SPECIAL ARTICLE
704
THE
DEATH
OF
BACTERIA
AS
A
FUNCTION
OF
UNBALANCED
GROWTH
By Seymour S. Cohen,* and Hazel D. Barner
H
IGHER organisms and microorganismssuch as bacteria may be distinguished
in many important respects. Among these
are significant differences in the relations
of cell growth and division to species
sun-vival, relations which may be expected to
bear on problems of chemotherapy.
In the life of bacteria, each cell is a unit
whose survival depends on its own ability
to multiply. Although bacteria may
main-tam their cellular integrity for considerable
periods without multiplication, a bacterium
which has lost the ability to multiply has
lost its potential for continuing survival.
Bacteria which have lost the power to
re-produce are commonly called “dead”;
agents which destroy their ability to form
colonies are termed “bactericidal.”
In the life of higher organisms, an
mdi-vidual contains numerous types of
differen-tiated cells, many of which have
perma-nently lost the ability to multiply. A nerve
cell, for example, has developed its
cyto-plasmic structure and function in such a
way that the multiplication of nerve cells
in the adult animal would be detrimental
to the specialized function of nervous tissue.
The cessation of multiplication in some of
the cells in higher forms of life permits the
fuffillment of their key function and
there-by facilitates the maintenance on
reproduc-tion of other cells. The survival of the
species in these cases involves a subtle
in-tercellular balance of growth and
reproduc-tion among many cells.
From the Children’s Hospital of Philadelphia and the Departments of Pediatrics and Biochemistry of the University of Pennsylvania School of Medi-cine.
Aided by a grant from the Commonwealth Fund.
Presented before the Centennial Medical Convo-cation of the Children’s Hospital of Philadelphia,
June 2-4, 1955.
o ADDRESS: 1740 Bainbridge Street, Philadelphia
46, Pennsylvania.
The exigencies of survival, derived from
these relations of growth and
multiplica-tion, are also reflected in the intracellular
economy of bacteria and of higher
organ-isms. It has been found in the last few years
that many bacteria are unable to degrade
the polymers which they may have
synthe-sized.1’ 2 The synthesis of protein and nibose
nucleic acid (RNA) in the cytoplasm of
such cells is “growth”; the increase in
bac-terial cytoplasm is normally matched by an
increase in nuclear substance. In the life
cycle of bacteria, the irreversible increase
in cell mass is compensated by cell division.
In the survival of these forms, growth and
division are balanced phenomena following
the irreversible synthesis of cytoplasnric
and nuclear polymers.
In the evolution of higher organisms,
soy-eral developments have severed the
connec-tions between cell growth and cell division.
As mentioned above, an increase in cell
size is functionally desirable in the nerve
cell and the growth of the cytoplasm of this
cell is not matched by a growth of nuclear
substance to be followed by cell division.
On the other hand in the cells of certain
organs such as liven, cytoplasmic growth is
relatively limited in volume although
ac-tual synthesis is fully as extensive. This is
made possible in several ways. Some
syn-thesized polymers may be released from
the cell, e.g., serum proteins. Other cell
polymers such as RNA may be degraded
while new RNA is elaborated, resulting in
“turnover,” a phenomenon not yet observed
in bacteria. Thus in the cells of higher
on-ganisms polymer synthesis does not always
involve cell growth. In higher organisms
the separation of synthesis, growth and
di-vision is essential to cellular differentiation
and in these organisms this separation may
be expected to contribute to the
406080
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Fic. 1. Structural relations of thymine and
related compounds.
bacteria the rigid relationship of synthesis,
growth of nucleus and cytoplasm and cell
division may be expected to reduce the
adaptability of these organisms. We may
inquire, for example, concerning the
conse-quences of inhibiting the nuclear or cell
division of bacteria under conditions of
con-tinuing polymer synthesis. We may
won-der whether the successful chemotherapy
of microbial infections in man has not
mad-vertently taken advantage of these
funda-mental differences between animals and
microorganism.
More concretely, we shall now explore
some of the results of dissociating the
nor-mally balanced relations of cytoplasmic and
nuclear syntheses in bacteria. As will be
seen below, the upsetting of the normal
balance of these relations does lead to the
death of bacteria.
INVESTIGATIONS AND RESULTS
For the past 2 years, we have studied
the physiology and survival of a mutant
strain of E. coli, 15T, which requires
thy-mine for multiplication. Thymine is a
pyri-midine (Fig. 1) and is a component of a
polymeric deoxynibosenucleic acid (DNA),
which is found only in the nucleus. As is
well known, DNA is an essential component
of chromosomes and is believed to be of
critical importance in the transmission of
genetic characteristics. Mitotic division in
higher cells is preceded by a doubling of
DNA and half of the total DNA is then
transmitted to each daughter of the
divid-ing cell. Growing bacteria deprived of
thy-mine may well be expected to develop
flaws of nuclear synthesis which should
seriously affect the ability of the bacteria
to divide.
It was found that when E. coli ‘ST was
incubated in a synthetic medium
contain-ing glucose, nitrogen and phosphate but
lacking in thymine, the bacteria rapidly
lost the power to form colonies when plated
in the presence of thymine.l A typical
ex-periment is presented in Figure 2. If
glu-cose or other metabolites were omitted
from the medium, the bacteria remained
viable; they must metabolize and grow in
order to die.
During growth in the absence of thymmne,
the bacteria at least doubled their protein
and RNA content and enlarged in size to
about 5 times their normal volume.4 Cells
in this condition excreted
ultraviolet-ab-sorbing substances which proved to be a
mixture of intermediates of nucleic acid
metabolism. It was found that the
nynimi-dine, uracil, was the major excretion
prod-uct of such cells and it was demonstrated
by means of experiments with radioactive
glucose that the uracil was synthesized
dur-ing growth in the absence of thymine.4 It
was further shown in this manner that the
mutant is unable to make more than 4 per
cent of its normal thymine requirement.
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COHEN DEATH OF BACTERIA DUE TO UNBALANCED GROWTH
The major block in this mutant appears to
be in the methylation of uracil on of a
una-cil derivative. Methyl groups may be made
normally because the organism does not
have a methionine requirement. Indeed
the synthesis of methyl groups is revealed
in a dramatic manner by the synthesis of a
new methylated amino purine during
growth in the absence of thymmne.
Despite the absence of thymine,
synthe-sis of DNA proceeds to the extent of about
20 per cent of that already present.
Accord-ing to Dunn and Smith,5 the new DNA
con-tains N-methyl adenine (methylamino
pu-nine) in place of thymine. The structures of
the relevant bases are presented in Figure
1. It would appear that the methyl group
normally affixed to uracil is transferred
in-stead to adenine.
It may be supposed that it is the
synthe-sis of an abnormal DNA containing
methyl-amino purine instead of thymine, which is
responsible for cell death. Some arguments
may be marshalled against this hypothesis.
First, cells which have irreversibly lost the
power to form colonies to the extent of 99
per cent of the cell population are
neverthe-less still able to make DNA when given
thymine. Thus the synthesis of the new
type of methylamino purine DNA does not
prevent the synthesis of thymine DNA.
Some other factors would seem to prevent
the resumption of normal division. Second,
from Figure 2 it can be seen that a lag of 30
minutes ensues before death begins in the
absence of thymine. Some DNA synthesis,
presumably of the new methylamino pu.
rine type, occurs in this interval without the
death of a significant number of cells. It
has been shown that if thymine is added
at this point, just before death begins, the
cells are all capable of dividing.
Indeed when division is begun in this
way, it is found that division is
synchno-nized in the entire culture.6 In the absence
of thymine, the cells have piled up at the
same stage in a multiplication cycle. On
ad-dition of thymine at 30 minutes, there is an
additional lag of 25 minutes in which time
DNA slightly more than doubles. This
syn-thesis comes to a halt and the cells proceed
to divide rapidly. Sometimes 2 divisions
oc-cur without a detectable lag between them
but after the second division the cells
con-tinue to divide synchronously. It therefore
appears that, as in higher cells, the doubling
of DNA precedes division.
By this technique one may distinguish
several phases within a single division cycle
and it is possible to obtain mass cultures of
bacteria, all in the same phase of the cycle.
We have exploited this situation by
study-ing the multiplication of T2 bacteriophage
in bacteria at different stages of DNA
syn-thesis and division. We have found with
this system that the major parameters of
the 1 step growth experiment (the latent
period, rise period, and burst size) were
essentially independent of the stage of
divi-sion of the bacteria.6
We have tentatively concluded that the
death of the mutant is in some manner a
manifestation of the lack of balance of
con-tinuing cytoplasmic growth and incomplete
nuclear synthesis. More specifically 2
hy-potheses may be offered. Firstly, the
syn-thesis of cytoplasmic structures has formed
a framework within which the nucleus
can-not operate when given thymine essential
for DNA synthesis. Or secondly, nuclear
di-vision has proceeded with a stock of DNA
inadequate for maintaining nuclear
func-tion and incapable of setting cell division
into play. Either situation may be supposed
to have provoked death by unbalanced
growth.
Such a mutant, which dies in the absence
of one essential requirement, is an unusual
organism. Most mutant isolation techniques
in common use at the present time are
in-capable of isolating microorganisms
pos-sessing such a lethal characteristic and it
may be asked if there are not many mutants
which die in this manner, before isolation
is possible. Most single requirements are
essential to both nuclear and cytoplasmic
syntheses and auxotrophs lacking these
re-quirements simply do not grow. Other
thy-mine or thymidine requiring bacteria, e.g.,
I-.
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2 HOURS
TABLE I
PROPERTIES OF BACTERIA UNDER CONDITIONS PRODUCING I)EATII
Agent Cell Size RNA
Synthesis
DNS
Synthesis
Accumulated
Products in Medium
Requiremenl for
Bacterial
Participation
Thymine-less death4
enlarged active inhibited uracil, orotic acid,
hy-poxanthine
must metabolize and grow
Ultraviolet irradiation7
enlarged or filamentous
active inhibited deoxyribonucleotides, thymidylic acid
must metabolize and grow
Nitrogen mustard
filamentous active inhibited ? ?
Penicillin filamentous somewhat
inhibited
? uracil nucleotides must metabolize
and grow
and under conditions of nutritional
defi-ciency the additional unsatisfied
require-ments may be expected to prevent
un-balanced growth and to permit cell
sur-vival.
We have shown that sulfanilamide, a
FIG. 3. The induction of thymine deficiency in E. coli, strain B, by growth in sulfanilamide and a number of metabolits, including amino acids, purines, vitamins, and thymine. Death is a
conse-ltlence of omitting thymine from this medium.
compound usually called “bacteriostatic,”
may under special conditions be used to
impose a thymine deficiency in strains of
E. coli other than ‘5T and to thereby
pro-yoke death by unbalanced growth.
Sulfanil-amide is a competitive antimetabolite to
p-aminobenzoic acid and therefore prevents
the synthesis of folic acid, which is present
in coenzymes essential to the transport of
one carbon fragment. These fragments
form parts of certain amino acids, e.g.,
methionine, serine, histidine, parts of
pu-rines, and parts of vitamins, e.g.,
pan-thotenic acid and the methyl group of
thymine. E. coli grown in the presence of
sulfonamide drugs therefore develop
multi-pie nutritional requirements and growth
stops in the absence of the appropriate
amino acids, purines, vitamins, etc. Growth
and division may be restored by supplying
a mixture of the metabolites indicated, as
in Figure 3. If the purines on amino acids
are omitted growth of the culture comes to
a halt. If these are supplied to support
growth while thymine alone is omitted the
cells die. Thus thymine-less death can be
induced in cells other than our unusual
mu-tant.#{176}
0 It appears possible that antifolic agents, e.g.,
Amethopterin#{174}, might be induced to be more
DEATH OF BACTERIA DUE TO UNBALANCED GROWTH
We have raised the question whether
various treatments which kill bacteria
(ul-traviolet radiation, nitrogen mustard and
penicillin) do not do so by inducing
Un-balanced growth. In Table I are
summar-ized the effect of such agents as compared
with the major characteristics of
thymine-less death. It will be noted that a
consider-able similarity exists among all of these in
that death is a concomitant of cell
synthe-sis under conditions of a profound
disturb-ance in nucleic acid metabolism. In the
case of penicillin, it has been shown that
death will not occur if growth is prevented.
A number of the methods of treatment
cur-rently employed to kill actively
multiply-ing tumor cells, (antifolic acid agents,
ra-diation, nitrogen mustards) induce effects
with mono than a superficial similarity to
the death from unbalanced growth
im-posed by thymine deficiency.
We have made a careful comparison
be-tween the characteristics of death in E. coli
‘ST- induced by low doses of ultraviolet
radiation and by thymine deficiency. It has
been shown that an irreversible killing of
the bacteria is equally dependent, in the
2 instances, on continuing cell syntheses.4’
The death of irradiated cells does not
oc-cur in the absence of sources of carbon and
nitrogen and continuing protein synthesis.
If these syntheses are prevented, as for
ex-ample by the inhibition of growth by means
of the amino acid antimetabolite, 5-methyl
tryptophan, various repair mechanisms can
come into play to restore and conserve the
viability of irradiated bacteria. It appears,
therefore, that the major type of killing
ob-served as a result of low doses of ultraviolet
irradiation, in the strains examined, may
indeed be termed death by unbalanced
growth. It appears that in this case, as in
thymine deficiency, the synthesis of
flu-clear DNA is inhibited, although the 2
positions of the metabolic chain, at which
interruption of the formation of the
essen-tial polymer occurs, are different in the 2
instances.
SUM MARY
Studies have been described with a
thy-mine-requiring strain of E. coli. This
or-ganism dies in the absence of its
require-ment. It has been shown that nuclear and
cytoplasmic syntheses are no longer
bal-anced under conditions of thymine
defici-ency and continuing unbalanced synthesis
results in the loss of the power to multiply.
Thymine deficiency and death of other
bac-tonal strains may be provoked by
sulfanil-amide in the presence of certain
metabol-ites which then support unbalanced
growth. The killing action of low doses of
ultraviolet irradiation also appears to
de-pend on growth and may be prevented by
inhibiting protein synthesis. It was shown
that many bactericidal treatments are
sim-ilar in that they appear to affect nucleic
acid metabolism and to require continuing
growth for their lethal actions to be
mani-fest. A selective inhibition of DNA
synthe-sis appears to be capable of inducing death
by unbalanced growth.
REFERENCES
1. Manson, L. A. : The metabolism of
ribo-nucleic acid in normal and bacteriophage-infected Escherichia coli.
J.
Bact., 66:703, 1953.2. Hogness, D. S., Cohn, M., and Monod,
J.:
Studies on the induced synthesis of beta
galactosidase in Escherichia coli; the
kinetics and mechanism of sulfur
incor-poration. Biochim. Biophys. Acta, 16: 99, 1955.
3. Barner, H. D., and Cohen, S. S. : The
in-duction of thymine synthesis by T2
infec-tion of a thymmne-requiring mutant of
Escherichia coli.
J.
Bact., 68:80, 1954.4. Cohen, S. S., and Barner, H. D. : Studies on
unbalanced growth in Escherichia coli.
Proc. Nat. Acad. Sc., 40:885, 1954.
5. Dunn, D. B., and Smith,
J.
D. : Occurrenceof a new base in the deoxyribonucleic
acid of a strain of bacterium coli. Nature,
175:336, 1955.
6. Bamer, H. D., and Cohen, S. S.:
Synchroni-zation of division of a thymineless mutant
of Escherichia coli. Federation Proc., 14:
177, 1955.
