A Synthetic Community System for Probing Microbial Interactions Driven by Exometabolites

Abstract

Though most microorganisms live within a community, we have modest knowledge about microbial interactions and their implications for community properties and ecosystem functions. To advance understanding of microbial interactions, we describe a straightforward synthetic community system that can be used to interrogate exometabolite interactions among microorganisms. The filter plate system (also known as the Transwell system) physically separates microbial populations, but allows for chemical interactions via a shared medium reservoir. Exometabolites, including small molecules, extracellular enzymes, and antibiotics, are assayed from the reservoir using sensitive mass spectrometry. Community member outcomes, such as growth, productivity, and gene regulation, can be determined using flow cytometry, biomass measurements, and transcript analyses, respectively. The synthetic community design allows for determination of the consequences of microbiome diversity for emergent community properties and for functional changes over time or after perturbation. Because it is versatile, scalable, and accessible, this synthetic community system has the potential to practically advance knowledge of microbial interactions that occur within both natural and artificial communities. IMPORTANCE Understanding microbial interactions is a fundamental objective in microbiology and ecology. The synthetic community system described here can set into motion a range of research to investigate how the diversity of a microbiome and interactions among its members impact its function, where function can be measured as exometabolites. The system allows for community exometabolite profiling to be coupled with genome mining, transcript analysis, and measurements of member productivity and population size. It can also facilitate discovery of natural products that are only produced within microbial consortia. Thus, this synthetic community system has utility to address fundamental questions about a diversity of possible microbial interactions that occur in both natural and engineered ecosystems.

Keywords: Burkholderia; Chromobacterium; Pseudomonas syringae; community ecology; microbial metabolomics; model microbial systems; synthetic communities.

FIG 1

The filter plate system reproduces known microbial interactions facilitated by exometabolites. (A) Control. The Chromobacterium violaceum mutant strain Cv026 cannot produce acyl-homoserine lactones (AHLs), while strain Cv017 can. AHLs trigger production of the purple pigment violacein. (B) AHLs from Cv017 diffused through wells to induce quorum sensing and violacein production in Cv026. (C) Colonies of Cv026, diluted from the Transwells in panel B and plated, reverted color in the absence of exogenous AHLs.

FIG 2

Global exometabolite changes in a three-member community compared across time and with mass spectral replication. Shown are results from principal-component analysis (PCA) of normalized, log₂-transformed mass spectral profiles. Profiles are colored by time and labeled by replicate (R1 to R4). Quality control series (QC), a composite of all experimental samples, are labeled by analysis order (T1 to T6). A total of 977 mass features were included after quality filtering.

FIG 3

Exometabolites exhibit directional changes over stationary phase in a three-member synthetic microbial community. Shown is a heat map of 977 mass feature changes over time within a three-member community, where samples are columns and features are rows. Each sample is the average of four time point replicates, each started independently from new cultures. Euclidean distance was calculated from Z-scored mass spectral profiles. Features with similar dynamics were clustered by Ward’s method. Letter designations for clusters were added post hoc to aid in discussion. “QC” is quality control series, an even composite of all experimental samples that was run at regular intervals on the mass spectrometer to assess instrument stability and feature consistency.

FIG 4

The filter plate system provides evidence of inhibition among members. Shown are changes in live cell counts of P. syringae over stationary phase, measured using flow cytometry of Syto9-stained cells recovered from the filter plates. Five wells per plate and two replicate plates per time point were used to assess P. syringae cell counts when grown in monoculture (A), the three-member community (B), coculture with B. thailandensis (C), and coculture with C. violaceum (D). Reduced counts in coculture and the three-member community (compared to counts in monoculture or with C. violaceum) suggest an antagonistic interaction with B. thailandensis.