Global characterization of the cyclic-di-GMP regulome by bi-directionally evolving a social trait in 1 Pseudomonas fluorescens

10 Microbial communities comprise densely packed cells where competition for space and 11 resources are fierce. These communities, generally referred to as biofilms, provide advantages to 12 individual cells against immunological and antimicrobial intervention, dehydration, and predation. High 13 intracellular pools of cyclic diguanylate monophosphate (c-di-GMP) cause individual cells to aggregate 14 during biofilm formation through the production of diverse extracellular polymers. Genes that encode c- 15 di-GMP enzymes or their regulators are commonly mutated during chronic infections due to enhanced 16 resistance to phagocytosis and antibiotics. Genome annotations predict the presence of numerous c-di- 17 GMP catalytic enzymes in most bacterial species, but the functionality and regulatory control of the vast 18 majority remain unconfirmed. Here, we begin to fill this gap by utilizing an experimental evolution 19 system in Pseudomonas fluorescens Pf0-1, which repeatedly produces a unique social trait through 20 bidirectional transitions between two distinct phenotypes converging on c-di-GMP modulation. Parallel 21 evolution of 33 lineages captured 147 unique mutations among 191 evolved isolates in genes that are 22 empirically demonstrated, bioinformatically predicted, or previously unknown to impact the intracellular 23 pool of c-di-GMP. Quantitative chemistry confirmed that each mutation causing the phenotypic shift either amplifies or reduces c-di-GMP production. We integrate our data with current models of known 25 regulatory and catalytic systems, describe a previously unknown relationship between branched-chain 26 amino acids and c-di-GMP production, and predict functions of several new proteins that either regulate 27 or catalyze c-di-GMP production. Sequential mutations that continuously disrupt or recover c-di-GMP 28 production across discrete functional elements suggest a complex and underappreciated 29 interconnectivity within the c-di-GMP regulome. expand the current model of c-di-GMP production by the Wsp signaling and demonstrate that branched-chain amino acid (BCAA) production is a recurrent target of mutations that positively correlate to measured c-di-GMP levels. We also describe several proteins with no previous associations to c-di- GMP that likely act as internal regulators. Lastly, we predict the interconnectivity of the c-di-GMP regulome through sequential mutations that disrupt or recover c-di-GMP regulation across functionally discrete systems. This work demonstrates how a simple experimental evolution system can systematically map out c-di-GMP regulatory and enzymatic elements and uncover remarkable molecular innovations. sequence in modulate activities of could readily manifest otherwise. Although direct functional studies are necessary to resolve the underlying mechanisms, this study reveals a complex and underappreciated interconnectivity within the c-di-GMP regulome. Harvesting natural selection in the laboratory could provide a unique path for discovering and disentangling complex molecular processes that are relevant 558 in a clinical setting.