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

Abstract

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.DNA methylation | SMRT sequencing | methylome D NA methylation involves the addition of a methyl group to either adenine or cytosine by a site-specific DNA methyltransferase. The role of this epigenetic mechanism in the regulation of multiple bacterial processes is described in several reviews (1-5). Two adenine methyltransferases, Dam in the γ-proteobacterium Escherichia coli and CcrM in the α-proteobacterium Caulobacter crescentus, have regulatory functions and are not part of a restriction-modification system. Notably, the synthesis of CcrM, but not Dam, is cell cycle-regulated. CcrM plays an essential role in Caulobacter cell-cycle control (6-8) and in Agrobacterium tumifaciens (9), and it is involved in infective processes in Brucella abortus (10) and in the plant-microbe symbiotic relationship in Mesorhizobium meliloti (11), whereas Dam is involved in virulence of numerous bacterial species (1).In α-proteobacteria, CcrM methylates adenines in GANTC sites (12). In Caulobacter, CcrM is expressed for only a brief time late in chromosome replication (12), and the Lon protease then rapidly degrades CcrM, with a half-life of about 10 min (13). Before the initiation of DNA replication, the chromosome is in the fully methylated state at GANTC sites. Upon bidirectional progression of the replication forks, newly replicated DNA becomes hemimethylated; each copy contains one parental methylated strand and one daughter unmethylated strand. It was predicted that newly replic...

Essential roles of methionine and S -adenosylmethionine in the autarkic lifestyle of Mycobacterium tuberculosis

“…The results supported previous data showing that only a single DNA methylase is active in H37Rv [Rv3263, which methylates the adenines in CTGGAG/CACCAG to form N6-methyladenine (23)]. The interpulse duration (IPD) ratio (Methods) represents the proportion of molecules in a population that are methylated at each site (24), and the mean IPD ratio summarizes this value across the genome. We measured the mean IPD ratio of triplicate samples of the H37Rv control, the ΔmetA mutant grown in the presence of methionine, and the mutant after 5 d of methionine starvation.…”

Genome Modification in Enterococcus faecalis OG1RF Assessed by Bisulfite Sequencing and Single-Molecule Real-Time Sequencing

“…SMRT sequencing has been used to characterize the methylomes of bacteria (47)(48)(49)(50)(51)(52)(53)(54). During SMRT sequencing, DNA polymerase kinetics are monitored in real time; modified bases in the sequencing template lead to altered polymerase kinetics relative to those obtained with an unmodified template (55,56).…”

“…This is because only subtle changes in polymerase kinetics are generated by m5C (53,55). Tet1 oxidation has been utilized to convert m5C to 5-carboxylcytosine, which enhances detection of m5C sites in bacterial genomes (49,(52)(53)(54). This protocol amendment was not utilized in our study.…”

High resolution mapping of modified DNA nucleobases using excision repair enzymes

“…These methods achieve resolution in the 100-1000 base pair range, precluding direct identification of specific sites of DNA modification. A new method based on ChIP-seq for mapping sites of singlestranded DNA enables global studies of putative DNA damage but does not identify specific modified nucleobases ).Finally, new single-molecule sequencing platforms detect a variety of modified nucleobases in their native contexts (Clarke et al 2009;Clark et al 2011;Kozdon et al 2013), but nanopore sequencers are not yet widely available, and real-time analysis of single molecule polymerase incorporation events suffers from a high error rate and high cost of genome-wide coverage, limiting comprehensive characterization of large eukaryotic genomes.Here we developed a method that couples the specificity and efficiency of excision repair enzymes with the scale and throughput of massively parallel DNA sequencing to identify single sites or local content of modified nucleobases throughout the genome. This new Excision-seq method provides advantages over existing methods by generating sequencing libraries that are enriched for sites of modification, while maintaining high resolution mapping information.…”

“…Finally, new single-molecule sequencing platforms detect a variety of modified nucleobases in their native contexts (Clarke et al 2009;Clark et al 2011;Kozdon et al 2013), but nanopore sequencers are not yet widely available, and real-time analysis of single molecule polymerase incorporation events suffers from a high error rate and high cost of genome-wide coverage, limiting comprehensive characterization of large eukaryotic genomes.…”