Identification And Characterization Of Two DNA Methytransferases In Mycobacterium Tuberculosis

DNA methylation is a kind of epigenetic modification that occurs in almost all the organisms including bacteria. DNA methylation is a biochemical process where a methyl group is added to the cytosine or adenine by DNA methyltransferases. In bacteria, DNA methyltransferases combined with its cognate restriction endonuclease form a restriction-modification systemto protect themselves from foreign DNA. On the other hands, orphan DNA methyltransferases are also found in many bacterial genomes. They play an important role in gene expression and regulation. Here we reported the comprehensive methylome of BCG on a single nucleotide level for the first time by using single-molecule real-time sequencing (SMRT) technology. It is made up of three m6A DNA methylation motifs. The extent of modification is different for the three motifs. Among them, the extent of modification at motifs CACGCAG and CTCCAG was more than 99%, while the extent of motif GATN4RTAC was only 77%. Besides, motif GATN4RTAC preferred to locate in the intergenic regions and promoters compared with other two motifs. Besides the reported MamA methyltransferase, two novel N6-methyladenine(m6A) methyltransferases were identified which we termed as HsdM and MamB respectively. We characterized the two novel DNA methyltransferases by using some technologies including gene expression, protein purification, gene knockout, restriction enzyme profiling and SMRT sequencing of recombination plasmids. The results showed that, HsdM is a typical type I DNA methyltransferase, and its recognition motif is GATN4RTAC, producing m6A on two strands. The knowout experiments showed that HsdS2 subunit was essential for HsdM DNA methyltransferase activity, while HsdSl subunit is non-essential. MamB is a type IIG DNA methyltransferases, and its targeted sequence motif is CACGCAG, producing m6A on single strand. In the study, we made a thorough analysis on the DNA methylome and the DNA methyltransferases of BCG stain and provided some clues on the molecular mechanism of tuberculosis epigenetic modifications.