c-di-AMP synthesis is essential under standard growth conditions

In the GBS genome, a single gene, thereafter named dacA, encodes a protein containing a DisA_N domain (PF02457 Pfam domain), the only known domain with c-di-AMP synthesis activity [11]. The dacA gene is localized in a highly conserved three-gene operon encoding DacA, a putative DacA activity regulator (Gbs0903) and the essential GlmM enzyme (Gbs0904) involved in synthesis of cell-wall metabolite precursors [9, 11, 18, 19]. All attempts to inactivate dacA using standard protocols were unsuccessful, suggesting that dacA is an essential gene. Therefore, a conditional ΔdacA mutant was constructed in a strain bearing an ectopic copy of dacA cloned on a replicative vector and transcribed from the anhydrotetracycline (aTc)-inducible promoter P_tetO (S1 Fig). The growth of the ΔdacA / P_tetO_dacA mutant is aTc dose-dependent on TH medium incubated in aerobic growth conditions (Fig 1A). The mutant does not grow in the absence of aTc, while its growth was similar to that of the WT strain in presence of 50 ng/ml aTc.

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Fig 1. c-di-AMP synthesis is conditionally essential in GBS.

(A) Serial dilutions of the WT and ΔdacA mutant containing the inducible P_tetO_dacA vector were spotted on TH agar supplemented or not with anydrotetracycline (aTc), and incubated 24 hours in standard growth condition (i.e. aerobic condition). (B) Growth of the ΔdacA / P_tetO_dacA mutant on TH, Granada, and Columbia + 5% horse blood (COH) incubated in aerobiosis or anaerobiosis for 24 hours. About 10⁴ bacteria were spread on each media and a disk containing 500 ng aTc was added on each plate before incubation. (C) Growth of the ΔdacA-1 and ΔdacA-2 mutants on TH incubated in aerobiosis or anaerobiosis. (D) Growth of the ΔdacA-2 mutant complemented with the WT dacA or the inactivated dacA* alleles under the control of the inducible promoter P_tetO on TH incubated in aerobiosis with aTc, and on Granada and COH in anaerobiosis with or without aTc.

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By testing Granada medium, a GBS-specific medium developed to detect the orange-red polyenic pigment granadaene under anaerobic conditions [35], we unexpectedly observed growth of the ΔdacA / P_tetO_dacA mutant in the absence of aTc (Fig 1B). This anaerobic growth is independent from the medium components since it was also observed when grown in TH or Columbia Horse Blood (COH) agar. In contrast, the growth is aTc-dependent in aerobic condition whatever medium used (Fig 1B). In addition, ΔdacA / P_tetO_dacA colonies are not pigmented and not hemolytic in anaerobic conditions, unless the aTc-dependent ectopic dacA copy was expressed (Fig 1B). This indicates that c-di-AMP synthesis is necessary for granadene production, the GBS pigment that is also a ß-hemolysin/cytolysin [36].

The anaerobic growth of the ΔdacA / P_tetO_dacA strain was exploited to construct ΔdacA mutants without an ectopic dacA allele. The first ΔdacA-1 mutant was selected after anaerobic growth following the loss of the vector containing the additional dacA copy (S1 Fig). The second ΔdacA-2 mutant was constructed from the parental ΔdacA::dacA integrant by selecting the deletion mutant directly under anaerobic conditions (S1 Fig). Both ΔdacA mutants grow in anaerobiosis, although the ΔdacA-1 mutant growth is slightly altered on TH compared to the WT strain, and they do not grow in aerobiosis (Fig 1C). Re-introduction of the P_tetO_dacA vector in the two ΔdacA mutants restored growth, pigmentation, and hemolysis in the presence of aTc (Fig 1D). In contrast, expression of an inactivated DacA*, bearing a R₂₁₃K substitution in the RHR conserved di-adenylate cyclase motif [37, 38], does not complement the ΔdacA phenotypes (Fig 1D). As expected, the purified recombinant DacA protein produces c-di-AMP from two molecules of ATP while the recombinant DacA* is devoid of di-adenylate cyclase activity (S2 Fig). Thus, c-di-AMP synthesis appears essential for growth in aerobiosis and necessary for optimal growth in anaerobiosis.

Mutation of the BusAB transporter is necessary in the absence of c-di-AMP

The genomes of the parental WT NEM316 strain, of the two ΔdacA::dacA integrants, and of the two corresponding ΔdacA mutants were sequenced (S1 Table). Compared to the published reference sequence [39] (RefSeq NCBI NC_004361), fifteen SNPs or INDELs are present in our WT strain and in all of its progeny (S2 Table). The genome sequence of the first ΔdacA::dacA integrant is identical to the parental WT strain, while the second integrant displays a SNP located in the cylD gene of the cyl operon encoding the ß-hemolysin/cytolysin [36, 40] (S3 Table).

Compared to their parental integrants, the two ΔdacA mutants have two additional mutations in the same genes: oppC (the gbs0146 locus) and busB (the gbs1838 locus) (Fig 2A and S3 Table). The first gene encodes the OppC oligopeptide transporter subunit [41] and the two mutants have independent frameshift mutations (+A in ΔdacA-1 and—A in ΔdacA-2) located at the beginning of the gene (Fig 2A). The second gene encodes a transmembrane protein homologous to the Lactococcus lactis BusB subunit [42]. In this species, BusB and its cytoplasmic partner BusA form an ABC transporter involved in osmolyte import (Fig 2A). In the ΔdacA mutants, busB has either a SNP resulting in a V₆₂D substitution localized in the first transmembrane domain of BusB (ΔdacA-1) or a single nucleotide deletion at position 120 (ΔdacA-2) (S3 Table). In addition to the busB and oppC mutations, the ΔdacA-1 mutant has an additional copy of TnGBS, a 47kb integrative and conjugative element already present three times in the parental strain [39, 43], integrated in an intergenic region (S3 Table).

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Fig 2. Mutation of the BusAB transporter is necessary in the absence of c-di-AMP.

(A) Schematic representations of the independent busB and oppC mutations in ΔdacA mutants and of the BusAB and Opp transporters. (B) Conditional expression of a WT copy of oppC and busB in the ΔdacA-2 mutant in anaerobiosis on TH with and without aTc. The ΔdacA-2 mutant with the empty vector P_tetO is used as a control.

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To assess the functional significance of the two shared mutated genes, we introduced a replicative vector containing a wild-type copy of oppC or busB under the control of the aTc inducible P_tetO promoter in the ΔdacA mutants (Fig 2B). Expression of a WT copy of busB, but not of oppC, inhibited the anaerobic growth of the ΔdacA mutants (Fig 2B). Therefore, a mutation in the osmolyte transporter BusB appears necessary to counteract the effect of dacA inactivation under anaerobic conditions. The independent occurrence of a loss of function mutation in oppC in the two mutants also suggested that this mutation was necessary but not sufficient.

Adaptation to the absence of c-di-AMP synthesis involves intertwined mutations

Attempts to delete dacA in ΔbusB, ΔoppC, and ΔbusB ΔoppC backgrounds were unsuccessful, suggesting the necessity of additional compensatory mutations. To identify these additional pathways, we selected ΔdacA clones able to grow in aerobiosis. In liquid cultures, the ΔdacA mutants display high growth variability that was recorded by following their aerobic growth in liquid medium (Fig 3A). When isolated colonies (n = 48) grown anaerobically were directly inoculated in liquid media, around 75% were unable to grow under aerobic conditions, the remaining cultures showing weak or intermediate growth defects (Fig 3B and 3C). However, after 4 serial cultures under anaerobic conditions, almost three quarters of these cultures were able to grow as the WT strain under aerobic conditions (Fig 3C). The growth of each culture remains highly variable, suggesting that different populations arose and co-exist during serial cultures. However, this is not due to a higher mutation rate of the ΔdacA mutants since rifampicin resistant colonies were obtained at a similar frequency with WT and ΔdacA mutant strains (S2 Fig).

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Fig 3. Adaptation to the absence of c-di-AMP synthesis involves intertwined mutations.

(A) Schematic representation of the experiment. Strains were propagated on solid media in anaerobiosis. Isolated colonies were picked in TH and incubated overnight (o/n) at 37°C in anaerobiosis. At each serial dilution step (0, 1, … n), aliquots were taken and the growth in aerobiosis was monitored in triplicate. (B) Representative growth curves of the ΔdacA-2 mutant in aerobiosis after two serial culture in anaerobiosis. The growth curves obtained from 4 independent ΔdacA colonies (number 5, 8, 9 and 16) illustrate the variability. Growth curves were classified as corresponding to no growth (OD < 0.01), weak (OD < 0.1), intermediate (OD > 0.1 at 20 hrs), or strong adaptation (OD > 0.1 at 10 hrs). Growth curves are the mean of triplicate +/- SEM for each independent colony. (C) Percentage of ΔdacA-2 cultures (n = 24) showing aerobic adaptation following serial cultures in anaerobic condition. Similar results were obtained with 24 cultures done with the ΔdacA-1 mutant. (D) Frequency of ΔdacA mutant able to grow in aerobiosis on TH agar plates after dilution of one overnight anaerobic cultures. Median with interquartile range were calculated from ten independent cultures. (E) Suppressors corresponding to ΔdacA mutant able to grow aerobically were isolated and their phenotype confirmed by spotting on TH agar. (F) Distribution of the mutation on the 2.2 Mb genome of ΔdacA-1 (left) and ΔdacA-2 (right) mutants (outer ring) and of ΔdacA suppressors (inner rings). The dacA deletion is highlighted in black. Mutations in the ΔdacA-1 and ΔdacA-2 mutants absent in the parental WT strain are color-coded in red, and mutations specific of suppressors are in orange. Identity of gene or operon independently mutated in more than two strains are shown. ABC transporters are indicated in bracket. (G) Conditional expression of a WT copy of mutated genes in the ΔdacA suppressors S39. Phenotypic effect of the expression of each gene was tested by adding aTc (50 ng/ml) in TH. Coloured boxes highlight growth inhibition upon expression of a WT allele in aerobiosis and anaerobiosis (red boxes), or aerobiosis only (orange). (H) Same experiment as in (G) in 9 ΔdacA suppressors. See S3 Fig for the corresponding images.

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Fourteen independent ΔdacA suppressors (5 from ΔdacA-1 and 9 from Δdac-2) were isolated on TH in aerobiosis after a single overnight incubation in liquid medium in anaerobiosis. In this condition, the proportion of colonies growing on TH in aerobiosis is highly variable, usually between 0.5 and 10⁻³ (Fig 3D). Each isolated suppressor grew on TH plates as the WT (Fig 3E), and the absence of c-di-AMP in whole bacterial extracts of ΔdacA mutants and of several suppressors was confirmed (S2 Fig), excluding that a cryptic di-adenylate cyclase was activated to compensate for the absence of dacA.

The genomes of the 14 ΔdacA suppressor strains were sequenced to identify the compensatory mechanisms, but the number of mutations were variable, with no single mutated gene common to all suppressors (S3 Table). All suppressors carry the two oppC and busB mutations present in the parental ΔdacA mutant and between 1 to 7 additional mutations (Fig 3F). The mutations are mostly SNPs (n = 24, including 19 leading to amino-acid substitution), followed by small indels (n = 12, including 8 in coding sequence), three deletions of 36–47 bp, and one 90-bp duplication (S3 Table). Independent mutations in the same gene or functional complex were identified in different suppressors (Fig 3F), including mutations in an operon encoding a second osmolyte ABC transporter homologous to the L. lactis OpuABC glycine betaine transporter [44], in the glutamine ABC transporter GlnPQ [45, 46], and in a putative secreted protein (Gbs1444) of unknown function (S4 Table). Also interesting is the presence of additional loss of function mutations in BusB in suppressor S5 originating from the ΔdacA-1 mutant with the BusB V₆₂D substitution (S3 Table).

To identify causative mutations restoring growth in the absence of c-di-AMP, we focused our analysis on nine different suppressor mutants. In each of these suppressors, a WT copy of the mutated genes expressed from the aTc-inducible promoter was introduced. As expected, induction of the WT copy of busB inhibited the growth of six of the nine suppressors in aerobiosis and anaerobiosis (Fig 3G and 3H, and S3A Fig). The expression of a WT busB allele was toxic only under aerobic growth in two suppressor mutants, and had no effect on one suppressor (Fig 3H). These results confirm that busB inactivation is necessary for bacterial growth in the absence of dacA, but reveal that additional mutations can alleviate busB toxicity in the absence of c-di-AMP.

Among the eight genes mutated at least once in the nine suppressors, four are toxic upon re-expression of their WT copy in five of the suppressors (Fig 3H and S3A Fig). The expression of Gbs1444 encoding a putative secreted protein is toxic in the three suppressors containing a mutation in this gene. The remaining three genes inhibiting growth upon their re-expression encode for ABC transporters: OpuCA, GlnPQ, and PstB (a phosphate ABC transporter homolog). We excluded a non-specific toxic effect of the tested genes by expressing them in the same condition in a WT background (S3B Fig).

Overall, at least one mutated gene in each of the nine suppressors studied was toxic upon conditional expression of a WT copy, suggesting that the corresponding mutation compensates the absence of c-di-AMP. Nevertheless, the pattern of mutations suggests strong epistasis, i.e. the effect of the toxic gene is dependent of the other mutations present in a given suppressor. For instance, in suppressors S30 and S34, mutation of busB is not sufficient for aerobic growth of the parental ΔdacA mutants, but should be combined with glnPQ or mscS (Fig 3H). In contrast, in suppressors S6, S35, and S47, the toxic effect of a functional busB allele in a ΔdacA background can be attenuated by mutations in gbs1444 or pstB (Fig 3H). The pattern of compensatory mutations and epistatic interactions suggest that c-di-AMP controls a highly regulated and interconnected essential pathway.

c-di-AMP binds conserved osmolyte and potassium transporters

Compensatory mutations might encode for proteins directly regulated by c-di-AMP. To identify these direct c-di-AMP regulated processes, interaction between c-di-AMP and candidate proteins were assayed by DRaCALA [47]. Fourteen proteins were selected as candidates, including the BusA, OppD, and OppE cytoplasmic ATPases subunits of osmoprotectant and oligopeptide transporters (Fig 2), the mutated proteins tested for their phenotypes upon re-expression in ΔdacA suppressors (Fig 3H), and three additional proteins containing a RCK_C/TrkA_C domain with a putative c-di-AMP binding motif [22–24]. The corresponding genes were cloned and expressed as a fusion protein in E. coli (S4 Fig), and whole-cell extracts were incubated with radiolabelled c-di-AMP.

C-di-AMP binds to four proteins: KtrA (Gbs1678), TrkH (Gbs1639), OpuCA (Gbs0235) and Gbs1201, thereafter named BusR (Fig 4A). The binding of radiolabelled c-di-AMP is specific since it could be displaced by addition of cold c-di-AMP but not of c-di-GMP, cAMP, cGMP, AMP or ATP (Fig 4B). Three of the four c-di-AMP binding proteins are homologs of conserved potassium (KtrA and TrkH) and osmolyte (OpuCA) transporters. Two of them, KtrA and OpuCA, are mutated in one or three of the nine suppressors, respectively (S4 Table). These two proteins are conserved c-di-AMP binding proteins, where binding is dependent on their RCK_C/TrkA_C [22, 23] or CBS [27, 28] domains, respectively. Among the four GBS proteins containing RCK_C/TrkA_C domains tested (Fig 4C), only one, EriC (Gbs1174), a chloride channel homolog, did not give a positive signal with c-di-AMP in our DRaCALA screen (Fig 4A). However, only the RCK_C domain of EriC was used in this experiment (S4 Fig) as we failed to express in E. coli the full-length protein with its eleven transmembrane domains. Therefore, these results do not rule out the possibility that a full length EriC might bind c-di-AMP.

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Fig 4. c-di-AMP binds three transporters subunits and a transcriptional factor.

(A) Interaction of radiolabelled c-di-AMP with targeted protein by DRaCALA. Full-length proteins were expressed in E. coli, except for EriC where only the RCK_C can be expressed. Whole E. coli extracts were mixed with radiolabelled c-di-AMP and spotted on a nitrocellulose membrane. C-di-AMP bounds to protein does not diffuse as far as free c-di-AMP. Quantification of the inner and outer circles intensities allows to calculate the fraction of bound c-di-AMP. (B) Specificity of the c-di-AMP interaction. Same as (A) with the addition of cold competitor to the reaction before spotting on membrane. (C) Color-coded representation of the domain organisation of selected proteins. Number of amino acids are indicated at the end of proteins. The RCK_C (red) and CBS (orange) domains are predicted c-di-AMP and nucleotides binding domains, respectively. The RCK_N domain (regulator of potassium conductance, white) is prevalent among potassium channels. The GntR domain (green) is a winged helix-turn-helix DNA binding domain. The ABC domain (blue) represent the ATPase domain of ABC transporter. The ClC domain (purple) is found in chloride ion channels, a family of voltage-dependent gating transporter with 11 transmembrane domains.

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It is also interesting to note that BusA and OpuCA are two highly similar subunits of osmolyte transporters (55% similarities, e = 6 e-59). The two proteins contain a CBS domain (Fig 4C). However, BusA, the cytoplasmic subunit of the BusAB transporter which is mutated in ΔdacA mutants, does not bind c-di-AMP in contrast to OpuCA (Fig 4A). This confirms that CBS domains may have a similar topology but different physiological ligands [26]. This also implies different mechanisms of regulation for the BusAB and OpuC osmolyte transporters.

BusR directly represses the busAB transporter necessary for c-di-AMP dependent osmotic regulation

The fourth c-di-AMP binding protein identified by DRaCALA is a putative transcriptional regulator of the GntR family containing a winged helix-turn-helix DNA binding domain (Fig 4C). BusR is highly similar to the annotated MngR trehalose transcriptional repressor in Chlamydia trachomatis (e value = 2 e-107) and to the L. lactis BusR transcriptional repressor (5 e-61). In L. lactis, the busR gene is localized immediately upstream of the busAB operon [48], whereas in GBS busR and busAB are separated by 655 kb and no transcriptional regulator is located in the vicinity of the busAB operon.

The homology with L. lactis suggests a putative conserved function of BusR on busAB transcription in GBS. Therefore, we purified recombinant GBS BusR and tested its binding on the P_busAB promoter of the busAB operon. Gel shift assays show that P_busAB migrates more slowly in the presence of BusR (Fig 5A) and footprint experiments show two BusR-protected regions in the P_busAB promoter, one overlapping the -35 and -10 elements and the +1 transcription start site (Fig 5B). Deletion of busR increases expression of the busAB operon compared to the WT or the ΔbusR_c complemented strain (Fig 5C). These results demonstrated that the c-di-AMP binding protein BusR is a transcriptional regulator directly repressing the busAB operon.

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Fig 5. BusR is a transcriptional repressor of the osmolyte transporter BusAB.

(A) Gel shift assay with increasing concentration of recombinant BusR and the radiolabelled P_busAB promoter. (B) Footprint experiment on the P_busAB promoter with BusR. The two DNase I protected boxes are numbered from the start codon of the busAB operon. The position of the transcriptional start site and of the -10 and -35 elements are highlighted. (C) Quantification of busAB transcript by RT-qPCR in the WT, the ΔbusR mutant, and the ΔbusR_c complemented strain. Means and SD are calculated from three independent RNA purification done from exponentially growing cultures in TH. (D) Spotting dilutions of WT, ΔgdpP, ΔbusA, ΔbusB, ΔbusR, and ΔgdpP ΔbusR cultures on TH with and without 800 mM NaCl incubated in aerobiosis and anaerobiosis.

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To test the functional link between c-di-AMP and the BusR-BusAB osmolyte import system, we analysed the phenotypes of the deletion mutants (ΔbusA, ΔbusB, ΔbusR, ΔgdpP, and ΔbusR ΔgdpP) in response to osmotic stresses. As observed in several bacteria, deletion of the c-di-AMP phosphodiesterase GdpP increases the intracellular c-di-AMP concentration in GBS (20- to 38-fold, S2C Fig). Furthermore, the ΔgdpP mutant is more susceptible to hyperosmotic stress compared to the WT strain (Fig 5D). Strikingly, ΔgdpP osmo-susceptibility is dependent on a functional BusR transcriptional regulator. Deletion of BusR has no or a weak effect on bacterial growth upon hyperosmotic challenge, while the two subunits of the BusAB transporter are as important as GdpP to resist the hyperosmotic stress (Fig 5D). The double ΔbusR ΔgdpP deletion abolishes the susceptibility of the ΔgdpP mutant (Fig 5D), showing that elevated c-di-AMP leads to hyperosmotic susceptibility by acting through the transcriptional repressor BusR.

c-di-AMP is dispensable for growth in osmolyte-depleted media

The pattern of compensatory mutations and the identification of c-di-AMP binding proteins point towards a coordinated regulation of potassium and osmolyte uptake as the essential function of c-di-AMP in GBS. We therefore tested the growth of the ΔdacA mutant in a chemically defined medium (CDM) with variable potassium and osmolyte concentrations (S5 Table). To this end, we used the ΔdacA-2 mutant with an empty vector, a dacA complementing vector, or a busB expressing vector to complement the busB loss-of-function mutation in this mutant. In this CDM, c-di-AMP synthesis is dispensable for bacterial growth regardless of the potassium concentration and incubation condition, except for anaerobic growth of the mutant expressing a WT copy of busB at high potassium concentrations (5 mM) (Fig 6A). Strikingly, addition of glycine betaine to CDM inhibits the growth of the ΔdacA-2 mutant expressing busB regardless the potassium concentration, except in aerobiosis at extremely low concentrations of potassium (Fig 6A).

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Fig 6. c-di-AMP is dispensable in osmolyte depleted medium.

(A) Growth of the ΔdacA-2 mutant with the empty vector control (P_tetO), and the dacA or busB conditional expression vectors (P_tetO_dacA or P_tetO_busB) on media with 50 ng/ml aTc. The rich TH medium was used as control and potassium and glycine betaine was added to synthetic medium (CDM) incubated in aerobiosis and anaerobiosis. (B) Same experiment as in (A) with the ΔdacA-A mutant and its isogenic WTb-A control.

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The inhibitory effect of glycine betaine is dependent on busB expression since glycine betaine does not inhibit the growth of the ΔdacA-2 mutant with the empty vector (Fig 6A) and has no effect on the ΔdacA-2 / P_tetO_busB mutant in the absence of aTc. Similarly, the inhibitory effect of glycine betaine is observed with carnitine, a related osmolyte [49], while choline, a common precursor of glycine betaine, has no effect (S5 Fig). In the same conditions, busB expression in a WT strain has no effect on growth (S5 Fig), showing that osmolytes such as glycine betaine or carnitine need the expression of busAB and the absence of c-di-AMP to be toxic. Overall, the presence of an osmolyte in the culture medium appears to be the main cause of growth inhibition in the absence of c-di-AMP synthesis. The concentration of potassium is also important under specific conditions ([K⁺]_high in anaerobiosis and [K⁺]_low in aerobiosis in presence of osmolyte), suggesting that growth inhibition results from a combination of dysregulated potassium and osmolyte uptake.

c-di-AMP is essential to avoid the inhibitory effect of osmolytes

To test if the growth condition is sufficient to alleviate the essential function of dacA, we repeated the construction of a ΔdacA mutant except that all steps were performed in CDM without osmolyte and with 0.5 mM potassium. In this condition, we readily obtained ΔdacA mutants and their respective WTb controls at high frequency (S1 Fig). On CDM, the growth of the new ΔdacA-A mutant was similar to the WT and WTb controls regardless the potassium concentration and incubation conditions (Fig 6B). Addition of glycine betaine inhibits ΔdacA-A at all tested potassium concentrations in aerobic condition and only at high potassium concentration in anaerobic condition (Fig 6B). Finally, the ΔdacA-A mutant was unable to grow on TH (Fig 6B). Two additional ΔdacA mutants (-B and–C), obtained from independent parental ΔdacA::dacA integrants, displayed the same phenotypes as the ΔdacA-A mutant. These results confirmed that c-di-AMP synthesis is essential in rich medium and dispensable in minimal medium, unless osmolytes are present. The inhibiting effect of osmolytes is dependent on aerobiosis and anaerobiosis and, to a lesser extent, on potassium concentrations, suggesting a link between osmotic regulation and metabolism.

The genome of the three new, independent pairs of ΔdacA and WTb strains were sequenced (S1 Table). None of the ΔdacA-A to -C mutants share a mutation with the previously sequenced ΔdacA-1, ΔdacA-2, and ΔdacA suppressors (S3 Table). The only exception is the cylD SNP in the ΔdacA-B that is also present in the ΔdacA-2 mutant and their common parental ΔdacA::dacA integrant (S3 Table). Still, the three ΔdacA-A to–C mutants each have one mutation compared to the WT strain. These mutations are localized in gbs0330, encoding the transcriptional repressor FabT (S3 Table), embedded in the fab operon encoding enzymes of the essential type II fatty acid synthesis pathway [50]. Unexpectedly, the WTb controls and two of the three parental ΔdacA::dacA integrants show the same fabT mutations (S3 Table). The independent fabT mutations imply a strong selective pressure most probably due to the nutritional supply in the medium and not to c-di-AMP depletion. Targeted sequencing of the fabT locus of the WT and ΔdacA::dacA integrants after growth in overnight cultures in TH and CDM 0.5 mM K⁺ confirmed that fabT mutations are selected at a high frequency only on CDM medium independently of c-di-AMP (S6 Fig).

GBS strains and growth conditions

The WT GBS strain used in this study is NEM316, the originally sequenced (RefSeq NC_004368.1) serotype III reference isolate [39]. The usual Todd Hewitt (TH, Difco Laboratories), Columbia supplemented with 10% horse blood (BioMérieux), and Granada medium (BioMérieux) were used for propagation and phenotypic tests. A chemically defined medium (CDM) containing inorganic salts, vitamins, amino acids, nucleobases, pyruvate and glucose (S5 Table) was adapted from reference [65]. Glycine betaine, potassium chloride, and sodium chloride (Sigma-Aldrich) are added when stated. Buffering at pH 7.3 was done by adding Hepes (50 mM). Liquid GBS cultures are done in static condition incubated in aerobiosis or anaerobiosis. Anaerobiosis is obtained in hermetic jars with AnaeroGen gas packs (Oxoid, ThermoFischer). Growth curves in aerobiosis were done in 96 wells microplates (150 μl) at 37°C with constant shaking and automatic recording of OD₆₀₀ every 20 minutes (BioTek Synergy). Erythromycin and kanamycin (Sigma-Aldrich) are used for plasmid selection at 10 and 500 μg/ml, respectively. Anhydrotetracycline (Sigma-Aldrich) is used for conditional expression from the P_tetO inducible promoter at 0–100 ng/ml [66]. Rifampicin (50 μg/ml) was used for the quantification of spontaneous resistant mutations.

Vector constructions

Bacterial strains and plasmids (S6 Table), oligonucleotides (S7 Table), and detailed vectors construction (S8 Table) are provided in the corresponding supplementary tables. The pTCV_P_tetO vector was used for anhydrotetracycline inducible expression in GBS [66], and the shuttle thermosensitive plasmid pG1 was used for chromosomal deletion, as described previously [67, 68]. Plasmids were constructed by standard restriction and ligation cloning or by Gibson assembly, purified on columns (Qiaprep, Qiagen) and all inserts were sequenced. Plasmids were introduced in GBS by electroporation, except for the ΔdacA mutants which were transformed by conjugation with the E. coli HB101/pRK24 donor strain, as described previously [69], to avoid liquid cultures.

For DRaCALA experiments, E. coli Bli5 strain was used with the pET-28a (N-terminal His-tag) and pIVEX (N-terminal His-MBP tag) vectors. Similar results were obtained with the two vectors, except for TrkH which is detected by Western only with the His-tag, and OpuCA which give a positive signal by DRaCALA only with the His-MBP tag. For OpuCA, the MBP tag might increase the solubility of the tagged protein, as observed previously with the OpuCA homologue in S. aureus [27]. For recombinant rDacA, rDacA*, and rBusR purification, E. coli Bli5 were used with pET28a expression vectors. For E. coli, antibiotics were used at the following concentrations: ticarcillin, 100 μg/ml; chloramphenicol 30 μg/ml; ampicillin 100 μg/ml; erythromycin, 150 μg/ml; and kanamycin 25 μg/ml.

GBS deletion and conditional mutants

GBS mutants were constructed with the corresponding thermosensitive pG1 vectors (for dacA, gdpP, busA, busB, and busR deletion) in three steps, involving: i) selection of transformants at permissive temperature (30°C) with erythromycin; ii) chromosomal integration of the deletion vector at the targeted loci at restrictive temperature (37°C); and iii) decombination and loss of the deletion vector at permissive temperature (30°C) without selective pressure. The final step can give back to a WT allele (defined as the WTb controls) or to deletion of the targeted loci (unmarked deletion). Confirmation of the WTb or deletion genotypes was done by PCR and Sanger sequencing for each mutant.

Attempts to delete dacA (i.e. in-frame deletion of the DacA cytoplasmic domain, codon 106 to 234 of the 283 amino-acids protein) following the standard protocol were unsuccessful, given only WTb colonies at the final step. Therefore, an additional copy of dacA was cloned into the conditional pTCV_P_tetO expression vector [66] and introduced into the ΔdacA::dacA intermediate strain (called the integrant) at 30°C with erythromycin and kanamycin (S1 Fig). The final step of losing the integrated vector was repeated in presence of 50 ng/ml aTc and the ΔdacA / P_tetO_dacA in-frame deletion mutant was obtained at high frequency.

To obtain ΔdacA mutants without the P_tetO_dacA expression vector, serial cultures in anaerobic condition were done without the selective pressure to maintain the vector (S1 Fig). The P_tetO_dacA vector was lost in a WTb background after two serial cultures but all ΔdacA / P_tetO_dacA retain the vector in the same condition, indicating that a leaky expression of the ectopic dacA copy is sufficient to keep a fitness advantage. By testing more than 200 non-pigmented clones after 6 serial cultures on Granada in anaerobic condition, we isolated one ΔdacA mutant (ΔdacA-1) which has lost the P_tetO_dacA vector. An independent ΔdacA-2 mutant was obtained from the ΔdacA::dacA integrant by performing all subsequent steps in anaerobiosis on Granada (S1 Fig). The frequency of ΔdacA mutant versus WTb strain was less than 1%, confirming that ΔdacA has a fitness disadvantage compared to the WT strain. Finally, the standard protocol was repeated to construct the ΔdacA-A, -B and -C mutants except that all steps were done in CDM, resulting in high frequencies of ΔdacA mutant.

Genome sequencing

Genomic DNA was purified from 10 ml of overnight cultures in TH or CDM, except for ΔdacA-1 and –2 mutants which were made from colonies on TH plates incubated in anaerobiosis. Bacterial pellets were treated with lysosome (20 mg/ml) and proteinase K before mechanical breaking of the cell by microbeads (FastPrep, MP Biomedicals), and genomic DNA purification (DNeasy Blood, Qiagen) and quantification (Qubit hsDNA, ThermoFisher Scientific). Five micrograms of DNA were used for libraries preparations. The first set of DNA (S1 Table) was treated and sequenced by the Sequencing Core Facilities of Institut Pasteur (Paris, France) with TrueSeq DNA LT kits and single-read sequencing (150 bp) on a MiSeq instrument (Illumina). The second set of DNA (S1 Table) was sheared (Covaris S220 instrument), treated with commercial enzymes and purification kits (Klenow, T4 ligase, T4 polynucleotide kinase, Phusion polymerase from New England Biolabs, and MinElute and QiaQuick columns from Qiagen), ligated to multiplex adapters (NEXTflex, Illumina), and purified (500 bp mean fragment). Paired-end sequencing (2 x 76 bp) was done on a NextSeq 550 apparatus (Illumina). After quality assessments, trimming and de-multiplexing, sequence reads were mapped on the 2.2 Mb reference sequence (RefSeq NC_004368.1) using Geneious software (Biomatters Ltd), resulting in a mean coverage of 131x and 609x for the MySeq and NextSeq instruments, respectively (S1–S4 Tables).

Differential Radial Capillary Action of Ligand Assay (DRaCALA)

Interaction between c-di-AMP and targeted GBS proteins was tested by DRaCALA [47] on whole E. coli protein extract. Expression of the candidate GBS protein was done in Bli5 containing pET-28a or pIVEX expression vector (S6 Table). Expression of the tagged-GBS protein was induced with IPTG (1 mM) for 6 hours at 30°C. Bacterial pellet from 1 ml culture is suspended in 100 μl binding buffer (40 mM Tris pH 7.5, 100 mM NaCl, 10 mM MgCl2, 0.5 mg/ml lysozyme, 20 μg/ml DNase), lysed by 3 freeze-thaw cycles, and directly used for DRaCALA and Western blot analysis using anti-His-tag antibodies. For DraCALA, 1 nM ³²P-labeled c-di-AMP, synthetized as described in reference [22], was added to the whole protein extract, incubated at room temperature for 5 min, and 2.5 μl was spotted onto nitrocellulose membrane. Membranes are revealed with radiographic films (Amersham Hyperfilm ECL, GE Healthcare) and signal intensity quantified with ImageJ (NIH). The c-di-AMP bound fraction was calculated as described [47]. For competition assay 200 μM of cold nucleotides (c-di-AMP, c-di-GMP, cAMP, cGMP, AMP, and ATP; BioLog Life Science Institute, Germany) were added to the protein extract altogether with radiolabelled c-di-AMP.

c-di-AMP synthesis activity

Recombinant rDacA (amino-acids 96 to 243, deleted from the transmembrane domain) and the mutated rDacA* (with a R₂₁₃K substitution) were expressed as 6xHis N-terminal tagged forms (pET28a vector) in Bli5 E. coli strain. Cultures were done at 37°C in LB until OD₆₀₀ = 0.7 before protein induction with IPTG (1 mM) for 3 hours. After centrifugation and one cycle of freezing (-20°C), pellets are suspended in 20 ml of buffer (50 mM Na₂HPO₄/NaH₂PO₄, 300 mM NaCl, pH7.0), and broken by one passage through a French press at 14000 p.s.i. Cell debris were eliminated by centrifugation and the recombinant proteins were purified by chromatography (5 ml TALON crude column, GE Healthcare) with a linear gradient from 0 to 150 mM imidazole in 50 mM Na₂HPO₄/NaH₂PO₄, 300 mM NaCl, pH7.0, at 5 ml/min for 20 min. Fractions containing the enzyme were pooled and the buffer was exchanged on PD10 column previously equilibrated with 10 mM Bis-Tris, 100 mM NaCl, pH 7.5. Diadenylate cyclase activities were tested at 37°C with 2.5 μM rDacA or rDacA* incubated with 1 mM ATP in 50 mM Tris pH 8.5, 100 mM NaCl and 10 mM MnCl₂. Formation of c-di-AMP was followed each 14 min by RR-HPLC using a reverse-phase column (Agilent ZORBAX Eclipse XDB-C18, 2.1 x 100 mm, 1.8 μm). Samples were analyzed by RR-HPLC with a flow rate of 0.25 ml/min and a linear gradient of 1–12% acetonitrile (CH3CN) in 20 mM triethylammoniumacetate buffer, pH 7.5. The ATP and c-di-AMP peak areas were used to quantify substrate and product formation.

c-di-AMP quantification

C-di-AMP quantification in GBS was done by LC-MS/MS (BIOLOG Life Science Institute), following company instructions. Late-exponential GBS cultures (OD₆₀₀ = 0.8) in TH Hepes 50 mM incubated in aerobiosis or anaerobiosis were centrifuged (15 min, 4°C, 2,500 g), and the pellet washed in PBS. Bacteria were suspended in extraction buffer (acetonitrile/methanol/water; 2/2/1), incubated 15 min on ice, heat extracted 10 min at 95°C, and incubated for an additional 15 min on ice. A final mechanical cell lysis step was done with 0.1 mm microbeads with shaking (2 x 30”, FastPrep-24, MP Biomedicals). After centrifugation (10 min, 4°C, 20,000 g), supernatant was transferred into a new tube and the extraction step was repeated twice on cell debris without the heating step. The three supernatants were pooled and store at -20°C overnight to complete protein precipitation. After centrifugation (20 min, 4°C, 20,800 g), the whole extract was evaporated to dryness (Eppendorf concentrator 5301) before quantification by LC-MS/MS. Protein concentration in the bacterial culture was done (Pierce BCA, Thermo Fischer) in parallel to the extraction to normalize c-di-AMP concentration to the total protein content.

BusR purification and BusR-DNA interaction

Full length recombinant rBusR (amino-acids 1 to 213 tagged with a N-terminal 6xHis) expressed in Bli5 E. coli strain was purified as rDacA, except that IPTG-induction was done at 20°C overnight, and with an additional purification step by gel filtration (Superdex 10/300 GL, GE Healthcare) after affinity chromatography in a final buffer containing 20 mM Hepes pH 7, 150 mM NaCl. Electrophoretic mobility shift assay (EMSA) was done with a 245 bp PCR fragment (primers pLD1 + pLD2) corresponding to the promoter region of the busAB operon (P_busAB). This 5’ region includes the transcription start site and the -10 and -35 boxes, as characterized by whole genome TSS mapping [70]. Primer pLD1 is radiolabelled with T4 polynucleotide kinase (New England Biolabs) and [γ-³²P]-dATP before PCR reaction. Protein-DNA interaction was done with rBusR, radiolabeled P_busAB (10⁴ c.p.m), 0.1 μg/μl of Poly(dI-dC) (Pharmacia), and 0.02 μg/μl BSA in binding buffer (25 mM Na₂HPO₄/NaH₂PO₄ pH 8, 50 mM NaCl, 2 mM MgCl₂, 1 mM DTT, 10% glycerol) for 20 min at room temperature. Samples were separated onto a 6% polyacrylamide gel for 1 hour at 4°C and analyzed by autoradiography. The same conditions were used for footprinting, with the addition of 62.5 ng/ml DNaseI (Worthington Biochemical) for 30 seconds at room temperature after incubation in the binding buffer. DNaseI treatments were stopped by the addition of 0.4 M sodium acetate, 50 μg ml⁻¹ sonicated calf thymus DNA, and 2.5 mM EDTA, before DNA purification by phenol extraction and ethanol precipitation. Purified DNA from each reaction were adjusted to load an equivalent number of radiolabeled product (5 × 10⁴ c.p.m. equivalent) on 6% polyacrylamide/7 M urea sequencing gels. Maxam and Gilbert reactions (A + G) on P_busAB was carried out as control and gels were analyzed by autoradiography.

RNA isolation and quantification

Total RNA were extracted from exponentially growing cells (OD₆₀₀ = 0.4) in TH at 37°C (FastRNA ProBlue, MP Biomedicals) and residual DNA removed with the TURBO DNase (Ambion / Thermo Fischer Scientific). RNA were quantified (Nanodrop 2000, Thermo Fischer) before reverse transcription (iScript cDNA synthesis, Bio-Rad). Quantitative PCR (qPCR) was carried out using specific primer pairs (S8 Table) and EvaGreen Universal qPCR Supermix (Bio-Rad) in a CFX96 apparatus (Bio-Rad). Relative quantification of specific gene expression was calculated with the ΔΔCq method, with gyrA as the housekeeping reference gene. Results are normalized against the WT strain and each assay was performed in triplicate on three independent cultures.