Stochastic Assembly of Bacteria in Microwell Arrays Reveals the Importance of Confinement in Community Development.

Ryan H Hansen,Andrea C Timm,Amber N Bible,Dale A Pelletier,Mitchel J Doktycz,Michael L Simpson,Jennifer L Morrell-Falvey,Collin M Timm,Scott T Retterer,Xiangzhen Li

doi:10.1371/journal.pone.0155080

Abstract

The structure and function of microbial communities is deeply influenced by the physical and chemical architecture of the local microenvironment and the abundance of its community members. The complexity of this natural parameter space has made characterization of the key drivers of community development difficult. In order to facilitate these characterizations, we have developed a microwell platform designed to screen microbial growth and interactions across a wide variety of physical and initial conditions. Assembly of microbial communities into microwells was achieved using a novel biofabrication method that exploits well feature sizes for control of innoculum levels. Wells with incrementally smaller size features created populations with increasingly larger variations in inoculum levels. This allowed for reproducible growth measurement in large (20 μm diameter) wells, and screening for favorable growth conditions in small (5, 10 μm diameter) wells. We demonstrate the utility of this approach for screening and discovery using 5 μm wells to assemble P. aeruginosa colonies across a broad distribution of innoculum levels, and identify those conditions that promote the highest probability of survivial and growth under spatial confinement. Multi-member community assembly was also characterized to demonstrate the broad potential of this platform for studying the role of member abundance on microbial competition, mutualism and community succession.

Highlights

Microbial communities impact our lives in dramatic ways
By varying the length scale of the well diameter (Fig 1C), we show that homogenous populations assemble into large (101–102 μm diameter) wells, while highly heterogeneous populations assemble into small (100–101 μm diameter) wells
Scanning Electron Microscopy (SEM) images of low and highaspect ratio wells demonstrate the capability to tune the level of spatial confinement into which the bacterial communities are assembled (Fig 2A and 2B)

Summary

Introduction

Microbial communities impact our lives in dramatic ways. Taking on the role of both friend and foe, these communities shape our environment and ecosystems, fuel business and agriculture, and simultaneously support and perplex our healthcare system. Most communities are as dynamic as they are diverse, continuously adapting in their composition, organization and function to survive and thrive in changing environmental landscapes. Bacterial Community Assembly in Microwells Reveals Role of Confinement authors would like to thank the J. Mougous Laboratory (University of Washington, Seattle, WA) for the supply of P. aeruginosa strains used in these studies and Dr B. Tseng Laboratory (University of Washington, Seattle, WA) for the supply of plasmids used to express GFP and mCherry in E. coli. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Methods

Results

Conclusion