Fig 1.
Nutrient–growth regulation and dysregulation.
(A) Natural nutrients control the growth or the stress-response state of a cell (reviewed in [2]). Growth stimulatory molecules are colored green, and growth inhibitory molecules are colored red. Broadly speaking, the presence of natural essential nutrients (e.g., nitrogen, glucose, sulfur, phosphorus) activates the TORC1 pathway. Glucose additionally activates the Ras/PKA pathway, although this activation is transient if essential natural nutrients are incomplete [10]. For simplicity, we have omitted other input pathways (e.g., in [11]). TORC1 and Ras/PKA pathways activate cell growth (green box) and inhibit stress response (red box). Conversely, the shortage of a natural nutrient or inhibition of TORC1 (via rapamycin) triggers stress responses, including cell-cycle arrest, autophagy, and oxidative metabolism. Note that the mRNA levels of greater than a quarter of yeast genes are linearly correlated with growth rate, independent of the nature of the nutrient limitation [4,12]. During slow growth, repressed genes include those involved in ribosome synthesis, translation initiation, and protein and RNA metabolism, whereas induced genes are involved in autophagy, lipid metabolism, and oxidative metabolism (including those annotated to peroxisomes and the peroxisomal matrix) [4,9,13,14]. (B) Left: when limited for a natural nutrient, an auxotroph responds properly with muted growth (red box) and survives with high viability. Right: when limited for the auxotrophic nutrient, an auxotroph suffers nutrient–growth dysregulation (orange box). Despite nutrient limitation, cells experience poor cell-cycle arrest, suffer reduced autophagy, and metabolize glucose via fermentation instead of respiration. Consequently, these cells suffer low viability. S1 Text offers additional discussions. Auxo, auxotroph; Bcy1, bypass of cyclic-AMP requirement protein 1; PKA, protein kinase A; TORC1, target of rapamycin complex 1.
Fig 2.
lys− cells suffer nutrient–growth dysregulation when limited for lysine.
(A) The poor viability of lys− cells during lysine starvation is rescued by inhibiting growth and is exacerbated by activating growth. Exponentially growing mCherry-expressing lys− (WY2490) cells were washed and starved for either only lysine or both lysine and glucose for 3 hours to deplete cellular storage and cultured and imaged in indicated environments (Methods, “Fluorescence microscopy”). Total fluorescence (normalized to the initial value) approximates total biomass [27]. In the absence of lysine, lys− cells died rapidly when glucose was abundant (2%, blue circles). This rapid death could be rescued if we inhibited growth by adding the TORC1 inhibitor rapamycin (1 μM, green crosses) or by simultaneously starving for glucose (“No carbon,” green squares). Rapid death was exacerbated if we activated growth by deleting the PKA inhibitor BCY1 (red diamonds). Error bars correspond to 2 standard deviations for 6 replicate wells. Plotted data are provided as S1 Data. (B) lys− cells engage in less autophagy during lysine starvation compared with during nitrogen starvation. Lane 1 is the protein ladder. lys− cells expressing GFP-Atg8 (WY2521) were grown to exponential phase (Lane 2) and washed free of nutrients. Cells were then starved for only lysine (Lane 3), only nitrogen (Lane 4), or both lysine and nitrogen (Lane 5) for 8 hours. Cell extracts were subjected to western blotting using anti-GFP antibodies (Methods, “Autophagy assay”). High GFP/GFP-Atg8 ratio indicates high autophagy activity. Data are representative of 3 trials. Atg8, autophagy-related protein 8; bcy1Δ, a mutant with deletion of bypass of cyclic-AMP requirement gene 1; GFP, green fluorescent protein; PKA, protein kinase A; TORC1, target of rapamycin complex 1.
Fig 3.
(A) The evolution of auxotrophs. Ancestral lys− cells (WY950; WY1335) were grown for tens of generations in minimal medium, either in lysine-limited chemostats [35] or via coculturing with a lysine releaser in a cross-feeding yeast community [30,36]. Out of 20 independent lines, we randomly isolated approximately 50 clones for whole-genome sequencing. Chemostat evolution and coculture evolution both yielded met− mutants: 1 (WY1604) out of 9 clones in chemostat evolution; 3 (WY1569, WY2376, WY1591) out of 42 clones in coculture evolution). These mutants, all isolated from independent lines, required an externally supplied organosulfur such as methionine to grow. A glutamine auxotrophic gln1 clone was also identified. In the experiment shown here, clones were grown to exponential phase in SD supplemented with amino acids, washed with SD, starved for 3 hours to deplete cellular storage, and spotted on indicated agar plates at 30°C. (B) The organosulfur synthesis pathway in S. cerevisiae. S. cerevisiae utilizes sulfate supplied in the medium to synthesize the organosulfur homocysteine, which is then used to make a variety of other organosulfurs, including methionine, cysteine, and GSH [37]. All orgS− mutants we have identified (orange) fail to synthesize homocysteine, and thus can be supported by any of the organosulfurs depicted here (blue; note the interconvertibility between organosulfurs). Anc, ancestor; met−, methionine-requiring mutant; gln1, glutamine metabolism gene 1 mutant; GSH, reduced glutathione; SD, synthetic minimal glucose medium.
Fig 4.
Lysine-limited lys− cells mainly release GSH and GSXs.
Ancestral lys− cells (WY1335) were cultured in lysine-limited chemostats at 8-h doubling time unless otherwise indicated. (A) Mass spectra traces of GSH ion. Gray and black lines correspond to known quantities of GSH added to fresh growth media. Blue and green correspond to filtered supernatants harvested at approximately 48 hours from chemostats at 4-hour and 8-hour doubling time, respectively, with different symbols denoting replicate chemostats. Plotted data are provided as S2 Data. (B) GSH and GSXs constitute the majority of the organosulfur niche. Supernatants were harvested at steady state cell density (approximately 26 hours) and filtered. (i) Bioassay quantification of total organosulfur was performed by comparing the final turbidity of met10− (WY1604) grown in supernatants versus in various known concentrations of GSH. (ii, iii) GSH in supernatant was quantified by HPLC and LC–MS. (iv) GSH + GSX in supernatants were quantified by first reducing GSX to GSH with TCEP and then measuring total GSH via LC–MS. Error bars mark 2 standard deviations of samples from 3 independent chemostats. Plotted data are provided as S4 Data. (C) Organosulfurs are likely released by live cells. Total organosulfur in chemostat supernatant (brown) far exceeded that expected from release by dead cells (purple). Organosulfurs were quantified using the met10 bioassay. To estimate dead-cell release, we measured dead-cell density using flow cytometry (Methods, “Flow cytometry”), and multiplied it with the average amount of organosulfur per cell (Methods, “Metabolite extraction”). Three independent experiments are plotted (S5 Data). GSH, reduced glutathione; GSX, glutathione S-conjugate; HPLC, High-Performance Liquid Chromatography; LC–MS, liquid chromatography–mass spectrometry; met−, methionine-requiring mutant; TCEP, tris(2-carboxyethyl)phosphine.
Fig 5.
Organosulfur release is associated with nutrient–growth dysregulation.
lys− cells (WY1335) were cultured in lysine-limited (green) or glucose-limited (gray) chemostats at 8-hour doubling time. The concentrations of glucose and lysine in the input fresh minimal medium are marked. (A) Higher percentage of dead cells in lysine-limited chemostats than in glucose-limited chemostats. Live- and dead-cell densities were measured via flow cytometry (Methods, “Flow cytometry”; S6 Data). (B) Higher organosulfur concentrations in lysine-limited chemostats than in glucose-limited chemostats. Organosulfur concentration was measured in terms of GSH equivalents using the met10 bioassay (S5 Fig), with dotted line marking the lower limit of the linear detection range (S6 Data). (C) Adenine-limited ade− cells release organosulfurs at comparable rates as lysine-limited lys− cells. Cells were cultured in chemostats limited for the respective auxotrophic metabolite at 8-h doubling time until a steady state was reached. Release rate was quantified using r = dil × [orgS]ss/[Live]ss (Eq 14 in [30]), where dil is the dilution rate (ln2/8/hour), [orgS]ss is the steady-state organosulfur concentration (measured in terms of GSH equivalents using the met10 bioassay; S5 Fig), and [Live]ss is the steady-state live-cell density. Different colors correspond to experiments done on different days, and each data point represents an independent chemostat (S5 Data). ade−, adenine-requiring mutant; GSH, reduced glutathione; lys−, lysine-requiring mutant; met, methionine-requiring mutant.
Fig 6.
lys−orgS− displays a frequency-dependent fitness advantage over lys− during lysine limitation if organosulfur is also limited.
(A, B) Fitness advantage of lys−orgS− over lys− requires low organosulfur and autophagy. Cell growth was imaged via fluorescence microscopy. (A) In excess lysine and GSH conditions, lys−orgS− (WY1604, orange) grew slower than isogenic lys− (WY2429, blue). All data can be found in S7 Data. (B) Isogenic lys− (WY2429, blue), lys−orgS− (WY1604, orange), and lys−orgS−atg5− (WY2370, green) were cultured in the absence of lysine without (circles) or with (crosses) 1 μM rapamycin and without or with 20 μM GSH. Total fluorescence of the field of view at the endpoint (110 hours) was normalized against that of time zero. lys−orgS− (yellow circle, second column) survived lysine starvation better than lys− (blue circle, first column) when GSH was low. When GSH was abundant, lys−orgS− (yellow circle, fifth column) behaved similarly to lys− (blue circle, fourth column), surviving lysine starvation poorly and rescued by the growth inhibitor rapamycin (yellow and blue crosses, fifth and fourth columns). The high viability of organosulfur-limited lys−orgS− and of rapamycin-treated cells were abolished when autophagy was prevented (WY2370, green circles and crosses). Full data are plotted in S10 Fig and provided as S8 Data. Error bars represent 2 standard deviations from 4 positions in a well. (C) Negative-frequency–dependent fitness advantage of lys−orgS− over lys−. Isogenic BFP-tagged lys−orgS− (WY2072 or WY2073) and mCherry-tagged lys− (WY2045) were placed in competition in a lysine-limited environment by coculturing with a lysine-releasing strain (WY1340) (Methods). The ratio of lys−orgS− to lys− over time was measured by flow cytometry (Methods, “Competition”). Smooth lines serve as visual guide. Different colors represent different starting ratios, while different symbols represent independent experiments. Plotted data can be found in S9 Data. atg5, autophagy-related gene 5 mutant; GSH, reduced glutathione; lys−, lysine-requiring mutant; orgS−, organosulfur-requiring mutant; rap, rapamycin.
Fig 7.
Nutrient–growth dysregulation and the evolution of an organosulfur-mediated interaction.
(A) During lysine limitation, lys− cells suffered reduced autophagy and viability compared to during natural limitation (Fig 2). (B) These cells released organosulfurs mainly comprising GSX and, to a lesser extent, GSH (Fig 4). We hypothesize that organosulfur release is a detoxification response to nutrient–growth dysregulation because organosulfur release was significantly reduced during glucose limitation (Fig 5B) or in excess lysine (S7 Fig). (C–D) The released organosulfurs created a metabolic environment that could support the growth of the newly arisen lys−orgS−. (E) The rare lys−orgS− mutant rose in frequency (Fig 6C) because of a fitness advantage gained by properly responding to sulfur limitation—a natural limitation; e.g., compared with lysine-starved lys− cells, lys−orgS− cells doubly starved for sulfur and lysine maintained high cell viability in an autophagy-dependent fashion (Fig 6B, left). (F) Eventually, lys−orgS− reached a steady-state frequency at which organosulfur supply and consumption were balanced (Fig 6C). GSH, reduced glutathione; GSX, glutathione-S-conjugate; lys−, lysine-requiring mutant; orgS−, organosulfur-requiring mutant.