Single-molecule real-time (SMRT) sequencing can be used to identify a wide variety of chemical modifications of the genome, such as methylation. Here, we applied this approach to identify N6-methyl-adenine (m6A) and N4-methyl-cytosine (m4C) modification in the genome of Bacillus pumilus BA06. A typical methylation recognition motif of the type I restriction-modification system (R-M), 5'-TCm6AN8TTGG-3'/3'-AGTN8m6AACC-5', was identified. We confirmed that this motif was a new type I methylation site using REBASE analysis and that it was recognized by a type I R-M system, Bpu6ORFCP, according to methylation sensitivity assays in vivo and vitro. Furthermore, we found that deletion of the R-M system Bpu6ORFCP induced transcriptional changes in many genes and led to increased gene expression in pathways related to ABC transporters, sulfur metabolism, ribosomes, cysteine and methionine metabolism and starch and sucrose metabolism, suggesting that the R-M system in B. pumilus BA06 has other significant biological functions beyond protecting the B. pumilus BA06 genome from foreign DNA.

中文翻译：

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在短小芽孢杆菌BA06中鉴定出一种新颖的DNA甲基化基序，并可能在基因表达的调节中起作用。

单分子实时（SMRT）测序可用于鉴定基因组的多种化学修饰，例如甲基化。在这里，我们应用这种方法来鉴定短小芽孢杆菌BA06基因组中的N6-甲基-腺嘌呤（m6A）和N4-甲基-胞嘧啶（m4C）修饰。确定了I型限制性修饰系统（RM）的典型甲基化识别基序5'-TCm6AN8TTGG-3'/ 3'-AGTN8m6AACC-5'。我们通过REBASE分析证实了该基序是一个新的I型甲基化位点，并且根据体内和体外的甲基化敏感性测定，已被I型RM系统Bpu6ORFCP识别。此外，我们发现删除RM系统Bpu6ORFCP会诱导许多基因的转录变化，并导致与ABC转运蛋白相关的途径中的基因表达增加，