摘要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 types,clonal groups,viscosity/virulence,and drug resistance.Their methy-lomes were further characterized using Pacific Biosciences single-molecule real-time 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 methyl-transferases were also validated.Additionally,we analyzed the genomic distribution of GATC and CCWGG methylation motifs shared by all strains,and identified differential distribution pat-terns of some hemi-/un-methylated GATC motifs,which tend to be located within intergenic regions(IGRs).Specifically,we characterized the in vivo methylation kinetics at single-base resolu-tion on a genome-wide scale by simulating the dynamic processes of replication-mediated passive demethylation and MTase-catalyzed re-methylation.The slow methylation of the GATC motifs in the replication origin(oriC)regions and IGRs implicates the epigenetic 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.