Cell Cycle Constraints and Environmental Control of Local DNA Hypomethylation in α-Proteobacteria

Abstract

Heritable DNA methylation imprints are ubiquitous and underlie genetic variability from bacteria to humans. In microbial genomes, DNA methylation has been implicated in gene transcription, DNA replication and repair, nucleoid segregation, transposition and virulence of pathogenic strains. Despite the importance of local (hypo)methylation at specific loci, how and when these patterns are established during the cell cycle remains poorly characterized. Taking advantage of the small genomes and the synchronizability of α-proteobacteria, we discovered that conserved determinants of the cell cycle transcriptional circuitry establish specific hypomethylation patterns in the cell cycle model system Caulobacter crescentus. We used genome-wide methyl-N6-adenine (m6A-) analyses by restriction-enzyme-cleavage sequencing (REC-Seq) and single-molecule real-time (SMRT) sequencing to show that MucR, a transcriptional regulator that represses virulence and cell cycle genes in S-phase but no longer in G1-phase, occludes 5'-GANTC-3' sequence motifs that are methylated by the DNA adenine methyltransferase CcrM. Constitutive expression of CcrM or heterologous methylases in at least two different α-proteobacteria homogenizes m6A patterns even when MucR is present and affects promoter activity. Environmental stress (phosphate limitation) can override and reconfigure local hypomethylation patterns imposed by the cell cycle circuitry that dictate when and where local hypomethylation is instated.

Fig 1. Regulation of C. crescentus cell cycle and methylation of the chromosome.

(A) Schematic of the C. crescentus cell cycle and the regulatory interactions that control G1-phase promoters. Transcription from G1-phase promoters is activated by phosphorylated CtrA (CtrA~P, in blue) and repressed by MucR1/2. CtrA~P accumulates in pre-divisional and swarmer (SW) cells, and is eliminated by regulated proteolysis in the stalked (ST) cell upon compartmentalization and at the swarmer to stalked cell differentiation. The red bar indicates that MucR1/2 proteins are present at all stages of the cell cycle. (B) Schematic of the chromosome replication and adenine methylation (m6A) during the C. crescentus cell cycle. Chromosome replication and methylation are shown for the same cell cycle stages depicted in panel A. In non-replicative SW cells (T10) the chromosome is methylated on both strands. Upon differentiation into ST cells (T40), chromosome replication start from the origin (blue star) located at the old cell pole; progression of the replisomes (orange dots) generates hemi-methylated chromosomes (black and green lines represent respectively the methylated and unmethylated strand; T70 and T100). The cell cycle-regulated adenine methyltransferase CcrM is synthesized only in late pre-divisional cells, where it methylates the newly synthesized chromosome strands. CcrM is then specifically proteolyzed by the Lon protease. The purple numbers indicate the position along the chromosome of the P169 (1), P1149 (2) and P2901 (3) promoters. T10, T40, T70 and T100 indicate the time after synchronization at which the samples for anti-MucR1 ChIP-Exo were taken (see text).

Fig 2. MucR occludes specific GANTC sites from methylation.

(A) Schematic of the loci carrying hypomethylated GANTCs occluded by MucR. The position of the hypomethylated GANTCs identified by Kozdon et al. [12] is indicated by purple asterisks. Red lines represent the occupancy of MucR1 and the values (x10⁴ per-base coverage) calculated by the super-resolution bioinformatic approach represent the average of the four time points (T10, T40, T70 and T100, as described in the Methods). The MucR-dependent transcription start sites, determined by TSS-EMOTE, are indicated by black (sense) and green (antisense) arrows. Dashed arrows indicate transcription start sites found in both WT and ΔmucR1/2 strains (CCNA_01083-CCNA_01084) or down-regulated in the ΔmucR1/2 strain compared to the WT (promoter of CCNA_02831). (B) Methylation percentage of the loci shown in panel A in the WT and ΔmucR1/2 strains, as determined by HinfI-qPCR.

Fig 3. Hypomethylation by MucR is impaired in G1-phase cells.

(A) Methylation percentage of the P169, P1149 and P2901 sequences in strains in which the methyltransferase CcrM is stabilised. The HinfI-qPCR analysis indicates that methylation is increased in cells carrying P_lac-ccrM or the lon mutation. In the case of the lon mutant, the methylation of P169, P1149 and P2901 is not affected by increased levels of MucR1 [R1: P_van-mucR1, R1 long: N-terminally extended dominant-negative MucR1 variant expressed from P_van on pMT335]. (B) Methylation percentage of the P169, P1149 and P2901 sequences in WT cells that constitutively express ccrM or heterologous GANTC-methylases from P_van on pMT335 (TA, Thermoplasma acidophilum; HP, Helicobacter pylori; hinf, Haemophilus influenzae). (C) Immunoblot showing the steady-state levels of CcrM, MucR1 and MucR2 in WT cells as well as the P_lac-ccrM, lon and ΔmucR1/2 strains. Molecular size standards are indicated on the right as blue lines with the corresponding values in kDa. (D) Genome-wide occupancy of MucR1 in synchronised WT cells at four different time points as determined by ChIP-Exo and super-resolution bioinformatic analysis: swarmer (T10), stalked (T40), early pre-divisional (T70) and late pre-divisional cells (T100). The x axis represents the nucleotide position on the genome, whereas the y axis shows the enrichment ratio (E.R.) for each promoter region as reported in the S4 Table (see Methods for a detailed description). (E) Heat map of the enrichment ratios of selected loci (those that contain hypomethylated GANTCs occluded by MucR, see Fig 2) at the four time points after synchronization. The pilA locus is shown as comparison as it is a well-characterized target of MucR [17]. The heat map shows that MucR1 is constitutively associated with these loci.

Fig 4. MucR and methylation by CcrM regulate transcription from target promoters.

Beta-galactosidase activity of P169 (WT promoter) and P169* (with all GANTCs mutated to GTNTCs) (A), P2901 (WT promoter) and P2901* (with the two GANTCs mutated to GTNTCs) (B) and P1149 (C) in WT and ΔmucR1/2 cells. Mutation of MucR1/2 increases expression from P169-, P2901- and P1149-lacZ independently from the presence of GANTCs. Values are expressed as percentages (activity of WT promoter in WT cells set at 100%). (D) Beta-galactosidase activity of P169-, P1149-, P2901-lacZ promoter probe constructs and two MucR-dependent control promoter reporters (P_hvyA-lacZ and P_pilA-lacZ) in WT and cells that constitutively express ccrM (ccrM::P_lac-ccrM or Δlon::Ω). Methylation of the target promoters by CcrM increases the LacZ activity. Values are expressed as percentages (activity in WT cells set at 100%). (E) Beta-galactosidase activity of P169-, P1149- and P2901-lacZ in WT cells that constitutively express ccrM or a heterologous GANTC-methylase from T. acidophilum (TA) on plasmid under control of P_van. Values are expressed as percentages (activity in WT carrying the empty vector set at 100%). (F) Beta-galactosidase activity of P169-, P1149- and P2901-lacZ in WT and ΔmucR1/2 cells that constitutively express ccrM (ccrM::P_lac-ccrM). Values are expressed as percentages (activity in WT cells set at 100%).

Fig 5. Hypomethylation control by MucR is conserved in α-proteobacteria.

(A) Schematic of the two MucR-dependent hypomethylated loci (SMa1635 and SMa2245) identified by SMRT-sequencing in the in S. meliloti WT genome. Position of the hypomethylated GANTCs is indicated by purple asterisks. The blue arrows indicate the DNA fragments cloned for LacZ promoter probe assays. (B) HinfI-qPCR analysis showing that SMa1635 and SMa2245 are hypomethylated in S. meliloti WT cells compared to mucR::Tn cells. (C) Constitutive expression of ccrM^Cc from P_lac on pSRK [47] in S. meliloti WT cells increases the methylation percentage of SMa1635 and SMa2245, indicating that hypomethylation of GANTCs by MucR is also impaired in S. meliloti G1-phase cells. (D) MucR occupancy at SMa1635, SMa2245 and SMc1552 (control) in WT and mucR::Tn S. meliloti cells, as determined by qChIP using antibodies to C. crescentus MucR2. SMa1635 and SMa2245 are bound by S. meliloti MucR, which suggests that hypomethylation of GANTCs at these loci is directly due to occlusion by MucR. (E) Beta-galactosidase activity of Pa1635–lacZ and Pa2245–lacZ in S. meliloti (fragments indicated by blue arrows in panel A). Both DNA fragments show a promoter activity that is strongly de-repressed in mucR::Tn cells compared to the WT strain. Values are expressed as percentages (activity in WT cells set at 100%). (F) Beta-galactosidase activity of Pa1635 and Pa2245 in C. crescentus WT and ΔmucR1/2 cells expressing mucR^Sm. Expression of mucR^Sm from P_van on pMT335 decreases beta-galactosidase activity of Pa1635 and Pa2245. Values are expressed as percentages (activity in WT cells carrying the empty vector set at 100%). (G) Beta-galactosidase activity of Pa1635 and Pa2245 in C. crescentus WT, P_lac-ccrM or lon cells. Values are expressed as percentages (activity in WT cells set at 100%).

Fig 6. Cell cycle and environmental signals affect methylation patterns.

(A) Competition between CtrA and CcrM. Schematic of the synthetic promoter carrying an attenuated E. coli phage T5 promoter followed by three GANTCs (purple asterisks) overlapping two CtrA-boxes (in yellow). HinfI-qPCR analysis shows that this sequence is hypomethylated in WT cells, whereas constitutive expression of ccrM (P_lac-ccrM) increases the methylation percentage. This indicates that DNA-binding proteins other than MucR can also occlude GANTCs from methylation. (B) Methylation percentage of P169 and P1149 in phosphate-limiting conditions compared to rich medium (PYE), determined by HinfI-qPCR analysis. Phosphate starvation (6h) significantly increases the methylation level of P169 but not P1149. (C) Beta-galactosidase activity of P169–lacZ and P169*–lacZ (GANTCs mutated to GTNTCs as in Fig 4A) in WT and ΔmucR1/2 cells in rich medium and phosphate-limiting conditions. Phosphate starvation induces transcription from P169–lacZ and P169*–lacZ independently from the presence of MucR1/2. Values are expressed as percentages (activity in WT cells grown in PYE set at 100%). (D) Beta-galactosidase activity of P1149–lacZ in WT and ΔmucR1/2 cells in rich medium and phosphate-limiting conditions. Phosphate starvation does not significantly affect the activity of P1149–lacZ. Values are expressed as percentages (activity in WT cells grown in PYE set at 100%).