Apo-structure of the S. aureus c-di-AMP cyclase DacA_CD

The S. aureus c-di-AMP cyclase enzyme DacA has three N-terminal transmembrane helices, which are followed by the cytoplasmically located enzymatic DisA_N domain. To gain further insight into the function and catalytic mechanism of the S. aureus c-di-AMP cyclase DacA, the soluble catalytic domain starting from amino acid F101 (hereafter referred to as DacA_CD) was expressed and purified as N-terminal His-tag fusion protein. Following removal of the His-tag, the protein was crystallized and the structure of the nucleotide-free DacA_CD domain was obtained by molecular replacement using the L. monocytogenes CdaA protein (PDB 4RV7) as a search model [35] (Fig 1 and S1 Table). The two proteins have an identify of 51.7% and the structures overlapped with a root-mean-square deviation (RMSD) of 0.438 Å. The S. aureus DacA_CD protein displayed the expected globular fold, with a central β-sheet made up of 7 antiparallel strands flanked by 5 helices (Fig 1A, left). While the L. monocytogenes CdaA_CD protein was found to be a tetramer in the asymmetric unit (although dimeric in solution) [35], the S. aureus DacA_CD was present as a symmetric dimer, with two protomers interacting via the backbone of α3, β2 and the loop connecting α3 and β3 (Fig 1A and 1B). At the interface, residue N166 from one protomer formed a hydrogen bond interaction with residue T172 from the second protomer and a van der Waals interaction was formed between residue P173 and residues N166 and I165 of the neighbouring protomer (Fig 1B). Further analysis using the PdbePISA program [39] revealed a total buried surface area of 1450 Ų at the dimer interface with a ΔG (int) of -8.3 kcal/mol and a ΔG (diss) of 2.1 kcal/mol, indicative of a strong protein/protein interaction.

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Fig 1. Crystal structure of the S. aureus DacA_CD protein in the nucleotide-free and nucleotide-bound states.

(A) Protein structure of the S. aureus DacA_CD protein with and without ApCpp, shown in ribbon representation. The apo S. aureus DacA_CD structure was solved by molecular replacement using the L. monocytogenes CdaA protein (PDB 4RV7) as template. The two monomers in the crystallographic unit are shown in teal and purple (for apo-DacA_CD) and in cyan and brown (for ApCpp-DacA_CD). The dimer is shown in two views with the second view in a 90° angle with the predicted active site residues D176, G177, T207 shown in stick representation in the 90° view (right). While the dacA sequence starting from codon F101 were included in the construct, only residues Y110 to G260 were visible in chain A and residues S111 to T261 in chain B. (B) Zoomed-in view of the S. aureus ApCpp-DacA_CD binding site (left) and DacA_CD dimerization interface (right). Electron densities corresponding to ApCpp and the metal ion are displayed. ApCpp and the DacA_CD interacting residues are shown in stick representation. Amino acid residues N166, T172 and P173 providing hydrogen bonding or van der Waals interactions are also shown in stick representation.

https://doi.org/10.1371/journal.ppat.1007537.g001

Structure of S. aureus DacA_CD bound to ApCpp

In order to condense two ATP molecules to c-di-AMP, two protomers need to be arranged in a head-to-head conformation [23, 35]. However, in the S. aureus DacA_CD dimer found in the crystallographic asymmetric unit, the active sites of the protomers are outward facing and hence are too far apart for c-di-AMP synthesis (Fig 1A). In addition, analysis of the crystal lattice did not reveal any symmetry-related molecule arranged with the catalytic sites in a head-to-head orientation. To determine whether nucleotide binding could trigger a conformational change of the protein leading to inter-protomer rearrangement, the S. aureus DacA_CD protein was crystallized in the presence of ApCpp, a non-hydrolyzable ATP substrate as well as 2 mM MnCl₂. Crystals were readily obtained and diffracted to 2.7Å (Fig 1 and S1 Table). The structure was solved by molecular replacement using the structure of the nucleotide-free S. aureus DacA_CD domain as a search model. The DacA_CD protein co-crystallized with ApCpp had the same dimeric arrangement as observed for the nucleotide-free protein, but extra densities for the ApCpp nucleotide were observed in each protomer (Fig 1). As expected, the ApCpp molecule was found in the catalytic pocket with residues D176 and G177 interacting with the ribose moiety, residue T207 interacting with the phosphate backbone and residue Y192 engaged in a π-stacking interaction with the adenine base (Fig 1B, left). The overall protein structures of the nucleotide-free and the nucleotide-bound proteins were very similar and overlapped with an RMSD of 0.34 Å (Fig 1A). Our data suggest that nucleotide binding does not trigger any major conformational changes leading to a rearrangement of the DacA_CD dimer. However, we did observe a near head-to-head configuration of DacA_CD by inspecting neighbouring molecules within the crystal lattice (S1A Fig). While the individual protomers in neighbouring units are too far apart to be engaged in a catalytic reaction, the data do suggest that the protein can form a catalytically active dimeric conformation by forming higher-order oligomers. Furthermore, overlaying the DacA_CD -ApCpp structure with a dimer of the B. subtilis DisA structure highlighted that there is sufficient space for two S. aureus DacA_CD dimers to interact in a head-to-head orientation as required for catalysis (S1B Fig).

Enzymatic activity and characterization of the S. aureus DacA_CD dimer interface

To determine whether the observed DacA_CD dimer could be physiologically relevant and enzymatically active, we next performed enzyme activity assays using different DacA_CD variants to test the relevance of the observed dimer interface. Two key interacting residues in the dimer interface are amino acids N166 and T172 (Fig 1B). To potentially disrupt this interaction, we mutated these two residues to lysines, yielding variant DacA_CD-K. To “lock” the dimer conformation, we also mutated these residues to cysteines (DacA_CD-C variant) to facilitate the formation of disulfide bonds between the two protomers. WT DacA_CD and the DacA_CD-K and DacA_CD-C variants were purified and the oligomeric state of the proteins was estimated by size exclusion chromatography. The WT DacA_CD and DacA_CD-C variant had similar elution profiles and eluted at a retention volume as expected for a DacA_CD dimer (S2 Fig). The main peak for the DacA_CD-K variant eluted with the same retention volume as the WT DacA_CD and DacA_CD-C variants indicating that the protein was still able to form a dimer. However, additional faster and slower eluting peaks were observed for the DacA_CD-K variant indicative of protein unfolding or aggregation (earlier eluting peak) and monomer formation (later eluting peak) (S2 Fig). These data indicate that introduction of lysine residues partially disrupts DacA_CD dimer formation. To further assess if the DacA_CD-C variant could form disulfide-bridge locked dimers, the purified protein was crystallized and its structure determined. The protein crystallized in the same dimeric conformation as the DacA_CD protein and electron densities consistent with the formation of two disulfide bonds were observed for the DacA_CD-C variant, which were absent in the DacA_CD protein (Fig 2 and S1 Table).

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Fig 2. Structure of the DacA_CD-C variant.

(A) X-ray structure of the DacA_CD-C variant. The structure of the tag-less DacA_CD-C variant was solved by molecular replacement using the WT S. aureus DacA_CD protein shown in Fig 1 as template. The DacA_CD-C protein was found as a dimer in the crystallographic unit. (B) Electron density of the dimer interface in DacA_CD-C, showing electron densities consistent with the formation of two disulfide bonds between two DacA_CD-C monomers at sigma 1.3.

https://doi.org/10.1371/journal.ppat.1007537.g002

Next, the purified proteins were tested for enzyme activity by quantifying the conversion of α-P³²-labelled ATP into P³²-labelled c-di-AMP. First, the metal dependency of the WT DacA_CD protein was assessed. To this end, DacA_CD was incubated with ATP spiked with a small amount of α-P³²-labelled ATP in buffers containing 1 or 10 mM of one of the divalent cations Mg²⁺, Co²⁺ or Mn²⁺. After 4 h the reactions were stopped and the input ATP and c-di-AMP reaction product quantified by phosphorimaging. This analysis revealed that the S. aureus DacA_CD protein is most active in the presence of Mn²⁺ and hence all subsequent experiments were performed in the presence of 10 mM MnCl₂ (Fig 3A). Next, a time course experiment was performed and the DacA_CD enzyme displayed an in vitro catalytic activity with an average catalytic rate of 2.28x10^-10 M/min (Fig 3B). This activity is slow, but consistent with previous observations [23]. To assess the activity of the different DacA_CD variants, enzyme activity assays were performed with the DacA_CD, DacA_CD-C, or DacA_CD-K (either obtained from the predicted monomer or dimer peak) proteins. The DacA_CD-C variant displayed very similar activity to the WT DacA_CD protein (Fig 3C), suggesting that locking the dimer through two disulfide bridges does not influence the activity of the enzyme. In contrast, the DacA_CD-K variant derived either from the late eluting (predicted monomer) or earlier eluting (predicted dimer) peaks displayed very low activity (Fig 3C). We reasoned that the activity loss could be due to an intrinsic instability of this protein variant. To test this, the stability of the DacA_CD-K variant was assessed using a thermofluor assay and compared to that of the WT DacA_CD and DacA_CD-C variant. The melting curves for the DacA_CD and DacA_CD-C proteins were sigmoidal, which is typical for well-folded proteins (Fig 3D). On the other hand, the DacA_CD-K variant displayed high levels of fluorescence from the beginning of the melting curve (Fig 3D), which is indicative of an unfolded protein and likely explains the low enzymatic activity of the DacA_CD-K variant. Taken together, these data show that WT DacA_CD and the ‘locked-dimer’ DacA_CD-C variant show similar enzymatic activities, suggesting that the stable dimer we observed in our crystal structures will likely transiently interact with other dimers to form higher oligomers for catalysis and c-di-AMP product formation.

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Fig 3. Activity assays of DacA_CD and its variants.

(A) Enzyme activity assay and metal-dependency of the WT DacA_CD protein. 5 μM DacA_CD was incubated with 100 μM M ATP spiked with a small amount of radiolabeled ATP in buffer containing 1 mM or 10 mM of Mg²⁺, Co²⁺ or Mn²⁺ metal ions. The reactions were incubated for 4 h at 37°C and % ATP to c-di-AMP conversion determined. The average values and standard deviations from three independent experiments were plotted. (B) DacA_CD cyclase activity time-course experiment. 5 μM DacA_CD was incubated with 100 μM ATP spiked with radiolabeled ATP in buffer containing 10 mM Mn²⁺ and the reactions were stopped at the indicated time points. The % ATP that was converted to c-di-AMP was determined and the average values and standard deviations from three independent experiments plotted. (C) Cyclase activity of WT DacA_CD, DacA_CD-C and DacA_CD-K variants. The indicated protein was incubated with ATP for 4 h as described in panel B and the average values of % ATP conversion and standard deviations from three independent experiments were plotted. (D) Thermofluor melting curves. 5 μM of the indicated protein was incubated with SYPRO Orange dye, the solution was heated from 25 to 95°C in 1°C increments and the fluorescence readings determined. The melting curve for WT DacA_CD is shown in black, for the DacA_CD-C variant in red, for the DacA_CD-K variant derived from the presumed dimer peak in blue and the DacA_CD-K variant derived from the presumed monomer peak in green.

https://doi.org/10.1371/journal.ppat.1007537.g003

S. aureus DacA_CD and GlmM form a complex in vitro

Results from in vivo crosslinking and bacterial two-hybrid experiments suggested that the phosphoglucosamine mutase GlmM can interact with the DacA homologs in B. subtilis and L. lactis [17, 18]. To assess whether the S. aureus DacA_CD protein can interact with the S. aureus GlmM protein, a plasmid for the overexpression of a C-terminally His-tagged S. aureus GlmM protein (GlmM-His) was generated. Next, expression of the His-DacA_CD and GlmM-His proteins was induced in E. coli, the cells lysed and the proteins purified by Ni-affinity and size exclusion chromatography, or E. coli lysates from His-DacA_CD and GlmM-His overexpressing strains mixed before affinity and size exclusion chromatography. When the two proteins were purified together, a new faster eluting peak corresponding to a species of higher molecular weight and indicating the formation of a complex, was obtained (Fig 4A). SDS-PAGE analysis of aliquots from the different elution fractions confirmed the co-elution of the His-DacA_CD and GlmM-His proteins in the high molecular weight peak (Fig 4A). To further confirm the interaction of DacA_CD with GlmM in vitro, a pull-down assay was performed. For this purpose, the His-tag was removed from DacA_CD and the purified tag-less protein subsequently mixed in equimolar amount with purified GlmM-His. The protein mixture was then applied to a Ni-NTA column and after two wash steps, bound proteins were eluted. The untagged DacA_CD protein quantitatively co-eluted with GlmM-His, confirming the protein/protein interaction (S3 Fig). As control, the tag-less DacA_CD protein was subjected to the same procedure in the absence of GlmM-His. In this case, the protein was found in the load and wash fractions. Taken together, these results show that DacA and GlmM can form a stable complex in vitro, and that the two proteins can be co-purified as a single species. To estimate the size and stoichiometry of the complex, the purified DacA_CD-GlmM complex was further analyzed by SEC-MALS as well as native mass spectrometry. Based on the SEC-MALS elution profile, the complex had an estimated molecular weight of 129 kDa ± 0.4% (S4 Fig) and based on the native mass spectrometry analysis, the major species in the spectrum corresponded to a complex of 139.003 kDa ± 10.41 Da (Fig 4B). These data are consistent with a DacA_CD-GlmM complex comprising one GlmM dimer (theoretical mass 99.462 kDa) interacting with one DacA_CD dimer (theoretical mass 19.374 kDa), with a theoretical molecular weight of 138.21 kDa (S2 Table).

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Fig 4. The S. aureus DacA_CD and GlmM proteins form a stable complex in vitro.

(A) Size exclusion profiles of the His-DacA_CD (blue line), GlmM-His (red line) proteins and the His-DacA_CD/GlmM-His complex (black line). The insert shows a Coomassie-stained gel with aliquots of fractions from the complex peak. The experiment was performed three times and a representative result is shown. (B) Native mass-spectrometry experiment of the DacA/GlmM complex. The spectrum was deconvoluted using the software Amphitrite [53]. Experimental and calculated spectra are shown in black and red, respectively. Charge state distributions corresponding to the different detected species are depicted in different colours. The molecular weights reported correspond to: DacA/GlmM dimer complex (purple line, 277.896 kDa, four copies of DacA_CD with four copies of GlmM); DacA/GlmM complex (brown line, 139.003 kDa, two copies of DacA_CD with two copies of GlmM–main DacA/GlmM complex); GlmM dimer (green line, 99.983 kDa); GlmM monomer (purple line—50.302 kDa); DacA_CD dimer (blue line—39.232 kDa); DacA_CD monomer (grey line—19.374 kDa) (S2 Table).

https://doi.org/10.1371/journal.ppat.1007537.g004

S. aureus GlmM is a negative regulator of DacA activity both in vitro and in vivo

A study with L. lactis indicated that GlmM might inhibit the activity of DacA upon binding [17]. To investigate this further, c-di-AMP production by the S. aureus DacA_CD enzyme was assessed in the absence or presence of GlmM. DacA_CD was incubated alone or mixed at a 1:2 molar ratio with GlmM, and conversion of ATP to c-di-AMP assessed after 4 h incubation at 37°C. No ATP conversion was observed in the presence of GlmM (Fig 5A). In a second experiment, a 2-fold molar excess of GlmM was added to a DacA_CD reaction, 30 min after initiating the reaction, and the sample subsequently incubated for a further 150 min (180 min total reaction time). As controls, 30 and 180 min DacA_CD enzyme reactions were also set up in the absence of GlmM. When GlmM was added 30 min following the initiation of the DacA_CD enzymatic cycle, the production of c-di-AMP was arrested (Fig 5B). Taken together, these data show that GlmM can effectively block the activity of the DacA_CD cyclase domain in vitro without the need for any additional cofactors.

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Fig 5. S. aureus GlmM negatively impacts the activity of DacA_CD in vitro and in vivo.

(A) DacA_CD enzyme activity in the presence of GlmM. Enzyme assays were set up as described in Fig 3 in the absence or presence of 10 μM GlmM added at the start of the reactions and reactions stopped after 4 h incubation. (B) Enzyme assays were set up with 5 μM DacA_CD and 10 μM GlmM added after 30 min incubation and the reactions incubated for further 150 min (180 min total). As a control, DacA_CD enzyme reactions were also set up in the absence of GlmM and incubated for 30 or 180 min. The average values of % ATP to c-di-AMP conversion and standard deviations from three independent experiments are plotted. (C) ELISA determination of c-di-AMP levels in E. coli. Cell extracts were prepared from E. coli strains containing the empty pBAD33 vector or producing DacA, DacA/YbbR, DacA/YbbR/GlmM or DacA/GlmM. The cellular c-di-AMP levels were determined by ELISA and the average values (μM c-di-AMP per ml E. coli culture with an A₆₀₀ of 10) and standard deviations of three independent experiments plotted.

https://doi.org/10.1371/journal.ppat.1007537.g005

To investigate this further and confirm that GlmM can also inhibit the activity of the full-length membrane-anchored DacA enzyme, full-length DacA was expressed in E. coli either alone, in the presence of YbbR or GlmM or in the presence of both, GlmM and YbbR, and c-di-AMP production assessed by ELISA. To this end, dacA, dacA-ybbR or the complete dacA-ybbR-glmM operon was cloned into plasmid pBAD33, allowing for arabinose inducible DacA, DacA/YbbR or DacA/YbbR/GlmM expression. For expression of DacA and GlmM only, the YbbR start codon was mutated and the dacA-no-ybbR-glmM operon inserted into plasmid pBAD33. Next, DacA and YbbR production from the different constructs was confirmed by western blot using protein-specific antibodies and GlmM production was assessed by visualizing proteins by Coomassie staining. With the exception of the empty vector control strain, similar DacA amounts were produced in all strains regardless whether or not YbbR, GlmM or YbbR and GlmM were co-expressed with the cyclase (S5 Fig). A clearly visible Coomassie stainable band of the expected size for GlmM was observed for the two strains overproducing GlmM (S5 Fig). YbbR production was also observed in the E. coli strain containing plasmids pBAD33-dacA-ybbR or pBAD33-dacA-ybbR-glmM. Mutating the YbbR start codon drastically reduced the production of YbbR but did not completely abolish its production, indicating that a second internal start codon might be utilized (S5 Fig). Next, the c-di-AMP production was assessed in the different strains. As expected, no c-di-AMP could be detected in E. coli containing the empty vector (Fig 5C). High amounts of c-di-AMP were detected upon DacA expression or expression of DacA/YbbR. On the other hand, c-di-AMP levels were drastically reduced upon co-expression of GlmM, which is consistent with the data reported by Zhu et al. [17] (Fig 5C). Taken together, these data show that GlmM is a negative regulator of the S. aureus c-di-AMP cyclase DacA both in vivo and in vitro. Of note, when GlmM activity was assessed in vitro in the presence of DacA_CD using a previously reported coupled enzyme assay [40], no significant enzyme inhibition was observed. The specific activity of the pure GlmM enzyme was estimated at 15.9 ± 3.1 μmol/min/mg of protein in the assay conditions used. Addition of DacA in a 5- to 50-fold excess over GlmM did not at all inhibit the phosphoglucosamine mutase activity and its presence in 400-fold excess only led to a slight 15% reduction of GlmM activity. Taken together, these data indicate that only GlmM can impact the activity of the c-di-AMP cyclase enzyme but likely not vice versa.

GlmM of Gram-negative bacteria do not regulate or interact with the S. aureus DacA protein

Next, we tested if GlmM proteins from unrelated Gram-negative bacteria can also interact and influence the activity of the S. aureus DacA protein. To this end, we overexpressed the S. aureus DacA protein from pBAD33-dacA along with C-terminally His-tagged versions of the E. coli (EC), Pseudomonas aeruginosa (PA) and as control S. aureus (SA) GlmM proteins in E. coli and measured c-di-AMP levels by ELISA (Fig 6). As expected, expression of the S. aureus GlmM-His protein (SA) blocked c-di-AMP production, while c-di-AMP levels were not reduced upon expression of the E. coli or P. aeruginosa GlmM proteins (Fig 6B). However, when we analyzed the GlmM protein levels by western-blot following induction with 1 mM IPTG, we noted that the S. aureus proteins was expressed at significant higher levels as compared to the other two GlmM proteins (Fig 6A). Therefore, we repeated the experiment using 10 mM IPTG for the induction of the E. coli and P. aeruginosa GlmM proteins but no (0 mM) or very low levels (0.001 or 0.0001 mM) of IPTG for the induction of the S. aureus GlmM protein. In this case, similar GlmM amounts were observed for all strains (Fig 6C). Again, only expression of the S. aureus GlmM protein (be it to a lesser extent) but not expression of the GlmM proteins from the Gram-negative bacteria led to a reduction in the cellular c-di-AMP levels (Fig 6D). Next, we investigated the interaction of the S. aureus DacA and P. aeruginosa GlmM proteins in vitro. The expression of the S. aureus His-DacA_CD and P. aeruginosa GlmM-His proteins were induced in E. coli, the cells lysed and the individual proteins purified by Ni-affinity followed by size exclusion chromatography. In addition, the lysates from the S. aureus His-DacA_CD and P. aeruginosa GlmM-His overexpressing strains were mixed before affinity and size exclusion chromatography. In contrast to the observation with the S. aureus GlmM protein (Fig 4A), the P. aeruginosa GlmM-His protein did not coelute with the S. aureus His-DacA_CD (Fig 6E). Taken together, these data highlight a specificity in the interaction between the S. aureus DacA and GlmM proteins, as no interaction was detected between the S. aureus DacA protein and the GlmM proteins from the Gram-negative bacteria E. coli and P. aeruginosa.

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Fig 6. GlmM proteins from E. coli or P. aeruginosa do not interact or impact the activity of the S. aureus DacA enzyme.

(A and C) Detection of DacA and GlmM-His by western blot. Protein samples were prepared from E. coli XL1-Blue pBAD33-dacA containing either an empty pTrcHis60 vector (-) or a plasmid for expression of the S. aureus GlmM-His (SA), the P. aeruginosa GlmM-His (PA) or the E. coli GlmM-His (EC). DacA or the GlmM-His proteins were detected by western-blot using an anti-DacA or anti-His antibody as indicated below the respective panel. DacA protein expression was induced with 0.2% arabinose and GlmM protein expression with 1 mM IPTG in (A) or with the amount of IPTG as indicated in (C). (B and D). ELISA determination of c-di-AMP levels in E. coli. Cell extracts were prepared from the E. coli strains used in panels A and C. The cellular c-di-AMP levels in μM per ml E. coli culture with an A₆₀₀ of 10 were determined by ELISA and the average values and standard deviations of three independent experiments plotted. (E) Coomassie stained gels. Aliquots from the size exclusion fractions of the complex (top), the P. aeruginosa GlmM_PA-His (middle) or the S. aureus His-DacA_SA (bottom) were separated on 12% PAA gels and proteins visualized by Coomassie staining. The experiment was performed twice, and a representative result is shown. GlmM and DacA proteins are indicated with an asterisk and a square, respectively in panels A, C and E.

https://doi.org/10.1371/journal.ppat.1007537.g006

S. aureus GlmM protein structure and small-angle X-ray scattering analysis of the DacA_CD/GlmM complex

In order to gain structural information on the S. aureus DacA_CD/GlmM complex, we first set out to determine the structure of the S. aureus GlmM protein in isolation. To this end, the C-terminal His-tagged S. aureus GlmM protein was purified, the His-tag was removed, and the purified protein crystallized in the presence of Mg²⁺ and glucosamine-6-P. The structure of GlmM was obtained by molecular replacement using the GlmM protein structure from Bacillus anthracis (PDB 3PDK) as a search model [41] (Fig 7 and S1 Table). While the protein was crystallized in the presence of metal ion and substrate, no corresponding extra electron density was observed. The S. aureus GlmM protein and the B. anthracis protein, which share 67% identity on a protein level, displayed a similar fold and dimeric arrangement and the structures overlapped with an RMSD of 0.996 Å. One GlmM monomer consisted of four α-β domains with the fourth most C-terminal domain linked by a flexible loop (Fig 7). Next, we attempted to crystallize and solve the structure of the S. aureus DacA_CD/GlmM complex. While crystals were obtained under several conditions, poor diffraction prevented the structural determination of the complex. In order to gain structural information of the DacA/GlmM complex, the single proteins and complex were subjected to small-angle X-ray scattering (SAXS) analysis (Fig 8, S6 and S7 Figs and S3 Table). The reconstructed model for the GlmM sample showed an elongated particle, consistent with the dimeric arrangement found in the crystal structure (Fig 8B) and with previously published findings [42]. In the case of DacA, the model was most consistent with the particle being a DacA_CD dimer, however an extra density was observed to one extremity (Fig 8A), which we speculate is due to the presence of extra N-terminal amino acids, not visible in the X-ray structure, and hence appear as a flexible and unstructured region of the protein. The reconstructed envelope of the complex displays a bigger particle size as compared to GlmM dimer alone, consistent with a DacA_CD dimer interacting with a GlmM dimer (Fig 8C). A sequential fitting of the GlmM dimer structure followed by the fitting of the DacA dimer structure into the SAXS envelope of the complex with Chimera [43] yielded the best fit for a model with a correlation score of 0.9196 in which a DacA dimer is positioned on top of a GlmM dimer (Fig 8C). In this model, the active sites of DacA protomers are occluded through binding to the GlmM protein (Fig 8D), while the GlmM active sites are still accessible. To validate the SAXS analysis, the reconstructed envelopes were used to calculate theoretical Collision Cross Section (CCS) values using the program EM∩IM [44]. These theoretical values were subsequently compared to those experimentally determined by Ion-Mobility Mass Spectrometry. The theoretical values agree with the CCS determination (S4 Table), showing that the structural models of DacA, GlmM and their complex obtained by SAXS correspond to the shapes of the proteins in solution. Taken together, our model provides an explanation as to how the interaction of DacA with GlmM inhibits c-di-AMP production without affecting the activity of the GlmM enzyme.

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Fig 7. S. aureus GlmM structure.

Structure of the S. aureus GlmM protein shown in ribbon representation. The protein was crystallized in the presence of GlcN-6P and MgCl₂ but no densities for the metal ion or substrate were present in the structure. The structure was solved by molecular replacement using the B. anthracis GlmM protein (PDB 3PDK) as template. The protein crystallized as dimer and the two different monomers are shown in orange and purple. The dimer is shown in two views with the second view rotated by 90°.

https://doi.org/10.1371/journal.ppat.1007537.g007

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Fig 8. Small Angle X-ray Scattering (SAXS) data for S. aureus proteins DacA_CD, GlmM and the DacA_CD/GlmM complex.

Reconstructed envelopes of the purified DacA_CD (A), GlmM (B) and DacA_CD/GlmM complex (C). DacA_CD (12 mg/ml), GlmM (8 mg/ml) and the DacA_CD/GlmM complex (12 mg/ml) were injected onto a Superdex 200 5/150 column coupled to a Small-Angle X-Ray beam. All data were analyzed and envelopes reconstructed using ScÅtter. Conversion of envelopes to maps and subsequent structure fitting were performed using Chimera. (D) Zoomed in views of the DacA_CD active sites in the DacA_CD/GlmM SAXS model. Catalytic residues on both DacA_CD protomers are shown in stick representations. The interaction of GlmM with DacA_CD covers the active sites, thus preventing DacA_CD dimers from interacting and forming catalytically active species.

https://doi.org/10.1371/journal.ppat.1007537.g008

Bacterial strains and plasmid construction

All strains used in this study are listed in S5 Table and primers used for plasmid construction are listed in S6 Table. E. coli strains were grown in Lysogenic broth (LB) supplemented when appropriate with the antibiotics indicated in S5 Table. Protein expression was induced with 0.5 mM IPTG unless specified otherwise in the text or 0.2% arabinose. Plasmids for the expression of the S. aureus DacA and GlmM proteins were constructed as follows: The DNA fragments coding for the S. aureus DacA catalytic domain starting from amino acid residue F101 (referred to as DacA_CD domain) and GlmM were amplified by a PCR using LAC* genomic DNA as template and primer pairs ANG1135/ANG1137 (for DacA_CD) or ANG2342/ANG2343 (for GlmM). The primers for cloning glmM contained DNA sequences for appending a C-terminal 6-His tag proceeded by a thrombin cleavage site. The PCR products were digested with NheI/EcoRI (for DacA_CD) or NcoI/XhoI (for GlmM-His) and then ligated with plasmid pET28b, which has been digested with the same enzymes. The resulting plasmids pET28b-His-dacA_CD and pET28b-glmM-His were initially recovered in E. coli XL1-Blue yielding strains ANG1858 and ANG3994 and subsequently introduced into E. coli BL21 (DE3) yielding the protein overexpression strains ANG1865 and ANG3997, respectively. Plasmids pET28b-His-dacA_CD-K and pET28b-His-dacA_CD-C for the expression of the DacA_CD variants with amino acid residues N166 and T172 replaced by lysines (DacA_CD-K variant) or cysteines (DacA_CD-C variant) were constructed by Splicing Overhang Extensions (SOE) PCR. Primer pairs ANG1135/ANG2616 and ANG2617/ANG1137 (DacA_CD-K variant) or ANG1135/ANG2618 and ANG2619/ANG1137 (DacA_CD-C variant) were used for the first PCR. Next, the respective PCR products were fused in a second PCR using primer ANG1135 and ANG1137. The resulting products were digested with NheI/EcoRI and ligated with pET28b that had been cut with the same enzymes. The resulting plasmids pET28b-His-dacA_CD-K and pET28b-His-dacA_CD-C were initially recovered in E. coli XL1-Blue giving strains ANG4319 and ANG4321 and subsequently introduced in E. coli BL21 (DE3) yielding strains ANG4320 and ANG4322. For construction of plasmids pBAD33-dacA, pBAD33-dacA-ybbR and pBAD33-dacA-ybbR-glmM the dacA, dacA-ybbR and dacA-ybbR-glmM regions were amplified by PCR from S. aureus LAC* chromosomal DNA using primer pairs ANG928/ANG2475, ANG928/ANG2476 and ANG928/ANG2477, respectively. The PCR products were cut with KpnI and HindIII and ligated with plasmid pBAD33, which had been cut with the same enzymes. The resulting plasmids pBAD33-dacA, pBAD33-dacA-ybbR and pBAD33-dacA-ybbR-glmM were recovered in E. coli XL1-Blue yielding strains ANG4120, ANG4121 and ANG4122. For construction of plasmid pBAD33-dacA-no-ybbR-glmM (strain ANG4263), the YbbR start codon was mutated by SOE PCR. To this end, the front and back parts of the dacA-no-ybbR-glmM insert were amplified using plasmid pBAD33-dacA-ybbR-glmM as template and primer pairs ANG1244/ANG2597 and ANG2598/ANG1245, respectively. The PCR fragments were fusing in a second PCR using primer pair ANG1244/ANG1245. The resulting product was digested with KpnI and HindIII and cloned into plasmid pBAD33 cut with the same enzymes. The sequences of inserts were verified by fluorescent automated sequencing at GATC Biotech. For the overexpression of the S. aureus DacA enzyme and His-tagged GlmM proteins from different bacteria, the empty control plasmid pTrcHis60, as well as plasmids pMLJ4 (GlmM-His E. coli), pMLJ11 (GlmM-His S. aureus) or pMLD137 (GlmM-His P. aeruginosa) were introduced into E. coli strain XL1-Blue pBAD33-dacA, yielding strains ANG5174, ANG5175, ANG5177 and ANG5178, respectively.

Protein expression, purification, and histidine-tag cleavage

One to two litre cultures of E. coli strains BL21 (DE3) containing plasmids pET28b-His-dacA_CD (ANG1865), pET28b-His-dacA_CD-K (ANG4320), pET28b-His-dacA_CD-C (ANG4322) or pET28b-glmM-His (ANG3997) were grown in LB medium supplemented with kanamycin (30 μg/ml) at 37°C with agitation. Once the cultures reached an A₆₀₀ of approximately 0.5–0.6, protein expression was induced with 0.5 mM IPTG and the cultures were subsequently incubated for a further 3 hours at 37°C. Cells were harvested by centrifugation, suspended in 20 ml of 50 mM Tris pH 7.5, 500 mM NaCl buffer and lysed by passing the cell suspensions twice through a French press cell at 1100 psi. For the purification of the His-DacA_CD/GlmM-His complex, the appropriate cell lysates were mixed after lysis and incubated on ice for 10 minutes. Next, the lysates were clarified by centrifugation at 20,000 × g for 40 min and then loaded by gravity flow onto columns containing 4 ml Ni-NTA resin (Qiagen) equilibrated prior to use with 50 mM Tris pH 7.5, 500 mM NaCl buffer. The columns were washed with 30 ml of 50 mM Tris pH 7.5, 500 mM NaCl, then 30 ml of 50 mM Tris pH 7.5, 500 mM NaCl, 50 mM imidazole buffer and the proteins eluted in 5 × 1 ml fractions with 50 mM Tris pH 7.5, 200 mM NaCl, 500 mM imidazole buffer. Elution fractions containing the purified protein or protein complex were pooled and then loaded onto a Superdex 200 10/60 Hiload column (GE Healthcare), equilibrated with 30 mM Tris pH 7.5, 150 mM NaCl buffer. When the GlmM-His protein was purified alone, DTT was added to the purified protein at a final concentration of 1 mM after the size-exclusion chromatography step to maintain the protein in a soluble state. To cleave the His-tag from proteins, 20 U thrombin (Sigma) was added per 10 mg protein after the Ni-NTA column purification step and the mixture was incubated overnight at 4°C with agitation. The tag-less proteins were subsequently purified by size exclusion chromatography as described above. The purified proteins or complex were concentrated using 10 kDa cutoff Amicon centricons (Millipore). Protein concentration was measured either by determining the absorbance at 280 nm or by the Bradford method. For the investigation of the interaction between the S. aureus His-DacA_CD enzyme and the P. aurugniosa GlmM-His, one litre cultures of E. coli strain BL21 (DE3) containing plasmid pET28b-His-dacA_CD (ANG1865) was grown in LB medium supplemented with kanamycin (30 μg/ml) at 37°C with agitation. Once the culture reached an A₆₀₀ of 0.5, protein expression was induced with 0.5 mM IPTG and the culture was subsequently incubated O/N at 16°C. Strain XL1-Blue pMLD137 (GlmM-His P. aeruginosa) was grown to an A₆₀₀ of 0.5 and protein expression was induced with 0.5 mM IPTG and the culture subsequently incubated for an additional 4 hours at 37°C. Cells were harvested by centrifugation, suspended in 20 ml of 50 mM Tris pH 7.5, 500 mM NaCl buffer supplemented with 0.1% v/v ß-mercaptoethanol and complete EDTA free protease inhibitor (Roche) and lysed by sonication for 2 min at 15 sec on and 30 sec off intervals at 60% amplitude. The proteins and complex were purified by nickel affinity chromatography as described above but using only 2 ml of Ni-NTA resin and eluting the proteins in 2.5 ml of 50 mM Tris pH 7.5, 200 mM NaCl, 500 mM imidazole buffer supplemented with 0.1% v/v ß-mercaptoethanol. Next, 500 μl of the eluate was separate on a Superdex 200 10/300GL column (GE Healthcare) using 30 mM Tris pH 7.5, 150 mM NaCl as running buffer. 0.5 ml fractions were collected, and aliquots of the indicated elution fractions separated on 12% PAA gels and proteins visualized by Coomassie staining.

SEC-MALS and native mass spectrometry analysis of the DacA_CD/GlmM protein complex

100 μl of the purified, tag-less DacA_CD/GlmM protein complex at 18 mg/ml was loaded onto a Superdex 200 Increase 10/300 column (GE Healthcare) pre-equilibrated with 30 mM Tris pH 7.5, 150 mM NaCl buffer and run at 0.5 ml/min in the same buffer. The column was coupled to a MALS detector and refractometer and the UV absorbance, laser scattering and refractive index change were monitored throughout the run. The data were analysed using the ASTRA 6.0 software and fitted according to the Zimm model for static light scattering. The experiment was performed in duplicate and a representative graph is shown. Prior to the native mass spectrometry experiment, the tag-less DacA_CD/GlmM was loaded onto a Superdex200 10/300 column (GE Healthcare) pre-equilibrated with 200 mM ammonium acetate pH 7.5. Samples were further buffer exchanged with Amicon Ultra centrifugal filtration units (Merck Millipore) using 200 mM Ammonium Acetate (Fisher Scientific). Samples were diluted to 13.5 μM. The samples were directly introduced into a first generation Waters Synapt QToF (Waters Corporation, UK) using nanoelectrospray gold-coated borosilicate glass capillaries prepared in-house [51]. Mass calibration was performed using 30 mg/mL Caesium Iodide (Fluka) solution. Machine parameters used were: capillary 1.4 kV, sampling cone 40 V, extraction cone 4 V, backing pressure 6.20 mbar, trap CE 15 eV, transfer CE 13 eV, bias 16 V, source wave velocity 300 ms^-1, source wave height 0.2 V, trap wave velocity 300 ms^-1, trap wave height 0.2 V, IMS wave velocity 260 ms^-1, IMS wave height 8.0 V, transfer wave velocity 260 ms^-1, transfer wave height 8.0 V. For collision cross section calculation calibrants were sprayed from 200mM Ammonium Acetate solution. The calibrants used were Cytochrome C (Calbiochem), β-Lactoglobulin (Sigma Aldrich), Bovine Serum Albumin (Sigma Aldrich), Concanavalin A (Sigma Aldrich), Serum P Albumin (Calbiochem) and Pyruvate Kinase (Sigma Aldrich). The CCS calibration curve was produced using the method described by Thalassinos et al. [52], with a calculated R² value of 0.9601. Spectra were analyzed using MassLynx v4.1 (Waters Corporation, UK) and the program Amphitrite [53].

Protein crystallization, structure solution and analysis

The tag-less, nucleotide-free DacA_CD protein and tag-less DacA_CD-C variant were crystallized in 200 mM LiCl₂, 100 mM Na-cacodylate pH 6.5, 30% PEG400, using a protein concentration of 7.5 mg/ml. To obtain the ApCpp-bound DacA_CD complex structure, the tag-less DacA_CD protein at a concentration of 7.5 mg/ml was incubated for 30 min on ice with 2 mM ApCpp (Jena Bioscience) and 2 mM MnCl₂ and the protein subsequently crystallized in 10 mM MgCl₂, 50 mM MES pH 5.8, 0.2M KCl and 3% PEG8000 buffer. The tag-less GlmM protein was crystallized in the presence of 2 mM MgCl₂ and 2 mM GlcN-6P (Sigma) in 2.0 M sodium malonate buffer. All proteins were crystallized by the vapour diffusion method. DacA_CD, DacA_CD-C and GlmM crystals were frozen in liquid nitrogen without the addition of further cryo-protectants and datasets collected at the I03 Beamline at the Diamond Light Source (Harwell Campus, Didcot, UK). Data integration was performed with DIALS [54], the reflections were scaled and merged with Aimless [55] and intensities were converted to structure factors using Ctruncate [56]. Analysis of the Matthews coefficient revealed that two protein molecules were present in the asymmetric unit for all proteins. The structures were solved by the molecular replacement method with Phaser [57] using the L. monocytogenes CdaA protein (PDB 4RV7) as a template for DacA_CD or the B. anthracis GlmM protein (PDB 3PDK) as a template for the S. aureus GlmM protein. Structure refinement was performed with Phenix [58] and model building with Coot [59]. Structure figures were created with Pymol and Chimera [43, 60].

Affinity pull-down experiments

For the pull-down experiment, 50 μM tag-less DacA_CD and 50 μM GlmM-His protein were mixed in 30 mM Tris pH 7.5, 150 mM NaCl buffer and incubated on ice for 10 min. Next, the protein mixture was applied by gravity flow to a column containing 1 ml of Ni-NTA equilibrated with 30 mM Tris pH 7.5, 150 mM NaCl buffer. As a control, 500 μl of a 50 μM tag-less DacA protein solution in 30 mM Tris pH 7.5, 150 mM NaCl were also applied by gravity flow onto a column. The flow-through fractions were collected for subsequent SDS-PAGE analysis. The columns were then washed with 15 ml of 30 mM Tris pH 7.5, 150 mM NaCl, 10 mM imidazole buffer and the wash fraction collected and proteins finally eluted with 2 ml 30 mM Tris pH 7.5, 150 mM NaCl, 500 mM imidazole buffer. Aliquots of the load, flow through, wash and elution fractions were run on a 12% SDS-PAGE gel and proteins visualized by Coomassie staining. The DacA_CD/GlmM-His pulldown experiment was performed in triplicate and a representative result is shown; the control experiment using the DacA_CD protein alone was performed once.

DacA_CD enzyme activity assay

To assess the metal dependency of the S. aureus DacA_CD enzyme, enzyme reactions were set up as follows: 2 μl of 10X reaction buffer (400 mM Hepes pH 7, 1 M NaCl, 100 mM or 10 mM of MgCl₂, CoCl₂ or MnCl₂) was mixed on ice with 13.6 μl of water and 2 μl of 1 mM ATP. Next, 2 μl of DacA_CD from a 50 μM stock solution and 0.4 μl of α-P³²-labelled ATP (Perkin Elmer—3.3 μM, 250 μCi) were then added to the sample yielding a final reaction volume of 20 μl. The reactions were incubated at 37°C for 4 h. Reactions were stopped by heating for 5 min at 95°C and the conversion of ATP to c-di-AMP assessed by spotting 1 μl of each reaction onto a TLC plate (Millipore). The nucleotides were separated using a 3.52 M (NH₄)₂SO₄ and 1.5 M KH₂PO₄ (mixed in a 1:1.5 v/v ratio) buffer system, the TLC plate were subsequently dried and the radioactive signal detected using a Typhoon FLA 700 imager. The bands corresponding to radioactive ATP and c-di-AMP were quantified using the ImageQuant software and the % ATP to c-di-AMP conversion calculated. As the highest enzyme activity was seen in the presence of MnCl₂, all subsequent enzyme reactions were set up in reaction buffer containing a final concentration of 10 mM MnCl₂. For the time course experiment, 100-μl reactions were set and 10-μl aliquots removed at the indicated time point and stopped by heating. The activity of the DacA_CD-K and DacA_CD-C variants was assessed by setting up 20-μl reactions as described above using a final protein concentration of 5 μM and incubating the reactions for 4 h at 37°C. To analyze the ATP conversion of DacA_CD when bound to GlmM, 2 μl of GlmM from a 100 μM stock solution was added to a standard enzyme reaction yielding a DacA_CD:GlmM molar ratio of 1:2 and the reactions were incubated for 4 h at 37°C. To evaluate the impact of the addition of GlmM when added to an ongoing DacA_CD enzyme reaction, 2 μl of GlmM from a 100 μM stock solution were added 30 min after initiating the reaction and the reaction was incubation for a further 2 h. As control, DacA_CD reactions set up in the absence of GlmM were stopped after 30 min and 3 h. The enzyme assays were performed in triplicate and the average values and standard deviations plotted.

GlmM enzyme activity assay

The phosphoglucosamine mutase activity of GlmM was determined by using a coupled radioactive assay in which the GlcN-1-P synthesized from GlcN-6-P by GlmM was quantitatively converted to N-acetylglucosamine-1- phosphate (GlcNAc-1-P) in the presence of pure GlmU enzyme [40]. The standard assay mixture (50 μl) contained 50 mM Tris/HCl (pH 8), 3 mM MgCl₂, 1 mM GlcN-6-P, 0.4 mM [¹⁴C]acetyl-CoA (1.9 GBq/mmol, 700 Bq), 0.7 mM glucose-1,6-diphosphate, pure E. coli GlmU (30 ng) and S. aureus GlmM (10–50 ng) enzymes (appropriate dilutions in 20 mM phosphate buffer, pH 7.2, containing 0.5 mM MgCl₂ and 0.1% 2-mercaptoethanol). When the DacA protein was also included in the assay, pure GlmM and DacA proteins were first pre-incubated together with different ratios at 37°C for 5 min before addition to the reaction mixture at t = 0. Then, mixtures were incubated at 37°C for 30 min and the reactions were stopped by the addition of 10 μl of glacial acetic acid. The radioactive substrate (acetyl-CoA) and product (GlcNAc-1-P) were separated by thin-layer chromatography (TLC) on pre-coated silica gel 60F₂₅₄ plates (Merck) using 1-propanol/ammonium hydroxide/water (6:3:1, v/v) as the mobile phase (R_f factors of these compounds were 0.72 and 0.17, respectively). Radioactive spots were located and quantitated with a radioactivity scanner (Rita Star, Raytest Isotopenmeβgeräte GmbH, Straubenhardt, Germany).

Assessment of protein stability by thermofluor analysis

To assess the protein stability, a thermofluor experiment was performed. Reactions were set up as follows: 2 μl of either 10X enzyme reaction buffer (400 mM Hepes pH 7, 1 M NaCl, 100 mM MnCl₂) or 10X protein purification buffer (300 mM Tris pH 7.5, 1.5M NaCl) were added to the appropriate wells of a 96-well qRT-PCR plate and mixed with 15 μl of H₂O. Next, 2 μl of 100 μM DacA_CD, DacA_CD-C, DacA_CD-K (monomer) or DacA_CD-K (dimer) protein stock solutions were added and finally 1 μl of 100X Sypro orange was added to yield a total reaction volume of 20 μl and a final concentration of Sypro orange of 5X. The reactions for each protein were set up in triplicate in a 96-well plate. An Applied Biosystems OneStepPlus real-time PCR system was used for the thermal unfolding reactions and the temperature was increased every 30 s by 1°C from 25°C to 95°C and the fluorescence intensities measured. The data were analyzed using the Applied Biosystems StepOne Plus software. Fluorescence readings of the triplicate samples were averaged and normalized for each protein as previously described [61] using the following equation: (F-F_min)/(F_max-F_min) where F is the fluorescence value at each temperature, F_min is the average minimum fluorescence value detected for the each protein and F_max the average maximum value detected for the each protein.

Small-angle X-ray scattering

SAXS data for the tag-less DacA_CD and GlmM proteins as well as the DacA_CD/GlmM complex were collected at the B21 beamline at the Diamond Light Source (Didcot, UK) using an Agilent 1200 HPLC system equipped with a Superdex 200 5/150 column (GE Healthcare). Prior to data collection, the column was equilibrated with 30 mM Tris pH 7.5, 150 mM NaCl. 50 μl of 12.8 mg/ml DacA_CD, 10 mg/ml GlmM or 15 mg/ml DacA_CD/GlmM protein solutions were loaded onto the size exclusion column and the data collected continuously during the sample elution. All datasets were analyzed using ScÅtter (http://www.bioisis.net) using the scattering frames corresponding to the elution peaks. Particle reconstruction was performed using DAMMIF [62], averaging 13 models in slow mode. Mean NSD values obtained from DAMFILT were 0.723 for the DacA_CD/GlmM complex, 0.763 for GlmM and 0.738 for DacA_CD.

Quantification of c-di-AMP by ELISA

E. coli XL1-Blue strains containing plasmid pBAD33 and derivatives thereof allowing expression of the S. aureus DacA, YbbR and/or GlmM protein from an arabinose inducible promoter were grown overnight at 37°C in 5 ml LB + 0.2% glucose + 20 μg/ml chloramphenicol. The next day, the cultures were diluted 1:200 into 20 ml fresh LB medium supplemented with 20 μg/ml of chloramphenicol and incubated for 2 h 45 min at 37°C. Next, arabinose was added to a final concentration of 0.2% and the cultures incubated for a further 3 h at 37°C. The A₆₀₀ was measured and cells equivalent to 10 ml at A₆₀₀ of 1 were collected by centrifugation, washed twice with phosphate buffered saline (PBS) pH 7.4 and frozen. For the co-expression of the S. aureus DacA protein and the GlmM-His proteins from different bacteria, E. coli XL1-Blue strains containing plasmid pBAD33-dacA and the empty vector pTrcHis60, or plasmids pMLJ4 (GlmM-His E. coli), pMLJ11 (GlmM-His S. aureus), or pMLD137 (GlmM-His P. aeruginosa) were grown overnight at 37°C in 5 ml LB + 0.2% glucose with 100 μg/ml ampicillin and 20 μg/ml chloramphenicol. The next day, the cultures were diluted 1:200 into 20 ml fresh LB medium supplemented with the appropriate antibiotics and incubated for 2 h 45 min at 37°C. Next, IPTG was added to a final concentration as indicated in the figure and legend and the cultures were incubated for 15 min at 37°C. Next, arabinose was added to a final concentration of 0.2% and the cultures incubated for a further 3 h at 37°C. The A₆₀₀ was measured and cells equivalent to 10 ml of A₆₀₀ of 1 were collected by centrifugation, the cells washed twice with phosphate buffered saline (PBS) pH 7.4 and frozen. For the ELISA assay, the cell pellets were thawed and suspended in 1 ml 50 mM Tris pH 8 buffer or 1 ml 50 mM Tris pH 8 buffer containing 1 mg/ml lysozyme. The cells were lysed by sonication for 2 to 3 min with 10 sec. on and 10 sec. off cycles at 40% amplitude and the samples were subsequently boiled for 10 min. The cell debris was removed by centrifugation at 20000 × g for 5 min and the soluble fraction transferred to a fresh tube. A competitive ELISA for the quantification of the cellular c-di-AMP levels was performed as previously described [12, 63]. Briefly, the ELISA was performed as follows: the wells of a NUNC Maxisorb ELISA 96-well plate were coated with the Streptococcus pneumoniae c-di-AMP binding protein CpaA by placing 100 μl of a 10 μg/ml protein solution in 50 mM Na₂CO₃, 50 mM NaHCO₃ pH 9.6 buffer into the appropriate wells and the plate was subsequently incubated overnight at 4°C. The coating solution was removed, the wells washed 3 times with 200 μl PBS pH 7.4 containing 0.05% Tween 20 (PBST) and then blocked with 150 μl of 5% BSA in PBS pH 7.4 solution and the plate was incubated at 18°C for 1 h. Next, the blocking solution was removed and each well was washed 3 times with 200 μl PBST. Reference samples for the c-di-AMP concentration standard curve and the E. coli samples were prepared as follows: For the standard curve, c-di-AMP (Biolog) solutions in 50 mM Tris pH 8 buffer were prepared ranging from 300 nM to 3.125 μM and mixed with an equal volume of a 50 nM biotin-c-di-AMP solution. For the E. coli samples, appropriate dilutions of the extracts were prepared in 50 mM Tris pH 8 and each sample mixed with an equal volume of the 50 nM biotin c-di-AMP solution. Next, 100 μl of the standard solutions and E. coli samples were added in triplicate to the CpaA protein-coated ELISA plate. The plate was incubated at 18°C for 2 h and the wells were subsequently washed 3 times with 200 μl PBST buffer. Next, 100 μl of High Sensitivity Streptavidin solution (ThermoFisher Scientific) diluted 1:5,000 in PBS was added to each well and the plate incubated at 18°C for 1 hr. The wells were washed 3 times with 200 μl PBST buffer and finally 100 μl of the developing solution, which was made up by dissolving a 10 mg o-phenylenediamine dihydrochloride tablet (OPD) (Merck) in 20 ml citrate buffer pH 5 buffer supplemented with 20 μl 30% H₂O₂, was added to each well. The plate was incubated at 18°C in the dark for 30 min and the reaction stopped by the addition of 100 to 200 μl 2 M H₂SO₄ to each well. The absorbance was subsequently measured at 480 nm and the readings for the c-di-AMP standards were used to generate a second order polynomial calibration curve and the quadratic equation was used to determine the c-di-AMP concentrations (in μM) per ml E. coli samples with A₆₀₀ = 10. For each experiment, the average value of the triplicate sample on the ELISA plate was determined. The experiment was performed three times with independent E. coli samples and as a final result the average value and standard deviations from all three experiments was plotted.

Detection of DacA and YbbR by western blot and GlmM by coomassie staining

The E. coli cultures used for the detection of c-di-AMP by ELISA were also used for the detection of DacA and YbbR by western blot and GlmM by Coomassie staining or GlmM-His by western blot with an His antibody. Following the 3 h induction step with 0.2% arabinose, bacteria from 1-ml culture equivalent to an A₆₀₀ of 1 were harvested and suspended in 100 μl 2× SDS sample buffer. The samples were boiled for 10 min and centrifuged for 5 min at 13,000 × g. Next, 10 μl of each sample was run on a 12% SDS-PAGE gel and to assess the overproduction of GlmM, proteins were visualized by Coomassie staining. The DacA and YbbR proteins were detected by western blot using protein-specific antibodies primary antibodies produced at Covalab and previously used [12, 45] and the GlmM-His proteins using an HRP-conjugated His-tag antibody (Sigma). To this end, the proteins were electro-transferred to PVDF membranes, the membranes blocked with 5% (w/v) milk in Tris buffered saline containing 0.1% (v/v) Tween-20 (TBST). The membranes were subsequently incubated with DacA or YbbR-specific polyclonal rabbit antibodies in 5% (w/v) milk in TBST buffer at a 1:5000 dilution. After washing the membranes with TBST buffer, they were incubated with the secondary HRP-conjugated anti-rabbit IgG antibody (Cell signaling Technology) used at a 1:10000 dilution. The HRP-linked His-tag antibody was used at a 1:5,000 or 1:10,000 dilution. Next, the membranes were washed and the blots developed using the Clarity Western ECL Blotting Substrate (Bio-Rad) and imaged using a ChemiDoc Touch system (Bio-Rad). The experiment was performed on three independently grown samples and a representative result is shown.

Accession numbers

Structure coordinates were deposited in the Protein Database, under PDB codes 6GYW (nucleotide-free DacA_CD), 6GYX (ApCpp-bound DacA_CD), 6GYY (DacA_CD-C mutant), 6GYZ (GlmM). SAXS models were deposited in the SASBDB database, under accession codes SASDE78 (DacA_CD/GlmM complex), SASDE88 (GlmM) and SASDE98 (DacA_CD).