High-throughput interaction screens illuminate the role of c-di-AMP in cyanobacterial nighttime survival

Abstract

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.

The circadian clock and darkness control natural competence in cyanobacteria

“…Sequencing reads of the barcodes were curated to keep only barcodes located within the middle 80% of each coding sequence; genes not represented by at least three bar codes in different positions or without at least 15 T 0 reads across replicates were removed from the analysis 26,53 . This curation resulted in 82,495 barcodes (also named strains) for the experimental conditions, 90,997 barcodes for the controls, and 99,872 barcodes for the T 0 samples distributed across 1,885 genes.…”

“…This curation resulted in 82,495 barcodes (also named strains) for the experimental conditions, 90,997 barcodes for the controls, and 99,872 barcodes for the T 0 samples distributed across 1,885 genes. For each gene, a fitness value that describes how the loss of function affects natural competence and a corresponding statistic were estimated in R as described previously 26,53 .…”

“…In order to globally identify all genes that enable natural competence, we transformed an S. elongatus RB-TnSeq library 25,26 with eDNA carrying selectable markers that recombine into the S. elongatus chromosome at neutral sites (NS) 27 . We reasoned that transposon mutants that are incompetent for transformation would be selectively missing from the transformed population.…”

“…Mutant colonies were collected as pools from selective and non-selective control conditions and the barcodes were sequenced. The relative abundance of mutants for each represented gene in both conditions was interpreted as a measure of fitness with a corresponding level of confidence (T-value) for each locus 26 (Fig. 1a, Supplementary Table 1).…”

Nitrogen chlorosis in unicellular cyanobacteria – a developmental program for surviving nitrogen deprivation

“…Furthermore, many genes for hypothetical proteins are found in this list and are strongly and rapidly induced upon resuscitation, among them sll0505, encoding a cyclic diadenylate (c-di-AMP) cyclase). The homologue from S. elongatus has been shown to be involved in night-time survival (Rubin et al, 2018), indicating possible parallels between these survival processes.…”