Simultaneous Discovery of Positive and Negative Interactions Among Rhizosphere Bacteria Using Microwell Recovery Arrays

Abstract

Understanding microbe-microbe interactions is critical to predict microbiome function and to construct communities for desired outcomes. Investigation of these interactions poses a significant challenge due to the lack of suitable experimental tools available. Here we present the microwell recovery array (MRA), a new technology platform that screens interactions across a microbiome to uncover higher-order strain combinations that inhibit or promote the function of a focal species. One experimental trial generates 10⁴ microbial communities that contain the focal species and a distinct random sample of uncharacterized cells from plant rhizosphere. Cells are sequentially recovered from individual wells that display highest or lowest levels of focal species growth using a high-resolution photopolymer extraction system. Interacting species are then identified and putative interactions are validated. Using this approach, we screen the poplar rhizosphere for strains affecting the growth of Pantoea sp. YR343, a plant growth promoting bacteria isolated from Populus deltoides rhizosphere. In one screen, we montiored 3,600 microwells within the array to uncover multiple antagonistic Stenotrophomonas strains and a set of Enterobacter strains that promoted YR343 growth. The later demonstrates the unique ability of the platform to discover multi-membered consortia that generate emergent outcomes, thereby expanding the range of phenotypes that can be characterized from microbiomes. This knowledge will aid in the development of consortia for Populus production, while the platform offers a new approach for screening and discovery of microbial interactions, applicable to any microbiome.

Keywords: consortia; high throughput screening; microbial communities; microbial interactions; microbiome; microdevice; microwell; plant growth promoting rhizobacteria.

Figure 1

Microwell recovery arrays (MRAs) for screening microbe-microbe interactions. (i) Green fluorescent protein (GFP)-expressing focal species are combined with a random combination of bacteria cells from an environmental microbiome in a stochastic seeding process. Different shapes represent unique microorganisms. (ii) Cells are trapped within their wells using a photodegradable polyethylene glycol (PEG) hydrogel membrane and monitored in parallel during co-culture using time lapse fluorescent microscopy (TLFM). (iii) The membrane is ablated over a target well showing highest or lowest levels of focal species growth using patterned light exposure, then (iv) isolates are extracted and recovered from an opened well. (v) Isolates are identified using 16S amplicon sequencing. (vi) Steps (iii–v) are repeated in iterative fashion to remove each community of interest.

Figure 2

(A) Model C58-GFP (green) – PAO1-mCherry (red) co-culture in the MRA. Arrows indicate rare outlier wells where C58-GFP outgrew PAO1-mCherry. (B) Scatter plot of green (C58-GFP) vs. red (PAO1-mCherry) well signals from a sample 549 well array at various time points. Outlier wells where C58 outgrew PAO1 are identified after the culture period (green). (C) Individual growth trajectories from a sample nominal well (well #1109), where PAO1 growth rate was significantly higher than that of C58 and an outlier well (Well #1223), where C58 outgrew PAO1.

Figure 3

YR343-GFP growth in mono-culture and co-culture within 10 μm microwells. (A) TLFM images of a sample 15 × 15 array of microwells after (i) seeding only YR343-GFP or (ii) seeding YR343-GFP with isolates from a Populus trichocarpa rhizobiome. (B) Growth curves generated from a sample 900 microwell array during YR343-GFP mono-culture, or (C) YR343-GFP co-culture with rhizosphere isolates. Outlier wells representing growth promoting and antagonistic communities, respectively were identified from the growth curves.

Figure 4

Sequential removal of growth-promoting and antagonistic communities from an array sub-section after co-culture. (A) Microwell array before and after co-culture. This 15 × 15 microwell array contained both a YR343 growth promoting community (blue) and YR343 antagonistic (red) community that were targeted for extraction. (B) Targeted removal of the microwell community in which YR343 grows to its highest observed end-point fluorescence (top row and blue outline), followed by targeted removal of a microwell community in which YR343-GFP grew poorly (bottom row and red outline). Purple area denotes UV exposure area used for membrane degradation. (C) Maximum likelihood phylogenetic tree based on partial 16S ribosomal RNA (rRNA) sequences (1,007 sites) of select reference strains and isolates extracted from promoted (P) and antagonized (A) wells. We collapsed the branches of the monophyletic group composed of Enterobacter sp. and Pantoea sp. strains and the clade of Stenotrophomonas sp. strains. Agrobacterium tumefaciens C58 was used as the outgroup (OG) organism and the following reference strains were included: Pantoea sp. YR343, Enterobacter cloacae E3442, Pseudomonas putida S13.1.2, and Stenotrophomonas maltophilia NCTC10259. We labeled nodes with corresponding bootstrap percentages.

Figure 5

Interactions identified in the MRA can be validated in 96-well plate format. (A) Left: YR343 growth curves after inoculation into conditioned media from the antagonistic isolate, the isolate consortia, or unconditioned media (UCM). The control (green line) is conditioned media that was not inoculated with YR343 to verify that there was no growth carry over or contaminating microbes present. Right: Corresponding carrying capacity and growth rates for each growth curve. (B) Left: Analogous YR343 growth curves after inoculation into conditioned media from a promoter isolate or the promoter isolate combination. Right: Corresponding carrying capacity and growth rates. All growth experiments occurred at 28°C, 215 rpm. Statistical differences were identified by comparison of growth metrics between YR343 culture in conditioned media from each isolate or isolate mixture and YR343 growth in UCM (Wilcoxon two-sample test, ^*p < 0.01, n = 6 independent experiments).