The discovery and study of toxin-antitoxin (TA) systems helps us advance our understanding of the strategies prokaryotes employ to regulate cellular processes related to the general stress response, such as defense against phages, growth control, biofilm formation, persistence, and programmed cell death. Here we identify and characterize a TA system found in various bacteria, including the global pathogen Mycobacterium tuberculosis. The toxin of the system (DarT) is a domain of unknown function (DUF) 4433, and the antitoxin (DarG) a macrodomain protein. We demonstrate that DarT is an enzyme that specifically modifies thymidines on single-stranded DNA in a sequence-specific manner by a nucleotide-type modification called ADP-ribosylation. We also show that this modification can be removed by DarG. Our results provide an example of reversible DNA ADP-ribosylation, and we anticipate potential therapeutic benefits by targeting this enzyme-enzyme TA system in bacterial pathogens such as M. tuberculosis.

We searched for novel ADP-ribosylation systems in bacterial genomes and identified an operon that encodes a conserved protein containing a distinct type of macrodomain () associated with an uncharacterized protein domain annotated as DUF4433 ( Figure 1 A). The DUF4433 and macrodomain operon are found in diverse bacterial species, including pathogens like Mycobacterium tuberculosis (Mtb) and Klebsiella pneumoniae, cyanobacteria, and extremophiles such as Thermus aquaticus (Taq). Interestingly, the orthologous operon from the opportunistic human pathogen Pseudomonas mendocina was identified as a TA system by a recent high-throughput screen (). Moreover, the genetic screens in Mtb indicate that the macrodomain ortholog (Rv0060) is an essential gene in this organism, whereas the toxin component (Rv0059) is dispensable (). Macrodomains are well-described protein modules that bind or hydrolyze the ADPr moiety attached to different substrates and control many important cellular processes (). Strikingly, despite no obvious homologies based on primary sequence comparisons, our initial 3D modeling attempts suggested that DUF4433 might be an ADP-ribosyltransferase related to PARPs and NAD-dependent toxins (). From this, we hypothesized this TA system operates via transfer of ADPr moieties onto target molecules and sought to uncover its exact molecular function.

(B) Images of bacterial growth at room temperature of BL21(DE3) with pBAD TaqToxin E160A and empty pET (Toxin E160A), pBAD TaqToxin and empty pET (Toxin), empty pBAD and pET TaqAntitoxin (Antitoxin), or pBAD TaqToxin and pET TaqAntitoxin (Toxin Antitoxin). Plates were supplemented with glucose and IPTG for induction of expression from pET vector, or arabinose and IPTG for expression from both pET and pBAD vectors.

(A) Schematic representation of the operon and surrounding genomic loci of the TA system in different bacteria. DUF, domain of unknown function; Macro, macrodomain. Scale bar represents length of 1 kb. Numbers correspond to the domain boundaries of the protein amino acid sequence according to Pfam.

ADP-ribosylation is a chemical modification of macromolecules via transfer of an ADP-ribose (ADPr) moiety from NADonto molecular targets (usually proteins). ADP-ribosylation regulates many processes in eukaryotes (), and recent studies suggest this modification might play important roles in bacterial metabolism ().

The recent discoveries of a number of distinct TA systems have highlighted how diverse these systems are, with different systems sensing different stimuli and targeting different biological processes (). This variety allows TA systems to subtly regulate distinct metabolic pathways to best survive different stress conditions (). Studying TA systems has greatly enhanced our understanding of the diversity of evolutionary strategies that regulate cellular processes in prokaryotes, but they are also recognized as potential drug targets and as useful tools in biotechnological applications ().

Toxin-antitoxin (TA) systems are sets of two or more closely linked genes that together encode a toxic protein as well as a corresponding neutralizing antidote. TA systems were first reported as small loci on plasmids known as “addiction modules,” where they ensure the conservation of the genomic makeup of bacterial populations by killing those daughter cells that have lost the TA encoding plasmids (). Subsequently, chromosomal TA systems were found to be widely distributed in bacteria and archaea () and have been shown to regulate antiphage defense, biofilm formation, dormancy, pathogenicity, persistence, and virulence () by reducing the metabolism of some cells within a population to a dormant state or inducing other adaptations that enable the bacteria to survive environmentally unfavorable conditions until conditions improve ().

Results and Discussion

Finn et al., 2016 Finn R.D.

Coggill P.

Eberhardt R.Y.

Eddy S.R.

Mistry J.

Mitchell A.L.

Potter S.C.

Punta M.

Qureshi M.

Sangrador-Vegas A.

et al. The Pfam protein families database: towards a more sustainable future. We focused on Mtb and Taq as representative species containing the TA proteins of interest. While antitoxin proteins were cloned and expressed routinely, we were unable to clone the toxin components by conventional cloning approaches. This was likely due to their toxicity in E. coli even at the minute levels of toxin transcription/translation. However, we were able to clone the Taq (but not the Mtb) wild-type (WT) toxin using a repressed arabinose-inducible promoter. First, we confirmed that the Taq proteins behave as a TA pair ( Figure 1 B) by showing that E. coli cells expressing the WT toxin did not grow unless the antitoxin was co-expressed. In addition, when we substituted a single completely conserved glutamate residue that is predicted to be critical for DUF4433 activity (), E160A in Taq protein, we observed the mutant construct was non-toxic. In short, neither the antitoxin nor the inactive toxin mutant alone impaired bacterial growth ( Figures 1 B and S1 A, available online).

Next, we checked whether this TA system could exert bacteriostatic effect. Cells co-transformed with both the Taq toxin and antitoxin genes, and allowed to express only the toxin for half an hour before expression was inhibited again, did not form colonies when plated out. However, when the same cells were plated on antitoxin-inducing plates, the cell growth was restored ( Figure S1 B). If the toxin expression was allowed to continue for more than 1 hr, cell growth could not be restored by plating them on antitoxin-inducing plates. As observed earlier, cells expressing an inactive toxin or with repressed toxin expression did not show toxicity ( Figure S1 B).

To exclude the possibility that the effect of the Taq toxin is specific to the E. coli strain used, we also induced Taq toxin expression in WT E. coli K-12 strain MG1655 and observed that induction of Taq toxin results in inhibition of growth on agar plates ( Figure S1 C).

To investigate the biochemical activities of the Taq TA system components, we used the same expression system and purified recombinant proteins from E. coli ( Figure 1 C). The tag used for purification did not affect the toxin’s toxicity in E. coli ( Figure S1 D).

32P-NAD+ ( Figure 2 TaqToxin/DarT ADP-Ribosylates ssDNA Oligonucleotides on Thymidines with Sequence Preference Show full caption (A) Autoradiography of denaturing polyacrylamide gel analyzing TaqToxin ADP-ribosylation modification reactions using short oligonucleotide (GJ1) or its complementary sequence (GJ1rc) as substrates in the presence of 32P-NAD+. (B) UV detection of ethidium bromide-stained denaturing polyacrylamide gels separating reactions as in (A) in the presence of NAD+. (C) Mass spectra of modified (top) and non-modified (bottom) 9-mer GJ4-Ts oligonucleotide. The double- and triple-charged molecular ions are clearly detected. The shift of m/z values of the molecular ions for the modified oligonucleotide corresponds to ADP-ribosylation. (D) Diagnostic ion magnification of tandem mass spectrometry (MS/MS) spectra of the oligonucleotides as in (C). Relevant fragments are indicated on the side, while the key fragment is highlighted in orange. The difference between modified and non-modified fragments corresponds to the size of the ADP-ribosyl moiety. To identify substrates for the ADP-ribosylation activity of the Taq toxin, we analyzed different fractions of bacterial cells, i.e., protein extracts, total bacterial RNA, or denatured genomic DNA (gDNA), as possible acceptors of this modification and incubated them with the Taq toxin in the presence ofP-NAD Figure 1 D). We detected no effect in reactions containing protein extracts or total RNA when compared to the buffer control. However, we observed that the reaction with denatured genomic DNA retained a radioactive signal at the origin of TLC plates, suggesting ADP-ribosylation. The effect seemed specific for single-stranded DNA (ssDNA), as we did not observe presumed ADP-ribosylation when we used non-denatured, double-stranded DNA ( Figure S1 E). We confirmed this observation by utilizing defined, short ssDNA fragments as substrates by three different in vitro assays ( Figures 2 A, 2B, and S1 F). Interestingly, whereas one short oligonucleotide was efficiently modified, an oligonucleotide of the reverse complementary sequence produced only a minor signal, hence suggesting sequence specificity of the toxin. In contrast to other ADP-ribosyl transferases, we did not detect toxin automodification under the various conditions tested ( Figure S1 G). Altogether, we concluded that ssDNA is a direct target of the toxin reaction.

To further explore the sequence specificity of the toxin, we used a selection of various oligonucleotides as substrates for the toxin. Oligonucleotides as short as eight bases could still be modified ( Figures S2 A and S2B). Global analysis of the oligonucleotides that could be efficiently modified revealed the presence of a TNTC motif. Substitutions of any of these key nucleotides abolished ADP-ribosylation of oligonucleotide ( Figure S2 C), whereas nucleotide substitutions outside the motif did not alter the modification efficiency ( Figure S2 D). An RNA oligonucleotide containing a UNUC motif could not be modified by the toxin ( Figure 2 E). Furthermore, the strict DNA specificity and the importance of the thymidine base were supported by the observation that the toxin did not modify the oligonucleotides where thymidines were substituted with deoxyuridines ( Figure S2 F).

To pinpoint the exact position of the nucleotide modification, we employed mass spectrometry. The mass shift between the modified and non-modified oligonucleotides indicated ADP-ribosylation ( Figure 2 C), and the modified base was unambiguously identified as the second thymidine in the TNTC motif ( Figures 2 D and S2 G). However, the exact atom that is modified remains to be determined. To our knowledge, this represents the first report of a thymidine base being ADP-ribosylated, and we propose naming the DUF4433 enzyme as DarT for DNA ADP-ribosyl transferase.

Knowing that DarT is a DNA ADP-ribosyl transferase, we wanted to observe its effect on several biological pathways in bacteria. First, we tried to establish if DNA ADP-ribosylation could induce DNA damage signaling via the SOS response. Indeed, we observed that TaqDarT induction in MG1655 cells induced the SOS response, as observed by increasing RecA levels over time ( Figure S2 H). As expected, in DH5α cells RecA levels remained constant due to the genetically abrogated SOS response of this strain ( Figure S2 H), which indicates that activation of the SOS response cannot be the sole reason for DarT-mediated growth inhibition.

We next considered that DNA ADP-ribosylation could also affect DNA replication, which we tested by measuring BrdU incorporation after TaqDarT induction. As expected, cells expressing WT TaqDarT, but not the E160A mutant, incorporated less BrdU ( Figure S2 I). The effect was particularly strong in DH5α cells, where almost no BrdU could be detected minutes after TaqDarT induction, whereas in MG1655 cells the effect became evident 1 hr after TaqDarT induction, maybe due to lower levels of TaqDarT expression in MG1655, or attenuation of the effect due to the activated SOS response. We concluded that DarT expression affects DNA replication.

Rack et al., 2016 Rack J.G.

Perina D.

Ahel I. Macrodomains: structure, function, evolution, and catalytic activities. Barkauskaite et al., 2015 Barkauskaite E.

Jankevicius G.

Ahel I. Structures and mechanisms of enzymes employed in the synthesis and degradation of PARP-dependent protein ADP-ribosylation. Figure 3 Antitoxin Macrodomain De-ADP-Ribosylates DarT-ADP-Ribosylated Oligonucleotides Show full caption (A) Autoradiographs of denaturing polyacrylamide gel (top) or TLC plates (bottom) separating antitoxin reactions with 32P-NAD+ ADP-ribosylated oligonucleotide as substrate. Macro, macrodomain construct. ADPr standard reaction corresponds to poly-ADPr glycohydrolase-treated PARP1 reaction. (B) Orthogonal view of TaqDarG-macro (cartoon) bound to ADPr (sticks). (C) Topological diagram of the DarG macrodomain structures. (D) Structural comparisons between TaqDarG-macro (yellow cartoon) bound to ADPr (green sticks) showing Lys80 (yellow sticks) and TARG1 (maroon cartoon; PDB: 4J5S ) showing a covalent lysyl-ADPr adduct (magenta and maroon sticks). (E) Close up of the active site of TaqDarG-macro showing the residues involved in ADPr binding. The ADPr ligand is shown with its 2F o -F c electron density contoured at 1σ. (F) UV detection of ethidium bromide-stained denaturing polyacrylamide gel separating de-ADP-ribosylation reactions of TaqDarT ADP-ribosylated oligonucleotide by different TaqDarG-macro mutants. Reaction time in minutes is indicated at the top. Unmodified and ADP-ribosylated oligonucleotides were used as markers of migration. (G) Images of bacterial growth at room temperature of BL21(DE3) with pBAD TaqDarT and pET vector encoding TaqDarG, DarG K80A, DarG-macro, or DarG-macro K80A. Plates were supplemented with glucose and IPTG for induction of expression from pET vector, or arabinose and IPTG for expression from both pET and pBAD vectors. We next focused on the antitoxin. Given the previously identified de-ADP-ribosylation activities of different macrodomains (), we tested whether the antitoxin containing the macrodomain could reverse DNA ADP-ribosylation. Incubation of ADP-ribosylated oligonucleotide with either the full-length antitoxins or truncations containing only the macrodomain resulted in the loss of modification ( Figures 3 A, top, and S3 A) and the release of free ADPr as described for other ADP-ribosylation-removing macrodomains () ( Figure 3 A, bottom). These results suggest that this TA pair acts via reversible DNA ADP-ribosylation, and we propose naming the antitoxin DarG for DNA ADP-ribosyl glycohydrolase.

10 -helical element ( Sharifi et al., 2013 Sharifi R.

Morra R.

Appel C.D.

Tallis M.

Chioza B.

Jankevicius G.

Simpson M.A.

Matic I.

Ozkan E.

Golia B.

et al. Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease. Sharifi et al., 2013 Sharifi R.

Morra R.

Appel C.D.

Tallis M.

Chioza B.

Jankevicius G.

Simpson M.A.

Matic I.

Ozkan E.

Golia B.

et al. Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease. Table 1 Data Collection, Phasing, and Refinement Statistics apo-TaqDarG-macro ADPr-TaqDarG-macro apo-MtbDarG-macro Data Collection Wavelength (Å)/beam line 0.98999/I02 0.97625/I04-1 0.97625/I04-1 Detector Pilatus 6M Pilatus 2M Pilatus 2M Space group C2 P2 1 2 1 2 1 P2 1 2 1 2 1 a (Å) 103.83 37.41 68.84 b (Å) 45.11 60.40 75.45 c (Å) 35.62 76.74 116.12 α (°) 90.00 90.00 90.00 β (°) 101.25 90.00 90.00 γ (°) 90.00 90.00 90.00 Content of asymmetric unit 1 1 4 Resolution (Å) 41.16–1.67 60.39–2.50 59.22–2.17 (1.71–1.67) (2.60–2.50) (2.23–2.17) R sym (%) a a R sym = Σ|/−</>|/Σ/, where / is measured density for reflections with indices hkl. 5.5 (69.1) 4.4 (15.3) 8.4 (230.2) I/σ(I) 18.2 (2.0) 25.0 (7.0) 15.2 (1.5) Completeness (%) 96.6 (79.0) 98.2 (85.8) 99.2 (98.4) Redundancy 6.5 (4.9) 6.7 (5.0) 13.2 (13.2) CC 1/2 (%) (77.3) (99.2) (65.8) Number of unique reflections 18,354 (1,097) 6,306 (593) 32,503 (2,345) Refinement R cryst (%) b b R cryst = Σ||Fobs| − |Fcalc||/Σ|Fobs|. 17.2 19.5 21.0 R free (%) c c R free has the same formula as R cryst , except that the calculation was made with the structure factors from the test set. 20.3 24.4 25.1 RMSD bond length (Å) 0.017 0.012 0.013 RMSD bond angle (°) 1.57 1.60 1.49 Number of Atoms Protein 1,250 1,228 4,712 Water 136 17 74 Chloride ion 3 1 3 Glycerol 12 0 0 ADPr 0 36 0 Average B Factor Protein (Å2) 14.3 33.4 44.7 Water (Å2) 40.3 43.8 66.3 Chloride ion (Å2) 38.1 56.5 81.7 Glycerol (Å2) 51.5 N/A N/A ADPr (Å2) N/A 40.1 N/A Ramachandran Plot Favored 96.5 98.0 97.3 Allowed 3.5 1.4 2.0 Disallowed 0 0.7 0.7 Data for the highest resolution shell are given in parentheses. To get a better understanding of the antitoxin function, we determined the high-resolution X-ray crystal structures of the Taq and Mtb DarG macrodomains (TaqDarG-macro and MtbDarG-macro) in a ligand-free or ADPr-bound form. ( Figures 3 B and S4 A–S4C; Table 1 ). TaqDarG-macro and MtbDarG-macro share the same overall structure with an RMSD (root-mean-square deviation) of 0.89 Å over 149 α-carbons and a 56.4% sequence identity. The DarG macrodomain adopts a typical macrodomain fold composed of a six-stranded mixed β sheet sandwiched between four α helices and one 3-helical element ( Figures 3 B and 3C). It is structurally most similar to TARG1 ( Figure 3 D), a eukaryotic enzyme that possesses protein de-ADP-ribosylation activity and shares the overall shape of the DarG-macro ligand-binding pocket as well as the position of the ligand within it (). ADPr-TaqDarG-macro displays an RMSD of 1.85 Å over 137 α-carbons and a sequence identity of 28% when compared to TARG1 (chain A of PDB: 4J5S ) ( Figure 3 D). Similarly, TARG1 and apo-MtbDarG-macro display an RMSD of 1.68 Å over 128 α-carbons with a sequence identity of 23%. The ligand-binding pocket of the DarG macrodomain is formed by four surface loops and the bound ADPr moiety in ADPr-TaqDarG-macro forms hydrogen bonds with N8, L9 T20, N22, V31, Q34, T79, G117, G119, N120, and G121 ( Figures 3 E and S4 A). W83 lies at the end of the active site that is close to the distal ribose of the ADPr moiety, and the equivalent position is occupied by A90 in TARG1. If this were the “entrance” of the ADP-ribosylated nucleotide to the active site, W83 would be in a position to stack with the thymine ring of the ADP-ribosylated thymidine moiety, putting it into the right position to allow K80 access to the thymidine-ribose bond ( Figure 3 E). K80 is in the equivalent position of the main catalytic lysine residue of TARG1 and is proposed to act as a nucleophile that attacks the ribose-C1″ position and releases the glutamate residue of the acceptor protein in TARG1, forming a covalent lysyl-ADPr intermediate that may be decomposed via hydrolysis by D125 to release the ADPr product (). The calculated electrostatic surface maps reveal that residues surrounding this area of the active site are mostly positively charged in DarG ( Figures S4 D–S4F) and could therefore potentially be involved in binding the negatively charged ssDNA substrate.

Sharifi et al., 2013 Sharifi R.

Morra R.

Appel C.D.

Tallis M.

Chioza B.

Jankevicius G.

Simpson M.A.

Matic I.

Ozkan E.

Golia B.

et al. Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease. To probe the requirements for the de-ADP-ribosylation activity of DarG, we devised constructs with substitutions of conserved and ADPr pocket-facing amino acids ( Figures 3 E, S4 A, and S4G). Most of the mutations reduced the activity of the macrodomain, suggesting possible contributions to substrate binding ( Figure 3 F). While some of the mutations (H82A and W83A) showed little or no effect on the de-ADP-ribosylation activity of TaqDarG after 21 min, others (N22A, K29E, G119E, and K80A) had marked inhibitory effects. Interestingly, mutation of K80, the equivalent of the main catalytic lysine residue in TARG1, showed the most significant effect on substrate turnover out of all the mutants tested and resulted in inactive TaqDarG, indicating that this feature is conserved between TARG1 and DarG (). N22A showed the most significant effect on substrate turnover after K80A, and because of its location and the effect of its mutation on the enzyme’s activity, it might be involved in the positioning and binding of the ADP-ribosylated thymidine moiety ( Figures 3 E and S4 A). The reduced catalytic activity observed in the G119E mutant is most likely due to the position of the residue in one of the loops involved in ligand binding. This loop undergoes a conformational change between residues 117 to 122 in order to grasp the ADPr moiety upon ligand binding, with a maximum distance variation of 7.66 Å between the α-carbon of Gly121 in both states ( Figures S4 B and S4C).

Yamaguchi et al., 2011 Yamaguchi Y.

Park J.H.

Inouye M. Toxin-antitoxin systems in bacteria and archaea. We tested the importance of TaqDarG’s catalytic residue K80 in rescue experiments. In contrast to the WT full-length TaqDarG or TaqDarG-macro, the K80A mutants of TaqDarG did not rescue the toxic effects of TaqDarT expression ( Figure 3 G). The TaqDarG K80A mutant seemed to allow minor growth of bacteria at 37°C ( Figure S3 B), but not to the same extent as WT TaqDarG or TaqDarG-macro. Taken together, this shows that the macrodomain is sufficient to act as an antitoxin to DarT and suggests that full-length DarG might additionally inhibit DarT through protein-protein interaction, as is common for type II TA systems (). In support of this, we observed a stable interaction between TaqDarT and TaqDarG, as judged by size exclusion chromatography ( Figure S3 C). We also observed a significant inhibition of the DNA ADP-ribosylation reaction in the presence of the TaqDarG K80A mutant. In contrast, TaqDarG-macro K80A did not inhibit the reaction ( Figure S3 D). We conclude that the protein-protein interaction might provide another layer of DarT regulation, in addition to the reversal of the DNA ADP-ribosylation by DarG macrodomain hydrolytic activity.

Figure 4 Reversible ADP-Ribosylation Is Conserved in Mycobacterium tuberculosis TA System Show full caption (A) Autoradiography of denaturing polyacrylamide gel (top) and TLC plate (bottom) separating ADP-ribosylation and de-ADP-ribosylation reactions of in vitro-translated toxins (indicated at the top) containing GJ1 oligonucleotide as substrate. De-ADP-ribosylation reactions were supplemented with the indicated antitoxins. (B) Model of DarTG-catalyzed reversible DNA ADP-ribosylation and its effects. Having uncovered the reversible DNA ADP-ribosylation activity of the TaqDarTG TA system, we wanted to test whether the same mechanism is conserved in Mtb. Since our attempts to clone WT MtbDarT were unsuccessful, we translated the toxin in vitro. Importantly, we confirmed that the Mtb DarTG proteins exhibit DNA ADP-ribosyltransferase and hydrolase activities toward the same substrates as the Taq proteins ( Figure 4 A). Taken together, our data show that the DNA ADP-ribosylating toxin and de-ADP-ribosylating antitoxin activities are conserved between Taq and Mtb, and likely among other orthologous TA systems.

Nakano et al., 2015 Nakano T.

Takahashi-Nakaguchi A.

Yamamoto M.

Watanabe M. Pierisins and CARP-1: ADP-ribosylation of DNA by ARTCs in butterflies and shellfish. To our knowledge, our data reveal the first example of a reversible DNA modification via ADP-ribosylation and show that this biochemistry can be employed by TA systems ( Figure 4 B). This suggests that DNA ADP-ribosylation might be more prevalent than previously thought. Previously, irreversible DNA ADP-ribosylation has been demonstrated only in a distinct family of toxins called pierisins (). Unlike pierisins, which modify guanidines, the DarTG system modifies thymidines reversibly with high substrate specificity. As such, DarTG is well suited to tightly control physiological processes in microbes by interfering with DNA replication or transcription.

Wen et al., 2014 Wen Y.

Behiels E.

Devreese B. Toxin-antitoxin systems: their role in persistence, biofilm formation, and pathogenicity. We have shown that the DarTG system is able to induce bacteriostatic effects ( Figure S1 B) and that DNA replication is affected by DarT expression ( Figure S2 I), which could be the underlying principle of growth arrest caused by DarT. This makes it tempting to speculate that the function of such a reversible TA system could be persistence induction, since the state could be reversed by enzymatic activity. However, other functions for DarTG cannot be excluded because it could also play a role in anti-phage defense, where ssDNA would be an attractive specificity-determining factor, or it could act as an addiction module used to preserve the integrity of genomic loci, as is sometimes suggested for TA systems ().

DarTG is hard to place within one of the current types of TA systems. On one hand, the DarG antitoxin interacts with and seems to inhibit the DarT toxin, as is common in type II systems. On the other hand, DarG also acts on the target of DarT, thereby resembling type IV TA system. However, while both of these systems comprise a protein antitoxin, DarG is an enzyme, which makes DarTG different from either type II or IV and may warrant the creation of a new TA system type.

An interesting observation is that DarTG is often inserted in type I restriction modification system operons ( Figure 1 A). This raises the possibility of DNA methylation and ADP-ribosylation crosstalk, which further studies should address. An alternative explanation could be that a TA insertion in the locus serves as a stabilizer for the type I restriction modification system operon locus, as discussed above.