Editors' ChoiceMicrobiology

A DNA-ribosylating toxin and its antidote

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Sci. Signal.  03 Jan 2017:
Vol. 10, Issue 460, eaam6835
DOI: 10.1126/scisignal.aam6835

A bacterial toxin-antitoxin system acts through reversible ADP-ribosylation of single-stranded DNA.

Toxin-antitoxin (TA) systems, in which a bacterium can express both a toxic protein and the antidote to the toxin, are widespread in bacteria, where they may be involved in regulating stress responses by limiting cellular proliferation and metabolism. Jankevicius et al. identified a TA system in which the toxin protein acts by ADP-ribosylating the bacterium’s own single-stranded DNA at specific sequences and the antitoxin counteracts this reaction by two distinct mechanisms. Several pathogenic and extremophile species, including Mycobacterium tuberculosis (Mtb) and Thermus aquaticus (Taq), contain an operon in which genes (named darT and darG by the authors) contained domains known to be involved in ADP-ribosylation. When Taq darT was inducibly expressed in Escherichia coli, the bacterium failed to grow, whereas expressing both darT and darG rescued normal growth. Furthermore, growth could still be rescued if darG was induced within an hour of inducing expression of darT. Neither expression of mutated darT nor expression of darG alone impaired growth, leading the authors to conclude that DarT was a toxin and DarG an antitoxin. When different bacterial macromolecular fractions were incubated with purified recombinant DarT, single-stranded DNA (ssDNA) was ADP-ribosylated but not proteins or RNA. In a set of defined ssDNA oligonucleotides, DarT only modified those containing a TNTC motif, in which N is any nucleotide. Mass spectrometry revealed that the second thymidine was ribosylated, in contrast to the only other known DNA ADP-ribosylation reaction, in which guanine is irreversibly modified. Within minutes of expressing DarT, cells incorporated less synthetic thymidine analog, suggesting that the bacteriostatic effect of DarT is due to interference with DNA replication. ADP-ribosylated oligonucleotides incubated with full-length DarG or with a specific catalytic portion called the macrodomain lost the modification and released ADP-ribose, suggesting that DarG can specifically reverse the modification carried out by DarT. Expression of the macrodomain of DarG in cells expressing DarT was sufficient to rescue growth at room temperature, but expression of the DarG macrodomain with mutations in catalytic sites was not. However, catalytically inactive DarG enabled limited bacterial growth at 37°C and also inhibited the ADP-ribosylation reaction in vitro when co-incubated with DarT and oligonucleotides, suggesting that DarG may have an additional noncatalytic role in preventing DarT toxicity. The same reactions were carried out by in vitro transcribed DarT and DarG proteins from Mtb. Although ADP-ribosylation of macromolecules in host cells is a known mechanism of virulence in pathogenic bacteria, this study identified a distinct variation of this modification that occurred on a specific site in bacterial single-stranded DNA and was reversible. Harms and Gerdes discuss the roles these reactions may play in their endogenous settings, for example by allowing pathogenic bacteria to enter a period of latency.

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