Animals live and evolved in a world teeming with microbes; as a result, animal- bacterial associations, ranging from pathogenic to mutualistic, are ancient and ubiquitous. Progress toward understanding mechanisms and evolution of bacterial signaling with eukaryotes has been limited by the paucity of experimentally tractable and phylogenetically relevant research models. Our lab has discovered that the development of multicelled colonies in the choanoflagellate Salpingoeca rosetta is regulated by a chemical signal from the bacterium Algoriphagus sp. Elucidating the molecular mechanisms underlying this signaling interaction promises to provide important insights into developmental signaling between bacterial symbionts and animals because (1) choanoflagellates are the closest living relatives of animals and (2) Bacteroidetes bacteria, of which Algoriphagus is a member, predominate the microbiomes of animal intestines, where they exert influences on development, health, and disease. We propose three aims to determine the mechanisms underlying molecular signaling by Algoriphagus bacteria and its regulation of choanoflagellate colony development. First, in collaboration with the Clardy lab (Harvard Medical School) we will isolate colony-inducing compounds from diverse bacteria, determine their structures and use a combination of organic chemistry and classical genetic manipulations of biosynthetic genes to perform structure/function analyses. Second, we will define the signaling interface between bacteria and choanoflagellates by tagging the bacterial inducer and visualizing dynamics of signal delivery. Third, we will identify the choanoflagellate receptor(s) for bacterial signaling molecules using biochemical and candidate gene approaches. This research has the potential to uncover the actual mechanisms involved in inter-kingdom signaling which will allow us to examine the evolution of modern host- bacterial interactions. PUBLIC HEALTH RELEVANCE: Bacterial symbionts profoundly influence their animal hosts by regulating development, maintaining a healthy immune system, and determining the efficiency of nutrient metabolism. To understand how bacteria regulate animal biology, new model systems that can reveal the fundamental processes underlying interactions between eukaryotes and their associated microbial communities must be developed. To this end, we propose to determine the molecular mechanisms that allow bacteria to regulate the development of multicellularity in choanoflagellates, the closest living relatives of animals.