Deconstructing and Reconstructing Cheese Rind Microbiomes for Experiments in Microbial Ecology and Evolution

Abstract 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