INTRODUCTION

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The bacterial cell cycle incorporates two important, discontinuous processes, DNA replication and cell division or septation. Many of the genes and proteins involved in these processes have been identified and characterized previously. In general, they are highly conserved across the eubacterial subkingdom, though much of what is known has been derived from studies of Escherichia coli (recently reviewed in detail in references 18, 26, and 29). During each cell cycle, the chromosome, usually in the form of a single circular molecule, needs to undergo a single complete round of replication. Regulation is mainly exerted at the level of initiation, and over a range of growth rates, this seems to occur when the cell attains a critical mass, relative to the number of copies of the origin of replication (oriC) it contains. Originally, this "initiation mass" (M_i) was thought to be constant (5), but more recent measurements suggest that it decreases slightly with increasing growth rate (52).

Following initiation, DNA replication proceeds bidirectionally from oriC. At all reasonably rapid growth rates (doubling time, <100 min), the time taken to replicate the chromosome (the C period) is more or less constant, at about 40 min. When the forks meet, in the terminus region, diametrically opposite to oriC on the E. coli chromosome map, termination occurs. This is followed by decatenation of the interlinked daughter chromosomes and, if necessary, resolution of chromosome dimers which may have arisen by homologous recombination.

During the remainder of the cycle, the chromosomes need to move apart (partition) so that when division occurs, they are segregated into the daughter cells. The mechanism of partitioning is poorly understood (reviewed in reference 50). One school of thought supposes that the nucleoids are moved apart by a relatively passive mechanism, mediated by the many transient interactions that occur between the DNA and the cell envelope, as a result of the coupled transcription and translation of secreted proteins. An alternative view is that partitioning is effected by an active, specific partitioning machinery, perhaps analogous to the mitotic apparatus of eukaryotic cells.

The period between the termination of DNA replication and septation, sometimes known as the D period in E. coli, is also relatively constant, at about 20 min (18). During this period, the cell prepares for division by assembling a multicomponent "divisome" comprising several proteins. The major component of this assembly is a tubulin-like protein, FtsZ, which forms a ring at the site of septation that is thought to contract to bring about division (reviewed in reference 26). The extent to which the division and DNA replication cycles are coupled or interconnected is not yet clear (34).

Aside from E. coli, little work has been done on cell division in any other bacterium except Bacillus subtilis. An important advantage of B. subtilis for cell cycle studies lies in its ability to sporulate. This occurs in response to starvation, and it begins with a reorganization of the cell cycle to produce a modified, asymmetric cell division (10, 45). Thus, the mechanisms controlling both the timing and positioning of the septum are under inducible control. The few studies that have been done on cell cycle progression in vegetatively growing B. subtilis (e.g., see references 2, 19, and 32) have detected only small differences from the E. coli paradigm. The most overt of these lies in the tendency of sister B. subtilis cells to remain connected in cell chains for a significant but variable period after formation of the division septum (19, 36). Thus, the D period of B. subtilis, as formally defined, is highly variable.

Recently, the subcellular localization of many proteins involved in cell division of both E. coli and B. subtilis has been determined by application of newly developed cytological methods (reviewed in reference 42). In the case of B. subtilis, interpretation of the results of these experiments would be greatly facilitated by a better knowledge of cell cycle progression in this organism. Several of the methods routinely used to measure cell cycle parameters in E. coli are unsuitable for B. subtilis because of the tendency of the cells of the latter to form chains. However, this problem can be overcome by the use of fluorescence microscopy (15). This work describes an analysis of cell cycle progression of B. subtilis under four different growth conditions. Several similarities and differences between cell cycle dynamics in B. subtilis and E. coli have been detected. The availability of four different well-characterized growth conditions should be of considerable use for future studies of the regulation and subcellular assembly of the machineries responsible for DNA replication, chromosome partitioning, and cell division.

	MATERIALS AND METHODS

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Bacterial strains. B. subtilis SG38 trpC2 amyE (11) was used for all the experiments described in this work. Strain L5481 dnaB19(Ts) xin-15 flaD1 (49) was used for the internal standard of DNA content in the digital image analysis. Strain 1.5 (trpC2 spoIIAC1 [12]) was used to prepare chromosomal DNA for use as a standard for the quantitative DNA-DNA hybridization.

Media and growth conditions. S medium contained (NH₄)₂SO₄ (0.2% w/v), K₂HPO₄ (1.4%), KH₂PO₄ (0.6%), sodium citrate (0.1%), Mg₂SO₄ (0.02%), MnSO₄ (0.056%), and glucose (0.5%). TS medium was S medium supplemented with L-glutamate (0.5%) and Difco yeast extract (0.001%). CH medium (which contains 10% casein hydrolysate) was as specified in detail previously (32a; modified as described in reference 35). CHG medium was CH medium containing glucose (0.5%). All media were supplemented with tryptophan (20 µg/ml). A single colony of SG38 was inoculated into 5 ml of each medium, diluted 5,000-fold into a further 5 ml, and incubated overnight at 30°C. For S medium, the overnight culture was supplemented with 15 µl of CH medium to prevent sporulation. In the morning, each culture was diluted back in fresh prewarmed medium to an optical density at 600 nm (OD₆₀₀) of 0.1. The cultures were incubated with shaking at 37°C until they reached an OD₆₀₀ of 0.7 to 0.8, at which point they were harvested for DNA analysis or fixed for microscopy. A plot of log OD₆₀₀ against time was used to ensure that the cultures were growing exponentially when harvested (not shown).

Measurement of cell length, cell width, nucleoid length, and DNA content by digital image analysis. The microscopic methods of Hauser and Errington (15) were used. Briefly, the cells were fixed with ethanol and the DNA was stained with DAPI (4',6-diamidino-2-phenylindole). Phase contrast and epifluorescence images of fields of cells were captured and analyzed electronically. To randomize the sampling, all cells within a field, except those in aggregates were analyzed. Cell length and cell width were measured directly from images. The DNA content of nucleoids in individual cells was measured by its fluorescence intensity relative to that of standard nucleoids containing single complete chromosomes. By this method, DNA content is underestimated because the DNA is more condensed in the sample nucleoids than in the standard nucleoids. The approximate extent of this underestimation is given by the ratio of the maximum pixel brightness of the nucleoid to that of the standard nucleoids (15). This ratio (1.2 to 1.5) was calculated for each field of cells from the mean maximum pixel brightness of all of the nucleoids (standard deviation, ~10%) and was used as a correction factor for the DNA content measurements. The average of the measurements obtained did not differ significantly from the average DNA content of the population measured by a direct chemical method (see below). The images in Fig. 5 were obtained as described by Glaser et al. (13).

Preparation of chromosomal DNA, probes, and filter hybridization. The cell samples were lysed and chromosomal DNA was extracted as described previously (15). For preparation of the standard DNA from strain 1.5, a 150-ml culture of the strain was induced to sporulate, as described by Partridge and Errington (35). Twenty-two hours after the initiation of sporulation, the sporlets (representing about 95% of the population) were harvested and isolated by differential centrifugation, as described by Magill and Setlow (27). The sporlets were lysed and their DNA was purified as described for the DNA samples from vegetative cells. The chromosomes of sporlets are generated by a mechanism similar to that of spores (41, 55) and so are expected, like spores (16), to contain single completed chromosomes, but their DNA is much easier to extract than that of mature spores. The concentrations of the chromosomal DNA stocks were estimated by absorbance at 260 nm, and then they were diluted in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) to a final concentration of 50 µg/ml. For filter hybridization (17), the DNA stocks were further diluted in denaturing buffer (1 N NaOH, 3 M NaCl in TE buffer), such that 20 µl contained 0.15 µg of DNA. (Samples of 0.05 µg of DNA were also hybridized to check that the probe was saturating.) The DNA was applied to a nylon membrane with a dot blot apparatus, denatured with 0.5 M NaOH-1.5 M NaCl (2 min), and then neutralized with 0.5 M Tris-Cl-1.5 M NaCl, pH 7.5 (5 min). The filter was briefly immersed (5 s) in 2× SSPE (1× SSPE is 0.18 M NaCl, 10 mM NaH₂PO₄, and 1 mM EDTA [pH 7.7]) and baked at 80°C for 120 min. After soaking in 6× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), prehybridization was for 2 h at 42°C in 5× SSPE, 50% (vol/vol) formamide, 0.001% (wt/vol) sodium dodecyl sulfate (SDS), 0.001% (wt/vol) Ficoll, 0.001% (wt/vol) polyvinylpyrrolidine, and sonicated veal thymus DNA (0.1 mg/ml). Hybridization was done in the same buffer containing the appropriate labelled probe. Table 1 shows the primers used in the PCRs to generate the 3-kbp probes from various regions of the B. subtilis chromosome. The probes were radiolabelled by using the Random Primed DNA Labelling Kit (Boehringer Mannheim) and [-³²P]dATP (Amersham). Following hybridization, the filters were washed twice for 15 min (ambient temperature) in buffer 1 (2× SSC, 0.1% SDS) and once for 30 min (65°C) in buffer 2 (5× SSC, 0.1% SDS). The membrane was cut into rectangles, each containing a single DNA dot, and placed into 2 ml of H₂O in a scintillation vial. Samples were counted for 5 min in the energy window for ³H (Cerenkov effect).

Determination of DNA content in cultures. The biochemical quantitation of the total DNA content of cultures was as described previously (15). The cell concentration was determined by directly counting ethanol fixed cells in a Petroff-Hausser counting chamber (average of three estimations). Each estimation was based on the average number of cells in 20 ruled squares, each with a volume of 50 pl.

Calculation of the cell length distributions. The distribution of cells as a function of cell length was modelled according to a newly derived model developed for this work (see the Appendix). The major difference between the new model and the Collins-Richmond approach (4) is that we assume that the spectra of lengths at birth and septation do not overlap significantly. It is known that in E. coli there is a narrow distribution of cell lengths at septation, and overlap is, therefore, unlikely. This allows the probability of a cell having a given length at birth to be calculated directly by using equation 8 in the Appendix; no iteration is required.

l/

Determination of average DNA content at any point in the cell cycle. We assume that an "ideal" cell is born with the average birth length, , and with average DNA content at birth, y_o chromosome equivalents, and that it septates (after a period of time equal to the generation time, ) with length equal to the average septation length, 2, and average DNA content, 2y_o chromosome equivalents. Migration of DNA replication forks are assumed to occur linearly with time, at a rate determined by the C period, _C. If no chromosomal growth occurs between termination of replication and initiation (i.e.,   _C), then the DNA content of an ideal cell as a function of time, y(t), can be calculated as follows: If dichotomous replication occurs (i.e., _C/2    _C), then:

Calculation of average cell length at nucleoid transition. The percentage of the cell population in each nucleoid state class (mononucleate monolobed, mononucleate bilobed, binucleate monolobed, binucleate bilobed, and tetranucleate monolobed) was calculated directly from the measured data. The cell length at which the transition between any two consecutive nucleoid states occurs was estimated by calculating (from the best fits [see the Appendix] shown in Fig. 1) the cell length at which the cumulative probability of a cell having a cell length less than the transition length is equal to the cumulative probability of a cell existing in the preceding state.

View larger version (26K): [in this window] [in a new window]   FIG. 1.   Characterization of exponentially growing cells in CHG (A), CH (B), TS (C), and S (D) media by digital image analysis. The cells were categorized according to cell length (frequency within the population) and to nucleoid conformation and number (lightest to darkest bars, monucluceate, mononucleate bilobed, binucleate, binucleate bilobed, and tri- or tetranucleate, respectively). The solid and dotted lines show ideal cell length distributions calculated as described in the Appendix, using values for the normalized variance of the birth probability distributions, (l/)2, of 0.03 and 0.06, respectively. Data are from >400 individual cells from each medium.

	RESULTS AND DISCUSSION

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Measurement of cell cycle parameters in four different media. To determine the average cell cycle for B. subtilis cells growing in various media, affording generation times between 30 and 73 min, we used the light microscopic methods of Hauser and Errington (15). Some of the data for one medium, CH, were described previously (15). In Fig. 1 and 2, data from 200 or more individual cells growing in each of the four different media are summarized. For each cell the state of the nucleoids (number and conformation) were compared with cell length and DNA content per cell (see Materials and Methods). To check the accuracy of the fluorimetric DNA estimations, we measured the DNA concentration of each culture by a direct chemical method and, having determined the cell number by direct microscopic counting, calculated the average DNA content per cell. As shown in Table 2, the values obtained differed only by about 10% from the average DNA content per cell obtained by fluorescence microscopy. An error of this magnitude would have little effect on the cell cycle modelling described below.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Relationship between DNA content and cell length for populations of cells grown in CHG (A), TS (B), and S (C) media. DNA content (expressed as chromosome equivalents per cell) was measured and plotted against cell length. The standard regression line for each plot is shown. Data are from about 200 individual cells from each medium.

Measurement of the B. subtilis C period. The ratios of chromosomal markers close to oriC and terC in the various cultures were determined by quantitative DNA-DNA hybridization (Table 3), and these values were used to calculate C periods (Table 3). The results were similar to each other, from 50 to 58 min, and were close to previously published values (8). It is interesting that the C period of B. subtilis is significantly longer than that of E. coli (about 42 min over this range of generation times [18]). Thus, DNA replication forks in B. subtilis apparently migrate at only about four-fifths of the rate in E. coli.

Cell cycle models. To derive models for cell cycle progression we needed to determine, in addition to the generation time and C period, the average length and DNA content at birth. For the cell length at birth, the data described above were treated with an algorithm developed for this purpose, which is described above and in the Appendix. The solid and broken lines shown in Fig. 1 show predicted ideal cell length distributions, using two possible values for the only free parameter, the normalized variance of the birth probability distribution (0.03 and 0.06). As shown, variation of this parameter had little effect on the shape of the plot. For the purposes of all subsequent analyses, a value of 0.03 was used.

View larger version (25K): [in this window] [in a new window]   FIG. 3.   Models for cell cycle events progression in B. subtilis growing at four different rates. Shown are the major events during the cell cycle: birth (b), initiation (i), termination (t), binucleation (bi), and septation (s). Dots indicate the initiation of DNA replication. The cell length is shown on the scale below the models.

Initiation of replication. The initiation of DNA replication is a key event in the bacterial cell cycle. The point in the cell cycle at which initiation occurred was calculated from the average DNA content at birth and the C period (Table 2). In all cases the observed DNA content of cells at the initiation length was consistent with the predicted value (compare Fig. 1 and 2 with Table 2). For cells in which the generation time was greater than or equal to the C period (S or TS media), initiation of DNA replication occurred in fully replicated chromosomes. The DNA content of such cells was two chromosome equivalents rather than one, because the products of the previous round of DNA replication did not segregate into separate cells until later in the cycle. It is interesting to compare this with E. coli, in which slow-growing cells (e.g., doubling time, 70 min) tend to initiate DNA replication soon after birth. Termination then occurs soon enough in the cycle for the nascent sister chromosomes to segregate immediately at the forthcoming division (18). B. subtilis thus tends to maintain a higher chromosomal copy number than E. coli.

Termination of DNA replication. The bidirectional replication of the B. subtilis chromosome ends when the two replication forks meet in the terminus region, located approximately opposite to the replication origin (57). The timing of termination and the cell length at which this occurs are determined by the time of initiation of replication and the duration of the C period (18). Therefore, it varied considerably from medium to medium (Table 2). For more slowly growing cells (with a generation time less than or equal to the C period) termination should coincide with the cell's attaining a DNA content of two chromosome equivalents. Thus, in cells growing in TS medium, in which the C period is more or less equal to the generation time, initiation and termination should occur almost simultaneously. In S medium, in which the generation time is well in excess of the C period, a significant gap should occur between termination of one round of replication and initiation of the next. During this interval the DNA content should remain constant at two chromosome equivalents, until the initiation mass is achieved. However, we did not detect a clear gap in the S medium cell cycle, presumably either because of the natural variation in the population of cells or the relative insensitivity of our measurements. For cell populations growing in CH and CHG with generation times less than the C period, termination of replication occurred after the succeeding cell cycle had started and, hence, at a DNA content in excess of two chromosome equivalents.

The D* period. In E. coli it has been shown that there is a relatively fixed interval (the D period) between the termination of a round of DNA replication and cell separation (18). Measurement of this period in B. subtilis is more complicated because septation and cell separation do not coincide; indeed the time between these two events varies markedly under different growth conditions (19). It seems that most of the variation lies in the time taken to hydrolyze the thick peptidoglycan layer (typical of gram-positive bacteria) that binds the sister cells together after septation. We have therefore found it useful to use the term D* for the period between termination of DNA replication and formation of the corresponding division septum (15). Our present data show (Table 2) that the D* period was relatively constant in three of the media tested but that, as was the case for the L_i, it was somewhat reduced in CHG medium. The possibility that the link between termination of DNA replication and septation is less tight in B. subtilis than in E. coli is supported by the observation (7a, 43, 56) that a delay in DNA replication in B. subtilis has much less of an effect on septation than that in E. coli (3).

Nucleoid separation. While measuring cell length and DNA content, we also recorded nucleoid appearance and number per cell (Fig. 1). At all growth rates tested, the cells showed either one or two discrete nucleoids (i.e., were mononucleate or binucleate), with the exception of a few of the largest cells in CH or CHG. In general, it appeared from the distributions shown in Fig. 1 that the transition from one to two nucleoids occurred progressively later in the cell cycle as the growth rate increased. Thus, in S medium, the cells were mononucleate throughout most of the cycle, whereas in CH and CHG, the cells were mainly binucleate. The average point of transition from the mononucleate to the binucleate state was determined for each medium as described in Materials and Methods. In the faster-growing cells (CH or CHG medium), binucleation occurred at or soon after termination, in accordance with previous observations for E. coli (6). However, in more slowly growing cells (S or TS medium), there was a significant delay between the two events. Interestingly, nucleoid separation (binucleation) occurred at a cell length of about 3 µm in both of these media, which was similar to the length at binucleation in CH medium (Table 2). It seemed possible that the failure to observe nucleoid separation in cells of less than 3.0 µm might reflect the minimum length of the nucleoid. To determine the length of a nucleoid corresponding to a single unreplicated chromosome we used a dnaB(Ts) mutant. At the nonpermissive temperature, this mutant completes ongoing rounds of DNA replication but does not initiate new rounds (49), so the cells arrest with single completed chromosomes. (Such cells were used as a standard for the DNA content measurements discussed above.) The average length of the nucleoid in these standard cells was 1.5 ± 0.25 µm (200 nucleoids measured). This value suggests an obvious explanation for our failure to detect nucleoid separation in cells of <3.0 µm.

Nucleoid length. Woldringh and coworkers have argued that chromosome partitioning is a relatively passive process driven by multiple transient associations between the DNA and the cell envelope, mediated by the coupled transcription and translation of secreted proteins (summarized in reference 54). In support of this view, they showed that in E. coli the nucleoids increase in length smoothly and continuously during growth (48). To test whether nucleoid expansion was also continuous in B. subtilis, we measured the total length of the nucleoid in the images of cells grown in the four different media and plotted these relative to cell length (Fig. 4). As for E. coli, a clear linear relationship was obtained in all four media. Thus, overall movement of the nucleoid in both organisms appears to be continuous.

View larger version (17K): [in this window] [in a new window]   FIG. 4.   Relationship between nuclear length and cell length over a range of growth rates for cells growing in CHG (A), CH (B), TS (C), and S (D) media. For each plot standard regression lines are shown. For binucleate cells, nuclear length corresponds to the sum of the two nuclei. Data are from 200 cells from each medium.

Nucleoid bilobation. Support for an underlying active partitioning mechanism was obtained from the observation of cell cycle-associated changes in nucleoid morphology. Figure 5 shows a compilation of images of cells at different points in the cell cycle, from each of the four different media. In general, small (i.e., newly separated) nucleoids had a relatively uniform oval shape. However, larger nucleoids tended to appear dumbbell shaped or bilobed. The frequency of bilobation was greater at higher growth rates: at the lowest growth rate in S medium, only 20% of the nucleoids were bilobed, whereas at the highest growth rate tested (CHG medium) most of the nucleoids were bilobed (Fig. 1). The time of transition to a bilobed state was measured for all four cell populations and related to cell length and DNA content, as shown in Table 2. For cells growing with doubling times greater than the C period, the nucleoids became bilobed shortly before they divided to produce two separate nucleoids in a binucleate cell. It is possible that in these cells the bilobed appearance was due to the presence of two completed but still overlapping nucleoids. As discussed above, complete nucleoid separation is probably delayed as a function of the overall dimensions of the cell at these low growth rates. However, in larger cells, growing with generation times of less than the C period, bilobation occurred relatively early in the cell cycle and well before termination of DNA replication. From the average DNA contents at the transition to the bilobed state (Table 2) it can be deduced (46) that the ongoing rounds of DNA replication would have progressed to about 60 (CH) or 75% (CHG). We suggest that in these faster-growing cells, at least, the bilobed conformation is a consequence of the active separation of the partially replicated daughter chromosomes. If so, the data indicate that this partitioning process is in progress or complete well before the round of DNA replication finishes. The bilobed state probably reflects the active separation of the oriC regions of the daughter chromosome segments, which is mediated in part by the Spo0J-dependent chromosome partitioning system (13, 24, 25, 51).

View larger version (39K): [in this window] [in a new window]   FIG. 5.   Compilation of representative images of the different cell types found in four different growth media S (A), TS (B), CH (C), and CHG (D). DAPI fluorescence images are overlaid on phase-contrast images. The nucleoids are the brightly stained objects within the dark cell bodies. All of the images are at the same magnification. Scale bar, 2 µm.