helmstetter 1967

J. Mol. Biol. (1967) 24, 417427
Rate of DNA
Synthesis during the Division
of Escherichia coli B/r
Cycle
CEURLES E. HELMSTETTER
Roswell Park Memorial Institute
Buffalo, New York, U.S.A.
(Received4 October1966)
The rate of DNA synthesis during the division cycle of Eacherichia coli B/r has
been measured in exponentially
growing cultures. This was accomplished
by
pulse-labeling
exponential
phase cultures with [14C]thymidine
and measuring
the amount of label incorporated
into cells of different ages in the cultures. The
amount of label in cells of different ages was found by binding the bacteria to the
surface of a membrane filter at the end of the labeling period and counting the
radioactivity
in the cells which were eluted continuously
from the membrane.
Since primarily
new-born
cells are eluted from a membrane-bound
culture of
E. coli B/r, the radioactivity
per effluent cell during each generation of elution
was a measure of the amount of [14C]thymidine
incorporated
into the oldest
through the youngest cells in the exponential
phase culture. This experimental
approach was considered to be superior to those employing synchronized
cells
for these studies, because all manipulations
were carried out after, rather than
before, the incorporation
took place.
It was found that there is an abrupt increase in the rate of DNA synthesis
during the division cycle of cells growing in glucose, glycerol, suocinate or acetate
minimal medium which occurs at a cell age, in fractions of a generation, of 0.4,
0.8, O-9 and 0.9, respectively. In succinate- and acetate-grown
cells, this increase
is preceded by a pronounced minimum in the rate of [14C]thymidine
incorporation during the last half of the division cycle. It is suggested that the period of
reduced thymidine incorporation
corresponds to a gap between rounds of DNA
replication,
and that the abrupt increase corresponds to the start of a new round
of DNA replication.
1. Introduction
Synchronously
dividing
populations
of Esch&chia
coli have frequently
been used to
study the relationship
between DNA synthesis and the division cycle of this organism.
However,
serious reservations
concerning
this approach
have been expressed;
the
principal
one being that the procedures
used to obtain synchrony
might alter the
pattern of DNA synthesis (Schaechter,
Bentzon
& Maalee, 1959; Abbo t Pardee,
1960; Maalee, 1962). These reservations
appear well justified
in view of the conflicting data which have been reported.
In early studies on synchronized
cultures of
E. coli in glucose-salts
medium, DNA synthesis was found to occur during only a
portion of the division cycle (Barner & Cohen, 1956; Maruyama,
1956; DeLamater,
1956). However,
in more recent experiments,
in which synchronization
procedures
that were considered to have less effect on cellular metabolism
were used, essentially
continuous
synthesis was found (Abbo & Pardee, 1960; Nagata, 1963; Cummings,
1965; Clark & Maalee, 1967). Abbo & Pardee (1960) presented evidence that the rate
of DNA synthesis increases exponentially
during the division cycle of synchronously
417
418
C. E.
HELMSTETTER
dividing E. coli B growing in glucose-salts
medium. They obtained the synchronous
population
by a filtration
procedure
which was designed to collect the smallest
cells from an exponential
phase culture. More recently, Clark & Maalae (1967) found
that the rate of DNA synthesis doubles in the middle of the division cycle during
synchronous
growth
of E. coli B/r in glucose medium.
In their experiments
synchronous growth was obtained by the membrane-selection
technique
of Helmstetter
& Cummings (1964), which selects the youngest cells from a growing culture.
In this report an alternative
method for studying the cell cycle with the membraneselection technique
is described. This alternative
approach is based on the idea that,
since the effect of any synchronization
treatment on cellular metabolism
is uncertain, it
would be preferable to study the kinetics of DNA synthesis in untreated,
exponentialphase cells. This could be accomplished
in theory by pulse-labeling
an exponential
phase culture with radioactive
thymidine
and measuring
the incorporation
of t,he
label into cells of different ages. In order to perform such an experiment,
a method
for sorting out cells of different ages would be required.
We have used the membraneselection technique for this purpose. In brief, the rate of DNA synthesis was measured
as a function
of cell age by: (1) pulse-labeling
an exponentially
growing
culture of
E. coli B/r with radioactive
thymidine,
(2) binding the labeled cells to a membrane
filter,
and
continuously
(3)
measuring
from
the
the
membrane
radioactivity
in
as described
the
in
new-born
a previous
cells
report
which
were
(Helmstetter
eluted
$
Cummings,
1964). Thus, the incorporation
of radioactive
thymidine
into cells of
different ages in the exponential-phase
culture was determined
by measurement
of
the radioactivity
in their progeny. Although
the utility of this approach for studying
the cell cycle depends upon the accuracy with which new-born
cells can be identified
with their parents, the advantage of the method over synchronous
growth studies is
that the uncertainty
as to the physiological
state of the cells at the time of labeling
has been eliminated.
2. Materials
(a) Bacteria
and Methods
and growth conditions
The organism
used was E. coli B/r (ATCC
12407).
The minimal
salts medium
contained
2 g NH,Cl,
6 g Na2HP04,
3 g KHzP04,
3 g NaCl
and 0.25 g MgSO,
in 1 liter of distilled,
demineralized
water.
Glucose,
glycerol,
sodium
succinste
or sodium
acetate
was added
at 1 g/liter
for the appropriate
experiments.
Each
experiment
was performed
on a
loo-ml.
culture
which
had incubated
for approximately
17 hr at 37°C with
shaking,
and
was at a concentration
of 1 x 10s cells/ml.
in exponential
growth.
After
these cells were
bound
to the membrane
filter,
they
were washed
and eluted
with
medium
which
had
previously
supported
growth
to 1 x lOa cells/ml.,
and had been filtered
through
a grade
HA Millipore
filter
(Millipore
Filter
Corp.).
This filtered
medium
will hereafter
be called
conditioned
medium.
(b) Apparatus
The procedure
for continuous
withdrawal
of new-born
cells from a culture
attached
to
the surface
of a membrane
filter has been described
previously
(Helmstetter
& Cummings,
1964).
The cells were bound
to a grade
GS (0.22 p pore-size),
152-mm
diameter
Milliporo
membrane
filter
which
was clamped
in a two-section
porcelain
filter
apparatus
(Carl
Schleicher
& Schuell
Co., type PA 15). The support
plate in this apparatus
was replaced
with a stainless-steel
support
screen
(Millipore
Filter
Corp.,
cat. no. YY2214264),
and the
porcelain
upper
section
with several
rubber
gaskets
which
were supplied
with the apparatus. After
filtering
the cells onto the membrane,
the assembly
was inverted
and placed
on top of an additional
porcelain
bottom
section
to maintain
constant
temperature
on
the membrane.
Conditioned
medium
was then poured
into the top. The elution
rate was
DNA
SYNTHESIS
IN
E.
COLI
419
governed
by connecting
the top to a reservoir
of conditioned
medium
through
a model
AL-4-E
Sigmamotor
pump
(Sigmamotor,
Inc.).
The stems of both
bottom
sections
had
been cut off to facilitate
handling
and to permit
the effluent
to drop
freely
from
the
membrane.
The entire
procedure
was performed
in a 37°C Full View incubator
(Precision
Scientific
Co.).
(c) Experimental
procedure
[‘%]Thymidine
(30 me/m-mole,
Nuclear
Research
Chemicals,
Inc.) was added to a IOO-ml.
culture
when
it had reached
a concentration
of 1 x lOa cells/ml.
in exponential
growth.
After
brief exposure
(1 to 2 min) to the label,
the culture
was poured
onto the membrane
and filtered
under
vacuum.
Filtration
was stopped
when almost
all of the fluid had passed
through
the filter,
and the remaining
portion
was poured
off. The cells, now bound
to the
membrane,
were washed
by passing
100 ml. of conditioned
medium
through
the filter
in
the same manner.
The beginning
of the washing
period
was considered
to be the termination of the labeling
period
and also the zero time of elution.
The filter apparatus
was then
inverted
and elution
was begun
by pouring
conditioned
medium
into the top and starting
the pump.
The total time required
for these operations
was approximately
45 sec. Samples
of the effluent
were collected
consecutively
during
constant
time-intervals,
and 0.5 ml.
was removed
for measurement
of the bacterial
concentration
with
a Coulter
Counter
model
B as described
previously
(Helmstetter
& Cummings,
1963).
Each sample
of the
effluent
was precipitated
with cold trichloroacetic
acid at 5% final concentration
and kept
in an ice bath for 30 min. The samples
were then collected
on 25mm
diameter
membrane
filters,
washed
with cold 5% trichloroacetic
acid containing
100 pg of unlabeled
thymidinc/
ml. and dried.
The radioactivity
of the membrane
was determined
in Liquifluor
(Nuclear
Chicago
Corp.)
with a Nuclear
Chicago
liquid
scintillation
system.
3. Experimental
(a)
Description
Design
of the procedure
Figure
1 is a schematic
outline
of the procedure
for
of different
ages in an exponentially
growing
culture
Step I
(pulselabeling)
o.gsogj
Step
to &
mMembrane]
the rate at which
a macromolecule.
cells
For
1
It
(binding)
r
get!Zatioy
(elution)
:Z,:fce”S
C
[I‘-achlnmt
Step
determining
synthesize
III
Start
of elution
+
FIG. 1. Outline
of the procedure
for determining
the
into cells of different
ages in an exponentially
growing
23
TtiT
rate of incorporation
culture.
of a labeled
molecule
420
C. E.
HELMSTETTER
simplicity,
four
representative
cells from
an exponential-phase
culture
are followed
through
the three steps of the procedure.
The ages of the cells are shown
as fractions
of an
interdivisional
period.
In step I the cells are pulse-labeled
with
a radioactive
precursor
of a hypothetical
macromolecule,
and the amount
of label
which
is assumed
to be incorporated
during
the pulse
is indicated
by the letter
in each cell. The cells are then
immediately
bound
to a nitrocellulose
membrane
by filtration
in step II. After
inverting
the membrane,
elution
is begun
by pouring
minimal
medium
on top of the membrane.
The subsequent
growth
of the four cells and their progeny
during
each generation
time of
elution
is shown
in step III.
The division
stages of the cells at four distinct
times
during
each generation
of continuous
elution
are shown
beginning
at the top and progressing
clockwise.
The arrows
indicate
the cells which
would
appear
in the effluent,
based on the
observation
that
primarily
new-born
cells are eluted
from
a membrane-bound
culture
of
E. co& B/r (Helmstetter
& Cummings,
1964). It is assumed
that the new-born
cells elutetl
are daughters
which
did not form
part
of the attachment
of their
parents
to the membrane.
The amount
of label in each new-born
cell is indicated.
The letter
“11” symbolizes
the filial relationship
between
the eluted
cell and the original
bound
cell; i.e., during
the
first generation
of elution
“n”
equals
one, during
the second
it equals
two, etc. At the
beginning
of elution
(twelve
o’clock
in step III),
the new-born
cell eluted
is a daughter
of
the cell which
wss at age 1.0 initially
and it contains
D/2 units of label. After
0.25 generation of elution,
each cell has progressed
0.25 unit in age, and the new-born
cell eluted from
the membrane
at this time
is a daughter
of the cell which
was initially
at age 0.75. It
contains
C/2 units of label. This sequence
of events
continues
until
all of the cells initially
attached
to the membrane
have
divided,
and is then
repeated
during
the second
and
succeeding
generations
of elution.
In summary,
the cells eluted
from
the membrane
during
each generation
are progeny
of the oldest
through
the youngest
cells init,ially
attached,
and each of these contains
half of the label
in its pa,rent.
Thus,
the rate of
incorporation
of a labeled
compound
into cells of different
ages in a population
is dct,ermined
by pulse-labeling
the cells and measuring
the amount
of label in their
progeny.
This experimental
procedure
is quite similar
to the radioautographic
method
for studying
the period
of DNA
synthesis
which was first described
by Howard
& Pelt (1953) and which
has been used in various
forms
by a number
of investigators.
In their experiments,
as in
those to be described
here,
cells were labeled
with
a precursor
of DNA,
but the DNA
synthetic
period
was analyzed
by observing
the subsequent
appearance
of labeled
mitotic
figures
by radioautography
rather
than by withdrawing
cells of a particular
age from the
culture.
For clarity,
it has been assumed
that when a cell divides
on the membrane
one daughter
is eluted
and the other
remains
attached.
This has been indicated
by showing
the cells
attached
to the membrane
by one end. Actually
most cells may be attached
longit,udinally,
and, as a result,
both
daughters
may
remain
attached
when
some of the cells divide.
Therefore,
the number
of new-born
cells which
remain
attached
may be greater
than the
number
which
are eluted;
but this effect would
not alter
interpretation
of the data presented in this report.
It has also been assumed
that the label is equally
distributed
between
the daughter
cells. This need not be the case and, in fact, the method
could
be used to
determine
the presence
of oriented
or non-equal
partition
of the label.
(b)
Theoretical
considerations
To facilitate
interpretation
of data
obtained
with
this
procedure,
a hypothetical
experiment
will
be described.
First,
the concentration
of cells .in the effluent
from
a
membrane-bound
population
of bacteria
with
a particular
age distribution
will be considered.
Figure
2(a) shows the ideal distribution
of cell ages in an exponentially
growing
culture.
If the ages of the bound
cells were also distributed
in this manner,
and if elution
proceeded
as shown
in Fig. 1, then ideally
the concentration
of cells in the effluent
during
the first two generations
of elution
would
appear
as shown
in Fig. 2(b). Since the cells
which
are eluted
during
each generation
are progeny
of the oldest through
the youngest
cells initially
bound
to the membrane,
the elution
curve
during
each generation
is the
reverse
of the age distribution
curve.
Again
it has been assumed
that when
a bacterium
divides
on the membrane
one daughter
is eluted
and the other
remains
attached.
If less
DNA
SYNTHESIS
than one-half
of the new-born
cells
would
be greater
in each succeeding
membrane
would
increase.
E. COIL1
IN
were eluted,
generation,
421
the concentration
since the number
of cells in the
of cells bound
effluent
to the
i”\ll~~~
0
0.50
Cell
0
I.0
050
age
of a generation)
(fraction
Elution
I.0
time
(a)
I.50
2.0
(generations)
(b)
FIG. 3. (a) Idealized
age distribution
in an exponential
phase culture
containing
no dispersion
in generation
times of individual
cells.
(b) Theoretical
concentration
of cells in the effluent
from a membrane-bound
culture
with an
age distribution
as shown in (a).
$.”
2.0
2:
2s
Fi?
2%
m
B
I.0
A
o
050
Cell
(fraction
of
I.02
age
0
0.50
El ution
I
I.0
time
I.50
2.0
(generations)
a genemtion)
(a)
(b)
FIG. 3. (a) Rate of synthesis
of two hypothetical
macromolecules
a8 a function
of cell age.
(b) Theoretical
radioactivity
per cell in the effluent
from a membrane-bound
culture
if it were
pulse-labeled
with radioactive
precursors
of the macromolecules
described
in (a).
Next
suppose
that the rate of synthesis
of two hypothetical
macromolecules
as a function of cell age is given
by Fig. 3(a). That is, the rate of synthesis
of molecule
A doubles
in the middle
of the division
cycle, whereas
the rate of synthesis
of molecule
B increases
exponentially
during
the division
cycle.
Figure
3(b) shows the results
expected
if this
population
were pulse-labeled
with radioactive
precursors
of molecules
A and B, and then
analyzed
with
bhe elution
procedure.
Curve
A in Fig.
3(b) shows
the radioactivity
per
effluent
cell due to incorporation
of the precursor
of molecule
A. For the first half of the
first generation
of elution,
the radioactivity
per effluent
cell is constant,
since these cells
would
be progeny
of cells which
were between
the age 1.0 and 0.50 at the time of pulselabeling.
During
the last half of the first generation
of elution,
the radioactivity
per cell
is one-half
the initial
value
since these cells would
be progeny
of cells which
were at age
0.50 through
0 initially.
This same pattern
is repeated
in the second generation
of elution,
but at corresponding
elution
times
the radioactivity
per effluent
cell is equal to one-half
that of the first generation.
Similarly,
the radioactivity
per effluent
cell due to incorporation
of the precursor
of macromolecule
B would
be expected
to decrease
exponentially
as is
shown
by curve
B of Fig.
3(b).
Consequently,
the rate of synthesis
of these
macromolecules
as a function
of increasing
cell age is shown
from right to left in each generation
of elution.
C. E.
422
HELMSTETTER
4. Results
(a) E&ion
of cells from
a membrane-bound
population
When an exponentially growing population of E. coli B/r is filtered onto the surface
of a membrane filter, and the membrane is inverted and conditioned medium is
passedthrough it, primarily new-born cells are eluted from the membrane (Helmstetter & Cummings, 1964). Figure 4 shows the number of bacteria in consecutive
samplesof the effluent from membrane-bound cultures of E. coli B/r in minimal-salts
medium containing glucose, glycerol, succinate or acetate as the carbon source. In
this and the next Figure, the time scalesare arranged so that the positions of the
peaks in the bacterial elution curves coincide, and the ordinates are graduated in
logarithmic scalesto ease visualization of the periodicity in the curves. As noted
I
I
I
I
1
4.0 -
20
I.0
Glucose
;c
4.0
80
40
120
2.0
4)
‘-0
I.0
x
0
Glycerol
;c
0
100
50
2
z
4.0
-z
v
20
Succinate
Id
0
210
70
2.0
i
I.0
Acetate
0.5
:;:-II_
0
210
105
Elution
time
315
(mln)
Fro. 4. Number
of cells in consecutive
samples of the effluent
from membrane-bound
cultures
of E’. coli B/r growing
in minimal-salts
medium
containing
glucose, glycerol,
succinate
or acetate
as carbon source.
In each experiment
a loo-ml.
culture
containing
1 x 108 cells/ml.
was filtered
o&o the membrane. The rate of elution with the four media was 6.5 ml./min,
65 ml./min,
5 ml./min
and 5 ml.,’
min, respectively.
The sampling
periods
are indicated
by the horizontal
bars in the histograms,
and were 4 min in glucose and glycerol,
and 5 min in succinate
and acetate medium.
DNA
SYNTHESIS
IN
E. COLI
423
above, the shape of the elution curves mainly reflected the age distribution
of the
bacteria initially bound to the membrane and the efficiency with which the nemborn cells were eluted from the membrane. The shapes of the curves are similar t,o
those anticipated based on the theoretical exponential curve shown in Fig. 2(b),
except for the effect of dispersion in division times and the increase in bacterial
concentrations with elution time. The upward trend of the curves indicates that fewer
than half of the new-born cells are eluted from the membrane, especially during the
first generation of elution.
(b) Incorporation
of [‘“Cjthymidine
into cells of different ages
The rate of incorporation of [14C]thymidine into cellsof different agesin populations
of E. coli B/r growing in glucose, glycerol, succinate or acetate
medium was determined by the method described. Figure 5 shows the radioactivity per efiuent cell
I
4.0:
,hL;
I
I
I
2.0
I
I
I
i
I
I
I
I
I
;Succinate
1
iAcetate
Elution
I20
I
I
timetmin)
Fro.
5. Radioactivity
per cell in the effluent
from membrane-bound
cultures
of E. coli B/r which
had been
pulse-labeled
with
[lW]thymidine.
Cells growing
in glucose,
glycerol,
succinate
and acetate
medium
were exposed
to [‘%]thymidine
at 0.1 PC/ml.
for
1 min,
0.1 PC/ml.
for 2 min,
0.1 PC/ml.
for 2 min
and
0.15 +/ml.
for 2 min,
respectively.
The cultures
were bound
to membrane
filters
as described
in Materials
and Methods
and washed
and eluted
with
conditioned
medium
containing
the same carbon
source.
424
C. E.
HELMSTETTER
during elution of the four membrane-bound
cultures which had been pulse-labeled
with [14C]thymidine.
The vertical broken lines indicate
approximately
the elution
time in generations.
The generation
times were determined
by measuring
the time
interval
between peaks of the bacterial elution curves obtained in each experiment.
In glucose-grown
cells, there was little change in the cts/min per effluent cell during
elution of the progeny of those cells which were between the end and the middle of
their division cycle during the pulse-labeling
period. The radioactivity
in the progeny
of cells which were between the middle and the beginning
of their division cycle was
1st
Generation
1
I
0
2nd
Generation
Gly,cerol
4.0
Ia0 2
.-5
O 5
3.0 eQ)
m
P
2,o 2
60
4.0
)li-
.
3.0
I.5
2-o
I.0
I.0
0.5
0
(fractions
05
I.0
2
0
0
0.5
I*0
Cell age
of a generation)
Fm. 6. Rate of [‘Wlthymidine
incorporation
during the division
cycle of E. coli B/r grown in
glucose, glycerol,
succinate
and acetate minimal
medium.
This Figure is a replot of the cts/min/106
effluent
cells in the first two generations
of elution
in
Fig. 5 as a function
of the age of the parents
(first generation)
and grandparents
(second generation)
of these cells at the end of the pulse-labeling
period.
The first generation
in this Figure
is a plot
of the data between
the first vertical
dashed line and the origin in Fig. 5 (i.e., the first generation
of elution
in reverse
order),
and the second generation
is data between
the second and the first
verticd
dashed lines in Fig. 5.
DNA
SYNTHESIS
IN E. COLI
423
also roughly constant, but equal to about O-6of the cts/minin the progeny of the older
cells. Therefore, the rate of [14C]thymidine incorporation into cells which were in the
last half of their division cycle was approximately double that into cells which were
in the tlrst half of their division cycle. Figure 6 showsthe results of the sameexperiments, but with the radioactivity of the effluent cellsduring the first two generations
of elution plotted as a function of the age of their ancestors at the time of pulselabeling. Therefore, this Figure showsthe rate of [14C]thymidine incorporation during
the division cycle directly. The results show that the rate of thymidine incorporation
increasesabruptly around the middle of the division cycle of glucose-grownE. coli B/r.
Similarly, the results in Figs 5 and 6 indicate that in glycerol-grown cells the rate of
incorporation of [l*C]thymidine increases abruptly between the middle and the
end of the division cycle. The results with succinate- and acetate-grown cells indicate
a period of reduced [14C]thymidine incorporation during the last half of the division
cycle, followed by a rapid increase in incorporation just before the end of the cycle.
Finally, it can be seen from Fig. 6 that the cts/min/106effluent cells in the second
generation of elution were not equal to O-5of that in the first generation, as would be
expected theoretically, but they were greater-about O-6 of the first generation.
I cannot explain this discrepancy at present, and in the interpretation of these experiments I rely more on the shapesof the incorporation patterns than on the absolute
values.
5. Discussion
The rate of thymidine incorporation into cells of different ages in exponentially
growing cultures of E. coli has been determined. This was accomplished by pulselabeling the cultures with [14C]thymidine and measuring the radioactivity in the
new-born cells released from the cultures after they were bound to the surface of
membrane filters. The rate of incorporation of [14C]thymidine into cells of different
ages was considered a measure of the rate of DNA synthesis during the bacterial
division cycle. It must be noted that the influence of relevant intracellular pools on
the incorporation of [14C]thymidine during the pulse-labeling period and during the
initial elution period has not been determined. However, Clark & Maaloe (1967)
measuredthe time course of thymidine incorporation into bacterial samplestaken at
various times during synchronous growth, and they found linear incorporation
curves which extrapolated to 30 secondson the time axis in each case. This result
suggeststhat there is little, if any, significant change in pool-size during the division
cycle of E. coli B/r.
It was found that the rate of DNA synthesis increasesabruptly at a time in the
division cycle which is characteristic of the carbon source in the medium in which the
cells were grown. We will consider that the age at which the rate of DNA synthesis
increasesin the average cell can be estimated by determining the age which correspondsto the mid-point of the increase in radioactivity per eflluent cell in the second
generation in Fig. 5 or Fig. 6. The secondgeneration was chosenbecausethe first and
third generations are incomplete. On this basis,the cell age in fractions of a generation
at which the rate of DNA synthesis increases is 0.4 in glucose-, 0.8 in glycerol-,
O-9 in succinate- and O-9 in acetate-grown cells. Thus, the increase occurs about 24
minutes before division in glucose-grown cells and about 10 minutes before division
in glycerol-, succinate- and acetate-grown cells. The results in glucose-grown cells
support the finding of Clark & Maalee (1967) that the rate of DNA synthesis doubles
426
C. E.
HELMSTETTER
around
mid-cycle
during
synchronous
growth
of E. coli rather than increasing
exponentially
during the cycle as reported by Abbo & Pardee (1960).
DNA synthesis was found to occur throughout
the division cycle of cells growing in
glucose minimal
medium.
This is in agreement
with previous experiments
which
demonstrated
essentially
continuous
DNS synthesis in E. coli growing exponentially
in glucose minimal
medium by the use of radioautography
following
brief exposure
to [3H]thymidine
or [3H]thymine
(Schaechter,
Bentzon
& Maaloe, 1959; Lark 8:
Lark, 1965) and by measuring the loss of viability
after incorporation
of [3H]thymine
(Pachler, Koch & Schaechter,
1965). The results also indicate that the abrupt increase
in thymidine
incorporation
near the end of the division cycle of glycerol-, succinateand acetate-grown
cells is preceded by a period of lesser incorporation
with respect to
the first half of the cycle. The decrease in thymidine
incorporation
was very slight
in glycerol-grown
cells, and more pronounced
in succinnte- and acetate-grown
cells.
This period of reduced incorporation
presumably
corresponds to the interval between
rounds of DNA replication
in E. coli suggested by Maalee & Hanawalt
(1961) and
Maalee (1961), and observed in the radioautographic
experiments
of Lark (1966).
Lark found a period devoid of DNA synthesis in slow-growing
cells by briefly exposing
cultures of E. coli ETto [3H]thymine
and measuring
by radioautography
the
fraction of cells which incorporated
the label. The results reported here show the same
basic effect of slow growth except that the gap in DNA synthesis in E. coli B/r is
evident at faster growth rates in comparison
t)o Lark’s findings with E. coli 15’l!-.
One major difference is that Lark’s data indicate that the gap occurs during the first
half of the division cycle, whereas the results described in this report show that it
occurs during the last half of the cycle of E. coli Bjr. Unfortunately,
this apparent
difference cannot be invest,igated
with the elution technique
since E. coli 15T- does
not bind properly to the membrane.
If the period of reduced thymidine
incorporation
corresponds
to a gap between
rounds of DNA replication,
then the time of the abrupt increase in the rate of incorporation
at the end of this period corresponds
to the time in the cell cycle when
each new round of DNA replication
commences. Clark & Maaloe (1967) investigated
t,he time of initiation
of DNA replication
in glucose-, glycerol- and succinate-grown
cells by measuring
the extent of incorporation
of thymidine
in the presence of
chloramphenicol
by cells in different
stages of synchronous
growth.
Since it was
assumed that protein synthesis is required
to initiate
DNA synthesis, the time of
synthesis of the protein required
for initiation
of DNA replication
would be determined by their experiments.
They found that replication
is initiated
in glucose
cells at the same time that the rate of DNA
sYynthesis doubles,
and that
initiation
occurs at about the same time prior to division
(20 to 25 minutes)
in
glycerol and succinate
cells. This time is slightly
earlier in the division
cycle of
glycerol and succinate cells than the time before division that a new round of DNA
replication
begins as reported
here. This could mean that the protein required
for
initiation
is synthesized a few minutes before a new round of DNA replication
begins
in slow-growing
cells, but to be certain of this apparent
time difference both experiments would have to be performed under the same conditions.
The major advantage of the method presented here is that the labeling took place
in an unaltered
exponential-phase
culture and all manipulations
were carried out
after, rather than before, the incorporation
had occurred. In fact, the reason I have
adopted
this approach
is that I have observed unusual and apparently
distorted
DNA
SYNTHESIS
IN
E.
427
COLI
patterns
of thymidine
and
u&line
incorporation
into synchronous
populations
obtained
from membrane-bound
cells. These observations,
along with a comparison
between results obtained using both applications
of the membrane-selection
technique,
will be presented elsewhere. With regard to the possible sources of error in the present
technique,
it should be noted that I have assumed that the attachment
of log-phase
cells to the surface of the membrane
did not distort the sequence in which cells of
different ages in the culture would have divided. In addition,
it was assumed that
there was no delay in cell division at the beginning
of elution, and that new-born
cells were &ted
from the membrane
at the moment of division. If a delay of either
kind occurred, the time in the division cycle at which a round of DNA replication
was found to begin in these experiments
would be earlier than the observe&
ime.
Although
we have no evidence to suggest the possibility
of errors of this kind, further
experiments
with this method will be necessary to establish
the validity
of these
assumptions.
I wish to express
my sincere
thanks
to Dr Ole Maalee
ment
during
my visit
in his laboratory,
where
many
were developed.
Special
thanks
are due to Dr Stephen
suggestions
from the very
beginning
of this investigation.
Pierucci
for many
helpful
discussions
and to Evadne
technical
assistance.
This
investigation
was supported
by U.S.
Public
CA 08232 from the National
Cancer
Institute.
for his hospitality
and encourageof the ideas for this investigation
Cooper
for his numerous
valuable
I am also indebted
to Dr Olga
Chin and Eras Revelas
for expert
Health
Service
Research
grant
REFERENCES
Abbo,
F. E. & Pardee,
A. B. (1960).
Biochim.
biophys.
Acta, 39, 478.
Barner,
H. D. & Cohen,
S. S. (1956).
J. Bact. 72, 115.
Clark,
D. J. & Maalee,
0. (1967).
J. Mol.
Biol. 23, 99.
Cummings,
D. J. (1965).
Biochim.
biophys.
Acta, 85, 341.
DeLamater,
E. D. (1956).
Symp.
Sot. Gen. Microbial.
6: 215.
Hehnstetter,
C. E. & Cummings,
D. J. (1963).
Proc. Nat. Acad. Sci., Wash. 50, 767.
Helmstetter,
C. E. & Cummings,
D. J. (1964).
Biochim.
biophys.
Acta, 82, 608.
Howard,
A. & Pelt, S. R. (1953).
Heredity,
6 (Suppl.),
261.
Lark,
C. (1966).
Biochim.
biophyiya. Acta, 119, 517.
Lark,
K. G. & Lark,
C. (1965).
J. Mol.
Biol.
13, 105.
Maaloe,
0. (1961).
Cold Spr. Harb.
Symp.
Quad.
Biol.
26, 45.
Maalee,
0. (1962).
In The Bacteria,
cd. by I. D. Gunsalus
8z R. Y. Stanier,
vol. IV,
New York:
Academic
Press.
Maalee,
0. & Hanawalt,
P. G. (1961).
J. Mol.
Biol. 3, 144.
Maruyama,
Y. (1956).
J. Boxt. 72, 821.
Nagata,
T. (1963).
Proc. Nat. Acad. Sci., Wash. 49, 551.
Pachler,
P. F., Koch,
A. L. & Schaechter,
M. (1965).
J. Mol.
Biol.
11, 650.
Schaechter,
M., Bentzon,
M. W. & Maalee,
0. (1959).
Nature,
183, 1207.
p. 1.