Sater Mohamad R Abdul, Lamelas Araceli, Wang Guilin, Clark Tyson A, Röltgen Katharina, Mane Shrikant, Korlach Jonas, Pluschke Gerd, Schmid Christoph D
Swiss Tropical and Public Health Institute, Socinstrasse 57, Basel, Switzerland.
Universität Basel, Petersplatz 1, Basel, Switzerland.
PLoS One. 2015 Dec 11;10(12):e0144612. doi: 10.1371/journal.pone.0144612. eCollection 2015.
The Gram-negative bacterium Neisseria meningitidis features extensive genetic variability. To present, proposed virulence genotypes are also detected in isolates from asymptomatic carriers, indicating more complex mechanisms underlying variable colonization modes of N. meningitidis. We applied the Single Molecule, Real-Time (SMRT) sequencing method from Pacific Biosciences to assess the genome-wide DNA modification profiles of two genetically related N. meningitidis strains, both of serogroup A. The resulting DNA methylomes revealed clear divergences, represented by the detection of shared and of strain-specific DNA methylation target motifs. The positional distribution of these methylated target sites within the genomic sequences displayed clear biases, which suggest a functional role of DNA methylation related to the regulation of genes. DNA methylation in N. meningitidis has a likely underestimated potential for variability, as evidenced by a careful analysis of the ORF status of a panel of confirmed and predicted DNA methyltransferase genes in an extended collection of N. meningitidis strains of serogroup A. Based on high coverage short sequence reads, we find phase variability as a major contributor to the variability in DNA methylation. Taking into account the phase variable loci, the inferred functional status of DNA methyltransferase genes matched the observed methylation profiles. Towards an elucidation of presently incompletely characterized functional consequences of DNA methylation in N. meningitidis, we reveal a prominent colocalization of methylated bases with Single Nucleotide Polymorphisms (SNPs) detected within our genomic sequence collection. As a novel observation we report increased mutability also at 6mA methylated nucleotides, complementing mutational hotspots previously described at 5mC methylated nucleotides. These findings suggest a more diverse role of DNA methylation and Restriction-Modification (RM) systems in the evolution of prokaryotic genomes.
革兰氏阴性菌脑膜炎奈瑟菌具有广泛的遗传变异性。到目前为止，在无症状携带者的分离株中也检测到了拟议的毒力基因型，这表明脑膜炎奈瑟菌不同定植模式背后的机制更为复杂。我们应用了太平洋生物科学公司的单分子实时（SMRT）测序方法，来评估两种遗传相关的A群脑膜炎奈瑟菌菌株的全基因组DNA修饰图谱。所得的DNA甲基化组显示出明显的差异，表现为共享的和菌株特异性的DNA甲基化靶基序的检测。这些甲基化靶位点在基因组序列中的位置分布显示出明显的偏向性，这表明DNA甲基化与基因调控相关的功能作用。对A群脑膜炎奈瑟菌菌株扩展集合中一组已确认和预测的DNA甲基转移酶基因的开放阅读框（ORF）状态进行仔细分析，结果表明脑膜炎奈瑟菌中DNA甲基化的变异性潜力可能被低估。基于高覆盖短序列读数，我们发现相位变异是DNA甲基化变异的主要贡献因素。考虑到相位可变位点，DNA甲基转移酶基因的推断功能状态与观察到的甲基化图谱相匹配。为了阐明目前脑膜炎奈瑟菌中DNA甲基化功能后果尚未完全表征的情况，我们揭示了甲基化碱基与我们基因组序列集合中检测到的单核苷酸多态性（SNP）之间存在显著的共定位。作为一项新的观察结果，我们报告在6mA甲基化核苷酸处也存在增加的突变性，补充了先前在5mC甲基化核苷酸处描述的突变热点。这些发现表明DNA甲基化和限制修饰（RM）系统在原核生物基因组进化中具有更多样化的作用。
