Cyclic di-AMP regulates genome stability and drug resistance in <i>Mycobacterium</i> through RecA-dependent and RecA-independent recombination

In <i>Escherichia coli</i>, RecA plays a central role in the rescue of stalled replication forks, double-strand break (DSB) repair, homologous recombination (HR), and induction of the SOS response. While the RecA-dependent pathway is dominant, alternative HR pathways that function independently of RecA do exist, but relatively little is known about the underlying mechanism. Several studies have documented that a variety of proteins act as either positive or negative regulators of RecA to ensure high-fidelity HR and genomic stability. Along these lines, we previously demonstrated that the second messenger cyclic di-AMP (c-di-AMP) binds to mycobacterial RecA proteins, but not to <i>E. coli</i> RecA, and inhibits its DNA strand exchange activity in vitro via the disassembly of RecA nucleoprotein filaments. Herein, we demonstrate that <i>Mycobacterium smegmatis ΔdisA</i> cells, which lack c-di-AMP, exhibit increased DNA recombination, higher frequency of mutation, and gene duplications during RecA-dependent and RecA-independent DSB repair. We also found that c-di-AMP regulates SOS response by inhibiting RecA-mediated self-cleavage of LexA repressor and its absence enhances drug resistance in <i>M. smegmatis ΔdisA</i> cells. Together, our results uncover a role of c-di-AMP in the maintenance of genomic stability through modulation of DSB repair in <i>M. smegmatis</i>.