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

Abstract

Understanding the consequences of microbe-microbe interactions is critical in efforts to predict the function of microbiomes and to manipulate or construct communities to achieve desired outcomes. The investigation of these interactions poses a significant challenge -in part due to the lack of suitable experimental tools. We present the Microwell Recovery Array, a high throughput approach designed to rapidly screen interactions across a microbiome and uncover higher-order combinations of strains that either inhibit or promote the function of a GFP-producing focal species. One experiment generates 10 4 unique microbial communities that contain a focal species combined with a unique combination of previously uncharacterized cells from plant rhizosphere. Cells are then sequentially extracted from individual co-culture wells that display highest or lowest levels of focal species function using a novel high-resolution photopolymer extraction system. Microbes present are subsequently identified and the putative interactions are validated. Using this approach, we screen the Populus trichocarpa rhizosphere for bacterial strains affecting the survival and growth of Pantoea sp. YR343, a plant growth promoting strain isolated from the P. trichocarpa rhizosphere. We were able to simultaneously isolate and validate multiple Stenotrophomonas strains that antagonize strain YR343 growth and a set of Enterobacter strains that promote strain YR343 growth. The latter demonstrates the unique ability of the platform to uncover multimembered consortia that generate emergent phenotypes. This knowledge will inform the development of beneficial consortia that promote the production of Populus biofuel feedstock, while the platform is adaptable to screening higher-order interactions in any microbiome of interest. Significance StatementAchieving a fundamental understanding of microbe-microbe interactions that occur within microbial communities is a grand challenge in microbiology due to the limited experimental tools available. In this report, we describe a new tool that enables one to screen microbial interactions across thousands of compositionally unique communities to discover collections of bacteria that antagonize or promote the survival and growth of bacteria with important functions. This approach has the unique ability to uncover higher-order combinations of bacteria that generate emergent

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

“…Geometric features of microwells such as well depth and diameter are also controlled through standard microfabrication methods. With these benefits, microwells have been useful for studying bacteria growth under spatial confinement 26 , and most recently for the discovery of symbiotic and antagonistic interactions between different bacterial species when confined together at the microscale 18 .…”

“…The separation process in both formats has been successfully used to isolate bacteria that display unique growth behavior for downstream genotyping after culture and microscopic observation, a critical capability for connecting cell genotype to phenotype. To date, genomic characterizations of bacteria extracted from these interfaces have included 16S amplicon sequencing to identify multi-species collections of bacteria from environmental microbiomes that generate emergent growth behavior 18 , and for whole-genome sequencing to successfully identify genetic mutations that cause rare growth profiles in cells present within mutant libraries 19 .…”

“…2. Microfabrication: Follow the protocol described by Barua et al 18 to design and fabricate the microwell array (Supplementary Figure 1). NOTE: Standard photolithographic techniques described by Timm et al 17 were applied to fabricate microwell arrays on parylene-coated silicon wafers.…”

“…Two-color fluorescence images revealed that both the membrane and the cells within the wells are removed for both irradiation patterns17 . Unlike the bulk hydrogel format, cell extraction here has only been observed in the shape of cell clusters18 .…”

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

“…This manuscript has been released as a pre-print at BioRxiv, (Barua et al, 2020). We thank Priscila Guzman (Divsion of Biology, Kansas State Univeristy) for making the C58 and PAO1 strains used in experiments.…”