INTRODUCTION

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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 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.

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.

View larger version (9K): [in this window] [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.

	MATERIALS AND METHODS

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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/pOU420Ap^s (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/pOU420Ap^s cultures, 20 µg of ampicillin and 50 µg of chloramphenicol were added per ml.

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.

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).

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).

	RESULTS

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Underreplication levels during exponential growth. By decreasing the growth temperature of the intR1 strain EC::71CW/pOU420Ap^s, 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.

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Growth curves (A) and DNA/mass ratios (B) of EC::71CW/pOU420Aps. The strain was grown exponentially for at least 10 generations in M9 glucose medium at 39°C (), 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.

View larger version (27K): [in this window] [in a new window]   FIG. 3.   Cell size (light scatter; left columns) and DNA content (fluorescence; right columns) distributions of EC1005 and EC::71CW/pOU420Aps growing exponentially in M9 glucose medium at 39°C (A) and of EC::71CW/pOU420Aps at different times after the shift to 37°C (B) or 38°C (C).

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 replicationthat is to say, an increase in the average initiation masswas detected by flow cytometry during periods of underreplication in EC::71CW/pOU420Ap^s 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.

1

1

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Kinetics of DNA synthesis during replication runout in EC::71CW/pOU420Aps. The strain was grown exponentially in M9 glucose medium at 39°C, and at an A550 of 0.029, part of the culture was shifted to 37°C. At an A550 of 0.29 at 39°C, and 5 h after the shift to 37°C (A550, 0.28), rifampin (200 µg/ml) and cephalexin (50 µg/ml) were added to the cultures (time zero). At different time points, samples were collected and analyzed by flow cytometry. (A) DNA histograms during replication runout at 39°C (left column) and 5 h after the shift to 37°C (right column). The figure shows the distribution of DNA content in samples collected before (time zero) and at the indicated times after addition of the drugs. (B) Progress of the coefficient of variance (CV-half) for the four-chromosome peak during the runout of replication at 39°C () or 5 h after the shift to 37°C (). Notice that more experimental data are taken into account in panel B than are shown in panel A.

Cell division during underreplication. The effects of underreplication levels on cell division were studied in EC::71CW/pOU420Ap^s 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.

View larger version (47K): [in this window] [in a new window]   FIG. 5.   Phase-contrast and fluorescence photomicrographs (A), cell length distributions (B), septum localization patterns (C), and nucleoid distributions (D) for EC::71CW/pOU420Aps. The strain was grown exponentially in M9 glucose medium at 39°C, and at an A550 of between 0.02 to 0.04, the culture was split and downshifted to 38 and 37°C. Three hours after the shift from 39°C (upper panels), 4 h after the shift to 38°C (central panels), and 5 h after the shift to 37°C (low panels), cells from the cultures growing at the different temperatures were collected and fixed in ethanol (70%, final concentration). (A) After DAPI staining (0.5 µg/ml), the cell populations were analyzed by combined phase and fluorescence microscopy (see Materials and Methods). One thousand cells from the total population and 250 septating cells from each culture were analyzed. Bar, 5 µm. (B) Cell length distributions in the total population (continuous lines) and of septating cells (broken lines). (C) Septating cells with (from the top to the bottom of the bars) one (), 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.

View larger version (36K): [in this window] [in a new window]   FIG. 6.   Three-dimensional plot of the length of the two future daughter cells versus the number of cells obtained from length measurements of septating cells of EC::71CW/pOU420Aps growing at 37°C. The data are the same as in the lower panels Fig. 5B (broken lines) and 5D.

	DISCUSSION

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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)/  2^D/] (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.