The Many Roles of the Bacterial Second Messenger Cyclic di-AMP in Adapting to Stress Cues

Bacteria respond to changes in environmental conditions through adaptation to external cues. Frequently, bacteria employ nucleotide signaling molecules to mediate a specific, rapid response. Cyclic di-AMP (c-di-AMP) was recently discovered to be a bacterial second messenger that is essential for viability in many species. In this review, we highlight recent work that has described the roles of c-di-AMP in bacterial responses to various stress conditions. These studies show that depending on the lifestyle and environmental niche of the bacterial species, the c-di-AMP signaling network results in diverse outcomes, such as regulating osmolyte transport, controlling plant attachment, or providing a checkpoint for spore formation.

ABSTRACT

Bacteria respond to changes in environmental conditions through adaptation to external cues. Frequently, bacteria employ nucleotide signaling molecules to mediate a specific, rapid response. Cyclic di-AMP (c-di-AMP) was recently discovered to be a bacterial second messenger that is essential for viability in many species. In this review, we highlight recent work that has described the roles of c-di-AMP in bacterial responses to various stress conditions. These studies show that depending on the lifestyle and environmental niche of the bacterial species, the c-di-AMP signaling network results in diverse outcomes, such as regulating osmolyte transport, controlling plant attachment, or providing a checkpoint for spore formation. c-di-AMP achieves this signaling specificity through expression of different classes of synthesis and catabolic enzymes as well as receptor proteins and RNAs, which will be summarized.

INTRODUCTION

Bacteria often utilize nucleotide signaling molecules to respond to changes in the environment, including stress stimuli. Diverse bacterial species share similar components of nucleotide signaling networks but have distinct lifestyles and must adjust responses to survive in these environmental niches. The main functions of a number of bacterial nucleotide-based second messengers have been characterized, such as cyclic AMP (cAMP), which controls carbon catabolite repression (1), the stringent starvation response of alarmone guanosine tetraphosphate or pentaphosphate [(p)ppGpp] (2), and cyclic di-GMP (c-di-GMP), which is involved in the motile-sessile switch (3). Recently, cyclic GMP-AMP (cGAMP) signaling was described to alert antiphage defense (4). In particular, cyclic di-AMP (c-di-AMP) has emerged as an essential nucleotide for viability in many bacteria and has been implicated in controlling many facets of bacterial physiology in many Gram-positive and some Gram-negative species. Since perturbations in c-di-AMP levels may lead to growth defects or inhibition, the nucleotide has been designated an essential poison (5).

Diadenylate cyclases (DAC) produce c-di-AMP from two molecules of ATP, and phosphodiesterases break down the molecule to AMP or phosphoadenylyl adenosine (pApA) (reviewed in references 6, to ,8). DAC domains are necessary for producing c-di-AMP and exist in nearly 76,000 species of bacteria and archaea (9). The homeostasis of c-di-AMP levels is also influenced by extracellular release, which can occur via multidrug resistance (MDR) family efflux pumps (10–13). The c-di-AMP signaling output is carried out by c-di-AMP receptor proteins and RNA molecules that alter functionality after binding the nucleotide. In each bacterial species, this signaling network is comprised of a different array of cyclases, c-di-AMP phosphodiesterases, and receptor molecules; therefore, not all functions of c-di-AMP signaling are conserved.

Several classifications of diadenylate cyclases and phosphodiesterases have been described. Diadenylate cyclases comprise a DAC domain combined with additional domains for protein localization and control of c-di-AMP production. Bacteria may encode one or multiple DAC domain-containing proteins. The most commonly characterized cyclase classes are a membrane-associated cyclase, CdaA, the DNA integrity-scanning protein DisA, and the sporulation-specific CdaS (6, 14–16). Phosphodiesterases are also categorized by distinct domains required for activity and regulation. GdpP and Pde2 types harbor DHH (Asp-His-His)/DHHA1 (DHH-associated) domains yet have different cellular localization and breakdown products. GdpP is membrane associated and converts c-di-AMP to pApA, while Pde2 is cytosolic and produces AMP (17–19). The HD (His-Asp) domain phosphodiesterase PgpH is a transmembrane protein and catabolizes c-di-AMP to pApA (20). AtaC is a newly described c-di-AMP phosphodiesterase that breaks down c-di-AMP to AMP and has structural similarity to the alkaline phosphatase PhnA (21). In addition to these intracellular phosphodiesterases, a c-di-AMP phosphodiesterase, CdnP, also cleaves extracellular c-di-AMP (22). The repertoire of c-di-AMP makers and breakers, as well as c-di-AMP binding proteins, forms a unique signaling network in each species.

Attributable to lifestyle, natural reservoirs, and environmental cues, bacteria use the second messenger c-di-AMP to regulate ion homeostasis and osmolarity as well as specialized functions (Table 1). For example, recently it has been reported that biofilm formation, sporulation, and nighttime survival are affected by c-di-AMP signaling. This review will discuss the role of the c-di-AMP signaling network as a mediator of bacterial stress responses.

TABLE 1.

Summary of specialized functions affected by c-di-AMP

Function	Speciesa	Phenotype	Reference(s)
Potassium transport	B. subtilis, B. thuringiensis, L. lactis, L. monocytogenes, M. pneumoniae, S. aureus, S. agalactiae, S. pneumoniae, S. elongatus	c-di-AMP inhibits K+ uptake and enhances K+ efflux	13, 27, 29, 31–35, 38, 45, 47, 50, 170
Osmotic stress	L. lactis, L. monocytogenes, S. aureus, S. agalactiae	c-di-AMP inhibits compatible solute uptake	13, 30, 36, 42
Acid stress	B. subtilis, L. lactis, S. aureus, S. pyogenes	Higher c-di-AMP levels increase acid stress resistance	70, 71, 73, 74
	S. pneumoniae	Higher c-di-AMP levels increase sensitivity to acid stress	61, 72
DNA repair	B. subtilis, D. radiodurans, M. smegmatis, M. tuberculosis	Lack of c-di-AMP increases sensitivity to DNA damage	77, 83, 84
	S. pneumoniae	Higher c-di-AMP levels increase sensitivity to DNA damage	18
Sporulation	B. subtilis	Lack of c-di-AMP blocks sporulation initiation and spore germination	89, 90, 93
	B. thuringiensis	Lack of c-di-AMP blocks sporulation initiation	94
β-Lactam drug resistance	B. subtilis, L. lactis, L. monocytogenes, S. aureus	Higher c-di-AMP levels are correlated with increased β-lactam resistance	17, 68, 92, 96, 97
Genetic competence	S. pneumoniae	Higher c-di-AMP levels are correlated with decreased transformation efficiency; lower c-di-AMP levels are correlated with increased cell death after addition of competence stimulating peptide	72
Nighttime survival	S. elongatus	Lack of c-di-AMP increases sensitivity to dark cycle	170

^aSpecialized functions affected by c-di-AMP have been reported in these species. Potassium transport is a broadly conserved function in c-di-AMP-producing bacteria.

MODULATING OSMOTIC STRESS

Bacteria respond to fluctuations in environmental osmotic pressure by precisely moderating intracellular levels of electrolytes (such as K⁺ and Cl⁻) and compatible solutes (such as glycine betaine, proline, glutamate, trehalose, and carnitine) (23, 24). When the external osmolarity increases, K⁺ is rapidly transported into the cells, accompanied by the synthesis or uptake of compatible solutes to avoid dehydration. On the other hand, K⁺ and compatible solutes are released when the environmental osmolarity decreases to prevent cell lysis. These osmoregulatory mechanisms are used to control turgor pressure, which is the force exerted by the intracellular water on the cell wall (25, 26).

Potassium transport.

Recently, a large number of reports have shown that c-di-AMP plays a critical role in bacterial responses to osmotic stress by controlling homeostasis of K⁺ and osmolytes (13, 27–45). c-di-AMP inhibits K⁺ import by binding directly to and/or regulating expression of Ktr/Trk, KimA, Kup, and Kdp uptake systems and facilitates export via CpaA and KhtT transporters (Fig. 1). Ktr systems of the Trk/Ktr/HKT family include the high-affinity KtrA-KtrB and low-affinity KtrC-KtrD K⁺ transporters (46). Specifically, c-di-AMP interacts with RCK_C (regulator of conductance of K⁺) domains through hydrogen bonds and hydrophobic interactions of the regulatory proteins KtrA and KtrC (29, 47). In Bacillus subtilis, Staphylococcus aureus, and Streptococcus pneumoniae, KtrA is a peripheral protein facilitating K⁺ uptake by transporter KtrB, which is impaired when c-di-AMP binds to KtrA (27, 29). Similar to KtrA, in B. subtilis and Listeria monocytogenes, binding of c-di-AMP to KtrC also prevents K⁺ uptake by KtrC-KtrD (31, 48, 49). Other bacterial species encode homologs of one or both Ktr systems that also bind c-di-AMP, such as Mycoplasma pneumoniae and Streptococcus agalactiae (30, 31, 50).

FIG 1.

Bacterial K⁺ homeostasis controlled by c-di-AMP. Summary of c-di-AMP-binding proteins and a riboswitch that affect K⁺ transport, only some of which may be present in each bacterial species. Color codes of c-di-AMP-binding domains in each protein are the following: yellow, RCK_C domain; red, HAK/KUP/KT family proteins; orange, USP domain; green, protein does not bind c-di-AMP. Binding of c-di-AMP blocks K⁺ uptake and induces K⁺ export in these respective transport pathways.

Another set of transporters is the HAK/KUP/KT family proteins, which are widely associated with K⁺ uptake in bacteria, fungi, and plants (51). In Lactococcus lactis, both KupA and KupB belong to the HAK/KUP/KT family and are capable of binding c-di-AMP. Both proteins are high-affinity K⁺ transporters, and their transport activities are inhibited upon binding of c-di-AMP (13, 35). In addition, KimA (previously named YdaO) is a recently recognized c-di-AMP binding K⁺ transporter of the HAK/KUP/KT family (31, 32, 45, 52). The expression of the N-terminal extracellular domain and transmembrane domain of this protein are sufficient for the uptake of K⁺ when expressed in an Escherichia coli model, whereas the C-terminal domain is required for c-di-AMP-dependent inhibition (31). Two independent copies of the c-di-AMP-responsive ydaO riboswitch control the expression of kimA and ktrAB, with one genetically upstream of kimA and the other upstream of ktrAB (45, 53). This ydaO riboswitch in the leader region of the kimA mRNA refolds upon binding of c-di-AMP and prevents the transcription of kimA (45, 53, 54). Overall, KtrA-KtrB, KupA, and KupB are high-affinity K⁺ transporters, KimA is a medium-affinity K⁺ transporter, and KtrC-KtrD is a low-affinity transporter but is high affinity in the presence of glutamate (31, 35, 55).

The Kdp complex is a backup system to scavenge K⁺ when environmental K⁺ concentrations are low, which has been well characterized in E. coli (56). This K⁺ uptake system consists of KdpA, KdpB, KdpC, and KdpF proteins encoded by a single kdpFABC operon, which is transcriptionally controlled by a two-component system, KdpDE (57, 58). In S. aureus, the expression of the kdpFABC operon is also upregulated by the two-component system KdpDE, which is inhibited when c-di-AMP binds to the sensor kinase KdpD at the universal stress protein (USP) domain (29, 33). Some S. aureus strains encode a second KdpD, named KdpD2, which also binds c-di-AMP via the USP domain. However, it remains unknown whether KdpD2 is functional in regulating K⁺ homeostasis (33). In Bacillus thuringiensis, the kdpFABC operon is not controlled by KdpDE but is negatively regulated by c-di-AMP through a c-di-AMP-responsive riboswitch. During K⁺ limitation, bacteria produce less c-di-AMP and, thus, derepress the inhibition by the riboswitch to enhance the transcription of the kdpFABC operon (38).

In addition to interacting with K⁺ uptake systems, c-di-AMP controls K⁺ homeostasis by modulating K⁺ export as well. In B. subtilis, the KhtT subunit of the KhtTU K⁺/H⁺ antiporter (also known as YhaTU) binds c-di-AMP, which triggers K⁺ export (32, 59). CpaA (cation/proton antiporter A) of both B. subtilis and S. aureus also has been shown to be activated by c-di-AMP binding (29, 45, 60). Mutations in a monovalent cation exporter, NhaK, were identified in a suppressor screen of a c-di-AMP null mutant of B. subtilis in a medium with 5 mM K⁺ (45). Unlike KhtT and CpaA, which contain RCK_C domains, NhaK does not bind c-di-AMP (29, 32, 45).

While K⁺ homeostasis is rigorously controlled by c-di-AMP via multiple transporters, in B. subtilis and S. pneumoniae, c-di-AMP levels are controlled by K⁺ abundance in the environment. In these organisms, the diadenylate cyclase CdaA is poorly expressed in media with low extracellular K⁺, which corresponds to low intracellular c-di-AMP concentrations. In contrast, c-di-AMP levels increase significantly under the high K⁺ conditions (45, 61). In S. pneumoniae, mutations in CabP, a KtrA homolog, decrease c-di-AMP accumulation, although the mechanism has not been elucidated (61). Thus, there exists an elaborate regulatory network that integrates environmental K⁺ concentrations, K⁺ transporters, and c-di-AMP homeostasis.

Osmolyte transport.

c-di-AMP is essential for bacterial viability in routine laboratory rich media in the Firmicutes. c-di-AMP null mutants of B. subtilis, L. monocytogenes, and S. aureus are unable to grow under such growth conditions (18, 28, 30, 39, 41). Suppressor mutations screened from mutants lacking c-di-AMP vary to certain extents. In B. subtilis, mutations in the cation efflux gene nhaK were identified, whereas in L. monocytogenes and S. aureus, mutations in several osmolyte or peptide transporters were detected (39, 41). All these genes play a role in bacterial responses to osmotic stress. These findings indicate that either c-di-AMP modulates bacterial osmolarity through different mechanisms in these species or these screens did not fully identify all suppressor mutations (41). OpuC is a high-affinity uptake system for carnitine and comprised of four proteins, OpuCA, OpuCB, OpuCC, and OpuCD, which are expressed by the opuCA-opuCD operon (36). In both S. aureus and L. monocytogenes, c-di-AMP binds directly to the cystathionine beta-synthase (CBS) domain of the ATPase OpuCA. Bacteria reduce carnitine uptake when c-di-AMP is overproduced (8, 36). In a suppressor screen of a diadenylate cyclase knockout mutant of L. monocytogenes, mutations in loci of the oligopeptide permease (opp), the osmolyte glycine betaine importer (gbu; homologous to opuAABC in B. subtilis), or the c-di-AMP binding proteins CbpB and PstA (62–66) restored the growth of the mutant in rich media (39). Notably, c-di-AMP-binding ydaO riboswitches have been found upstream of genes encoding osmolyte transporter domain proteins in some species from Clostridia, Fusobacteria, and Bacillales classes and are predicted to control their expression, which further exemplifies the multiple mechanisms of osmotic stress regulation by c-di-AMP (53, 67). Suppressor mutations in c-di-AMP-null S. aureus identified that the activities of both OpuD and AlsT contribute to the growth defect of the mutant when grown in rich medium (41). OpuD is the main glycine betaine uptake system, whereas AlsT functions as a glutamine transporter, and bacterial c-di-AMP production is reduced in the presence of glutamine in media (40, 41). In Streptococcus agalactiae, two deletion strains of diadenylate cyclase could be obtained in this bacterium, accompanied by mutations in oppC and busB (30). As described above, OppC is a subunit of oligopeptide transporter OppABCDF. BusB and its cytoplasmic partner, BusA, form an ABC transporter for the uptake of glycine betaine. Further investigation suggests that BusB must be inactive for viable growth of S. agalactiae in the absence of c-di-AMP (30). Interestingly, the expression of busAB is repressed by a transcription factor, BusR, and the regulatory role of BusR is inhibited when c-di-AMP directly binds to BusR at the RCK_C domain (30). The expression of busAA has been shown to be downregulated in a ΔgdpP mutant of L. lactis, but after salt addition, expression is upregulated in this mutant (68). Furthermore, in a suppressor screen with a ΔgdpP strain of L. lactis under osmotic stress conditions, mutation in BusR was detected (13), elucidating another instance of modulation of gene expression and protein activity by c-di-AMP to control osmolyte uptake. The response of c-di-AMP-mediated osmolyte transport is summarized in Fig. 2.

FIG 2.

Bacterial osmolyte homeostasis controlled by c-di-AMP. Summary of osmolyte transporters that are affected by c-di-AMP by direct binding to the transporter (OpuCA), transcriptional regulator (BusR), or riboswitch (ydaO), which has been found upstream of genes encoding osmolyte transporters. Color codes of c-di-AMP-binding domains in each protein are the following: yellow, RCK_C domain; purple, CBS domain.

SURVIVING ACID STRESS

Adaptation to acid stress is mostly resolved by H⁺ extrusion, which is constrained by maintaining the proton motive force and membrane potential (69). Therefore, the uptake of additional cations, including the dominant intracellular cation K⁺, offsets H⁺ release (69). Response to pH changes mediated by c-di-AMP has been explored in multiple bacterial species (61, 70–73). The observations are species specific. An S. aureus strain with a G206S substitution in the diadenylate cyclase DacA has reduced bacterial c-di-AMP production and is highly susceptible to acidic pH, whereas ΔgdpP mutants in S. aureus, L. lactis, and B. subtilis are more resistant than their parental strains (70, 73, 74). Surprisingly, a ΔybbR (ybbR is also named cdaR) mutant of S. aureus, which generates more c-di-AMP than the parental strain, is highly sensitive to acid stress. Meanwhile, suppressors of the ΔybbR mutant screened under acidic conditions exhibited mutations in DacA or other genes that also resulted in the reduction of bacterial c-di-AMP levels (70), supporting that c-di-AMP is involved in the response to acid stress. Streptococcus pyogenes encodes a sole diadenylate cyclase, which is nonessential for bacterial growth in rich media (71, 75). A mutant deficient in this enzyme is unable to grow in media adjusted to pH 6.0, while c-di-AMP phosphodiesterase mutants exhibited growth similar to that of the wild type (WT) under this condition (71). However, in S. pneumoniae, a mutant with a V76G mutation in CdaA produces less c-di-AMP and is more resistant to acidic pH, while strains lacking the phosphodiesterases Pde1 and/or Pde2 are more susceptible (61, 72). The regulation of K⁺ and osmolyte transport by c-di-AMP may intersect with the bacterial response to acid stress; however, the molecular basis remains unclear.

PROTECTING DNA INTEGRITY

Bacterial DNA damage responses include surveying mechanisms to check for DNA breaks, which recruit repair complexes to the lesion sites. The first diadenylate cyclase was identified during a structural study of a DNA integrity-scanning protein, DisA, which marks the recognition of c-di-AMP as a second bacterial messenger (14). In vitro, the conversion of ATP to c-di-AMP by DisA is inhibited substantially by branched DNA, such as Holliday junctions or stalled replication forks, suggesting a role in maintaining fidelity prior to DNA replication and segregation (14).

The regulation of DNA repair by c-di-AMP and DisA encompasses a multilayered response. In many species, including B. subtilis, B. thuringiensis, Mycobacterium smegmatis, Mycobacterium tuberculosis, Thermotoga maritima, and Fusobacterium nucleatum, disA is genetically arranged in an operon with radiation-sensitive gene A, radA (also known as sms), which takes part in DNA repair and recombination (76–78). In B. subtilis and M. smegmatis, RadA forms a complex with DisA that inhibits its diadenylate cyclase activity, which signals the presence of DNA damage (14, 76, 79). RadA aids in homologous recombination by complexing with recombinase RecA (80, 81). DisA also interacts directly with and diminishes the activity of RecA, which is required to form the DNA repair intermediates that are recognized by DisA (82, 83). However, in M. smegmatis, DisA is required for recA mRNA translation, so there is a fine-tuning of RecA protein levels and activity by c-di-AMP (82, 83). In addition, in this species and M. tuberculosis, RecA nucleoprotein filaments are disrupted by c-di-AMP binding (83). These results, combined with those for live-tracing DisA molecules, suggest that DisA scans the genome for recombination intermediates, which, along with RadA, inhibits c-di-AMP production (Fig. 3) (79). RadA was shown to potentiate RecA recombinase activity, and the RecA repair complex disassembles once c-di-AMP is produced after the repair (79).

FIG 3.

Role of c-di-AMP in DisA-mediated DNA damage response. DisA is a DNA-scanning protein that produces c-di-AMP in the absence of DNA damage, which signals as a checkpoint prior to DNA replication and for entry into sporulation in sporulating species. When DisA binds to branched DNA and complexes with the DNA repair protein RadA, diadenylate cyclase activity undergoes allosteric inhibition. RadA recruits RecA for recombination and repair of the damaged DNA. c-di-AMP production by DisA is resumed after DNA integrity is restored.

The disruption of c-di-AMP homeostasis can affect the survival of bacteria exposed to DNA-damaging agents. Examinations of specific diadenylate cyclase or c-di-AMP phosphodiesterase mutants have shown differences in resistance to nonionizing radiation, such as UV light, ionizing radiation of gamma rays, and chemical agents with various mechanisms of action. In S. pneumoniae, ΔgdpP mutants are slightly more susceptible to UV treatment than WT bacteria, while the Δpde2 mutant and the phosphodiesterase-null mutant are 100-fold more susceptible (18). Deinococcus radiodurans, an extremophile that is highly resistant to ionizing radiation, is more susceptible to gamma rays and UV treatment in the absence of both CdaA and the regulatory protein CdaR, demonstrating that c-di-AMP synthesis is an important factor in achieving high levels of resistance to radiation (84). Since D. radiodurans can survive other environmental stress conditions, such as dessication and oxidative stress, and the response mechanisms are linked (85, 86), it is possible that c-di-AMP is integral to multiple stress resistance traits in this species. After exposure to DNA-damaging chemicals, methyl methane sulfate (MMS) or hydrogen peroxide, B. subtilis mutants of the diadenylate cyclases disA and cdaA, respectively, were susceptible to these reagents (77). MMS methylates purines, which stalls replication forks, while oxidative damage leads to single-strand breaks and base deamination; therefore, the survival differences of each diadenylate cyclase-deficient strain likely relates to the defense and repair mechanisms for each chemical. A strain lacking gdpP that has high levels of c-di-AMP is more tolerant of both chemicals than the WT (77). Collectively, control of c-di-AMP signaling is utilized to combat various DNA-damaging pathways.

It has been well established that c-di-AMP acts as a second messenger for DNA integrity during sporulation of B. subtilis. In this process, B. subtilis undergoes complex morphological changes where a single rod-shaped cell divides asymmetrically and eventually lyses to release a mature spore, which remains dormant to survive stress conditions (87, 88). The initiation of sporulation requires replicated chromosomal DNA integrity, which is monitored by DisA. In a deficient resuspension medium lacking glucose that induces sporulation, DisA expression and complexation as an octamer on DNA increases (89, 90). As the DisA complex surveys DNA, c-di-AMP production indicates the lack of DNA damage and sporulation initiates (14, 89, 90). However, upon DNA damage, a drop in c-di-AMP levels blocks sporulation initiation when, at branched DNA sites, DisA stops producing c-di-AMP and the expression of c-di-AMP phosphodiesterase GdpP is elevated (90). After the recruitment of repair mechanisms, sporulation can proceed (89). It has been reported that the addition of exogenous c-di-AMP to B. subtilis leads to a rapid entry into sporulation (90). In addition to DisA, B. subtilis encodes the diadenylate cyclases CdaA, which affects cell wall stress, and CdaS, which is specifically expressed during sporulation under the control of sigma factor σ^G (16, 91, 92). After the introduction of nutrients to spores, the ΔcdaS mutant has 2-fold impaired germination efficiency compared to that of the WT strain (93). Therefore, while c-di-AMP production by DisA affects spore formation in B. subtilis, c-di-AMP produced by CdaS appears to be utilized in spore germination. However, prior to asymmetric division in B. thuringiensis, sigma factor σ^H directs cdaS expression, which is needed for the production of spores (94, 95). Together, c-di-AMP signaling is required for maintaining DNA integrity and regulating spore formation and germination. Studies of c-di-AMP regulation of additional spore-forming bacteria, such as other Bacillus spp. and Clostridium spp., may reveal how different species utilize DisA and CdaS during this complex process.

ANTIBIOTIC RESISTANCE

Infections from antibiotic-resistant organisms are an increasing threat, as many pathogens are developing resistance to last-line drugs, and there is a current lag in the development of new antibiotics. Understanding the molecular basis for antibiotic resistance mechanisms may uncover pathways with which to direct novel therapeutics. c-di-AMP signaling has been reported to play a role in antibiotic resistance to cell wall- and membrane-targeting antibiotics.

β-Lactam antibiotics inhibit penicillin-binding proteins that are involved in peptidoglycan cross-linking. In general, mutations that decrease diadenylate cyclase activity increase susceptibility to β-lactams, while mutations inactivating c-di-AMP phosphodiesterases enhance resistance (17, 68, 92, 96–98). In addition to peptidoglycan, Gram-positive bacteria incorporate lipoteichoic acids (LTA) in the cell wall. Methicillin-resistant S. aureus (MRSA) strains carry the mecA gene encoding penicillin-binding protein 2a (PBP2a), which has low affinity for β-lactams. In MRSA as well as methicillin-sensitive S. aureus strains devoid of LTA, suppressor mutations were obtained in gdpP (17). These LTA-deficient suppressor strains have improved cell size and growth in rich culture media in the absence of osmoprotectants, similar to the WT (17). The deletion of gdpP in LTA-containing cells increases resistance to cell wall-targeting antibiotics, including the β-lactams oxacillin and penicillin G, and the bacteriocin lysostaphin, which cleaves pentaglycine bridges. These results may be explained by an overrepresentation of higher-order linked muropeptides in the ΔgdpP strain compared to that in the WT (17). Evolution of a mecA⁺ population with heterogenous oxacillin resistance selected for homogenous, high-level resistant isolates with gdpP mutations that correlate with increased PBP2a expression (98). Similarly, in isogenic MRSA paired strains, the strain carrying a change in dacA with lower c-di-AMP levels has a higher growth rate and reduced oxacillin resistance (96). Therefore, in MRSA, c-di-AMP signaling may affect β-lactam resistance via cell wall composition, osmotic regulation, and/or by controlling PBP2a protein levels. Increased β-lactam resistance resulting from mutations inactivating c-di-AMP phosphodiesterase GdpP homologs also has been reported in B. subtilis, L. lactis, and L. monocytogenes (68, 92, 97).

Resistance to daptomycin, a cell membrane-disrupting drug of last resort for vancomycin-resistant enterococci, has also been associated with increased c-di-AMP levels caused by mutations in gdpP or the liaFSR genes, encoding a three-component membrane stress response system (99). After the evolution of E. faecalis in daptomycin, one set of mutants had a single-nucleotide change in gdpP paired with a mutation in the response regulator gene liaR (99). However, a ΔliaR mutant also exhibited elevated levels of c-di-AMP but does not confer daptomycin resistance (99); therefore, a combination of c-di-AMP signaling and cell membrane modifications likely contribute to this mechanism in E. faecalis.

CONTROLLING PNEUMOCOCCAL COMPETENCE

The process of natural transformation, the ability of cells to acquire extracellular DNA, is governed by genetic competence programs. In the naturally competent organism S. pneumoniae, the competence program controls the expression of 288 genes (100), including genes encoding DNA uptake and recombination machinery. There are 100 different pneumococcal capsular serotypes, of which only a small subset is included in the widely available capsule-based vaccines (101–103). Thus, these vaccines provide only limited protection against pneumococcal infections caused by vaccinated serotypes. The mechanism of acquiring extracellular DNA through natural transformation leads to antibiotic resistance and allows targeted serotype strains to switch their capsular machinery and evade immune surveillance (104–106).

The pneumococcal competence program is induced by the recognition of the mature form of a secreted pheromone, competence-stimulating peptide (CSP), by a two-component system, ComDE (107–109). The activation of the response regulator ComE drives the expression of early competence genes and the alternative sigma factor, ComX, which controls late gene expression (110). The lysis mechanisms autolysis and fratricide are induced during the competent state to release DNA from a subpopulation that is available for acquisition by the surviving cells (111–117). The regulation of competence induction is tightly controlled based on the inputs of secreted protein pheromone, growth phase, and stress stimuli (118). In particular, DNA damage-dependent and -independent stress, such as antibiotic treatment, pH changes, and oxygen availability, can initiate the competence state to improve adaptation to these stress conditions (119–129). The competence regulon also affects colonization and virulence in this pathogen (130–132).

Genes in the competence program are upregulated in c-di-AMP phosphodiesterase mutant bacteria in mid-log phase (72). Despite this induction of the competence state, ΔgdpP Δpde2 pneumococci have a defect in transformation for both plasmid and integrative DNA that is partially c-di-AMP dependent. Pneumococci with a single-amino-acid substitution (V76G) in CdaA, which is designated cdaA*, contain lower concentrations of c-di-AMP than WT cells (72). CSP addition inhibits growth of the cdaA* mutant population through ComD but has minor effects on WT and phosphodiesterase-deficient cultures. As mentioned earlier, in pneumococci, c-di-AMP blocks K⁺ uptake by binding a Trk family KtrA homolog, CabP, which complexes with the transmembrane K⁺ transporter TrkH (27). Deletion of trkH and cabP in cdaA* bacteria reduced the growth inhibition by CSP, suggesting that increased [K⁺] in cdaA* is detrimental during competence induction.

In S. pneumoniae and Streptococcus mutans, environmental K⁺ concentrations and K⁺ uptake transporters, TrkH and Trk2, respectively, are required for competence development and transformation (72, 133). Therefore, the changes in competence state in pneumococci with elevated c-di-AMP levels is partially due to c-di-AMP hindrance of K⁺ transport. In addition, the mechanism of action of the compound COM blockers, inhibitors of pneumococcal competence, is perturbing the proton motive force via decreasing H⁺ and/or K⁺ flux (134). The c-di-AMP signaling network, as a regulator of K⁺, the competence state, and essential pathways, may also serve as an attractive target for therapeutics that may be conserved among Streptococcus spp. and likely other naturally competent bacteria.

BIOFILM FORMATION

Bacterial biofilms are multicellular aggregates that are surrounded by adhesins, polysaccharide, extracellular DNA, and other matrixes. Biofilm architecture inherently allows the communities to be more resistant to antimicrobials in infections and cleaning agents on surfaces (135). Adherence and colonization in animal hosts or plant tissues may be facilitated by biofilm pathways. Biofilm formation initiates with attachment to surfaces, followed by the formation of microcolonies and maturation, before dispersion from the biofilm (136). External stress stimuli, including osmolarity, oxygen levels, and temperature, can influence biofilm development (137, 138). The second messenger c-di-GMP controls the switch between planktonic and sessile lifestyles by affecting gene expression and protein activity to reduce flagellar movement and enhance biofilm formation (139). However, in several species of the Firmicutes, biofilm formation can be controlled by c-di-AMP. The biofilm phenotypes that have been observed to be altered by high or low intracellular c-di-AMP levels are summarized in Table 2.

TABLE 2.

Summary of biofilm phenotypes

Bacterial strain	Genotypea	[c-di-AMP]	Biofilm formation	Matrix/biotic colonizationb	Reference(s)
Bacillus subtilis NCIB3610	ΔgdpP ΔpgpH	Increased	Decreased	ND	154
	ΔdisA	Decreased	Decreased	No plant attachment	155
Streptococcus suis serotype 2	ΔgdpP	Increased	Increased	Decreased adherence to Hep-2 epithethial cells	147
Streptococcus mutans UA159	ΔgdpP	Increased	Increased	Increased EPS, increased colonization of Drosophila	144
	ΔcdaA	Absent	Decreased	Decreased EPS, decreased colonization rat oral cavity	143
	ΔcdaA	Absent	Increased	Increased EPS (cell lysis?)	146
Streptococcus mutans XC	Δpde2	Increased	Increased	ND	145
Streptococcus gallolyticus subsp. gallolyticus UCN34	ΔgdpP	Increased	Decreased	Decreased adherence on HT-29 human intestinal cells	37
Streptococcus pyogenes HSC5	Δpde2	Increased	Increased	ND	148
	ΔcdaA	Absent	Decreased	ND	148
Staphylococcus aureus USA300 SEJ1	ΔgdpP	Increased	Increased	ND	17
	ΔgdpP	Increased	Decreased	Decreased eDNA matrix	141
Staphylococcus aureus USA300 HG003	gdpP::Tn	Increased	Decreased	Decreased eDNA matrix	141

^aHomolog of diadenylate cyclase and c-di-AMP phosphodiesterase listed.

^bND, not described.

The effect of c-di-AMP on biofilm formation was first observed in S. aureus, a human pathogen with recalcitrance to antibiotics due to its persistent biofilms (140). A c-di-AMP phosphodiesterase gdpP null strain has 3-fold more biofilm adherence than WT bacteria when grown in rich medium with addition of NaCl (17). However, in another study, this same ΔgdpP strain in rich medium supplemented with glucose was observed to have a decrease in biofilm formation and decreased extracellular DNA (eDNA) matrix (141). These biofilm-dependent phenotypes were suggested to be due to enhanced cell wall integrity in the ΔgdpP mutant. Further, biofilm induction with glucose correlates with lower c-di-AMP levels (141). During growth of S. aureus in cell culture media with human plasma, biofilm-dependent autolysis is observed in both WT and ΔgdpP cultures, but the rate is significantly higher in ΔgdpP biofilms (142). It is possible that the stark contrast in biofilm culture cell lysis among ΔgdpP cells is responsible for the reported differences in biofilm formation studies in S. aureus, depending on the incubation time and culture conditions.

Association between c-di-AMP and biofilm regulation has been explored in multiple species of the genus Streptococcus and in Enterococcus faecalis. However, the results are rather diverse. In S. mutans, deletion of the c-di-AMP phosphodiesterase homolog of gdpP improves biofilm formation and increases the expression of gtfB, which encodes a major glucan-producing enzyme needed for biofilm polymer production, while the removal of the diadenylate cyclase reduces extracellular polysaccharides (EPS) and biofilm formation (143, 144). This mechanism is via a c-di-AMP-binding Trk-family protein, CabPA, that directly interacts with a transcriptional regulator, VicR, which controls the expression of gftB. In another study, only the deletion of the phosphodiesterase Pde2 homolog, but not GdpP, increased biofilm formation compared to that of the WT in S. mutans (145). The discordant results accredited to GdpP or Pde2 could be due to differences in culture medium compositions or the utilization of different strains (UA159 versus XC). Conversely, another report showed that cdaA deletion increased EPS/cell ratio and biofilm; however, there is increased cell lysis in the culture conditions tested that may affect interpretation (146). In Streptococcus suis, a swine pathogen that can cause invasive zoonotic diseases, a ΔgdpP mutant has increased biofilm formation compared to that of the WT (147). A strain lacking c-di-AMP in the human pathogen S. pyogenes does not form biofilms, and only the deletion of pde2, not gdpP, leads to an increase in biofilm formation (148). Conversely, in Streptococcus gallolyticus, a pathogen associated with infective endocarditis and colorectal cancer, the deletion of gdpP decreases biofilm formation (37). In E. faecalis, treatment with a small compound, ST056083, which reduces diadenylate cyclase activity, also reduces biofilm growth and EPS matrix, indicating that c-di-AMP enhances the biofilm formation of this pathogen (149–151).

In B. subtilis, biofilm communities are composed of morphologically distinct subpopulations, such as cells that specialize in sporulation, motility, and matrix production (152). The second messenger c-di-AMP affects biofilm formation and plant attachment, but, similar to the observations in other species, disparate results exist, likely due to differences in medium composition (reviewed in references 153–155). Potassium homeostasis, which is rigorously controlled by c-di-AMP in B. subtilis, affects biofilm initiation. KinC, a membrane protein kinase, initiates biofilm formation upon sensing low intracellular potassium, which may be partially facilitated by the potassium efflux protein YugO (156, 157). The addition of potassium results in less biofilm, while potassium leakage induces biofilm formation (156, 157). Therefore, it is most likely that the abrogation of c-di-AMP production, which increases intracellular potassium concentrations, would not support biofilm formation. However, there are four sensory kinases (KinA, KinB, KinC, and KinD) and other mechanisms affecting biofilm initiation that could be influenced by c-di-AMP signaling and lead to divergent biofilm phenotypes.

Colony morphology is an indication of biofilm matrix production. In one report, the accumulation of c-di-AMP due to the deletion of both phosphodiesterases, gdpP and pgpH, but not the single mutants, disrupts complex colony formation, similar to mutants of the epsA-O operon, which produces EPS and the biofilm matrix protein tasA (154). Biofilm formation is restored in the phosphodiesterase-deficient mutant when the biofilm repressor SinR is also absent, suggesting that the effect of high c-di-AMP levels on biofilm formation is through SinR (154). However, another study found a loss of colony architecture with deletion of disA and an increase in colony wrinkling with the phosphodiesterase mutants (155). It is possible there is a link between the specific functions of DisA in DNA damage repair and colony architecture that does not involve the other diadenylate cyclases, CdaA and CdaS.

Plant root attachment in B. subtilis is facilitated by biofilm formation. It was reported that the ΔdisA strain has reduced attachment, while the phosphodiesterase-deficient strains have increased attachment (155). Coculture with WT cells enhanced the attachment of disA mutants, which was independent of EPS production but partially dependent on c-di-AMP secretion through the MDR-like efflux proteins YcnB and YhcA (155). It will be interesting to determine how intracellular and extracellular c-di-AMP is sensed to affect microbial communities and biofilm composition.

INTERACTION BETWEEN SIGNALING NETWORKS OF c-di-AMP AND (p)PPGPP

To adapt to nutrient deprivation, bacteria produce the alarmone (p)ppGpp to induce stringent starvation responses that regulate growth, DNA replication, RNA polymerase, and protein synthesis (reviewed in reference 158). (p)ppGpp is synthesized and hydrolyzed by Rel Spo homolog (RSH) proteins, where the synthase domain transfers two phosphates from ATP to either GTP or GDP to generate pppGpp or ppGpp, respectively [referred to as (p)ppGpp]. Strikingly, (p)ppGpp and c-di-AMP signaling networks converge to coordinate bacterial viability and stress responses (Fig. 4).

FIG 4.

Interactions between networks of c-di-AMP and (p)ppGpp. c-di-AMP hydrolysis by phosphodiesterases with transmembrane domains, GdpP and PgpH, is regulated by (p)ppGpp. c-di-AMP activates (p)ppGpp synthesis by RelA, but not RelPQ, through an unknown mechanism. The regulatory role of CodY can be derepressed by GTP and indirectly inhibited by (p)ppGpp. The CodY regulon affects the c-di-AMP signaling network independent of (p)ppGpp.

The first notion of the link between c-di-AMP and (p)ppGpp was a report that the deletion of gdpP in L. lactis enhances resistance to glucose starvation at acidic pH, suggesting that the deletion alters the bacterial (p)ppGpp levels (74). In vitro studies have confirmed that ppGpp inhibits the hydrolysis of c-di-AMP by two distinct c-di-AMP phosphodiesterases, GdpP and PgpH (20, 73, 159). GdpP does not hydrolyze (p)ppGpp (159), and (p)ppGpp regulates PgpH activity at an allosteric site (20). In bacterial cells, a mutant defective in (p)ppGpp synthesis reduces bacterial c-di-AMP levels, which supports that (p)ppGpp inhibits c-di-AMP phosphodiesterase activity in vivo (159).

c-di-AMP is essential for multiple bacteria when grown under rich medium conditions (16, 18, 39, 92, 159, 160). In L. monocytogenes lacking c-di-AMP, nearly 300 suppressor strains were sequenced, and 1.76% of these possessed mutations in the synthase domain of the relA gene, disrupting (p)ppGpp production (160). In Firmicutes, (p)ppGpp is synthesized by three synthases: RelA, RelP, and RelQ. RelA is a dual-function enzyme that has (p)ppGpp hydrolase activity in addition to synthase, whereas RelP and RelQ only function as synthases (160). The diadenylate cyclase is dispensable when the (p)ppGpp synthase-encoding genes are deleted in L. monocytogenes (160). Therefore, a main driver of the essentiality of c-di-AMP is the toxic accumulation of (p)ppGpp under rich medium conditions in the absence of c-di-AMP. In line with this, c-di-AMP affects the homeostasis of (p)ppGpp. Deletion of gdpP in S. aureus elevates (p)ppGpp levels significantly, which can be abolished by mutation of the synthesis domain of RelA but not by deletion of relP or relQ. Thus, the activation of (p)ppGpp synthesis by c-di-AMP is likely dependent on RelA (159). However, c-di-AMP does not directly bind RelA in vitro, so the activation is through an undiscovered indirect mechanism (159).

CodY is a GTP- and branched-chain amino acid-responsive global transcriptional regulator that represses nutrient uptake and amino acid biosynthesis genes. However, when (p)ppGpp accumulates during nutrient starvation, GTP levels decrease, which disrupts CodY complexation with DNA (161). Interestingly, the lack of diadenylate cyclase essentiality in the ΔrelAPQ mutant grown in rich media could be reversed by deletion of codY (160). Additionally, diadenylate cyclase is no longer essential for bacteria grown in minimal media, which favors the inactivation of CodY (160). Furthermore, a recent study of urinary tract infection (UTI) caused by E. faecalis demonstrated that the expression of gdpP was highly elevated in a CodY-dependent manner after transition from a culture medium to urine (162). These observations suggest that there is a link between CodY regulon and the c-di-AMP network, which is independent of (p)ppGpp. Together, c-di-AMP is essential for bacterial growth in rich media due to an aberrant accumulation of (p)ppGpp, leading to toxic changes in the expression of the CodY regulon (160). More details for this perspective are still to be elucidated.

NIGHTTIME SURVIVAL IN CYANOBACTERIA

Cyanobacteria are photoautotrophic organisms that play important roles in carbon, oxygen, and nitrogen biogeochemical cycles and have garnered interest in the sphere of biotechnology (163–165). As light is a required energy source, light-dark cycles are integral to cyanobacterial metabolism (166). The DAC domain is found in cyanobacterial genomes along with c-di-AMP riboswitches upstream of putative osmolyte transporters and biosynthesis enzymes (53, 167–169). To date, the effect of c-di-AMP signaling in response to changes in light stimuli has been explored in the unicellular cyanobacteria Synechocystis sp. strain PCC 6803 and Synechococcus elongatus PCC 7942 (168, 170).

Under white-light growth conditions, perturbing c-di-AMP homeostasis with either cyclase overexpression or phosphodiesterase overexpression in Synechocystis causes a growth defect and aggregation compared to WT cultures (168). The growth defect of these strains is ameliorated in lower light (15 μmol photons m⁻² s⁻¹ compared to 35 μmol m⁻² s⁻¹) (168). A cdaA deletion strain could not be constructed, alluding to the essentiality of c-di-AMP in this organism (168). Nevertheless, a cdaA mutant is viable in the cyanobacterial species S. elongatus (170).

In response to changes in lighting, c-di-AMP levels are augmented in S. elongatus. c-di-AMP accumulates 3-fold within 20 min after the onset of darkness (170). In addition, in 12-h light/12-h dark cycles, there is a trend toward higher intracellular concentrations of c-di-AMP during the dark cycle. In line with these results, the cdaA mutant has a growth defect during light/dark cycles, specifically during the dark phase, but not in constant light, suggesting that c-di-AMP is required for nighttime survival (170). The sensitivity of the cdaA mutant to darkness is likely due to the toxic accumulation of reactive oxygen species in the absence of photosynthesis-adjacent reduction mechanisms, which are regulated by the circadian clock transcription factor RpaA (171). In the cdaA mutant, deletions of genes encoding electron sinks or resistance to oxidative damage further reduce survival in light, and mutations affecting circadian clock components, including in a protein that inactivates RpaA, rescue growth in light/dark cycles (170). Since these new studies establish that c-di-AMP homeostasis is critical to managing stress stimuli in diurnal lifestyles, it is important to determine the molecular mechanisms underlying responses to light and dark cycles to improve biotechnological and environmental uses of cyanobacteria.

FINAL CONCLUSIONS AND FUTURE DIRECTIONS

The study of the signaling di-nucleotide c-di-AMP is an evolving, active area of interest due to its regulation of vital physiological functions and its occurrence in a myriad of microorganisms. In addition, the colonization and virulence of several c-di-AMP-producing pathogens are affected by altering c-di-AMP homeostasis (18, 20, 71, 97, 147, 172–176), although it is not clear if changes in pathogenesis are due to bacterial responses to stress stimuli and/or surveillance of secreted c-di-AMP by the innate immune system. Untangling these aspects in vivo can be confounded by genetic alterations of c-di-AMP homeostasis affecting multiple functions, including overall bacterial fitness. However, the inactivation of stress response pathways combined with mutations affecting c-di-AMP levels may lead to more defined answers about the role of c-di-AMP in bacterial pathogenesis.

Despite many fascinating reports of how c-di-AMP is utilized in a variety of stress responses, including regulating osmotic stress and surviving DNA damage, the signaling pathways from stress stimuli to receptors are not fully elucidated. Likewise, in a newer field more studies are required to clear up inconsistencies in observations that are likely due to unexplored aspects of the c-di-AMP signaling network.

Biographies

Tiffany M. Zarrella is currently a postdoctoral fellow in the laboratory of Anupama Khare at the National Cancer Institute, where she studies interactions between Pseudomonas aeruginosa and Staphylococcus aureus. She received her B.S. in biochemistry from Syracuse University and earned her Ph.D. at Albany Medical College in the laboratory of Guangchun Bai, where she researched the role of the bacterial signaling nucleotide cyclic di-AMP in controlling stress responses and competence in Streptococcus pneumoniae. She has an interest in better understanding the molecular mechanisms underlying bacterial responses to environmental stimuli.

Guangchun Bai is an assistant professor in the Department of Immunology and Microbial Disease at Albany Medical College. He received his M.D. and Ph.D. from Fourth Military Medical University, China. He completed his postdoctoral fellowship with Kathleen McDonough at the Wadsworth Center, New York State Department of Health. His current research is focused on deciphering the role of cyclic di-AMP in bacterial physiology and pathogenesis in Streptococcus pneumoniae and Mycobacterium tuberculosis. He has worked in the field of cyclic di-AMP signaling for nearly 10 years.