects (37). The filamentation effect of
the inducing factor is sometimes cured by high sodium
chloride concentrations, by pantoyl lactone or by photo-
reactivation. Diverse effects such as the presence of
deleterious chemicals at low concentrations or nutritional
deprivation cause filamentation. This suggests that
division septa are readily diSpensable cell structures
and/or that their formation is the most sensitive
physiological process in the cell.

Antibacterial agents that induce filamentation
include dyes (37), antibiotics (29, 37), metabolite
analogues and inhibitors (38, 83, 97), and heavy metals
(37) including platinum compounds (74). Nutritional
deficiences causing filament formation involve biotin
(89, 94), iron (65), and magnesium and potassium ions
(87, 110). On the other hand, filamentous forms are
greatly reduced in slowly‘ growing chemostat cultures
(48) and filamentation of B, 22;; is induced in rich

medium plus L-lysine (112). Physical effects such as

30

high (100) and low (88) temperature, radiation (42) or
high pressure (118) can cause filamentation. Sometimes
the cause of filamentation is not easy to define, e.g.
spontaneous filamentation of Proteus at the edges of
colonies (41).

Filamentation due to unconditional expression of
genetic mutants is rarely studied (86). The most studied
filamentation mutants are conditional mutants. The
conditioning agents may be chemical (62) or physical
agents such as ultra-violet irradiation (108, 113) and
temperature (36, 103).

In Escherichia coli temperature sensitive (Ei)

 

filamentous mutants have been classified into two pheno-
types but each phenotypic class contains several genotypes
(36). One phenotype forms filaments with continued DNA
synthesis and another forms filaments without prolonged
DNA synthesis at the restrictive temperature.

The variety of factors inducing filamentation may
reflect the apparent complexity of septation. This idea
of complexity is supported by the mapping of filamentation
mutants of B, subtilis (102) andB, 92;; (36, 99) at several
widely separated loci.

One cause of filamentation appears to act on
septation indirectly at the control level. Thus inhibition
of DNA synthesis by a variety of treatments (ultra-violet

irradiation, mitomycin C, thymine starvation, nalidixic

31

acid or certain conditional mutations) leads to filamenta-
tion (5). This is because septation cannot occur until a
round of DNA replication is completed in B, ggli, Repli-
cation completion signals the start of septation. The B.
221$.t3 mutant of Reeve, Groves and Clark appears to affect
this signal directly (68). However, some BngNA mutants
continue to divide at restrictive temperatures thus
yielding anucleate cells of uniform size (35) or of
variable size (39). Since this division is inefficient,
long filaments are eventually produced. Some of these
continued division B22 mutants may carry a second mutation
responsible for this behavior (36).

A second cause of filamentation acts directly at
the level of septation or at least cross wall formation.
Thus low levels of penicillin can induce filamentation in
‘B. gala, though sometimes the filaments are unstable unless
high concentrations of salt are present (93). Penicillin
induced filamentation appears to be related to the fact
that the stage of the cell cycle most sensitive to
penicillin is septation (59). Hartman, H61tje and
Schwarz suggest this is because a critical enzyme
(endopeptidase) probably concerned with cross-wall
formation is more sensitive to penicillin than an equiva-
lent enzyme (glycosidase) probably concerned with elonga—

tion (31). Decreased peptidoglycan content has been noted

32

as the result of nutritionally induced filamentations in
B, ggl£’(lll, 112).

A third cause of filamentation appears to act
directly at the membrane level. Bgffilament formation
in B, ggl£_due to a mutation at the E5; locus is associated
with increased azide and PEA resistance. PEA is believed
to affect DNA membrane attachment sites (117).

Finally the possibility exists that some mutations
may cause filamentation by affecting translation. A
relationship between aminoacyl-tRNA synthetase activity
and cell division has been reported (95, 96) in a EE?
filamentation mutant of B. subtilis (56).

The ubiquity of agents inducing filamentation and
the several known sites of action suggest that the process
of septation is very complex. Thus, while filament forming
mutants are tempting to study with respect to Sporulation
and cell division septations, the actual nature of the
lesion causing aseptation is difficult to determine.
Indeed, the mode of action of a filamentation mutation
has not yet been worked out. Nevertheless, whether the
mutation is intimately related to septation or its control
or whether it is more distantly related perhaps via
intermediary metabolism may be immaterial from the point
of view of comparing cell division septation and sporulation
septation. Thus although the relationship of the mutation

and septation may be indirect it could still be important.

10.

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ARTICLE 1

CELL DIVISION SEPTATION AND SPORULATION SEPTATION

IN BACILLUS MEGATERIUM

 

BY

A. D. Hitchins and H. L. Sadoff

Reprinted from Spores 2, pp. 148-152 (1972), American

Society for Microbiology, Washington, D. C.

44

___ _ _ _ _ __ _ _ __WMMWM'JEFT - “I" "“ ' ‘ ’ F.

SPORES V
Copyright 0 I972 American Society for Microbiology I
Printed In U.S.A.

Cell Division Septation and Sporulation Septation !
in Bacillus megaterium1

ANTHONY D. HITCHINS AND HAROLD L. SADOFF
Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48823

 

. SPORES V
Copyright 6 1972 American Society for Microbiology
Printed in U.S.A.

Cell Division Septation and Sporulation Septation
in Bacillus megateriumI

ANTHONY D. HlTCHlNS AND HAROLD L. SADOFF
Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48823

A temperature-sensitive mutant (TH 14) of Bacillus megaterium ATCC 19213
was Isolated by replica plating colonies formed by survivors of nitrosoguanidine
mutagenesrs and a subsequent D-cycloserine selection step. The mutant and its
parent grew at snmilar rates [mean generation time (MGT) about 2 hr] at 26 C the
permisswe temperature. At 39 C, the restrictive temperature, the parent grew with
an MGT of about 1 hr, but the mutant, after a short period of rapid growth formed
filamentswrth an MGT of 2 hr. The filaments were aseptate and, since deoxy-

tton of the. mutant at 30 and 39C were 0.6 and 10‘5, respectively, whereas the
corresponding values for the parent were 0.8 and 0.1. No sporulation septa or later
stages of sporulation were detectable in stationary-phase filaments. These and
other results suggest that (i) the filamentation and asporogenic characteristics are
due to the same mutant gene and (ii) the early stages of sporulation represent a

modified cell division.

 

We are interested in comparing and contrasting

the processes of cell division septation and
sporulatton septation and their control. The two
processes have much in common, suggesting that
the early stages of sporulation may represent a
modified cell division. This idea was proposed by
Roblnow (8) in 1960 and has been reviewed (3,
4. 10). However, Kretschmer (6) has suggested
that sporulation septation is due to the reinitia-
tlon of membrane synthesis at a polar zone that
IS normally responsible for cell elongation.
_ SIX'Drulation septa differ from cell division septa
1n hemg asymmetrically located in the cell and in
thetr lack of peptidoglycan. These two differences
are Important for the subsequent stages in de-
velopment of a spore.

If the early stages of sporulation are analogous
to cell dtvision, then mutants defective in some
Phase of cell division [deoxyribonucleic acid
(DNA)_ replication or its initiation, nuclear
reparation, septation] might be asporogenic due
0 blocksoccurring before stage II of sporulation.

e Cleoscrtbe here the isolation and properties of a
CBondttlonal temperaturesensitive (ts) mutant of

aczllus megaterium that is defective in Septation
(A. D. Hitchins and H. L. Sadoff. Bacteriol.
Proc., p. 24, 1971). A similar ts filamentation
mUIant of B. subtilis has been reported (7), but

I o .
St Publication number 571! of the Agricultural Experimental
anon, Michigan State University.

it has not been used to compare cell division
septatlon and sporulation septation.

MATERIALS AND METHODS

Strains. B. megaterium ATCC 19213 was the parent
strain used in this study. A temperature-sensitive
mutant, TH 14, was derived from the parent strain,
and a streptomycin-resistant TH 14 strain was also
obtained.

Media. The defined sucrose-salts (SS) medium of
Slepecky and Foster (11) was used, but in some ex-
periments sucrose was replaced by other carbon
sources. The concentrations of the carbon sources
were 0.3% (w/v), and these were either autoclaved or
filter-sterilized as appropriate. Nutrient broth and
nutrient agar were used with or without a supplement
of the trace metal salts of SS medium.

Cultures. Supplemented (metal salts) nutrient agar
or SS medium agar slants were used for maintenance
of all strains. Stock spore suspensions were prepared
on SS agar or in SS liquid medium. Cultures were
grown in lOO-ml volumes of SS medium at 30 C from
heated (70 C, 30 min) spore inocula. lnosine and
alanine (25 rig/ml) were included to initiate germina-
tion, and growth was monitored by turbidity meas-
urements at 600 nm in either a Beckman DU spec-
trophotometer (l-cm cuvettes) or a Beckman Spec-
tronic-ZO colorimeter (l l-mm tubes).

Isolation of mutants. The following procedures
were utilized to isolate the B. megaterium mutant
TH 14. Cells were grown to mid—exponential phase in
glycerol-salts medium at 30C and then treated with
75 pg of N—methyl-N’-nitro-N-nitrosoguanidine per

148

 

SEPTATION IN B. MEGATERIUM

ml (NTG; Aldrich Chemical Co.) for 30 min at room
temperature. Approximately 5% of the cells survived
this mutagenesis when counts were made on nutrient
agar. The cells were washed twice in glycerol-salts
medium, inoculated into fresh medium, and incu-
bated at 30 C for 16 hr. This permitted the expression
and multiplication of any conditional division mu-
tants surviving the NTG treatment. A 10-ml sample
of the culture was added to 50 ml of glycerol-salts
medium and shaken for 2 hr at 37 C. After 2 hr of
growth, D-cycloserine (100 pig/ml) was added to the
culture to kill the dividing, nonmutant cells. This
selection process was carried out over 4 hr after
which a 5-ml sample of the culture was washed twice
and inoculated into SS medium at 30C to obtain
spores. These were subsequently washed and stored as
an aqueous suspension at 4 C.

Appropriate dilutions of the spore suspension
were spread on glycerol-salts agar (L~alanine and
mum, 100 pg/ml each) to yield 100 colonies per
plate at the permissive temperature. Two replicas of
each plate were made on glycerol-salts agar without
germmants. One plate of each pair was incubated at
the permissive temperature, and the other was incu-
bated at the restrictive temperature. The comparison
of each pair of plates led to the isolation of six tenta—
llVely ts mutants out of about 1,500 colonies. Subse-
quent testing showed that five isolates, including
Tl'l 14, were ts on nutrient agar and one was ts only
Withglycerol as carbon source.

,ngllt microscopy. Cells were examined and photo-
"Wographs were taken with a phase-contrast micro-
SCOpe. The lengths of 25 or 50 cells per sample were
measured with an ocular micrometer; one length unit
'5 eqUivalent to 1,250 nm. Duplicate determinations of
cell numbers were made with a Petrofi-Hauser count-
mg chamber. To 1 ml of cell suspension were added
0.2 ml of 10% formaldehyde and 0.8 ml of glycerol
(50%, v/v). Samples were then diluted in glycerol
{(30% V/V). Nuclei were stained by the Giemsa tech-

rque.

Electron microscopy. Culture samples of the mutant
were collected on prewetted membrane filters (Milli-
Dore Corp.) and washed with 10 ml of glutaraldehyde
(3'70) tn 0.] M sodium cacodylate (Sigma)-HCI buffer,
pH 7.2. A l-mm layer of warm Noble agar (1%) was
Poured over the filter. The filter with agar was kept in
COld glutaraldehyde (3%) and buffer overnight. After
being washed three times with buffer over a period of
Several hours, the filter with agar was cut into small
squares. Specimens were postfixed in the osmium
tetroxrde fixative of Kellenberger, Ryter, and Séchaud
(5) for 19 hr at room temperature. The specimens were
Dosttreated for 2 hr with 0.5% uranyl acetate in
Kellenberger’s buffer and then dehydrated as follows:

» 70, and three changes of 100% ethanol followed
by three changes of propylene oxide. Dehydrated
Spectmens were taken through a graded series of
Propylene oxide-Epon mixtures into pure Epon. After
mlymerization, the samples were sectioned with a
diamond knife on a Porter-Blum MT-2 (Sorvall lnc.,
Norwalk, Conn.) ultramicrotome. Sections were
mounted on 300-mesh copper grids and viewed with
a ”“1198 300 electron microsc0pe at an accelerating

149

voltage of 60 kv. The fine structure of the parent strain
cells (grown at 30 C in SS medium) has been reported
(2).

DNA. DNA was determined by both the methods
of Ceriotti (1) and of Roodyn and Mandel (9). For
the latter determination, the procedure of Yang and
Brubaker (12) was followed except that 1.79 mM
uracil-2-“C (Calbiochem; 0.056 pCl/meIC) was
used. Sample sizes were reduced (1.0 to 0.5 to 0.2
ml) with increasing cell densities to facilitate filtration.

Viable counts. These were determined by spread-
ing 0.1-ml samples of dilutions on nutrient agar.
Heat-resistant colony-forming units were determined
by heating samples (70 C, 30 min) in totally submerged
sealed glass ampoules.

RESULTS

Filamentation. The temperature-sensitive mu-
tant of B. megaterium designated TH 14 formed
filaments at 37, 39, and 41 C, whereas the parent
strain grew normally producing rod-shaped cells
at these temperatures. At 26 or 30 C, the mutant
cells were normal in appearance. Occasionally
filaments occurred at 30 C but only at a low fre-
quency (less than 10‘?)

Substrates. At restrictive temperatures, fila—
mentation occurred in nutrient broth or in de-
fined medium containing a single carbon source.
The substrates tested were: sucrose, sodium cit-
rate, ribose, L-glutamate, glycerol, sodium succi-
nate, L-alanine, sodium pyruvate, and sodium
acetate.

Growth. The mutant and parent strains had
mean generation times (MGT) of 2 hr at 26C
and 1.5 hr at 30C in SS medium. At 39 C, the
parent strain had an MGT of 1 hr in the same
medium. When the mutant strain was grown at
26 C and then was shifted to 39 C, its grth rate
(measured by turbidity change) increased for
about the first hour but then gradually returned
to the rate characteristic of 26C (Fig. 1). Short
filaments were detectable by the third hour after
shift-up, and these increased in length with con—
tinued incubation (Fig. 2). .

Cell size and number. After a temperature shift-
up, the average cell length of mutant TH 14 be-
gan to increase at 2 hr, but the growth response
appeared to be biphasic (Fig. 3). There was a
rapid initial increase which lasted for 2 to 3 hr,
but thereafter the total count increased more
slowly. The frequency distribution of cell Sizes
showed that the filament p0pulation rs very
heterogeneous with regard to size and that l to
10% normal size cells are always present (Table
l). The frequency and total count data showed
that some cell division of mutant TH 14 may be
possible at the restrictive temperature. .

DNA synthesis and septation. DNA synthesrs
occurred throughout filamentous growth (Fig. 1).

 

 

150

Acot'l

E

 

HITCHlNS AND SADOFF

‘coo

DNA

 

 

    
 

 

 

 

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2 v—
» E
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f. e mu 1'”. no um r”. no
2 1 1 I 4 l 1 n 1 l 1 l I
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C
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3 -
2
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not." 806 Mutant 30¢
L l I l l 1 L L A 1
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rm: (hr)

FIG. 1. Growth and DNA biosyuthesis by wild type and ts mutant TH 14 at 26 and 39 C. The SS medium
(200 ml) contained 0.1%, (w/v) sucrose. Culture turbidity was measured at 600 nm. DNA was measured by the
int/ole method (I) and plotted as absorbancy (A ) units at 490 nm. One A490 unit is equivalent to 5 pg of DNA
per ml of culture. Culture samples (20 ml) were added to 0.8 ml of 12.5 N perchloric acid at 4 C to stop growth.
Symbols: 0, growth of wild type; 0, growth ofmutant; A, wild-type DNA; A, mutant DNA.

Multiple nuclei could be seen in filaments stained
by the Giemsa method. Electron microscopy of
ultrathin sections of filaments at various stages of
develOpment confirmed that filaments are multi-
nucleate and aseptate (Fig. 4) and that no ab-
normal forms of septation occurred.

Temperature shift-down and subculture. When
filamentous cells at the restrictive temperature
were shifted-down to the permissive temperature,
division did not begin until an hour after shift-
down (Fig. 5). The overall effect of subculture of
filamentous cells by dilution into prewarmed
fresh medium is that filaments increased in
length. However, first there was a lag in the
average length increase followed by a decrease.

Viability and cross feeding. Table 2 shows that

the colony-forming ability of spores of the mutant
TH 14 decreased markedly at temperatures above
30 C, whereas that of wild—type spores was little
affected. The cell viability of mutant TH 14 had a
temperature response similar to that of its spores.
Colonies formed by the mutant at the restrictive
temperature grew slowly and remained small com-
pared to wild-type colonies. No cross-feeding ef-
fect was observed at 37 or 41 C between mutant
and wild—type strains.

Sporulation. The frequency of sporulation of
wild—type and mutant strains is shown in Table 3.
The mutant was asporogenous at restrictive
temperatures even when allowance was made for
the decreased sporulation of the wild-type strain
at such temperatures. The heat-resistant colony-

lift 55 "i"
it“s?” V
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ill Elfin”

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11M:

SEPTATION IN B. MEGATERIUM 151

 

x Total
/— "l"
X
/

'fil

GROWTH
6.. TOTAL CELLS/u.
\

0.: _

LENGTH (Mi) 3

0|

 

 

 

o l l I l l I l l 1 I l
'2345678910H
nouns

in :59- 3. Formation offilaments by is mutant TH 14
3 medium. The culture was shifted from 30 ’0
at 605” time zero. Symbols: 0, culture optical density
1 nm; X, total cell count per ml; 0, average cell
”'8’”! (1 unit equals 1,250 nm).

2;":108 unit count suggests that the mutant is
l' Otally asporogenic sensu strictu but is weakly
° '808porogem'c.

Electron microscopy of ultrathin sections of
stationary-phase filaments showed that they lack
sporulation septa or any other later stages of
sporulation. A comparison of the DNA yields of
permissive (sporulating) and restrictive (asporo-
genic) cultures of the mutant suggests that the

A600 asporogeny is not due to an inability to complete

the last round of DNA replication which forms
the mother cell and germ cell genomes.

TABLE 1. Change of frequency“ distribution of cell
lengths with incubation time at 39 C

 

Cell length categoryb

 

 

 

Time (hr) _
1—5‘ 6-10 ill—15 16—20l21—25’26—30 31-35 36—51

0.0 100 0 0 0 0 0 0 0

l 3 98 2 0 0 0 0 0 0
3.0 58 42 0 0 O 0 0 0
5.0 12 60 26 2 0 O O 0

7 0 4 20 34 26 12 4 O 0

9 5 8 8 18 20 10 16 10 10

 

° Frequency distribution is expressed in per

cent; 50 cells equal 100%.
5 Cell length is measured in arbitrary units (1

unit equals 1,250 nm).
‘ Parent cells and permissively grown cells of

TH 14 fit into the 1—5 category.

 

 

 

HITCH INS AND SADOFF

J .4 “ .l . '4,
- 14,- L} : .1:-
‘. ' )

FIG. 4. Electron micrographs of ultrathin sections of stationary-phase filaments of mutant TH 14. The scale bar
represents 1,000 nm. The micrographs show that the filaments are aseptate and multinucleate. One of the filaments

is attached to a cell.

 

 

 

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‘3
_I I I I I I I I L I I i
2 4 6 8 IO I
HOURS

FIG. 5. Eflect of shift-down of the growth tem-
perature from 39 to 30 C on filaments. The arrows

The shift-down of stationary-phase filaments to
permissive temperatures results in the formation
of a low percentage of sporangia. Only the shorter
filaments form such sporangia and in this event
the spores are terminally located. Preliminary
work with six revertant isolates, appropriately

TABLE 2. Efl'ect of growth temperature on the
viability of spores and cells of the parent
and mutant strains

 

 

Determination 30 C 37 C 39 C 41 C
Spare viability“
Parent ............... 100 96.0“)0 87.00000
Mutant TH 14 ........ 100 1.0000 0.00003
Cell viability
Mutant TH 14 ........ 100 0.2800 0.0080
Mutant TH 14 str'. . . 100 0.0008 (0.”!

 

 

 

 

0' Viability is expressed as per cent survivors, the value at
30 C being taken as 100.

indicate time of shift down. Symbols: X, growth; 0,
average cell length. One length writ equals 1,250 nm.

 

SEPT ATION IN B. MEGATERIUM i53

| TABLE 3. Sporangia frequency“ of parent and mutant
-. .. . strains at various growth temperatures

 

 

 

 

Determination 30 C 38 C ( .39 C 42 C
Parent ........ 78 (9)5 47 (ll) 7 (7) O (0)
Mutant ;

TH 14 ....... 157 (I4) 0 (0) 0 (0)c 0 (O)

 

 

“Sporangia frequency expressed as percentage
of cells containing phase-bright spores.

‘ Values in parentheses are percentages of
cells containing phase-dark or incompletely phase-
bright spores.

‘Heat-resistant colony-forming count equals
108 per ml of culture.

 

mrked with streptomycin resistance, showed that
reversron to temperature resistance is accom-
pamed by reversion to sporogeny.

DISCUSSION

The B. megaterium TH 14 ts mutant forms
multinucleate, aseptate filaments and has a re-
duced rate of grOWth at restrictive temperatures.
ASD0r08eny is associated with this phenotype,
and thus these two properties probably result

6 I from the same mutation. The asporogenic prop-
. ' -— fifty was not utilized for selection and isolation of
, the mutant. The asporogenic phenotype is ts like

the filamentous phenotype, and the loss of the ts

fihmentation property, by spontaneous reversion,
— ...' results in the simultaneous reappearance of the
'0fmulatttl'flli-‘Yi’f: sporogem’c character. The slight “leakiness” of
t/liltucleate. 0 . h0th_ the filamentation and asporogenic proper-

ties ls consistent with this genetic relationship.

" . The ts mutation may be in a gene that functions
rational-PM“; In both cell division septation and sporulation
res multst'nirfw" Septation, but the role of the gene product is
sporangiaomflii unknown. It could act at either the control or
parangia and m I»: Strl-lctural level. The results which are presented
13”,. located. its support. the Idea that early stages of sporulation
:nt isolates) 3W are e(lulvalent to a modified cell divismn.

 

0m remh"?"‘”:,
md rel/5 0! "5’ 5”

CI 37C )5: .
196.0000]
/L0000/
300 “‘3"
I 3;”; ‘(flil'ci

.th

ACKNOWLEDG MENTS

We thank Prudence J. Hall and H. Stuart Pankratz for help
with the electron microscopy.

This work was supported by Public Health Service grant
M01863 of the National Institutes of Allergy and Infectious
Diseases and forms part of the requirements for the Ph.D. degree
in microbiology at Michigan State University. One of us (A. D.
Hitchins) is a graduate research assistant.

LITERATURE CITED

1. Ceriotti, G. 1952. A microchemical determination of desoxv-
ribonucleic acid. J. Biol. Chem. 198:297—303.

2. Ellar, D. J., D. G. Lundgren, and R. A. Slepecky. 1967. Fine
structure of Bacillus megaterium during synchronous
growth. J. Bacteriol. 94:1189—1205.

3. Hanson, R. S., J. A. Peterson, and A. A. Yousten. 1970.
Unique biochemical events in bacterial sporulation.
Annu. Rev. Microbiol. 24:53—90.

4. Hitchins, A. D., and R. A. Slepecky. 1969. Bacterial sporula-
tion as modified procaryot'u: cell division. Nature (London)
223:804—807.

5. Kellenberger, E., A. Ryter, and J. Séchaud. 1958. Electron
microscope study of DNA-containing plasms. II. Vegetative
and mature phage DNA as compared with normal bac-
terial nucleoids in difi'erent physiological states. I. Biophys.
Biochem. Cytol. 4:671—678.

6. Kretschmer. S. 1971. Sporulationsverhalten von Bacillus
megaterium w'arhrend Kohlenstofi'hunger. Z. Allg. Mikro-
biol. 11:113—120.

7. Mach, F., and H. Engelbrecht. 1970. Isolation und erste
Charakterisierung einer temperatursensitiven filamentbsen
Mutante von Bacillus subtilis SB 19. Z. Allg. Mikrobiol.
10:383—395.

8. Robinow, C. F. 1960. Morphology of bacterial spores, their
development and germination, p. 207-248. In I. C. Gunsalus
and R. Y. Stanier (ed.), The bacteria, vol. 1. Academic
Press Inc, New York.

9. Roodyn, D. B., and H. G. Mandel. 1960. A simple membrane
fractionation method for determining the distribution of
radioactivity in chemical fractions of Bacillus cereus.
Biochim. Biophys. Acta 41:80-88.

10. Slepecky, R. A. 1969. Synchrony and the forrmtion and
germination of bacterial spores, p. 77—100. In G. M.
Padilla, G. L. Whitson. and I. L. Cameron (ed.), The cell
cycle; gene-enzyme interactions. Academic Press Inc.,
New York.

ll. Slepecky, R. A., and J. W. Foster. 1959. Alterations in metal
content of spores of Bacillus megaterium and the efl'ect on
some spore properties. 1. Bacterial. 78:117—123.

12. Yang, G. C. H., and R. R. Brubaker. I971. Efi‘ect of Ca2+ on
the synthesis of deoxyribonucleic acid in virulent and
avirulent Yerslnia. Infect. Immunity 3:59-65.

 

 

 

I ll“.

"n: huu'r‘ig.tft‘\lil!

I‘ll. ‘0. .tl

 

ARTICLE 2

FURTHER PROPERTIES OF A THERMOSENSITIVE ASPOROGENOUS

FILAMENTATION MUTANT OF BACILLUS MEGATERIUM

 

BY

A. D. Hitchins and H. L. Sadoff

(Manuscript in preparation)

51

Further Properties of a Thermosensitive Asporogenous

Filamentation Mutant of Bacillugpmegaterium1

 

ANTHONY D. HITCHINS AND HAROLD L. SADOFF

Department of Microbiology and Public Health,

 

Michigan State University,

 

East Lansing, Michigan, 48823

 

1Journal Article No. 6050 from the Michigan Agri-

cultural Experiment Station.

52

(ABSTRACT)

 

Thermosensitive mutant, TH14, of Bacillus megaterium

 

ATCCl9213 is defective in cell division septation and spore
formation at the restrictive temperature (39 C). As a
consequence, the mutant forms multinucleate aseptate
filaments and is aSporogenic. The mutation does not result
in any qualitative compositional changes in membrane pro-
teins which are extractable in hot sodium dodecylsulfate
plus 2-mercaptoethanol. At the restrictive temperature,
the mutant has a reduced content of a small molecular
weight protein(s) equivalent in size to cytochrome g. .A
membrane protein(s) with a molecular weight of nearly
80,000 appears to be partially derepressed in the mutant
grown at restrictive temperature. In addition, numerous
unidentified Spherical inclusions of fairly uniform size
(diameter circa 100 nm) are present in the cytoplasm at

the restrictive temperature. Filamentous growth of the
mutant is less sensitive to penicillin than growth in the
rod form. Growth in either form is equally sensitive to
D-cycloserine at the concentrations used for selection of
the mutant. Temperature shift-up experiments suggest that
l to 2 rounds of deoxyribonucleic acid (DNA) replication

occur before the phenotypic expression of the mutation

53

54

occurs. The septations corresponding to the DNA replica-
tions can be either 2 division septations or 1 division
septation plus a subsequent sporulation septation. This
conclusion coupled with previously reported work supports
the hypothesis that the early stages of sporulation
represent/a modified cell division.

Bacterial cell division mutants whose expression
is thermosensitive (ts) have proved to be useful for under-
standing division (l,l4,15,23,26,28).‘ In contrast,
although it is about 12 years since ts bacterial sporulation
mutants (Spg_E§) were first isolated by Lundgren and Beskid
(18), use of ts mutants in studying sporulation has been
rare (30). Nevertheless, the ability to control the
phenotypic eXpression of a mutant gene by temperature
shifts is particularly useful in studying the role of
that gene in processes consisting of sequential events
such as sporulation (11). This is especially true when
the gene's function is known.

Recently, two ts cell division mutants have been
used to study sporulation. One of these is a mutant with
ts initiation of deoxyribonucleic acid (DNA) synthesis
(20). The other, which is the subject of this report,

is a ts mutant (TH 14) of Bacillus megaterium which grows

 

normally at 30 C but forms aseptate filaments at 39 C,
the restrictive temperature. This mutant is also ts

asporogenous being blocked at sporulation Stage I (DNA

55

in axial filament configuration) since no sporulation septa
(Stage II) are detectable at restrictive temperature (10).
Apparent lack of recognizable Stage I forms in the mutant
at the restrictive temperature can be attributed to the
difficulty of defining the stage in the parent strain (6).
According to the recently recommended terminology of Young
and Wilson (34) ts mutant TH14 can be designated §EQ.E§.£
with respect to its ts asporogenic prOperty.
Thermosensitive blocking of cell division septation
and sporulation septation in the same mutant suggested that
the two processes are closely related (10). Such a rela-
tionship supports the hypothesis that the early stages of
sporulation (Stages I and II) represent a modified cell
division (ll). In this paper we compare the membrane
proteins of the parent strain and the mutant at permissive
and restrictive temperatures (A. D. Hitchins and H. L.
Sadoff. Bacteriol. Proc., p. 37, 1972) and report some

other prOperties of the mutant.

MATERIALS AND METHODS

Bacterial strains. The parent strain was Bacillus

 

megaterium ATCCl9213. The mutant strain (TH14) was derived

 

from the parent strain by chemical mutagenesis (10).

Previously described techniques. Determinations of

 

heat resistant colony forming units, percentage of sporu—
lation and deoxyribonucleic acid (DNA) biosynthesis and
the preparation of ultrathin sections for electron micro-
scopy are described in the preceding communication (10).

Culture conditions. Cell and filament-form cultures

 

for isolation of membrane proteins were prepared by allowing
preheated spores (log/ml; 70 C for 30 min) to germinate and
grow out at 30 C and 39 C respectively. Spores were pro-
duced on solid medium as described previously (10). The
medium used was the SS-medium of Slepecky and Foster (29)
containing sucrose (0.3% w/v) and supplemented with
germinants (L-alanine and inosine, 100 ug/ml each) and
L-leucine (50-300 ug/ml). Cells and filaments were
harvested by centrifugation (10,000 x g for 30 min) when
the culture turbidity (correSponding to an absorbency
reading at 600 nm) was 0.7 to 1.0 (equivalent to about

8 8

3 x 10 to 4 x 10 cells/ml). Drained cell and filament

pellets from lOO—ml cultures were Stored at ~10 C. When

56

57

labeled membrane proteins were required, 100-m1 amounts

of the above medium were further supplemented with either
L-leucine-4, 53H (1 mCi; 30-50 Ci per mmole) or L-leucine-
U-14C (50 uCi; 222 mCi/mmole). Cultures for other eXperi-
ments were prepared using vegetative inocula grown as

described previously (10).

Membrane preparation. Protoplast ghosts were pre-

 

pared by 1ysozyme lysis of rods or filaments with lysozyme
under hypotonic conditions. The method was based on that
of Salton and Freer (25). Frozen pellets of rods or

10 cells) were thawed

filaments (equivalent to 3 to 4 x 10
and resuspended in 10 ml Tris-HCl buffer (trishydroxymethy—
laminomethane, 0.05 M and pH 7.5 at 30 C) containing protease
inhibitors (ethylenediaminetetraacetic acid and phenylmethyl-
sulfonyl chloride both at a concentration of 10-3 M) and
eggwhite lysozyme (E.C. 3.2.1.17; 250 ug/ml). Viscosity
increased due to release of DNA from the lysing cells.
Viscosity was decreased by addition of beef pancreas
deoxyribonuclease (E.C. 3.1.4.5; 200 ug/ml). After 1 to

1.5 hr protoplast ghosts were sedimented by centrifugation

at 10,000 x g for 30 min. The supernatant containing the
cell cytoplasmic fraction was stored at -10 C. ProtOplast
ghosts were resuSpended in 5 m1 Tris-HCl buffer (0.05 M

and pH 7.5 at 5 C) and separated from unlysed cells and

particulate debris by differential centrifugation in 12-ml

tubes in a HB4 swing-out head of a Sorvall centrifuge at

58

5,000 x g for l min. The resulting pellet from each
differential sedimentation was resuspended in 5 ml buffer
and recentrifuged. The supernatant solutions from the
initial centrifugation and three subsequent runs were
pooled. The final pellet which was mainly whole cells
and debris was discarded. The pooled supernatant solu-
tions were centrifuged at 40,000 x g for 30 min. The
protoplast ghost pellet was resuspended and washed twice
by centrifugation in fresh cold Tris-HCl buffer. The
final protOplast ghost pellet was drained and stored
overnight at -10 C or immediately treated with membrane
dissolution mixture.

Membrane dissolution, Washed protoplast ghost

 

pellets (membrane equivalent to that from about 4 x 1010

cells) were suspended in 0.3 m1 of membrane dissolution
mixture. This mixture contained sodium dodecylsulfate
(SDS; 1% w/v) and 2-mercaptoethanol (2% v/v) in sodium
phosphate buffer (0.01 M and pH 7.0). The ghosts were
heated in this mixture at 95 C for 10 min. Insoluble
material was removed by centrifugation at 10,000 x g for
20 min at room temperature. The supernatant containing
extracted membrane proteins was stored at room temperature
in airtight vessels.

SDS—polyacrylamide gel electrOphoresis (PAGE). The

 

SDS-PAGEmethod for analysis of membrane proteins was based
on published methods (27,33). The gel polymerizing mixture,

in glass tubes (100 x 6 mm internal diameter), contained

59

acrylamide (10% w/v), N,N'-methylenebisacrylamide (0.368%
w/v), ammonium persulfate (0.07% w/v), sodium phosphate
buffer (0.1 M and pH 7.0), SDS (0.1% w/v) and N,N,N',N'-
tetramethylethylenediamine (0.03% v/v). The electrophoresis
buffer consisted of sodium phosphate buffer (0.1 M and pH
7.0) containing SDS (0.1% w/v). Gels were prerun at 4
ma/gel tube for 1 hr to remove ammonium persulfate and
other low molecular weight materials.

Samples applied to each gel contained 1 ul of
tracking dye (Bromphenol blue; 0.1% w/v), 1 drop of
glycerol and 5—15 ul of the membrane protein solution
(protein content 8-16 mg/ml). Protein was determined by
the method of Lowry g£_§1. (17). ElectrOphoresis was run
at 8 ma/gel until the tracking dye front reached the end
of each tube. Gels were stained for protein with Coomassie
brilliant blue by the method of Weber and Osborn (33)
except that destaining was done by diffusion at 37 C
into repeated changes of destaining fluid. Stained gels
were scanned using a Gilford densitometer at a wavelength
of 600 nm. Bovine serum albumin, fraction V (68,000
molecular weight), and horse heart cytochrome 3 (11,700
molecular weight) were used as size marker proteins (33).

Gels containing mixtures of membrane proteins
labeled with L-leucine 4,5-3H and L-leucine-U-14C respec-'
tively were frozen at -10 C and sliced into l-mm discs

using a multiple razor blade gel slicer (Diversified

60

Scientific Instruments, San Leandro, Calif.). Slices of
gel were digested with 0.2 ml hydrogen peroxide (30% by
volume) at 70 C for 12 hr in glass scintillation vials.

The dry residues were dissolved in 0.2 m1 of deionized
distilled water. Bray's (4) scintillation fluid (5 ml)
was added and the mixture shaken well. Vials were counted
for 10 min in a Packard Tri—Carb scintillation spectrometer
(model 3320). Tritium was counted at a gain of 61.5% while
14C was counted at a gain of 11%. The over-spill of the
tritium count into the carbon channel was about 1% of the

14

tritium channel count and that of C into the tritium

channel was about 3% of the carbon chennel count. In cal-

14

culating C/3H ratios or vice versa the relatively insig-

 

nificant over-spill was ignored.

Sources of chemicals. Antibiotics were obtained
as follows: sodium penicillin G and D-cycloserine
(Nutritional Biochemicals Corp., Cleveland, Ohio),
rifampicin (Mann Research Labs., New York, N. Y.) and
chloramphenicol (Parke Davis and Co., Detroit, Mich.).
The sources of the proteins used were: lysozyme and
beef pancreas deoxyribonuclease (Sigma Chemical Co.,

St. Louis, Mo.), horse heart cytochrome 3 (General
Biochemicals Inc., Chagrin Falls, Ohio) and bovine

fraction V albumin (Nutritional Biochemicals Corp.).

61

L-alanine and inosine were obtained from CalBiochem,
San Diego, Calif. and L-leucine from Nutritional Bio—
chemicals Corp. L-leucine-4,5-3H was from International
Chemical and Nuclear Corp., Waltham, Mass. and L—leucine-
U-14C was from CalAtomic, Los Angeles, Calif. The following
chemicals were bought from Eastman Organic Chemicals,
Rochester, N. Y.: ethylenediaminetetraacetic acid (EDTA),
acrylamide, N,N'—methylenebisacrylamide, N,N,N',N'-
tetramethylethylenediamine and 2-mercaptoethanol. Other
chemicals were obtained as follows: phenylmethylsulfonyl
chloride (CalBiochem), sodium dodecylsulfate (Sigma
Chemical Corp.), trishydroxymethylaminomethane (Nutritional
Biochemicals Corp.), ammonium persulfate (Fisher Scientific
Co., Fair Lawn, N. J.), bromphenol blue sodium (Allied
Chemical, New York, N. Y.) and Coomassie brilliant blue

R-250 (Mann Research Labs.).

RESULTS

Spore germination and outgrowth. Fig. 1 shows

 

the germination and outgrowth of mutant and parent spores
at permissive (30 C) and restrictive (39 C) temperatures.
Initiation of germination of the spore suspension, which
occurs during the initial decline of turbidity, was
slightly slower with the mutant at 39 C than in the
parent at 39 C. The times for the turbidity values to
decrease to 50% of the initial value were 10 and 5.5 min
respectively. As expected from the results of growth
experiments (10), mutant spore outgrowth and growth at
the restrictive temperature were thermosensitive and
resulted in filamentation. Also predictable from the
results of previous experiments (10), the mutant grew
slower at 39 C when forming filaments than at 30 C when
growing in the rod form. Thus, the turbidometrically
measured mean generation time (MGT) of the mutant was

1 hr at 30 C and 2 hr at 39 C. In contrast, the parent
strain had a similar MGT (about 1 hr) at both temperatures.
In rod-form cultures the second division was occurring by
the time the turbidity had reached a value of 0.7.

Membrane proteins. Preliminary electrophoresis

 

studies employing gels stained with Coomassie blue showed

62

63

that at least 25 protein fractions were contained in the
solubilized membrane preparations. No qualitative
differences between the protein fractions of parent and
mutant membranes at permissive and restrictive tempera-
tures (Fig. 2) could be detected by visual examination and
spectrophotometric scanning of the stained gels. However,
one protein fraction (A) with a molecular weight of about
80,000 was increased in amount in the mutant at the
restrictive temperature relative to other proteins. ‘Another
fraction (Z) was decreased in amount in the mutant at the
restrictive temperature.

The preliminary observations with stained gels
were confirmed by SDS-PAGE fractionation of three appro-
priate combinations of differentially labeled (14C and 3H)
membrane proteins prepared from parent and mutant cells
grown at permissive and restrictive temperatures. The
double labeling profiles obtained in four eXperiments
are shown in Figs. 3, 4, 5 and 6. 'Table 1 summarizes
the results from 5 profiles obtained using 3 different
mixtures. The results are expressed as the average
radioisotope ratios of all fractions and 3 subfractions
(A, Z and all fractions except A and Z). Table 2 shows
the radioisotope ratios of the A and Z fractions relative
to all other fractions as calculated from the results in
Table 1. Variation of the relative amounts of either the

A or the Z fraction in the parent and the mutant strains

64

at permissive or restrictive growth conditions is shown

in Table 3. The relative amounts of the A and the Z
fractions were calculated from the data in Table 2. In
Table 3, it can be seen that both the A and the Z frac-
tions increased by about 10% in the parent stain when

the temperature was changed from the permissive to the
restrictive condition. In contrast, when the corresponding
change was made with the mutant strain, the A fraction

increased by about 50% while the Z fraction decreased by

 

about 30%. Fraction A was about the same molecular

weight as horse heart cytochrome 2 (11,700). 3
Inclusion bodies. Amorphous inclusion bodies were

found in the filaments formed by the mutant at restrictive

temperature. The bodies were circular in cross section

with fairly uniform diameters of about 100 nm. This

suggested they were spherically shaped. They are

illustrated in Figs. 7 and 8. The bodies were relatively

electron tranSparent suggesting that they did not contain

material with uranyl ion binding sites. The bodies were

detectable within 3 hr after shift-up of cells to the

restrictive temperature but may be formed earlier than

this. A notable feature of the inclusions was their

relatively large concentration at one of the poles of

the filaments.
Antibiotic sensitivity. Fig. 9 shows that filaments

growing at 39 C were more resistant to penicillin than

normal cells of the mutant growing at 26 C. No such

65

difference was detectable with D-cycloserine at con-
centrations of 25, 50 and 100 ug/ml which totally inhibited
growth. Rifampicin (l, 10 and 50 ug/ml) inhibited growth
of filaments and rods of the mutant totally. Preliminary
results suggest that chloramphenicol (10 ug/ml) inhibited
division of filaments after shift-down of growth temperature.
Temperature shift-up. Fig. 10 shows the effect of
temperature shift-up on sporulation of samples taken at
different times during the exponential and stationary
phases of growth of a culture at permissive temperature.
Temperature shift-up before 40% of total growth had
occurred inhibited by 80% the formation of microscopically
detectable spores. Shifts-up during the time when growth
increased from 40 to 70% of the total growth allowed an
increasing proportion of spores to be produced. During
the mid-stationary phase of growth, temperature shifts
(increases) again caused a decline in spore formation.
When a temperature shift-up was carried out on cultures
that had reached 40 to 100% of the total possible growth,
the spores formed were less heat resistant than those
produced after a shift-up late in the stationary phase.
The heat-resistant colony-forming unit curve confirms
the mid—stationary phase decline in postshift-up spore
formation shown by the percent spores curve (Fig. 10).
Maximal postshift-up sporulation was observed when the
temperature change occurred in the late exponential, early

stationary and late stationary growth phases.

66

DNA synthesis. DNA synthesis continued during

 

filamentation at 39 C. Fig. 11 shows growth and DNA
synthesis by the mutant strains at the permissive (26 C)
and restrictive (39 C) temperatures. The extent of growth
and the yield of DNA were the same whether conditions were
restrictive or permissive. After temperature shift-up the
rates of both growth and DNA biosynthesis were increased
by about the first hour. Thereafter both the rates of
growth and DNA synthesis decreased to the corresponding

permissive rates.

DISCUSSION

Despite the general similarity between the membrane
proteins of the parent and mutant strains, two well defined
differences were noted. In comparing the proteins several
assumptions were made. (1) It was assumed that the pro-
teins were equally extractable from the membranes of
either strain grown at permissive or restrictive condi-
tions. The extraction procedure was rigorous and commonly
used in these kinds of comparisons (7,14,26). (2) The
complete disaggregation of protein subunits was assumed.
The high extraction temperature used should have helped
to ensure this but could have produced some artifacts
due to the scission of covalent peptide bonds (26).

(3) It was assumed that the extent of quenching of
fluorescence was constant in all SDS-PAGE protein frac-
tions (26). Variations in quenching would have had the
greatest effect on the assay of tritium-labeled membrane
proteins. The similarities between the distribution of
radioactivity (both 3H and 14C) and the scans of the
stained electropherograms suggest that quenching was
constant in the various fractions. (4) It was assumed
that no membrane proteins were lost during membrane

isolation procedures. The method of Salton and Freer

67

68

(25) was selected because it minimized such losses. They
found that protoplast ghosts of Gram-positive bacteria
(including Bacillus Sp.) prepared in Tris-HCl buffer
retained their carotenoids and cytochromes much better
than ghosts prepared in phosphate buffer.

As expected, electrOphoresis resolved the soluble
membrane preparation into a large number of fractions.
An outstanding feature of the SDS-PAGE protein profiles
was that one component (fraction Z) formed 50 to 60% of
the total extractable membrane protein (Fig. 3, 4, 5 and
6). A major component of such proportions is not seen in
most published membrane protein profiles (7,14,15,26,28).
However, some of the profiles of Siccardi 23 a1. (28) do
resemble those reported here. Presumably, the protein
profile would be dependent on the method of protoplast
ghost membrane preparation and the method of Salton and
Freer (25) was not used for the published profiles referred
to above (7,14,15,26,28). It is unlikely that the fraction

Z is due to heat induced rupture of peptide bonds during
3 i ’14 '

extraction since its H and C ratio differs from the

average ratio. To achieve such a result, the rupture
would therefore have had to be differential with respect

3 14

to H and C labled proteins. Another possibility is that

membrane proteins were lost during the protoplast ghost

3 M) in the

preparation due to the presence of EDTA (10-
Tris-HCl buffer. Such treatment is known to osmotically
unstabilize membranes by removing divalent cations such

as magnesium (19). However, in the absence of evidence

to the contrary, it is unlikely that EDTA at

69

10‘3

M would cause massive and selective removal of proteins
from membranes. Therefore, it must be assumed that the Z
fraction represents a small protein or a protein subunit

present in high concentration in the membrane of B.

megaterium.

 

Fraction Z was relatively repressed in the mutant
at the restrictive temperature (Table 2). The protein(s)
in the fraction corresponds in size to cytochrome E but
whether the fraction is homogeneous has not been determined.
If it were cytochrome g, the interesting possibility would
arise that its repression in the mutant at the restrictive
temperature could be due to iron limitation, perhaps due
to some defect in iron transport.* Iron transport in the
particular strain used in this study is known to be mediated
by the siderochrome, schizokinen, a hydroxamate of citric
acid (22). Iron limitation has been reported to cause
filamentation (24) but these observations have been disputed

(2). Cytochrome (a, b, g) repressed or deficient mutants

,_ , L- ......_.y.

of B. subtilis are asporogenic (31). ,However, such mutants
are not filamentous. Sometimes the effect on the cytochromes
in such mutants is a secondary result of blocks in the tri-
carboxylic acid cycle or of thiamine deficiency. Again,
these latter mutants are not filimentous.

'A reduced cytochrome 3 content in cells could
conceivably cause energy limitation due to a decreased
rate of oxidative phosphorylation. This could account

for the reduced growth rate of the mutant at the restrictive

70

temperature. It seems improbable, however, that energy
limitation could act selectively at the level of cell
division septation though DNA synthesis in aerobically

growing Escherichia coli is stimulated by uncouplers of

 

oxidative phosphorylation (12).

The membranes were obtained from cells and fila-
ments which had been harvested at a time when only 50 to
70% of the total possible growth had occurred.‘ This
variability in time of harvest could have affected the
cytochrome content of the membrane, for it is known that
the cytochrome (a,‘b, 3) content of B, cereus cells
increases markedly at the end of growth (16).

Fraction A was relatively derepressed in the mutant
grown at the restrictive temperature.’ It is not known
whether fraction A was homogenous and heterogeneity would
partially mask more marked changes in a given protein's
concentration.

Though changes in fractions A and Z were the most
noticeable it is possible that changes in the levels of
other membrane proteins occurred which were not easily
resolvable from the background noise of such experiments.
Changes in A and Z were readily detectable because of
their relative concentration or because of their more
obvious locations in the gels near the origin and

tracker dye front respectively.

71

The differences in membrane composition between
filaments and rods could be due to (a) the direct effect
of the filamentation mutation on a control or structural
gene, or (b) due to pleiotropic effects. One possible
pleiotropic effect would result if there were differences
in the protein composition of lateral membrane and septum
membrane. Lack.of septum membrane would result in absence
of septum specific proteins. This possibility cannot
apply to a fraction like Z which forms such a major part
of the total membrane protein. Neither could it apply to
protein fraction like A which increases at the restric-
tive temperature.

The Spherical inclusion bodies were observed in
mutant cells which.had been grown at the restrictive
temperature. They appear to be due to the filamentation
mutation. Their chemical composition is unknown. They
could be poly-beta-hydroxybutyrate (PHB) granules but if
so, do not resemble normal PHB granules. .PHB is the
reserve material of this organism and PHB granules were
visible in the filaments by phase—contrast microscopy but
they were not concentrated at the polar regions of
filaments like the granules.

The granules do not resemble DNA-. They probably
do not contain ribonucleic acid (RNA) like the aggregates
occurring in platinum-compound induced filaments of E.

coli (13) since they are relatively electron transparent.

72

Electron microscopically, they do not resemble the protein-
containing bodies described in a ts mutant and a normal
strain of two Bacillus species (3,5). The greatest con-
centration of inclusions occurred at the polar regions
of the filaments. There was some indication that they
may be localized only at one pole. However, this has to
be confirmed as does the relationship of the pole concerned
to the last cell division septum.

Bacterial cells are most sensitive to penicillin
at the septation stage of the cell division cycle (21).
The relative insensitivity to penicillin of the aseptate
filaments compared with rods of the mutant is consistent
with this fact. In E. 221i it has been shown that the
glycosidase, which is probably concerned with cell wall
elongation is less sensitive to penicillin than the
endopeptidase, which is probably concerned with crosswall
formation (8).

D—Cycloserine (DCS) was the selective agent used
in the isolation of this mutant (10)-» Penicillin was not
used because many Bacillus species have inducible or
constitutive penicillinases. The fact that growth of the
filaments at 39 C and of the mutant at 26 C, which occur
at similar rates, were totally inhibited by the concentration
used for mutant selection shows that filaments are not more
resistant than rods to DCS as long as their rates of growth

are similar. However, since the mutant grows slower than

73

the parent at the restrictive temperature, DCS probably
exerted its selective action by being less active against
slower growing mutant cells than against faster growing
parent cells. This sort of selection is reported to occur
with penicillin (32).

The equal sensitivity of growth of rods and filaments
of the mutant to rifamycin (1-50 ug/ml) shows that the
mutation involved does not confer ts-rifamycin resistance.
Thus, filamentation does not appear to be due to an RNA
polymerase mutation. In Bacillus species, mutants resistant
to 10 ug/ml of rifamycin have been obtained.

A preliminary experiment indicated that the division
of filaments after temperature shift-down was sensitive to
chloramphenicol, suggesting that aseptation at restrictive
temperature does not involve reversible heat inactivation
of a ts protein (1,23). This conclusion is supported by
the fact that there is no sudden burst of division upon
temperature shift-down of filaments (10).

In the temperature shift-up experiment, the pro-
portion of spores formed could be used to measure the
proportion of cells making sporulation septa if it was
assumed that a constant proportion of cells forming septa
completed sporulation. By comparing the time in the
growth cycle at which shift-up prevented spore formation
(Fig. 10) with the amount of DNA synthesized in the

permissive culture at that time (Fig. 11) an approximate

'74
estimate could be made of the time at the restrictive
temperature required for the filamentation mutation to take.
effect. This time will be expressed in numbers of DNA
replications. The shapes of the two growth curves in the
shift-up and DNA experiments differ in the transition
region from exponential phase to stationary phase. There-
fore, the estimate has been made using both time of end
of growth and culture turbidity. On the basis of time
of end of growth, 10, 25 and 50% sporulation can occur
after temperature shift-up when 1.5, 1.6 and 2.0 doublings
of DNA could still occur. Thus, at least one but not more
than two doublings of DNA must still be possible after
temperature shift-up in order for sporulation to occur.
It seems fair to conclude that after shift—up the round
of DNA synthesis in progress is completed, the corresponding
division septation occurs and a whole new round of DNA
replication occurs which is then followed by sporulation
septation. When shift-up is earlier in the exponential
phase, two cell divisions and hence septations occur before
the filamentous growth rate is established (10).

A decrease in the ability to sporulate after shift-
up occurs when the temperature increase is carried out at
the time of sporulation septation and engulfment (6,9).
Possibly these stages are very sensitive to the 30 to 39 C
shift because the extensive membrane synthesis and

morphogenesis they involve requires a membrane whose

75

composition is exactly adapted to the existing temperature.
The spores formed after the early temperature shifts (30
to 39 C) before the culture at 30 C is committed to
sporulation, seem to be relatively heat sensitive. The
reason for this is not known. Low frequencies of
sporulation occurred after temperature shifts (increases)
earlier in the growth cycle. This could be due to a
combination of factors: exponential phase sporulation

by the permissive culture, the leakiness of the ts TH14

mutation and sporulation by revertants.

ACKNO WLEDGMENTS

Thanks are due to H. Stuart Pankratz and Victoria M.
Koo Hitchins for help with the electron microscopical
observations.

This work was supported by Public Health Service
grant Al 01863 of the National Institute of Allergy and
Infectious Diseases and forms part of the requirements

for the Ph.D. degree in microbiology at Michigan State

University.

76

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'79

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34.

'80

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Washington, D. C.

 

81

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’84

FIGURE 1. Germination and growth of parent strain
and ts mutant TH14 strain spores. Symbols: 0, parent at
39 C; 0, parent at 30 C; A, TH14 at 39 C (filaments); A,
TH14 at 30 C. Cultures behaving like these were used for
cytoplasmic membrane preparation (see MATERIALS AND METHODS).
Turbidity was measured at 600 nm.

85

.>._._D_mm3._.
5 l..

 

 

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>._._n:mm3._.

IO

 

 

HOURS

86

FIGURE 2. Densitometric scan of gels with membrane
protein bands separated by SDS-PAGE. Migration of the bands
was from left (-) to right (+). Symbols for sources of
membranes: 39, restrictive temperature; 30, permissive
temperature; TH14, mutant strain; parent, parent strain;

A and Z are the protein fractions referred to in the text.
The gels for the lower two scans (parent) were not
electrophoresed as long as those for the upper two scans
(TH14). Optical density is in arbitrary units.

87'

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(WI-1009) AIISNEO "lVOlldO

88

FIGURE 3. Radiolabeled membrane protein SDS-PAGE
profiles of ts mutant TH14. Symbols: continuous line,
profile Of tagged (L-leucine-4,5-3H) proteins from
permissively grown (30 C) cell membranes; dotted line,
profile of tagged (L-leucine-U-14C) proteins from
restrictively (39 C) grown filament membranes; A and
Z protein fractions are defined in the text. SDS-
protein complexes migrated from left (-) to right (+).

89

 

 

 

 

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90

FIGURE 4. Radiolabeled membrane protein SDS-PAGE
profiles of ts mutant TH14. Symbols and other legend as
in Figure 3. Unlike Figure 3 the profile of the L- -leucine-
U-14C tagged restrictive proteins has been normalized
relative to the profile of the L—leucine-4, 5- 3H tagged
permissive proteins. Normalization involves multiplying
each 14C value by the average radioisotope ratio for all
gel slice fractions (see Table l).

91

 

 

 

 

 

 

 

 

 

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92

FIGURE 5. Radiolabeled membrane protein SDS-PAGE
profiles of parent strain. Symbols: dotted line, profile
of tagged (L-leucine—U-14C) proteins from cells grown at
30 C; continuous line, profile of tagged (L-leucine-4,5-3H)
proteins from cells grown at 39 C; A, Z, + and - are as in
Figure 3 legend. The profile of the L-leucine-U-14C tagged
proteins has been normalized as described in the Figure 4
legend.

_93

 

50

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l

COUNTS / IO MIN. (x no“)

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FRACTION NUMBER

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94

FIGURE 6. Radiolabeled membrane protein SDS-PAGE
profiles of a mixture of parent and ts mutant TH14 membrane
proteins. Symbols: continuous line, profile of tagged
(L-leucine-4,5-3H) proteins from membranes of parent cells
grown at 39 C; dotted line, profile of tagged (L-leucine-
U-14C) proteins from membranes of mutant filaments grown
at 39 C; A, Z, + and - are as in the Figure 3 legend.

COUNTS / Io IIIINJX I0'3I

I5

8 .

GI

95

 

 

 

 

 

 

 

 

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96

FIGURE 7. Inclusion bodies in filament of ts mutant
TH14. Symbols: I, inclusion bodies; D, DNA. The scale bar
represents 1000 nm. Longitudinal and transverse sections
of filaments 5 hr into the stationary phase are shown. The
mutant and.parent strains form short chains of cells.
Inclusions are especially concentrated at filament poles.

97

 

98

FIGURE 8. Inclusion bodies at pole of a filament
of ts mutant TH14. Symbols as in Figure 7. The inclusions
are not completely amorphous but substructure is not easily
resolvable. There appears to be some capsular material.
The scale bar represents 100 nm.

99

 

100

FIGURE 9. Effect of penicillin on growth of rods and
filaments of ts mutant TH14. Growth at 26 and 39 C was
measured turbidometrically at a wavelength of 600 nm.
Benzylpenicillin (sodium salt) was used. Symbols: X,
control and l unit/ml; 0, 10 units/m1; A, 100 units/ml;

A, control; 0, 1, 10 and 100 units/ml.

101

 

26C

 

 

39C

 

 

 

 

ALIOIBUOJ.

00' ""

HOURS

102?

FIGURE 10. Effect of temperature shift-up on
sporulation of ts mutant TH14. Samples (10 ml) of a permis-
sively growing culture at 30 C were shifted to 39 C. Symbols:
0, percent sporangia; A, percent heat resistant (70 C for
30 min) colony-forming units (cfu); O, turbidity at 600 nm.
Heat resistant cfu count normalized relative to the percent
sporangia determination by arbitrarily assumin the maximum
heat resistant cfu count corresponds to l x 10 cfu/ml.

For percent sporangia determinations, 500 cells were counted
at 0, l, 2 and 3 hr (no spores were seen at these times)
while 200 cells were counted at all other times. Sporangial
determinations include sporangia with nonrefractile spores
and free Spores when present. The permissive culture at

30 C formed 74% sporangia compared with 73% at 39 C in the
13 hr shift-up sample.

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104

FIGURE 11. Growth and DNA synthesis of ts mutant
TH14. Symbols: 0, culture turbidity at 26 C (permissive
temperature); A, culture at 39 C (restrictive temperature);
0, DNA synthesis at 26 C; A, DNA synthesis at 39 C. Growth
monitored by measuring culture turbidity at 600 nm. DNA
content of the cultures measured as uracil-2-14C incorporation
(cpm/ml culture) into the alkali-stable fractions of cells
as described previously (10). Prewarmed (26 C) culture medium
(200 ml) containing uracil (200 ug/ml) was inoculated with
actively growing cells from an overnight culture grown in
the same conditions. Uracil-2—14C (20 uCi total) was added
and after 30 min at 26 C the culture was split into two
halves. One-half was kept at 26 C and the other shifted

to 39 C.

105

ammnhusu 42 \Zmuv 20F<moamouz_

 

 

 

 

 

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HOURS

CONCLUSION

Results of studies of the properties of a thermo-

sensitive filament forming mutant of Bacillus megaterium

 

have been presented. These suggest that the mutation has
a common effect on cell division septation and sporulation
septation. This emphasizes the predicted relationship
between these two processes.

How does the mutation exert its effect on septation?
This question cannot be answered yet. However, there are
several noteable properties of the mutant that should be
considered. Gross DNA synthesis and segregation appear to
be unaffected by the mutation. The aseptation effect of
the mutation is not apparent until after a time-delay
equivalent to 1 to 2 rounds of DNA replication, i.e. 2
division septations. There is also a delay in recovery
from the effect of the mutation although this phenomenon
has not been analyzed in terms of cell division.

The mutation caused a decrease in eXponential growth
rate at restrictive temperature compared with the parent
strain and this effect became apparent before the filamenta-
tion effect._ Inclusion bodies of undetermined composition
accumulated in the mutant by the time filamentation started.
The mutation caused quantitative changes in at least 2

membrane proteins.

106

107

One hypothesis of the mode of action of the ts
mutation is that a metabolic process is altered which causes
a reduction in growth rate at the restrictive temperature
relative to the parent strain. The limitation leads to
accumulation of the inclusion bodies and to aseptation.

An alternative hypothesis would be that the control process
for septation is upset by the mutation. This results in
partial growth inhibition and, when septation signals stop,
filaments are formed. Materials needed for septation then
accumulate. Obviously, a study of the nature of the
inclusions will be critical to understanding the mode

of action of the mutation.

APPENDIX

Bacterial Sporulation as a Modified Procaryotic

Cell Division

BY

A. D. Hitchins and R. A. Slepecky

Reprinted from Nature (Lond.) 223: 804-807 (1969)*

*

This review was partially prepared at the Department
of Microbiology and Public Health of Michigan State University
while one of the authors (A. D. H.) was a student there.

108

(Reprinted from Nature, Vol. 223, N0. 5208, pp. M7, August 23, 1969)

'

Bacterial Sporulation as a Modified Procaryotic Cell Division

by

A. o. HITCHINS‘
R. A. SLEPECKY

Biological Research Laboratories,
Syracuse University, ~
New York |32|0

\

SEVERAL authors”, observing that both bacterial sporula-
tion and cell division involve compartmentalization of a
cell by means of septum formation, have stated that
sporulation is a modified or atypical cell division. Because
this idea has been mentioned rarely in reviews concerning
sporulation1-‘v1°-", and only briefly in the context of
sporulation synchrony", we present here an evaluation
of a hypothesis which explains sporulation in terms of the
more general phenomenon of cell division. This hypothesis
suggests that sporulation is a modified cell division and an
expression of unbalanced growth resulting from .- luff
down growth condition. It gives a logical framework .m
which to arrange much of what is known about sporulation.

Table l. HYPOTHBTICAL RELATIONSHIP OI SPORULATION AND CELL DIV“ th

Stags - Sporulation event Proposed relationship to cell division
I Pre‘septation: DNA in Slow separation of DNA replicated in in te
axial filament form log and early stationary phases
II Septation Asymmetric cell division results in two
unemBIsl sized cells each with a part of
‘ 'thfi) N A (mother cell and smaller germ
cc
Membrane synthesis out of step with cell
wall formation (stage IV). Envelopment
begins encystment of germ cell
Peptidoglycan synthesis: equivalent to
cell wall formation. DPA is a product
of secondary metabolism
Cost and cortex encyst the germ cell. Coat
protein a result of secondary metabolism
Net result of asymmetric cell division is
one can cell encysted in products of the
mother cell

III Envelopment

IV Cortex and germ cell wall
formation. Dipicolinic
acid (DPA) made

V Coat formation

VI Maturation of spore

VII Lysis of mother cell and Lysls as a result of lack of further division
release of free spore

Sporulation requires an orderly sequence of seven well
defined morphological steps for its completion. Table 1
compares the morphological stages of sporulation as cur-
rently described", with sporulation interpreted as a modi-
fied cell division. Sporulation seems to be the resultant of
two factors. Factor one is” the innate ability of spore
forming cells to complete a round of DNA replication and
to partition the replicated molecules by means of a

’Present address: Department of Mlcrobio . d Public Heslt ,
Michigan State University, East lensing. simplified; , h

. freeing the encysted small cell.

This article reviews the Idea that the bacterial spore Is a small cell,
formed by an asymmetric cell division and enveloped by the larger
sister cell. Both processes seem to be morphological expressions of
unbalanced growth resulting from shift down growth conditions.

‘septum as late as 2—3 h after the end of growth. In

essenCe this means cell division without net growth. The
second factor is the efi‘ect of change of metabolic pattern,
resulting from exhaustion of growth limiting substrate,
on this cell division. The metabolism of the cells changes
from that typical of logarithmic growth, through a shift
down growth pattern, to a pattern of endogenous metabol-
ism which includes turnover and consequent reallocation
of cellular materials. This results in unbalanced synthesis;
there is an accumulation of new end products (secondary
Inetahowm) and, most importantly, chemical modifica-
tions and changes in the relative timing of synthesis of
components common to the vegetative and sporulating
cells.

Perhaps such metabolic imbalance exerts its first effects
on the DNA of the sporulating (dividing) cell. The first
\ isll)le effect of the change, however, is the marked size
inequality of the resulting daughter cells (asymmetric
division). This differential effect on the daughter cells
becomes progressively amplified so that the later stages
of sporulation bear little apparent relationship to cell
division. These stages include envelopment of the small
cell by the large one and encystment of the small cell in
products of the large cell. Finally, the large cell lyses,

During the first stage of sporulation, the DNA assumes
a linear configuration along the long axis of the future
sporangium (axial filament stage). This has usually been
considered unique to sporulating cells, but gimilar con-
figurations have been seen during normal cell division"-"
and in a non-spore-forming organism”. Several facts
suggest that axial filaments could be formed as a result
of slow separation of newly replicated DNA molecules"-“.
First, the last round of DNA replication, which presum-

ably starts in the late log phase of growth, continues

during the axial filament stage”. Second, the spore germ
cell receives half of the DNA originally present in the
sporangium‘m‘. Thus stage I can be regarded as the
completion of the DNA replication stage of cell division.

- How spores with a reported chromosome centent of two“-“

fit into this interpretation is not clear.

109

z-‘ormation ofSepturr.

As in cell division, compartmentalization of the dupli-
cated genome of the sporangium occurs during stage II
when a septum is formed due to membrane invagination.
At the fine structure level, sporulation'septation seems to
be similar to cell division septation'Mv“, but differs in
that no cell wall material is formed between the two
membranes of the septum. But when sporulation is upset.
as in rejuvenation experiments“ or in certain mutant
strains"»", cell wall material may be deposited. It should
be noted that there is some recent morphological evidence
for the presence of cell wall material between forespore
septa in clostridia'f. In addition there is chemical evidence
for peptidoglycan formation" during preseptation.

This is probably due to turnover which is known to
occur in non-growing bacteria in some conditions", but
not cell wall synthesis may occur in Clostridium pecti-
novorum which increases markedly in size during pre-

septation”. Much more precise correlation of the timing

of peptidoglycan synthesis with that of septation has been
demonstrated by the detection of significant incorporation
of diaminopimelic acid during septation (unpublished
results of R. Greene and R. A. S.) in pulse labelling experi-
ments during synchronous sporulation". These data
indicate that some rearrangement of cell wall material, if
not complete resynthesis, is necessary for septum initia-
tion. Whether the presence of a cell wall is necessary for
septation is not clear, however, even though worl‘. with
sporangial protoplasts has shown that subsequent enx slop.
ment (stage III) requires the presence of an inter»: (97 ll
wall".

Antibiotics known to inhibit cell division by inhibiting
cell wall synthesis also inhibit sporulation septation”.
This fact confirms the involvement of peptidoglycan in
septation initiation but, more importantly, highlights the
similarity of the sporulation and division septation pro-
cesses”. This similarity is further accentuated by the
fact that phenethylalcoh’ol (PEA) inhibits both cell divi-
sion and sporulation septationl’vu although the two
processes differ in sensitivity. This effect of PEA is
especially noteworthy because the compound is believed
to affect the site(s) controlling DNA replication and cell
division”. '

Aseptate” and multiseptate“-" sporulation mutants
and the peptidoglycan-less septa of wild type strains pose
a problem for the hypothesis. If sporulation septation and
division are related; how can sporulation septation be
prevented or altered by mutations that do not also affect
cell division septation i One answer is that such mutations
are conditionally expressed. A possibly analogous situa-
tion exists in the abnormal cell division septation of
Bacillus mutants". These are conditional mutants lull
unfortunately the effect of the mutations on spnrlilm in”
septation was not reported, Conditional sporulation :iupl .1-
tion mutants might, however, be expected to be morn
frequently isolated than non-conditional mutants accord-
ing to the hypothesis, for routine methods for selecting
sporulation mutants depend on the mutant being able to
form a population" by normal cell division.

Possibly the most important difference between cell
division septation and sporulation septation is the asym-
metric positioning of the sporulation septum very near
to one of the sporangial poles. It must be noted, however,
that asyrmnetric septation is not specific to bacteria
which produce spores. It occurs in minicell formation",
division of filaments“, division of a blue—green alga”
and in yeasts". The asymmetry of the sporulation septa-
tion can probably be regarded as the first morphological

expression of unbalanced growth resulting from the estab- -

lishment of shift down growth conditions" ‘ during stage
I‘i‘°-“. Sporulating cells have a physiology similar to
cells growing on acetate" in that during stage I sporulat-

ing cells adapt to by-products of growth, acetate and.

pyruvate". As these by-products are utilized, endogenous
metabolism (as evidenced by macromolecular turn-

110

over)"-“v“-" begins to dominate sporulation physiology.
The gradual slowing of growth to the zero or near zero rate
characteristic of the stationary phase, and the cell’s
increasing dependence on endo nous metabolism, could
lead to unbalanced synthesis. this could then result in
an asymmetric division as s ted for certain casts".
Microcycle .s rulation“ illustrates the effect at the
condition of e medium can have on outgrowing spam in
determining the choice between division or sporulation
septation. If sporulation septation is essentially a modi-
fied cell division, then it is a division that can occur in
conditions of virtually no net growth. Such relative
independence of growth and cell division is sometimes
apparent in vegetative cells“.

One can only speculate as to the nature of the underly-
ing macromolecular events which lead to the asymmetric
septation. Presumably a relationship between membrane
formation and DNA replication is involved. In conditions
that limit growth, one of the daughter DNA molecules of
the last replication may be less active in the control of
synthesis. The dominant DNA molecule might affect the

.positioning and availability of membrane attachment

sites" for the other DNA, molecule as suggested in the
site-territory model“ for the control of DNA replication.
Attachment sites seem to be more important than the
actual DNA molecules, because which of the two daughter
DNA molecules—the “old” or the “new”-——enters the
f uturu grim cell of the spore is_randomly determined“ in B.
cums jlinb as is the segregation of daughter DNA molecules
in B. subtilis cell division“. But the latter report is
contmdicted by a previous finding" and the recently
reported demonstration of DN A excretion (about 40 per
cent) during B. subtilis sporulation“ suggests that
daughter DNA molecule segregation patterns may be
more dificult to determine than previously thought.

Germ Cell Genome

In addition, because the germ cell is several times smaller
in volume than the sporangial cell, this may necessitate a
difi'erent supercoiling of the DNA molecule, which in turn
could affect the functioning of the germ cell DNA. It has
been suggested on the basis of photochemical data that
spore DNA is less hydrated than vegetative DNA“ and
that the configuration is altered during germination" of
the spore to reform a vegetative cell. It is not clear at
what stage of sporulation the spore’s new DNA configura-
tion would be established.

‘ The apparent inactivity of the future spore or germ c 11
genome seems to be only temporarylor partial. Thus the
newly formed germ cell increases in size“ presumably as a
result of net synthesis. - Protein synthesis is known to
«incur in the germ cell compartment after septation“ but
“'lh I i .r this is net'synthesis is not clear, for the amount of
pix-inn degradation occurring there is unknown". An
interesting. suggestion is that the engulfed germ cell is in -
a step-up growth condition, perhaps caused by amino-acids
ful‘liled by the engulfing cell (H. L. Sadoff, personal com-
munication). The location of the cortical membrane or
germ cell wall suggests that it is synthesized by the germ
cell. The larger size of the mother cell coupled with its
probable synthesis of the coat layers, cortex and its
membrane envelopment of the germ cell, however, suggests
that it synthesizes more actively than the germ cell.
Studies on the relative types and amounts of metabolism
by the germ and mother cells are hampered by the tech-
nical difficulty of separating the two cells“. Germ cells
have been isolated some time after engulfment, however
(D. Karp, D. Lang and D. G. Lundgren, personal com-
munication), and this system should help to clarify some
of the preceding problems. . '

Sporulation further resembles cell division when th
two compartments resulting from sporulation septation
behave like normal cells; they can both divide in certain
conditions. Thus the mother cell ‘ will divide when re.
juvenatedwy“. There are positive“ and negative“ reports

-1)f germ cell ch’u‘.'t.mizo.-z- just at: r seitation but before
its envelopinrnt by the mot. :er own "the germ cell of the
mature spore can either diwde or resporulate without
normal division after germination“. The two compart-
ments also resemble cells in that each can form a proto-
plast if the sporangial wall is removed just after septation
but before engulfment‘-“-". Association of mesosomes
with septation in sporulation and cell division’ suggests
that the septation processes are related. The changes in
mesosome structure during growth and‘sporulation are
also very similar", but the role of mesosomes and mem-
branes in . DNA replication and distribution between
daughter cells is not clear“.

Events subsequent to septation can be interpreted as
further expressions of the mother cell’s unbalanced syn-
thesis as first manifested by the asymmetric septation.
Envelopment (stage III) of the germ cell protoplast by
the mother cell protoplast can be regarded as the con-
sequence of mother cell membrane synthesis being out of
step with net cell wall synthesis, which is delayed until
stage IV (cortex formation); membrane synthesis within
the confines of the sporangial wall inevitably leads to
cnguiiment. This tends to be supported by the fact that,
aftei treatment with lysozyme, the germ cell protoplast is
not enveloped by the mother cell protoplast even though
still attached to it“. No data are available on the tim. A
of membrane synthesis in relation to septation uml
envelopment although very active incorporation of labeh. «l
phosphate into phospholipid during stage I has been
reported“. The use of Nomarski interference contrast
optics enables easy quantitation of Bacillus megaterium
septation and envelopment“, and so it should now be
possible to correlate more precisely biochemical events,
such as phospholipid synthesis, with the earliest morpho-
logical events of sporulation.

Germ Cell Wall

Cortex formation (stage IV) involves peptidoglycan
formation, and it has been suggested that this is equivalent
to cell wall formation by the mother cell‘. The germ cell
wall (cortical membrane) peptidoglycan appears before
the cortex in B. subtilis". The delay in the synthesis of
cortex is presumably a consequence of a continuation of
unbalanced synthesis in the mother cell. '

The spore coat is believed to be synthesized by the
mother cell‘°v”-“, for it is located there. As a result,
some mother cell cytoplasm lying between the coat and
the envelopment membrane is. incorporated into the
maturing spore“. The fate of the envelopment membrane
is not known. Continuation of unbalanced growth 188113
to lytic disruption of the mother cell, but the germ ('1 ll,
being in a rejuvenated state and surrounded by a resistant
coat, survives. Coat and cortex formation can be regar (L (l
as an encystment of the germ cell by the mother cell’.
Thus the mature spore consists of a small cell (the germ
cell) with its cell wall surrounded by products of un-
balanced growth of a second cell (the mother cell).

Many of the molecules involved in sporulation are
similar to those involved in vegetative growth. These
include proteins", lipids“v v",
can" ‘1 “. This information 1s consistent with sporulation
being a modified cell division. It implies that many of
the cell’ s genes function in both sporulation and growth.
These molecular differences reported for peptidoglycan“,
proteinsu and DNA" seem to be minor modifications.

The morphological changes that occur during sporula-
tion seem to be principally the result of changes in the
relative timing and quantity of membrane and cell wall
materials that are synthesized. In this way, different
shapes may be made from similar materials. The mem-
brane seems to play a particularly important part in shape
changes, for it is likely that the cortex and coat shapes
are determined by the membrane topography. The im-
portance of unbalanced cell wall synthesis 1n shape changes

has been reported 1n a non- sporulating organism“ and the ..

DNA" and peptidogly- -

lll

eiiect of partial or complete lack of cell wall on shape in
L forms is well known“.

On the other hand, some substances are sporulation-
specific and their production probably involves, except
perhaps in the case of the exosporium, expression of
genes not concerned with vegetative growth. These sub
stances"-“-" include the coat, dipicolinic acid (DPA),
antibiotics, toxins, the exosporium, parasporal bodies,
and certain enzymes. Most of these substances are prob-
ably synthesized by the mother cell except perhaps the
DPA. The exosporium, parasporal body, toxins, and
possibly the antibiotics are not produced by all sporulating
organisms. These sporulation specific substances can be
regarded as products of secondary metabolism“ con-
sequent on the unbalanced growth conditions. Some
genes expressed during vegetative growth are not ex-

pressed or are only slightly expressed during sporulation

as evidenced by the reduced amounts or absence of certain
enzymes in spores“.

Both vegetative and sporulation genes are expressed
during sporulation, and so the RNA should be similar in
sporulating and vegetative cells except for the mRN A
involved in the transcription of sporulation~ or vegetative-
specific enes.’ Available evidence‘"-11 does not refute
this. Ri somal RNAs from spores and vegetative cells
..f I.‘ ml {life are reported to be genetically similar”.

W. i .1 the hypothesis that sporulation is a modified
.ml (li\'ls'l( 11 is acceptable for the following reasons. First,
the spore contains an independent cell (the germ cell)
which must have arisen by cell ' division because, in
accordance with Virchow’s postulate, a cell can only be
formed by the division of a pre-existing cell. Second, the
morphological and compositional evidence available is
consistent with the hypothesis which emphasizes the
similarities of spore and vegetative cell composition and
structure as well as accommodating the differences. The
hypothesis incorporates the modern concepts of un-
balanced growth, metabolic shifts, turnover and secondary
metabolism in attempting to explain the dynamics of
sporulation. Some of the evidence, especially that regard-‘
ing the earliest stage, is only fragmentary or inferential.
Likewise-the details of how unbalanced growth conditions
later lead to unbalanced synthesis when there is no net
growth remain obscure. Several possible mechanisms have
been suggested'-"-“ and will not be discussed here. The
slowness of sporulation (6—8 h) and the similarity of the
early stages to cell division, however, suggest that there
is a gradual modification of the vegetative metabolic
pattern, rather than a relatively sudden switch from a
vegetative to a sporulation genome. Thus the sporulating
cells become committed to each stage of sporulation as it
is apprnmeiwd, rather than becoming committed to the
whole prowess all at one time“. Most significantly, the
hypothesis suggests new areas of investigation. One such
key area is the precise relationship of asymmetric septation
to cell division septation. Although understanding the
asymmetric septation may have to await advances in our
knowledge of cell division, it is conceivable that knowledge
of sporulation septation may be useful in understanding
cell division septation. Sporulation septation studies are
not as dependent on synchrony, although this is possible",
as cell division septation studies where there are over-
lapping cycles of septation in Asynchronous cultures.
Indeed, the hypothesis suggests that cell division itself,
rather than sporulation, is the simplest example of bac-
terial difl'erentiation at the cellular level of complexity.
Other sporulation phenomena where the hypothesis may
prove a useful guide to investigation include log phase
sporulation“ and oligosporogeny".

Received March 4; revised June 12,1969.

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