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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.

SUBMITTER: Rubin BE

PROVIDER: S-EPMC5897029 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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

Rubin Benjamin E BE Huynh TuAnh Ngoc TN Welkie David G DG Diamond Spencer S Simkovsky Ryan R Pierce Emily C EC Taton Arnaud A Lowe Laura C LC Lee Jenny J JJ Rifkin Scott A SA Woodward Joshua J JJ Golden Susan S SS

PLoS genetics 20180402 4

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 pho ...[more]

PMID: 29608558

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Project description:Second messengers are intracellular molecules regulated by external stimuli known as first messengers that are used for rapid organismal responses to dynamic environmental changes. Cyclic di-AMP (c-di-AMP) is a relatively newly discovered second messenger implicated in cell wall homeostasis in many pathogenic bacteria. C-di-AMP is synthesized from ATP by diadenylyl cyclases (DAC) and degraded by specific c-di-AMP phosphodiesterases (PDE). C-di-AMP DACs and PDEs are present in all sequenced cyanobacteria, suggesting roles for c-di-AMP in the physiology and/or development of these organisms. Despite conservation of these genes across numerous cyanobacteria, the functional roles of c-di-AMP in cyanobacteria have not been well-investigated. In a unique feature of cyanobacteria, phylogenetic analysis indicated that the broadly conserved DAC, related to CdaA/DacA, is always co-associated in an operon with genes critical for controlling cell wall synthesis. To investigate phenotypes regulated by c-di-AMP in cyanobacteria, we overexpressed native DAC (sll0505) and c-di-AMP PDE (slr0104) genes in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis) to increase and decrease intracellular c-di-AMP levels, respectively. DAC- and PDE-overexpression strains, showed abnormal aggregation phenotypes, suggesting functional roles for regulating c-di-AMP homeostasis in vivo. As c-di-AMP may be implicated in osmotic responses in cyanobacteria, we tested whether sorbitol and NaCl stresses impacted expression of sll0505 and slr0104 or intracellular c-di-AMP levels in Synechocystis. Additionally, to determine the range of cyanobacteria in which c-di-AMP may function, we assessed c-di-AMP levels in two unicellular cyanobacteria, i.e., Synechocystis and Synechococcus elongatus PCC 7942, and two filamentous cyanobacteria, i.e., Fremyella diplosiphon and Anabaena sp. PCC 7120. C-di-AMP levels responded differently to abiotic stress signals in distinct cyanobacteria strains, whereas salt stress uniformly impacted another second messenger cyclic di-GMP in cyanobacteria. Together, these results suggest regulation of c-di-AMP homeostasis in cyanobacteria and implicate a role for the second messenger in maintaining cellular fitness in response to abiotic stress.

Project description:c-di-AMP is an important second messenger molecule that plays a pivotal role in regulating fundamental cellular processes, including osmotic and cell wall homeostasis in many Gram-positive organisms. In the opportunistic human pathogen Staphylococcus aureus, c-di-AMP is produced by the membrane-anchored DacA enzyme. Inactivation of this enzyme leads to a growth arrest under standard laboratory growth conditions and a re-sensitization of methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. The gene coding for DacA is part of the conserved three-gene dacA/ybbR/glmM operon that also encodes the proposed DacA regulator YbbR and the essential phosphoglucosamine mutase GlmM, which is required for the production of glucosamine-1-phosphate, an early intermediate of peptidoglycan synthesis. These three proteins are thought to form a complex in vivo and, in this manner, help to fine-tune the cellular c-di-AMP levels. To further characterize this important regulatory complex, we conducted a comprehensive structural and functional analysis of the S. aureus DacA and GlmM enzymes by determining the structures of the S. aureus GlmM enzyme and the catalytic domain of DacA. Both proteins were found to be dimers in solution as well as in the crystal structures. Further site-directed mutagenesis, structural and enzymatic studies showed that multiple DacA dimers need to interact for enzymatic activity. We also show that DacA and GlmM form a stable complex in vitro and that S. aureus GlmM, but not Escherichia coli or Pseudomonas aeruginosa GlmM, acts as a strong inhibitor of DacA function without the requirement of any additional cellular factor. Based on Small Angle X-ray Scattering (SAXS) data, a model of the complex revealed that GlmM likely inhibits DacA by masking the active site of the cyclase and preventing higher oligomer formation. Together these results provide an important mechanistic insight into how c-di-AMP production can be regulated in the cell.

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

Publications

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

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