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[CANCER RESEARCH 28, 1802-1809,September 1968]
Control of Cell Division: Models from Microorganisms
Arthur B. Pardee
Program in Biochemical Sciences, Moffett
Laboratory,
Princeton
Control of Cell Division
One approach to the discovery of a difference between nor
mal and malignant cells is to investigate the regulation of cell
division. Normal tissues are regulated so that their cells are in
a steady-state balance between duplication and destruction.
Malignant cells appear to duplicate unceasingly and are not
in balance with the rest of the organism; they appear to have
lost a control mechanism for cell division. Our problem is to
determine how normal control mechanisms function, how they
are deranged in malignant cells, and how they can be restored.
The working hypothesis of this article is that the funda
mental biochemical events which regulate cell division are
similar in both bacteria and higher organisms. This hypothesis
will be useful at present to the extent that bacteria provide
a logical framework for ideas and experiments regarding an
imal cell division.
Research with microorganisms has frequently furnished val
uable models for workers with higher organisms. Well-known
examples are biochemical pathways, gene structure and func
tion, and control mechanisms at the levels of both enzyme syn
thesis and catalytic activity. Even for hormone action, a phe
nomenon which does not appear in bacteria, fundamental in
sights have been provided by microbiology through ideas of
metabolic control. Studies of cell division with microorganisms
might similarly provide a valuable source of concepts and a
frame of reference for workers with animal cells. The obvious
differences, morphologic and temporal, between the two sys
tems may well be only variations on a basic theme.
This article- will attempt to present an organized picture of
current beliefs regarding bacterial replication. Our knowledge
of bacterial division is increasing rapidly, though it is far from
complete. Conflicting reports are published; some of these no
doubt will prove important, but now they are interesting
mainly to specialists. These differing results probably reflect
the complexity of cell division, which must depend on the in
fluences of poorly appreciated experimental variables originating
from all parts of the cells and from the environment. The
author has tried to piece the data together into a sort of "bestguess" guide. In no sense is a critical review of the entire lit
erature intended. This would obscure the main concepts in a
mass of detail. No attempt will be made to give a historic
perspective, or even to give credit to individuals where it is
certainly due. References will be to a limited set of very recent
articles which can provide the next level of understanding and
references to earlier work.
Fortunately, several of the most active groups have recently
summarized their efforts (9, 14, 16, 22). This author's views
1802
University, Princeton, New Jersey 08540
of a few years ago on the bacterial division cycle are sum
marized (12). The reviews furnish a guide to the literature
before 1966.
Bacterial Division
Most investigations of bacterial division have been carried
out with the closely related Gram-negative staining organisms
Escherichia coli and Salmonella typhimurium. These organisms
are implied unless otherwise stated. Gram-positive Bacillus
species have been used for some fundamental studies on chro
mosome replication and for morphologic investigations. It is
too early to say whether important differences of cell division
regulation exist between different bacteria. Major points of
bacterial division and chromosomal duplication are illustrated
schematically in Chart 1.
Bacteria increase their mass and cytoplasmic components
(total RNA and protein) approximately exponentially with
time if they are uncrowded and well nourished. A transverse
barrier or septum appears periodically at the middle of the
rod-shaped cell. Light and electron microscopy show that in
E. coli this is created by an inward-growing furrow of the cell
membrane which lies just inside the more rigid cell well. In
Gram-positive organisms the septum appears simultaneously
across the entire cytoplasm; wall material is formed on the
septum. Following this, the daughter cells separate. They are
nearly of equal size (coefficient of variation ± 10%) in a
constant environment. A compensation mechanism must re
store unusually sized cells to the average upon the following
division, since a negative correlation between the size of mother
and daughter cells is observed. The precise distribution of size
suggests a close relation between total cell mass and the timing
and spacing of septum formation. The actual separation of
the cells is less precisely timed (±20%), probably because of
randomness of the movements which shake the cells apart
(16).
Nuclear bodies can be observed by light microscopy with
staining or phase, or in the electron microscope. There are
often two or four to an organism, depending on nutrition. The
septum is formed between the central pair. These nuclear bodies
contain bacterial DNA. They are believed to be attached to
the cell membrane, or in the case of Bacillus to complex mem
brane structures named mesosomes (9). There is no evidence
of nuclear membranes or of any of the complex mitotic ap
paratus found in animal cells. In this connection, it should be
remembered that an entire bacterium is scarcely larger than
an animal mitochondrion (1 to 3 cu /*).
Freely growing bacteria divide as often as once every 40
min in a synthetic medium which contains a single well-utilized
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Models from Microorganisms
BACTERIAL
CYCLE
min = 40 min
Orni
/
Growth
Membrane
Replicase
Attachment
DNA
Origin
30
Chart 1. The bacterial
min
cycle, a schematic
20
representation.
carbon source such as glucose. They can divide twice as frer
quently in very well-supplemented media, or many times more
slowly with inferior carbon sources such as acetate. The cell
size and number of nuclear bodies decreases several-fold as the
medium becomes poorer. The main requirement for division
is neither a constant time nor a critical mass that is the same
under all conditions. A subtler control is suggested by the
differences in composition and the complex adjustments in
macromolecular syntheses that bacteria undergo when they are
transferred from one medium to another (16).
Bacteria, unlike most cells of higher animals, do not reach
a limit of division even in colonics on solid media. Bacteria
stop dividing only when they reach high concentrations in
liquid media. The cells become smaller when they reach this
terminal stage of their growth; they then resemble bacteria
which are growing on a poor carbon source. When they are
resuspended in fresh medium, they start growing again only
after a time lag. The basis for these changes, especially of
failure to divide, is not well understood, but in some instances
lack of oxygen or nutrients, or accumulation of toxic products
including hydrogen ions is responsible.
The Cycle of DNA Replication
Duplication of bacterial DNA must be coordinated with
cell division so that each daughter cell obtains a full comple
ment of hereditary material. Timing of DNA duplication during
the bacterial cycle is represented schematically in Chart 1;
the DNA is illustrated at about VLOOO
its relative length. One
anticipates some sort of coupling mechanism which permits the
cell division mechanism to sense the progress of DNA replica
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1968
min
tion, and vice versa. DNA replication should be, in part, con
trolled by the events of cell division. Cell components other
than DNA do not have to be so closely coordinated with cell
division; they exist in numerous copies and can be distributed
approximately equally by chance.
Bacterial DNA is found in the nuclear bodies, as shown for
example by radioautography of bacteria which have incor
porated thymidine-3H. In the resting state, each nuclear body
consists of a single molecule of double-stranded DNA of length
about 1.3 mm (about 1,000 times as long as the bacterium)
and molecular weight about 3 X 10*. Radioautographs of care
fully lysed E. coli show the DNA to be circular, at least part
of the time. These morphologic studies are completely sup
ported by genetic mapping which shows the E. coli chromo
some to carry all of its over 100 known genetic markers in a
single, circular order. Bacillus subtilis has a similar chromo
some; the evidence for a circular structure has so far been
found only in germinating spores (25).
A chromosome starts to replicate at a definite, heritable
origin. Replication is semiconservative, each of the two new
strands being base-paired by hydrogen bonds to an old strand
of the opposite polarity. Recent studies using density labeling
suggest that each new strand is covalently linked to the term
inus of an original strand (25). All three double strands re
main together at the origin, forming a Y-shaped fork which
opens toward the replication point. The replicase is thought to
be attached to the cell membrane; the old DNA moves into
this replication point and the replicas emerge, so that a second
Y-shaped fork completes a loop within the larger circular chro
mosome (see Chart 1). The DNA at the replication fork seems
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Arthur B. Pardee
to be more easily denatured than the bulk of the DXA (10).
As the chromosome moves through the replication point, genes
double in number one after the other in the order of their
sequence on the chromosome. Finally, when the end of the
chromosome is reached, a new round of replication commences
after some special events of initiation.
Evidence for sequential gene replication is of three main
sorts. First, the quantity of a given gene can be measured in
0. subtilis by transformation with free DNA, the number of
transformants being proportional to the number of genes (after
applying suitable controls) (22). Genes at the origin exist in
twice as many copies as genes at the terminus; intermediate
genes are present at intermediate concentrations in an unsynchronized culture,; owing to the random location of the Yshaped replication points. Second, in cultures undergoing syn
chronous replication, genes are shown by transformation and
density-labeling to double in a definite order (22). Third, as
each element on the chromosome is replicated, its maximum
ability (potential) for producing a corresponding enzyme (or
lysogenic virus) doubles; this can be observed as an increased
rate of enzyme (or virus) synthesis upon induction of a syn
chronously dividing bacterial culture (20). Furthermore, these
increases in potential depend on DNA replication (4). These
results indicate a definite direction of replication around a
circular chromosome, but it is not clear whether the direction
is the same in all substrains of an organism, nor indeed whether
the origin of replication is the same or different in all substrains. This question is stimulating some very active research.
DNA replication can also be initiated by bacterial conjuga
tion, in which DNA is transferred to a recipient bacterium (ß,
8). Here the origin and direction of replication are clearly
fixed in any one strain, but both origin and direction differ
from one strain to another. Bacteria with different conjugation
origins also show different timing of enzyme potential changes
(20).
The rate of DNA synthesis appears to double at about 20
min before division in synchronized cultures. This is attributed
to initiation at this time of a new round of chromosome dupli
cation, with a doubling of the number of replication points.
The rate of synthesis per growing point appears to be con
stant, as if the limiting factor were the rate at which DNA
could move past a single replicating enzyme (7). This result
and the constancy of rate of DNA synthesis in a variety of
media which permit fairly rapid growth suggest that neither
the supply of nutrients nor their rate of conversion to the
deoxynucleoside triphosphates is limiting under these condi
tions. However, several workers using other conditions of syn
chrony have found an exponentially increasing rate of DNA
synthesis, as if the supply of immediate precursors were in
creasing throughout the division cycle (12).
The time required for chromosomal replication depends on
nutrition, but to a smaller extent than does the time required
for cell division (14). The two events are not occurring in
parallel but are synchronized at division. DNA replication can
continue through the entire E. coli cycle when division times
are an hour or less. When the growth rate is slower (division
times of more than two hours), the time required for DNA
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replication increases, as if building blocks become limited. The
time of chromosome replication is not sufficiently long to
occupy the entire division cycle but takes place only during the
some part (12). Similar results have been obtained with B.
subtilis (18). In contrast to rapidly growing E. coli, and like
animal cells, synchronized cultures of Alkaligenes fecalis can be
made to synthesize DNA during only part of the division
cycle at rapid growth rates (13).
Partition of DNA between Daughter
Cells
The replication of bacterial DNA does not require cell divi
sion. Under many conditions bacteria grow into long filaments
when they fail to divide. These conditions include poor nutri
tion, Mg+ + deprivation, presence of toxic substances such as
penicillin or crystal violet, mutagens, inhibitors of DNA syn
thesis, or very mild irradiation with ultraviolet light or X-rays
(2, 12). Septa are not formed; their synthesis is more sensitive
than almost any other process in the bacteria. If DNA syn
thesis continues, the nuclear bodies are distributed along the
entire length of the filamentous cells in many cases. One con
cludes that DNA initiation, synthesis, and nuclear body for
mation require neither cell division nor septum formation. Also,
longitudinal membrane growth requires none of the events of
DXA synthesis and replication.
In spite of the ready dissociation of DNA synthesis from
cell division, DNA is precisely partitioned between daughter
cells under normal conditions. This is noted from the constancy
of both the quantity of DNA and the number of nuclear bodies
per cell in each medium (1C). Furthermore, old and new strands
of DNA are not passed on at random to the daughter cells but
in a definite order according to when they were synthesized
(5, 14). Episomes (nonchromosomal DNA molecules that carry
genetic information) are also partitioned in a definite way be
tween daughter cells, and their number per cell remains con
stant (9).
This separation of various DNA components, precise in time
antl quantity, cannot be arranged by chance, as with cytoplasmic contents. The most attractive model assumes that the
mechanism for segregation of genetic material at bacterial di
vision is very like the one observed with cells of higher or
ganisms, in which the chromosomes are physically attached to
a mitotic apparatus that separates them. There is no indication
of a mitotic apparatus in bacteria. However, bacterial mem
branes replace parts of animal cells for other functions such
as sites for oxidative phosphorylation. The DNA molecules
are visualized as being attached to the longitudinal bacterial
membrane (!)). The observed partitioning of DNA of various
ages can be accounted for by a model in which attachment of
a DNA molecule occurs at the time its replication commences
(5). This membrane elongates as the bacterium grows, thereby
separating the attachment points of the newly formed sister
chromosomes. These attachment points would have to serve
as loci around which the daughter DNA strands condense at
the time of septum formation in order for the long DNA
strands to be completely segregated by the short distance be
tween the attachment points. But if the origins of daughter
chromosomes are connected during DNA replication, which can
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occupy the entire division cycle, the points of attachment that
separate during this cycle cannot be at these origins. One can
imagine how one of the new replicas passes through a new
attachment site and remains with this site at completion, while
the other replica remains attached by the replicase (Chart 1).
Linking of DNA to membranes was originally suggested by
.studies of bacterial conjugation in which membrane contact
between bacteria appears to trigger DNA replication (6, 8),
Evidence for attachment of DNA to membranes has been ob
tained by electron microscopy of B. subtilis; membrane bodies
known as mesosomes appear to be the points of connection
(9). These might also be the growing points of DNA replica
tion, with the replicase enzyme holding DNA at the position
where it is being synthesized. As further evidence, presumed
DNA growing points are found in a membrane fraction of
disrupted bacteria.
In support of this spatial fixation, specific DNA strands are
often conserved throughout many generations at the extreme
ends of growing chains of B. subtilis cells (5). These same
regions conserve their membrane material (9). These two re
sults taken together suggest a firm union between DNA and
definite membrane sites. Conservation of membrane and DNA
in the same progeny cells has now been demonstrated (3).
The relatively exact partition of cell mass between daughter
cells (16) might also be explained by the growth character
istics of the longitudinal membrane. If the growing point of
this membrane is at the center of the cell and between two
points of DNA attachment, if growth is equally rapid toward
both ends from this point, and if septum formation occurs
at this point, the cell would divide equally.
Initiation
of DNA Replication
Biosynthesis of a macromolecule requires a special initiation
reaction in addition to the sequential attachment of building
blocks that make up the bulk of the synthesis. This is as true
of bacterial chromosome duplication as it is of RNA or pro
tein synthesis. Initiation appears to be that part of replication
where regulatory influences determine the timing of macromolecule synthesis and the quantity of completed macromole
cule. The subsequent synthesis, so much more prominent in
quantity and duration, proceeds relatively automatically (1C).
This concept is analogous to the regulation of small-molecule
synthetic pathways by end-product inhibition, where regula
tion of the initial step determines the others. Thus, the key
event in regulation of chromosome replication should be sought
in initiation. This must be studied with intact cells at present,
since DNA synthesis by extracts or purified enzymes is not
sufficiently physiologic to be significant to the problem.
The most significant finding is that protein synthesis must
occur before each initiation (16). In bacteria deprived of an
essential amino acid, the round of DNA replication in progress
is completed but a new round does not commence. Reinitiation
upon amino acid addition starts up DNA synthesis. This occurs
after different times in individual cells; probably cytoplasmic
events which occur prior to initiation were stopped at different
stages in individual bacteria. The different consequences of
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1968
amino acid starvation or adding chloramphenicol, 5-fluorouracil, or phenethyl alcohol suggest that DNA initiation re
quires synthesis of two proteins with different sensitivities to
these inhibitors; these might be new membrane attachment,
initiator, or replicator proteins (14).
General nutritional supply, measured by growth rate, has a
marked effect on initiation. When a culture reaches the end
of its growth, the chromosomes complete their replication but
do not initiate the next round. Slowly growing, poorly nour
ished bacteria complete a round of DNA replication and then
there is a delay before the next round is initiated. In succinate
medium alternate replications of the two chromosomes in one
E. coli cell have been reported (14). At intermediate growth
rates each replication of a chromosome is initiated very soon
after the previous one is completed; DNA replication appears
continuous. In very rich medium, a dichotomous replication
occurs in B. subtilis: about half way through the first round
a second round of replications commences at both origins, and
the growing chromosome has three forks and thus four copies
of each genetic locus near the origin (22).
Studies with specific inhibitors have indicated that proteins
are required for initiation, as mentioned above. Phenethyl al
cohol blocks initiation at the same point as does amino acid
starvation (14). This inhibitor seems to act by increasing per
meability of the bacterial membrane (21), again suggesting a
connection between membrane and DNA replication. Acridine
dyes appear selectively to inhibit replication of episomes rela
tively more than chromosomal replication. These dyes "cure"
the bacteria of their episomes (6).
Chemical activators of DNA initiation have not been reported,
except for the influence of rich medium in initiation of dichoto
mous replication. However, bacteria whose DNA synthesis is
blocked by thymine starvation initiate a new round of DNA
synthesis at only one of the two potential new origins when
thymine is restored. This differs from initiation at both origins
following amino acid starvation. Initiation in thymine-starved
bacteria is inhibited by chloramphenicol or 5-fluorouracil and,
therefore, seems to require RNA-dependent protein synthesis.
Cytosine arabinoside, which inhibits DNA synthesis, does not
cause premature initiation, suggesting that some metabolic im
balance of thymine-deprived cells initiates, perhaps by inducing
the essential protein (14). These effects strongly hint at some
process similar to enzyme induction-repression in the DNA
initiation event.
The Replicón Hypothesis
The most plausible current model of DNA initiation is based
on the represser-operator model for regulation of protein syn
thesis through messenger RNA synthesis (8). An entire DNA
molecule (chromosome or episomal element) is considered to
be a unit of DNA replication. This is named a "replicón." Initi
ator and replicator genes on the replicón are thought to be in
volved in initiation by analogy with the represser and operator
genes for regulation of enzyme synthesis on an operon. The
initiator gene carries the information for structure of an initi
ator protein which enters the cytoplasm after it is synthesized.
When the replicator gene receives this initiator protein, DNA
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Arthur B. Pardee
synthesis is initiated and is propagated down the chromosome.
The effect of the initiator is positive in the sense that it is
required for starting DNA synthesis.
The principal evidence for the replicónhypothesis is obtained
with mutant episomes defective in DNA replication (8). Nor
mal episomes replicate independently of chromosomes, yet in
rhythm with cell division. The mutant episomes replicate more
slowly than the cell divides if the temperature is raised from
30°Cto 42°C.The bacteria are not killed because all of their
essential genes, including those required for chromosome repli
cation, are on the chromosome and are heat-stable. But loss of
the episome can be detected by loss of the genes it alone carries.
Thus, when an episome carrying the yS-galactosidase gene was
lost from a host with a lac~ chromosome, lactose-negative bac
terial colonies were easily identified on selective agar plates.
With such a system, conditions affecting replication of episomes
could be investigated readily.
The main conclusion was that initiation of DNA replication
requires the synthesis of a replicon-specific cytoplasmic protein.
Involvement of a protein was inferred from the sharp heat
lability of replication (characteristic of protein denaturation).
This protein has a positive role, since DNA replication stops
in its absence. (By contrast, destruction of a heat-labile repressor permits enzyme formation.) That the protein is cyto
plasmic was indicated by cooperation between an episome with
a heat-stable, replication-controlling system and a heat-labile
episome within the same bacterium. Specificity is shown by the
inhibition at increased temperature of episomal but not chro
mosomal replication in the bacteria with a heat-labile episomal
replicón.
Observations on episomes and injected DNA fragments sup
port the idea that DNA units must carry special structures in
order to replicate independently. Episomes are thought to repli
cate under the control of their own replicator and structural
genes. Their transfer from one bacterium to another by con
jugation requires DNA replication which is controlled by these
genes, also called sex factors (6).
In contrast to episomes, chromosome segments injected by
high-frequency recombination donor bacteria usually cannot
replicate unless they are integrated into the recipient chromo
some by genetic recombination. These segments cannot serve
as templates for DNA synthesis in the same cell where the com
plete chromosome is replicating. They appear to lack the repli
cator gene which is at the terminal end of a replicón (6).
The establishment of lysogenic phages as prophages in their
bacterial hosts and subsequent harmonious replication requires
inhibition of the phages' autonomous replication apparatus.
This inhibition in lysogenized bacteria creates immunity to
superinfecting phages of the same strain. However, when superinfection is by related phage of a different immune type, both
the superinfecting and the lysogenic phages replicate autono
mously. The explanation is similar to the replicónmodel : a posi
tively acting initiator substance is required for autonomous
phage replication; production of this initiator is inhibited in
the lysogenized situation; and the superinfecting phage pro
duces initiator that is used by both phages in the same cell
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(23). Lysogenized phage also start autonomous replication after
exposure to "induction" treatments such as irradiation.
Other results suggest very definitely that episome replication
depends on some property of the host, as would be expected
from its replication in rhythm with host division. The number
of episomes that a bacterium can carry is limited, which sug
gests a competition between episomes for sites in the cell. Mu
tations of the bacterial host chromosome, as well as episomal
mutations, can cause the higher-temperature
elimination of
episomes (8).
The replicónhypothesis is the most plausible and useful one,
at present, because it suggests experiments to test it. It presents
major questions: What chemical change occurs upon initiation?
Is it a change in DNA, such as local denaturation (10), or a
scission of covalent bonds which connect terminal and original
DNA strands (25) ? Or is it the combination of an enzyme or
membrane protein with a starting site? Is the protein which
is required for initiation (16) the product of the initiator struc
tural gene? Is the initiator protein identical to the membrane
attachment site?
Cytoplasmic
Coinitiators
According to the replicón hypothesis, the initiator protein
made during one round of DNA replication activates the next
round. One can imagine a short burst of initiator synthesis,
perhaps created by duplication of the initiator gene. The entire
chromosome, including the initiator gene, is thought to repli
cate in the absence of protein synthesis when the bacteria are
starved of amino acids. The initiator gene in its replicated form
might produce initiator only after amino acids are again sup
plied. This could account for the source of protein required for
initiation.
This simple model runs into several problems. The prema
ture, dichotomous initiation that occurs in very rich medium,
and also the long delay between termination and the next initi
ation observed in poor medium strongly suggest specific nu
tritional requirements for initiation. The coordination of dupli
cation in each cell cycle of all of the replicons in a cell suggests
some common cytoplasmic factor that interacts with all of the
replicon-specific initiator proteins at the same time. Also, a
general cytoplasmic change, the degradation of 10% of the cell
proteins, has been noted at the time of DNA replication (19).
Still consistent with the replicón hypothesis would be a
replicon-specific initiator protein whose function or synthesis
is activated periodically by a low molecular weight compound.
The concentration of this compound, which we will name a coinitiator, could change during the cell cycle in a manner that
depends on both chromosome replication and nutrition.
The only molecules so far proposed as co-initiators are DNA
precursors (13). The concentrations in bacteria of purine
deoxynucleotides are low except when purine deoxynucleosides
are supplied in the medium, or upon thymine starvation. Pyrimidine deoxynucleotides are found, but in amounts (3% of
DNA) insufficient to suggest that DNA synthesis is initiated
by their availability as precursors. However, their role as coinitiators is possible because their concentration rises a few
minutes before stepwise DNA synthesis commences in synchro-
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nized A. fecalis, as would be predicted. Periodic deoxyribonucleotide synthesis is still observed when DNA synchrony is
destroyed by adding excess of all four deoxynucleosides (13).
The pool levels are therefore not determined by DNA synthesis,
and DNA initiation does not depend solely on variations of
these pools. The requirement of all four deoxynucleosides sug
gests that the control of initiation might be dependent on a
balance or cooperation effect between these compounds, similar
to multivalent repression of enzyme synthesis. Although no di
rect evidence is available regarding the chemical nature of coinducers, deoxy-compounds, or others, the data on nutritional
effects and pools appear sufficient to retain this concept in the
scheme of DNA initiation.
Co-initiators would have to rise and fall during the replication
cycle in order to be effective periodic triggers of DNA initi
ation. One often-suggested basis for these changes depends on
the cells reaching a critical mass at the time of replication (this
mass depending on nutrition). The concentrations of some intracellular metabolite could change as the cell grows because
the mass increases more rapidly than the surface (more so in
a spherical cell tlian in a rod-shaped one). If the rate of syn
thesis of cell envelope precursors was proportional to the mass
and their rate of use was proportional to the surface, their
concentration could increase through the cell cycle and trigger
replication at a critical level. Other evidence regarding a con
nection between cell envelope and DNA replication will be
discussed in the next section.
A different basis for rising and falling pools of co-initiator
depends on periodic enzyme synthesis during the division cycle.
The doubling of a cell's potential (maximal ability) to form an
enzyme as the corresponding gene is replicated can be modified
by enzyme induction, repression, and inactivation. Based on
this, models for self-generating (autogenous) cycles of enzyme
synthesis have been suggested (18, 20); this predicts that en
zyme oscillations could in turn create marked metabolite oscil
lations with periods equal to the DNA replication cycle. In this
way, co-initiator concentrations could periodically reach a maxi
mum, the timing depending on nutritional effects on repression
mechanisms and also on the replication of a structural or regu
lator gene once per DNA replication.
Timing of Septum Formation
The periodic formation of cross walls is an essential prelude
to separation of daughter cells and their DNA. What mecha
nism initiates septum formation periodically in time?
Cell division follows DNA completion by 20 min in a variety
of media (7). This close coordination of DNA replication and
cell division suggests that completion of DNA replication might
trigger septum formation (15). This is supported by many
observations that, when DNA synthesis stops (because of in
hibitors, irradiation, or thymine starvation), septum formation
and cell division also stop. Filamentous bacteria are formed.
The spatial separation of two nuclear bodies upon completion
of DNA replication might in some way trigger septum forma
tion. The requirement of two nuclear bodies has been suggested
on the basis of electron micrographs which show that the sep
tum is between them (12). It has also been invoked to explain
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1968
the one-generation delay in increased rate of cell division follow
ing a nutritional shift-up (15). An exception to this pattern is
found with a newly described mutant in which cross walls create
"minicells" containing about one-tenth the normal amount of
cytoplasm and no DNA (1). Apparently the positioning of
septa is deranged; nuclear bodies do not have to exist on both
sides of the division point.
The coupling between DNA and septum formation is not a
tight one, since it can be perturbed by various nutritional con
ditions, inhibitors, and mutations. For instance, rapidly growing
cells have more than one nuclear body per cell; that is, septa
are formed regularly but less often between nuclear body di
visions. The observations on uncoupling of DNA synthesis from
cell division fall into a similar pattern to those described above
for uncoupling initiation of DNA replication, and suggest a
similar hypothesis. Perhaps DNA replication initiates septum
formation by creating some cytoplasmic change upon its own
completion. Transmission of this impulse could depend on
cytoplasmic conditions for its effectiveness in starting cell
division.
A relation between DNA and septum formation can be studied
using lon~ mutants (2). These mutants fail to form septa after
very mild ultraviolet- or X-irradiation. Irradiation does not
appear to act directly on the septum-forming membrane region,
although it is quite specific in not noticeably altering DNA
synthesis or other metabolism. The nucleic acid-type action
spectrum and the higher sensitivity of these mutants when they
also lack a DNA-repair mechanism suggest that DNA is the
target. This is shown clearly when bacterial division is made
more sensitive by incorporation of 5-bromouracil into DNA
(24). A transient damage to DNA in these mutants seems to
prevent septum formation for a half-dozen generations, where
upon the filaments lyse.
The lon~ mutants also provide a clue to cytoplasmic events
involved in initiation of septum formation. The damage in
irradiated mutants can be reversed by shifting the bacteria to
poorer media; some compounds in the rich medium seem to
prevent repair of damaged septum formation. Increased tem
perature or pantoyl lactone can also reverse the inhibition. In
jection of an episomal lon+ gene into irradiated lon~ bacteria
repairs the defect, indicating a dominant irons effect of lon+
(24). Most interesting, lon+ bacteria release substances into
the medium which help irradiated lon~ bacteria to recover.
There are at least two of these two factors; they can be ex
tracted from lon+ cells (2). All of these results suggest that
irradiation of lon~ DNA causes an imbalance of metabolism
which prevents septum formation. Depending on the genetic
factor Ion and nutrition, the original balance is restored or the
imbalance perpetuates itself, resulting in filament formation
and eventual lysis.
A clue to the kinds of metabolites that might be involved is
gained from the observation that lon~ bacteria are usually mucoid; that is, they overproduce cell wall components (17). Possi
bly cell envelope precursors which are in excess in the Ionbacteria inhibit the recovery of the irradiated cells (24). Further
indications that cell wall metabolism plays a role in septum
formation is gained from observations that inhibitors of cell
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Arthur B. Pardee
wall synthesis, such as penicillin or crystal violet, cause filament
formation. Septa also do not form in spheroplasts, which are
bacteria in hypertonic, osmotically protecting medium whose
cell wall has been partly dissolved by lysozyme or malformed
by exposure to penicillin.
Perhaps the most profitable working hypothesis is that peri
odic DNA completion in turn drives an oscillation of metabolites
in the cytoplasm which triggers septum initiation. Alternatively,
the attachment of DNA to the longitudinal bacterial membrane
might, upon completion, transmit a signal from one of the replicons directly via the cell membrane to the septum forming site.
Summation and Prospect
The main concepts regarding bacterial division are the follow
ing. Bacteria divide periodically at intervals precisely deter
mined by their environment as well as their genetic composition.
Unlike animal cells, intracellular or intercellular controls do not
cause division to stop. Cell division is in coordination with DNA
replication so that the genetic material is exactly partitioned
between daughter cells. The spatial separation of daughter
chromosomes appears to depend on their connection to the
longitudinal bacterial membrane. Growth of this membrane
might move apart the DNA attachment points, and its extent
of growth might be a regulating factor. Formation of an inter
vening septum can account for partition of genetic material
between daughter cells.
Although DNA synthesis appears to be continuous through
the cell cycle under some conditions, it is a periodic event whose
end can be separated in time from the beginning of its next
round. The single bacterial chromosome is conceived of as a
unit of replication which has been named replicón. A special
initiation event which requires protein synthesis starts each
cycle of replicónsynthesis, which then continues automatically
down the chromosome. Each replicón is proposed to contain
genetic elements that create a positive feedback loop, which
together with oscillations of cytoplasmic co-initiators is sug
gested to control the timing of DNA initiation.
DNA initiation does not depend on cell division. Rather, the
timing of cell division depends on the DNA cycle. Septum for
mation, which is the first morphologic step in cell division, might
be initiated by a signal from the completion step of DNA repli
cation, which probably occurs on the membrane. Transmission
of this signal is modified by nutrition and inhibitors, as is the
signal for DNA initiation. Events initiated by the DNA cycle
indirectly trigger both a new round of DNA replication and
initiation of cell division via septum formation.
Many parallels can be found between those observations and
the mass of information regarding division of animal cells (13).
The fundamental difference, aside from cell structures, seems
to lie in a regulation by which the impulse that initiates DNA
replication is blocked in mature, normal animal cells. The in
hibition seems to originate mainly from contact with neighbor
ing cells. Its absence in malignant cells suggests both a role of
cell membrane in regulation, similarly to bacteria, and differ
ences in cell membranes of normal and cancer cells.
A major step in originating and developing any hypothesis
is to collect and analyze the available information. For analysis,
data must be divided and subdivided into a scheme where re
1808
lated facts are brought into suggestive arrangements. It is hoped
that this organization of the simpler, more readily perceived
phenomena of bacterial division will serve initially to guide
some who work with more complex systems.
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Control of Cell Division: Models from Microorganisms
Arthur B. Pardee
Cancer Res 1968;28:1802-1809.
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