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Journal of Bacteriology, December 1998, p. 6364-6374, Vol. 180, No. 23
Department of Microbiology, Biomedical
Center, Uppsala University, S-751 23 Uppsala, Sweden
Received 5 May 1998/Accepted 30 September 1998
The key processes of the bacterial cell cycle are controlled and
coordinated to match cellular mass growth. We have studied the
coordination between replication and cell division by using a
temperature-controlled Escherichia coli intR1 strain. In
this strain, the initiation time for chromosome replication can be displaced to later (underreplication) or earlier (overreplication) times in the cell cycle. We used underreplication conditions to study
the response of cell division to a delayed initiation of replication.
The bacteria were grown exponentially at 39°C (normal DNA/mass ratio)
and shifted to 38 and 37°C. In the last two cases, new, stable, lower
DNA/mass ratios were obtained. The rate of replication elongation was
not affected under these conditions. At increasing degrees of
underreplication, increasing proportions of the cells became elongated.
Cell division took place in the middle in cells of normal size, whereas
the longer cells divided at twice that size to produce one daughter
cell of normal size and one three times as big. The elongated cells
often produced one daughter cell lacking a chromosome; this was always
the smallest daughter cells, and it was the size of a normal newborn
cell. These results favor a model in which cell division takes place at
only distinct cell sizes. Furthermore, the elongated cells had a lower
probability of dividing than the cells of normal size, and they often
contained more than two nucleoids. This suggests that for cell division
to occur, not only must replication and nucleoid partitioning be
completed, but also the DNA/mass ratio must be above a certain
threshold value. Our data support the ideas that cell division has its
own control system and that there is a checkpoint at which cell
division may be abolished if previous key cell cycle processes have not
run to completion.
The cell cycles of all organisms
contain key events that occur only once during each doubling in mass,
replication of the genome, separation of the daughter genomes, and cell
division. These events are carefully coordinated with each other and
controlled to match the increase in cell mass. In eukaryotic cells,
there seem to be two independent, alternating processes, genome
replication (S phase) and separation of the daughter genomes (M phase);
each S phase requires a preceding M phase and vice versa
(27).
During the bacterial cell cycle (for reviews, see references
15, 24, 30, and 38), there are
three recognized periods at a slow growth rate, B, C, and D
(20). Chromosome replication occurs during period C, and
nucleoid partitioning and cell division take place during period D. The
length of C plus D can be much longer than the generation time, To address the questions of the number of control points in the cell
cycle of Escherichia coli and their molecular basis, we have
constructed so-called int strains (4, 17, 23,
29). In these strains, a 16-bp deletion in the leftmost 13-mer of
the chromosomal replication origin, oriC, which is crucial
for the initial opening of the origin at initiation of replication, has inactivated oriC (8). Plasmid replicons have been
inserted into oriC. Thereby, chromosome replication is
controlled by the plasmid replicon and independent of the normal cell
cycle controls. The copy number of plasmid R1 is about the same as that
of oriC at moderate and high growth rates (18)
and is essentially regulated by two transcription rates, that of the
mRNA for the rate-limiting initiator protein (RepA) and that of the
antisense RNA (CopA) that negatively controls the efficiency by which
the RepA mRNA is translated (28). We have constructed
intR1 strains in which both of these transcription rates are
controlled by temperature such that the chromosome content is normal at
39°C, lower (underreplication) at lower temperatures, and higher
(overreplication) at temperatures above 39°C (4, 6). This
corresponds to increasing or decreasing the initiation mass, which is
the same as moving the time of initiation of replication to later or
earlier times in the cell cycle compared to the normal situation (see
Fig. 1).
These int strains have been used to study how nucleoid
partitioning and cell division are controlled (1-3, 5, 6,
13). By using the temperature-controlled intR1 strains
under conditions of overreplication, we have been able to show that
cell division does not seem to be triggered by chromosome replication
but rather that it has its own, independent means of control
(3). This system was also used to switch off initiation of
replication, allowing ongoing rounds of replication to proceed to
completion, and afterward to switch on a single or multiple rounds of
replication in order to study the correlation between chromosome
replication and cell division (6). Several other groups have
studied the effects on cell division of inhibition and switching on of
DNA replication by using temperature-sensitive dna
mutants (see reference 20, p. 1635 to 1636).
However, with our intR1 system, it is possible to
study populations growing exponentially at reduced DNA/mass
ratios, i.e., without switching off replication during the experiment. Hence, there will not be any disruption of the cell cycle, and the physiology of the cells will be less disturbed than
it is in the switch-off/switch-on experiments referred to above. This
allows studies of more aspects of the cell cycle, e.g., the correlation
between the timing of initiation of chromosome replication and nucleoid
processing-cell division, the requirement of factors other than
completed replication for cell division, the causes for inhibition of
cell division at increased initiation mass, and the location of cell
divisions in elongated and filamented cells, etc.
We have used the intR1 system to study the effects of
underreplication (reduced DNA/mass ratio) on key cell cycle events
during exponential growth. The ratio between CopA RNA and RepA mRNA
sets the DNA/mass ratio at levels that deviate from the normal value. Shifts from 39°C to lower temperatures lead to exponential growth with new, reduced DNA/mass ratios, because the rate of synthesis of
CopA RNA is increased. A reduction in the DNA/mass ratio corresponds to
movement of initiation of replication to later-than-normal times (Fig.
1). The question of how nucleoid
partitioning and cell division respond to this arises. Will the length
of the D period be kept constant and the cells divide at continuously
increasing sizes as the delay in replication increases
("Under-replication-I" in Fig. 1), or do the cells omit cell
division and wait until they are twice the normal septating size
("Under-replication-II" in Fig. 1)? Our data are essentially in
accord with the latter.
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Effects of Chromosome Underreplication on Cell
Division in Escherichia coli
and
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
. To
accommodate this, there may be up to three overlapping replication
cycles at a rapid rate of growth. Hence, alternations between periods C
and D cannot occur in bacteria as they do in eukaryotic cells.
Therefore, under different growth conditions, replication initiates and
partitioning occurs at different times in the cell cycle and can take
place at any time from birth to cell division. Since there may be
overlapping replication cycles, there cannot be a requirement for
completed nucleoid partitioning before the next replication cycle is
initiated. Cell division does not seem to be required for the two other
key events, and thus, inhibition of cell division leads to the
formation of filaments with 2, 4, 8, 16, etc., separated nucleoids.
Damage to the DNA or other effects that slow down replication induce systems, such as the SOS system, that transiently delay cell division in order to allow replication to be completed before the cell divides.
Inhibition of initiation of chromosome replication reduces the
frequency of, but does not totally block, cell division, which leads to
the formation of elongated or filament cells; the residual cell
division causes the formation of DNA-less cells. This and other pieces
of information indicate that initiation of chromosome replication,
nucleoid partitioning, and cell division have independent control
systems that link these processes to cell mass (for reviews, see
references 15, 24, 30, and 38).
This requires that within a single cell cycle there are checkpoints
that ensure that chromosome replication is finished before nucleoid
partitioning and cell division take place.
View larger version (9K):
[in a new window]
FIG. 1.
Over- and underreplication during the cell cycle. B, C,
and D designate the different cell cycle periods. Under-replication-I
and -II, working hypotheses about the effects of chromosome
underreplication on cell division.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Strains.
The E. coli K-12 strains used in this
study were EC1005 (metB1 nalA relA1 spoT1 
r
F
) (19) and its intR1 derivative
EC::71CW/pOU420Aps (6).
Media and growth conditions. Bacteria were grown in M9 medium supplemented with 0.2% glucose, 50 µg of methionine per ml, and 1 µg of thiamine per ml (M9 glucose medium) (32). To EC::71CW/pOU420Aps cultures, 20 µg of ampicillin and 50 µg of chloramphenicol were added per ml.
The cultures were incubated in thermostatically controlled water baths (Heto) with a maximum deviation of 0.2°C from the set temperature. Growth in mass was monitored by the absorbence of the culture at 550 nm (A550), using a Novaspec II spectrophotometer (LKB). Exponentially growing cultures were obtained from overnight cultures after the appropriate dilution to allow at least 10 generations of growth in fresh medium before the temperature shifts. At the moment of the temperature shifts, the cultures had an A550 of between 0.02 to 0.04, and culture samples for microscopic analysis were taken at an A550 of between 0.25 and 0.45 (3, 4, and 5 h after the shift from the cultures at 39, 38, and 37°C, respectively).Flow cytometry. Flow cytometry was essentially performed as described by Skarstad et al. (35). Samples from the bacterial cultures were collected, and the cells were directly fixed in ethanol by mixing 0.1 ml of a culture growing in M9 glucose medium with 1.75 ml of ice-cold 74% ethanol (70% ethanol [final concentration]). Fixed cells were stored at 4°C. For staining, 7.6 × 106 to 1.7 × 105 cells were pelleted by centrifugation. The cells were washed in 1 ml of cold staining buffer (10 mM Tris, pH 7.4; 10 mM MgCl2), and after centrifugation the cells were carefully resuspended in 70 µl of the same buffer. An aliquot of the cell suspension (65 µl) was mixed with an equal volume of a staining solution (40 µg of ethidium bromide per ml and 200 µg of mithramycin A per ml, dissolved in the staining buffer). The analysis was performed with a Bryte HS flow cytometer using the software Win-Bryte version 1.06 (Bio-Rad). The measurements were carried out with the flow cytometer being triggered on light scattering. Thus, particles below a certain size were not recorded, and in this way it was possible to separate sample debris from small cells and the fluorescence of all particles above the threshold for light scattering was detected. Plastic calibration beads (Bio-Rad) with a diameter of 1.5 µm were used to monitor instrument performance and to standardize the scale for between-sample comparisons.
Cell size (light scattering) and DNA content (fluorescence) distributions obtained by flow cytometry were analyzed either directly (see Fig. 3 and 4) or after obtaining average values in the population for this parameters (see Fig. 2B and Table 1). To calculate the average cell size and DNA content values, the histogram curves were integrated by using the software Win-Bryte version 1.06.Analysis of chromosome replication kinetics by flow cytometry. To analyze the kinetics of chromosome replication by flow cytometry, 200 µg of rifampin and 50 µg of cephalexin were added per milliliter to the cultures and samples were collected at different time points after addition of the drugs. Rifampin blocks the initiation of chromosome replication from oriR1, as well as from oriC, while allowing ongoing rounds of replication to be completed (9, 14). Cephalexin blocks cell division (7, 21). Addition of cephalexin to the cultures at the same time as rifampin allows a correct determination of the number of replication origins per cell (35). Inhibition of replication initiation, but not of elongation, results in cells containing fully replicated chromosomes after the replication runout. The DNA histograms will then display peaks corresponding to fully replicated chromosomes. Therefore, by flow cytometry it is possible to follow the appearance of peaks in the fluorescence histograms during the replication runout. In this way the speed of replication fork movement can be estimated (33, 34).
To compare the rates of chromosome replication progression of different cell populations, the evolution of the coefficients of variance for chromosome peaks during replication runout were analyzed. We determined the coefficients of variance (CVs) for the upper half of the peaks (CV-half values) corresponding to four chromosomes per cell, and these values versus the time of sampling were plotted. The curves showed two parts, a fast decrease in the beginning that was followed by a stabilization at a certain level of resolution. The values for the slope of the first part of the curves and the intersection point, in the x axis, between both parts of the curves were compared for the different populations under study (see Fig. 4B).Microscopic studies. Cells, fixed in the same way as for flow cytometry analysis (70% ethanol [final concentration]) were pelleted and then resuspended in 0.9% NaCl. Aliquots (5 to 10 µl) were spread on microscope slides covered with thin layers of 1% agar containing 0.9% NaCl and 0.5 µg of 4',6-diamidino-2-phenylindole (DAPI) per ml. With this staining technique, extensive nucleoid condensation and fusion are avoided (2, 37).
The cells were analyzed under a Nikon Optiphot-2 combined phase-contrast and fluorescence microscope. The images were digitalized by using a charge-coupled device camera (Sony Instruments) connected to a computerized image analysis system (software and hardware from Bergström Instruments). Cell length measurements, analysis of septating cells, and determination of the number of nucleoids per cell were performed manually from the digitized images, using length measurement as well as profile tools provided with the software. The digitized images were transferred to Adobe Photoshop 3.0 and printed with a dye sublimation printer.| |
RESULTS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Underreplication levels during exponential growth. By decreasing the growth temperature of the intR1 strain EC::71CW/pOU420Aps, it was possible to reduce its normal DNA/mass ratio, which at 39°C was close to that in the parental strain replicating from oriC, and to stabilize new ratios during a period of exponential growth.
EC::71CW/pOU420Aps and its oriC parental strain, EC1005, were grown exponentially in M9 glucose medium at 39°C for at least 10 generations. At an A550 of between 0.02 and 0.04, the cultures were split, and the parental strain was shifted to 37°C and the intR1 strain was downshifted to 38, 37, or 36°C. The growth in mass was monitored by measuring the A550 (Fig. 2A; Table 1). Cells were collected from the cultures growing at 39°C and at different times after the temperature shift to 38, 37, or 36°C, fixed in ethanol, and analyzed by flow cytometry and microscopy (Fig. 2B and 3; Table 1) (see also Fig. 5). The DNA/mass ratios in the cultures at different incubation temperatures were estimated from the flow-cytometric data (Fig. 2B; Table 1).
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), and at time zero different parts of the culture were
shifted to 38°C (
), 37°C (
), or 36°C (
). The growth in
mass was monitored by measuring absorbance
(A550), and the DNA/mass values were obtained
from flow-cytometric measurements (see Materials and Methods). DNA/mass
ratios are expressed relative to the values obtained at 39°C.
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Replication speed during underreplication.
The aim of the
present work was to analyze the effects of a delay of the initiation of
replication on cell division. Although a delay in the time of
initiation of replication
that is to say, an increase in the average
initiation masswas detected by flow cytometry during periods of
underreplication in EC::71CW/pOU420Aps after
temperature downshifts (Fig. 3), a larger C/ ratio could be an
additional cause for the decrease in the DNA/mass ratio (10,
12). If an extension of the C period were the case, a delay in
the time of cell division could be ascribed to the replication period
being longer than in the control situation whenever a completed chromosome replication and correct partitioning are required for cell
division. Hence, it was important to determine the length of the C period.
0.726 min1 at
39°C and 0.794 min1 at 37°C, and the crossing point
was at 57 min in both cases. Similar results were obtained in an
analysis of the two-chromosome peaks (data not shown).
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Cell division during underreplication. The effects of underreplication levels on cell division were studied in EC::71CW/pOU420Aps at different and stabilized DNA/mass ratios. With this analysis, we addressed two main questions regarding the control of cell division in bacteria: when and where cell division takes place.
Three hours after the temperature shifts from 39°C, 4 h after the shift to 38°C, and 5 h after the shift to 37°C, samples from the different cultures were collected and the cells were fixed in 70% ethanol. These cell populations were studied under the microscope after DAPI staining (see Fig. 5A). We analyzed 1,000 cells in the total population, or 250 cells in the case of septating cells, by phase-contrast and fluorescence microscopy and image analysis, and we determined the cell length, position of the incipient septum, and number of nucleoids per cell (see Materials and Methods). Microscope images showed a homogeneous population of cells growing at 39°C, with an average normal cell size distribution and one or two nucleoids per cell (Fig. 5A, upper panel). After the temperature downshifts, the cell size and nucleoid distribution of the population became more heterogeneous. Cultures growing at 38 and 37°C 4 and 5 h after the shift, respectively, contained filamented cells as well as cells of normal size (Fig. 5A, middle and lower panels). At 38°C, many cells were slightly bigger than the average size at 39°C, and some filaments were present. The proportion of filamented cells in the population increased as the replication level decreased. Thus, at 37°C, many real filaments could be observed. Filamented cells frequently contained more than one or two nucleoids, generally having around four, which were clearly separated, and large DNA-free areas. At 37°C, DNA-less cells of normal size were frequently found. Therefore, the abolishment of cell division during underreplication that had been detected by flow cytometry was easily observed by microscopy (Fig. 3 and 5A; Table 1). This effect could be quantified by determining the increase of the average mass/cell ratio and cell length in the population (Table 1).
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), two (
), three (
), four (
), or five and
more (
)
nucleoids. (D) To represent the localization of the septum, the lengths
of the two future daughter cells produced in each septation event were
plotted against each other, with the shorter cell always being chosen
for the x axis. The dotted lines represent different
daughter cell length ratios. Closed circles represent the cells which
would give two nucleated daughter cells after division, and open
circles represent the cells which would give one nucleated and one
nucleoid-free daughter cell. In the latter case, the anucleate cell was
always the shorter one.
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DISCUSSION |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We have used a so-called intR1 strain to vary the initiation mass of E. coli from normal to higher values by performing small changes in growth temperature. Shifting a culture growing exponentially at 39°C to 38 or 37°C resulted in a transition to new stable DNA/mass ratios, namely, about 80 and 60% of the initial value, respectively. This corresponds to a delay in the time of initiation of replication by 15 or 27% of the generation time (see below) (Fig. 1). Under these conditions, the septating cells were of two main classes, those that divided in the middle to give daughter cells of about normal size and those that were twice as big and divided to produce one daughter of normal size and one three times as big (1:3 ratio); some of the divisions resulted in one daughter (always the smallest) being chromosomeless. Our data are in accord with the mode denoted under-replication-II in Fig. 1. Under conditions of underreplication there was a considerable fraction of the cells that were elongated and contained more than two nucleoids. Hence, the direct coupling of nucleoid partitioning and cell division was lost. This suggests that cell division requires not only completed replication and nucleoid partitioning but also a DNA/mass ratio above a certain threshold value. Our main results, obtained under conditions of exponential growth, are similar to those reported by Bernander et al. (6) when cell division was studied after synchronous initiation of replication in the same intR1 strain after runout of replication by shifting exponentially growing populations to 36°C.
It should be stressed that Fig. 1 gives an idealized picture of the cell cycle in int strains, since initiation of replication of the intR1 chromosome is random over time (23); hence, the time of initiation varies within the population, but Fig. 1 describes the average situation. The randomness also explains why there was not a distinct shift from the normal cell division pattern to a new one during underreplication. There will always be some cells in which initiation of replication occurs early and that divide essentially normally.
The delay in initiation of replication caused by the transition from
39°C to 38 or 37°C can be calculated as follows. The average
number of genome equivalents per cell, G, present in an exponentially growing population is determined by the equation G = (/C · ln2)[2(C + D)/
2D/] (11),
in which C, D, and are the lengths of the C and D periods and the generation time, respectively. A delay by x
min of initiation of replication would give the equation G = (/C · ln2)[2(C + D x)/ 2(D x)/].
The values for the delay x cannot be calculated exactly because D is not known. However, the data from Fig. 4 show that the lengths of the C period at 39 and 37°C were approximately the same. Hence, assuming that C and D are 57 (Fig. 4) and 20 min, respectively, the value of x can be calculated to be 10 and 30 min at 38 and 37°C, respectively, or 15 and 27% of the generation times at these two temperatures. It should also be stressed that the measured generation times are overestimates of the real ones because DNA-less cells were formed during underreplication.
During underreplication, the cell length distributions of the septating cells were slightly broader than under normal conditions; this broadening took place only at the large side of the cell length distributions. This indicates that cell division could be postponed when replication and nucleoid partitioning were slightly delayed and that when the delay were longer, cell division would be omitted and would not take place until one doubling in mass later (Fig. 5D). The molecular nature of this putative cross talk between replication-nucleoid partitioning and cell division is unknown. Previous experiments showed that the SOS- and SfiA-mediated division inhibition was not the main reason for the cessation of division that was observed after runout of chromosome replication in this intR1 strain (6). Under these conditions, some of the divisions occurred at twice the normal mother length; in this case, one of the daughter cells was of normal newborn size. This favors the idea that cell division has got its own, independent control system. It had been expected that the cells would divide in the middle. The 1:3 division pattern of elongated cells suggests that there is a size window during which cell division is allowed. The maximum size that still allowed cell division was about 25% bigger than the maximum size of dividing cells at the normal DNA/mass ratio (Fig. 5D). If cell division has not occurred at this size, it is omitted and a second opportunity occurs one mass doubling later. However, the ability to divide in the middle is lost, and cell division now takes place at new sites one-fourth of the cell length from the poles. Hence, cell division in some way seems to measure the distance from one of the poles; according to Donachie et al. (16), cell division at all growth rates initiates at a specific cell length rather than at a specific cell mass. As a matter of fact, even when very long cells divided, one of the daughters was of normal cell size, and although the number of measured cells was small, our data suggest that division occurs at a 1:7 ratio between the lengths of the two daughter cells. It may be appropriate to mention that Raskin and de Boer (31) showed that the MinE protein forms a ring in the middle of the cell very early in the cell cycle. They found that there were two MinE rings in some elongated cells and that these rings were placed one-fourth of the cell length from each cell pole, as if the ring in the middle had disappeared. As a matter of fact, we observed that filamented cells occasionally contained two septa at the cell quarters, with the normal distance to each pole, and that only about 4% of the septating cells at 37°C were twice the normal mother size and still divided in the middle.
Omission of one cell division led to the appearance of elongated cells with more than two separated nucleoids. Hence, there was no tight coupling between completed replication-nucleoid partitioning and cell division; rather, for cell division to occur, not only must cells be of an appropriate size and two separated nucleoids be present, but also the DNA/mass ratio must be above a certain threshold value.
During underreplication, in a significant number of divisions of filamented cells the smallest daughter did not contain chromosomal DNA. Several groups have studied the cell size distribution of anucleate cells formed after total inhibition of DNA replication by using temperature-sensitive dna strains (20, 22, 25, 26, 36). The data are somewhat conflicting; some groups found that the anucleate cell size distribution was narrow and comparable to that of normal newborn cells (22, 36), whereas Mulder and Woldringh (25, 26) reported a more random distribution. In our case, the anucleate daughter cells were of normal size, supporting the idea of a nonrandom positioning of the septa (see also reference 20, p. 1635 to 1636). We have earlier favored the idea that the size of the nucleoid determines the size of the daughter cells (2), but the size of the DNA-less cells contradicts this and strengthens the conclusion that cell division has its own control system also with respect to the position of the division site.
When E. coli is grown in different media its generation time varies. The dimensions of the cells, both diameter and length, increase with increasing growth rate (16, 39). In the present study, however, although there was a reduction in growth rate during underreplication, the cell diameter did not change measurably (0.84 ± 0.08 µm at 39°C versus 0.88 ± 0.09 µm at 37°C) (Fig. 5A). Hence, it appears that the diameter of the cells is set by the medium rather than by the growth rate.
In conclusion, our studies with intR1 strains under conditions of both overreplication (3) and underreplication (this paper) indicate that cell division has its own control system, with respect both to its coupling to the cell mass and to the positioning of the division sites, that is independent of chromosome replication. The nature of this control system is not yet known.
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ACKNOWLEDGMENTS |
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This work was supported by the Swedish Natural Science Research Council and the Swedish Cancer Society. Grants from the Knut and Alice Wallenberg foundation enabled us to purchase the microscope and the image analysis equipment. E.B. acknowledges a fellowship from the Swedish Institute.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, Biomedical Center, Uppsala University, Box 581, S-751 23 Uppsala, Sweden. Phone: 46 18 471 45 26. Fax: 46 18 53 03 96. E-mail: kurt.nordstroem{at}mikrobio.uu.se.
Present address: Departamento de Bioquímica y
Biología Molecular y Genética, Facultad de Ciencias,
Universidad de Extremadura, 06080 Badajoz, Spain.
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REFERENCES |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Journal of Bacteriology, December 1998, p. 6364-6374, Vol. 180, No. 23
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