Stress-Associated and Growth-Dependent Mutagenesis Are Divergently Regulated by c-di-AMP Levels in Bacillus subtilis

A previous proteomic study uncovered a relationship between nutritional stress and fluctuations in levels of diadenylate cyclases (DACs) and other proteins that regulate DAC activity, degrade, or interact with c-di-AMP, suggesting a possible role of this second messenger in B. subtilis stress-associated mutagenesis (SAM). Here, we investigated a possible role of c-di-AMP in SAM and growth-associated mutagenesis (GAM). Our results showed that in growing cells of B. subtilis YB955 (hisC952, metB25 and leuC427), the DACs CdaA and DisA, which play crucial roles in cell wall homeostasis and chromosomal fidelity, respectively, counteracted spontaneous and Mitomycin-C-induced mutagenesis. However, experiments in which hydrogen peroxide was used to induce mutations showed that single deficiencies in DACs caused opposite effects compared to each other. In contrast, in the stationary-phase, DACs promoted mutations in conditions of nutritional stress. These results tracked with intracellular levels of c-di-AMP, which are significantly lower in cdaA- and disA-deficient strains. The restoration of DAC-deficient strains with single functional copies of the cdaA and/or disA returned SAM and GAM levels to those observed in the parental strain. Taken together, these results reveal a role for c-di-AMP in promoting genetic diversity in growth-limiting conditions in B. subtilis. Finally, we postulate that this novel function of c-di-AMP can be exerted through proteins that possess binding domains for this second messenger and play roles in DNA repair, ion transport, transcriptional regulation, as well as oxidative stress protection.