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. 2023 Jul 14:14:1076570.
doi: 10.3389/fmicb.2023.1076570. eCollection 2023.

Pleiotropy drives evolutionary repair of the responsiveness of polarized cell growth to environmental cues

Enzo Kingma  1 , Eveline T Diepeveen  1 , Leila Iñigo de la Cruz  1 , Liedewij Laan  1
Affiliations

Pleiotropy drives evolutionary repair of the responsiveness of polarized cell growth to environmental cues

Enzo Kingma et al. Front Microbiol. .

Abstract

The ability of cells to translate different extracellular cues into different intracellular responses is vital for their survival in unpredictable environments. In Saccharomyces cerevisiae, cell polarity is modulated in response to environmental signals which allows cells to adopt varying morphologies in different external conditions. The responsiveness of cell polarity to extracellular cues depends on the integration of the molecular network that regulates polarity establishment with networks that signal environmental changes. The coupling of molecular networks often leads to pleiotropic interactions that can make it difficult to determine whether the ability to respond to external signals emerges as an evolutionary response to environmental challenges or as a result of pleiotropic interactions between traits. Here, we study how the propensity of the polarity network of S. cerevisiae to evolve toward a state that is responsive to extracellular cues depends on the complexity of the environment. We show that the deletion of two genes, BEM3 and NRP1, disrupts the ability of the polarity network to respond to cues that signal the onset of the diauxic shift. By combining experimental evolution with whole-genome sequencing, we find that the restoration of the responsiveness to these cues correlates with mutations in genes involved in the sphingolipid synthesis pathway and that these mutations frequently settle in evolving populations irrespective of the complexity of the selective environment. We conclude that pleiotropic interactions make a significant contribution to the evolution of networks that are responsive to extracellular cues.

Keywords: adaptation; cell architecture; cell polarity; fluctuating environment; laboratory evolution; phenotypic plasticity.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Deletion of bem3△nrp1△ causes defects in pre- and post-diauxic growth. (A) Time-lapse series of the diauxic shift. The WT and bem3△nrp1△ strain were subjected to a switch from 2% glucose media to 3% ethanol media after 8 h in 2% glucose media. The images show that while the WT strain is able to resume growth, the bem3△nrp1△ cells increase in size without producing daughter cells. Scale bars represent 10 μm (B) Growth curves of a WT (blue) and the bem3△nrp1△ mutant (black) when grown in 2% glucose media. This data was used to obtain a measure for TPRE–shift. Red dots indicate the point of diauxic shift, dashed lines represent the Standard Error of the Mean (SEM). (C) Doubling time of the WT strain and the bem3△nrp1△ mutant during growth before the diauxic shift is entered (TPRE–shift). (D) Growth curves of a WT (blue) and the bem3△nrp1△ mutant (black) when grown in 0.1% glucose media. This data was used to obtain a measure for TPOST–shift. Red dots indicate the point of diauxic shift, dashed lines represent the SEM. (E) Doubling time of the WT strain and the bem3△nrp1△ mutant during growth after passing through diauxic shift is entered (TPOST–shift). (F) Comparison of the OD at which the bem3△nrp1△ mutant and the WT strain enter the diauxic shift and their OD at stationary phase when grown in YP + 0.1% glucose. The plot shows that while both strains enter diauxic shift at around the same density, the final density of the populations differ. *p-value < 0.05, **p-value < 0.005, Welch t-test.
FIGURE 2
FIGURE 2
Overview of the scheme for experimental evolution. A polarity mutant that displays sensitivity to environmental stress is obtained after the deletion of the genes BEM3 and NRP1. To assess the role the environment plays during the evolution of a network that is responsive to environmental signals, this mutant is evolved in an environment in which the stress level fluctuates batch culture and an environment where the stress level is constant continuous culture.
FIGURE 3
FIGURE 3
Experimental evolution of bem3△nrp1△ mutants in a constant and variable environment. (A) (Top) In a continuous culture, both nutrient concentration and cell density remain constant over time. (Bottom) Scatter plot of TPRE–Shift against TPOST–Shift for 14 evolved bem3△nrp1△ lines and 2 wild-type populations after 70 generations of evolution in a continuous culture. Dashed lines indicate the values of TPRE–Shift and TPOST–Shift of ancestral bem3△nrp1△ strain. Error bars show the SEM. (B) (Top) In a batch culture there are periodic fluctuations over time in nutrient concentration and cell density. (Bottom) Scatter plot of TPRE–Shift against TPOST–Shift for 8 evolved bem3△nrp1△ lines and 2 WT lines after 300 generations of evolution in a batch culture. Dashed lines indicate the values of TPRE–Shift and TPOST–Shift of ancestral bem3△nrp1△ strain. Error bars show the SEM. (C) Time-lapse of evolved lines CCE1 and CCE2 (continuous culture) during a sudden switch from 2% glucose media to 3% ethanol media (dashed red line). The images show that evolved line CCE1 contains cells that have a response to this environmental change that is phenotypically similar to the response of the WT strain. Evolved line CCE2 has a response that resembles the response of the ancestral bem3△nrp1△, but with a smaller increase in cell size (see Figure 1A). Scale bars represent 10 μm.
FIGURE 4
FIGURE 4
The mutational spectrum of different phenotypic subgroups that emerged after experimental evolution of bem3△nrp1△ populations. The mutant-specific mutations found in each gene for evolved continuous culture lines that decreased their respiration rate, evolved continuous culture lines that increased their respiration rate and evolved batch culture lines. Genes are grouped according to their cellular process GO-term. All genes shown were only mutated in the bem3△nrp1 populations and not in the wild-type populations, with the exception of WHI2 and SWI1, which were also found to be mutated in the wild-type populations evolved in the continuous culture.
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Grants and funding

LL and EK gratefully acknowledge funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant agreement No. 758132).

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