show Abstracthide AbstractThe 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. Overall design: 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.