## 3 Discussion
Therefore, the promutagenic effects induced by H\({}_{2}\)O\({}_{2}\) in the _cdaA_-deficient strain could be indirect and derived from factors that compromise the homeostasis of the cell envelope. Accordingly, cell envelope stressors, including, antibiotics, heat, ethanol as well as H\({}_{2}\)O\({}_{2}\) activate the expression of genes under control of the extracytoplasmic function factor \(\sigma^{\text{M}}\) (ECM) [8; 26]. The transcription of _disA_ and _radaA_ is simultaneously induced following activation of the \(\sigma^{\text{M}}\) response [27]. Notably, _radaA_, whose encoding product has beenimplicated in repair of DNA damage and recombination, can also physically interact with DisA and counter its DAC activity [27]. This interaction may impact not only the levels of c-di-AMP but also the scanning activity of DisA [12; 13] and presumably the processing of ROS-promoted lesions by RadA. Therefore, as previously reported [15], the accumulation of oxidative DNA lesions and/or recombination intermediates can cause replication/transcription conflicts that elicit low-fidelity replication events and that promote RifR mutagenesis in the _cdaA_ strain. Our results showed that _disA_ and _cdaA_ play, independently, a minor role in the mutagenic events promoted by M-C; however, the double disruption of these genes significantly increased the mutagenesis levels, above to those exhibited by the strains bearing single disruptions in _disA_ or _cdaA_ (Figure 4). These results suggest that DisA and CdaA, influence the response to damage inflicted by bifunctional alkylating agents via independent pathways. Two possible scenarios must be considered to explain these results. Firstly, it has been reported that RuvB, which is part of the LexA regulon [28], antagonizes the DAC and DNA scanning activities of DisA, which eliminate potentially mutagenic lesions from replicating chromosomes [29]. We speculate that, in the absence of DisA, DNA interstrand crosslinks induced by M-C accumulate and activate the SOS-response [30], promoting RecA/RuvB recombination events [31], error-prone replication/repair and mutagenesis [15]. Secondly, the mutagenic effects promoted by M-C can be also attributed to _yqhB_, whose transcription is regulated by the general stress and SOS responses [21; 30; 32]. YqhB counteracts the noxious effects inflicted by oxidative and electrophilic stresses [21]. Results from bioinformatic analyses indicate that the stress protein YqhB, (i) adopts a dimeric structure and (ii) possesses a CBD domain that interacts with c-di-AMP and activates its oxidoreductase distance function ([PERSON] and [PERSON], Unpublished Results). Therefore, it is feasible to anticipate a direct impact on GAM in cells deficient in these cellular pathways. Previous reports suggest that the unfavorable metabolic conditions operating in starved and non-growing bacteria promote SAM [18; 19]. Here, we show that fluctuations in the levels of c-di-AMP, via inactivation of _disA_ and _cdaA_, impacted SAM and result in a dramatic decline in the reversion frequencies of the mutant alleles _hisC_, _metB_ and _leuC_ (Figure 5). These effects could be exerted through NrdR a transcriptional repressor of the _urdEF_ operon, which encodes _B. subtilis_ ribonucleotide reductase (RNR) [21; 33]. A previous report revealed that c-di-AMP can allosterically interact with NrdR and inactivate its repressor activity [24; 34]. In starved cells, inactivation of NrdR triggers _nrdEF_ transcription, RNR synthesis, and increases in the levels of the dNTP pools, which affects the fidelity of DNA synthesis [33]. In support of these contentions, a recent report, showed that the genetic disruption of _nrdR_ and the subsequent derepression of RNR, promoted _B. subtilis_ SAM [33]. DarB, another c-di-AMP target, could be implicated in _B. subtilis_ SAM. It was shown that the c-di-AMP-DarB complex interacts with RelA to regulate the synthesis of the alarmone (p)ppGpp [35].