Precision Methylome and in vivo Methylation Kinetics Characterization of Klebsiella Pneumoniae.

Klebsiella pneumoniae (K. pneumoniae) is an important pathogen that can cause severe hospital- and community-acquired infections. To systematically investigate its methylation features, we determined the whole genome sequences of 14 K. pneumoniae strains covering varying serotypes, multilocus sequence typing (MLST), clonal groups (CGs), viscosity/virulence, and drug resistance. Their methylomes were further characterised using PacBio-SMRT and bisulfite technologies. We identified 15 methylation motifs (13 N6-methyladenine (6mA) and two 5-methylcytosine (5mC) motifs), among which eight were novel. Their corresponding DNA methylases (MTases) were also validated. Additionally, we analysed the genomic distribution of GATC and CCWGG methylation motifs shared by all strains, and identified differential distributive patterns of some hemi/un-methylated GATC motifs, which tend to be located within intergenic regions (IGRs). Specifically, we characterised the in vivo methylation kinetics at single base resolution on a genome-wide scale by simulating the dynamic processes of replication-mediated passive demethylation and MTase-catalysed re-methylation. The slower methylation rates of the GATC motifs in the replication origins (oriC) and IGRs implicate an epigenetic mechanism in the regulation of replication initiation and transcription. Our findings illustrate the first comprehensive dynamic methylome map of K. pneumoniae at single base resolution, and provide a useful reference to better understand epigenetic regulation in this and other bacterial species.