The Escherichia coli baby cell column: a novel cell synchronization method provides new insight into the bacterial cell cycle

Abstract

We describe a new method for synchronizing bacterial cells. Cells that have transiently expressed an inducible mutant 'sticky' flagellin are adhered to a volume of glass beads suspended in a chromatography column though which growth medium is pumped. Following repression of flagellin synthesis, newborn cells are eluted from the column in large quantities exceeding that of current baby machine techniques by approximately 10-fold. Eluted cultures of 'baby cells' are highly synchronous as determined by analysis of DNA replication, cell division and other events, over time after elution from the column. We also show that use of 'minutes after elution' as a time metric permits much greater temporal resolution among sequential chromosomal events than the commonly used metric of cell size (length). The former approach reveals the existence of transient intermediate stages that are missed by the latter approach. This finding has two important implications. First, at a practical level, the baby cell column is a particularly powerful method for temporal analysis. Second, at a conceptual level, replication-related events are more tightly linked to cell birth (i.e. cell division) than to absolute cell mass.

Fig. 1. Cell attachment and synchronization principle

A. Synchronization principle. Cells are tethered to glass beads, and continuously washed with growth medium. Upon each cell division, a newborn cell is released from its attached sister and collected in the eluted medium. An individual 1 min sample is then grown and analysed as a synchronous population (Experimental procedures). B. The sticky flagellin tethering construct. fliC^st is integrated into the chromosome at the galE locus. Expression of the 57 bp fliC^st mutation is controlled by the inducible tac promoter and constitutive lacI^Q repressor.

Fig. 2. Baby cell column

A. Complete system set-up consisting of column, pump and source of aerated medium. B. Diagram of assembled column with schematic of glass bead section. Cell-covered glass beads are packed between support screens of upper and lower flow adapters as shown. Arrows indicate flow of medium through the column. Plug flow is enhanced by a 0.22 μm membrane placed under the top support screen (See Experimental procedures for details).

Fig. 3. Column yield over time

A. Standard experimental column. One litre of exponentially growing cells was mixed with glass beads, incubated 5 min, and packed into a baby cell column. Flow of growth medium was started, and consecutive samples were analysed for cell number. Dotted vertical lines represent approximate generation times after cells were attached to the beads. The theoretical curve shows the expected number of cells eluted from a bound culture with perfect synchrony exhibiting the age distribution shown in the inset (Experimental procedures). B. Control column. In a control experiment, cells were added to the beads over a 150 min period [in 10 equal (1 g) increments] before packing into the column.

Fig. 4. Synchrony of baby cell cultures

A. Determination of the percentage of cells in S phase in a mixed population. A flow cytometry histogram of a newborn (t = 0) cell population from the baby cell column is shown with approximate G₁ (B period), S (C period) and G₂M (D period) areas (left panel). Estimation of the S-phase fraction was accomplished by quantifying the G₁ and G₂M fractions and subtracting them from the total population (right panels; see Experimental procedures for details). Similar values for the length of S phase were obtained by two other histogram calculation methods (data not shown). B. DNA histograms of cells from an asynchronous exponential culture (left panel), cells immediately after elution from the baby cell column (t = 0; middle panel), and cells incubated for an additional 120 min after elution (t = 120; right panel). C. S-phase fractions of synchronized cultures grown through two generations. Individual 1 min (5 ml) elution samples were incubated for varying lengths of time (0–300 min). Samples were then analysed by flow cytometry and S-phase fractions were calculated. D. Cumulative curves for the two rounds of DNA replication. Entry (solid lines) and exit (dashed lines) curves for each round are shown (see Results for details). E. Cell-length histograms for an asynchronous exponential culture (top panel) and a baby cell column culture analysed at t = 0 (bottom panel). Using the same cultures that had been analysed for DNA content, cell lengths were determined for approximately 100 cells per time point by microscopic observation. Gaussian curves based on the mean and standard deviations for each population are shown. F. Cell-length histograms for the first cell cycle are shown.

Fig. 5. Evaluation of cell length as a determinant of cell age

A. Progression of morphological classes in synchronized cells. Synchronized cell populations from the baby cell column were analysed for number of discernible origin and DnaX foci by FISH and GFP fluorescence respectively (described in detail elsewhere; Bates and Kleckner, 2005). Cells were grown in AB Alanine medium, resulting in a doubling time of ~123 min. Lifespans of each stage were calculated by cumulative curve analysis: (1,0) = 11 min, (1,1) = 28 min, (2,1-A) = 6 min, (2,2) = 26 min, (2,1-B) = 4 min, (2,0) = 48 min (2,0 cumulative curve shown, dotted line). B. Progression of the same morphological classes in populations determined by cell length. All cells in the above synchronized cell experiment (3000 ct) were pooled and binned according to cell length and the resulting distribution is shown (top; window size = 0.25 μm). The origin/DnaX content of cells belonging to the 13 most predominant cell-length bins (shaded bars) were plotted using cell length as the time determinant (bottom). Beginning (0% through cell cycle) and ending (100% through cell cycle) cell-size bins were estimated to be 2 μm and 5 μm respectively. C. Comparison of the kinetics of DNA replication and accumulation of cell mass. For cells growing with a doubling time of ~153 min (same data set as Fig. 4), the percentage of cells in the process of DNA replication (black symbols) and the percentage of cells that are ≥3 μm in length (gold symbols) are shown (top). Cumulative curves for these stages describe their rate of entry (bottom).