Microbial epigenetics

Directly detect epigenetic modifications with HiFi sequencing

HiFi sequencing directly detects DNA modifications in native DNA from variations in the polymerase kinetics of DNA base incorporation, without the need for arduous chemical conversion protocols like bisulfite treatment.

Detect genome-wide m6A and m4C R-M system motifs at coverage levels recommended for assembly
Obtain complete genomes with annotations for epigenetic modification
Reveal phase variation of R-M genes that regulate batteries of genes involved in pathogenesis, host adaption, and antibiotic resistance

Learn how other scientists have used SMRT sequencing to connect prokaryotic methylation to new biology:

Workflow: from DNA to microbial methylome

Sample + library preparation

Start with unamplified genomic DNA
Prepare SMRTbell libraries following the protocol for microbial whole genome sequencing.

Sequencing

Simultaneously generate whole genome and epigenome data by sequencing on a Revio or Vega system

Data analysis

Use SMRT Link Microbial Genome Analysis to assemble and annotate active R-M system motifs
View detailed reports on modifications and associated motifs

Spotlight

BACTERIAL METHYLATION PROFILES HELP ASSIGN ANTIBIOTIC-RESISTANCE GENES TO HOST ORGANISMS IN MIXED ENVIRONMENTAL SAMPLES

This study used PacBio long-read metagenomics plus bacterial methylation signatures to connect sul4-containing contigs to likely host genomes in complex wastewater communities, grounded in the observation that bacterial taxa carry species-specific methylation patterns because their methyltransferase repertoires differ. For the 12 sul4 contigs, they found an average of 6 methylated motifs of m6A or m4C per contig, with a range of 1 to 13. All but one contig had at least one methylated motif. The methylation patterns grouped the sul4 contigs into three broad categories, which suggested multiple distinct bacterial hosts.

Markkanen, M., et. al. (2026) Sulfonamide resistance gene sul4 is hosted by common wastewater sludge bacteria and found in various newly described contexts and hosts. Microbiology Spectrum. 14:e00857-25, https://doi.org/10.1128/spectrum.00857-25

$Fig 3 Methylation profiles of sul4 contigs and contigs without sul4. Contigs are represented as columns, and the methylation motifs are shown as rows on the right-hand side of the heatmap. The intensity of the purple color in the heatmap shows the fraction of the methylated sites for each of the motifs predicted by the MultiMotifMaker algorithm (54). The modification type (m6A or m4C) is indicated by color coding on the left-hand side. The top-row annotation indicates the MAG in which each contig is included, if applicable, and the taxa assigned to the MAGs are shown in the text.$

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Spotlight

A role for epigenetics in the regulation of replication and transcription

Comprehensive characterization and comparison of the methylomes of 14 K. pneumoniae strains revealed differential concentration of GATC motifs and methylation penetrance in intergenic regions. Modelling of MTase-catalysed methylation suggests slower rates of motifs in replication origins and IGRs, implicating a role in the initiation of replication and transcription.

Fu, J., et. al. (2022). Precision Methylome and In Vivo Methylation Kinetics Characterization of Klebsiella pneumoniae. Genomics, proteomics & bioinformatics, 20(2), 418–434. https://doi.org/10.1016/j.gpb.2021.04.002

Learn more

Fu, J. et.al. (2021) Precision methylome and in vivo methylation kinetics characterization of Klebsiella pneumoniae. Genom, Prot, and Bioinf. doi: 10.1016/j.gpb.2021.04.002.