Effects of chromosome underreplication on cell division in Escherichia coli

Abstract

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 degreesC (normal DNA/mass ratio) and shifted to 38 and 37 degreesC. 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.

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.

FIG. 2

Growth curves (A) and DNA/mass ratios (B) of EC::71CW/pOU420Ap^s. 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.

FIG. 3

Cell size (light scatter; left columns) and DNA content (fluorescence; right columns) distributions of EC1005 and EC::71CW/pOU420Ap^s growing exponentially in M9 glucose medium at 39°C (A) and of EC::71CW/pOU420Ap^s at different times after the shift to 37°C (B) or 38°C (C).

FIG. 4

Kinetics of DNA synthesis during replication runout in EC::71CW/pOU420Ap^s. 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.

FIG. 5

Phase-contrast and fluorescence photomicrographs (A), cell length distributions (B), septum localization patterns (C), and nucleoid distributions (D) for EC::71CW/pOU420Ap^s. 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.

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/pOU420Ap^s growing at 37°C. The data are the same as in the lower panels Fig. 5B (broken lines) and 5D.