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 methylomes 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 methyltransferases were also validated. Additionally, we analyzed the genomic distribution of GATC and CCWGG methylation motifs shared by all strains, and identified differential distribution patterns 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 resolution 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.

肺炎克雷伯菌（K. pneumoniae）是一种重要的病原体，可引起严重的医院和社区获得性感染。为了系统地研究其甲基化特征，我们确定了 14 种肺炎克雷伯氏菌菌株的全基因组序列，涵盖不同的血清型、多位点序列类型、克隆群、粘度/毒力和耐药性。他们的甲基化组使用 Pacific Biosciences 单分子实时和亚硫酸氢盐技术进一步表征。我们确定了 15 个甲基化基序 [13 N 6-甲基腺嘌呤 ( 6 mA ) 和两个 5-甲基胞嘧啶 ( 5mC) motifs]，其中八个是新颖的。它们相应的 DNA 甲基转移酶也得到了验证。此外，我们分析了所有菌株共享的 G A T C 和 C C W G G 甲基化基序的基因组分布，并确定了一些半甲基化/未甲基化 G A T C 基序的差异分布模式，这些基序往往位于基因间区域 (IGR)。具体来说，我们通过模拟复制介导的被动去甲基化和 MTase 催化的再甲基化的动态过程，在全基因组范围内以单碱基分辨率表征体内甲基化动力学。G A T的缓慢甲基化复制起点 ( oriC ) 区域和 IGR中的 C 基序涉及复制起始和转录的表观遗传调控。我们的研究结果以单碱基分辨率展示了肺炎克雷伯氏菌的第一个综合动态甲基化组图谱，并为更好地了解该细菌和其他细菌物种的表观遗传调控提供了有用的参考。