Synchronization principle and design of the baby cell column
In our synchronization system, exponentially growing cells are attached to microscopic glass beads that are packed within a chromatography column ( and ; below). Growth medium is pumped through the beads, allowing attached cells to continue to grow and divide as in a normal batch culture. Because of specific features of the system, described below, each attached dividing cell yields one daughter that remains attached to its bead and one daughter that exits the column. An aliquot of released cells collected over a suitably short time interval provides a highly synchronous population of newborn cells, which can be further incubated and/or analysed.
Cell attachment and synchronization principle
This system requires both strong attachment of loaded cells to the growth surface and selective lack of attachment of newborn cells. For attachment, cells are anchored to the glass beads via their flagella. E. coli
flagella have an intrinsic tendency to stick to glass. This tendency was further enhanced by use of a special ‘sticky’ allele of the flagellin gene, fliCst
(), previously used to tether cells onto the surface of glass microscope slides for examination of flagellar movement (Scharf et al., 1998
). When mixed with clean, uncoated glass beads, fliCst
cells spontaneously attach themselves to the bead surface.
For specific release of newborn cells, flagellar synthesis was manipulated. Expression of the fliCst gene was placed under control of the isopropyl β-D-thiogalactopyranoside (IPTG)-inducible tac promoter, with a cognate lacIQ repressor present to give particularly tight repression (; Experimental procedures). Cells are pregrown in the absence of IPTG and then, shortly before the start of the experiment, subjected to a brief pulse of IPTG to transiently induce fliCst expression. This pulse appears to result in the development of one or two flagella in most cells, as suggested by microscopic inspection (data not shown). Given this situation, an attached cell will release a newborn cell lacking flagella at the first or second division on the column. After the induction pulse, cells are attached to a volume of glass beads and then introduced into the column. IPTG is not present in the growth medium used to elute the cells. Use of a pulse of flagellin synthesis is essential to proper functioning of the column; if fliCst expression is constitutive, the initial yield of baby cells is very low, and after two to three generation times the column becomes clogged, as revealed by reduction in flow (data not shown). These effects are presumably attributed to reattachment of flagellated newborn cells to the glass beads. Thus, in our optimal protocol, unwanted overflagellation of cells is avoided by inducing flagellin synthesis in a short pulse, with prior and ensuing repression of the fliCst promoter with the hyperactive lacIq repressor.
The baby cell column () utilizes a standard chromatography set-up with one minor specialized modification. In standard gel chromatography, resistance of the gel bed to the flow of buffer usually generates even (plug) flow of liquid throughout the horizontal plane of the column. However, glass beads comprise a highly porous bed, resulting in a more parabolic flow, i.e. the medium tends to run straight down the centre of the column. To correct for this situation, a 0.2 μm-pore membrane is fitted onto the top adapter, providing a resistance to flow that forces the medium to spread radially before moving down the column (). In our standard the set-up, ~8 ml of cell-coated beads are loaded into a 16 mm chromatography column, which is then attached to a constant-flow peristaltic pump delivering medium at 5 ml min−1. The medium in the reservoir is aerated by brisk stirring. The entire assembly is placed into a constant temperature room to maintain growth temperature. The components for the system are readily available, e.g. off-the-shelf small diameter glass beads, a standard chromatography column and routine laboratory accessories (; Experimental procedures).
Characterization of column yield
To document the basic behaviour of the system, a baby cell column was run in AB proline medium at 30°C, which supports a doubling time of ~153 min for the CM735-derived strain used in this analysis. Under these conditions, cells grow with a linear, ‘eukaryotic-like’ cell cycle, with sequential G1
, S, G2
and ‘M’ phases (i.e. with no more than a single round of replication occurring at any given time). Such conditions allow for straightforward analysis of cell cycle events (below; Bates and Kleckner, 2005
). Beads were loaded into the column and elution was started at 5 ml min−1
. Immediately, a 1 min sample was taken and then again at 5 min intervals thereafter. As 1 min represents 1/153 = 0.65% of the length of a single cell cycle, the sampled cells should all be at very similar stages in the cell cycle.
Column yield over time
The number of cells per unit eluted volume was determined in each of the successively collected samples. Cell yield was initially ~8 × 109 cells min−1 but by t = 40 min dropped to ~2.5 × 108 cells min−1 and then remained relatively steady for two or more division times, excluding minor fluctuations (; below). The initial adjustment period prior to t = 40 min was always seen and likely represents elution of cells that were not tightly attached to beads.
Once the column achieved steady state, two interesting minor fluctuations were detected. First, yields were seen to vary by 30%, in cyclic fashion, with a periodicity of ~150 min, which corresponds closely to the doubling time of cells grown in batch culture under similar conditions (153 min; ). This cyclic variation is explained by the fact that an exponentially growing batch culture, such as the one used here to attach to the glass beads, exhibits an age distribution of two newborn cells (just divided) for every dividing cell (just about to divide) (, inset). Upon addition of the starting exponential culture to the glass beads (about 6 min prior to taking the first sample), all cells became attached to the beads essentially at the same time, and the cells in this attached culture proceeded through the cell cycle at a constant rate. Given the newborn-skewed age distribution of the starting culture, the number of attached cells in the process of division (and thus the number of eluted cells) increased as the attached culture matured, to a maximum exactly one generation time (~153 min) after the initial attachment (~t = −6 min). Thus, the number of eluted cells experienced peaks at generation intervals after attachment (, t = ~150 and t = ~310 min). If the culture attached to the column comprised a perfect exponential population (, inset), the baby cell yield would exhibit a saw-tooth pattern as shown (, theoretical curve; Experimental procedures). The closeness to which the experimental curve matches the theoretical curve implies that the attached cells are growing exponentially, without perturbation, through at least two full division cycles.
A second feature, underlying these fluctuations, is a slow, steady increase in total cell yield as shown by a corresponding increase in the maximum cell yield during the second division cycle as compared with the first (5.7 × 108 and 4.6 × 108 cells min−1 respectively). This characteristic suggests that the column-attached population is growing in size as the column ages, presumably because some newborn cells retain flagella and remain attached to the column rather than eluting. The observed increase in column yield, of about 20% over two generations, would suggest that about 10% of all newborn cells reattached to the column. The cell yield did not further increase after about three generations on the column (data not shown). This feature suggests that the increase observed during earlier generations reflects the reattachment of cells that carried residual flagella produced during the induction period, with no further increase after these flagella were diluted out.
To further examine these fluctuations, we performed a control experiment in which beads were added to the exponential culture in 10 equal (1 g) increments every 15 min, such that cell attachment was spaced out over a period of 150 min. In this protocol, small peaks of newborn cells would be expected every 10 min, which, at the resolution of these experiments, would result in an essentially constant yield of baby cells over time. As expected, after an initial adjustment period, cell yields were generally steady at ~3–4 × 108 cells min−1 and no regular periodic fluctuations could be detected; moreover, a slow gradual increase in total cell yield was again observed, in accord with the envisioned general effect (, red line).
Once the column reaches steady state, the number of cells released by the column during a single generation time corresponds to the number that are both stably affixed to the column and are actively dividing to release newborn cells. For the two generations shown by the data in , this value is 5.6 × 1010 and 6.8 × 1010 respectively. These values are very similar to the total number of cells that are affixed to the column (i.e. the number of cells initially loaded in the column minus the number of all cells eluted through the first 20 min ‘adjustment period’; Experimental procedures). We infer that essentially all of the cells remaining on the column after the adjustment period are actively dividing and releasing newborn cells.
The yield of newborn cells per minute collection time achieved by this method is 3–5 × 108
per 1 min sample (). This is about 10-fold higher than would be achieved by the Helmstetter baby machine under comparable conditions (for comparison, see Helmstetter et al., 1992
). Improvement results from the use of glass beads which, being both three-dimensional and relatively small (180 μm in diameter), present a large total surface area. In our standard set-up, the 10 g volume of beads creates ~1.7 × 103
of growth surface. Moreover, these beads are contained in a total volume of about 8 ml, thus allowing a very high number of cells to be grown in a small volume of medium.
Effective column lifetime
We normally begin collecting cell samples after two generation times, i.e. at t = ~300 min under these conditions, to ensure that all loosely attached cells have been removed, and elution rates have stabilized. The column continued to exhibit the properties described above and cells continued to exhibit good synchrony with respect to all assayed events for at least two doubling times thereafter, i.e. until ~6 h after assembly and for a total of approximately four generation times (below; data not shown). After this point, column yields tended to decay, slowly until t = 9 h and then significantly after 10 h, with collected cells also exhibiting significant loss of synchrony at these late times (data not shown). Deterioration of the column might be attributable either to eventual detachment of flagellated cells (via loss of contact or flagellum breakage) or, deterioration of growth conditions, leading to a less-productive population and/or cell death. We favour the former possibility because cells collected from a 10 h old column exhibited their first doubling at the same time as cells collected from the column soon after it was assembled (data not shown). Had growth conditions deteriorated on the column, an adjustment period prior to the next division would have been expected, with a corresponding delay and/or increased asynchrony in the timing of the next division.
Degree of synchrony of DNA replication in baby cell populations
As one way of assessing the synchrony of cell populations produced by the baby cell column, the timing of DNA replication was examined. A column was assembled under the conditions described above and run undisturbed for 5.5 h. A series of 1 min samples were collected in rapid succession and grown for different amounts of time in the elution medium without further dilution (~3 × 108 cells in 5 ml) at 30°C, thus giving a series of sample populations representing different times after elution from the column. This approach provided a larger number of cells for analysis at each time point than would have been achieved by taking only a single elution sample and removing aliquots at different appropriate times after collection. While not necessary for all analyses, this protocol is useful for some types of experiments, because it permits an unlimited number of time points, each of which can be analysed by a number of different types of assays.
For the current purpose, each sample was analysed for DNA content by flow cytometry, and the fraction of cells exhibiting DNA contents intermediate between 1 (G1 peak) and 2 (G2M peak) chromosomes was determined from the resulting distributions (, S peak; Experimental procedures). In an asynchronous batch culture grown under similar conditions, 24% of cells exhibited 1-chromosome equivalent of DNA, 39% exhibited 2-chromosome equivalents of DNA, and the remaining 37% exhibited intermediate DNA contents, implying that they were in the process of bulk DNA replication (, left panel). As expected, almost no cells exhibited more than two chromosomes at this growth rate. The sample of cells that had just emerged from the baby cell column exhibits a very different distribution (, middle panel): 84% of cells contained a single chromosome equivalent, implying that the large majority of cells had not yet initiated replication and thus were in G1. Of the remaining cells, 9% exhibited two chromosome equivalents and 7% exhibited intermediate DNA contents. Once again, essentially no cells exhibited more than two chromosome equivalents of DNA. Progression of DNA replication was then monitored by determining the percentage of cells showing intermediate DNA contents in the entire series of eluted cultures (). Cells undergoing DNA replication appeared in two dramatic waves that were appropriately separated by ~150 min, the length of a single cell cycle.
Synchrony of baby cell cultures
The area under any such primary curve is proportional to the amount of time that cells spend in the corresponding stage; for a long-lived stage, cells accumulate for a long time and then disappear, whereas for a transient stage, very few cells are ever present in that stage at any given time and the corresponding peak is very small. The lifespan of a stage is given quantitatively by determining the area under the curve (which has units of percentage cells × time) and dividing by the percentage of cells that progress through this stage, to give a duration for the stage (in units of time). If all cells in an eluted sample were behaving as a single synchronous population, the perentage of cells progressing through each stage would be 100%. However, there appears to be a subset of 15–20% of cells that are not part of the main synchronous population, which comprises each primary curve. For example, in the current experiment, ~16% of cells in the t
= 0 sample contained more than one chromosome equivalent of DNA. A similar level of ‘inappropriate’ cells is seen for any stage examined (Bates and Kleckner, 2005
). We infer that these cells represent inefficiencies in cell release and/or elution and that they can be considered as a separate ‘subpopulation’ of essentially asynchronously growing cells. This asynchronous population must be excluded from the calculation of lifespans. Thus, in the current experiment, the areas under the primary curves () were divided by 84%.
The calculated lifespans of the DNA replication periods in the two rounds of replication analysed in were 47 min and 49 min respectively. There is little published data available for cell cycle parameters in E. coli
K-12 strains at 30°C. However, the values obtained here are qualitatively reasonable, as they are within 30% of earlier measurements obtained by flow cytometry analysis of cells of the same background growing at a comparable doubling time but at 37°C (64 min; Allman et al., 1991
Additional information can be obtained by integrating under the primary non-cumulative curves (; Experimental procedures). The resulting cumulative curves describe the percentage of cells that had entered DNA replication as a function of time for each wave. Parallel curves displaced by the lifespan of the corresponding period describe the percentage of cells that had exited DNA replication as a function of time. Cumulative curves for the two successive rounds of DNA replication revealed that they were separated by almost exactly 150 min, as given by the interval between the times at which 50% of cells had entered replication in the two waves ().
Cumulative curves also describe the degree of synchrony of the majority cell population: the steeper the slope the more synchronous the culture. In a culture that was perfectly synchronized (100% of eluted cells are newborn), and in which replication initiation occurred at the same moment after birth in every cell, the cumulative curve would be vertical. In the experiment described here, cells entered the first replication period at the rate of about 10% of cells per 5 min, with 50% of cells entering within a 30 min span (). Strikingly, the cumulative curve for the second wave of replication was only slightly less steep than that for the first wave, implying that the same high degree of synchrony was maintained through at least two cycles. Even tighter synchrony, e.g. with 50% of cells entering a stage in as few as 15 min, has been observed in some experiments (e.g. Bates and Kleckner, 2005
Analysis of cell-length distributions in synchronous cultures
The cell samples analysed for DNA replication in the previous section were also analysed for cell lengths, determined by microscopic observation of 100 cells at each time point (Experimental procedures). In the newborn culture (, bottom panel) mean cell length was significantly smaller, and cell lengths were more tightly distributed (mean = 2.1 μm, SD = 0.3 μm) than in an asynchronous culture grown under similar conditions (top panel; mean = 2.9 μm, SD = 1.1 μm).
Overall, cell mass, and thus cell length, increase linearly with time in the cell cycle, doubling from the time of birth to the time of the next cell division, at which point both mass and length decrease by half. This progression is clearly visible in the baby cell populations (): average cell length increased from a mean of 2.1 μm at t = 0 to a maximum of 4.5 μm at 150 min. At 180 min, the average cell length abruptly dropped to 2.3 μm, indicating that most cells in the culture divided between 150 and 180 min, in accord with the 153 min doubling time of this strain in batch culture. These results further illustrate the overall synchrony of cells in samples from the baby cell column.
Time after cell birth versus cell mass as a time metric
As cell length increases essentially linearly with time during the cell cycle, many previous studies have analysed the relative and absolute timing of events by using cell length as a time metric. In the baby cell column approach, the time metric is ‘time after emergence from the column’, which corresponds by definition to the time after the previous cell division or ‘birth’. We were interested to compare temporal resolution provided by the two approaches. For this purpose, we examined the appearance and disappearance of cytologically detectable foci representing the replication origin, oriC
, as revealed by fluorescence in situ
hybridization (FISH) and of foci representing complexes of DnaX protein, the beta-clamp subunit of the replicative polymerase of E. coli
, as revealed by immunostaining of a DnaX-GFP fusion protein. Primary data were provided by a study described in detail elsewhere (Bates and Kleckner, 2005
When the cell types were plotted as a function of time after elution from the column, a clear progression was revealed (): cells were born with a single origin focus and no DnaX foci (1,0; open circles); a single DnaX focus then appeared (1,1; light grey circles); origins then separated (2,1-A; red circles, left peak); left and right replisomes and their corresponding DnaX foci then separated (2,2; dark grey circles); one DnaX focus then disappeared (2,1-B; red circles, right peak) and the second DnaX focus disappeared (2,0; green circles). Early classes then reappeared as the cells entered the next cell cycle. The (1,1) (2,2) and (2,0) classes were prominent, implying that they were long-lived; the (1,0) and the two (2,1) classes were less prominent, implying that they were more transient. These same differences are also reflected quantitatively in the lifespans of the different stages (figure legend of ). For cells in the final stage (2,0), an appropriate cumulative curve showed that 50% of cells entered this stage within a span of ~21 min (, dashed green line), implying a satisfactory high degree of synchrony in this particular experiment. Additional data suggest that the baby cell column permits temporal resolution of events that are separated by an interval as short as 5 min (Bates and Kleckner, 2005
Evaluation of cell length as a determinant of cell age
For comparison, all of the cells in all time points were pooled and binned according to cell length (, top). Then, for each bin, the percentage of cells having each of the six (origin, DnaX) phenotypes was plotted as a function of the mean cell length of that bin (, bottom). The general tendency for the prominent (1,1) (2,2) and (2,0) classes to occur in succession can again be seen. However, stages and relationships involving the three more transient stages are not discernible: the relationship between the transient (1,0) stage and the ensuing (1,1) stage is obscured and (2,1) cells are so broadly distributed at such a low level that no specific peak can be defined. These findings show that use of the baby cell column permits higher temporal resolution of sequential events, making it possible to reveal stages that are too closely spaced and/or too transient to be detected in asynchronous cultures with cell length as the time metric.
Timing of replication and related events is more tightly coupled to the prior cell division than to cell mass
These findings also have a further, more fundamental, implication: as cell length is exactly indicative of cell mass, our results imply that the timing of origin separation and DnaX appearance and disappearance are more tightly linked to the timing of the prior cell division than to absolute cell mass. Moreover, additional studies, involving comparisons of timing at different growth rates, further suggest that the assayed origin and DnaX events are linked to the DNA replication cycle (Bates and Kleckner, 2005
). This finding is interesting because it has often been proposed that replication initiation is linked specifically to absolute cell mass (e.g. Donachie and Blakely, 2003
). Our results would suggest, in contrast, that replication initiation is more tightly temporally coupled to the prior cell division than to cell mass.
To provide more direct evidence for this possibility, we analysed the available data in another way, using cumulative curves. The steepness of a particular cumulative curve reflects not only degree of synchrony within the population but also uniformity in the time of occurrence of an event following division (above). Thus, for a given population, two events whose timing is more or less tightly linked to the prior cell division will show, respectively, steeper or shallower cumulative curves. The cumulative curves for onset of DNA replication are quite steep, with 50% of cells entering S phase within a 29 min span in both the first and second rounds (, bottom, black curves). In fact, the cumulative curves for most events analysed thus far exhibit very similar slopes (Bates and Kleckner, 2005
) as seen not only for DNA replication but also for the origin and replisome changes described above, other chromosomal events, and division-related events such as appearance of a visible septum and the occurrence of division itself (Bates and Kleckner, 2005
). By implication, all of these events occur with similarly tight coupling to the preceding cell division. To similarly assess the linkage of cell mass to the timing of the prior division, we derived a cumulative curve for entry of cells into the ‘stage’ at which they had reached a length of 3 μm or longer (3 μm was chosen arbitrarily; it represents the average length of a cell that is about halfway through the cell cycle). The primary curve shows that cells accumulate in this ≥3 μm stage more gradually than they accumulate in the DNA replication stage and then exit at the time of cell division (~153 min; , top; gold curve). Correspondingly, the cumulative curve for this stage has a much shallower slope than that for initiation of DNA replication, with 50% of cells achieving 3 μm in length within a 58 min span (, bottom; gold curve). The cumulative curve for the second round of DNA replication (after cell division) is nearly as steep as the first round, therefore the shallow slope of the ≥3 μm curve is not attributed to a deterioration of synchrony in the cell populations (, bottom). We conclude that the time of initiation of DNA replication is more tightly coupled to the time of the prior division than is the time at which the cell reaches a particular length (mass). This possibility is also supported by other lines of evidence and is in accord with the predictions of a new model for control of replication initiation in which the act of cell division licenses the occurrence of the next round of replication initiation (Bates and Kleckner, 2005
Extensions and future prospects
One advantage of using glass beads as a matrix for cell attachment is the ease with which the scale of the system can be increased. In theory, scale is limited only by the capacity of the column and the ability to pump medium through the column at a sufficiently high rate, with appropriate redistribution across the upper surface of the bead matrix. Scaling up will be advantageous for some types of analyses. The set-up described here produces ~3 × 108
in a 5 ml volume (above). However, some experiments, e.g. DNA footprinting, require very large amounts of cells (~109
, Cassler et al., 1995
), thus necessitating pooling of several samples even at the relatively high current yield. Preliminary experiments have been carried out using a column of 5 cm in diameter and 50 g of glass beads, as compared to the 1.6 cm column and 10 g of beads used in the current set-up. The larger assembly resulted in a two- to threefold increase in cell yield per minute of collection time that, while still an improvement, was somewhat less than the factor of five that might have been expected. Resulting cells were still highly synchronous, but were slightly less synchronous than seen with the smaller column as judged by flow cytometry analysis. Both of these deficiencies are attributable to inefficient flow of medium through the wider column and we are currently working on a solution to this problem.
Analysis of fast growth rates
The baby cell column can be run with virtually any type of growth medium. This includes rich media such as Luria–Bertani broth, although flow rates must be increased substantially under these conditions to maintain steady-state growth of attached cells (data not shown). This requirement is likely attributed to a greater need to exchange spent media within the column, which contains less oxygen and a higher concentration of metabolic wastes under conditions of fast growth. The flexibility of the baby cell column with respect to growth medium also represents an additional improvement over the Helmstetter baby machine, because the poly-D-lysine-mediated attachment of cells to nitrocellulose membranes used in that method is highly sensitive to the presence of charged molecules in the media, such as casamino acids (Helmstetter and Cummings, 1964
) and thus is not so suitable for analysis of fast growth rates, at least in E. coli
K-12 where this attachment method is required (Introduction
). It will be very interesting to understand the relationship between patterns observed under slow-growth (linear cell cycle) conditions (e.g. Bates and Kleckner, 2005
) and events under rapid-growth conditions, which result in multiple overlapping replication cycles.
Applicability to other strains of E. coli and to other bacteria
The baby cell column should be easily applicable to any strain of E. coli.
The only requirements are: (i) presence of a complete and fully functional flagellar assembly, which can be determined by a standard motility assay (Berg, 2004
); (ii) integration of the Ptac-fliCst; lacIQ
construct somewhere in the genome, which can be accomplished by homologous recombination without the need for linked antibiotic resistance markers (Experimental procedures
); and (iii) absence of any untoward tendency for ‘stickiness’ of the overall cell surface. We have performed successful baby cell experiments in both CM735 (this study; Bates and Kleckner, 2005
) and W1485 strain backgrounds, which were chosen primarily because they exhibit particularly good motility. Additionally, both strains perform well in flow cytometry experiments for analysis of DNA replication (e.g. Allman et al., 1991
). Most other common laboratory strains (e.g. MG1655) also meet the above requirement and thus should be amenable to analysis via the baby cell column. This approach should be equally applicable to relatives of E. coli
(e.g. Salmonella typhimurium
) that are flagellated and exhibit common requirements for gene expression. Where necessary, external stickiness attributed to extracellular peptidoglycan can be eliminated by introduction of a gal-
mutation; in fact, homologous integration of the Ptac-fliCst; lacIQ
construct has exactly this effect.
Possible extension to other organisms
It is intriguing to consider the possibility that an analogous approach, involving elution from a glass bead column, might also be applicable to microorganisms without flagella or to other types of cells in dispersed cell culture by using an alternative method for attachment of the starting population. For example, cells marked with one or two antibodies to a cell surface component could be attached to beads coated with an appropriate secondary antibody. Dilution of the primary antibody by cell growth would yield newborn cells that could no longer attach to the column. Such ‘crossover’ applications have been demonstrated for the Helmstetter baby machine, which has been applied to yeast (Helmstetter, 1991
) and, more recently, even to mammalian cells (Thornton et al., 2002