Three-dimensional biofilm colony growth supports a mutualism involving matrix and nutrient sharing.

Heidi A Arjes,Kerwyn Casey Huang,Jason Peters,Yangbo Xiao,Haiwen Gui,Lisa Willis,Carol Gross

doi:10.7554/elife.64145

Abstract

Life in a three-dimensional biofilm is typical for many bacteria, yet little is known about how strains interact in this context. Here, we created essential gene CRISPR interference knockdown libraries in biofilm-forming Bacillus subtilis and measured competitive fitness during colony co-culture with wild type. Partial knockdown of some translation-related genes reduced growth rates and led to out-competition. Media composition led some knockdowns to compete differentially as biofilm versus non-biofilm colonies. Cells depleted for the alanine racemase AlrA died in monoculture but survived in a biofilm colony co-culture via nutrient sharing. Rescue was enhanced in biofilm colony co-culture with a matrix-deficient parent due to a mutualism involving nutrient and matrix sharing. We identified several examples of mutualism involving matrix sharing that occurred in three-dimensional biofilm colonies but not when cultured in two dimensions. Thus, growth in a three-dimensional colony can promote genetic diversity through sharing of secreted factors and may drive evolution of mutualistic behavior.

Highlights

52 In natural environments, many bacteria grow in dense, three-dimensional multicellular communities held together by extracellular matrix, often called biofilms
Cells depleted for the alanine racemase AlrA died in monoculture, but co-cultures survived via nutrient sharing in a biofilm but not in liquid. This rescue was enhanced in biofilm co43 culture with a parent unable to produce extracellular matrix, due to a mutualism 44 involving nutrient and matrix sharing
Including alrA, we identified several examples of mutualism involving matrix sharing that occurred in a three-dimensional biofilm colony but not when growth was constrained to two dimensions

Summary

Introduction

52 In natural environments, many bacteria grow in dense, three-dimensional multicellular communities held together by extracellular matrix, often called biofilms. Biofilms allow for genetic differentiation and division of labor that can mutually benefit distinct genotypes. The rate at which mutations fix in a population of a given size is higher in microbial colonies compared to well-shaken, liquid cultures [5], suggesting that spatial confinement supports an increase in genetic variation. Spatial confinement dramatically increases the frequency of interactions between nearby cells and the potential for coupled evolutionary outcomes, enhancing random genetic drift [6]. Mechanisms that support genetic diversity in the context of a three-dimensional bacterial colony or biofilm remain underexplored [7]

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Discussion

Conclusion