Proxi-RIMS-seq2 applied to native microbiomes uncovers hundreds of known and novel m5C methyltransferase specificities.

Methylation patterns in bacteria can be used to study restriction-modification or other defense systems with novel properties. While m4C and m6A methylation are well characterized mainly through PacBio sequencing, the landscape of m5C methylation is under-characterized. To bridge this gap, we performed RIMS-seq2 (rapid identification of methyltransferase specificity sequencing) on microbiomes composed of resolved assemblies of distinct genomes through proximity ligation. This high-throughput approach enables the identification of m5C methylated motifs and links them to cognate methyltransferases directly on native microbiomes without the need to isolate bacterial strains. Methylation patterns can also be identified on bacteriophage DNA and compared with host DNA, strengthening evidence for phage-host interactions. Applied to three different microbiomes, the method unveiled over 1900 motifs that were deposited in REBASE. The motifs include a novel eight-base recognition site (CATm5CGATG) that was experimentally validated by characterizing its cognate methyltransferase. Our findings suggest that microbiomes harbor arrays of untapped m5C methyltransferase specificities, providing insights into bacterial biology and biotechnological applications.