Bet-hedging and epigenetic inheritance in bacterial cell development

Abstract

Upon nutritional limitation, the bacterium Bacillus subtilis has the capability to enter the irreversible process of sporulation. This developmental process is bistable, and only a subpopulation of cells actually differentiates into endospores. Why a cell decides to sporulate or not to do so is poorly understood. Here, through the use of time-lapse microscopy, we follow the growth, division, and differentiation of individual cells to identify elements of cell history and ancestry that could affect this decision process. These analyses show that during microcolony development, B. subtilis uses a bet-hedging strategy whereby some cells sporulate while others use alternative metabolites to continue growth, providing the latter subpopulation with a reproductive advantage. We demonstrate that B. subtilis is subject to aging. Nevertheless, the age of the cell plays no role in the decision of its fate. However, the physiological state of the cell's ancestor (more than two generations removed) does affect the outcome of cellular differentiation. We show that this epigenetic inheritance is based on positive feedback within the sporulation phosphorelay. The extended intergenerational "memory" caused by this autostimulatory network may be important for the development of multicellular structures such as fruiting bodies and biofilms.

Fig. 1.

Typical B. subtilis microcolony development. (A) Still frames (phase contrast) of the outgrowth of a single cell into a sporulating microcolony. (Insets) Magnifications. (B) Log of microcolony biomass (black line) and average growth rates (gray triangles) are plotted in time. The growth rate is calculated as an exponential fit to cell length in time (arbitrary units, AU), measured from the birth point of the cell until the next cell division event or cell fate decision (see SI Materials and Methods). Biomass was calculated as a function of cell length (AU). (C) Each circle represents the point of birth in time of an individual cell. The average growth rate of this cell during its life is represented on the y axis (AU). (D) Cell fates plotted onto the birth points of C. Every birth point shown indicates the birth of a cell for which it is certain that either this cell or all of its descendents will follow a specific fate. Blue circles show the growth rates of individual cells committed to spore formation; green triangles show diauxic growth fate cells; and red triangles indicate lysing cells.

Fig. 2.

Aging in B. subtilis. Effects of consecutive divisions as a new pole cells (blue circles, showing rejuvenation) or old pole cells (red circles, showing aging). Error bars represent the SEM. Trend lines are shown in green (the actual progressions may not be linear). See also SI Fig. 8 and ref. .

Fig. 3.

Lineage-related expression of cell fates. (A) Average gfp expression from the spoIIA promoter of subpopulations of cells with different cell fates. For clarity, the fluorescence of only the first 13 h of the microcolony existence, before the later second round of growth and spore formation, is depicted. The SEM is indicated by error bars. (Inset) Magnification of the period between 460 and 520 min. (B) Fluorescence image of a B. subtilis microcolony after ≈12 h of growth when the microcolony consisted of 408 cells.

Fig. 4.

Inheritance in the decision to sporulate. (A) Parsimony mapping of the fluorescence character (GFP) onto the true lineage tree of 356 cells. Red tips, cells with fluorescence value above 40 fluorescence units. Black tips, cells below this threshold. (B) Parsimony mapping of fate information onto the true lineage tree of 531 cells. Every end point in the tree represents one offspring cell; red tips, spore-forming cells. Detailed parsimony character mappings are shown in SI Fig. 12.

Fig. 5.

Phosphorelay is required to generate subfamilies. (A and B) Schematic diagram of the regulatory cascade present in strains IIA/Δ0A/spo0A and IIA/spo0A/sad67, respectively. (C and D) Time-lapse microscopy of strains IIA/Δ0A/spo0A (P_spac-spo0A) and IIA/spo0A/sad67 (P_spac-sad67). Membranes are stained by FM5–95 (red), and GFP is depicted in green.