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Exocyclic GpC DNA methyltransferase from Celeribacter marinus IMCC12053
Korean J. Microbiol 2019;55(2):103-111
Published online June 30, 2019
© 2019 The Microbiological Society of Korea.

Junghee Kim, and Hyun-Myung Oh*

Institute of Liberal Arts Education, Pukyong National University, Busan 48547, Republic of Korea
Correspondence to: E-mail:; Tel.: +82-51-629-6869; Fax: +82-51-629-6949
Received January 9, 2019; Revised April 8, 2019; Accepted April 10, 2019.

DNA methylation is involved in diverse processes in bacteria, including maintenance of genome integrity and regulation of gene expression. CcrM, the DNA methyltransferase conserved in Alphaproteobacterial species, carries out N6-adenine or N4-cytosine methyltransferase activities using S-adenosyl methionine as a co-substrate.

Celeribacter marinus IMCC12053 from the Alphaproteobacterial group was isolated from a marine environment. Single molecule real-time sequencing method (SMRT) was used to detect the methylation patterns of C. marinus IMCC12053. Gibbs motif sampler program was used to observe the conversion of adenosine of 5′-GANTC-3′ to N6-methyladenosine and conversion of N4-cytosine of 5′-GpC-3′ to N4-methylcytosine. Exocyclic DNA methyltransferase from the genome of strain IMCC12053 was chosen using phylogenetic analysis and N4-cytosine methyltransferase was cloned. IPTG inducer was used to confirm the methylation activity of DNA methylase, and cloned into a pQE30 vector using dam-/dcm- E. coli as the expression host. The genomic DNA and the plasmid carrying methylase-encoding sequences were extracted and cleaved with restriction enzymes that were sensitive to methylation, to confirm the methylation activity. These methylases protected the restriction enzyme site once IPTG-induced methylases methylated the chromosome and plasmid, harboring the DNA methylase. In this study, cloned exocyclic DNA methylases were investigated for potential use as a novel type of GpC methylase for molecular biology and epigenetics.

Keywords : adenosine, cytosine, DNA methyltransferase, exocyclic amine group

Bacterial DNA methylation is one of the key steps in the maintenance of genome integrity and regulation of gene expressions (Gonzalez et al., 2014). DNA methylates due to formation of N6-methyladenine and 5-methylcytosine by an enzyme DNA methyltransferase (MTase), which plays important roles in gene transcription, bacterial growth, and proliferation (Li et al., 2017). Genes involved in DNA methylation and DNA methyltransferases (MTases) have been studied for the last 70 years (Jurkowska and Jeltsch, 2016). Deoxyadenosine methyltransferase (Dam) and CcrM (cell cycle-regulated MTase) have been widely studied and researched. Dam is important for the expression of pap operon, DNA replication initiation, and DNA repair (Adhikari and Curtis, 2016). CcrM is well-conserved among the Alphaproteobacterial lineage (Gonzalez et al., 2014), and CcrM-mediated DNA-methylation is essential in cell-cycle regulation (Mohapatra et al., 2014).

Caulobacter crescentus NA1000 is a model microorganism for gene expression study of CcrM protein (Kozdon et al., 2013), and CcrM homologs as other alphaproteobacterial DNA-(adenine N6)-MTases have gained attention (Maier et al., 2015). CcrM protein carries out cytosine-N4-MTase activity by using S- adenosyl methionine as a co-substrate, and also transforms N6-methyl adenosine (5′-GANTC-3′) (Jeltsch et al., 1999). N6- or N4-methyltransferase activities of CcrM-type proteins are grouped as exocyclic amino moiety enzymes compared to 5-methyl cytosine modifications, which directly modify the heterocyclic aromatic ring of the pyrimidine derivative (Loenen and Raleigh, 2014).

Celeribacter marinus IMCC12053 and Novosphingobium pentaromativorans US6-1 were isolated from a marine environment (Baek et al., 2014), and the strain IMCC12053, which is a known host for marine bacteriophage P12053L, was selected (Kang et al., 2012). N. pentaromativorans US6-1 was originally isolated as an oil spill degrading bacterium (Sohn et al., 2004) and the genome was elucidated form mining aromatic-hydrocarbon-degrading genes (Luo et al., 2012; Choi et al., 2015). Both the strains C. marinus IMCC12053 and N. pentaromativorans US6-1 were known to have adenosine-specific DNA methylase and N4-cytosine specific DNA methylase, according to our previous study (Yang et al., 2016). The CcrM homologs, IMCC12053_18853 (GenBank Acc. ALI55832) from strain IMCC12053 and AIT78768 (GenBank Acc. AIT78768) from US6-1, showed GpC and CpG motif-specific exocyclic amine methylating activities, respectively (Yang et al., 2016).

In the present study, we report the cloning and characterization of exocyclic DNA MTase from C. marinus IMCC12053, using N. pentaromativorans US6-1 genes as a control.

Materials and Methods

Methylation pattern analysis

Single molecule real time (SMRT) sequencing data produced by PacBioRS II were used for finding patterns from genomes of C. marinus IMCC12053 (Yang et al., 2017) (Table 1) and N. pentaromativorans US6-1 (data not shown). In this study, SMRT method (Eid et al., 2009) was exploited to find patterns of DNA methylation from the strains. Methylated patterns were analyzed by Gibbs motif sampler program (Lawrence et al., 1993), and we could obtain methylation patterns covering GpC (Yang et al., 2017) or CpG regions (Unpublished data by Yang, Jhung Ahn). IMCC12053_18853 (GenBank Acc. ALI55832) from C. marinus IMCC12053 and other related sequences of CcrM homologs were used for phylogenetic analyses using amino acids or nucleotide alignments. Amino acid sequences were aligned using hmm-aligner from hmmer 3.0 program (Eddy, 2011) and RAxML (ver. 7.3.4) (Stamatakis, 2006) were used for constructing the generation tree. Nucleotide alignments and subsequent analysis using MEGA7.0 program (Kumar et al., 2016) and MrBayes 3.2 (Ronquist et al., 2012) were used for constructing the Bayesian inference tree.

Sequence motif elements flanking m4C (N4-methylcytosine) and m6A (N6-methyladenosine) modifications in the IMCC12053 genome Gibbs program (v3.1) was used for collecting DNA sequences (10 base pairs long window size) and motif elements occurring greater than 50% of the time was used for visualization of the patterns. Methylated bases are shown in black letters.

Motif element Left end Right end No. motifs Avg. score (-10 LogP) Standard deviation
-9 0 43 25.7 4.81
-8 1 44 24.7 4.30
   -7 2 70 25.1 5.81
    -6 3 10 23.4 2.76
     -5 4 439 25.4 5.34
      -3 6 172 25.7 5.38
       -2 7 19 25.3 5.69
        -1 8 9 28.1 9.55
         0 9 19 27.8 6.24
-9 0 65 51.9 24.2
-8 1 35 51.9 22.7
   -4 5 1537 59.8 17.7
    -3 6 1110 59.3 16.9
     -2 7 11 26.2 5.81
      -1 8 9 21.8 2.33
       0 9 8 25.5 4.17

Strains and media

C. marinus IMCC12053 and N. pentaromativorans US6-1 cultures were used for extracting genomic DNA. Cloning vectors used for open reading frames from the genomic DNA were pGEMT-Easy and pGEMT Vector Systems (Promega). Expression of the DNA methylase was confirmed by sub-cloning into pQE30 (Qiagen). Chemically competent E. coli strains DH5α (Enzynomics) and HIT™-GM2163 (dam-/dcm-E. coli K12 strain, HIT™-GM2163 Value 108, RBC) were used for the study. HiYield Plus™ Plasmid Mini Kit (RBC) was used for plasmid DNA purifications. Luria-Bertani (LB) medium (Difco) was used with or without 100 μg/ml ampicillin. LB agar plates were also used with 100 μg/ml ampicillin plus 15 g/L Bacto Agar (Difco).

Genomic DNA extraction and polymerase chain reaction (PCR)

Genomic DNAs were purified using PureHelix Genomic DNA Prep Kit (Column type) (NanoHelix) and genomic DNAs were the template for PCR. Sequence-specific forward and reverse oligonucleotides for DNA methylases were as follows: 5′-TGACGACGAAAACACGTGAGGC-3′/5′-AGTTCATCTCCGCGCGGATTTG-3′ for C. marinus strain IMCC12053 and 5′-TGGGGCAGGTACTCGTCAAGG-3′/5′-ACGGCTCGGTGGCAAGCAGG-3′ for N. pentaromativorans strain US6-1.

SimpliAmp Thermal cycler (Life Technologies) was used for PCR. Initial denaturation was done for 30 sec at 95°C, amplification comprising the three-step temperature cycles of 30 sec at 95°C, 30 sec at 58–60°C, and 60 sec at 72°C was iterated 25 times. Final primer extension was done by incubating tubes for 60 sec at 72°C. Amplified PCR fragments were confirmed on 1% agarose gel by SYBR Safe DNA Gel Stain (Invitrogen). DNA fragments were cloned into pGEMT-Easy or pGEMT vectors. Cloned sequences were finally confirmed using Big Dye Sanger sequencing by Macrogen Inc.

Restriction enzyme digestion for sub-cloning open reading frames

DNA methylase ORF (GenBank Acc. ALI55832) from Celeribacter marinus IMCC12053 was digested with KpnI, PstI, and ORF (GenBank Acc. ALI55832) for Novosphingobium pentaromativorans US6-1 (GenBank Acc. AIT78768) was treated with SphI and SalI. Cloning vector pQE30 was used for the sub-cloning of the DNA fragments. The vector was treated with KpnI/PstI or SphI/SalI, followed by treatment with shrimp alkaline phosphatase (SAP) for 30 min at 37°C. SAP reaction was stopped by heat-inactivation at 65°C for 5 min. T4 DNA ligase (Promega cat. M180A) and accompanying standard sticky end ligation was done for ligating vectors and DNA inserts. Ligation reaction mixtures were transformed into competent DH5α (Enzynomics).

DNA methylase assay using methylation-sensitive restriction digestion

Once ALI55832 (IMCC12053_18853 from C. marinus IMCC12053) and AIT78768 (from N. pentaromativorans US6-1) were sub-cloned into pQE30 and E. coli DH5α, each plasmid was transformed into E. coli HIT™-GM2163 that had dam-/dcm- phenotypes. DNA methylase carrying the expression vector would methylate pQE30/methylases and chromosomal DNAs, once the cultures were treated with 100 μM IPTG for 3 h at 37°C in a shaking incubator (250 rpm).

Methylated and pristine genomic DNAs were purified using PureHelix Genomic DNA Prep Kit (Column type) (NanoHelix) using 4 ml of E. coli cultures. Plasmids were also prepared using a HiYield plus plasmid mini Kit (RBC), using 3 ml of the cultures.

Enzymes for methylation sensitive restriction digestion included MboI, MspI, and MluI (NEB). Methylated and non- methylated genomic DNAs and pQE30 plasmids carrying DNA methylases were treated at 37°C for 2 h; thereafter, these were analyzed on a 1% or 1.2% agarose TAE gel electrophoresis system and the gels were photographed after staining with SYBR safe DNA gel stain.

Results and Discussion

Methylation pattern recognition using PacBio RS II data

According to SMRT (single-molecule, real-time) analysis and Gibbs motif sampler program analysis (Yang et al., 2017), C. marinus IMCC12053 showed N4-cytosine methylase activity (GpC methylase) in addition to N6-adenosine methylase activity (5′-GANTC-3′) (Table 1).

CDD (Conserved Domain Database) search program of NCBI (Marchler-Bauer et al., 2015) indicated that IMCC12053_18853 (GenBank Acc. ALI55832) could be annotated as CcrM (CCNA_00382) from Caulobacter vibrioides NA1000 and these proteins could be observed for other genomes (Table 2). Using N. pentaromativorans US6-1 genome analysis as a control, we could also annotate AIT78768 as another CcrM (Table 2) with N4-cytosine methylase activity (CpG methylase, Supplementary data Table S1).

Comparison of DNA methyl transferases among some strains from Alphaproteobacteria subdivision

Organism Locus tag Description CDD result Comment
marinus IMCC12053
IMCC12053_1885 DNA modification methylase CcrM

IMCC12053_605 Predicted N6-adenine-specific DNA methylase

IMCC12053_2040 DNA modification methylase

IMCC12053_3116 DNA modification methylase

AIT78768 DNA modification methylase restriction endonuclease subunit M

AIT79233 Type II restriction enzyme, methylase subunit YeeA Annotated as “lactate dehydrogenase”

baekdonensis B30
B30_03115 Adenine-specific methyltransferase CcrM

B30_06546 Predicted N6-adenine-specific DNA methylase

indicus P73
P73_0385 Adenine-specific methyltransferase CcrM

P73_4804 DNA-cytosine methyltransferase

P73_2233 Predicted N6-adenine-specific DNA methylase

P73_4799 C-5 cytosine-specific DNA methylase

vibrioides NA1000
CCNA_00382 Adenine-specific methyltransferase CcrM

CCNA_00869 Type II restriction enzyme, methylase subunits adenine methyltransferase with an associated restriction enzyme domain

CCNA_01085 DNA-cytosine methyltransferase

CCNA_03741 DNA-cytosine methyltransferase

Phylogenetic analysis of DNA methylases from Celeribacter marinus IMCC12053 and related bacteria

We selected DNA methylases from C. marinus IMCC12053 and other genomes for constructing phylogenetic trees. To begin with, C. marinus IMCC12053 DNA methylases were four in number (Table 1) and these sequences were used as seed sequences for hmm search from hmmer 3.0 program. Using archaeal and bacterial sequences as outgroup sequences, CcrM related amino acids were aligned for a phylogenetic tree using RAxML (ver. 7.3.4) (Stamatakis, 2006). Alphaproteobacterial CcrM sequences were grouped with high boot strap value (94%) with CcrM from Caulobacter crescentus NA1000; others with EC were methylases with N4-cytosine MTase activities. RAxML analysis was done with 29 amino acid sequences with 431 distinct alignment patterns and substitution matrix was LG. Gaps and undetermined characters were excluded from the alignmnet, 161 amino acids were used for the RAxML with 100 rapid bootstrap values (Fig. 1).

Fig. 1.

Phylogeny of CcrM methyltransferases from Alphaproteobacteria with other groups of bacteria and archaea. Nine uppermost enzyme sequences represents Alphaproteobacterial CcrM sequences and they grouped with high boot strap value (94%) with CcrM from Caulobacter crescentus NA1000; others with EC number are methylases with N4-cytosine methyltransferase activities and they are listed in REBASE database. Tree was drawn with RAxML (version 7.3.4) with 29 amino acid sequences with 431 amino acid alignment with 100 rapid bootstrap for ML search. Gaps and undetermined characters were 62.69% and 161 amino acids were used for each sequence. Substitution matrix was LG using fixed base frequencies was exploited for the calculation of to an accuracy of 0.1000000000 Log likelihood units.

As shown in Fig. 1, 9 CcrM sequences from the upper-most part of the tree includes C. marinus IMCC12053 and N. pentaromativorans US6-1. These sequences formed a distinct clade with C. crescentus NA1000 with a boot strap value of 94%.

Using nucleotide sequence of IMCC12053_18853 (GenBank Acc. ALI55832 from CP012023.1) as a seed sequence for blastn and MEGA7.0 program for alignments, optimal condition determination, and nexus format file export for MrBayes. The 29 sequences collected, were used for Bayesian inference tree construction using MrBayes 3.2. IMCC12053_18853 (from Celeribacter marinus IMCC12053) formed a distinct sister-group with C. crescentus NA1000, N. pentaromativorans US6-1, Brucella abortus BAB8416, and Celeribacter baekdonensis B30 with 68% probability value (Supplementary data Fig. S1). Phylogenetic analyses with amino acid and nucleotide alignments verified that DNA MTase gene IMCC12053_18853 may play a role as a canonical CcrM gene, as seen in C. crescentus NA1000 (Adhikari and Curtis, 2016).

Cloning and expression of IMCC12053_1885

For MTase activity confirmation, we used pristine methylation-free chromosomal or plasmid DNA that was prepared using E. coli GSM2163 as a host, but IPTG-induction (100 μM IPTG at 37°C for 3 h) was expected to induce MTase protein expression as well as chromosomal and plasmid DNAs. MTases from strains IMCC12053 and US6-1 were digested using Msp I (5′-CCGG-3′; CpG not sensitive) and Mbo I (5′-GATC-3′; dam blocked and CpG impaired by overlapping) and MTase expression of US6-1 blocked Mbo I site by CpG methylase activities (Supplementary data Fig. S2). From the digestion pattern of GpC or CpG MTases on chromosomal sequences, IMCC12053_1885 (GenBank Acc. ALI55832) was a MTase different from MTase of US6-1. This result corresponded to PacBio RS II data of previous study (Yang et al., 2016).

IPTG-induced and Uninduced plasmid DNA encoding IMCC12053_1885 were digested with CpG blocked restriction enzymes (Supplementary data Table S1) including Mlu I (5′-ACGCGT-3′) and Pvu I (5′-CGATCG-3′) according to a previous method (Eberhard et al., 2001). Linearized plasmids were treated with Pst I (5′-CTGCAG-3′) showed the same pattern for methylated (I) and non-methylated (U) (Fig. 2A). Mlu I/Pst I digestion showed a slight increase in larger fragment size (that is from a single cut not by Mlu I but by Pst I) in the IPTG-induced plasmid preparation. Mlu I/Pvu I digestion produced more evident single fragment (indicated by arrows in Fig. 2A) in the induced plasmid lane. NebCutter 2.0 (Vincze et al., 2003) was used for generating in silico digested gel image files (assuming 1.2% TAE agarose) using the pQE30/IMCC12053_1885 sequence (refer to supplementary text) (Fig. 2B). NebCutter 2.0 was used only to calculate CpG methylation and we know that IMCC12053_1885 is not a CpG MTase (Supplementary data Fig. S2), but CpG-blocked Pvu I and Pst I (not sensitive to any methylation) successfully linearized pQE30/IMCC12053_1885. Moreover CpG-blocked Mlu I (5′-ACGCGT-3′) also had a GpC dinucleotide recognition to be impaired by IMCC12053_1885 MTase when 100 μM IPTG was added to the culture for expression of MTase activity. In real electrophoresis gels, partially methylated or hemi-methylated fragments from plasmids before and after induction may be observed as seen in Fig. 2A that corresponds to Fig. 2C.

Fig. 2.

Methylation sensitive digestion of plasmid DNA preparations of dam-/dcm- competent E. coli GM2163 transformed with IMCC12053_1885 (A). For linearization, two enzymes were used for digestion. Hemi-methylated or partially methylated bands can be identified in Mlu I/Pvu I and GpC methylation (†) occurred in Mlu I (ACGCGT). Mlu I (A*CG*CGT) and Pvu I (*CGAT*CG) are both blocked by CpG methylation (*). Pst I was not sensitive to CpG/dam/dcm methylation and was used for linearization use only. SYBR stained 1.2% agarose gel with 1 kb Marker from DM3200 (SMOBIO). *U, Uninduced; I, Induced. NEBcutter 2.0 was used for expected DNA framgments from a circular pQE30 vector carrying IMCC12053_1885 sequence (see Supplementary data for the sequence) (B). Actual interpretation of the electrophoresis patterns considering GpC MTase impairment of Mlu I (C).

In this study, we cloned and investigated an MTase from C. marinus IMCC12053 and it was different from CpG or GpC MTases. Previously reported CpG MTases were from Spiroplasma sp. strain MQ1 (M. SssI) (Renbaum et al., 1990), and GpC MTases were from Chlorella virus NYs-1 (M. CviPI) that recognized dinucleotide GpC and methylated 5-methyl cytosines in DNA (Xu et al., 1998). Unlike M. SssI and Chlorella virus NYs-1 MTases with CpG and GpC activities, GpC MTase from C. marinus IMCC12053 is a N6-methyl adenosine or N4-methyl cytosine forming enzyme (Yang et al., 2016). Using phylogenetic analysis, we screened and cloned the gene for exocyclic MTase, IMCC12053_1885 (GenBank Acc. ALI55832) with exocyclic GpC MTases. DNA MTases could be used to protect or impair restriction sites in DNA (Roberts et al., 2015), by changing chemical and physical properties of the DNA (Pérez et al., 2012). Other CpG/GpC MTase studies include methylated-DNA-specific probing and labeling DNA with isotopes (Herring et al., 2009; Harrison and Parle-McDermott, 2011). Most importantly, CpG and GpC MTases are important in gene expression and epigenetic studies (Harrison and Parle-McDermott, 2011; Jang et al., 2017). Recently, GpC MTase, not CpG MTase, was used as a potential regulator of gene expression in mitochondria (van der Wijst et al., 2017), suggesting that alphaproteobacterial DNA MTases may be of importance in the evolution and epigenetics in both bacterial and eukaryotic kingdoms.

적 요

DNA 메틸화는 유전체의 무결성의 유지 및 유전자 발현 조절과 같은 박테리아의 다양한 과정에 관여한다. Alphaproteobacteria 종에서 보존된 DNA 메틸 전이 효소인 CcrM은 S-아데노실 메티오닌을 공동 기질로 사용하여 N6-아데닌 또는 N4-시토신의 메틸 전이 효소 활성을 갖는다. Celeribacter marinus IMCC 12053는 해양 환경에서 분리된 알파프로테오박테리아로서 GpC 시토신의 외향고리 아민의 메틸기를 대체하여 N4-메틸 시토신을 생산한다. 단일 분자 실시간 서열 분석법(SMRT)을 사용하여, C. marinus IMCC12053의 메틸화 패턴을 Gibbs Motif Sampler 프로그램을 사용하여 확인하였다. 5′-GANTC-3′의 N6-메틸 아데노신과 5′-GpC-3′의 N4-메틸 시토신을 확인하였다. 발현된 DNA 메틸전이 효소는 계통 발생 분석법을 사용하여 선택하여 pQE30 벡터에 클로닝 후 dam-/dcm- 대장균을 사용하여 클로닝된 DNA 메틸라아제의 메틸화 활성을 확인하였다. 메틸화 효소를 코딩하는 게놈 DNA 및 플라스미드를 추출하고 메틸화에 민감한 제한 효소로 절단하여 메틸화 활성을 확인하였다. 염색체와 메틸라아제를 코드하는 플라스미드를 메틸화시켰을 때에 제한 효소 사이트가 보호되는 것으로 관찰되었다. 본 연구에서는 분자 생물학 및 후성유전학을 위한 새로운 유형의 GpC 메틸화 효소의 잠재적 활용을 위한 외향고리 DNA 메틸라제의 특성을 확인하였다.


This work was supported by the Korea National Research Fund (NRF-2017R1D1A1B03034706).

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