Many metagenomic sequencing studies have observed the presence of closely related bacterial species or genotypes in the same microbiome. Previous attempts to explain these patterns of microdiversity have focused on the abiotic environment, but few have considered how biotic interactions could drive patterns of microbiome diversity. We dissected the patterns, processes, and mechanisms shaping the ecological distributions of three closely related Staphylococcus species in cheese rind biofilms. Paradoxically, the most abundant species (S. equorum) is the slowest colonizer and weakest competitor based on growth and competition assays in the laboratory. Through in vitro community reconstructions, we determined that biotic interactions with neighboring fungi help resolve this paradox. Species-specific stimulation of the poor competitor by fungi of the genus Scopulariopsis allows S. equorum to dominate communities in vitro as it does in situ. Results of comparative genomic and transcriptomic experiments indicate that iron utilization pathways, including a homolog of the S. aureus staphyloferrin B siderophore operon pathway, are potential molecular mechanisms underlying Staphylococcus-Scopulariopsis interactions. Our integrated approach demonstrates that fungi can structure the ecological distributions of closely related bacterial species, and the data highlight the importance of bacterium-fungus interactions in attempts to design and manipulate microbiomes.

Cheese rind microbiomes are useful model systems for identifying the mechanisms that control microbiome diversity. Here, we describe the methods we have optimized to first deconstruct in situ cheese rind microbiome diversity and then reconstruct that diversity in laboratory environments to conduct controlled microbiome manipulations. Most cheese rind microbial species, including bacteria, yeasts, and filamentous fungi, can be easily cultured using standard lab media. Colony morphologies of taxa are diverse and can often be used to distinguish taxa at the phylum and sometimes even genus level. Through the use of cheese curd agar medium, thousands of unique community combinations or microbial interactions can be assessed. Transcriptomic experiments and transposon mutagenesis screens can pinpoint mechanisms of interactions between microbial species. Our general approach of creating a tractable synthetic microbial community from cheese can be easily applied to other fermented foods to develop other model microbiomes. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Isolation of cheese rind microbial communities Support Protocol 1: Preparation of plate count agar with milk and salt Basic Protocol 2: Identification of cheese rind bacterial and fungal isolates using 16S and ITS sequences Basic Protocol 3: Preparation of experimental glycerol stocks of yeasts and bacteria Basic Protocol 4: Preparation of experimental glycerol stocks of filamentous fungi Basic Protocol 5: Reconstruction of cheese rind microbial communities in vitro Support Protocol 2: Preparation of lyophilized and powdered cheese curd Support Protocol 3: Preparation of 10% cheese curd agar plates and tubes Basic Protocol 6: Interaction screens using responding lawns Support Protocol 4: Preparation of liquid 2% cheese curd Basic Protocol 7: Experimental evolution Basic Protocol 8: Measuring community function: pH/acidification Basic Protocol 9: Measuring community function: Pigment production Basic Protocol 10: RNA sequencing of cheese rind biofilms

Summary In vitro studies in plant, soil, and human systems have shown that microbial volatiles can mediate microbe–microbe or microbe–host interactions. These previous studies have often used artificially high concentrations of volatiles compared to in situ systems and have not demonstrated the roles volatiles play in mediating community‐level dynamics. We used the notoriously volatile cheese rind microbiome to identify bacteria responsive to volatiles produced by five widespread cheese fungi. Vibrio casei had the strongest growth stimulation when exposed to all fungi. In multispecies community experiments, fungal volatiles caused a shift to a Vibrio‐dominated community, potentially explaining the widespread occurrence of Vibrio in surface‐ripened cheeses. RNA sequencing identified activation of the glyoxylate shunt as a possible mechanism underlying volatile‐mediated growth promotion and community assembly. Our study demonstrates how airborne chemicals could be used to control the composition of microbiomes and illustrates how volatiles may impact the development of cheese rinds.

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CAG/CTG trinucleotide repeat expansions cause several degenerative neurological and muscular diseases. Koch et al. show that the chromatin remodeling...