Cross-kingdom chemical communication drives a heritable, mutually beneficial prion-based transformation of metabolism

Abstract

In experimental science, organisms are usually studied in isolation, but in the wild, they compete and cooperate in complex communities. We report a system for cross-kingdom communication by which bacteria heritably transform yeast metabolism. An ancient biological circuit blocks yeast from using other carbon sources in the presence of glucose. [GAR(+)], a protein-based epigenetic element, allows yeast to circumvent this "glucose repression" and use multiple carbon sources in the presence of glucose. Some bacteria secrete a chemical factor that induces [GAR(+)]. [GAR(+)] is advantageous to bacteria because yeast cells make less ethanol and is advantageous to yeast because their growth and long-term viability is improved in complex carbon sources. This cross-kingdom communication is broadly conserved, providing a compelling argument for its adaptive value. By heritably transforming growth and survival strategies in response to the selective pressures of life in a biological community, [GAR(+)] presents a unique example of Lamarckian inheritance.

Figure 1. S. hominis induces a stable circumvention of glucose repression

A) Growth of S. hominis adjacent to [gar⁻] S. cerevisiae on Gly + GlcN plates induces the yeast cells to acquire a glucosamine-resistant trait. Both organisms are plated in five-fold serial dilutions. B) This trait is stable after hundreds of generations of growth on non-selective media followed by re-testing on Gly + GlcN plates (top). See also Table S1.

Figure 2. S. hominis induces the [GAR⁺] prion

A) The GlcN-resistant trait induced by S. hominis has the same dominant, non-Mendelian pattern of inheritance as spontaneous [GAR⁺] in genetic crosses to [gar⁻] strains. B) Both the [GAR⁺] prion and the GlcN-resistant trait induced by S. hominis can be lost by transient reductions in Hsp70 activity caused by expression of a plasmid-borne dominant negative variant (K69M) of the Ssa1 protein. See also Fig. S1 and Fig.

Figure 3. S. hominis induces [GAR⁺] via a diffusible factor

Exposure to S. hominis-conditioned medium for 4 h leads to a large induction of the [GAR⁺] prion. Control ‘mock’ treated cells showed no such effect. The small number of colonies that do appear matches the frequency of spontaneous [GAR⁺] appearance in this W303 strain. 10-fold serial dilutions are plated on GLY + GlcN medium. See also Table S2.

Figure 4. [GAR⁺] inducing capacity is shared among many bacteria

A) Yeast cells (as the second row in Fig. 1A) can be induced to acquire [GAR⁺] by many bacteria and with a wide range of efficiencies. B) Clustering of sequenced, inducing bacteria (and their closest non-inducing relatives) by 16S RNA sequence does not reveal conservation of inducing capacity by clade. Bright red dots represent strains that strongly induce [GAR⁺]; light red dots represent strains with intermediate inducing ability; grey dots represent strains with weak or no inducing ability. See also Fig. S3 and Table S3-S6.

Figure 5. [GAR⁺] reduces ethanol production yet confers benefit to yeast cells

A) Representative growth curves of [GAR⁺] and [gar⁻] cells in rich medium (YPD) are indistinguishable. B) [GAR⁺] cells produce much less ethanol than isogenic [gar⁻] cells. Representative curves are shown. Similar results were obtained with both colorimetric and electrochemical detection. C) Similar effects occur in Chardonnay grape juice. Error bars represent the standard deviation obtained from three biological replicates. D) [GAR⁺] circumvents glucose repression of other carbon sources. E) [GAR⁺] confers advantage in mixtures of glucose and other carbon sources. F) Starting with equal numbers of cells, [GAR⁺] yeast are outcompeted by isogenic cells that do not harbor the prion in glucose alone. In contrast, [GAR⁺] strongly outcompetes [gar⁻] in a mixed carbon source environment (YP with 1.9% galactose and 0.1% glycerol in G); Molasses in D)). Calculated selection coefficients (S) are noted for [GAR⁺] in these conditions. Error bars are the standard deviation determined from three independent biological replicates. H) [GAR⁺] cells survive longer in aged cultures than isogenic [gar⁻]. Cultures were grown for 3 weeks in minimal grape must medium and viability was judged by the cells ability to export methylene blue and the formation of colony forming units. Error bars represent the standard deviation obtained from three biological replicates.

Figure 6. Single cell dynamics of [GAR⁺] induction

A) Schematic of microfluidic encapsulation of single yeast and bacterial cells in droplets and subsequent experiments. B-D) Scatterplots of mOrange vs. GFP fluorescence for ~10⁶ droplets containing either [gar⁻] yeast, [GAR⁺] yeast, or [gar⁻] yeast and E. coli strain MG1655 (an inducing bacterium) after 48 h incubation. See also Figure S4.

Figure 7. The switch to [GAR⁺] alters population dynamics and confers adaptive advantages in natural fermentations

A) Fermentations of natural grape juice seeded with [GAR⁺] yeast have a reduced fermentative yield (a composite measurement of ethanol production, CO₂ loss, and residual sugar - see Boulton et al, 1996) compared to isogenic [gar⁻] yeast. B) Lees collected at the end of fermentations seeded with [GAR⁺] cells contains at least one hundred fold more bacteria than is normally obtained with [gar⁻] cells. C) Chromatography analysis of post-fermentation supernatant following inoculation with [gar⁻] and [GAR⁺] yeast cells (see Extended Experimental Procedures). [gar⁻] fermentations exhibit tartaric, malic, and lactic acids, indicating completion of fermentation, while [GAR⁺] fermentations exhibit little malic acid. D)Yeast that acquire [GAR⁺] have extended viability, judged by methylene blue staining after prolonged culture and confirmed by changes in colony forming capacity (see Extended Experimental Procedures). Each point represents the percent of dye-permeable yeast in a field of 100 cells. E). [GAR⁺] cells are more resistant to ethanol than [gar⁻] cells. 10⁶ cells were incubated for 24 h at 30 °C in varying starting concentrations of ethanol in SD-CSM. Viability was judged by recovery of colony forming units. Error bars represent the standard deviation of six biological replicates. P-values by T-test: ***(P<0.0001), **(P<0.0005), *(P<0.005). F) [GAR⁺] cells have a strong growth advantage in limiting tryptophan relative to [gar⁻] cells. Approximately 10⁶ cells were challenged with the indicated concentration of tryptophan in synthetic medium for 24h and then plated to rich medium. Colony forming units in limiting tryptophan were compared to a tryptophan-replete control (50 ug/mL). Error bars represent the standard deviation obtained from three biological replicates.