PROJECT SUMMARY In nature, bacteria typically exist within multispecies communities. Bacterial communities play vital roles in shaping the environment and their plant and animals. In spite of the prevalence and importance of microbial communities, important gaps still remain in our understanding of how bacteria interact within these microbiomes. Our lab’s NIGMS-relevant research is focused on investigating the chemical, molecular, and genetic mechanisms bacteria use to chemically and physically interact within multispecies communities. We focus on the soil microbiome as our model system: the soil is not one of the most phylogenetically diverse microbial environments on the planet, but soil microbes are also the source of the majority of our antibiotics and many other therapeutics. Therefore, understanding the mechanisms bacteria use to interact within this natural environment will not only provide a systems-level understanding of complex natural microbiome interactions but also provide us with potential chemical tools and therapeutic leads to manipulate bacterial behavior. These bacterially generated compounds are celled specialized or secondary metabolites. These secreted chemical cues can act as cell-cell communication signals that influence the physiology and metabolism of neighboring bacteria. They play key roles in bacterial differentiation, or the development of transcriptionally distinct, heterogeneously expressed subpopulations of cells. We focus on the soil and probiotic bacterium Bacillus subtilis, which can differentiate into cells that are making biofilm matrix, swimming, or sporulating, among others. We are interested in understanding the transcriptional specificity, ancestral lineages, and spatial distributions of this cellular heterogeneity as well as what roles specialized metabolites play in its development. We also aim to discover specialized metabolites involved in interspecies cell-cell communication to expand our understanding of chemical interactions in native microbiomes and obtain chemical tools to modulate bacterial physiology. Finally, we seek to identify the genetic and molecular mechanisms these extracellular signals use to impact bacterial transcription, heterogeneity, and metabolic activity. This research is significant because it will reveal fundamental information about the chemical and genetic mechanisms bacteria use to interact with one another. Our results will deepen our molecular understanding of cell-cell interactions within microbial communities as well as enable the development of targeted interventions to manipulate microbial behavior in environmentally and therapeutically important bacteria.