A GCDGC-specific DNA (cytosine-5) methyltransferase that methylates the GCWGC sequence on both strands and the GCSGC sequence on one strand

Abstract

5-Methylcytosine is one of the major epigenetic marks of DNA in living organisms. Some bacterial species possess DNA methyltransferases that modify cytosines on both strands to produce fully-methylated sites or on either strand to produce hemi-methylated sites. In this study, we characterized a DNA methyltransferase that produces two sequences with different methylation patterns: one methylated on both strands and another on one strand. M.BatI is the orphan DNA methyltransferase of Bacillus anthracis coded in one of the prophages on the chromosome. Analysis of M.BatI modified DNA by bisulfite sequencing revealed that the enzyme methylates the first cytosine in sequences of 5'-GCAGC-3', 5'-GCTGC-3', and 5'-GCGGC-3', but not of 5'-GCCGC-3'. This resulted in the production of fully-methylated 5'-GCWGC-3' and hemi-methylated 5'-GCSGC-3'. M.BatI also showed toxicity when expressed in E. coli, which was caused by a mechanism other than DNA modification activity. Homologs of M.BatI were found in other Bacillus species on different prophage like regions, suggesting the spread of the gene by several different phages. The discovery of the DNA methyltransferase with unique modification target specificity suggested unrevealed diversity of target sequences of bacterial cytosine DNA methyltransferase.

Fig 1. Position of batIM and its activity in vivo.

(A) A map of the chromosome of B. anthracis Ames ancestor. The outermost blue bars represent the region of prophages. Gray bars represent the position of coding sequences: outer, coding sequences on the leading strand; inner, coding sequences on the lagging strand. Prophage genes were colored blue and the batIM gene was colored red. (B) Measurement of 5mC using UPLC-MS/MS for genomic DNA isolated from B. anthracis strains and M.BatI induced E.coli strains. ND, not detected.

Fig 2. M.BatI fully-methylates 5’-GCWGC-3’ and hemi-methylates 5’-GCSGC-3’.

(A) Digestion of genomic DNA of E. coli after inducing M.BatI production. Genomic DNA isolated from BYF823 (batIM-) or BYF822 (batIM+) induced with aTc 0 or 100 ng/ml was treated with specific restriction enzymes or untreated as controls. (B) Digestion of genomic DNA of wildtype (WT) and batIM knockout (Δ) strains of B. anthracis. (C) Sequence logo of sites around cytosines detected, by bisulfite sequencing, to be highly methylated. (D) Boxplots of methylation frequency of each sequence included in 5ʹ-GCNGC-3ʹ. (E) Fully-methylated and hemi-methylated motifs produced by M.BatI. Methylated cytosines were depicted with “Me” in a circle.

Fig 3. Target sequence confirmation of M.BatI by digestion of M.BatI methylated dsDNA.

(A) Structure of 60 bp dsDNA. The site with overlapping sequences was positioned at one-third from the right end. (B) Design of overlapping sites in each dsDNA and the expected outcomes by methylation and digestion reactions. Each site included a target site of another restriction enzyme (squares). Expected positions of methylation were depicted with “Me” in a circle. (C) Digestion tests of dsDNA. Each dsDNA was first treated with M.BatI and digested with one of the four restriction enzymes.

Fig 4. Toxicity of M.BatI variants in E. coli.

The number of colony forming units was determined after induction at 0 h with aTc for E. coli strains expressing (A) no M.BatI, (B) wildtype M.BatI, (C) M.BatI C86G, (D) M.BatI A98V, and (E) M.BatI D75N. Black, no induction; Green, aTc 1 ng/ml; Blue, aTc 10 ng/ml; Orange, aTc 100 ng/ml.

Fig 5. Genomic DNA modification by M.BatI with different levels of induction and mutations.

(A) Digestion of genomic DNA induced with different concentrations of aTc. (B) Digestion of genomic DNA modified by M.BatI variants.

Fig 6. Genomic context of batIM and its homologs.

Red, batIM and its homologs; pink, genes annotated as terminase, integrase, or recombinase encoding genes; yellow, annotated as head, tail, or other structural proteins encoding genes.