Genomic mapping of phosphorothioates reveals partial modification of short consensus sequences

Abstract

Bacterial phosphorothioate (PT) DNA modifications are incorporated by Dnd proteins A-E and often function with DndF-H as a restriction-modification (R-M) system, as in Escherichia coli B7A. However, bacteria such as Vibrio cyclitrophicus FF75 lack dndF-H, which points to other PT functions. To better understand PT biology, we report two novel, orthogonal technologies to map PTs across the genomes of B7A and FF75 with >90% agreement: real-time (SMRT) sequencing and deep sequencing of iodine-induced cleavage at PT (ICDS). In B7A, we detect PT on both strands of GpsAAC/GpsTTC motifs, but with only 18% of 40,701 possible sites modified. In contrast, PT in FF75 occurs as a single-strand modification at CpsCA, again with only 14% of 160,541 sites modified. Single-molecule analysis indicates that modification could be partial at any particular genomic site even with active restriction by DndF-H, with direct interaction of modification proteins with GAAC/GTTC sites demonstrated with oligonucleotides. These results point to highly unusual target selection by PT modification proteins and rule out known R-M mechanisms.

Convergence of DNA methylation and phosphorothioation epigenetics in bacterial genomes

“…The observation of PT modifications in 2,058 of the 37,698 GATC sites (5%) in E. coli HST04 (dndBCDE) is similar to previous SMRT analyses in E. coli B7A (12% of GAAC/GTTC) and V. cyclitrophicus FF75 (14% of CCA) (23), with the 228 ± 4 PT per 10 6 nt measured by LC-MS/MS analysis corroborating the quantitative nature of SMRT mapping. However, the map of the G PS ATC sites across the genome (GenBank accession no.…”

“…The chemical diversity of DNA modifications in bacteria has recently grown beyond methylation with the discovery of 7-deazaguanine modifications (2) and PT modifications of the DNA backbone (12,15). We have shown that PTs function in R-M systems with DndFGH (22,23,31) but that PTs are also present in bacteria that lack restriction genes. Although it is well-established that many bacteria possess multiple R-M systems and MTases, each with unique methylation consensus sequences (3, 32), we have discovered a proximity of two diverse DNA modifications in the same core consensus sequence, with interactions that point to a functional cooperation of the two modification systems.…”

“…For example, the application of liquid chromatography-coupled tandem quadrupole mass spectrometry (LC-MS/MS) to quantify nuclease-resistant PT-modified dinucleotides has revealed significant diversity in the quantity, sequence setting, and bacterial distribution of PT modifications, such as the d(G PS A) and d(G PS T) motifs in 1:1 proportions in the enteric pathogen Escherichia coli B7A (15). Subsequent application of single-molecule, real-time (SMRT) sequencing technology to map genomic PT sites in E. coli B7A revealed that these d(G PS A) and d(G PS T) motifs are located within conserved 4-bp complementary G PS AAC/G PS TTC contexts, with no apparent wider consensus sequence (23). However, only 12% of the 40,701 possible GAAC/GTTC sites on the chromosome were found to be PT-modified, even in the presence of active DndFGH (23).…”

“…SMRT sequencing of DNA modifications exploits the kinetic signatures, defined as pulse width and interpulse duration (IPD), arising when the DNA polymerase copies past the modification (27). Building on SMRT applications to nucleobase methylation (27), 5-hydroxymethylcytosine (28), and nucleobase damage (29), we recently demonstrated that SMRT sequencing is applicable to PT mapping, with identification of G PS AAC/G PS TTC and C PS CA in E. coli B7A and Vibrio cyclitrophicus FF75, respectively (23). Both 6m A and d(G PS A) modifications of template DNA generate kinetic signatures with regard to the A base.…”

“…Subsequent application of single-molecule, real-time (SMRT) sequencing technology to map genomic PT sites in E. coli B7A revealed that these d(G PS A) and d(G PS T) motifs are located within conserved 4-bp complementary G PS AAC/G PS TTC contexts, with no apparent wider consensus sequence (23). However, only 12% of the 40,701 possible GAAC/GTTC sites on the chromosome were found to be PT-modified, even in the presence of active DndFGH (23). These unique features differentiate dndBCDE-dndFGH from typical methylation-based R-M systems and suggest that it represents a type of bacterial defense system with highly unusual target selection by Dnd proteins.…”