Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst, Massachusetts, USA.
Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, South Carolina, USA.
J Bacteriol. 2024 Jun 20;206(6):e0008324. doi: 10.1128/jb.00083-24. Epub 2024 May 9.
Bacteria rely on DNA methylation for restriction-modification systems and epigenetic control of gene expression. Here, we use direct detection of methylated bases by nanopore sequencing to monitor global DNA methylation in Alphaproteobacteria, where use of this technique has not yet been reported. One representative of this order, , relies on DNA methylation to control cell cycle progression, but it is unclear whether other members of this order, such as , depend on the same systems. We addressed these questions by first measuring CcrM-dependent DNA methylation in and showing excellent correlation between nanopore-based detection and previously published results. We then directly measure the impact of Lon-mediated CcrM degradation on the epigenome, verifying that loss of Lon results in pervasive methylation. We also show that the AlkB demethylase has no global impact on DNA methylation during normal growth. Next, we report on the global DNA methylation in for the first time and find that CcrM-dependent methylation is reliant on Lon but impacts the two chromosomes differently. Finally, we explore the impact of the MucR transcription factor, known to compete with CcrM methylation, on the methylome and share the results with a publicly available visualization package. Our work demonstrates the utility of nanopore-based sequencing for epigenome measurements in Alphaproteobacteria and reveals new features of CcrM-dependent methylation in a zoonotic pathogen.IMPORTANCEDNA methylation plays an important role in bacteria, maintaining genome integrity and regulating gene expression. We used nanopore sequencing to directly measure methylated bases in and . In , we showed that stabilization of the CcrM methyltransferase upon loss of the Lon protease results in prolific methylation and discovered that the putative methylase AlkB is unlikely to have a global physiological effect. We measured genome-wide methylation in for the first time, revealing a similar role for CcrM in cell-cycle methylation but a more complex regulation by the Lon protease than in Caulobacter. Finally, we show how the virulence factor MucR impacts DNA methylation patterns in .
细菌依赖于 DNA 甲基化来实现限制-修饰系统和基因表达的表观遗传控制。在这里，我们使用纳米孔测序直接检测甲基化碱基，以监测α变形菌中的全基因组 DNA 甲基化，而这项技术在该领域尚未被报道过。该目下的一个代表，依赖于 DNA 甲基化来控制细胞周期进程，但目前尚不清楚该目下的其他成员，如，是否依赖于相同的系统。为了解决这些问题，我们首先测量了和中 CcrM 依赖性 DNA 甲基化的情况，并证明了纳米孔检测与先前发表的结果之间具有极好的相关性。然后，我们直接测量 Lon 介导的 CcrM 降解对表观基因组的影响，证实了 Lon 的缺失会导致广泛的甲基化。我们还表明，AlkB 去甲基酶在正常生长过程中对 DNA 甲基化没有全局影响。接下来，我们首次报告了中全基因组 DNA 甲基化的情况，并发现 CcrM 依赖性甲基化依赖于 Lon，但对两条染色体的影响方式不同。最后，我们探索了转录因子 MucR 的影响，已知该因子与 CcrM 甲基化竞争，对甲基组的影响，并与一个公开的可视化包共享结果。我们的工作证明了纳米孔测序在α变形菌中进行表观基因组测量的实用性，并揭示了一种动物病原体中 CcrM 依赖性甲基化的新特征。
DNA 甲基化在细菌中起着重要作用，它可以维持基因组的完整性并调节基因表达。我们使用纳米孔测序直接测量和中的甲基化碱基。在中，我们表明，Lon 蛋白酶的失活导致 CcrM 甲基转移酶的稳定，从而导致大量甲基化，并发现假定的甲基酶 AlkB 不太可能对全局生理产生影响。我们首次测量了中全基因组的甲基化情况，发现 CcrM 在细胞周期甲基化中起相似的作用，但与钙杆菌相比，Lon 蛋白酶的调节更为复杂。最后，我们展示了毒力因子 MucR 如何影响 DNA 甲基化模式。
