article: 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 signaling nucleotide cyclic di-adenosine monophosphate (c-di-AMP) has been implicated in a wide range of biological processes since its discovery less than a decade ago [1]. The molecule is active in multiple pathways including potassium transport, regulation of central metabolism, cell wall homeostasis, gene expression, and DNA damage responses [2]. In addition, c-di-AMP is the only second messenger that is essential to most of the organisms in which it is studied [3]. Despite the biological importance of c-di-AMP, in vivo studies of this signaling nucleotide have focused on a narrow array of Firmicutes and to a lesser extent Actinobacteria. Expanding the investigation of c-di-AMP to a broader diversity of microbial phyla may reveal new functions for this molecule. Organisms in the photosynthetic phylum Cyanobacteria are key players in global carbon, oxygen, and nitrogen cycling, and are promising platforms for renewable production of industrial chemicals [4–6]. While c-di-AMP has not been reported in the phylum, the existence of enzymes and riboswitches predicted by homology to interact with the molecule suggests its presence [2,7–9]. Signaling nucleotides previously studied in cyanobacteria (cGMP, c-di-GMP, (p) ppGpp, and cAMP) serve important roles in environmental responses and have revealed activities specific to photosynthetic organisms, such as regulating phototaxis, photosystem repair, and dark survival [8,10]. Characterization of c-di-AMP metabolism and its role in cyanobacteria may similarly lead to a better understanding of the phylum, as well as the functions of c-di-AMP. C-di-AMP research, as a young field with many unknowns, has particularly benefited from untargeted genetic interaction screens. Screens focused on secondary mutations that relieve the phenotypes of c-di-AMP-related mutants (alleviating interactions) have successfully identified genetic interactions with c-di-AMP synthases and phosphodiesterases [11,12]. However, these screens have been limited to assaying positive interactions, were non-quantitative, and have been performed under only a single condition. Quantitative genetic interaction screens to identify aggravating as well as alleviating interactions would provide a powerful tool to elucidate c-di-AMP function. Such screens have been conducted for other processes using next-generation sequencing paired with transposon mutagenesis (Tn-Seq) [13–15]. Tn-Seq for genetic interactions relies upon the generation of a new mutant library for each screen in the background of a knockout of interest [16]. While informative, the need to generate and characterize a new transposon-mutant library for each interaction screen is both costly and labor intensive [17]. These features have limited the applicability of Tn-Seq for interaction screens. A variation on Tn-Seq, RB-TnSeq, provides a cheaper and less laborious approach for screening transposon mutant libraries, in which the fitness of mutants in the population is tracked by sequencing and counting of 20-base pair “barcodes” in each transposon [18]. This approach has not yet been applied to interaction screens, where it offers great potential for mitigating their logistical weaknesses and enlarging their scope. Here, we used genetic and biochemical approaches to elucidate the presence and roles of c-di-AMP in the cyanobacterium Synechococcus elongatus PCC 7942. To further probe the molecule’s functions we developed and implemented an approach we named interaction RB-TnSeq, or IRB-Seq, to quantitatively measure genetic interactions with the c-di-AMP cyclase under multiple conditions.
  The putative c-di-AMP-producing and degrading enzymes in S. elongatus were identified computationally. Two domain organizations, DHH-DHHA1 (PF01368 and PF02272) and 7TM-7TMR_HD (PF07698), have been found to degrade c-di-AMP [19]. The software package HMMER [20] identified one of each in S. elongatus. Based on homology, the DHH-DHHA1 domain-containing protein, RecJ (Synpcc7942_1886), likely serves as a single-stranded DNA exonuclease. However, it may also act as a non-specific phosphodiesterase for c-di-AMP and linear 5′-phospho diadenylate (pApA), as documented in the literature [19]. The 7TM-TMR_HD-containing protein, Synpcc7942_0779, is a reciprocal best hit with the Listeria monocytogenes c-di-AMP degrading enzyme PgpH, with homology focused around the HD domain active site. Therefore, Synpcc7942_0779, and to a lesser extent RecJ, are likely phosphodiesterases for c-di-AMP in S. elongatus. A single protein in the organism contains a DisA_N domain (PF02457), currently the only domain known to be responsible for c-di-AMP production [21]. Although presence of this putative synthase has been noted several times in cyanobacteria, it has yet to be experimentally validated [2,8, 9,22]. This sequence, annotated at time of publication as “protein of unknown function DUF147”, predicts three transmembrane segments and a cytoplasmic DisA_N domain (Fig 1A). The arrangement of domains identifies the protein as a member of the most abundant family of DisA_N domain-containing proteins, CdaA [2,23]. Thus, we refer to this previously unannotated gene, which encodes the putative cyclase of c-di-AMP, as cdaA (Synpcc7942_0263). To validate this bioinformatic prediction of the c-di-AMP cyclase, we quantified c-di-AMP levels in wild type (WT) and a cdaA-null mutant using LC-MS. The WT value of 18. 8 μM (Fig 1B) is higher than reported in other organisms, including Staphylococcus aureus [24] and Bacillus subtilis [25], where the nucleotide plays biologically important roles. While c-di-AMP is essential for many other c-di-AMP-producing organisms, a fully segregated insertion mutant for cdaA, which has no other paralog in the genome of S. elongatus, is viable under constant-light standard laboratory conditions (S1 Fig). The residual signal in the cdaA mutant, potentially attributable to c-di-AMP (Fig 1B), was within the background noise in mass spectrometry quantification and unlikely to represent presence of the molecule (S2 Fig). In further support of CdaA’s role in c-di-AMP synthesis, the S. elongatus cdaA was expressed in Escherichia coli, which does not naturally produce c-di-AMP; high levels of the molecule were detected (S3 Fig). These data strongly suggest that S. elongatus produces c-di-AMP, and that CdaA catalyzes its synthesis. Concentrations of other signaling nucleotides studied in cyanobacteria change in response to light [8]. To investigate whether this may be the case with c-di-AMP in S. elongatus, we sampled every six hours over one 12-h light: 12-h-dark cycle (LDC). The c-di-AMP concentration was variable in WT both among samples within time points and between them, with an apparent, although non-significant, trend upwards at nighttime (Fig 1C). Given that signaling nucleotide responses to light have been shown to occur within minutes in cyanobacteria [26], we performed higher resolution sampling around the light-to-dark and dark-to-light transitions. While light did not have a large or immediate effect on c-di-AMP, a three-fold increase in the nucleotide’s levels was observed 15 minutes after onset of darkness (Fig 1D). Based on the observed increase in c-di-AMP levels upon onset of darkness, as well as previous research showing the importance of the closely linked signaling nucleotide ppGpp to dark survival in S. elongatus [10], we examined whether c-di-AMP is necessary to survive LDCs. In both solid and liquid media, the cdaA mutant has decreased growth in LDCs, but not in constant light (Fig 2A and 2B). This phenotype was successfully complemented by the addition of a WT copy of the cdaA gene to a neutral site (S4 Fig). Furthermore, although the cdaA mutant’s absorbance spectrum was not significantly different from WT’s before sensitizing LDCs, bleaching was apparent 30 h after the start of an LDC, and worsened thereafter (S5 Fig). Therefore, the cdaA mutation and the associated decrease of c-di-AMP levels are likely responsible for the LDC-specific deleterious phenotype. To explore the nature and cause of the LDC sensitivity, we examined populations over one LDC. While the cdaA mutant grew similarly to WT over the light portion of the LDC, viability of the mutant, as measured by colony forming units in outgrowths in continuous light from specific time points, rapidly decreased upon the onset of darkness. This difference was significant after two hours of exposure to darkness (P <. 05; t test), and became more pronounced over the course of the night (Fig 2C). Recent work showed that reactive oxygen species (ROS) correlate with death in darkness for S. elongatus [27]. Indeed, reactive oxygen species peaked in the mutant after the onset of darkness at more than two fold the level in WT and remained higher than WT through the course of the night (P < 10−5; Mann-Whitney-Wilcoxon Test) (Fig 2D). These data suggest that a still-cryptic mechanism actively kills the cdaA mutant specifically in the dark stage of the LDC and potentially as a consequence of high oxidative stress.
  C-di-AMP controls many aspects of bacterial physiology and is essential in the bacterial phylum Firmicutes, where most research on the molecule has taken place. The essential nature of c-di-AMP has been connected to its role in central metabolism [45], its linkage to levels of another signaling nucleotide (p) ppGpp [11], and its role in potassium homeostasis [46]. Importantly, the absence of c-di-AMP is lethal in Firmicutes in rich media, whereas minimal media are permissive for a null mutant. A possible explanation for S. elongatus’ viability with the cdaA mutation is that, as a photoautotroph, it is far more independent of nutrients in the medium than the previously studied heterotrophs. Regardless of its cause, the essential nature of c-di-AMP in many organisms has limited the ability to study this nucleotide [21]. Future genetic exploration of cdaA will be facilitated by the viability of the S. elongatus cdaA mutant. The IRB-Seq approach developed for this study enables high-throughput quantitative genetic interaction screens with minimal sequencing prep. In the approach, a second mutation is added directly to an existing RB-TnSeq library, removing the need of previous approaches to recreate a library in a new background and re-determine all insertion loci for each screen (Fig 3) [13–15]. Traditional sequencing preparation used in classical Tn-Seq studies is also avoided because survival of mutants is quantified by PCR and sequencing of 20 bp “barcodes” present in each transposon which serve as identifiers for each clone in the mutant library (BarSeq) [18]. IRB-Seq requires only one PCR and ~1/100th of an Illumina HiSeq 4000 lane per sample to provide a genome-wide quantitative measure of genetic interactions ranging from strong alleviation to full synthetic lethal. The advantage of a quantitative alternative to previous suppressor screens used in c-di-AMP research [11,12] is particularly apparent in this study. The cdaA mutant’s lack of fitness phenotype under constant light, and moderate phenotype under sensitizing LDCs, would have made traditional suppressor screens difficult, and likely ineffective. In addition, the high-throughput nature of this approach to genome-wide genetic interaction screens makes it feasible to conduct IRB-Seq screens in replicate and under many different sensitizing and permissive conditions. While this work does not determine the mechanism of the CdaA mediated c-di-AMP increase after darkness, several possibilities can be considered in light of the literature. One potential explanation is a transcriptional increase around dusk of c-di-AMP’s cyclase, CdaA, potentially controlled by the circadian clock. However, the immediate change in c-di-AMP level upon onset of darkness (Fig 1D), combined with a lack of dusk- or dark-induced expression of cdaA [47–49], make circadian or transcriptional control unlikely. Nor does the quantity of CdaA' s substrate, ATP, increase in darkness [50,51], and thus is not likely to explain the increases in c-di-AMP. Therefore, it is more likely that the spike in c-di-AMP level results from dark-dependent changes in the activity of CdaA or c-di-AMP’s phosphodiesterase. Elucidating the survival of Cyanobacteria in LDCs is important for improved understanding of both a phylum of tremendous ecological importance and an environmental challenge relevant to all photosynthetic organisms. For ease of research, however, most experiments on cyanobacteria have been conducted in simplifying constant-light conditions. The work in LDCs that exists shows oxidative stress management as a key component for surviving LDCs [27,29]. Similarly to the LDC-sensitive circadian clock mutants of rpaA and kaiA, lethality in the cdaA mutant occurred specifically upon the onset of night following high oxidative stress at dusk (Fig 2). The death in the mutant begins concurrent with a spike in c-di-AMP level in the WT (Fig 1D). This correlation suggests a role for the molecule in the day-night transition, a seemingly crucial period for surviving LDCs in the mutants where it has been studied. This role is further supported by the finding that a number of the top cdaA synthetically interacting genes determined by IRB-Seq are involved in oxidative stress mitigation. Thus, c-di-AMP appears to be involved in LDC survival through management of oxidative stress, a characteristic shared with LDC-sensitive clock mutants. A possible basis for the cdaA mutant’s sensitivity to LDCs and oxidative stress may be potassium transport. C-di-AMP has previously been associated with multiple potassium transporters [2]. In S. aureus c-di-AMP negatively regulates the Kdp potassium uptake system through its binding to the histidine kinase KdpD [38]. In this study KdpD was suggested by DRaCALA to bind c-di-AMP in S. elongatus. Additionally, two proteins from the Trk family of potassium transporters (Synpcc7942_1080 and Synpcc7942_1081) were found among the top synthetic interactions, suggesting that the cdaA mutant is sensitized to potassium transport mutations. Altered potassium transport previously has been shown in cyanobacteria to sensitize the cells to the oxidative stress-producing conditions of high light and heavy metal exposure [52,53]. Therefore, the canonical role of c-di-AMP in potassium homeostasis may, in S. elongatus, be involved in the non-canonical function of LDC survival through oxidative stress regulation. IRB-Seq is an inexpensive and straightforward approach to high-throughput quantitative interaction screens and is suitable for addressing an array of questions in different organisms. With 25 published RB-TnSeq libraries [28,54] and many more under development, there exists ample starting material for IRB-Seq screens, assuming the ability to deliver a second mutation into the host with high efficiency. In this case, natural transformation was sufficient to generate the double-mutant library, but other techniques such as conjugation may be necessary for species that are not naturally competent. The experimental pipeline and analysis tools developed here make this assay feasible for genetic interactions of any gene of interest. Furthermore, the addition of multiple mutations into the library, or reporters paired with cell sorting, should enable screening for more complex genetic interactions as well as identification of effects on gene expression by genetic perturbations.
  WT in this study and the background for all mutants was Synechococcus elongatus PCC 7942, stored in our lab as AMC06. For all experiments, growth was conducted in BG-11 media with appropriate antibiotics at 30°C [55]. Unless otherwise indicated, liquid culture growth was under constant light in 250 mL flasks containing 100 mL of culture shaken at 150 rpm (Thermo Fisher MaxQ 2000 Orbital Shaker). For mutagenesis by transformation, standard S. elongatus protocols [55] were altered for dark-sensitive mutants. The typical overnight incubation in darkness after DNA addition was instead performed under 70 μmol photons·m−2·s−1. For experiments using cdaA loss-of-function mutants, with the exclusion of IRB-Seq and IRB-Seq validation experiments, the plasmid for insertion mutagenesis (8S16-L9, conferring kanamycin (Km) resistance) was from the previously published unigene set [56]. For IRB-Seq and IRB-Seq validation experiments, which required a loss-of-function strain with an antibiotic resistance other than Km, a cdaA-deletion plasmid (AM5403) carrying the spectinomycin- (Sp) and streptomycin- (Sm) resistance gene aadA was constructed using CYANO-VECTOR procedures and devices [57]. This deletion removed a region from 8 bp upstream to 575 bp downstream of the translation start site of cdaA. Phenometrics ePBR photobioreactors (version 1. 1; Phenometrics Inc.) were used in LDC liquid culture assays for measurement of: survival, OD750, c-di-AMP quantity, and ROS. The bioreactors were inoculated with 400 mL of culture at OD750 = 0. 1. They were then grown under 150 μmol photons·m−2·s−1 with filtered air bubbled in at a rate of 50 mL/min. After 12 h, the bioreactor cultures were re-diluted to OD750 = 0. 1 and exposed to a 12: 12 h LDC for purposes of low-light entrainment of the circadian clock; dark-sensitive mutants were previously shown to survive at this light intensity in the bioreactors [27]. After this entrainment cycle the light intensity was increased to the restrictive condition of 500 μmol photons·m−2·s−1 for the remaining LDCs, at which point assays for physiology were conducted. High-resolution measurement of survival, c-di-AMP quantity, ROS, and absorbance spectrum were all conducted immediately following the increase of the bioreactors’ LDC light intensity to 500 μmol photons·m−2·s−1. Survival was measured using a 1: 5 dilution series over 8 wells with the first well containing a 1: 10 dilution of the bioreactor culture. The 8 dilutions were plated in 4 μL spots on solid medium and incubated under continuous illumination at 150 μmol photons·m−2·s−1 for 5 d, at which point they were photographed. The photographs were then analyzed for colony forming units (CFUs) and for surface area of the colonies using ImageJ [58]. For quantification of c-di-AMP, 1. 5 mL of culture was sampled, pelleted at 4 C at 20,000 g for 3 min, and resuspended in 500 μL methanol with 20 μL of 0. 5 μM heavy-labeled c-di-AMP. The mixture was then stored at -20 C until quantification (See C-di-AMP quantification). ROS was quantified on 1 mL of culture using the fluorescent marker H2DCFDA (Life Technologies catalog no. D399) as described previously [27]. Absorbance spectra were taken using a Tecan Infinite M200 plate reader. LDC-specific sensitivity assays were also conducted on plates. A 1: 5 dilution series over 8 wells was made for cdaA and a WT with the first well containing culture at OD750 = 0. 02. These dilutions of cdaA and a WT were then plated in 4 μL spots on duplicate plates. One of these plates was grown under 125 μmol photons·m−2·s−1 constant light for 5 d, while the other was grown under 125 μmol photons·m−2·s−1 LDCs for 8 d. Although LDCs at this light intensity are permissive in the bioreactors, they constitute a restrictive condition for dark-sensitive mutants on plates, where there is no self-shading by the culture. For one-on-one competition experiments single mutants were prepared for cdaA (AM5403) with Sp and Sm resistance, the putatively interacting mutants of interest carrying Km resistance, and WT controls for both Km resistance (8S15-K12), and Sp and Sm resistance (AM1303). Double mutants with the cdaA loss-of-function background and the putatively interacting second mutation were also prepared. The mutants were grown to OD750 ~ 0. 4 and washed three times with BG-11 without antibiotics via centrifugation at 4,696 G for ten minutes and diluted to an OD of 0. 015. Cultures were then mixed in a 1: 1 ratio according to the desired competition being assayed and grown for 8 days under ~100 μmol photons·m−2·s−1. Composition of competing populations was determined by leveraging the different antibiotic resistances of the strains. To assay survival of each genotype at zero, three, and eight days, mixed culture was serially diluted using a 1: 10 dilution series over 6 wells. The dilutions were plated in 20 μL spots in duplicate onto ~2 mL of BG-11 agar with either Sp and Sm, or Km on a 24-well clear plate. Colony number from the dilution series across the two antibiotic regimes was used to calculate CFUs in the original competition culture. Differentiation of the CFUs for the double mutants (Km-, Sp and Sm-resistant) and single cdaA mutants (Sp- and Sm-resistant) was accomplished via subtraction of the CFUs growing on Km from those growing on Sp and Sm.
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.
keywords: deletion mutation
  oxidative stress
  light
  electromagnetic radiation
  mutation
  genetic elements
  molecular biology techniques
  genetic interactions
  bacteria
  research and analysis methods
  cyanobacteria
  molecular biology
  molecular biology assays and analysis techniques
  physics
  cell biology
  genetic screens
  library screening
  gene identification and analysis
  genetics
  transposable elements
  biology and life sciences
  physical sciences
  genomics
  mobile genetic elements
  organisms
title: High-throughput interaction screens illuminate the role of c-di-AMP in cyanobacterial nighttime survival