Inherited adaptation of genome-rewired cells in response to a challenging environment

Abstract

Despite their evolutionary significance, little is known about the adaptation dynamics of genomically rewired cells in evolution. We have confronted yeast cells carrying a rewired regulatory circuit with a severe and unforeseen challenge. The essential HIS3 gene from the histidine biosynthesis pathway was placed under the exclusive regulation of the galactose utilization system. Glucose containing medium strongly represses the GAL genes including HIS3 and these rewired cells are required to operate this essential gene. We show here that although there were no adapted cells prior to the encounter with glucose, a large fraction of cells adapted to grow in this medium and this adaptation was stably inherited. The adaptation relied on individual cells that switched into an adapted state and, thus, the adaptation was due to a response of many individual cells to the change in environment and not due to selection of rare advantageous phenotypes. The adaptation of numerous individual cells by heritable phenotypic switching in response to a challenge extends the common evolutionary framework and attests to the adaptive potential of regulatory circuits.

Figure 1. Adaptation and propagation on plates.

From a culture of naive cells in galactose, an equal amount of cells were plated on Glu-his (a) and rich medium (b) plates. Images were captured after 12 days (a) and 48 h (b) of incubation, respectively. Note the high proportion of cells that adapted and formed colonies on glucose as well as the variable sizes of the colonies. Cells from a single adapted colony on the adaptation plate (a) were taken and equal amounts were further plated on Glu-his (c) and rich medium (d) plates. Both images [(c) and (d)] were taken at 48 h post-plating. Note the similar growth and uniform colony size of adapted cells on both medium types. Several adaptation experiments on Glu-his plates [as in panels (a) and (b)], each in replicates, were carried out and the proportion of adaptation was determined as the colony number on Glu-his divided by that on rich medium (e). Note the overall high proportion of adaptation (average of 0.5) and the variation, which was similar among replicates and among experiments.

Figure 2. Adaptation dynamics of chemostat populations.

Cell density (OD at 600 nm) as a logarithmic function of time for repeated chemostat experiments conducted under the same nominal conditions. As a comparison, the light and dark blue curves corresponds to chemostat experiments described in (Stolovicki et al., 2006). The histidine-lacking medium was switched from galactose to glucose as a sole carbon source at t=0, leaving all other nutrients the same. A steady-state typical of galactose metabolism was established prior to the switch into glucose. Bar—ten generations and the population growth phases are denoted in purple (I-IV). Inset: The fraction of adapted colonies during phase II (blue dots) on the background of the population density (gray) showing that the population became fully adapted by the end of phase II.

Figure 3. Population characteristics upon exposure to glucose.

The maximal fraction of adapted colonies (dots) along phase I of the chemostat (switching to glucose at t=0). The colonies initiated from individual cells that were sampled from the chemostat and grown on Glu-his plates for 20 days. Note the logarithmic scale. The chemostat population density was plotted in the background (gray line).

Figure 4. Distribution of colony sizes.

(a) A representative microscopy image of colonies grown on a Glu-his plate after a few days. These colonies were formed by seed cells sampled from the liquid culture at the onset of transition to Glu-his. Note the coexistence of a wide range of colony sizes down even to a single cell. Scale of image ∼320 μm. (b) The distributions of colonies area (in arbitrary units) based on numerous microscopy images, such as in panel (a), including a few hundred colonies each. Curves at different colors are for different times after the transition of the batch culture into Glu-his (black, t=0; red, t=4 h; and blue, t=12 h). Each two curves that have the same color but different symbols are from the same plates but were measured earlier (46–57 h; squares) and later (∼120 hrs; dots) after plating. The curves are normalized to unit area and presented as probability density for comparison (x-axis divided by10⁴). The bar denotes the approximate distance on the x-axis of a colony with ∼70 cells.

Figure 5. Growth curves of parallel populations in batch cultures.

Adapted (red) versus naive (nonadapted, black) cells that were switched from Gal-his to Glu-his and as a control adapted cells (blue) that were propagated just in Glu-his. All strains exhibited similar growth rates in the Glu-his environment. The different lines are measurements of different clones averaged over three replicates, showing less than 7% variability in their growth rates.

Figure 6. Simulated distributions of colony size.

Monte-Carlo simulations of colonies that were initiated from single cells and proliferated under two opposing processes: a decaying probability to continually divide and an increasing probability to acquire adaptation and unlimited divisions, both as an exponential function of exposure time to Glu-his (see Materials and methods section for details). The different curves are for different values of the parameter t_g, controlling these processes and representing the exposure time to Glu-his of the seed cells (see Materials and methods section). The colony sizes are measured in a number of cells in a colony. The curves are normalized to unit area and presented as probability density for comparison. Inset: the maximal fraction of adapted simulated colonies (i.e., colonies containing at least one adapted cell and, thus, can keep cell division indefinitely) in a log scale, as a function of the exposure time to Glu-his of their seed cells.