Identification of Cyclic-di-GMP-Modulating Protein Residues by Bidirectionally Evolving a Social Behavior in Pseudomonas fluorescens.

Modulation of the intracellular cyclic di-GMP (c-di-GMP) pool is central to the formation of structured bacterial communities. Genome annotations predict the presence of dozens of conserved c-di-GMP catalytic enzymes in many bacterial species, but the functionality and regulatory control of the vast majority remain underexplored. Here, we begin to fill this gap by utilizing an experimental evolution system in Pseudomonas fluorescens Pf0-1, which repeatedly produces a unique social behavior through bidirectional transitions between two distinct phenotypes converging on c-di-GMP modulation. Parallel evolution of 33 lineages captured 147 unique mutations among 191 evolved isolates in genes that are empirically demonstrated, bioinformatically predicted, or previously unknown to impact the intracellular pool of c-di-GMP. Quantitative chemistry confirmed that each mutation causing the phenotypic shift either amplifies or reduces c-di-GMP production. We identify missense or in-frame deletion mutations in numerous diguanylate cyclase genes that largely fall outside the conserved catalytic domain. We also describe a novel relationship between a regulatory component of branched-chain amino acid biosynthesis and c-di-GMP production, and predict functions of several other unexpected proteins that clearly impact c-di-GMP production. Sequential mutations that continuously disrupt or recover c-di-GMP production across discrete functional elements suggest a complex and underappreciated interconnectivity within the c-di-GMP regulome of P. fluorescens.