Predicting bacterial interaction outcomes from monoculture growth and supernatant assays

Abstract How to derive principles of community dynamics and stability is a central question in microbial ecology. Bottom-up experiments, in which a small number of bacterial species are mixed, have become popular to address it. However, experimental setups are typically limited because co-culture experiments are labor-intensive and species are difficult to distinguish. Here, we use a four-species bacterial community to show that information from monoculture growth and inhibitory effects induced by secreted compounds can be combined to predict the competitive rank order in the community. Specifically, integrative monoculture growth parameters allow building a preliminary competitive rank order, which is then adjusted using inhibitory effects from supernatant assays. While our procedure worked for two different media, we observed differences in species rank orders between media. We then parameterized computer simulations with our empirical data to show that higher order species interactions largely follow the dynamics predicted from pairwise interactions with one important exception. The impact of inhibitory compounds was reduced in higher order communities because their negative effects were spread across multiple target species. Altogether, we formulated three simple rules of how monoculture growth and supernatant assay data can be combined to establish a competitive species rank order in an experimental four-species community.


Figure S2
. Correlation plots between the three growth parameters maximum growth rate (µmax), area under the curve (AUC, integral), and inverse of the time to mid-exponential phase (1/Tmid).Each color corresponds to a bacterial species, while the symbol of the data point corresponds to the type of medium (circles = LB; rectangles = GIM).The r-values indicate the Pearson correlation coefficient.The trendline is based on a linear regression analysis (dashed = LB; solid = GIM).Data are from 3 independent experiments, each featuring 3-4 replicates per condition, resulting in a total of 9-10 replicates per condition. .Boxplots show the relative growth based on µmax of each species in the conditioned medium (70% fresh medium + 30% spent supernatant) of the other species both in (A) LB and (B) GIM medium, compared to a control treatment, depicted by the black dashed line (70% fresh medium + 30% NaCl solution [0.8%]).Relative growth was calculated by dividing the absolute µmax (estimates from curve fits) in the supernatant treatments by µmax in the control treatment (see Figure S5).Asterisks depict significant differences (alpha = 0.05) of a species' growth in the particular supernatant compared to its growth in the control medium using a linear mixed model with experimental block as random variable.Boxplots depict the median (line within the box) with the first and third quartiles.The whiskers cover 1.5x of the interquartile range or extend from the lowest to the highest value if all values fall within the 1.5x interquartile range.Data are from 3 independent experiments, each featuring 3-4 replicates per condition, resulting in a total of 9-10 replicates per condition.of each species in the conditioned medium (70% fresh medium + 30% spent supernatant) of the other species both in (A) LB and (B) GIM medium, compared to a control treatment, depicted by the black dashed line (70% fresh medium + 30% NaCl solution [0.8%]).Relative growth was calculated by dividing the absolute AUC (estimates from curve fits) in the supernatant treatments by the AUC in the control treatment (see Figure S7).Asterisks depict significant differences (alpha = 0.05) of a species' growth in the particular supernatant compared to its growth in the control medium using a linear mixed model with experimental block as random variable.Boxplots depict the median (line within the box) with the first and third quartiles.The whiskers cover 1.5x of the interquartile range or extend from the lowest to the highest value if all values fall within the 1.5x interquartile range.Data are from 3 independent experiments, each featuring 3-4 replicates per condition, resulting in a total of 9-10 replicates per condition.Growth curves show the mean OD600 measurements in conditioned medium (70% fresh medium + 30% spent supernatant) of the other species and a NaCl control treatment (70% fresh medium + 30% NaCl solution [0.8%]) in (A) LB and (B) GIM medium.Each colored curve stands for the conditioned medium of a specific species (see legend above panels) and the black line is the control.Shaded areas depict the standard deviation.Data are from 3 independent experiments, each featuring 3-4 replicates per condition, resulting in a total of 9-10 replicates per condition.

Figure S3 .
Figure S3.Boxplots show the absolute readouts of growth based on 1/Tmid of each species in the conditioned medium (70% fresh medium + 30% spent supernatant) of the other species and a NaCl control treatment (70% fresh medium + 30% NaCl solution [0.8%]) in (A) LB and (B) GIM medium.Boxplots depict the median (line within the box) with the first and third quartiles.The whiskers cover 1.5x of the interquartile range or extend from the lowest to the highest value if all values fall within the 1.5x interquartile range.Data are from 3 independent experiments, each featuring 3-4 replicates per condition, resulting in a total of 9-10 replicates per condition.

*Figure S5 .Figure S6 .
Figure S5.Boxplots show the absolute readouts of growth based on µmax of each species in the conditioned medium (70% fresh medium + 30% spent supernatant) of the other species and a NaCl control treatment (70% fresh medium + 30% NaCl solution [0.8%]) in (A) LB and (B) GIM medium.Boxplots depict the median (line within the box) with the first and third quartiles.The whiskers cover 1.5x of the interquartile range or extend from the lowest to the highest value if all values fall within the 1.5x interquartile range.Data are from 3 independent experiments, each featuring 3-4 replicates per condition, resulting in a total of 9-10 replicates per condition.

*Figure S7 .
Figure S7.Boxplots show the absolute readouts of growth based on AUC (area under the growth curve) of each species in the conditioned medium (70% fresh medium + 30% spent supernatant) of the other species and a NaCl control treatment (70% fresh medium + 30% NaCl solution [0.8%]) in (A) LB and (B) GIM medium.Boxplots depict the median (line within the box) with the first and third quartiles.The whiskers cover 1.5x of the interquartile range or extend from the lowest to the highest value if all values fall within the 1.5x interquartile range.Data are from 3 independent experiments, each featuring 3-4 replicates per condition, resulting in a total of 9-10 replicates per condition.

Figure S9 .Figure S10 .
Figure S9.Boxplots depict the CFU of the focal species (indicated in the header of each panel) in monoculture and in co-culture with each of the other species after 24 h in (A) LB and (B) GIM medium.The CFU-values of monocultures are corrected (divided by two) to account for the fact that the focal species inoculum was double in mono-compared to co-cultures.Boxplots show the median (line within the box) with the first and third quartiles.The whiskers cover 1.5x of the interquartile range or extend from the lowest to the highest value if all values fall within the 1.5x interquartile range.Data are from 6 individual experiments with 2 replicates per condition, resulting in a total of 7-12 replicates per condition (note in a few cases sample size was <12 because obtaining countable colonies for both species in co-culture was very difficult).

Figure S11 .
Figure S11.Variation in 4-species community dynamics in response to K toxin potency and diffusion.All simulations ran for 30,000 time steps with the four pathogens starting at equal fractions.Toxin potency is defined by the toxin threshold value, which is the number of toxin molecules required to kill a cell.Hence, toxin potencies increase with lower threshold values.Low diffusion (cell diffusion 0.0 μm 2 s −1 , toxin diffusion ∂ = 0.1 μm 2 s −1 ) corresponds to a structured environment.High diffusion (cell diffusion 5.0 μm 2 s −1 , toxin diffusion ∂ = 10.0 μm 2 s −1 ) corresponds to an unstructured environment as implemented in our experiments with shaken cultures.Lines show the mean and the standard deviation across 20 independent simulations.

Table S1 .
Values of pH for 30% supernatant + 70% original medium for all four species as well as in both media.SN = supernatant.