Project Summary/Abstract The intestinal microbiota plays a major role in human health and is therefore the focus of significant interest as a target for therapeutic interventions. However, our understanding at the mechanistic level of the ecological and evolutionary forces shaping this diverse and densely colonized ecosystem is still tenuous. Notably, although we know that horizontal gene transfer is pervasive in the gut community, we understand only superficially the different roles of the majority of these exchanged genes and how this repertoire affects community dynamics. Similarly, little is known about the mechanisms underlying community resiliency. This question is particularly intriguing for the Bacteroidales, the most abundant Gram-negative gut microbiome members, which can stably colonize for decades. This proposal will investigate the importance of biofilm formation by the Bacteroidales for community ecology and resilience, focusing on the conjugative megaplasmid pMMCAT which enables biofilm formation in the strains that acquire it. This plasmid is exceptional because of the high frequency of intrapersonal transfer to multiple Bacteroidales species and its ubiquity, with conserved architecture, across global human populations. Some studies suggest that mucosal biofilms in healthy humans are rare, but there is little information about other locations or unattached biofilms. I hypothesize that this megaplasmid, shared among many species in a community, enables the formation of multi-species biofilms and plays a role in community cooperation, notably through increasing community resilience. To test this hypothesis, or otherwise understand alternative roles of pMMCAT, I will systematically characterize the phenotypes conferred by this plasmid in culture and in gnotobiotic mice in a single strain (aim 1) or in different Bacteroidales consortia where some strains carry it (aim 2). To understand the ecological role of pMMCAT, I will evaluate if its conferred phenotypes are synergized or inhibited by the co-resident strains and whether all, only some, or none of the other strains benefit. To visualize biofilms in the colon prior to and following a stress pulse, I will use two different methods to preserve the spatial structure of the gut community and use a biofilm matrix-specific stain. I will subsequently examine the evolutionary dynamics of pMMCAT transfer in these consortia (aim 3). To this end, I will directly quantify and visualize plasmid transfer in culture and in a gnotobiotic mouse. I will also quantify the cost of carrying pMMCAT and expressing biofilm formation genes. I will evaluate the impact of the introduction of a cheater strain that doesn’t pay this fitness cost of producing the biofilm matrix public good. Finally, I will track the evolution of pMMCAT over the course of twelve years in four human volunteers previously found to have pMMCAT-harboring strains. This dissection of the dynamics and mechanisms underlying plasmid-enco...