The functions of DNA methylation by CcrM in Caulobacter crescentus: a global approach.

DNA methylation is involved in a diversity of processes in bacteria, including maintenance of genome integrity and regulation of gene expression. Here, using Caulobacter crescentus as a model, we exploit genome-wide experimental methods to uncover the functions of CcrM, a DNA methyltransferase conserved in most Alphaproteobacteria. Using single molecule sequencing, we provide evidence that most CcrM target motifs (GANTC) switch from a fully methylated to a hemi-methylated state when they are replicated, and back to a fully methylated state at the onset of cell division. We show that DNA methylation by CcrM is not required for the control of the initiation of chromosome replication or for DNA mismatch repair. By contrast, our transcriptome analysis shows that >10% of the genes are misexpressed in cells lacking or constitutively over-expressing CcrM. Strikingly, GANTC methylation is needed for the efficient transcription of dozens of genes that are essential for cell cycle progression, in particular for DNA metabolism and cell division. Many of them are controlled by promoters methylated by CcrM and co-regulated by other global cell cycle regulators, demonstrating an extensive cross talk between DNA methylation and the complex regulatory network that controls the cell cycle of C. crescentus and, presumably, of many other Alphaproteobacteria.

Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle.

The Caulobacter DNA methyltransferase CcrM is one of five master cell-cycle regulators. CcrM is transiently present near the end of DNA replication when it rapidly methylates the adenine in hemimethylated GANTC sequences. The timing of transcription of two master regulator genes and two cell division genes is controlled by the methylation state of GANTC sites in their promoters. To explore the global extent of this regulatory mechanism, we determined the methylation state of the entire chromosome at every base pair at five time points in the cell cycle using single-molecule, real-time sequencing. The methylation state of 4,515 GANTC sites, preferentially positioned in intergenic regions, changed progressively from full to hemimethylation as the replication forks advanced. However, 27 GANTC sites remained unmethylated throughout the cell cycle, suggesting that these protected sites could participate in epigenetic regulatory functions. An analysis of the time of activation of every cell-cycle regulatory transcription start site, coupled to both the position of a GANTC site in their promoter regions and the time in the cell cycle when the GANTC site transitions from full to hemimethylation, allowed the identification of 59 genes as candidates for epigenetic regulation. In addition, we identified two previously unidentified N(6)-methyladenine motifs and showed that they maintained a constant methylation state throughout the cell cycle. The cognate methyltransferase was identified for one of these motifs as well as for one of two 5-methylcytosine motifs.

An essential transcription factor, SciP, enhances robustness of Caulobacter cell cycle regulation.

A cyclical control circuit composed of four master regulators drives the Caulobacter cell cycle. We report that SciP, a helix-turn-helix transcription factor, is an essential component of this circuit. SciP is cell cycle-controlled and co-conserved with the global cell cycle regulator CtrA in the α-proteobacteria. SciP is expressed late in the cell cycle and accumulates preferentially in the daughter swarmer cell. At least 58 genes, including many flagellar and chemotaxis genes, are regulated by a type 1 incoherent feedforward motif in which CtrA activates sciP, followed by SciP repression of ctrA and CtrA target genes. We demonstrate that SciP binds to DNA at a motif distinct from the CtrA binding motif that is present in the promoters of genes co-regulated by SciP and CtrA. SciP overexpression disrupts the balance between activation and repression of the CtrA-SciP coregulated genes yielding filamentous cells and loss of viability. The type 1 incoherent feedforward circuit motif enhances the pulse-like expression of the downstream genes, and the negative feedback to ctrA expression reduces peak CtrA accumulation. The presence of SciP in the control network enhances the robustness of the cell cycle to varying growth rates.