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Thymidine hypermodification pathways, intermediates, cofactors, and products. Pathways of thymidine hypermodification (A). The thymidine hypermodifications discussed in this work utilize 5-hmdU, which is incorporated into DNA through steps occurring before and during DNA replication (as diagrammed in the grayed box) and proceed via a 5-PmdU common intermediate. Solid arrows and bolded enzyme names indicate experimentally verified activities, parentheses contain accession IDs for enzymes, and predicted molecular weights follow the abbreviations for the indicated nucleotide/nucleoside products. (B) HPLC/MS traces of nucleotide mixtures derived from mock treated 5-hmdU substrate DNA (lower trace) and M6 gp54 and ATP. Note, these samples were prepared by enzymatic hydrolysis of DNA in the absence of phosphatase activity. (C) HPLC/MS traces of nucleoside mixtures derived from reactions of 5-hmdU with M6 NedU biosynthetic enzymes and cosubstrates. Traces of no enzyme substrate DNA, synthetic N -GlyT and native M6 gDNA included for comparison. (D), HPLC/MS traces of nucleoside mixtures derived from reactions of 5-hmdU with ViI 5-NeOmdU biosynthetic enzymes. Traces from synthetic N -SerT (an isomer of O-SerT) and native ViI genomic DNA nucleosides included for comparison to enzymatically produced intermediates and final products, respectively. (E) HPLC/MS traces of nucleoside mixtures derived from reactions with PaMx11 5-AcNmdU biosynthetic enzymes. No enzyme DNA substrate control, and synthetic 5-NmdU standard shown for comparison.
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The DNAs of bacterial viruses are known to contain diverse, chemically complex modifications to thymidine that protect them from the endonuclease-based defenses of their cellular hosts, but whose biosynthetic origins are enigmatic. Up to half of thymidines in the Pseudomonas phage M6, the Salmonella phage ViI, and others, contain exotic chemical mo...
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... diverse hypermodified thymidines occur in the bacteriophages W-14, SP10, ViI, M6 and others (8- 11), the structures of which are shown in Figure 1A. ...
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... these latter two phages, the modifying substituents are connected to thymine through an ether (5-NeOmdU) or a C-C bond (5-NedU). The structures of these four hypermodified thymidines are shown at the pathway termini in Figure 1A. The chemical diversity of these modifications suggests an underlying diversity of enzymatic mechanisms involved in their formation. ...
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... intracellular development, phages W-14, SP10, ViI and M6 initially synthesize DNA containing 5-hmdU fully replacing thymidine using mechanisms similar to those of the Bacillus 'hmU phages' such as SPO1, 2C, SP8 and e, which encode a suite of metabolic functions that eliminate dTTP from the deoxynucleotide triphosphate (dNTP) pool of their host and replace it with 5-hydroxymethyl-2 -deoxyuridine triphosphate (5-hmdUTP) (12). This pre-replicative pathway is depicted in the grayed boxed region of Figure 1A. Central to this pathway is 2 -deoxyuridine monophosphate (dUMP) hydroxymethylase, a virally encoded thymidylate synthase homolog producing 5-hydroxymethyl-2 -deoxyuridine monophosphate (5-hmdUMP) from dUMP (13)(14)(15). ...
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... DNA recovered from purified virions of SP10, W-14, M6 and ViI contain no 5-hmdU. Instead, these thymidine hypermodifying phages convert 5-hmdU in sequence-specific contexts to either a hypermodified base ( Figure 1A) or canonical thymine prior to packaging of the viral DNA into the phage capsids (8,(19)(20)(21). Conversion of 5-hmdU to the hypermodified base in W-14 and SP10 was reported to proceed by pyrophosphorylation of 5-hmdU in the DNA polymer (9,22). ...
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... previous work by Aravind et al., comparative genomics was combined with highly sensitive homology detection to bioinformatically identify candidate DNA nucleobase kinases active on 5-hmdU in W-14 and SP10 genomes (23) (see also Figure 1A). Homologs of these genes were also found to be encoded in other phages, including M6 and ViI. ...
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... of these genes were also found to be encoded in other phages, including M6 and ViI. We cloned, expressed and purified gp54, the putative 5-hmdU DNA kinase of M6 (Supplementary Figure S1). Catalytic activity of M6 gp54 was assessed by incubating with genomic DNA containing 5-hmdU isolated from the Bacillus bacteriophage SP8 in the presence of 1 mM ATP. Phage SP8 genomic DNA fully substitutes thymidine with 5-hmdU and, thus, presents this non-canonical base in a variety of sequence contexts. ...
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... seen in Figure 1B, following treatment of the SP8 genomic DNA with purified M6 gp54, a new peak containing a species of 418 u (nominal mass) nucleotide eluting at ∼5 min was produced. A corresponding decrease in the amount 5-hmdUMP, which elutes at ∼10 min and exhibits a nominal mass of 388 u was observed, as can be determined from comparing to the integrated peak areas of the dA as an internal control. ...
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... evidence from the lysate reconstitution experiments suggesting that phage M6 gp51 and ViI gp247, both annotated as clade 2 aGPT-Pplases, catalyze group transfer to a 5-PmdU DNA substrate, we set out to reconstitute these enzymes' activities from purified components in vitro. Purified M6 gp51 incubated with free glycine and 5-hmdU DNA previously treated with 5-hmU DNA kinase produced a new peak in the LC-MS traces with nominal mass of 315 u, demonstrating that the monophosphorylated thymine is chemically competent for further enzymatic modification ( Figure 1C). To unambiguously show that glycine was being appended to the nucleobase, when the experiment was repeated with glycine-1,2-13 C 2 , a product of 317 u was observed (Supplementary Figure S9) confirming the addition of isotopically labeled glycine. ...
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... M6 gp51 modification reactions using glycine doubly deuterated at the alpha carbon (i.e. glycine-2,2-d 2 ) produced a species of 317 u showing retention of both deuterons ( Supplementary Fig- ure S10). These data suggest that an N-C bond is formed by appending the nucleophilic -amine of glycine to the base to produce 5-N α -glycinylthymidine (N α -GlyT) on DNA. ...
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... data suggest that an N-C bond is formed by appending the nucleophilic -amine of glycine to the base to produce 5-N α -glycinylthymidine (N α -GlyT) on DNA. To test this possibility, N α -GlyT was synthesized by reductive amination of 5-formyl-2 -deoxyuridine as described in the Supplementary methods and Supplementary Figure S11. As seen in Figure 1C and Supplementary Figure S11, this compound had identical mass and retention time to the nucleoside enzymatically produced by M6 gp51, suggesting that this intermediate is derived from 5-PmdU in the pathway leading to 5-NedU, as depicted in Figure 1A. ...
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... test this possibility, N α -GlyT was synthesized by reductive amination of 5-formyl-2 -deoxyuridine as described in the Supplementary methods and Supplementary Figure S11. As seen in Figure 1C and Supplementary Figure S11, this compound had identical mass and retention time to the nucleoside enzymatically produced by M6 gp51, suggesting that this intermediate is derived from 5-PmdU in the pathway leading to 5-NedU, as depicted in Figure 1A. ...
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... test this possibility, N α -GlyT was synthesized by reductive amination of 5-formyl-2 -deoxyuridine as described in the Supplementary methods and Supplementary Figure S11. As seen in Figure 1C and Supplementary Figure S11, this compound had identical mass and retention time to the nucleoside enzymatically produced by M6 gp51, suggesting that this intermediate is derived from 5-PmdU in the pathway leading to 5-NedU, as depicted in Figure 1A. ...
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... test this possibility, N α -GlyT was synthesized by reductive amination of 5-formyl-2 -deoxyuridine as described in the Supplementary methods and Supplementary Figure S11. As seen in Figure 1C and Supplementary Figure S11, this compound had identical mass and retention time to the nucleoside enzymatically produced by M6 gp51, suggesting that this intermediate is derived from 5-PmdU in the pathway leading to 5-NedU, as depicted in Figure 1A. ...
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... acid transferase activity was also demonstrated in reactions with purified ViI gp247 ( Figure 1D). A similar reaction, but with L-serine-( 13 C 3 , 15 N) produced a nucleoside with the expected 4 u additional mass (Supplementary Figure S12). ...
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... acid transferase activity was also demonstrated in reactions with purified ViI gp247 ( Figure 1D). A similar reaction, but with L-serine-( 13 C 3 , 15 N) produced a nucleoside with the expected 4 u additional mass (Supplementary Figure S12). The native ViI hypermodification contains an ether linkage between an ethanolamine moiety and the C5 methyl of thymidine, suggesting serine is initially appended to the base via its sidechain hydroxyl group to produce the nucleobase 5-O-serinylthymidine (O-SerT). ...
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... based on mass alone, we could not initially rule out incorporation of serine by ViI gp247 into DNA through substitution reaction via the relatively more nucleophilic serine -amine to produce 5-N -serinylthymidine (N -SerT). To rule out this latter possibility, a synthetically more accessible nucleoside standard consisting of serine modified thymidine containing an N-C linkage was synthesized by reductive amination of 5-formyluridine as described in the Supplementary Methods and in the scheme illustrated in Supplementary Figure S13. As seen in Figure 1D, this synthetic compound had a different retention time to the ViI enzymatic product, despite having identical mass, thus ruling out this isomer and indicating 5-O-serinylthymidine as the likely intermediate produced by ViI gp247 on the pathway to 5-NeOmdU as shown in Figure 1A. ...
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... rule out this latter possibility, a synthetically more accessible nucleoside standard consisting of serine modified thymidine containing an N-C linkage was synthesized by reductive amination of 5-formyluridine as described in the Supplementary Methods and in the scheme illustrated in Supplementary Figure S13. As seen in Figure 1D, this synthetic compound had a different retention time to the ViI enzymatic product, despite having identical mass, thus ruling out this isomer and indicating 5-O-serinylthymidine as the likely intermediate produced by ViI gp247 on the pathway to 5-NeOmdU as shown in Figure 1A. ...
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... rule out this latter possibility, a synthetically more accessible nucleoside standard consisting of serine modified thymidine containing an N-C linkage was synthesized by reductive amination of 5-formyluridine as described in the Supplementary Methods and in the scheme illustrated in Supplementary Figure S13. As seen in Figure 1D, this synthetic compound had a different retention time to the ViI enzymatic product, despite having identical mass, thus ruling out this isomer and indicating 5-O-serinylthymidine as the likely intermediate produced by ViI gp247 on the pathway to 5-NeOmdU as shown in Figure 1A. ...
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... alignment of aGPT-Pplases highlighting this conserved cysteine and nearby glutamate is shown in Figure 3A. Site-directed mutation of either the cysteine or the proximal glutamate to alanine resulted in a loss of activity in both M6 gp51 and ViI gp247 in our in vitro assay (Supplementary Figure S14). If an aGPT-Pplase2 active site cysteine does form a covalent bond with C6 of 5-PmdU with concomitant formation of an exocyclic methylene, then one might expect that in the absence of the natural co-substrate the methylene might be accessible to exogenously added nucleophile or reductant. ...
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... shown in sequence 1 of Fig- ure 2, M6 and related phages encode two ORFs, gp52 (PLP) and gp53 (rSAM), in between the genes encoding gp51 (aGPT-Pplase2) and gp54 (P-loop kinase), which we have shown here to synthesize N α -GlyT. The co-occurrence of these genes within the presumptive 5-NedU biosynthetic gene cluster across multiple related phage genomes (see also Supplementary Figure S15) strongly suggests that they are functionally linked. Sequence comparisons indicate that gp52 likely encodes a PLP-dependent enzyme whereas gp53 is a member of the radical SAM (rSAM) superfamily. ...
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... resulting protein solution was brown in color, a characteristic likely due to a [Fe 4 S 4 ] cofactor associated with the enzyme. UV-Vis spectra of purified M6 gp53 before and after reconstitution showed an increase at ∼410 nm characteristic of iron-sulfur cluster containing proteins (Supplementary Figure S16). Iron quantitation of purified M6 gp53 yielded ∼9 Fe per protein, consistent with two 4Fe4S clusters, an observation supported by the occurrence of a second possible iron-sulfur binding motif in the M6 gp53 protein sequence. ...
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... M6 gp53 was incubated with N α -GlyT-containing DNA oligos under reducing conditions and monitored for the formation of novel bases using LC-MS. As shown in Figure 1C, under these conditions, a new nucleoside was formed having a slightly shorter retention time but having the same mass (315 u) as the N α -GlyT substrate. Control experiments where SAM or reductants were omitted prevented the reaction from occurring (Supplementary Figure S17). ...
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... shown in Figure 1C, under these conditions, a new nucleoside was formed having a slightly shorter retention time but having the same mass (315 u) as the N α -GlyT substrate. Control experiments where SAM or reductants were omitted prevented the reaction from occurring (Supplementary Figure S17). ...
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... fractions containing these proteins displayed a yellowish hue and their UV-Visible absorbance spectra showed peaks in the 410-425 nm range characteristic of PLP-dependent enzymes ( Supplementary Fig- ure S18). As shown in Figure 1C, incubation of C α -GlyTcontaining DNA with purified M6 gp52 produced a nucleoside with identical retention time and mass to those of the native M6 5-NedU. ...
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... fractions containing these proteins displayed a yellowish hue and their UV-Visible absorbance spectra showed peaks in the 410-425 nm range characteristic of PLP-dependent enzymes ( Supplementary Fig- ure S18). As shown in Figure 1C, incubation of C α -GlyTcontaining DNA with purified M6 gp52 produced a nucleoside with identical retention time and mass to those of the native M6 5-NedU. ViI gp226 was analogously able to decarboxylate a substrate containing O-SerT leading to a nucleoside with nominal mass 301 u ( Figure 1D). ...
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... shown in Figure 1C, incubation of C α -GlyTcontaining DNA with purified M6 gp52 produced a nucleoside with identical retention time and mass to those of the native M6 5-NedU. ViI gp226 was analogously able to decarboxylate a substrate containing O-SerT leading to a nucleoside with nominal mass 301 u ( Figure 1D). Decarboxylation of stable isotope labeled O-SerT containing three 13 C and one 15 N resulted in the loss of a single carbon as evidenced by the formation of a 304 u nucleoside (Supplementary Figure S19). ...
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... gp226 was analogously able to decarboxylate a substrate containing O-SerT leading to a nucleoside with nominal mass 301 u ( Figure 1D). Decarboxylation of stable isotope labeled O-SerT containing three 13 C and one 15 N resulted in the loss of a single carbon as evidenced by the formation of a 304 u nucleoside (Supplementary Figure S19). In contrast, gp52 did not react with the N α -GlyT isomer produced by M6 gp51 (Supplementary Figure S7). ...
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... PaMx11-like phages of Pseudomonads are closely related to M6 and encode a similar gene cluster (sequences 6-8 in Figure 2; see also Supplementary Figure S15) that likely installs a hypermodified thymine derivative in the genomes of these viruses. The presence of 5-HMUDK and clade 2 aGPT-Pplase genes in these genomes at locations syntenic to phage M6 indicates that these phages likely utilize N α -glycinylthymidine as a precursor in the synthesis of a hypermodified base. ...
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... identify the hypermodified thymine generated by the PaMx11 gene cluster, we purified the putative FAD-dependent oxidoreductases gp47 of PaMx11 and its associated acetyltransferase, gp48. Chromatographic fractions containing PaMx11 gp47 displayed a yellowish hue and UV-Vis absorption spectra displayed absorbance peaks at 365 nm and 450 nm characteristic of FAD-dependent enzymes (Supplementary Figure S21) (47). We next incubated substrate oligonucleotides containing N α -GlyT residues with purified PaMx11 gp47 and/or gp48 either alone or in combination. ...
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... on the predicted functions of the enzymes, we supplemented the reactions with FAD and acetylCoA to maximize the likelihood of observing unique nucleosides. As shown in Figure 1E, the combination of both enzymes produced a new nucleoside with a ∼28.5 min retention time and a 299 u nominal mass. Reactions containing gp48 alone exhibited no reaction with N α -GlyT-containing oligos, whereas gp47 produced a new nucleoside with a ∼9.5 min retention time and a 257 u mass ( Figure 1E). ...
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... shown in Figure 1E, the combination of both enzymes produced a new nucleoside with a ∼28.5 min retention time and a 299 u nominal mass. Reactions containing gp48 alone exhibited no reaction with N α -GlyT-containing oligos, whereas gp47 produced a new nucleoside with a ∼9.5 min retention time and a 257 u mass ( Figure 1E). Treatment of this product oligo with gp48 recapitulated the 299 u nucleoside and, thus, established this enzyme as the likely ultimate step in the biosynthetic pathway of the unique hypermodified thymine in phage PaMx11. ...
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... example, we found contigs whose gene content and organization ( Figure 2, sequences 2 through 5) were identical or nearly identical to phage M6 (Figure 2, sequence 1) and therefore likely to make 5-NedU. Similarly, we found contigs (Figure 2, sequences 7 and 8) corresponding to phage PaMx11 (Figure 2, sequence 6) as well as SP10 (Figure 2, sequences 11 through 13). Among the contigs were interesting fusions between a 5-HMUDK and other hypermodification genes (Figure 2, sequences 14 through 16) such as a PLP-dependent enzyme and a triple fusion of 5-HMUDK, PLP-dependent decarboxylase, and a clade 2 aGPT-Pplase. ...
Citations
... On the other hand, 2-oxoglutarate/Fe(II)-dependent oxygenase (2-OG oxygenase) including JBP1 and JBP2 are responsible for conversion of thymidine into 5hmU at the DNA polymer level in kinetoplastids 15 . Similarly, some phages can also utilize TET/JBP family proteins to install 5hmU in their chromatin after DNA synthesis 16 . However, the exact route of 5hmU generation in dinoflagellates remained an unsolved mystery. ...
Dinoflagellate chromosomes are extraordinary, as their organization is independent of architectural nucleosomes unlike typical eukaryotes and shows a cholesteric liquid crystal state. 5-hydroxymethyluridine (5hmU) is present at unusually high levels and its function remains an enigma in dinoflagellates chromosomal DNA. Here, we demonstrate that 5hmU exhibits content variations in different dinoflagellates and is generated at the poly-nucleotide level through hydroxylation of thymidine. Importantly, we identified the enzyme, which is a putative dinoflagellate TET/JBP homologue, catalyzing 5hmU production using either in vivo or in vitro biochemical assay. Based on the near-chromosomal level genome assembly of dinoflagellate Amphidinium carterae, we depicted a comprehensive 5hmU landscape and found that most 5hmU peaks share a conserved TG-rich motif, and are significantly enriched in repeat elements, which mark partially overlapping regions with 5-methylcytosine (5mC) sites. Moreover, inhibition of 5hmU via dioxygenase inhibitor leads to transcriptional activation of 5hmU-marked transposable elements (TEs), implying that 5hmU appears to serve as epigenetic marks for silencing retrotransposon. Together, our results revealed the biogenesis, genome-wide landscape and molecular function of dinoflagellate 5hmU, providing mechanic insight into the function of this enigmatic DNA mark.
... Certain Salmonella phages possess the ability to undergo thymidine modifications, resulting in hyper-modification of their DNA. This serves as a protective mechanism against cleavage by host restriction endonucleases [44]. Upon examination of the SW16-7 genome, as per the PFAM database, we discerned five CDSs (CDS81, CD145, CD15, CDS82, CDS11) that could potentially participate in thymidine modifications. ...
Salmonella enterica serovar Weltevreden is a foodborne pathogen commonly transmitted through fresh vegetables and seafood. In this study, a lytic phage, SW16-7, was isolated from medical sewage, demonstrating high infectivity against S. Weltevreden, S. London, S. Meleagridis, and S. Give of Group O:3. In vitro inhibition assays revealed its effective antibacterial effect for up to 12 h. Moreover, analysis using the Comprehensive Antibiotic Resistance Database (CARD) and the Virulence Factor Database (VFDB) showed that SW16-7’s genome does not contain any virulence factors or antibiotic resistance genes, indicating its potential as a promising biocontrol agent against S. Weltevreden. Additionally, a TSP gene cluster was identified in SW16-7’s genome, with TSP1 and TSP2 showing a high similarity to lysogenic phages ε15 and ε34, respectively, in the C-terminal region. The whole-genome phylogenetic analysis classified SW16-7 within the Ackermannviridae family and indicated a close relationship with Agtrevirus, which is consistent with the ANI results.
... Recently, 7-deazaguanine deri vati v es hav e been found in DNA as components of restriction / modification systems in bacteria ( 4 , 5 ), and anti-restriction systems in phages ( 4 , 6 , 7 ). Epigenetic modifications are common among phages (8)(9)(10)(11) to resists to various bacterial defense systems (11)(12)(13)(14)(15)(16). ...
Bacteriophages and bacteria are engaged in a constant arms race, continually evolving new molecular tools to survive one another. To protect their genomic DNA from restriction enzymes, the most common bacterial defence systems, double-stranded DNA phages have evolved complex modifications that affect all four bases. This study focuses on modifications at position 7 of guanines. Eight derivatives of 7-deazaguanines were identified, including four previously unknown ones: 2′-deoxy-7-(methylamino)methyl-7-deazaguanine (mdPreQ1), 2′-deoxy-7-(formylamino)methyl-7-deazaguanine (fdPreQ1), 2′-deoxy-7-deazaguanine (dDG) and 2′-deoxy-7-carboxy-7-deazaguanine (dCDG). These modifications are inserted in DNA by a guanine transglycosylase named DpdA. Three subfamilies of DpdA had been previously characterized: bDpdA, DpdA1, and DpdA2. Two additional subfamilies were identified in this work: DpdA3, which allows for complete replacement of the guanines, and DpdA4, which is specific to archaeal viruses. Transglycosylases have now been identified in all phages and viruses carrying 7-deazaguanine modifications, indicating that the insertion of these modifications is a post-replication event. Three enzymes were predicted to be involved in the biosynthesis of these newly identified DNA modifications: 7-carboxy-7-deazaguanine decarboxylase (DpdL), dPreQ1 formyltransferase (DpdN) and dPreQ1 methyltransferase (DpdM), which was experimentally validated and harbors a unique fold not previously observed for nucleic acid methylases.
... In the meantime, phages are able to acquire new counter-attacks to keep up in this armed race [36]. Phages infecting Salmonella can overcome anti-phage defenses based on nucleic acid degradation by modifying their thymidine nucleotide bases [37,38]. Abortive infection can also be neutralized through mutations or deletion of specific phage regions that are recognized by the bacterial immunity system [31,39]. ...
Bacteriophages, which specifically infect and kill bacteria, are currently used as additives to control pathogens such as Salmonella in human food (PhageGuard S®) or animal feed (SalmoF-REE®, Bafasal®). Indeed, salmonellosis is among the most important zoonotic foodborne illnesses. The presence of anti-phage defenses protecting bacteria against phage infection could impair phage applications aiming at reducing the burden of foodborne pathogens such as Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) to the food industry. In this study, the landscape of S. Typhimurium anti-phage defenses was bioinformatically investigated in publicly available ge-nomes using the webserver PADLOC. The primary anti-phage systems identified in S. Typhi-murium use nucleic acid degradation and abortive infection mechanisms. Reference systems were identified on an integrative and conjugative element, a transposon, a putative integrative and mo-bilizable element, and prophages. Additionally, the mobile genetic elements (MGEs) containing a subset of anti-phage systems were found in the Salmonella enterica species. Lastly, the MGEs alone were also identified in the Enterobacteriaceae family. The presented diversity assessment of the anti-phage defenses and investigation of their dissemination through MGEs in S. Typhimurium constitute a first step towards the design of preventive measures against the spread of phage resistance that may hinder phage applications.
... installation by employing a reduced flavin as a waystation for the one-carbon from CH2THF to the base. However, the final step in the MnmEG reaction entails trapping the olefin by the amino group of glycine in contrast to hydride transfer from reduced flavin in the TrmFO reaction(56). Further, in mammalian MnmEG homologs, taurine substitutes for glycine(31).Besides tRNA modification, MnmG is also involved in other regulatory pathways. The gene encoding for MnmG is also potentially involved in regulating the pathogenicity and virulence of certain microbes. ...
The evolutionarily conserved bacterial proteins MnmE and MnmG collectively install a carboxymethylaminomethyl (cmnm) group at the fifth position of wobble uridines of several tRNA species. While the reaction catalyzed by MnmEG is one of the central steps in the biosynthesis of the methylaminomethyl (mnm) post-transcriptional tRNA modification, details of the reaction remain elusive. Glycine is known to be the source of the carboxy methylamino moiety of cmnm, and a tetrahydrofolate (THF) analog is thought to supply the one-carbon that is appended to the 5th position of U. However, the nature of the folate analog remains unknown. This manuscript reports the in vitro biochemical reconstitution of the MnmEG reaction. Using isotopically labelled methyl and methylene THF analogs, we demonstrate that methylene THF is the true substrate. We also show that reduced FAD is required for the reaction and that DTT can replace the NADH in its role as a reductant. We discuss the implications of these methylene-THF and reductant requirements on the mechanism of this key tRNA modification catalyzed by MnmEG.
... The top hits for similar genomes consisted of several Pseudomonas phages with 95 to 98% nucleotide identity (73 -96% query cover). Interestingly, phage SN1 has 96.76% nucleotide identity (91% query cover) with Pseudomonas phage M6 genome, which contains hypermodified thymines (reviewed in reference [10]). Half of the thymine residues in the M6 genome contain moieties synthesized through postreplicative modifications of 5-hydroxymethyl uridine. ...
... Half of the thymine residues in the M6 genome contain moieties synthesized through postreplicative modifications of 5-hydroxymethyl uridine. In M6-like phages, including SN1, the thymidine modification pathway includes several genes located upstream of the DNA polymerase gene (10). This cassette consists of genes that code for pyrimidine hyroxymethylase (Locus tag SN1_071), Nmad5 (SN1_019), aGPT-Pplase1 (SN1_020), nucleotide kinase (SN1_021), rSAM (SN1_022), pyridoxal-59-phosphate (PLP) dependent enzyme (SN1_023), and aGPT-Pplase2 (SN1_024). ...
Phage SN1 infects Sphaerotilus natans and Pseudomonas aeruginosa strains. Its genome consists of 61,858 bp (64.3% GC) and 89 genes, including 32 with predicted functions. SN1 genome is very similar to Pseudomonas phage M6, which contains hypermodified thymidines. Genome analyses revealed similar base-modifying genes as those found in M6. Phage SN1 was isolated in 1979 from activated sludge samples obtained from a wastewater treatment plant (Lincoln, Nebraska, USA) using S. natans ATCC 13338 as the host (1, 2). An early study showed that the siphophage SN1 has unusual bases in its genome as confirmed by cellulose thin-layer chromatography (1). Its genomic DNA also showed resistance to type II restriction endonucleases (2). Host range studies indicate that phage SN1 can also infect Pseudomonas aeruginosa strains PAO33 and OT684 (2). Here, phage SN1 was amplified with its host S. natans ATCC 13338 in nutrient broth (3 g/L beef extract, 5 g/L peptone) and agitated at 30°C (2). Cell debris were removed by filtration (0.45 mm) and filtrates were stored at 4°C until use. Phage SN1 also infected P. aeruginosa PAO1 (HER1153) in TSB/TSA medium at 30°C using both plaque assays and lysis of liquid cultures. Species identification of the above two host strains was confirmed by 16S sequencing.
... Purines and modified pyrimidines have been identified in bacteriophage DNA, but it is the pyrimidines that show the greatest diversity: uracil (U), 5-hydroxymethyluracil(hm5U), 5-hydroxymethyldeoxyuracil (hm5dU), α-glutamylthymine, α-putrescinylthymine, 5-dihydroxypentyluracil replace thymine in the DNA of Bacillus SP10 and Delftia W-14 bacteriophages, and 5-(2-aminoethoxy)methyluridine (NeOm5dU) and 5-(2-aminoethyl)uridine (Ne5dU) in the DNA of ViI and M6 phages [3], 5-methylcytosine (m5C), 5-hydroxycytosine, 5-hydroxymethylcytosine (hm5C) and glucosylhydroxymethylcytosine completely replace cytosine in the genomes of Escherichia coli T bacteriophages [4]. Biosynthetic pathways have been described for some of these bases: dU, hm5dU, NeOm5dU, Ne5dU, hm5C and glucosyl-m5C [3,5]. ...
Bacteriophage genomes are the richest source of modified nucleobases of any life form. Of these, 2,6 diaminopurine, which pairs with thymine by forming three hydrogen bonds violates Watson and Crick’s base pairing. 2,6 diaminopurine initially found in the cyanophage S-2L is more widespread than expected and has also been detected in phage infecting Gram-negative and Gram-positive bacteria. The biosynthetic pathway for aminoadenine containing DNA as well as the exclusion of adenine are now elucidated. This example of a natural deviation from the genetic code represents only one of the possibilities explored by nature and provides a proof of concept for the synthetic biology of non-canonical nucleic acids.
... Using mixtures of recombinant crude extracts derived from cultures expressing predicted hypermodification enzymes, Lee et al. were able to reconstitute the conversion of 5hmU to 5-NeOmdU in vitro (64). Further biochemical characterization of the 5hmU kinase did not lead to formation of a pyrophosphorylated thymidine (85). Nonetheless, monophosphorylated thymidine (PmdU) was shown to be chemically competent to serve as a substrate in the hypermodification reaction (85). ...
... Further biochemical characterization of the 5hmU kinase did not lead to formation of a pyrophosphorylated thymidine (85). Nonetheless, monophosphorylated thymidine (PmdU) was shown to be chemically competent to serve as a substrate in the hypermodification reaction (85). ...
... Using recombinant lysate-catalyzed hypermodification reactions in various combinations, Lee and coworkers were able to work out what enzymes were necessary and sufficient to hypermodify 5hmU (85). This hypermodification pathway is summarized in Fig. 7. ...
The DNA in bacterial viruses collectively contains a rich, yet relatively underexplored, chemical diversity of nucleobases beyond the canonical adenine, guanine, cytosine, and thymine. Herein, we review what is known about the genetic and biochemical basis for the biosynthesis of complex DNA modifications, also called DNA hypermodifications, in the DNA of tailed bacteriophages infecting Escherichia coli and Salmonella enterica. These modifications, and their diversification, likely arose out of the evolutionary arms race between bacteriophages and their cellular hosts. Despite their apparent diversity in chemical structure, the syntheses of various hypermodified bases share some common themes. Hypermodifications form through virus-directed synthesis of noncanonical deoxyribonucleotide triphosphates, direct modification DNA, or a combination of both. Hypermodification enzymes are often encoded in modular operons reminiscent of biosynthetic gene clusters observed in natural product biosynthesis. The study of phage-hypermodified DNA provides an exciting opportunity to expand what is known about the enzyme-catalyzed chemistry of nucleic acids and will yield new tools for the manipulation and interrogation of DNA.
Enzymatic modification of DNA nucleobases can coordinate gene expression, protection from nucleases, or mutagenesis. We recently discovered a new clade of phage-specific cytosine methyltransferase (MT) and 5-methylpyrimidine dioxygenase (5mYOX, e.g., TET) enzymes that produce 5-hydroxymethylcytosine (5hmC) as a precursor for additional post-replicative enzymatic hypermodifications on viral genomes. Here, we identify phage MT- and 5mYOX-dependent glycosyltransferase (GT) enzymes that catalyze linkage of diverse glycans directly onto 5hmC reactive nucleobase substrates. Using targeted bioinformatic mining of the phage metavirome databases, we discovered thousands of new biosynthetic gene clusters (BGCs) containing enzymes with predicted roles in cytosine sugar hypermodification. We developed a pathway reassembly platform for high-throughput functional screening of GT-containing BGCs, relying on the endogenous E. coli metabolome as a substrate pool. We successfully reconstituted a subset of phage BGCs and isolated novel and highly diverse sugar modifications appended to 5hmC, including mono-, di-, or tri-saccharide moieties comprised of hexose, N-acetylhexosamine or heptose sugars. Structural predictions and sugar product analyses suggest that phage GTs are related to host lipopolysaccharide, teichoic acid, and other small molecule biosynthesis enzymes and have been repurposed for DNA substrates. An expanded metagenomic search revealed hypermodification BGCs within gene neighborhoods containing phage structural proteins and putative genome defense systems. These findings enrich our knowledge of secondary modifications on DNA and the origins of corresponding sugar writer enzymes. Post-replicative cytosine hypermodification by virus-encoded GTs is discussed in the context of genome defense, DNA partitioning and virion assembly, and host-pathogen co-evolution.
While nucleic acid-targeting effectors are known to be central to biological conflicts and anti-selfish element immunity, recent findings have revealed immune effectors that target their building blocks and the cellular energy currency-free nucleotides. Through comparative genomics and sequence-structure analysis, we identified several distinct effector domains, which we named Calcineurin-CE, HD-CE, and PRTase-CE. These domains, along with specific versions of the ParB and MazG domains, are widely present in diverse prokaryotic immune systems and are predicted to degrade nucleotides by targeting phosphate or glycosidic linkages. Our findings unveil multiple potential immune systems associated with at least 17 different functional themes featuring these effectors. Some of these systems sense modified DNA/nucleotides from phages or operate downstream of novel enzymes generating signaling nucleotides. We also uncovered a class of systems utilizing HSP90- and HSP70-related modules as analogs of STAND and GTPase domains that are coupled to these nucleotide-targeting- or proteolysis-induced complex-forming effectors. While widespread in bacteria, only a limited subset of nucleotide-targeting effectors was integrated into eukaryotic immune systems, suggesting barriers to interoperability across subcellular contexts. This work establishes nucleotide-degrading effectors as an emerging immune paradigm and traces their origins back to homologous domains in housekeeping systems.
