Schmidt Nils, Stappert Nils, Nimura-Matsune Kaori, Watanabe Satoru, Sobotka Roman, Hagemann Martin, Hess Wolfgang R
Institute of Biosciences, Department of Plant Physiology, University of Rostock, D-18059 Rostock, Germany.
University of Freiburg, Faculty of Biology, Genetics and Experimental Bioinformatics, Schänzlestr. 1, D-79104 Freiburg, Germany.
DNA Res. 2024 Dec 1;31(6). doi: 10.1093/dnares/dsae035.
Epigenetic DNA modifications are pivotal in eukaryotic gene expression, but their regulatory significance in bacteria is less understood. In Synechocystis 6803, the DNA methyltransferase M.Ssp6803II modifies the first cytosine in the GGCC motif, forming N4-methylcytosine (GGm4CC). Deletion of the sll0729 gene encoding M.Ssp6803II (∆sll0729) caused a bluish phenotype due to reduced chlorophyll levels, which was reversed by suppressor mutations. Re-sequencing of 7 suppressor clones revealed a common GGCC to GGTC mutation in the slr1790 promoter's discriminator sequence, encoding protoporphyrinogen IX oxidase, HemJ, crucial for tetrapyrrole biosynthesis. Transcriptomic and qPCR analyses indicated aberrant slr1790 expression in ∆sll0729 mutants. This aberration led to the accumulation of coproporphyrin III and protoporphyrin IX, indicative of impaired HemJ activity. To confirm the importance of DNA methylation in hemJ expression, hemJ promoter variants with varying discriminator sequences were introduced into the wild type, followed by sll0729 deletion. The sll0729 deletion segregated in strains with the GGTC discriminator motif, resulting in wild-type-like pigmentation, whereas freshly prepared ∆sll0729 mutants with the native hemJ promoter exhibited the bluish phenotype. These findings demonstrate that hemJ is tightly regulated in Synechocystis and that N4-methylcytosine is essential for proper hemJ expression. Thus, cytosine N4-methylation is a relevant epigenetic marker in Synechocystis and likely other cyanobacteria.
表观遗传DNA修饰在真核生物基因表达中起关键作用，但其在细菌中的调控意义尚不清楚。在集胞藻6803中，DNA甲基转移酶M.Ssp6803II修饰GGCC基序中的第一个胞嘧啶，形成N4-甲基胞嘧啶（GGm4CC）。编码M.Ssp6803II的sll0729基因缺失（∆sll0729）导致叶绿素水平降低，从而产生蓝色表型，而抑制突变可使其恢复。对7个抑制克隆的重新测序显示，在编码原卟啉原IX氧化酶HemJ（对四吡咯生物合成至关重要）的slr1790启动子的鉴别序列中存在常见的GGCC到GGTC突变。转录组学和qPCR分析表明，∆sll0729突变体中slr1790表达异常。这种异常导致粪卟啉原III和原卟啉IX积累，表明HemJ活性受损。为了证实DNA甲基化在hemJ表达中的重要性，将具有不同鉴别序列的hemJ启动子变体导入野生型，然后缺失sll0729。sll0729缺失在具有GGTC鉴别基序的菌株中分离，导致类似野生型的色素沉着，而新制备的具有天然hemJ启动子的∆sll0729突变体表现出蓝色表型。这些发现表明，hemJ在集胞藻中受到严格调控，N4-甲基胞嘧啶对于hemJ的正常表达至关重要。因此，胞嘧啶N4-甲基化是集胞藻以及可能其他蓝细菌中的一种相关表观遗传标记。
