RNA-seq gene expression profiling comparing WT and DccrM Ptac-ccrM Agrobacterium tumefaciens C58 cells cultivated in ATGN+/-IPTG.

The cell cycle-regulated DNA methyltransferase CcrM is conserved in most Alphaproteobacteria, but its role in bacteria with complex or multicentric genomes remains unexplored. Here, we compare the methylome, the transcriptome and the phenotypes of wild-type and CcrM-depleted Agrobacterium tumefaciens cells with a dicentric genome with two essential replication origins. We find that DNA methylation has a pleiotropic impact on motility, biofilm formation and viability. Remarkably, CcrM promotes the expression of the repABCCh2 operon, encoding proteins required for replication initiation/partitioning at ori2, and inhibits gcrA, encoding a conserved global cell cycle regulator. Imaging ori1 and ori2 in live cells, we show that replication from ori2 is often delayed in cells with a hypo-methylated genome, while ori2 over-initiates in cells with a hyper-methylated genome. We thus propose that methylation by CcrM stimulates RepABC-dependent chromosomal origins, uncovering a novel and original connection between CcrM-dependent DNA methylation and genome maintenance in an Alphaproteobacterial pathogen. To investigate the impact of DNA methylation by CcrM on the transcriptome of an A. tumefaciens C58 derivative strain with a dicentric chromosome, we engineered a conditional ccrM mutant. A second copy of ccrM (Atu0794) under the control of the IPTG-inducible Ptac promoter was introduced into the dicentric chromosome of A. tumefaciens and then the native ccrM gene was deleted following a standard double recombination procedure, generating strain JC2307 (DccrM Ptac-ccrM) on ATGN minimal medium containing IPTG. We then performed gene expression profiling analyses using RNA-seq data comparing viable A. tumefaciens JC2307 cells incubated for 7 hours in ATGN without IPTG with that of control mutant cells cultivated in ATGN with IPTG and WT cells cultivated in ATGN without IPTG. Comparative gene expession profiling analysis of RNA-seq data for three types of cell cultures in exponential phase: DccrM Ptac-ccrM + IPTG, WT and DccrM Ptac-ccrM -IPTG.

细胞周期调控型DNA甲基转移酶CcrM（cell cycle-regulated DNA methyltransferase CcrM）在大多数α-变形菌门（Alphaproteobacteria）中保守存在，但其在具有复杂基因组或多中心基因组的细菌中的功能仍未被探索。本研究针对拥有两个必需复制起点的双中心基因组的根癌农杆菌（Agrobacterium tumefaciens）野生型与CcrM缺失菌株，比较了其甲基化组（methylome）、转录组（transcriptome）及表型特征。研究发现，DNA甲基化对细菌的运动能力、生物被膜形成与细胞活力具有多效性影响。值得注意的是，CcrM可促进repABCCh2操纵子（repABCCh2 operon）的表达，该操纵子编码ori2复制起始与分配所需的蛋白；同时可抑制编码保守全局细胞周期调控因子（global cell cycle regulator）的gcrA基因的表达。通过对活细胞内ori1与ori2的成像实验，我们发现，在低甲基化基因组的细胞中，ori2介导的复制过程常出现延迟；而在高甲基化基因组的细胞中，ori2则会过度起始复制。据此，我们提出CcrM介导的甲基化可激活依赖RepABC的染色体复制起点，揭示了α-变形菌门病原体中CcrM依赖型DNA甲基化与基因组维持之间一种全新的关联机制。为探究CcrM介导的DNA甲基化对具有双中心染色体的根癌农杆菌C58衍生菌株转录组的影响，我们构建了条件性ccrM突变株。将受异丙基-β-D-硫代半乳糖苷（IPTG，isopropyl β-D-thiogalactoside）诱导的Ptac启动子（Ptac promoter）调控的第二份ccrM拷贝（Atu0794）整合至根癌农杆菌的双中心染色体中，随后通过标准双重组操作（double recombination procedure）删除内源ccrM基因，最终在含有IPTG的ATGN基本培养基（ATGN minimal medium）上获得菌株JC2307（ΔccrM Ptac-ccrM）。随后，我们利用RNA测序（RNA-seq）数据开展基因表达谱分析，比较了三类样本的基因表达差异：在不含IPTG的ATGN基本培养基中孵育7小时的活根癌农杆菌JC2307菌株、在含IPTG的ATGN基本培养基中培养的对照突变株，以及在不含IPTG的ATGN基本培养基中培养的野生型菌株。本次分析针对三类处于指数生长期的细胞培养物：ΔccrM Ptac-ccrM + IPTG组、野生型组与ΔccrM Ptac-ccrM -IPTG组的RNA-seq数据开展了比较基因表达谱分析。