Pre-dispositions and epigenetic inheritance in the Escherichia coli lactose operon bistable switch

Abstract

The lactose operon regulation in Escherichia coli is a primary model of phenotypic switching, reminiscent of cell fate determination in higher organisms. Under conditions of bistability, an isogenic cell population partitions into two subpopulations, with the operon's genes turned on or remaining off. It is generally hypothesized that the final state of a cell depends solely on stochastic fluctuations of the network's protein concentrations, particularly on bursts of lactose permease expression. Nevertheless, the mechanisms underlying the cell switching decision are not fully understood. We designed a microfluidic system to follow the formation of a transiently bimodal population within growing microcolonies. The analysis of genealogy and cell history revealed the existence of pre-disposing factors for switching that are epigenetically inherited. Both the pre-induction expression stochasticity of the lactose operon repressor LacI and the cellular growth rate are predictive factors of the cell's response upon induction, with low LacI concentration and slow growth correlating with higher switching probability. Thus, stochasticity at the local level of the network and global physiology are synergistically involved in cell response determination.

Figure 1

Following the lactose operon switch in single cells of growing microcolonies. (A) Genetic network in the strain LCY1. Black lines represent regulatory interactions, with pointed ends for activation and blunt ends for inhibition; dark green arrows represent protein expression from the genes. (B) Microfluidic device for the observation of individual cells in a growing population of bacteria under dynamic environmental conditions.

Figure 2

Establishment of bimodality in microcolonies. (A) Fluorescent images (YFP channel) of a microcolony of LCY1 just before TMG introduction (inset; high light intensity) and 2 h after (low light intensity). (B) Fluorescence intensity of all individual cells (black symbols) and mean fluorescence of the population (solid yellow line) as a function of time. The green and red lines are representative examples of single-cell trajectories. The dashed gray line indicates the time of TMG introduction. (C) Distribution of YFP fluorescence of single cells 2 h after TMG introduction (microcolony shown in (A)) fitted with a mixture of two Gaussian distributions (in red) by the ‘mclust’ function of the R software. The solid black line indicates the minimal fluorescence a cell should exhibit to be considered as an induced cell.

Figure 3

Epigenetic inheritance is evidenced by genealogical clustering of induced cells. (A) Typical ‘Lineage history tree’ established for the microcolony presented in Figure 2A. Individual cells are plotted as horizontal lines where the color corresponds to fluorescence intensity (YFP channel) as a function of time (horizontal axis). At division time, a vertical line is drawn to connect the mother cell and its two daughters. The dashed line corresponds to TMG introduction. The black circle indicates an example of division of a cluster's common ancestor before TMG introduction. (B) Distribution of proportion of induced cells in the cells’ progeny; on the left calculated from the data and on the right calculated from random re-sampling of the data (the descendants of each ancestor are randomly drawn from the final cell population, irrespective of their genealogy).

Figure 4

Epigenetic inheritance of cell fate determinants. (A) Typical genealogical tree with pairs of sister cells present at TMG introduction (red) and pairs of sister cells in the population of mothers (blue) and grandmothers (green) of cells present at TMG introduction. The vertical black line corresponds to TMG introduction. (B) Correlation of responses between sister cells and first and second cousin cells (from four different experiments). The mean fluorescence of the progeny of a cell is plotted against the mean fluorescence of its sister's progeny, for cells present at TMG introduction (red) or cells in the population of mothers (blue) and grandmothers (green, inset) of cells present at TMG introduction.

Figure 5

LacI concentration and growth rate influence switching probability. (A) Mean initial fluorescence (arithmetic mean of normalized CFP and normalized YFP) (left) and growth rate (right) for the cells that have less than half of their progeny induced and those that have more than half of their progeny induced. (B) Fluorescent images (CFP and YFP channels) of a microcolony of LCY2 (λPR-CFP, P_LlacO1-YFP) at TMG introduction. (C) Switching probability of a cell as a function of its initial growth rate and initial fluorescence (arithmetical mean of normalized CFP and normalized YFP); each dot represents one cell; black dots correspond to cells that have more than half of their progeny induced and white dots to cells that have less than half of their progeny induced; the background color corresponds to the switching probability calculated with the generalized linear model. For two cells, we plotted a segment corresponding to the error measurement of the growth rate (±2 s.e.).

Figure 6

Final states depend on LacI concentration and growth rate. (A) Bifurcation diagram as a function of repression factor ρ (equilibrium points of LacY as a function of ρ) for a fixed growth rate of 0.017 min⁻¹ and an extracellular TMG concentration of 29 μM. (B) Bifurcation diagram as a function of growth rate (equilibrium points of LacY as a function of growth rate) for a fixed repression factor of 700 and an extracellular TMG concentration of 29 μM. The blue lines represent the stable equilibria (points S1 and S2 in Supplementary Figure S11), the red line represents the unstable equilibrium (point U in Supplementary Figure S11) and the black line represents the minimal LacY initial concentration leading to induction in conditions of bistability (point B in Supplementary Figure S11); the dashed lines determine the region of bistability.