Identification and biosynthesis of thymidine hypermodifications in the genomic DNA of widespread bacterial viruses

Abstract

Certain viruses of bacteria (bacteriophages) enzymatically hypermodify their DNA to protect their genetic material from host restriction endonuclease-mediated cleavage. Historically, it has been known that virion DNAs from the Delftia phage ΦW-14 and the Bacillus phage SP10 contain the hypermodified pyrimidines α-putrescinylthymidine and α-glutamylthymidine, respectively. These bases derive from the modification of 5-hydroxymethyl-2'-deoxyuridine (5-hmdU) in newly replicated phage DNA via a pyrophosphorylated intermediate. Like ΦW-14 and SP10, the Pseudomonas phage M6 and the Salmonella phage ViI encode kinase homologs predicted to phosphorylate 5-hmdU DNA but have uncharacterized nucleotide content [Iyer et al. (2013) Nucleic Acids Res 41:7635-7655]. We report here the discovery and characterization of two bases, 5-(2-aminoethoxy)methyluridine (5-NeOmdU) and 5-(2-aminoethyl)uridine (5-NedU), in the virion DNA of ViI and M6 phages, respectively. Furthermore, we show that recombinant expression of five gene products encoded by phage ViI is sufficient to reconstitute the formation of 5-NeOmdU in vitro. These findings point to an unexplored diversity of DNA modifications and the underlying biochemistry of their formation.

Keywords: DNA modification; bacteriophage; hypermodification; pyrimidine.

Fig. 1.

Examples of phage hypermodified pyrimidines and the generalized DNA thymidine (T) hypermodification pathway of phages SP10 and ΦW-14.

Fig. 2.

Genomic maps of the regions surrounding the 5-HMUDK gene of bacteriophages listed in Table 1. GenBank accession numbers as well as the name and host of the bacteriophages are indicated. Notably, 5-HMUDK coassociates with dUMP hydroxymethyltransferase (dU hmt), a member of the thymidylate synthase superfamily.

Fig. 3.

Restriction digests of modified and unmodified bacteriophage genomic DNAs. Genomic DNA extracted from the indicated bacteriophages was incubated with restriction enzymes AccI, EcoRI, HinfI, and NdeI. The predicted number of cut sites in each bacteriophage sequence is shown in parentheses next to the given enzyme; λ contains canonical DNA bases only. Phage SP8 DNA contains 5-hmdU, replacing thymidine. Phages SP10 and ΦW-14 contain hypermodified thymidines.

Fig. 4.

HPLC traces and MS analysis of bacteriophage M6 and ViI nucleosides. The trace in Top was obtained from bacteriophage λ to show the retention of canonical nucleosides. M6 and ViI show a fifth major peak corresponding to the hypermodified base. The protonated molecular ion detected for each hypermodified base is indicated as well as a hypothetical combination of atoms to account for the observed masses. dA, 2′-deoxyadenosine; dG, 2′-deoxyguanosine; dC, 2′-deoxycytidine; dT, thymidine.

Fig. 5.

Proposed structures of phage M6 (A) and ViI (B) modifications. 5-NedU and 5-NeOmdU are shown to be the actual modifications in this work.

Fig. 6.

CID pathways of protonated 5-hmdU, 5-mNmdU, 5-NedU, 5-heNmdU, and 5-NeOmdU. Proposed CID pathways are based on well-documented fragmentations for uracil derivatives.

Fig. 7.

In vitro reconstitution of 5-NeOmdU from recombinant bacterial lysates. Substrate DNA containing 5-hmdU incubated in mixed lysates from E. coli expressing ViI gp67, gp160, gp226, gp243, and gp247 contains a nucleotide product (301; denoted by the asterisk) of identical mass and retention time as the native modification of ViI. dA, 2’-deoxyadenosine; dG, 2’-deoxyguanosine; dC, 2’-deoxycytidine; dT, thymidine; rA, ribo adenosine.