## 3 Discussion  
Here we investigated the role of DACs and c-di-AMP in the mutagenic processes occurring during growth as well as in stationary-phase cells of _B. subtilis_ subjected to amino acid starvation. To this end, we independently or simultaneously disabled _disA_ and/or _cdaA_ encoding the two DACs that synthesize c-di-AMP in vegetative cells. Overall, the results revealed that c-di-AMP can play divergent roles counteracting GAM but stimulating SAM, thus, maintaining the fidelity of the genome or promoting genetic diversity as affected by the metabolic status of the cell. As _cdaA_ is the first gene of the _cdaA-cdaR-glmM-glmS_ operon [9; 21], we reasoned that replacement of this gene with an antibiotic cassette could abolish expression of the downstream essential genes _glmM_ and _glmS_, which are required for _B. subtilis_ cell wall synthesis [21; 22]. To avoid polar effects on these genes, we generated a construct in the integrative vector pMUTIN-4 that disrupted _cdaA_ through a single cross-over recombination event and allowed expression of the _cdaR-glmM-glmS_ cistrons from an IPTG-inducible _Pspac_ promoter [23]. Following this approach, we successfully disrupted _cdaA_ in the parental strain YB955 and an isogenic DisA-deficient strain (Figure S1). However, during selection of the transformant colonies and culturing in complex media, both strains required IPTG for a robust growth (Figure S1). Thus, this strategy avoids polar effects on _glmM-glmS_ and generated viable strains with disruptions in _cdaA_ and/or _disA_, in the genetic background _B. subtilis_ YB955, a prophage-cured strain that contains the _hisC952_, _metB5_, and _leuC427_ alleles [18]. It must be noted that previous reports showed that _B. subtilis_ strains deficient for _disA_ and _cdaA_ as well as a triple _disA/cdaA/cdaS_ mutant were able to grow in minimal medium with low concentrations of potassium [9; 10]. c-di-AMP has been shown to regulate important cellular processes in _B. subtilis_, including potassium and cell wall homeostasis, DNA-damage checkpoint events during sporulation and spore/germination outgrowth and protection against genotoxic compounds [8; 9; 10; 11; 12; 13; 14; 15; 16; 24]. Our results provide evidence for a novel function of this metabolite in modulating bacterial mutagenesis. Notably, the levels of c-di-AMP inversely correlated with the degree of spontaneous RifR mutagenesis in growing _B. subtilis_ cells. Consistently, the _disA_ strain, which showed higher levels of this metabolite, produced less mutations than the _cdaA_ strain. Furthermore, the disruption of both, _disA_ and _cdaA_, which dramatically reduced the production of c-di-AMP, resulted in the highest levels of spontaneous RifR mutagenesis in growing _B. subtilis_ cells. These results suggest that the levels of c-di-AMP may activate cellular processes that modulate spontaneous mutagenic events in growing _B. subtilis_ cells. A previous report showed that CdaA but not DisA, protects vegetative cells of _B. subtilis_ from hydrogen peroxide [16], a mutagenic agent that causes oxidative DNA damage and promotes several types of base substitutions [25]. Here we found that DisA counteracted the mutagenesis induced by H\({}_{2}\)O\({}_{2}\); contrastingly, CdaA promoted this type of mutagenesis. The checkpoint properties of DisA can be activated by genetic insults of oxidative nature or its repair intermediates [14; 15]. Accordingly, during _B. subtilis_ spore outgrowth, the absence of AP-endonucleases, which are base excision repair components that process ROS-promoted DNA lesions, activates the checkpoint function of DisA and protects the outgrowing cells [14]. This is in agreement with what we showed here; the loss of DisA can negatively impact the ability of _B. subtilis_ to withstand the mutagenic effects promoted by H\({}_{2}\)O\({}_{2}\). In contrast to DisA, CdaA is a membranal dimeric protein with no predicted functions in DNA repair, which plays critical roles in osmotic regulation and cell wall maintenance [9; 10; 24].