## 2 Results
### DisA and CdaA Are Required for SAM in B. subtilis  
A previous study reported that _B. subtilis_ starved cells displaying an altered response to c-di-AMP and its associated metabolic factors, exhibited an unbalanced pool of dNTPs [20]. This observation prompted us to determine if c-di-AMP influenced SAM. Then, the _B. subtilis_ strains, harboring disruptions in _disA_ and/or _cdaA_ as well as the chromosomal auxotrophies _hisC952_, _metB5_ and _leuC427_, were used to determine frequencies of reversion to prototrophy under starving and growth-limiting conditions. Compared to the parental strain YB955, the inactivation of _disA_ or _cdaA_ led to a decreased production of colonies with a His\({}^{+}\), Met\({}^{+}\) or Leu\({}^{+}\) phenotype; of note, this effect was exacerbated in the strain disrupted for both, _cdaA_ and _disA_ (Figure 5). These effects can be attributed to the single or simultaneous disruption of _disA_ and _cdaA_; in support of this contention, the independent expression of these genes from an IPTG-inducible promoter reestablished the _hisC_, _metB_, and _leuC_ reversion frequencies to the levels of the strain YB955 (Figure 5). Furthermore, as expected, the production of His\({}^{+}\), Met\({}^{+}\) and Leu\({}^{+}\) prototrophs, in the _disA_/_cdaA_ strain, reached levels such as those exhibited by the strains with single disruptions, following reintegration of _disA_ or _cdaA_, respectively (Figure 5). Analyses of survival rates of the strains tested showed that despite small fluctuations, the colony forming units count kept constant, during the ten-day period of the experiments, confirming that the differences observed in the SAM assays were due to the loss of DisA, CdaA, or both proteins and not to variations in cell viability (Figure 6). Figure 4: Mytomycin-C induced mutation frequencies to rifampicin resistance (Rif\({}^{\text{R}}\)) of _Bacillus subtilis_ YB955 (open bar), _cdaA_ (dark grey bar), _cdaA_ (black bar) and _cdaA_/_cdaA_ (hatched bar) were determined during exponential growth. Complemented strains _cdaA AmyE:_Phs-disA_ (light stippled bar), _cdaA AmyE:_Phs-cdaA_ (horizontal striped bar), _cdaA/_cdaA AmyE:_Phs-cdaA_ (vertical striped bar) and _cdaA_/_cdaA AmyE:_Phs-disA_ (dark stippled bar), were also tested. Data show the average from three independent experiments, each one by sextuplicate \(\pm\) standard deviations. All strains were supplemented with IPTG 0.25 mM. Different letters (a, b, c) represent significant statistical differences between means, the same letter denotes no significant difference, two letters in the same bar (a, b) mean statistically equal to both, group a and group b, as determined by a one-way ANOVA and multiple mean comparison by Tukey HSD test (all _p_-values < 0.05). Figure 5: Frequencies of stress-associated reversions for _his, met_, and _leu_ mutant genes of _B. subtilis_ strains YB955 (open circles), _AcdaA_ (dark grey triangles), _AlisA_ (open triangles) _AcdaA_ / _AlisA_ (black triangles) _AdisA amyE::Phs-disA_ (filled diamonds), _AcdaA_ / _AdisA amyE::Phs-disA_ (open diamonds), _\(\Delta\)cdaA_ _amyE::Phs-cdaA_ (filled squares) and _AcdaA_ / _AlisA amyE::Phs-cdaA_ (open squares) as described in Materials and Methods. Data represent counts from six plates averaged from three independent experiments normalized to initial cell titers \(\pm\) standard deviations. All strains were supplemented with IPTG 0.25 mM. *** represent significant statistical differences compared to the parental strain, as determined by a one-way ANOVA and Tukey HSD test (all _p_-values \(<\) 0.001). Figure 6: Determination of cell viability throughout SAM aversion assays of _B. subtilis_ strains YB955, \(\Delta\)_cdaA_, _AdisA_ and \(\Delta\)_cdaA_ / _AlisA_, and of reintegrated strains _AcdaA amyE::Phs-cdaA_, \(\Delta\)_cdaA_ / _AlisA amyE::Phs-cdaA_, \(\Delta\)_cdaA amyE::Phs-disA_ and \(\Delta\)_cdaA_ / _AlisA amyE::Phs-disA_.