幽门螺杆菌是引发胃癌的主要细菌致病菌，其基因高度多样，携带大量且变异丰富的限制修饰系统。目前关于细菌DNA甲基化的进化和功能的认识仍然有限。在本研究中，我们运用包含了所有已知地理种群的541个基因组数据集，对幽门螺杆菌的甲基体多样性进行了全面分析。96个甲基转移酶的频率及其相应识别序列的丰度受地理种群结构的严重影响，并且在20%的二型甲基转移酶中呈正相关或负相关关系。低密度模体更可能受到自然选择的影响，表现为基因组不稳定性和组成偏差较高。重要的是，直接相关性表明，在正选择的作用下，甲基化模式可以被积极丰富，表明特定位点在甲基化相关表型中具有重要功能。最后，我们发现了特定血统的选择性压力调节了ACGT模体的收缩和扩展，揭示了甲基化的遗传负荷可能依赖于局部生态因素。综上所述，自然选择可能塑造了甲基转移酶的丰度和分布，以及它们特定的识别序列，从而通过基因变异无法实现对基因组编码功能的微调。©2023 Springer Nature Limited.

The bacterial pathogen Helicobacter pylori, the leading cause of gastric cancer, is genetically highly diverse and harbours a large and variable portfolio of restriction-modification systems. Our understanding of the evolution and function of DNA methylation in bacteria is limited. Here, we performed a comprehensive analysis of the methylome diversity in H. pylori, using a dataset of 541 genomes that included all known phylogeographic populations. The frequency of 96 methyltransferases and the abundance of their cognate recognition sequences were strongly influenced by phylogeographic structure and were inter-correlated, positively or negatively, for 20% of type II methyltransferases. Low density motifs were more likely to be affected by natural selection, as reflected by higher genomic instability and compositional bias. Importantly, direct correlation implied that methylation patterns can be actively enriched by positive selection and suggests that specific sites have important functions in methylation-dependent phenotypes. Finally, we identified lineage-specific selective pressures modulating the contraction and expansion of the motif ACGT, revealing that the genetic load of methylation could be dependent on local ecological factors. Taken together, natural selection may shape both the abundance and distribution of methyltransferases and their specific recognition sequences, likely permitting a fine-tuning of genome-encoded functions not achievable by genetic variation alone.© 2023. Springer Nature Limited.