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Journal of General Microbiology (1 990), 136, 599-606.
Printed in Great Britain
599
Review Article
Cell growth and abrupt doubling of membrane proteins in Escherichia coli
during the division cycle
HERBERT
E. KUBITSCHEK-~
Biological and Medical Research Diuision, Argonne National Laboratory, 9700 South Cass Auenue, Argonne,
IL 60439, USA
Regulation of cell growth is a major biological mystery.
Even the kinetics of cell growth, although studied for
many years, remain elusive. This state of affairs reflects,
in large part, the fact that cell growth represents the
interaction of a large set of complex and interrelated
biochemical processes that allow the cell to survive and
respond to a tremendous variety of challenges and
stimuli. Thus, it might seem at first that studies of cell
growth during the cellular division cycle may be of little
help. Nevertheless, interest in cell growth patterns has
continued to increase because such studies reveal the
presence of major processes that contribute to the
regulation of cell growth and may also provide clues to
their biochemical identification.
Methods and criteria
Cell growth has been studied far more extensively in
Escherichia coli than in any other prokaryotic or
eukaryotic cell type. Several experimental approaches
have been used, including studies of growth in individual
cells, synchronized cultures, or exponential-phase populations. Because each of these approaches has produced a
variety of results and conclusions for the kinetics of cell
growth during the cell cycle in this micro-organism, no
common consensus on the pattern of cell growth has
developed, much less an understanding of its regulation.
The slow progress in this area reflects both the
complexity of growth and the technological difficulties in
its study, as well as the involvement of semantic
differences. For example, in these different studies,
‘growth’ has variously been defined and measured as
increase in cell length, surface area, volume, mass, or
content of cellular RNA and protein.
The accumulated evidence now makes it possible to
resolve some of the earlier conflicting results, especially
as we have become more cognizant of the kinds of
difficulties associated with these experiments. In addiTShortly after completing work on this paper, Dr Herbert E.
Kubitschek died on 28 November 1989, at the age of 69.
tion to reproducibility, there are three major requirements for experiments on cell growth. (1) Resolution
must be sufficient to distinguish among the three major
growth models that have been proposed - exponential,
linear, or bilinear growth. For example, the experimental
resolution must be far better than the largest difference,
about 6%, between exponential and linear growth (Fig
1). (2) Growth characteristics of cells in synchronized
cultures must remain unchanged from their values ii;
steady-state or exponential phase cultures: that is9
growth should not be perturbed by procedures used tc
synchronize cells. (3) Data analysis must concern an
appropriate growth parameter. In particular, as discussed later, averages over measured values of divided
and undivided cells in synchronous cultures are inappropriate because the two groups represent different
generations.
The earliest method for measuring cell growth was to
observe individual cells by light microscopy as they
progressed through the division cycle. Results were not
very meaningful until the late 1950s, however, when the
concept of maintaining balanced growth conditions was
established (Campbell, 1957). Nevertheless, even when
these conditions were adopted, very different patterns o i
length increase were found for the single strain E. cdr
B/r, including exponential (Schaechter et al., 1962),
bilinear (Cullum & Vicente, 1978) and interrupted
(Hoffman & Frank, 1965). These disparate patterns
reflected insufficient optical resolution, approximately
0.4-0.5 ym, in the measurement of cells 1-2 pm in length.
This lack of sufficient optical resolution led N .
Nanninga, C. L. Woldringh and their colleagues at the
University of Amsterdam to study cell growth and
division by a technique capable of much higher
resolution : electron micrography. However, with this
technique, they could not follow the growth of individuai
cells; consequently, cell growth kinetics were obtained
with a method based upon analysis of steady-state
population distributions of cell size, as pioneered by
Collins & Richmond (1962). This method provided
greater consistency for the measurement of the kinetics
0001-5987
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600
H . E. Kubitschek
2.0
1.5
--.
E
1.0
0.5
No. of generations
1.0
Fig. 1. Cell growth models. During linear growth relative cell mass
(rn/rno) increases at a constant rate, as shown by line A. Curve B
represents exponentialgrowth, differing from A by less than 6%at any
point during the cycle. Line C represents bilinear growth; the rate of
growth is constant, doubling at some point within the cycle. Actually,
linear growth is the particular case of bilinear growth for which the
growth rate change occurs at the end of the division cycle. Curve D is
the locus of rate change points for all bilinear curves, and shows the
maximum deviations between bilinear growth and the other models.
of cell length extension, which was exponential during
the division cycle (Koppes et al., 1978a, b ; Kubitschek &
Woldringh, 1983). Furthermore, if cell width were
constant, these results implied that cell volumes should
increase essentially exponentially. However, Trueba &
Woldringh (1980) found that cell diameters did not
remain constant but diminished by as much as 8% during
part of the cycle, corresponding to a change in crosssectional area of about 15%. In this case, cell volume
should increase less rapidly than exponentially. In
support of this view, Kubitschek (1981) found that when
both lengths and diameters were measured for individual
cells, then cell volume appeared to increase bilinearly,
i.e. with a step-up in growth rate during the cycle.
The Collins-Richmond analysis, however, requires
data of much greater precision than do more direct
measurements of cell growth because the shape of the
population distribution depends not only upon cell
growth during the cycle, but also upon the distributions
of cell size at birth and division. Analysis requires
estimations of these contributions from birth and
division, which must be either assumed or derived from
distributions of deeply constricted cells. Because these
different techniques are so strongly influenced by
statistical variation, experimental errors can only be
reduced by the labour-intensive approach of collecting
far more data than is required by other methods (Koch,
1966; Kubitschek & Woldringh, 1983).
The third method of determining cell growth kinetics
uses synchronous cultures. Ideally, a synchronously
growing cohort of cells of a given age or size is selected
from a steady-state or exponential-phase culture, and the
growth of this cohort is then used to represent the growth
pattern of the average individual cell. The use of
synchronized cultures, however, has led to observations
of a variety of growth patterns for E. coli. For length
extension alone, these patterns include, in addition to
those discussed above, linear (Donachie et af., 1976;
Pierucci, 1978), approximately exponential (Marr et af.,
1969), or even a more complex response (Meyer et al.,
1979).
When cell volume increase was examined in synchronized cultures, the growth patterns were mainly of two
kinds and appeared to depend upon the synchronizaton
technique. Cell volume increase (already concluded to be
approximately exponential from length measurements
alone) was also approximately exponential when cells
were synchronized by membrane elution or elutriation
centrifugation and their sizes were measured micrographically (Olijhoek et af., 1982; Olijhoek, 1983). On the
other hand, cell volume increase was found to be linear
when cells were synchronized by sucrose gradient
centrifugation and their sizes determined with a Coulter
electronic counter analyser system (Kubitschek, 1968a,
1986, 1990). This apparent dependence upon methodology highlighted a requirement for consistency of experimental results with those from cultures in steadystate or exponential growth for adequate comparisons to
be made.
The second criterion, the absence of perturbation of
cell growth, is commonly recognized, and tests for
perturbation are usually performed. However, these tests
are customarily based only upon the timing of increase in
cell numbers (Nanninga & Woldringh, 1985; Kubitschek, 1987), and as such are incomplete because cell
volumes may also be perturbed in synchronized cultures.
Several observations support the existence of cell
growth perturbation in cultures synchronized by membrane elution (Kubitschek, 1987, 1990). First, when cells
of E. coli B/r were filtered on membranes, synthesis of
protein D and other altered outer-membrane proteins
was induced, changing the chemical composition of the
outer membranes of these cells (Boyd & Holland, 1977).
Second, buoyant densities of E. coli cells synchronized by
membrane elution were significantly higher than those of
cells in their unsynchronized parent culture (Kubitschek
et al., 1983, 1984). Third, rates of increase of cell length
and volume changed periodically during the cell cycle
after membrane elution (Olijhoek & Nanninga, cited by
Nanninga et al., 1982). Fourth, in slowly growing
membrane-eluted cultures of E. coli B/rA, DNA replication was consistently initiated at or near the beginning of
the cell cycle, with a large gap in synthesis for most of the
final quarter or third of the cycle (Helmstetter &
Pierucci, 1976; Koppes et al., 19786). In exponentialphase cultures, however, the gap in DNA synthesis
occurred, instead, during the first part of the division
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Cell growth and membrane protein doubling
cycle as measured by DNA :DNA hybridization (Chandler et al., 1975), decay of incorporated 251 (Kubitschek
& Newman, 1978), and flow cytometry (Skarsted et al.,
1985). These comparisons provide very strong evidence
for perturbation of cell growth by the membrane elution
technique. Thus, although this technique has yielded
fundamental information on DNA replication, one
cannot conclude that it provides synchronized cultures
with cell characteristics representative of exponentialphase growth, as stated recently (Cooper, 1988a).
The third criterion is that the analysis of cell growth
must concern the proper cohort in a synchronousculture.
Cell growth during the division cycle has sometimes been
interpreted as the increase in mean, modal or total cell
volume in the synchronized culture (Scott et al., 1980;
Olijhoek et al., 1982). Although each of these parameters
represents growth of the average individual cell early in
the cycle, each ceases to represent the average as soon as
any cells divide, because the newly divided cells advance
to the following generation and are no longer representative of the kinetics of cell growth of the first generation
(Kubitschek, 1986). Because ages and volumes of newly
divided cells are smaller than those of the remaining
undivided cells, if both groups are averaged together,
then average cell size and age will decrease as more and
more cells divide near the end of the cycle. Furthermore,
the mean cell volume cannot then double during the
cycle, as occurs for undivided cells during steady-state or
exponential phase growth. Thus, average individual cell
growth is correctly represented only by the growth of the
undivided cells alone, for which both age and volume
continually increase in the same manner as they do in
steady-stateor exponential-phase cultures. The misuse of
mean, modal or total cell volume to describe cell growth
is readily apparent from graphs of cell growth: these
incorrect calculations yield a single, sinuous curve
connecting the points of successive division cycles. In
contrast, proper analysis of the growth of undivided cells
provides distinct curves of volume increase for each
generation.
Even when the three criteria (resolution, absence of
perturbation, and appropriate cohort) appear to be
satisfied, experimental results may still differ for
different techniques. To ascertain which set of results is
correct, the guiding principle is again to compare the
experimental results with those obtained in steady-state
or exponential-phase cultures. If a direct comparison
cannot be made, perturbation may be revealed by
examining some alternative measurable characteristic.
The timing of DNA replication of E. coli B/rA in slowly
growing exponential phase cultures is an example. As
observed by autoradiography of individual cells from an
exponential-phaseculture, initiation of DNA replication
starts very near the beginning of the division cycle and is
601
associated with a long D period (Koppes et al., 1978b).
However, when measurements were made upon exponential-phase cultures by methods of DNA :DNA
hybridization, by survival of cells exposed to decay of
incorporated 251,or by flow cytometry, initiation was
found to occur much later in the cycle, following a large
gap in DNA synthesis and associated with much shorter
D periods. Again, the concurrence obtained with the
latter three widely different techniques strongly supports
a large initial gap in DNA replication during the division
cycle of this strain of bacterium.
The source of the alternative kinetics observed with
electron micrography is not yet identified. Possibly, the
different kinetics concern the consequences of fixation.
Although electron micrography provides excellent dimensional resolution, the cells are flattened and deformed in cross-section during specimen preparation
(Woldringh et al., 1977). Also, although the fixation
technique was carefully designed to match optical
dimensions of cell length and width, the unusual pattern
produced for initiation of DNA synthesis in E. coli B/rA
continues to raise questions about the consequences of
the fixation technique. These questions also apply to
more recent electron micrographic results obtained for
cells in steady-state cultures exposed to shifts in growth
rate. In particular, in an experimental and analytical tour
de force, Zaritsky et al. (1982) examined a number of
models of cell growth for their agreement with the
experimental results during a shift in growth rate. Their
results supported a model of linear increase in cell surface
area and a gradual increase in growth rate during the
growth shift. As they pointed out, however, assumptions
of constancy of cell mass at initiation and constant
periods of chromosome replication might invalidate
their conclusions.
The three major criteria for E. coli cell-growth studies,
as discussed above, appear to have been achieved for
synchronous cultures by measurements of cell volume
with a Coulter counter analyser system in cultures
synchronized by selection of cells from velocity gradients. Results of tests for perturbation of growth were
negative, with the same doubling periods for cell
numbers and cell volumes in synchronous cultures and
parent cultures, and the same sizes of cells at birth and at
division (Kubitschek, 1986,1987,1990).The accuracy of
the Coulter counter analyser system also was examined
by comparing cell volumes observed with the counter to
those obtained with a common biophysical technique,
the measurement of excluded volume in packed cells.
The results supported the same cell volumes within
experimental errors (Kubitschek & Friske, 1986) and
therefore establish that the counter analyser measured
the cell volume within the outer membrane, as expected
(rather than, say, that within the inner cell membrane).
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H . E. Kubitschek
Growth models: comparison and implications
In the cultures just described, cell volumes increased
linearly in E. coli B/rA at doubling tmes of 25 and 40 min,
in 15.coli 15THU at doubling times of 25 and about
iO5 min, and in E. coli 12 WP2s at a doubling time of
about 105 min (Kubitschek, 1986, 1990). In two ways,
these results effectively ruled out an exponential increase
in cell volume during the cycle. Regressions fitted to the
data had slopes and intercepts that were in significant
disagreement with those predicted by the exponential
growth model. In addition, the nonrandom distribution
of residuals (i.e. differences between observed and
theoretical values) invalidated the model of exponential
growth. Bilinear growth was not completely ruled out,
but in one case at least it was shown that if growth is
bilinear, then growth rate doubling must occur very near
cell division, within 0.03 generations, and the pattern
would appear almost indistinguishable from linear
growth (Kubitschek, 1986).
These results also effectively ruled out an exponential
increase in cell mass, the fundamental growth parameter.
It has been shown that cell buoyant density in E. coli is
essentially independent of age (Kubitschek et al., 1983);
and thus cell mass, which is the product of cell volume
times density, must remain proportional to volume.
Studies of buoyant cell density and the criteria that must
be satisfied for experimental determination thereof were
reviewed earlier (Kubitschek, 1987).
Despite this evidence against exponential growth,
Cooper (1988a) argued that cell growth in E. coli must be
exponential because protein and RNA are known to
accumulate essentially exponentially in cells during the
division cycle. That argument, however, neglects the
contribution of soluble material in the cell pools to the
total cell mass. In the eukaryote Schizosaccharomyces
pornbe, for example, this ratio is approximately 15%
during midcycle, as observed long ago by Mitchison
(1957). Although the fraction of pool mass is only a few
percent of the total in E. coli, the kinetics of cell growth
depend strongly upon pool size variation during the
cycle. Assuming that macromolecular synthesis increases exponentially during the division cycle, linear
cell growth predicts that soluble pools will increase to
midcycle maxima and then decrease to their initial
values again at the end of the cell cycle, whereas
exponential cell growth predicts the absence of such a
midcycle maximum. The linear growth model was
supported by the behaviour of cell pools for uracil,
histidine and methionine in E. coli THU (Kubitschek &
Pai, 1988).
What is the implication of linear cell mass increase
during the cycle? Clearly, constant rates of mass increase
require constant rates of accumulation of compounds
from the surrounding medium. To test that possibility for
individual compounds, Kubitschek (1968 b) examined
rates of accumulation of glucose, acetate, phosphate,
sulphate, leucine, glycine and thymidine in synchronous
cultures of E. coli. In every case, the relative constancy of
the net uptake rates both supported the linear growth
model and provided evidence against the increasing
rates of uptake during the cycle predicted by the
exponential model. Uptake of potassium in synchronous
cultures also supported the linear growth model (Kubitschek et al., 1971).
Accumulation rate experiments inherently have much
greater resolution than measurements of cell mass or
volume and consequently are more capable of discriminating between alternative cell growth models. Despite
this advantage, I am aware of only one other attempt to
measure uptake rate, rather than incorporation, of a
protein or RNA precursor in E. coli, an experiment by
Cooper (1988~).He treated cells with a pulse of
radioactive leucine, then washed away the free radioactivity with warm medium before inverting the membrane
and sampling at intervals. However, these samples could
not have represented leucine uptake rates during the cell
cycle as stated because unincorporated leucine would
have almost immediatedly been dissipated by diffusion
from the cells or have become incorporated into protein
before the cells were eluted from the membrane. Thus,
those results cannot reveal the sizes of the leucine pools
later in cells of different ages or the fraction of the pool
leucine that becomes incorporated into the cell at
different ages.
Models have been proposed for both exponential and
linear growth. In his passive continuum model, Cooper
(1979,1982) proposed that cell mass increase is essentially exponential because the increase in cell mass is the
sum of the different macromolecular and micromolecular components of the cell and the rates of synthesis of
these components are assumed to be essentially proportional to their masses. This proportionality has been
found to hold, to good approximation, for RNA and
protein synthesis. Cooper’s (1984) model also postulates
that, at most, a very few ‘events’ occur during the cell
cycle, and therefore that ‘cell division is the end of a
process and the beginning of none’. That is, cell growth
between divisions is predicted to be simple and
continuous (Cooper, 1988b). The model thus predicts
that sudden transitions and new modes of regulation at
the whole-cell level do not occur.
In contrast, the model for linear cell growth is a
transport model of growth regulation (Kubitschek,
1968a, 6). It postulates that increase in cell mass during
steady-state growth is limited by and proportional to the
net rate of transport of materials into the cell, which in
turn is proportional to the number of functional transport
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Cell growth and membrane protein doubling
sites on the cell surface. Cell growth rate constancy
requires that the (functional) number of these sites
remain constant during the cell cycle and that the
number be doubled at or near cell division, thus
maintaining the same average numbers of sites on
daughter cells.
Two predictions of this latter model have now been
supported. The first is that some cell proteins associated
with the surface should double abruptly; abrupt doubling
has been observed for a number of envelope proteins of
E. coli (Ohki, 1979; Churchward & Holland, 1976; Boyd
& Holland, 1979) and for binding proteins for lactose and
maltose (Shen & Boos, 1973; Dietzel et al., 1978), as
discussed below. Second, assuming that cellular macromolecular synthesis increases essentially exponentially
during the division cycle, linear growth predicts that
soluble pools of RNA and protein precursors should
increase to midcycle maxima, then decrease again to
their initial values by the end of the division cycle.
Mesured values of pool sizes for uracil, histidine and
methionine in E. coli THU supported these predicted
pool kinetics (Kubitschek & Pai, 1988).
Abrupt doubling of transport proteins
Perhaps the most distinctive difference between the
exponential and the linear growth models concerns the
latter’s prediction of a set of transport proteins that
double abruptly during the cell cycle. This difference will
almost certainly distinguish between the two models and
has already begun to do so, as described below. First,
however, in opposition to that interpretation, it can be
argued that although a number of candidates have been
found, the evidence for the sharpness of the doubling is
insufficient because these doublings occurred at many
different times during the cell cycle and the relationships
of some of these proteins to transport was not established. Furthermore, stepwise increases contrast with the
results of Lutkenhaus et al. (1979), who observed by
means of two-dimensional electrophoresis essentially
continuous synthesis of 750 individual proteins of E. coli.
For an even more precise estimate of synthesis rates, they
also measured the radioactivity of some 30 of these
proteins synthesized during the cell cycle and found that
the variability of each was 15% or less. These results
would negate the linear growth model unless the proteins
measured are structural or ‘housekeeping’ proteins not
involved in the regulation of cell growth. The transport
sites model predicts that only a small fraction of the cell
proteins should double abruptly. In agreement, most or
all proteins observed to double abruptly are envelope or
periplasmic proteins.
Another objection to the evidence presented below, for
603
abrupt doubling of specific proteins during the cell cycle,
might be that this evidence is obtained from experiments
that have not been shown to satisfy the critical tests for
absence of growth perturbation. Indeed, in some cases,
data were obtained from cultures synchronized by
membrane elution and, consequently, are probably
perturbed. However, although the precise kinetics of
abrupt doubling of specific proteins may not be
established, there is no question about the occurrence of
the phenomenon. In each study, the time interval
required for doubling of envelope/periplasmic protein
was much shorter than the cell generation time.
The first membrane proteins found to increase
abruptly in E. coli K12 were cytochrome b, and L-aglycerolphosphate dehydrogenase (Ohki, 1972). The two
proteins appeared to double simultaneously during the
cell cycle, but the cell age at doubling depended upon the
method used for synchronization and occurred at ages of
either 0.6 or 0.2 generations. This dependence of time of
synthesis upon the synchronization method appears to be
a common feature in measurement of abruptly doubling
proteins and suggests that different methods of synchronization perturb the timing of doubling to different
degrees. The following year, using nutritionally synchronized cultures, Shen & Boos (1973) found that the
periplasmic galactose-binding protein was synthesized
only during or near cell division. Within a few years,
several other proteins were found to be produced in a
cycle-dependent manner. These included envelope proteins [the a-receptor protein (Ryter et al., 1975) and
protein D (Gudas et al., 1976; Boyd & Holland, 1977)],
inner-membrane proteins [L-a-glycerolphosphate dehydrogenase (Ohki & Mitsui, 1974) and lactose permease
(Ohki & Sato, 1975)], and another cytoplasmic protein
[the maltose-binding protein (Dietzel et al., 1978)l.
Churchward & Holland (1976) studied the synthesis of
29 envelope proteins in cultures synchronized by the
membrane elution technique. They found that rates of
synthesis of the great majority of these proteins increased
stepwise and that these steps occurred early in the cycle,
(However, one envelope protein, the D protein of M i
76000, was synthesized periodically near the time of
division.) To be a prediction of the linear growth model,
it would seem that abrupt doublings in rates of synthesis
of these surface proteins would be limited by the
numbers of functional transport sites.
Ohki (1979) identified 11 proteins, each of which
doubled 10-20 min before cell division, and also described their cellular locations ; these proteins were
primarily inner-membrane proteins, but a few were
outer-membrane or cytoplasmic. Ohki assigned these
proteins to cell-cycle control A. He assigned the maltoseand galactose-binding proteins and other periplasmic
proteins to cell-cycle control B because these proteins
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604
H . E. Kubitschek
were synthesized 11 min later. His studies clearly
demonstrated that there may be more than one cell-cycle
control mechanism that produces abrupt protein doubling; they suggest, however, no more than two.
Boyd & Holland (1979) also found that the rate of
synthesis of outer-membrane proteins, and in particular
of a 36.5 kDa porin, doubled during the final part of the
cell cycle. Doubling of phospholipid synthesis, observed
by Pierucci (1979) and Pierucci et al. (1981), was
confirmed by Joseleau-Petit et al. (1984), and again later
by the same group (Joseleau-Petit et al., 1987), using the
relatively new technique of automatic synchronization
(Kepes & Kepes, 1980). The confirmation also provides
support for this synchrony technique, which uses a
forcing method rather than selection to obtain synchronization. The abrupt synthesis of the 1-receptor protein
was confirmed by Ohki & Nikaido (cited in Ohki, 1979),
and later, by a very different method, by Vos-Scheperkepter et al. (1984), who showed that the 1amB protein
also doubled before division.
There is little or no evidence that abrupt doubling is
related to DNA replication per se. When DNA synthesis
was blocked, rates of synthesis of total and outermembrane and of cytoplasmic proteins continued to
increase in parallel (Boyd & Holland, 1979). Nor is there
evidence that the control for abrupt doubling operates at
the level of gene expression: while total RNA and trp
mRNA synthesis continued at almost normal rates at a
nonpermissive temperature in the cell-cycle control
mutant tsC42, the mutant failed to synthesize lac mRNA
at the nonpermissive temperature (Ohki, 1979). However, Benner et al. (1985) provided evidence that the
control of the maltose transport system is at the
transcriptional level. Furthermore, results for doubling
of the rate of phospholipid synthesis are compatible with
models in which some event is linked to replication
termination or nucleoid segregation (Joseleau-Petit et al.,
1987).Thus, the mechanism(s)for cell-cycle regulation of
abrupt doubling of selected proteins have not yet been
definitively identified.
The isolation of a cell-cycle control mutant by Ohki &
Mitsui (1974) is an important step in the elucidation of
this mystery. The defect of the tsC42 mutant (divE42)is
phase specific. The divE42 gene functions only about
20 min before cell division and is essential for cell growth
and membrane synthesis (Ohki & Sato, 1975). When
shifted to 42"C, the mutant increased in protein by a
factor of about 1.8 before ceasing growth, and at this
stage the cells were large and homogeneous in size. When
returned to a permissive temperature, cells divided
synchronously after an immediate burst of synthesis of
particular enzymes, such as succinate dehydrogenase. In
synchronous cultures, the cell cycle stage at which mutant
cells were arrested coincided with the time of synthesis of
the particular proteins whose synthesis was controlled by
divE42, leading Ohki (1979) to propose that a defect in
the divE42 gene is involved in both cell cycle regulation
of specific protein synthesis and of cell division. The
defect produced by this mutation was later identified as a
single alteration of the D stem of the isoacceptor serine
t R N A y (Tamura et al., 1983).
Finally, these results for the divE42 gene suggest that
the abrupt doubling of specific proteins in the absence of
gene doubling may also have a role in cell division. Other
evidence has recently been obtained by A. Robin & D.
Joseleau-Petit (personal communication), who found
that theftsZ gene was transcribed bilinearly, but with a
doubling in rate around the time of initiation of DNA
synthesis rather than near the end of the cell cycle; they
used an f t s Z : :lac2 fusion carried on a A prophage in
cultures under automatic synchronization. Thus, the
phenomenon of abrupt doubling of proteins near the end
of the cell cycle may offer crucial information on the
development of cell division, as well as on the regulation
of cell growth.
I thank Fred J. Stevens for his discussion and comments on the
development of this paper.
This work was supported by the US Department of Energy, Office of
Health and Environmental Research, under Contract no. W-3 1-109ENG-38.
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