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Bifidobacteria represent a dominant constituent of human gut microbiomes during infancy, influencing nutrition, immune development, and resistance to infection. Despite interest in bifidobacteria as a live biotic therapy, our understanding of colonization, host-microbe interactions, and the health-promoting effects of bifidobacteria is limited. To address these major knowledge gaps, we used a large-scale genetic approach to create a mutant fitness compendium in Bifidobacterium breve. First, we generated a high-density randomly barcoded transposon insertion pool and used it to determine fitness requirements during colonization of germ-free mice and chickens with multiple diets and in response to hundreds of in vitro perturbations. Second, to enable mechanistic investigation, we constructed an ordered collection of insertion strains covering 1,462 genes. We leveraged these tools to reveal community- and diet-specific requirements for colonization and to connect the production of immunomodulatory molecules to growth benefits. These resources will catalyze future investigations of this important beneficial microbe. 

Lactobacilli andAcetobacter sp.are commercially important bacteria that often form communities in natural fermentations, including food preparations, spoilage, and in the digestive tract of the fruit fly Drosophila melanogaster. Communities of these bacteria are widespread and prolific, despite numerous strain-specific auxotrophies, suggesting they have evolved nutrient interdependencies that regulate their growth. The use of a chemically-defined medium (CDM) supporting the growth of both groups of bacteria would facilitate the identification of the molecular mechanisms for the metabolic interactions between them. While numerous CDMs have been developed that support specific strains of lactobacilli orAcetobacter, there has not been a medium formulated to support both genera. We developed such a medium, based on a previous CDM designed for growth of lactobacilli, by modifying the nutrient abundances to improve growth yield. We further simplified the medium by substituting casamino acids in place of individual amino acids and the standard Wolfe’s vitamins and mineral stocks in place of individual vitamins and minerals, resulting in a reduction from 40 to 8 stock solutions. These stock solutions can be used to prepare several CDM formulations that support robust growth of numerous lactobacilli andAcetobacters. Here, we provide the composition and several examples of its use, which is important for tractability in dissecting the genetic and metabolic basis of natural bacterial species interactions.