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

Abstract

Life in a three-dimensional structure such as a biofilm is typical for many bacteria, yet little is known about how strains with different genotypes interact in this context. Here, to systematically explore gene knockdowns across various three-dimensional contexts, we created arrayed libraries of essential-gene CRISPRi knockdowns in the model biofilm-forming bacterium Bacillus subtilis and measured competitive fitness during colony co-culture with a wild type-like parent on different media and at different knockdown levels. Partial knockdown led to a wide range of fitness phenotypes, with targeting of translation-related genes often leading to lower growth rates and rapid out-competition by the parent. Several knockdowns competed differentially in biofilms versus non-biofilm colonies, in some cases due to lack of a particular nutrient in one medium. 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 co-culture with a parent unable to produce extracellular matrix, due to a mutualism 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. These findings demonstrate that growth in a three-dimensional biofilm can promote genetic diversity through sharing of secreted factors, and illustrate the role of matrix production in determining trajectories for biofilm evolution that may be relevant to pathogens and other environmental bacteria.

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

“…The fit parameters from the titration row were used to map the library data and assign GFP fractions; the data were normalized so that the average of the internal parent-RFP and parent-GFP co-culture controls was 1. The image analysis and GFP/RFP scripts are available on Data Dryad: doi: 10.5061/dryad.79cnp5htm ( Arjes, 2021 ).…”

“…The maximum growth rate of each culture was defined as the maximum derivative of ln(OD 600 ). The script used for analyzing the growth curves and calculating growth rates is available on Data Dryad: doi: 10.5061/dryad.79cnp5htm ( Arjes, 2021 ).…”

“…The entire spot was captured in a grid of images using a Nikon Ti-E inverted microscope with a 40 air objective (NA: 0.95) integrated with µManager ( Edelstein et al, 2014 ). Images were stitched together using custom Matlab (MathWorks, Santa Clara, CA, USA) code available on Data Dryad: doi: 10.5061/dryad.79cnp5htm ( Arjes, 2021 ). GFP and RFP stitched images were merged using Adobe Photoshop, adjusting each channel identically across all images.…”

“…Parent-RFP and parent-GFP were cultured and mixed in ratios from 0 to 100% in 20% increments as described above. [50].…”

“…Cultures of the alrA knockdown strain (HA420) and the parent strain (HA2) were separately cultured from a colony in liquid LB at 37 C until both strains reached OD 600 ~1.0. HA420 and HA2 cultures were mixed 1:1, and the mixture along with the HA420 [50]. GFP and RFP stitched images were merged using Adobe Photoshop, adjusting each channel equally.…”