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ABSTRACT Although bacteria exist in complex microbial communities in the environment, their features and behavior are most often studied in monoculture. While environmental enrichments or complex co-cultures with tens or hundreds of members might more accurately represent the natural communities of bacteria, we sought to create simple pairs of organisms to learn what conditions create successful co-culture and how bacteria change transcriptionally when a partner species is present. We grew two pairs of organisms in co-culture,Pseudomonas aeruginosaandEscherichia coliandLacticaseibacillus rhamnosusandBacteroides thetaiotaomicron. At first, both co-cultures failed, with one organism outcompeting the other. However, through manipulating media and environmental conditions, we created co-cultures with stable member ratios over many generations for each community. We then show that changes in the expression of metabolic genes are present in all studied species, with key catabolic and anabolic pathways often upregulated in the presence of another organism. These changes in gene expression fail to occur in conditions that will not lead to successful co-culture, suggesting they are essential for adapting to and surviving in the presence of others. IMPORTANCEIn 1882, Robert Koch and Fanny Hesse developed the agar plate, which enabled microbiologists to separate individual microbial cells from each other and create monocultures of a single strain of bacteria. This powerful tool has been used in the almost 150 years since to develop a robust understanding of how bacterial cells are structured, how they manage and process their information, and how they respond to the environment to produce behaviors that match their circumstances. We were curious about how the behavior of bacteria, as measured by their gene expression, changes between well-studied monoculture conditions and co-culture. We found that only specific growth conditions permit co-culture and that bacteria change their metabolic strategies in the presence of a partner.