The generalist accumulates extracellular nitrite
We first tested whether the generalist accumulates substantial extracellular nitrite under our experimental conditions, and thus creates a niche for the specialist. To accomplish this, we grew the generalist alone in bioreactors with anoxic ACS medium amended with 12 mM nitrate as the growth-limiting substrate and measured the extracellular concentrations of nitrate and nitrite over time. We performed these experiments at pH 6.5 (strong nitrite toxicity) and 7.5 (weak nitrite toxicity).
We observed a substantial accumulation of extracellular nitrite regardless of the pH (Fig. 3A, B). When grown at pH 6.5 (strong nitrite toxicity), extracellular nitrite accumulated to a concentration comparable to the initial nitrate concentration (measured maximum extracellular nitrite concentration, 11.8 mM; measured initial nitrate concentration, 12.0 mM) and was subsequently consumed to below the detection limit (Fig. 3A). When grown at pH 7.5 (weak nitrite toxicity), extracellular nitrite again accumulated to a concentration comparable to the initial nitrate concentration (measured maximum extracellular nitrite concentration, 11.7 mM; measured initial nitrate concentration, 12.9 mM) and was subsequently consumed to below the detection limit (Fig. 3B). During growth at pH 6.5, substantial nitrite consumption did not begin until a prolonged period of time after nitrate consumption was complete, resulting in a relatively long duration of nitrite availability (Fig. 3A). During growth at pH 7.5, in contrast, substantial nitrite consumption began immediately after nitrate consumption was complete, resulting in a relatively short duration of nitrite availability (Fig. 3B). The longer duration of nitrite availability at pH 6.5 indicates that the duration of the niche created by the generalist for the specialist depends on pH.
To routinely quantify the duration of nitrite availability, we grew the generalist alone with varying amounts of nitrate as the growth-limiting substrate. We then quantified the length of time from when the growth rate with nitrate was maximum to when the growth rate with nitrite was maximum. This cell density-based proxy measure is valid because the growth of the generalist is directly linked to the consumption of nitrate and nitrite (Fig. 3A, B). The cell density of the generalist was initially linearly correlated with nitrate consumption at both pH 6.5 (strong nitrite toxicity) (two-sided Pearson correlation test; r = −0.96, p = 1.5 × 10–8, n = 15) (Fig. 3A) and 7.5 (weak nitrite toxicity) (two-sided Pearson correlation test; r = −1.00, p = 2.2 × 10–16, n = 30) (Fig. 3B). After nitrate was depleted, the cell density of the generalist became linearly correlated with nitrite consumption at both pH 6.5 (strong nitrite toxicity) (two-sided Pearson correlation test; r = −0.97, p = 3 × 10–4, n = 7) (Fig. 3A) and 7.5 (weak nitrite toxicity) (two-sided Pearson correlation test; r = −0.97, p = 6.8 × 10–10, n = 16) (Fig. 3B). We further validated our cell density-based approach by testing for concordance with our IC-based direct measures of the duration of nitrite availability. We observed a significant positive and linear relationship between the cell density- and IC-based measures (two-sided Pearson correlation test; r = 0.999, p = 0.023, n = 3) (linear regression model; slope = 1.19, intercept = −2.31, r2 = 0.99) (Supplementary Fig. S2), which further validates our cell density-based approach to routinely estimate the duration of nitrite availability.
Using our cell density-based approach, we found that the duration of nitrite availability was significantly longer at pH 6.5 (strong nitrite toxicity) than at 7.5 (weak nitrite toxicity) regardless of the initial nitrate concentration (two-sample two-sided t-tests; Holm-adjusted p < 1.7 ×; 10–3, n = 5) (Fig. 3C). Moreover, at both pH 6.5 and 7.5, the duration of nitrite availability increased linearly with the initial concentration of nitrate (two-sided Pearson correlation tests; r > 0.92, Holm-adjusted p < 0.005, n = 7) (Fig. 3C). Taken together, our results indicate that the generalist does indeed accumulate substantial amounts of extracellular nitrite (Fig. 3A, B), thus creating a potential niche for the specialist, and that the duration depends on the initial nitrate concentration and pH (Fig. 3C). We note that the underlying mechanism causing the longer duration at pH 6.5 is unclear, but is likely linked to the increased toxicity of nitrite at this pH [16]. Regardless, the relevant point here is that we can use the pH to experimentally manipulate the duration of nitrite availability and measure the consequences on the growth of the specialist.
Origin of a nitrite cross-feeding interaction
We next tested whether the extracellular accumulation of nitrite by the generalist enables the origin of a nitrite cross-feeding interaction with the specialist. We used coexistence as a strict condition for evaluating whether we could successfully recreate the origin. To test for coexistence, we performed reciprocal initial ratio experiments, which provide definitive empirical evidence of coexistence [35]. Briefly, if the generalist and specialist can each increase in frequency from rare, then they can coexist. We tested this by serially transferring co-cultures of the generalist and specialist initiated at different ratios of specialist-to-generalist (rS/G). We then quantified changes in the log rS/G over the first three co-culture transfers, which is the fewest number of transfers that still allow us to perform statistical trend testing (n = 4 for the Mann–Kendall trend test). We used the fewest number of transfers to minimize the probability that genetic and phenotypic changes would emerge over the timecourse of the experiment.
We first tested whether the generalist can increase in frequency from rare. To achieve this, we used large initial log rS/Gs (mean measured initial log rS/Gs of 3.23 and 1.98 at pH 6.5 [strong nitrite toxicity] and 2.98 and 2.12 at pH 7.5 [weak nitrite toxicity]). After the co-cultures reached the stationary phase, we measured the final log rS/Gs and transferred the co-cultures into a fresh medium. At both pH 6.5 and 7.5, we found that the log rS/Gs decreased over the first three transfers (Mann–Kendall trend test; tau = −1, p = 0.042) (Fig. 4). Thus, the generalist can indeed increase in frequency from rare regardless of nitrite toxicity.
We next tested whether the specialist can also increase in frequency from rare. To achieve this, we used small initial log rS/Gs (mean measured initial log rS/Gs of −3.19 and −2.65 at pH 6.5 [strong nitrite toxicity] and −3.33 and −2.13 at pH 7.5 [weak nitrite toxicity]). At pH 6.5 (strong nitrite toxicity), we found that the log rS/Gs increased over the first three transfers (Mann–Kendall trend test; tau = 1, p = 0.042) (Fig. 4A). At pH 7.5 (weak nitrite toxicity), however, we found that the log rS/Gs did not significantly change over the first three transfers (Mann–Kendall trend test; tau = 0.67, p = 0.31) (Fig. 4B). Thus, our data indicate that the generalist and specialist can indeed coexist, but only conclusively at pH 6.5 when the duration of nitrite availability is relatively long (Fig. 3).
We could simulate the observed co-culture dynamics using a two-species Monod-type model where the generalist consumes nitrate, produces conditionally toxic nitrite, and finally consumes nitrite, while the specialist only consumes nitrite. We parametrized the model using our available growth data (Supplementary Fig. S4) and assumed that the generalist and specialist have equivalent kinetics for nitrite consumption (with the exception that the specialist has no competitive interaction between the nitrate and nitrite reductases). We then assembled both cell types at the initial log rS/Gs used in our experiments and simulated six serial transfers without adaptation of the model parameters.
The model accurately recreates the origin of the cross-feeding interaction where coexistence depends on nitrite toxicity (Fig. 4; dashed lines). The model suggests further factors that promote coexistence. In general, higher nitrate (and in turn higher nitrite) concentrations or conditions where nitrite is increasingly toxic (lower pH) accelerate the increase in the frequency of the specialist from rare (Supplementary Fig. S5). For example, at moderate nitrite toxicity, the specialist can increase in frequency from rare if sufficient nitrite is present (Fig. 3C; nitrite concentration correlates with intermediate durations of nitrite availability regardless of pH), but is likely outcompeted if the nitrite concentration is insufficiently high.
Long-term dynamics of the nitrite (NO2
–) cross-feeding interaction
We next evaluated the long-term dynamics of the nitrite cross-feeding interaction after its origin. Because we only experimentally observed coexistence at pH 6.5 (strong nitrite toxicity), all our further analyses focus on dynamics at this pH. We found that control cultures containing only the generalist displayed significantly reduced durations of nitrite availability after only three transfers (two-sample two-sided t-test, p = 0.003) (Supplementary Fig. S6). If the generalist altered its phenotype to reduce nitrite toxicity, then the emergence of such a phenotype may have important implications for the long-term persistence of the cross-feeding interaction (Fig. 1). We therefore serially transferred the co-cultures to a fresh medium for a total of 12 transfers. Our goal was to continue serial transfers such that genetic and phenotypic changes were more likely to emerge and accumulate.
We found that the long-term dynamics of the nitrite cross-feeding interaction depends on the initial composition of the co-culture (Fig. 4A). When the generalist was initially rare (measured initial log rS/Gs of 3.53, 1.93, and 0.26), the composition persisted without any further statistically observable changes to the rS/G between the third and twelfth transfers (Mann–Kendall trend tests; tau = –0.55 to 0.17, p > 0.6), and thus followed model predictions (Fig. 4A). However, when the specialist was initially rare (measured initial log rS/Gs of –3.19, –2.65, and –0.88), the relative abundances of the specialist continuously decreased between the third and twelfth transfers (Mann–Kendall trend tests; tau = –0.61 to –0.89, p < 0.03), displaying a unimodal dynamic that deviated from model predictions (Fig. 4A). For example, when the measured initial log rS/G was −3.19, the frequency of the specialist increased from 6.4 × 10–4 to 0.41 (640-fold) over the first three transfers, but then decreased to 8 × 10–2 (50-fold) over the remaining nine transfers (Fig. 4A and Supplementary Fig. S7). Overall, we observed a significant positive relationship between the initial and final log rS/G after 12 transfers (two-sided Pearson correlation test; r = 0.87, p = 0.025, n = 6).
Initial rS/G controls long-term co-culture dynamics and the duration of nitrite availability
Why would the initial co-culture composition determine the long-term dynamics of the nitrite cross-feeding interaction? We hypothesized that the initial composition determines the initial environment, which in turn determines the initial selection pressures acting on the generalist. For example, if the initial frequency of the specialist were large, then there could be a sufficient specialist to rapidly consume all the nitrite released by the generalist. Nitrite would not be available to the generalist, eliminating any benefits to a generalist with improved nitrite consumption and promoting the persistence of the cross-feeding interaction. Indeed, this is what we experimentally observed (Fig. 4A). In contrast, if the initial frequency of the specialist were small, then there might not be a sufficient specialist to rapidly consume all the nitrite released by the generalist. Nitrite would then be available to the generalist, providing benefits to a generalist with improved nitrite consumption and potentially disrupting the cross-feeding interaction. Again, this is what we experimentally observed (Fig. 4A).
If the above hypothesis were valid, then it leads to a clear expectation. As the initial rS/G increases, the generalist is less likely to accumulate phenotypes with improved nitrite consumption traits. To test this, we isolated 70 individual generalists from six co-cultures initiated at different rS/Gs (420 total isolates) after 12 serial transfers and measured the maximum observed growth rate with nitrite and the duration of nitrite availability. We did not observe a relationship between the initial log rS/G and the maximum observed growth rate with nitrite (two-sided Pearson correlation test; r = −0.51, p = 0.30, n = 6) (Fig. 5A), but we did observe a significant positive relationship with the duration of nitrite availability (two-sided Pearson correlation test; r = 0.90, p = 0.014, n = 6) (Fig. 5B). Thus, generalists serially transferred in co-cultures with larger initial rS/Gs are less likely to acquire significantly shorter durations of nitrite availability (Fig. 5B).
Initial rS/G controls the phenotypic diversification of the generalist
We finally tested whether the initial rS/G determines the phenotypic changes in nitrite production and consumption acquired by the 420 individual generalist isolates. To test this, we delineated the individual generalist isolates based on their growth properties with nitrate and nitrite, including maximum observed cell densities, maximum observed growth rates, and the duration of nitrite availability. We provide the complete methods for delineating different individual-level phenotypes in the Supplementary Information.
Overall, we found that the generalist isolates displayed five distinct phenotypes (designated as phenotypes A–E) (Supplementary Fig. S8). Phenotype A immediately switched between nitrate and nitrite consumption (Supplementary Fig. S8A). Phenotype B had a short time delay between nitrate and nitrite consumption and its cell density remained constant during that time (Supplementary Fig. S8B). Phenotype C (ancestral phenotype) had a long time delay between nitrate and nitrite consumption and its cell density remained constant during that time (Supplementary Fig. S8C). Phenotype D had a short time delay between nitrate and nitrite consumption and its cell density declined during that time (Supplementary Fig. S8D). Phenotype E had a long time delay between nitrate and nitrite consumption and its cell density declined during that time (Supplementary Fig. S8E). We further used genome re-sequencing of five or six randomly chosen representatives of each phenotype (A–E) to test whether these phenotypes correlate with genetic changes. We found that all isolates displaying phenotypes A, D, and E had genetic changes while all isolates displaying phenotypes B and C did not (Supplementary Table S2). Thus, our data suggest that phenotypic diversification of the generalist likely occurred via both genetic and non-genetic changes.
We observed relationships between the initial rS/Gs and the frequencies of specific phenotypes acquired by the generalist (Fig. 6). Phenotype A decreased in frequency as the initial rS/G increased (two-sided Pearson correlation test; r = −0.94, p = 6.2 × 10–3, n = 6) (Fig. 6). This phenotype has no observable time delay between nitrate and nitrite consumption (Supplementary Fig. S8A). Thus, as the specialist became dominant, generalists that immediately switched from nitrate to nitrite consumption became scarce. Conversely, phenotype C increased in frequency as the initial rS/G increased (two-sided Pearson correlation test; r = 0.95, p = 4.2 × 10–3, n = 6) (Fig. 6). This phenotype has the longest time delay between nitrate and nitrite consumption (Supplementary Fig. S8C). Thus, as the specialist became dominant, generalists with a long time delay between nitrate and nitrite consumption became dominant. Taken together, our data indicate that the initial rS/G controls the phenotypic diversification and trajectory of the generalist.
Initial rS/G determines the fitness gains of evolved generalist phenotypes
Can the presence of generalist phenotypes with altered nitrite consumption traits explain the displacement of the specialist after cross-feeding is fully established? To test this, we grew generalist phenotype A alone in bioreactors with anoxic ACS medium amended with 12 mM nitrate and measured the extracellular concentrations of nitrate and nitrite over time (Fig. 7A). In contrast to the ancestral generalist (Fig. 3A, B), phenotype A consumed nitrate and nitrite simultaneously and recovered the cell yield per unit substrate consumed (Fig. 7A and Supplementary Fig. S4E, F). We could describe phenotype A with a model where competitive inhibition between nitrate and nitrite is relaxed (Supplementary Fig. S4E, F). We then combined it with models describing the ancestral generalist (phenotype C) and the specialist and simulated serial transfers (Fig. 7B). This time, we allowed a small proportion (1 × 10–8) of the ancestral generalist to transform (mutate) into phenotype A prior to each transfer. Enabling the evolution of phenotype A resulted in dynamics similar to those that we observed experimentally (Fig. 4A and 7B). Thus, accounting for evolution resolved discrepancies between experiments and model simulations at small initial rS/Gs (Fig. 4A and 7B). We finally asked how the frequency of the specialist affects the fitness of phenotype A. As long as the frequency was larger than that of the generalist, the fitness of phenotype A relative to the ancestral generalist was below 1 (Fig. 7C), conditions that we only observed during the first transfer (Fig. 4A). This further explains the reduction or absence of phenotype A after 12 transfers in co-cultures that were initiated at large rS/Gs (Fig. 6).
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