Proxi-RIMS-seq2 applied to native microbiomes uncovers hundreds of known and novel m5 C methyltransferase specificities.

Methylation patterns in bacteria can be used to study Restriction-Modification (RM) or other defense systems with novel properties. While m4 C and m6 A methylation is well characterized mainly through PacBio sequencing, the landscape of m5 C methylation is under-characterized. To bridge this gap, we performed RIMS-seq2 on microbiomes composed of resolved assemblies of distinct genomes through proximity ligation. This high-throughput approach enables the identification of m5 C 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 viral DNA and compared to host DNA, strengthening evidence for virus-host interaction. Applied to three different microbiomes, the method unveils over 1900 motifs that were deposited in REBASE. The motifs include a novel 8-base recognition site (CAT m5 CGATG) that was experimentally validated by characterizing its cognate methyltransferase. Our findings suggest that microbiomes harbor arrays of untapped m5 C methyltransferase specificities, providing insights to bacterial biology and biotechnological applications.