PROJECT SUMMARY The objective of my laboratory is to characterize how molecular communication between bacteria and their animal hosts leads to specific and reproducible colonization. To accomplish this goal, the laboratory studies the Vibrio fischeri-squid system, in which the animal’s “light organ” is colonized exclusively by one bacterial species. This system is advantageous because bacteria colonize through the natural route of infection, all animals are colonized within three hours of bacterial inoculation into the seawater, the bacteria can be subject to detailed genetic manipulation, the precise site of infection can be imaged directly in the live animal host, and chemical analysis of the animal host enables detailed molecular investigations. Focusing on how squid are reproducibly colonized by the specific symbiont, to the exclusion of the millions of competing bacteria in seawater, has revealed key roles for bacterial aggregation and biofilm formation in promoting specific host- microbe interactions. Questions that our group is asking include: (1) How does a symbiont regulate a beneficial biofilm? Biofilms provide microbes with a protected environment in which they can act collectively and resist innate immune insults and antimicrobial compounds. V. fischeri elaboration of a symbiotic biofilm is required for entry into the host, providing an opportunity to study this process in the context of a natural host colonization model. Our past work identified BinK as a key negative regulator of biofilm formation and the planktonic-to-biofilm transition in the host. In this study, we examine how BinK interprets signals from the host and how that information is transmitted to V. fischeri. We examine mechanisms of signal transduction and seek to identify and characterize a ligand that regulates BinK activity. (2) What novel bacterial factors play critical functions in colonization processes? We have had success in applying global genetic approaches to identify bacterial colonization factors in V. fischeri. With a focus on novel and understudied bacterial genes for which the V. fischeri-squid system has the potential to elucidate protein functions, we identified a protein that has a substantial impact on biofilm formation and squid colonization. The protein is annotated as a putative RNA-binding protein, and we will characterize the molecular mechanisms by which this protein acts and determine how it impacts symbiotic biofilm formation. (3) How do small molecules influence microbiome specificity and colonization? We have begun to identify compounds that are present in the host and that are co-regulated with symbiotic behaviors. We will integrate genetic approaches to elucidate signaling pathways in the context of host colonization. A major strength of the V. fischeri-squid system is the ability to interrogate bacterial behavior in the intact animal host, and completion of these projects will enable a deeper understanding of the mechanisms underlying a...