The broadly conserved signaling nucleotide cyclic di-adenosine monophosphate (c-di-AMP) is essential for viability in most bacteria where it has been studied. However, characterization of the cellular functions and metabolism of c-di-AMP has largely been confined to the class Bacilli, limiting our functional understanding of the molecule among diverse phyla. We identified the cyclase responsible for c-di-AMP synthesis and characterized the molecule’s role in survival of darkness in the model photosynthetic cyanobacterium Synechococcus elongatus PCC 7942. In addition to the use of traditional genetic, biochemical, and proteomic approaches, we developed a high-throughput genetic interaction screen (IRB-Seq) to determine pathways where the signaling nucleotide is active. We found that in S . elongatus c-di-AMP is produced by an enzyme of the diadenylate cyclase family, CdaA, which was previously unexplored experimentally. A cdaA -null mutant experiences increased oxidative stress and death during the nighttime portion of day-night cycles, in which potassium transport is implicated. These findings suggest that c-di-AMP is biologically active in cyanobacteria and has non-canonical roles in the phylum including oxidative stress management and day-night survival. The pipeline and analysis tools for IRB-Seq developed for this study constitute a quantitative high-throughput approach for studying genetic interactions. Author summary Cyclic di-adenosine monophosphate (c-di-AMP) is a molecule that has significant roles in many microorganisms. This work shows the existence of c-di-AMP for the first time in photosynthetic microorganisms, cyanobacteria, and demonstrates its role in survival during the light-to-dark shifts that occur in day-night cycles. Despite the obvious importance of adaptation to these daily cycles for organisms that are fundamentally reliant on light, such as cyanobacteria, understanding of diurnal physiology is lacking because most cyanobacterial research is conducted during growth in constant light. To identify other players in c-di-AMP’s function we developed a low-cost and efficient method for finding interactions between genes. The technique combines one mutation, in this case for the gene that encodes the enzyme for synthesis of c-di-AMP, with thousands of other individual mutations to find pairwise interactions that affect fitness of the resulting mutants. Mutants are tagged with DNA barcodes to allow their survival to be easily tracked in a population of cells. The method enables us to place the function of c-di-AMP within the context of pathways previously known to be involved in day-night survival. Taken together, this work expands the known roles of c-di-AMP, improves our understanding of cyanobacterial survival in day-night cycles, and presents an improved approach for determining genetic interactions.
【초록키워드】 Mutation, Genetic, Stress management, oxidative stress, metabolism, DNA, survival, cells, viability, management, Research, death, pathway, genetic interaction, mutants, microorganisms, Bacteria, Potassium, fitness, mutant, cyanobacteria, adaptation, synthesis, Quantitative, interactions, proteomic, function, cellular, Signaling, nucleotide, Interaction, Analysis, Pathways, genetic interactions, Transport, characterization, Organisms, biochemical, technique, approaches, enzyme, growth, organism, molecule, cyclic, monophosphate, cdaA, cyclase, darkness, phylum, ENCODE, Affect, Genes, approach, IMPROVE, produced, responsible, resulting, identify, conserved, involved, addition, conducted, characterized, functional, determine, occur, implicated, expand, Synechococcus, 【제목키워드】 survival, Interaction,