Campbell Maxwell, Barton Ian Scott, Roop R Martin, Chien Peter
Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst, MA.
Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, NC.
bioRxiv. 2024 Mar 2:2024.03.01.583015. doi: 10.1101/2024.03.01.583015.
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 show 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.
细菌依靠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。在此，我们使用纳米孔测序直接检测甲基化碱基，以监测α-变形菌纲中的全基因组DNA甲基化情况，此前尚未有关于该技术在此类细菌中的应用报道。该菌纲中的一个代表性菌种，依靠DNA甲基化来控制细胞周期进程，但尚不清楚该菌纲中的其他成员，如，是否依赖相同的系统。我们通过首先测量中的CcrM依赖性DNA甲基化来解决这些问题，并表明基于纳米孔的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Lon的缺失会导致广泛的甲基化。我们还表明，在正常生长过程中，AlkB去甲基化酶对DNA甲基化没有全局性影响。接下来，我们首次报道了中的全基因组DNA甲基化情况，发现CcrM依赖性甲基化依赖于Lon，但对两条染色体的影响不同。最后，我们探究了已知与CcrM甲基化相互竞争的MucR转录因子对甲基化组的影响，并将结果与一个公开可用的可视化软件包共享。我们的工作证明了基于纳米孔的测序在α-变形菌纲表观基因组测量中的实用性，并揭示了一种人畜共患病原体中CcrM依赖性甲基化的新特征。
