Caulobacter cell cycle. A Caulobacter crescentus cell divides asymmetrically into two morphologically distinct cells: a stalked cell (ST) and a chemotactic swarmer cell (SW) with a single polar flagellum and type IV pili. The distal end of the stalk is capped with an adhesive holdfast. Differentiation from SW to ST occurs just before the initiation of DNA replication (replicating DNA is represented by the theta structure).

Caulobacter division time and elongation rate. (A) The size of a single attached cell over time is a “sawtooth” oscillation with a period of the cell division time. (B) The majority of swarmer cells are flushed out of the microfluidic channel. However, there is a finite probability of attachment of the daughters downstream of their mothers. Growth of the mother cell m (black trajectory) and daughter cell d (blue trajectory) can thus be simultaneously measured. The division times are labeled as described in Methods: the daughter attaches in generation g, so that the daughter's first generation is g + 1 with division time t_d,g+1, and the mother's division time in the same generation is t_m,g+1. (C) Division time in Caulobacter is tightly controlled: the average division time of 82 ST cells over 12 generations is 58.3 ± 9.5 min (total number of division events n = 727). The mean division time as a function of generation is shown in black, with error bars indicating the standard deviations. The trajectories of four single cells (green, blue, yellow, and red) are overlaid on the mean curve. (D) Cell elongation rates are also narrowly distributed and approximately constant over the course of the experiment, with an average elongation rate of 0.029 ± 0.006 μm²/min. Mean elongation rates are shown in black, and four individual cells trajectories are shown in color (green, blue, yellow, and red).

Microfluidic device. (A) Microfluidic channels measuring 200 μm wide by 50 μm deep by 2 cm long were made with PDMS and sealed with a glass coverslip. Cells adhere to the glass surface via the adhesive holdfast. (B) A microscope image of cells in the chamber shows a young stalked cell (1), an early predivisional cell (2), and a late predivisional cell (3). Cells are aligned in the direction of medium flow. (C) The fluid flowing in the channel at a constant rate is laminar. The two-dimensional cross section of a laminar flow profile is parabolic, and the arrows represent the decreasing medium flow velocity from the center of the channel to the sides. On cell division, the majority of daughter cells are flushed out the far end of the channel. However, daughter cells occasionally attach to the glass coverslip before separation and remain attached downstream of their mothers when division is complete, rotated by ∼180° relative to their original orientation. (D) Simulations of diffusion under flow in the microfluidic culture channel show that small molecules emitted from the cell do not accumulate in the channel. The concentration of a simulated small molecule with diffusion coefficient of 10⁻⁹ m²/s was fixed at the bacterial surface. The concentration of the simulated small molecule is color graded from high (black) to low (white). For the flow rate used in our experiments (12 μl/min), the small molecule concentration quickly drops off outside the cell.

Caulobacter cell cycle control network and the DivJ sensor histidine kinase. (A) CtrA is essential for Caulobacter viability, controlling the transcription of 144 of 553 known cell cycle-regulated genes and acting as a negative regulator of DNA replication. The stability of CtrA is differentially regulated in swarmer versus stalked cells by the DivK response regulator, the histidine kinase DivJ, and the histidine phosphatase, PleC. (B) DivJ (blue) is localized to the stalked pole of the cell, and PleC (red) is localized to the flagellar pole. The asymmetric localization of DivJ and PleC ensures that on cell division, the level of CtrA (orange) drops in the ST cell while remaining high in the nonreplicative SW. (C) Phase contrast micrographs of wild-type (WT) Caulobacter and a Caulobacter strain carrying a Tn5 transposon insertion at the divJ locus (ΔdivJ). Mutations in the gene encoding DivJ result in cell morphology defects such as elongation, multiple constriction sites, and multiple or mislocalized stalks. (D) Transposon disruption of divJ results in a marked increase in the variance of the ST division time distribution (red). The mean ST division time in the divJ∷Tn5 strain is 76.6 ± 32.0 min (coefficient of variation = 0.42). The wild-type distribution (blue) has a mean of 58.3 min and standard deviation of 9.5 min (n_WT = 727, n_ΔdivJ = 200). (E) The distribution of elongation rates is change in ΔdivJ (red). The mean elongation rate is increased from 0.029 μm²/min in the wild-type strain to 0.038 μm²/min in the mutant strain, whereas the standard deviation increases from 0.006 μm²/min to 0.016 μm²/min. The wild-type distribution is shown in blue.

Mother/daughter division control correlation. (A) Comparison of mother cell and daughter ST cell division times shows a significant positive correlation. The Spearman's rank correlation coefficient for 20 cell pairs across 12 generations is ρ₀ ≈ 0.41 (n = 122). (B) The probability distribution P(ρ) generated from 10,000 random permutations of mothers with nondaughters across all days is Gaussian with a mean correlation coefficient 〈ρ〉 = 0.08 with standard deviation σ_ρ = 0.09 (light gray). A more restricted probability distribution P(ρ) created from 10,000 random permutations of daughters with nonmothers, limited to within the same days, is also Gaussian with mean coefficient 〈ρ〉 = 0.21 and standard deviation σ_ρ = 0.07 (dark gray), thus indicating day-to-day variation in mean population interdivision time. The observed mother/daughter correlation coefficient of 0.41 is significant relative to these two probability distributions at the level of 3.6σ (p ≈ 0.0002) and 3σ (p ≈ 0.001), respectively. The division time correlation coefficient of 18 proximal, unrelated cell pairs is 0.29 (n = 169; p > 0.1), indicating that the effect of cell proximity on division behavior is not statistically significant. (C) Mother cells that stop dividing in the microfluidic channel are more likely to produce daughter cells that also exhibit early division arrest. The growth trajectories of a selected mother cell (black) and daughter cell (gray) that stop dividing over the course of the experiment are shown. In this figure, the daughter stops dividing two generations before its mother. (D) Probability that a daughter cell still divides in generation g, given that the mother arrests in generation g_m. At the generation of arrest of the mother, ∼50% of daughter cells are no longer dividing. The data fit the Fermi-Dirac distribution function P(g − g_m) = 1/(1 + exp[(g − g_m)/g₀]), where g₀ ≈ 0.45. Data were taken from 31 offspring of 26 terminal mother cells.