Cross-feeding maintains diversification in an experimentally evolved microbial community

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Abstract

Microbial communities are incredibly diverse. Yet, the eco-evolutionary processes originating and maintaining this diversity remain understudied. Here, we investigate the effect of interspecies interactions on the generation and maintenance of diversity in microbial communities. We studied a commensal interaction in a community of Acinetobacter johnsonii and Pseudomonas putida where the latter species cross-feeds on leaked resources produced by A. johnsonii . By evolving four experimental replicates in monoculture and co-culture for 200 generations, we observed the diversification of P. putida into two distinct morphotypes that differed from their ancestor by single-point mutations. One of the most prominent mutations hit the fleQ gene encoding the master regulator of flagella and biofilm formation. We experimentally confirmed that fleQ mutants were unable to swim and formed less biofilm than their ancestor, but they also produced higher yields. Interestingly, the fleQ genotype swept to fixation in monocultures but not in co-cultures. In co-cultures, the two lineages stably coexisted for approximately 150 generations. We hypothesized that A. johnsonii modulates the coexistence of the two lineages through frequency-dependent selection. However, invasion experiments in monoculture and co-culture did not support this hypothesis, suggesting other mechanisms maintain coexistence. Overall, our study shows that interspecies interactions play an important role in maintaining the diversity of newly formed lineages in microbial communities.

Importance

In nature, bacteria live in microbial communities and interact with other species, for example, through the exchange of resources leaked into the external environment (i.e., cross-feeding interactions). The role that these cross-feeding interactions play in the formation of new bacterial ‘species’ remains unexplored. Using a simple bacterial system in which one species cross-feeds resources to a second species (commensal species), we showed that the commensal species diversified into two subpopulations that persisted only when the cross-feeder partner was present. We further observed loss-of-function mutations in flagellar genes in both monocultures and co-cultures. Instead, mutants that retained the ability to swim and form biofilms were exclusively observed in co-cultures. These findings suggest that motility and surface attachment traits are important during cross-feeding, even in a well-shaken environment. Given that nutrient leakage is pervasive in microbial communities, the findings from this study have the potential to extend beyond our specific bacterial system.

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