RNA-seq gene expression profiling comparing ∆vsrA and ∆scmA∆vsrA Caulobacter crescentus NA1000 cells. RNA-seq gene expression profiling comparing ∆vsrA and ∆scmA∆vsrA Caulobacter crescentus NA1000 cells

The precise biological function of solitary cytosine MTases in bacteria remains mostly elusive. In this study, we dissected the potential influence of the solitary cytosine MTase CCNA_01085 (named ScmA), in the Caulobacter crescentus Alphaproteobacterial model. We constructed a ∆scmA mutant and used it to study the impact of ScmA-dependent methylation on C. crescentus phenotypes, on its fitness and on its transcriptome. These analyses revealed that DNA damaging events often take place in ∆scmA cells. Interestingly, these were shown to be dependent on the presence of the CCNA_2930 Vsr-like repair endonuclease naturally expressed in WT C. crescentus cells (named VsrA). Thus, we hypothesize that DNA damage can be induced by VsrA during DNA repair anomalies. Consequently, scmA+ cells largely outcompete ∆scmA cells during standard growth conditions. Interestingly, the vsrA gene is not genetically linked with the scmA gene on the C. crescentus chromosome despite the functional link that we discovered, revealing an original gene positioning architecture compared to previously characterized systems where Vsr-like repair endonucleases are usually encoded by genes located right next to MTase genes. Overall design: To investigate the impact of DNA methylation by ScmA (CCNA_01085) not linked to VsrA-induced DNA damage on the transcriptome of Caulobacter crescentus NA1000 strain, we constructed mutant strains deleted for VsrA and both VsrA and ScmA. We then performed gene expression profiling analyses using data obtained from RNA-seq of ∆vsrA/∆scmA∆vsrA cells grown exponentially (OD660=~0.4) in M2G medium.

细菌中孤立型胞嘧啶DNA甲基转移酶（MTase）的确切生物学功能，目前仍大多尚不明确。本研究以α-变形菌纲模式菌株新月柄杆菌（Caulobacter crescentus）为研究对象，解析了孤立型胞嘧啶DNA甲基转移酶CCNA_01085（命名为ScmA）的潜在调控作用。我们构建了scmA基因敲除（∆scmA）突变株，以此探究ScmA依赖型甲基化对新月柄杆菌表型、生存适应性及转录组的影响。上述分析结果显示，∆scmA突变株细胞中常发生DNA损伤事件。值得注意的是，研究发现这类DNA损伤依赖于野生型新月柄杆菌细胞中天然表达的CCNA_2930 Vsr样修复核酸内切酶（命名为VsrA）。据此我们提出假说：VsrA可在DNA修复异常过程中诱发DNA损伤。因此，在标准培养条件下，携带功能性scmA基因（scmA+）的细胞在竞争性生长中显著优于∆scmA突变株细胞。有趣的是，尽管我们发现了二者的功能关联，但vsrA基因与scmA基因在新月柄杆菌染色体上并不存在遗传连锁关系；相较于此前已报道的系统——这类系统中Vsr样修复核酸内切酶的编码基因通常紧邻甲基转移酶基因——本研究揭示了一种全新的基因定位模式。实验整体设计：为探究不依赖VsrA诱发DNA损伤的ScmA（CCNA_01085）介导的DNA甲基化对新月柄杆菌NA1000菌株转录组的影响，我们分别构建了仅敲除vsrA的单突变株，以及同时敲除vsrA与scmA的双突变株。随后，我们对在M2G培养基中培养至指数生长期（OD660≈0.4）的∆vsrA与∆scmA∆vsrA菌株细胞进行RNA测序（RNA-seq），以此开展基因表达谱分析。