Convergence of DNA methylation and phosphorothioation epigenetics in bacterial genomes

Abstract

Explosive growth in the study of microbial epigenetics has revealed a diversity of chemical structures and biological functions of DNA modifications in restriction-modification (R-M) and basic genetic processes. Here, we describe the discovery of shared consensus sequences for two seemingly unrelated DNA modification systems, ^6mA methylation and phosphorothioation (PT), in which sulfur replaces a nonbridging oxygen in the DNA backbone. Mass spectrometric analysis of DNA from Escherichia coli B7A and Salmonella enterica serovar Cerro 87, strains possessing PT-based R-M genes, revealed d(G_PS^6mA) dinucleotides in the G_PS^6mAAC consensus representing ∼5% of the 1,100 to 1,300 PT-modified d(G_PSA) motifs per genome, with ^6mA arising from a yet-to-be-identified methyltransferase. To further explore PT and ^6mA in another consensus sequence, G_PS^6mATC, we engineered a strain of E. coli HST04 to express Dnd genes from Hahella chejuensis KCTC2396 (PT in G_PSATC) and Dam methyltransferase from E. coli DH10B (^6mA in G^6mATC). Based on this model, in vitro studies revealed reduced Dam activity in G_PSATC-containing oligonucleotides whereas single-molecule real-time sequencing of HST04 DNA revealed ^6mA in all 2,058 G_PSATC sites (5% of 37,698 total GATC sites). This model system also revealed temperature-sensitive restriction by DndFGH in KCTC2396 and B7A, which was exploited to discover that ^6mA can substitute for PT to confer resistance to restriction by the DndFGH system. These results point to complex but unappreciated interactions between DNA modification systems and raise the possibility of coevolution of interacting systems to facilitate the function of each.

Keywords: DNA methylation; DNA phosphorothioation; bioanalytical chemistry; epigenetics; restriction–modification.

Fig. 1.

LC-MS/MS analysis of isolated and synthetic d(G_PS^6mA). (A) Naturally occurring d(G_PS^6mA) R_P in E. coli B7A (Upper) and S. enterica serovar Cerro 87 (Lower) was eluted at 29.5 min, identical to the retention time of synthetic d(G_PS^6mA) (C) in the R_P configuration. (B) The major fragment ions of d(G_PS^6mA) observed in LC-MS/MS analysis are shown in the structural insert, with the protonated molecular ion [M+H] appearing at m/z 611.

Fig. 2.

Dam enzymatic activity with different oligonucleotide substrates. (A) LC-MS/MS analysis of the Dam-methylated products using duplex oligonucleotides possessing normal GATC (dsDNA-noPT), single-stranded G_PSATC (dsDNA-ssPT), and double-stranded G_PSATC (dsDNA-dsPT) as substrates. The oligonucleotides are listed in Table S1. Dam recognizes G_PSATC in both R_P and S_P configurations. (B) Time course of the Dam enzymatic reaction with dsDNA-noPT, dsDNA-ssPT, and dsDNA-dsPT; 4 units of Dam were incubated with 20 pmol of a DNA substrate in 10 µL of 1× dam methyltransferase buffer supplemented with 80 µM SAM for 0 to 90 min at 37 °C, followed by inactivation at 65 °C for another 15 min. ^6mA was measured by LC-MS/MS at the indicated times (SI Materials and Methods).

Fig. 3.

PT and methylation site mapping across the E. coli HST04 genome. From outer to inner circles: Circles 1 and 2, (forward, reverse strands) 2,058 G_PSATC sites in E. coli HST04 (dndBCDE) detected by SMRT in ORFs (black) and nonencoding regions (green). No site is detected in noncoding RNA. These sites were almost completely converted to methylation-PT double modification sites in E. coli HST04 (dndBCDE, dam). Circles 3 and 4, methylation sites E. coli HST04 (dndBCDE, dam) (excluding sites in circles 1 and 2) in ORFs (gray), noncoding RNA (blue), and nonencoding regions (red). Circles 5 and 6, predicted protein-coding sequences colored according to cluster of orthologous groups of proteins (COG) function categories. Circle 7, tRNA/rRNA operons. Circle 8, GC content. Circle 9, GC skew.

Fig. S1.

Estimated probability functions of IPD ratios for three types of modifications. G_PSATC in E. coli HST04(dndBCDE), G_PS^6mATC, and G^6mATC in E. coli HST04(dndBCDE, dam) are denoted by black, green, and blue, respectively.

Fig. 4.

Growth and cell viability analysis of bacterial strains. (A and B) Effect of pWHU705, expressing DndFGH of H. chejuensis KCTC2396, on the growth of E. coli HST04 at 37 °C (A) and 15 °C (B) in liquid LB broth (Upper) and on LB-agar plates (Lower). E. coli HST04 harboring empty pBluescript SK+ or pACYCDuet-1 plasmid was used as the control. (C) Cell viability assessment to compare protection mediated by DNA methylation and PT modification against DndFGH restriction. Exponentially growing cells at 37 °C were appropriately diluted and plated onto fresh LB-agar plates, which were incubated at 37 °C or 15 °C for cfu counting. Error bars indicate the SDs of three independent experiments.

Fig. S2.

Growth analysis of WT B7A and the in-frame deletion mutant. Growth was assessed at 37 °C (A) and 15 °C (B) both in LB broth (Upper) and on LB-agar plates (Lower).

Fig. S3.

Summary of the interaction and interference between DNA methylation and PT modification. The diagram illustrates the idea of a DNA methyltransferase and the DndABCDE proteins sharing the same site in DNA to generate a hybrid modification motif, d(G_PS^6mA). In a bacterium possessing Dam MTase and the DndABCDE proteins similar to those from H. chejuensis KCTC 2396, both sets of proteins recognize the same GATC motif to generate hybrid G_PS^6mATC structures, with G_PSATC and G^6mATC able to replace each other to protect against restriction by DndFGH, respectively. A similar situation may exist for the d(G_PS^6mA) hybrid modification in the GAAC motif in E. coli B7A and S. enterica serovar Cerro 87.