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

Abstract

Microbial communities comprise densely packed cells where competition for space and resources are fierce. These communities, generally referred to as biofilms, provide advantages to individual cells against immunological and antimicrobial intervention, dehydration, and predation. High intracellular pools of cyclic diguanylate monophosphate (c-di-GMP) cause individual cells to aggregate during biofilm formation through the production of diverse extracellular polymers. Genes that encode c-di-GMP enzymes or their regulators are commonly mutated during chronic infections due to enhanced resistance to phagocytosis and antibiotics. Genome annotations predict the presence of numerous c-di-GMP catalytic enzymes in most bacterial species, but the functionality and regulatory control of the vast majority remain unconfirmed. 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 either amplifies or reduces c-di-GMP production. We integrate our data with current models of known regulatory and catalytic systems, describe a previously unknown relationship between branched-chain amino acids and c-di-GMP production, and predict functions of several new proteins that either regulate or catalyze 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.