In densely populated colonies of the bacterium Pseudomonas fluorescens Pf0-1, diverse mutations in the rsmE gene are naturally selected by solving the problem of overcrowding. Here, we show that RsmE-regulated secretions function together to create and protect space of low cell density.

Microbial communities comprise densely packed cells where competition for space and resources is fierce. Aging colonies of Pseudomonas fluorescens are known to repeatedly produce mutants with two distinct phenotypes that physically work together to spread away from the overcrowded population.

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.

Cells in microbial communities on surfaces live and divide in close proximity, which greatly enhances the potential for social interactions. Spatiogenetic structures manifest through competitive and cooperative interactions among the same and different genotypes within a shared space, and extracellular secretions appear to function dynamically at the forefront. A previous experimental evolution study utilizing Pseudomonas fluorescens Pf0-1 colonies demonstrated that diverse mutations in the rsmE gene are repeatedly and exclusively selected through the formation of a dominant spatial structure. RsmE’s primary molecular function is translation repression, and its homologs regulate various social and virulence phenotypes. Pseudomonas spp. possess multiple paralogs of Rsm proteins, and RsmA, RsmE, and RsmI are the most prevalent. Here, we demonstrate that the production of a mucoid polymer and a biosurfactant are exclusively regulated through RsmE, contradicting the generalized notion of functional redundancy among the Rsm paralogs. Furthermore, we identify the biosurfactant as the cyclic lipopeptide gacamide A. Competition and microscopy analyses show that the mucoid polymer is solely responsible for creating a space of low cellular density, which is shared exclusively by the same genotype. Gacamide A and other RsmE-regulated products appear to establish a physical boundary that prevents the encroachment of the competing genotype into the newly created space. Although cyclic lipopeptides and other biosurfactants are best known for their antimicrobial properties and reducing surface tension to promote the spreading of cells on various surfaces, they also appear to help define spatial structure formation within a dense community.