No abstract

DNA double-strand breaks (DSB) in bacteria can be repaired by non-homologous end-joining (NHEJ), a two-component system relying on Ku and LigD. While performing a genetic characterization of NHEJ in Sinorhizobium meliloti, a representative of bacterial species encoding several Ku and LigD orthologues, we found that at least two distinct functional NHEJ repair pathways co-exist: one is dependent on Ku2 and LigD2, while the other depends on Ku3, Ku4 and LigD4. Whereas Ku2 likely acts as canonical bacterial Ku homodimers, genetic evidences suggest that Ku3-Ku4 form eukaryotic-like heterodimers. Strikingly, we found that the efficiency of both NHEJ systems increases under stress conditions, including heat and nutrient starvation. We found that this stimulation results from the transcriptional up-regulation of the ku and/or ligD genes, and that some of these genes are controlled by the general stress response regulator RpoE2. Finally, we provided evidence that NHEJ not only repairs DSBs, but can also capture heterologous DNA fragments into genomic breaks. Our data therefore suggest that NHEJ could participate to horizontal gene transfer from distantly related species, bypassing the need of homology to integrate exogenous DNA. This supports the hypothesis that NHEJ contributes to evolution and adaptation of bacteria under adverse environmental conditions.

Antibiotic resistance of Mycobacterium tuberculosis is exclusively a consequence of chromosomal mutations. Translesion synthesis (TLS) is a widely conserved mechanism of DNA damage tolerance and mutagenesis, executed by translesion polymerases such as DinBs. In mycobacteria, DnaE2 is the only known agent of TLS and the role of DinB polymerases is unknown. Here we demonstrate that, when overexpressed, DinB1 promotes missense mutations conferring resistance to rifampicin, with a mutational signature distinct from that of DnaE2, and abets insertion and deletion frameshift mutagenesis in homo-oligonucleotide runs. DinB1 is the primary mediator of spontaneous −1 frameshift mutations in homo-oligonucleotide runs whereas DnaE2 and DinBs are redundant in DNA damage-induced −1 frameshift mutagenesis. These results highlight DinB1 and DnaE2 as drivers of mycobacterial genome diversification with relevance to antimicrobial resistance and host adaptation.

DNA repair systems allow microbes to survive in diverse environments that compromise chromosomal integrity. Pathogens such as Mycobacterium tuberculosis must contend with the genotoxic host environment, which generates the mutations that underlie antibiotic resistance. Mycobacteria encode the widely distributed SOS pathway, governed by the LexA repressor, but also encode PafBC, a positive regulator of the transcriptional DNA damage response (DDR). Although the transcriptional outputs of these systems have been characterized, their full functional division of labor in survival and mutagenesis is unknown. Here, we specifically ablate the PafBC or SOS pathways, alone and in combination, and test their relative contributions to repair. We find that SOS and PafBC have both distinct and overlapping roles that depend on the type of DNA damage. Most notably, we find that quinolone antibiotics and replication fork perturbation are inducers of the PafBC pathway, and that chromosomal mutagenesis is codependent on PafBC and SOS, through shared regulation of the DnaE2/ImuA/B mutasome. These studies define the complex transcriptional regulatory network of the DDR in mycobacteria and provide new insight into the regulatory mechanisms controlling the genesis of antibiotic resistance in M. tuberculosis.