Identification of cyclic-di-GMP-modulating protein residues by bi-directionally evolving a social trait in Pseudomonas fluorescens

Modulation of the intracellular cyclic di-guanosine monophosphate (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 trait 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 predictably amplifies or reduces c-di-GMP production. We integrate our mutation, phenotype, and quantification data with current models of known regulatory and catalytic systems, describe a previously unknown relationship between a regulatory component of branched-chain amino acid biosynthesis and c-di-GMP production, and predict functions of 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 . Importance Microbial communities comprise densely packed cells where competition for space and resources is fierce. In our model system, mutant cells with a dry (D) phenotype are selected from a population with a mucoid (M) phenotype, and vice versa, because M and D cells physically work together to spread away from the overcrowded colony. D cells produce high levels of c-di-GMP and M cells produce low levels, so each mutation impacts c-di-GMP production. C-di-GMP is a second messenger which regulates diverse bacterial phenotypes that cause tremendous clinical and environmental problems. Many bacteria possess dozens of enzymes that are predicted to produce c-di-GMP, but most are considered to be non-functional. Here, we take advantage of the bi-directional selection of M and D phenotypes to identify key residues that could force these enzymes to turn on or off. Several unexpected proteins also participate in this process, but very little is known about them. ### Competing Interest Statement The authors have declared no competing interest.