Coculturing Bacteria Leads to Reduced Phenotypic Heterogeneities

Abstract

Isogenic bacterial populations are known to exhibit phenotypic heterogeneity at the single-cell level. Because of difficulties in assessing the phenotypic heterogeneity of a single taxon in a mixed community, the importance of this deeper level of organization remains relatively unknown for natural communities. In this study, we have used membrane-based microcosms that allow the probing of the phenotypic heterogeneity of a single taxon while interacting with a synthetic or natural community. Individual taxa were studied under axenic conditions, as members of a coculture with physical separation, and as a mixed culture. Phenotypic heterogeneity was assessed through both flow cytometry and Raman spectroscopy. Using this setup, we investigated the effect of microbial interactions on the individual phenotypic heterogeneities of two interacting drinking water isolates. Through flow cytometry we have demonstrated that interactions between these bacteria lead to a reduction of their individual phenotypic diversities and that this adjustment is conditional on the bacterial taxon. Single-cell Raman spectroscopy confirmed a taxon-dependent phenotypic shift due to the interaction. In conclusion, our data suggest that bacterial interactions may be a general driver of phenotypic heterogeneity in mixed microbial populations.IMPORTANCE Laboratory studies have shown the impact of phenotypic heterogeneity on the survival and functionality of isogenic populations. Because phenotypic heterogeneity plays an important role in pathogenicity and virulence, antibiotic resistance, biotechnological applications, and ecosystem properties, it is crucial to understand its influencing factors. An unanswered question is whether bacteria in mixed communities influence the phenotypic heterogeneity of their community partners. We found that coculturing bacteria leads to a reduction in their individual phenotypic heterogeneities, which led us to the hypothesis that the individual phenotypic diversity of a taxon is dependent on the community composition.

Keywords: Raman spectroscopy; axenic culture; coculture; flow cytometry; microbial interactions; phenotypic heterogeneity; single cell; synthetic ecosystems.

FIG 1

Illustration of the experimental setup. Bacteria in apical and basal phase can interact via metabolites in their shared medium while they are physically separated by the membrane of the cell culture inserts. Four synthetic communities were created: two axenic cultures, a coculture, and a mixed culture. There were biological replicates (n = 3) for each synthetic community.

FIG 2

(A) Phenotypic alpha diversity D₀ for both individual bacterial taxa in communities of axenic cultures, cocultures, and mixed cultures. The taxa are denoted as taxon E (Enterobacter sp.) and taxon P (Pseudomonas sp.), respectively. The populations are indicated with names in the form of “X treated with Y,” where X is the taxon in the sample (E, P, or EP) and Y is what was present on the other side of the membrane (E, P, or fresh medium). There were biological replicates (n = 3) for each community. The dashed lines indicate the average trend of the replicates. P values indicate the significance of the differences between the axenic culture and coculture populations at 72 h for Enterobacter (P_E) and Pseudomonas (P_P) (one-sided Wilcoxon rank sum test). (B and C) Contrast analysis of the phenotypic fingerprints was performed to compare the phenotypic community structure of axenic cultures and coculture members with respect to fluorescence intensity. Each plot is a comparison between the axenic culture and coculture of the same taxon at the same time point, averaged over the three biological replicates. The color gradient indicates whether density in the coculture increased (purple) or decreased (dark green) relative to their respective axenic culture at the specified time point. (B) The upper row presents contrast results for Enterobacter. (C) The lower row presents contrast results for Pseudomonas. If the difference between the two communities is <0.01, no contrast value is shown on the graphs, which causes the appearance of different clusters. Note that different scales were used for the different taxa.

FIG 3

PCoA ordination of the Bray-Curtis dissimilarities between the phenotypic fingerprints for both individual bacterial taxa in communities of axenic cultures, cocultures, and mixed cultures. The ordination is shown in three graphs, split according to time, since this allows for easier interpretation of how the different communities are relating to each other at each time point. The taxa are denoted as taxon E (Enterobacter sp.) and taxon P (Pseudomonas sp.), respectively. The populations are indicated with names in the form of “X treated with Y,” where X is the taxon in the sample (E, P, or EP) and Y is what was present on the other side of the membrane (E, P, or fresh medium). There were biological replicates (n = 3) for each community. The variance explained by the overall temporal effect (r²_Time), as well as the effect of coculturing compared to axenic culture (r²_E), is provided (PERMANOVA). The effect of coculturing was not significant for Pseudomonas and is therefore not indicated.

FIG 4

Predicted relative abundances in the mixed cultures. The random forest classifiers that were used to infer community composition were constructed using the fingerprints of the coculture members at the corresponding time point as input data. Green lines indicate the predicted relative abundances of Enterobacter; blue lines indicate the predicted relative abundances of Pseudomonas. The different shades correspond to biological replicates (n = 3).

FIG 5

Visualization of the separability of the single-cell Raman spectra for Enterobacter and Pseudomonas in axenic culture and coculture at 72 h. There are 51 single-cell measurements for each population. The taxa are denoted as taxon E (Enterobacter sp.) and taxon P (Pseudomonas sp.), respectively. The populations are indicated with names in the form of “X treated with Y,” where X is the taxon in the sample (E, P, or EP) and Y is what was present on the other side of the membrane (E, P, or fresh medium). PCA was carried out for the entire data set (A), for the spectra of Enterobacter separately (B), and for the spectra of Pseudomonas separately (C). The variance explained by the effect of coculturing compared to axenic culture (r²_E) is provided for Enterobacter (PERMANOVA). Beta dispersions were differing significantly between the two groups, and therefore no PERMANOVA could be performed for Pseudomonas.