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

In Escherichia coli, 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 either as 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 binds to mycobacterial RecA proteins, but not E. coli RecA, and inhibits its DNA strand exchange activity in vitro via the disassembly of RecA nucleoprotein filaments. Herein, we demonstrate that Mycobacterium smegmatis {Delta}disA 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 M. smegmatis {Delta}disA cells. Together, our results uncover a role of c-di-AMP in the maintenance of genomic stability through modulation of DSB repair in M. smegmatis. SignificanceCyclic di-AMP is a second messenger present in bacteria and archaea and is implicated in a variety of functions in the cell, including DNA repair, cell wall metabolism, virulence, and gene expression. We show here that it maintains genome stability in Mycobacterium by regulating RecA-dependent and -independent DNA recombination pathways. It also regulates SOS response by inhibiting the self-cleavage of LexA by mycobacterial RecA. Absence of c-di-AMP leads to higher drug resistance in Mycobacterium.