RBTnSeq analysis of Higher order interactions in microbial community

This study aims to investigate how microbial interactions change along with microbial community complexity (number of interacting species). Such information is central to our understanding and further manipulation of microbial communities. Here, we used a bottom-up approach to investigate microbial interaction mechanisms from pairwise cultures up to 4-species communities from a simple microbiome (Hafnia alvei, Geotrichum candidum, Pencillium camemberti and Escherichia coli). Specifically, we characterized the interaction landscape for each species combination involving E. coli by identifying E. coli's interaction-associated genes using an RB-TnSeq-based interaction assay. Altogether, we demonstrate the complex architecture of microbial interactions even within a simple microbiome, and provide a mechanistic and molecular explanation of higher-order interactions that emerge as community gets more complex.