The DarT/G toxin-antitoxin system encodes a pair of enzymes that mediate the addition of an ADP-ribose moiety onto thymidine in ssDNA in a reversible, sequence specific manner. Although originally characterised in Thermus aquaticus, the system is present in a number of important pathogens including all members of the mammal adapted M. tuberculosis complex, notably including human and bovine TB. Utilizing CRISPRi technology to silence DarG antitoxin expression, we have shown that DarT performs ADP-ribosylation of gDNA in cellulo in M. bovis BCG, leading to a rapid arrest of DNA replication and cell division, and that is ultimately toxic to the bacterium. In MTBC, darT and darG are transcriptionally linked to the dnaB gene, which encodes the replicative helicase that interacts with ssDNA at the chromosome origin (OriC) to initiate then drive DNA branch migration during replication. We demonstrate in vitro and in cellulo that MTBC DarT heavily ADP-ribosylates TTTW motifs in the AT-rich DnaB-loading region of OriC, suggesting that the DarTG system may work as a reversible regulator of replication. Furthermore, unregulated ADP-riboslyation by DarT induces the DNA damage SOS response, including the ImuA’ImuB/DnaE2 mutasome which has been implicated in DNA damage-induced mutagenesis and acquisition of resistance to antibiotics.
Immunopurification and NGS sequencing of ADP-riboslyated gDNA fragments has given further insight into the role of ADP-riboslyation in M. tuberculosis physiology, confirming ADP-ribosylation of OriC and demonstrating ADP-ribosylation at additional genomic loci, prominently including genes involved in the SOS response, DNA metabolism, and ribosomal proteins. This identifies the potential for ADP-ribosylation to act as a genome-wide epigenetic and cell signalling factor.
We aim to further understand the role of DarTG in bacterial physiology including DNA replication, the DNA damage response, persistence and drug-resistance in Mycobacterium tuberculosis.