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

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 CRISPRi 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.

Spatial alanine metabolism determines local growth dynamics ofEscherichia colicolonies

“…In contrast to these metabolites, alanine can not only be used as carbon source, but also as a nitrogen source in the cross-feeding-dependent region, and we note that it is currently not clear what limits growth in the higher regions of the colonywhether it is carbon, nitrogen, or other elements such as iron, sulfur, or phosphorous. Very recently, it was shown that alanine can be cross-fed in colonies of the Gram-positive bacterium Bacillus subtilis, and that this effect required three-dimensional colonies for unknown reasons (Arjes et al, 2021). In our E. coli model system, three-dimensional growth is required to create an anaerobic region replete with carbon and nitrogen sources that causes alanine secretion, and we speculate that this effect may also be required in B. subtilis, and in many other species.…”

“…Several cross-feeding interactions have been described in detail for multi-species communities (Henson et al, 2019;Kim et al, 2017;Moree et al, 2012;Pande et al, 2016;Røder et al, 2020;Smith et al, 2019;Watrous et al, 2013;Yang et al, 2009). Even though cross-feeding interactions are likely a ubiquitous process in single-species bacterial multicellular structures (San Roman & Wagner, 2018), only a few metabolic interactions between subpopulations have been documented for single-species biofilms (Arjes et al, 2021;Cole et al, 2015;Evans et al, 2020;Lin et al, 2018;Liu et al, 2015). For E. coli colonies grown on agar-solidified medium, and for bacterial communities in general, it is still unclear how many subpopulations interact metabolically, and on which length and time scales these interactions take place, and how significant these many interactions are for the community growth and stability.…”

“…In contrast, single-species biofilms offer precisely controllable model systems with reduced complexity for understanding basic mechanisms of metabolic differentiation and the interaction of subpopulations in communities. Investigations of phenotypic heterogeneity in single-species assemblages have already revealed fundamental insights into metabolic interactions of subpopulations with consequences for the overall fitness and growth dynamics of the assemblage (Arjes et al, 2021;Cole et al, 2015;Evans et al, 2020;Lin et al, 2018;Liu et al, 2015Liu et al, , 2017Wolfsberg et al, 2018). However, even for single-species biofilms, the extent of metabolic heterogeneity and metabolic dependencies of subpopulations are unclear.…”

Cryptic surface-associated multicellularity emerges through cell adhesion and its regulation

“…Theoretical models on surface growth are paralleled by a large body of empirical literature where surface-associated organisms are studied by either growing collectives in isolation (e.g., biofilms, colonies, pellicles, swarms, fruiting bodies) [110][111][112][113], thereby focusing on their multicellular organization [36,[114][115][116][117][118], or by competing several genotypes against each other on a surface [30,[119][120][121][122][123][124]. Competition experiments revealed that spatial interactions can strongly affect the outcome of competition, oftentimes leading to the coexistence of multiple genotypes [43,120,125].…”

Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron

“…Surprisingly, during liquid growth after passaging BT2397 (tnr3) through a colony, we observed reversion to wild-type phenotypes, indicating that growth conditions can play a major role in B. theta phenotypes. In the future, it will be interesting to screen the condensed collection in diverse media conditions to uncover additional phenotypes, as a previous screen of a CRISPRi library of Bacillus subtilis essential-gene depletions uncovered a wider distribution of phenotypes in a minimal medium compared with LB (48).…”