RNA-seq gene expression profiling comparing WT (NA1000) and isogenic ∆scmA Caulobacter crescentus 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. To investigate the impact of DNA methylation by ScmA (CCNA_01085) on the transcriptome of Caulobacter crescentus NA1000, we constructed a ∆scmA mutant. We then performed gene expression profiling analyses using data obtained from RNA-seq of WT/∆scmA cells grown exponentially (OD660=~0.4) in M2G medium.

细菌中孤立型胞嘧啶DNA甲基转移酶（DNA Methyltransferase, MTase）的确切生物学功能，目前大多尚未阐明。本研究以α-变形菌纲模式生物新月柄杆菌（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）的潜在影响。我们构建了ΔscmA基因缺失突变株，并以此为工具，探究了ScmA依赖型甲基化对新月柄杆菌表型、生存适应性以及转录组的影响。上述分析结果显示，ΔscmA突变株细胞中频繁出现DNA损伤事件。值得注意的是，研究发现这类DNA损伤事件的发生，依赖于野生型（Wild Type, WT）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：在DNA修复异常过程中，VsrA可诱发DNA损伤。因此，在标准培养条件下，携带功能性scmA基因的细胞（scmA+）在生存竞争中显著优于ΔscmA突变株细胞。有趣的是，尽管我们发现了vsrA与scmA基因存在功能关联，但在新月柄杆菌的染色体上，二者并不存在遗传连锁关系。相较于此前已表征的基因系统——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排布——本研究揭示了一种独特的基因位置排布模式。为探究ScmA（CCNA_01085）介导的DNA甲基化对新月柄杆菌NA1000转录组的影响，我们构建了ΔscmA突变株。随后，我们以在M2G培养基中指数生长期（OD₆₆₀≈0.4）培养的野生型与ΔscmA突变株细胞的RNA测序（RNA-seq）数据为基础，开展了基因表达谱分析。