Impact of direct physical association and motility on fitness of a synthetic interkingdom microbial community

Mutualistic exchange of metabolites can play an important role in microbial communities. Under natural environmental conditions, such exchange may be compromised by the dispersal of metabolites and by the presence of non-cooperating microorganisms. Spatial proximity between members during sessile growth on solid surfaces has been shown to promote stabilization of cross-feeding communities against these challenges. Nonetheless, many natural cross-feeding communities are not sessile but rather pelagic and exist in turbulent aquatic environments, where partner proximity is often achieved via direct cell-cell adhesion, and cooperation occurs between physically associated cells. Partner association in aquatic environments could be further enhanced by motility of individual planktonic microorganisms. In this work, we establish a model bipartite cross-feeding community between bacteria and yeast auxotrophs to investigate the impact of direct adhesion between prokaryotic and eukaryotic partners and of bacterial motility in a stirred mutualistic co-culture. We demonstrate that adhesion can provide fitness benefit to the bacterial partner, likely by enabling local metabolite exchange within co-aggregates, and that it counteracts invasion of the community by a non-cooperating cheater strain. In a turbulent environment and at low cell densities, fitness of the bacterial partner and its competitiveness against a non-cooperating strain are further increased by motility that likely facilitates partner encounters and adhesion. These results suggest that, despite their potential fitness costs, direct adhesion between partners and its enhancement by motility may play key roles as stabilization factors for metabolic communities in turbulent aquatic environments.

mean value and the whiskers extend to ± S.D as confidential interval, while circles indicate the biological replicates. Statistical analyses (t-test one-and two-way ANOVA) were performed using either JupyterLab (ANACONDA) or Microsoft Excel. Correlation analysis were performed using the data analysis add-in of Microsoft Excel and plotted using the regplot function of the seaborn package JupyterLab (ANACONDA). The shadow part represents 95% confidence interval while the lines represent the linear regression fit. Mean growth rate values for the specific time interval were calculated as the difference between the log2 of the OD600 measured respectively at 25 h and 16 h divided by the time interval expressed in hours. Figure S1. Characterization of S. cerevisiae and E. coli co-aggregation A-D Confocal microscopy images of mixtures of S. cerevisiae expressing mTurquoise2 (blue) with E. coli that is (A) Fim + (wt), (B) Fim + in presence of 4% mannose, or lacking either (C) the tip (ΔfimH) of the mannose-binding fimbriae filaments or (D) the entire filament (ΔfimA). In all cases E. coli (magenta) is expressing mCherry. Scale bar = 20 µm. E-H Auto-and cross-correlation analysis of co-aggregation between S. cerevisiae and (E) E. coli Fim + , (F) E. coli Fim + in presence of mannose, (G) E. coli ΔfimH and (H) E. coli ΔfimA, each with three biological replicates (represented by different lines). Autocorrelation analysis between neighboring pixels in one fluorescent channel (mTurquoise2) reflects the characteristic size of yeast cells or/and aggregates, whereas the cross-correlation analysis between two different channels (mCherry and mTurquoise2) reflects the characteristic size and number of mixed bacteria-yeast aggregates. These analyses were performed for the entire images, with each image contained at least twenty yeast cells and one hundred bacterial cells. I Quantification of aggregation, calculated as area under the curve for auto-and cross-correlations analysis from the plots shown in E-H. One-way ANOVA tests, followed by an HSD Tukey test as post hoc analysis were performed from three biological replicates.

Figure S3. Absence of aggregation in monocultures
A-C Confocal microscopy images of single cultures of (A) E. coli FimA + ΔtyrA, (B) E. coli ΔfimA ΔtyrA, both expressing mCherry (magenta), or (C) S. cerevisiae Δtrp3 expressing mTurquoise2 (blue) grown in YNB glucose supplemented with CSM. Scale bar = 20 µm. Figure S4. Growth of partners in cross-feeding communities with or without adhesion A,B Final cell counts from co-cultures as in Figure 2A,B for (A) S. cerevisiae and (B) E. coli. p values were obtained from a two-tailed t-tests assuming equal variances of the data sets, each with three biological replicates (indicated by circles). Cohen´s d values were calculated to quantify the effect size. C Final OD from co-cultures in Figure 2A,B. p values were obtained from a two-tailed t-tests assuming equal variances of the data sets, each with three biological replicates (indicated as circles). Cohen´s d values were calculated to quantify the effect size.

Figure S5. Activation status of the fim operon
A Assay used to determine the promoter orientation. The promoter region (618 bp) of the fim operon was amplified by PCR using primers P1 and P2, as indicated. This region contains a unique SnaBI restriction site. The digestion of the PCR fragments with SnaBI results in specific fragment pairs according to the state (On/Off) of the promoter, thus displaying a specific pattern of bands once the digestion is run via electrophoresis on a 2% agarose gel. B Quantification of the fim status of the E. coli partner based on band intensities as shown in (A), which is comparable to values obtained in LB cultures. Three biological replicates were used and are indicated as circles.

Figure S8. Relative growth fitness effects of different fluorescent markers
Final cell fraction of (sfGFP+) of E. coli cells in co-cultures with S.cerevisiae Δtrp3 that were inoculated with equal amounts of E. coli ΔtyrA ΔfimA either expressing mCherry (magenta-pOB2) or sfGFP (green-pNB1) and grown in YNB glucose minimal media for 72 h. One sample t-test assessing for the difference from a 50% mean performed with six biological replicates represented as circles.

Figure S9. Effect of initial cell density on fitness benefit of E. coli fimbriation
Fraction of Fim+ cells (labeled with mCherry) in the total E. coli population measured by flow cytometry in coculture with sfGFP-labelled fimbrialess (ΔfimA) E. coli and S. cerevisiae Δtrp3. Co-cultures were inoculated at different initial cell densities, with initially equal amounts of Fim + (labelled with mCherry) and ΔfimA (labelled with sfGFP) cells and grown for 96 h in YNB-glucose. Error bars represent standard deviations of six biological replicates represented as circles. One-way ANOVA test, followed by an HSD Tukey test as post hoc analysis were performed to assess for difference between samples. A one-sample t-test was performed to assess differences from an average fraction of 50%. Cohen´s d values were used to quantify the effect size.

Figure S10. Test for direct cytoplasmic material exchange between partners
Following cell imaging and the ensuing segmentation to distinguish the two partners, the intensity in the channel corresponding to the fluorescence of the other partner was measured in the area of each organism. Blue and orange box plots in the lower panel represent samples from clumping or non-clumping communities respectively. In total, thirty-five yeast cells have been measured per each condition and above one-hundred for E. coli. ns from a two-tailed t-test assuming equal variances of the data sets represented by more than six biological replicates.

Figure S11. Effects of partner adhesion on fitness for a community with S. cerevisiae Δtrp4 strain
Fraction of Fim + cells (labelled with mCherry) in the total E. coli population co-cultured with ΔfimA E. coli (labelled with sfGFP) and S. cerevisiae Δtrp4 (labelled with mTurquoise2). Co-cultures were inoculated with equal amounts of Fim + and ΔfimA cells and grown for 96 h either in YNB-glucose or in YNB-glucose supplemented with 4% mannose. Scatter plots represent the distribution of three biological replicates (indicated by circles). Whiskers represent the standard deviation. Both two-tailed t-test assuming equal variances of the data sets and one sample t-test to assess differences from a 50% average were performed. Cohen´s d values were used to quantify the effect size.

Figure S12. Verification of the auxotrophies
Growth curves from cultures of the E. coli ΔtyrA ΔtrpC cheater strain (labeled "C") both in mono culture in YNB + glucose, either with no supplements or supplemented with tyrosine and tryptophan, and in co culture in YNB + glucose with either S. cerevisiae Δtrp3 or E. coli ΔtyrA (labeled "P").

Figure S13. Cell count of cheater E. coli in the cross-feeding community
Number of cheater (labeled "C") E. coli cells measured by flow cytometry in the cross-feeding co-cultures with yeast and an E. coli partner (labeled "P") that is either fimbriated (straight lines) or fimbrialess (dotted line) as in figure 4C. Error bars represent standard deviations of three biological replicates. ****p ≤0.0001 from a twotailed t-test assuming equal variances of the data sets.

Figure S14. Dependence of the composition and growth of communities on the cheater fraction
A-F Dependence of the growth rate in exponential phase (A,D), of the total final OD600 (B,E) and of the final yeast cell count (C,F) from cocultures as in Figure 3C,E on the initial fraction of the cheater (labelled "C") at inoculation and either a fimbriated (left) or fimbrialess (right) E. coli partner (labelled "P"). G-I Same data but plotted against the final cheater fraction at the time of the measure, 25 h in (G) and 72 h in (H) and (I). Linear regression analysis and two-tailed t-test assuming equal variances of the data sets were performed. Each condition was assessed for three biological replicates, indicated as dots while shadings indicate a confidence interval of 95%. Regression line slopes, indicated as β, have been included along with the standard error.

Figure S15. Effects of direct physical association between partners in presence of fimbrialess cheater
Fraction of a fimbrialess cheater (ΔfimA) in communities containing either Fim + or ΔfimA E. coli partner at the initial 50% abundance of cheater, grown in YNB-glucose (orange), YNB-glucose supplemented with 4% mannose (red) and in in YNB-glucose supplemented with CSM and with S. cerevisiae prototroph (green). *p ≤0.05, ns=not significant in a two tailed t-test assuming equal variances of the data sets for three biological replicates, represented as circles.

Figure S16. Protective effects of fimbriation against cheater in community with Δtrp4 yeast strain
Fraction of fimbriated cheater in communities containing either Fim + (solid line boxes) or ΔfimA (dashed line boxes) E. coli partner at the initial 50% abundance of the cheater, grown either in YNB-glucose or YNB-glucose supplemented with 4% mannose, as indicated. ****p ≤0.0001, ns=not significant in a two tailed t-test assuming equal variances of the data sets for six biological replicates represented as dots.

Figure S17. Cross-correlation between spatial arrangement of community members in presence of cheater strain
Area under the curve from cross correlations analysis (see Fig. S1) between different community members in sessile communities grown as in Figure 3G. Two-sided t-test assuming equal variance between data sets were performed. Each data set included four biological replicates.  Figure 4B. Error bars represent standard deviations of six to twelve biological replicates. ****p ≤0.0001 in a two tailed t-test assuming equal variances of the data sets. Cohen´s d was calculated to quantify the effect size.

Figure S19. Characterization of motility phenotypes
A-D Particle tracking of E. coli strains used to assess influence of motility, for motility wildtype (A), ΔcheY (B), ΔmotA (C), and ΔfliC (D). Each color represents the trajectory of a single bacterium. E-H Quantification of fraction of swimmers (E), swimming speed (F), tumbling rate (G) and average residence time at the surface (H) for each strain. Of note, rare reorientation events in ΔcheY strain that are detected as tumbling are rather caused by cell collisions with other cells, surface defects or alike. Error bars represent the standard deviations of three biological replicates, each measuring at least fifty cell trajectories. One-way ANOVA and two-sided t-test assuming equal variance between data sets were performed.

Figure S20. Fimbriation has no impact of motility
Swimming speed, measured as in Fig. S19, of fimbriated or fimbrialess E. coli cells grown in YNB fructose supplemented with CSM. ns from a t-test assuming equal variances between the samples for three biological replicates, indicated as dots, each measuring at least fifty cell trajectories.

Figure S21. Activation status of the fim operon does not depend on motility
Quantification of the fim status of all the E. coli MG1655 partners used in this study, as described in Fig. S5, including the non-motile and non-chemotactic. p value from a one-way ANOVA test with three biological replicates per each strain indicated as circles.

Figure S22. Dependence of fitness of motile cells on shaking rate in physically interacting community
A Fraction of motile E. coli cells (labeled with mCherry) compared to the total E. coli population in co-culture with sfGFP-labeled non-motile (ΔfliC) E. coli cells and with yeast at different shaking rates, as indicated. Communities were inoculated with different initial optical density (OD) as indicated and grown for 96 hours in YNB-glucose minimal medium. Error bars represent standard deviations of three biological replicates. One sample t-was performed. Cohen´s d was calculated to quantify the effect size. B Correlation analysis between motile strain cell fractions in FliC+ and ΔfliC co-cultures inoculated with an initial OD600 of 0.001 and the different shaking rates at which they were cultured. The linear regression analysis was performed with a sample size of 29 biological replicates. Figure S23. Effects of motility on communities containing cheater with or without cross feeding A Cheater fraction within the total E. coli population in cross-feeding communities containing a non-motile and fimbriated E. coli cheater (labeled with sfGFP) co-cultured with yeast and with an mCherry-labeled E. coli partner displaying different status of fimbriation and motility, as indicated. Communities were inoculated with an initial 50% cheater fraction and an initial optical density of 0.001, and grown for 96 hours under shaking (300 r.p.m) or without shaking (0 r.p.m) in YNB-fructose minimal medium. Error bars are standard deviations of six biological replicates represented as circles. ****p<0.0001, ***p<0.001, **p<0.01 from paired t-test. B Cheater fraction in communities in absence of cross feeding, having either motile or non-motile fimbriated cheater strains (labeled "C"), in combination with indicated E. coli partner cells, inoculated with a total initial OD of 0.001 and grown in YNB fructose supplemented with CSM under shaking (300 r.p.m). p value from a one-way ANOVA test.  Table S2. Two-way ANOVA analysis Table representing the results from a two-way ANOVA analysis followed by a Tukey HSD post hoc analysis for data presented in Fig. 2F.