Control of the Diadenylate Cyclase CdaS in Bacillus subtilis: AN AUTOINHIBITORY DOMAIN LIMITS CYCLIC DI-AMP PRODUCTION*

The Gram-positive bacterium Bacillus subtilis encodes three diadenylate cyclases that synthesize the essential signaling nucleotide cyclic di-AMP. The activities of the vegetative enzymes DisA and CdaA are controlled by protein-protein interactions with their conserved partner proteins. Here, we have analyzed the regulation of the unique sporulation-specific diadenylate cyclase CdaS. Very low expression of CdaS as the single diadenylate cyclase resulted in the appearance of spontaneous suppressor mutations. Several of these mutations in the cdaS gene affected the N-terminal domain of CdaS. The corresponding CdaS mutant proteins exhibited a significantly increased enzymatic activity. The N-terminal domain of CdaS consists of two α-helices and is attached to the C-terminal catalytically active diadenylate cyclase (DAC) domain. Deletion of the first or both helices resulted also in strongly increased activity indicating that the N-terminal domain serves to limit the enzyme activity of the DAC domain. The structure of YojJ, a protein highly similar to CdaS, indicates that the protein forms hexamers that are incompatible with enzymatic activity of the DAC domains. In contrast, the mutations and the deletions of the N-terminal domain result in conformational changes that lead to highly increased enzymatic activity. Although the full-length CdaS protein was found to form hexamers, a truncated version with a deletion of the first N-terminal helix formed dimers with high enzyme activity. To assess the role of CdaS in sporulation, we assayed the germination of wild type and cdaS mutant spores. The results indicate that cyclic di-AMP formed by CdaS is required for efficient germination.     

Structural basis for the inhibition of the Bacillus subtilis c-di-AMP cyclase CdaA by the phosphoglucomutase GlmM

Cyclic-di-adenosine monophosphate (c-di-AMP) is an important nucleotide signaling molecule that plays a key role in osmotic regulation in bacteria. c-di-AMP is produced from two molecules of ATP by proteins containing a diadenylate cyclase (DAC) domain. In Bacillus subtilis, the main c-di-AMP cyclase, CdaA, is a membrane-linked cyclase with an N-terminal transmembrane domain followed by the cytoplasmic DAC domain. As both high and low levels of c-di-AMP have a negative impact on bacterial growth, the cellular levels of this signaling nucleotide are tightly regulated. Here we investigated how the activity of the B. subtilis CdaA is regulated by the phosphoglucomutase GlmM, which has been shown to interact with the c-di-AMP cyclase. Using the soluble B. subtilis CdaA_CD catalytic domain and purified full-length GlmM or the GlmM_F369 variant lacking the C-terminal flexible domain 4, we show that the cyclase and phosphoglucomutase form a stable complex in vitro and that GlmM is a potent cyclase inhibitor. We determined the crystal structure of the individual B. subtilis CdaA_CD and GlmM homodimers and of the CdaA_CD:GlmM_F369 complex. In the complex structure, a CdaA_CD dimer is bound to a GlmM_F369 dimer in such a manner that GlmM blocks the oligomerization of CdaA_CD and formation of active head-to-head cyclase oligomers, thus suggesting a mechanism by which GlmM acts as a cyclase inhibitor. As the amino acids at the CdaA_CD:GlmM interphase are conserved, we propose that the observed mechanism of inhibition of CdaA by GlmM may also be conserved among Firmicutes.     

An extracytoplasmic protein and a moonlighting enzyme modulate synthesis of c-di-AMP in Listeria monocytogenes

The second messenger cyclic di-AMP (c-di-AMP) is essential for growth of many bacteria because it controls osmolyte homeostasis. c-di-AMP can regulate the synthesis of potassium uptake systems in some bacteria and it also directly inhibits and activates potassium import and export systems, respectively. Therefore, c-di-AMP production and degradation have to be tightly regulated depending on the environmental osmolarity. The Gram-positive pathogen Listeria monocytogenes relies on the membrane-bound diadenylate cyclase CdaA for c-di-AMP production and degrades the nucleotide with two phosphodiesterases. While the enzymes producing and degrading the dinucleotide have been reasonably well examined, the regulation of c-di-AMP production is not well understood yet. Here we demonstrate that the extracytoplasmic regulator CdaR interacts with CdaA via its transmembrane helix to modulate c-di-AMP production. Moreover, we show that the phosphoglucosamine mutase GlmM forms a complex with CdaA and inhibits the diadenylate cyclase activity in vitro. We also found that GlmM inhibits c-di-AMP production in L. monocytogenes when the bacteria encounter osmotic stress. Thus, GlmM is the major factor controlling the activity of CdaA in vivo. GlmM can be assigned to the class of moonlighting proteins because it is active in metabolism and adjusts the cellular turgor depending on environmental osmolarity.     

Intracellular Concentrations of Borrelia burgdorferi Cyclic Di-AMP Are Not Changed by Altered Expression of the CdaA Synthase

The second messenger nucleotide cyclic diadenylate monophosphate (c-di-AMP) has been identified in several species of Gram positive bacteria and Chlamydia trachomatis. This molecule has been associated with bacterial cell division, cell wall biosynthesis and phosphate metabolism, and with induction of type I interferon responses by host cells. We demonstrate that B. burgdorferi produces a c-di-AMP synthase, which we designated CdaA. Both CdaA and c-di-AMP levels are very low in cultured B. burgdorferi, and no conditions were identified under which cdaA mRNA was differentially expressed. A mutant B. burgdorferi was produced that expresses high levels of CdaA, yet steady state borrelial c-di-AMP levels did not change, apparently due to degradation by the native DhhP phosphodiesterase. The function(s) of c-di-AMP in the Lyme disease spirochete remains enigmatic.     

YybT Is a Signaling Protein That Contains a Cyclic Dinucleotide Phosphodiesterase Domain and a GGDEF Domain with ATPase Activity

The cyclic dinucleotide c-di-GMP synthesized by the diadenylate cyclase domain was recently discovered as a messenger molecule for signaling DNA breaks in Bacillus subtilis. By searching bacterial genomes, we identified a family of DHH/DHHA1 domain proteins (COG3387) that co-occur with a subset of the diadenylate cyclase domain proteins. Here we report that the B. subtilis protein YybT, a member of the COG3387 family proteins, exhibits phosphodiesterase activity toward cyclic dinucleotides. The DHH/DHHA1 domain hydrolyzes c-di-AMP and c-di-GMP to generate the linear dinucleotides 5′-pApA and 5′-pGpG. The data suggest that c-di-AMP could be the physiological substrate for YybT given the physiologically relevant Michaelis-Menten constant (K_m) and the presence of YybT family proteins in the bacteria lacking c-di-GMP signaling network. The bacterial regulator ppGpp was found to be a strong competitive inhibitor of the DHH/DHHA1 domain, suggesting that YybT is under tight control during stringent response. In addition, the atypical GGDEF domain of YybT exhibits unexpected ATPase activity, distinct from the common diguanylate cyclase activity for GGDEF domains. We further demonstrate the participation of YybT in DNA damage and acid resistance by characterizing the phenotypes of the ΔyybT mutant. The novel enzymatic activity and stress resistance together point toward a role for YybT in stress signaling and response.     

Mutation in the C-Di-AMP Cyclase dacA Affects Fitness and Resistance of Methicillin Resistant Staphylococcus aureus

Faster growing and more virulent strains of methicillin resistant Staphylococcus aureus (MRSA) are increasingly displacing highly resistant MRSA. Elevated fitness in these MRSA is often accompanied by decreased and heterogeneous levels of methicillin resistance; however, the mechanisms for this phenomenon are not yet fully understood. Whole genome sequencing was used to investigate the genetic basis of this apparent correlation, in an isogenic MRSA strain pair that differed in methicillin resistance levels and fitness, with respect to growth rate. Sequencing revealed only one single nucleotide polymorphism (SNP) in the diadenylate cyclase gene dacA in the faster growing but less resistant strain. Diadenylate cyclases were recently discovered to synthesize the new second messenger cyclic diadenosine monophosphate (c-di-AMP). Introduction of this mutation into the highly resistant but slower growing strain reduced resistance and increased its growth rate, suggesting a direct connection between the dacA mutation and the phenotypic differences of these strains. Quantification of cellular c-di-AMP revealed that the dacA mutation decreased c-di-AMP levels resulting in reduced autolysis, increased salt tolerance and a reduction in the basal expression of the cell wall stress stimulon. These results indicate that c-di-AMP affects cell envelope-related signalling in S. aureus. The influence of c-di-AMP on growth rate and methicillin resistance in MRSA indicate that altering c-di-AMP levels could be a mechanism by which MRSA strains can increase their fitness levels by reducing their methicillin resistance levels.     

Two-Step Synthesis and Hydrolysis of Cyclic di-AMP in Mycobacterium tuberculosis

Cyclic di-AMP is a recently discovered signaling molecule which regulates various aspects of bacterial physiology and virulence. Here we report the characterization of c-di-AMP synthesizing and hydrolyzing proteins from Mycobacterium tuberculosis. Recombinant Rv3586 (MtbDisA) can synthesize c-di-AMP from ATP through the diadenylate cyclase activity. Detailed biochemical characterization of the protein revealed that the diadenylate cyclase (DAC) activity is allosterically regulated by ATP. We have identified the intermediates of the DAC reaction and propose a two-step synthesis of c-di-AMP from ATP/ADP. MtbDisA also possesses ATPase activity which is suppressed in the presence of the DAC activity. Investigations by liquid chromatography -electrospray ionization mass spectrometry have detected multimeric forms of c-di-AMP which have implications for the regulation of c-di-AMP cellular concentration and various pathways regulated by the dinucleotide. We have identified Rv2837c (MtbPDE) to have c-di-AMP specific phosphodiesterase activity. It hydrolyzes c-di-AMP to 5′-AMP in two steps. First, it linearizes c-di-AMP into pApA which is further hydrolyzed to 5′-AMP. MtbPDE is novel compared to c-di-AMP specific phosphodiesterase, YybT (or GdpP) in being a soluble protein and hydrolyzing c-di-AMP to 5′-AMP. Our results suggest that the cellular concentration of c-di-AMP can be regulated by ATP concentration as well as the hydrolysis by MtbPDE.     

Identification,Characterization, and Structure Analysis of the Cyclic di-AMP-binding PII-like Signal Transduction Protein DarA

The cyclic dimeric AMP nucleotide c-di-AMP is an essential second messenger in Bacillus subtilis. We have identified the protein DarA as one of the prominent c-di-AMP receptors in B. subtilis. Crystal structure analysis shows that DarA is highly homologous to P_II signal transducer proteins. In contrast to P_II proteins, the functionally important B- and T-loops are swapped with respect to their size. DarA is a homotrimer that binds three molecules of c-di-AMP, each in a pocket located between two subunits. We demonstrate that DarA is capable to bind c-di-AMP and with lower affinity cyclic GMP-AMP (3′3′-cGAMP) but not c-di-GMP or 2′3′-cGAMP. Consistently the crystal structure shows that within the ligand-binding pocket only one adenine is highly specifically recognized, whereas the pocket for the other adenine appears to be promiscuous. Comparison with a homologous ligand-free DarA structure reveals that c-di-AMP binding is accompanied by conformational changes of both the fold and the position of the B-loop in DarA.     

Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates

To reveal mechanisms of DNA damage checkpoint initiation, we structurally and biochemically analyzed DisA, a protein that controls a Bacillus subtilis sporulation checkpoint in response to DNA double-strand breaks. We find that DisA forms a large octamer that consists of an array of an uncharacterized type of nucleotide-binding domain along with two DNA-binding regions related to the Holliday junction recognition protein RuvA. Remarkably, the nucleotide-binding domains possess diadenylate cyclase activity. The resulting cyclic diadenosine phosphate, c-di-AMP, is reminiscent but distinct from c-di-GMP, an emerging prokaryotic regulator of complex cellular processes. Diadenylate cyclase activity is unaffected by linear DNA or DNA ends but strongly suppressed by branched nucleic acids such as Holliday junctions. Our data indicate that DisA signals DNA structures that interfere with chromosome segregation via c-di-AMP. Identification of the diadenylate cyclase domain in other eubacterial and archaeal proteins implies a more general role for c-di-AMP in prokaryotes.     

Listeria monocytogenes Multidrug Resistance Transporters and Cyclic Di-AMP,Which Contribute to Type I Interferon Induction,Play a Role in Cell Wall Stress

The intracellular bacterial pathogen Listeria monocytogenes activates a robust type I interferon response upon infection. This response is partially dependent on the multidrug resistance (MDR) transporter MdrM and relies on cyclic-di-AMP (c-di-AMP) secretion, yet the functions of MdrM and cyclic-di-AMP that lead to this response are unknown. Here we report that it is not MdrM alone but a cohort of MDR transporters that together contribute to type I interferon induction during infection. In a search for a physiological function of these transporters, we revealed that they play a role in cell wall stress responses. A mutant with deletion of four transporter genes (ΔmdrMTAC) was found to be sensitive to sublethal concentrations of vancomycin due to an inability to produce and shed peptidoglycan under this stress. Remarkably, c-di-AMP is involved in this phenotype, as overexpression of the c-di-AMP phosphodiesterase (PdeA) resulted in increased susceptibility of the ΔmdrMTAC mutant to vancomycin, whereas overexpression of the c-di-AMP diadenylate cyclase (DacA) reduced susceptibility to this drug. These observations suggest a physiological association between c-di-AMP and the MDR transporters and support the model that MDR transporters mediate c-di-AMP secretion to regulate peptidoglycan synthesis in response to cell wall stress.     

Two DHH Subfamily 1 Proteins in Streptococcus pneumoniae Possess Cyclic Di-AMP Phosphodiesterase Activity and Affect Bacterial Growth and Virulence

Cyclic di-AMP (c-di-AMP) and cyclic di-GMP (c-di-GMP) are signaling molecules that play important roles in bacterial biology and pathogenesis. However, these nucleotides have not been explored in Streptococcus pneumoniae, an important bacterial pathogen. In this study, we characterized the c-di-AMP-associated genes of S. pneumoniae. The results showed that SPD_1392 (DacA) is a diadenylate cyclase that converts ATP to c-di-AMP. Both SPD_2032 (Pde1) and SPD_1153 (Pde2), which belong to the DHH subfamily 1 proteins, displayed c-di-AMP phosphodiesterase activity. Pde1 cleaved c-di-AMP into phosphoadenylyl adenosine (pApA), whereas Pde2 directly hydrolyzed c-di-AMP into AMP. Additionally, Pde2, but not Pde1, degraded pApA into AMP. Our results also demonstrated that both Pde1 and Pde2 played roles in bacterial growth, resistance to UV treatment, and virulence in a mouse pneumonia model. These results indicate that c-di-AMP homeostasis is essential for pneumococcal biology and disease.     

Radiation-sensitive Gene A (RadA) Targets DisA,DNA Integrity Scanning Protein A,to Negatively Affect Cyclic Di-AMP Synthesis Activity in Mycobacterium smegmatis

Cyclic di-AMP has been recognized as a ubiquitous second messenger involved in the regulation of bacterial signal transduction. However, little is known about the control of its synthesis and its physiological role in bacteria. In this study, we report a novel mechanism of control of c-di-AMP synthesis and its effects on bacterial growth in Mycobacterium smegmatis. We identified a DisA homolog in M. smegmatis, MsDisA, as an enzyme involved in c-di-AMP synthesis. Furthermore, MsRadA, a RadA homolog in M. smegmatis was found to act as an antagonist of the MsDisA protein. MsRadA can physically interact with MsDisA and inhibit the c-di-AMP synthesis activity of MsDisA. Overexpression of MsdisA in M. smegmatis led to cell expansion and bacterial aggregation as well as loss of motility. However, co-expression of MsradA and MsdisA rescued these abnormal phenotypes. Furthermore, we show that the interaction between RadA and DisA and its role in inhibiting c-di-AMP synthesis may be conserved in bacteria. Our findings enhance our understanding of the control of c-di-AMP synthesis and its physiological roles in bacteria.     

Mycobacterium tuberculosis Rv3586 (DacA) is a diadenylate cyclase that converts ATP or ADP into c-di-AMP

Cyclic diguanosine monophosphate (c-di-GMP) and cyclic diadenosine monophosphate (c-di-AMP) are recently identified signaling molecules. c-di-GMP has been shown to play important roles in bacterial pathogenesis, whereas information about c-di-AMP remains very limited. Mycobacterium tuberculosis Rv3586 (DacA), which is an ortholog of Bacillus subtilis DisA, is a putative diadenylate cyclase. In this study, we determined the enzymatic activity of DacA in vitro using high-performance liquid chromatography (HPLC), mass spectrometry (MS) and thin layer chromatography (TLC). Our results showed that DacA was mainly a diadenylate cyclase, which resembles DisA. In addition, DacA also exhibited residual ATPase and ADPase in vitro. Among the potential substrates tested, DacA was able to utilize both ATP and ADP, but not AMP, pApA, c-di-AMP or GTP. By using gel filtration and analytical ultracentrifugation, we further demonstrated that DacA existed as an octamer, with the N-terminal domain contributing to tetramerization and the C-terminal domain providing additional dimerization. Both the N-terminal and the C-terminal domains were essential for the DacA's enzymatically active conformation. The diadenylate cyclase activity of DacA was dependent on divalent metal ions such as Mg(2+), Mn(2+) or Co(2+). DacA was more active at a basic pH rather than at an acidic pH. The conserved RHR motif in DacA was essential for interacting with ATP, and mutation of this motif to AAA completely abolished DacA's diadenylate cyclase activity. These results provide the molecular basis for designating DacA as a diadenylate cyclase. Our future studies will explore the biological function of this enzyme in M. tuberculosis.     

Plant Nucleotide Cyclases: An Increasingly Complex and Growing Family

Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, cGMP, has long been known to be critical to many different processes in higher plants while guanylyl cyclases (GCs), enzymes that catalyse the formation of cGMP from GTP have largely remained elusive. This is somewhat surprising considering that the unicellular green alga Chlamydomonas reinhardtii contains >90 annotated GCs. We have recently shown (PLoS ONE 2(5): e449) that a recombinant cytoplasmic domain of the Arabidopsis brassinosteroid receptor AtBRI has GC activity in vitro. This finding may suggest that other leucine-rich receptor kinases such as the phystosulfokine receptor may also confer GC activity as it has a high degree of similarity in the domain that has been delineated as essential for catalysis. In addition, the discovery of increasing complexities in the molecular architecture of higher plant nucleotide cyclases (NCs) is entirely compatible with findings in Chlamydomonas where such domains appear in >20 different combinations suggesting a role in highly diverse and complex signaling events.Key Words: nucleotide cyclase, guanylyl cyclase, cGMP, signal transduction, Arabidopsis thaliana, Chlamydomonas reinhardtii     

Highly efficient enzymatic preparation of c-di-AMP using the diadenylate cyclase DisA from Bacillus thuringiensis

Cyclic 3′,5′-diadenosine monophosphate (c-di-AMP) is a newly recognized bacterial nucleotide second messenger molecule. In addition, it has been shown to be a potential vaccine adjuvant. Although multiple methods are available for c-di-AMP synthesis, the yields are low and the purification procedures are laborious. Here, we report an enzymatic method for more efficient and economical c-di-AMP synthesis using a diadenylate cyclase DisA from Bacillus thuringiensis BMB 171 (btDisA). After overexpression and purification of btDisA, the enzyme-catalyzed reaction conditions were further investigated. Under the optimum conditions, in which 100 mM CHES (pH 9.5) containing 2 μM btDisA, 10 mM ATP, and 10 mM MgCl₂ was incubated at 50 °C for 4 h, a high conversion rate of c-di-AMP was obtained. Coupling this process with HPLC purification and lyophilization yielded 100 mg of highly pure c-di-AMP that was harvested in white powder form from a 50 mL enzyme-catalyzed reaction system. The protocol is not only directly applicable for preparing abundant amounts of c-di-AMP for extensive biochemical and immunological use, but can also be scaled up to meet the requirements for medical applications.     

Functional Analysis of a c-di-AMP-specific Phosphodiesterase MsPDE from Mycobacterium smegmatis

Cyclic di‑AMP (c-di-AMP) is a second signaling molecule involved in the regulation of bacterial physiological processes and interaction between pathogen and host. However, the regulatory network mediated by c-di-AMP in Mycobacterium remains obscure. In M. smegmatis, a diadenylate cyclase (DAC) was reported recently, but there is still no investigation on c-di-AMP phosphodiesterase (PDE). Here, we provide a systematic study on signaling mechanism of c-di-AMP PDE in M. smegmatis. Based on our enzymatic analysis, MsPDE (MSMEG_2630), which contained a DHH-DHHA1 domain, displayed a 200-fold higher hydrolytic efficiency (k_cat/K_m) to c-di-AMP than to c-di-GMP. MsPDE was capable of converting c-di-AMP to pApA and AMP, and hydrolyzing pApA to AMP. Site-directed mutations in DHH and DHHA1 revealed that DHH domain was critical for the phosphodiesterase activity. To explore the regulatory role of c-di-AMP in vivo, we constructed the mspde mutant (Δmspde) and found that deficiency of MsPDE significantly enhanced intracellular C₁₂-C₂₀ fatty acid accumulation. Deficiency of DAC in many bacteria results in cell death. However, we acquired the M. smegmatis strain with DAC gene disrupted (ΔmsdisA) by homologous recombination approach. Deletion of msdisA reduced bacterial C₁₂-C₂₀ fatty acids production but scarcely affected bacterial survival. We also provided evidences that superfluous c-di-AMP in M. smegmatis could lead to abnormal colonial morphology. Collectively, our results indicate that MsPDE is a functional c-di-AMP-specific phosphodiesterase both in vitro and in vivo. Our study also expands the regulatory network mediated by c-di-AMP in M. smegmatis.     

Cyclic Di-AMP Impairs Potassium Uptake Mediated by a Cyclic Di-AMP Binding Protein in Streptococcus pneumoniae

Cyclic di-AMP (c-di-AMP) has been shown to play important roles as a second messenger in bacterial physiology and infections. However, understanding of how the signal is transduced is still limited. Previously, we have characterized a diadenylate cyclase and two c-di-AMP phosphodiesterases in Streptococcus pneumoniae, a Gram-positive pathogen. In this study, we identified a c-di-AMP binding protein (CabP) in S. pneumoniae using c-di-AMP affinity chromatography. We demonstrated that CabP specifically bound c-di-AMP and that this interaction could not be interrupted by competition with other nucleotides, including ATP, cAMP, AMP, phosphoadenylyl adenosine (pApA), and cyclic di-GMP (c-di-GMP). By using a bacterial two-hybrid system and genetic mutagenesis, we showed that CabP directly interacted with a potassium transporter (SPD_0076) and that both proteins were required for pneumococcal growth in media with low concentrations of potassium. Interestingly, the interaction between CabP and SPD_0076 and the efficiency of potassium uptake were impaired by elevated c-di-AMP in pneumococci. These results establish a direct c-di-AMP-mediated signaling pathway that regulates pneumococcal potassium uptake.     

Complex Structure and Biochemical Characterization of the Staphylococcus aureus Cyclic Diadenylate Monophosphate (c-di-AMP)-binding Protein PstA,the Founding Member of a New Signal Transduction Protein Family

Signaling nucleotides are integral parts of signal transduction systems allowing bacteria to cope with and rapidly respond to changes in the environment. The Staphylococcus aureus PII-like signal transduction protein PstA was recently identified as a cyclic diadenylate monophosphate (c-di-AMP)-binding protein. Here, we present the crystal structures of the apo- and c-di-AMP-bound PstA protein, which is trimeric in solution as well as in the crystals. The structures combined with detailed bioinformatics analysis revealed that the protein belongs to a new family of proteins with a similar core fold but with distinct features to classical PII proteins, which usually function in nitrogen metabolism pathways in bacteria. The complex structure revealed three identical c-di-AMP-binding sites per trimer with each binding site at a monomer-monomer interface. Although distinctly different from other cyclic-di-nucleotide-binding sites, as the half-binding sites are not symmetrical, the complex structure also highlighted common features for c-di-AMP-binding sites. A comparison between the apo and complex structures revealed a series of conformational changes that result in the ordering of two anti-parallel β-strands that protrude from each monomer and allowed us to propose a mechanism on how the PstA protein functions as a signaling transduction protein.