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Abstract Experimental studies of microbial evolution have largely focused on monocultures of model organisms, but most microbes live in communities where interactions with other species may impact rates and modes of evolution. Using the cheese rind model microbial community, we determined how species interactions shape the evolution of the widespread food- and animal-associated bacterium Staphylococcus xylosus. We evolved S. xylosus for 450 generations alone or in co-culture with one of three microbes: the yeast Debaryomyces hansenii, the bacterium Brevibacterium aurantiacum, and the mold Penicillium solitum. We used the frequency of colony morphology mutants (pigment and colony texture phenotypes) and whole-genome sequencing of isolates to quantify phenotypic and genomic evolution. The yeast D. hansenii strongly promoted diversification of S. xylosus. By the end of the experiment, all populations co-cultured with the yeast were dominated by pigment and colony morphology mutant phenotypes. Populations of S. xylosus grown alone, with B. aurantiacum, or with P.solitum did not evolve novel phenotypic diversity. Whole-genome sequencing of individual mutant isolates across all four treatments identified numerous unique mutations in the operons for the SigB, Agr, and WalRK global regulators, but only in the D. hansenii treatment. Phenotyping and RNA-seq experiments highlighted altered pigment and biofilm production, spreading, stress tolerance, and metabolism of S. xylosus mutants. Fitness experiments revealed antagonistic pleiotropy, where beneficial mutations that evolved in the presence of the yeast had strong negative fitness effects in other biotic environments. This work demonstrates that bacterial-fungal interactions can have long-term evolutionary consequences within multispecies microbiomes by facilitating the evolution of strain diversity. 

ABSTRACT Diversification can generate genomic and phenotypic strain-level diversity within microbial species. This microdiversity is widely recognized in populations, but the community-level consequences of microbial strain-level diversity are poorly characterized. Using the cheese rind model system, we tested whether strain diversity across microbiomes from distinct geographic regions impacts assembly dynamics and functional outputs. We first isolated the same three bacterial species ( Staphylococcus equorum , Brevibacterium auranticum , and Brachybacterium alimentarium ) from nine cheeses produced in different regions of the United States and Europe to construct nine synthetic microbial communities consisting of distinct strains of the same three bacterial species. Comparative genomics identified distinct phylogenetic clusters and significant variation in genome content across the nine synthetic communities. When we assembled each synthetic community with initially identical compositions, community structure diverged over time, resulting in communities with different dominant taxa. The taxonomically identical communities showed differing responses to abiotic (high salt) and biotic (the fungus Penicillium ) perturbations, with some communities showing no response and others substantially shifting in composition. Functional differences were also observed across the nine communities, with significant variation in pigment production (light yellow to orange) and in composition of volatile organic compound profiles emitted from the rinds (nutty to sulfury). IMPORTANCE Our work demonstrated that the specific microbial strains used to construct a microbiome could impact the species composition, perturbation responses, and functional outputs of that system. These findings suggest that 16S rRNA gene taxonomic profiles alone may have limited potential to predict the dynamics of microbial communities because they usually do not capture strain-level diversity. Observations from our synthetic communities also suggest that strain-level diversity has the potential to drive variability in the aesthetics and quality of surface-ripened cheeses.