DOI QR코드

DOI QR Code

Exocyclic GpC DNA methyltransferase from Celeribacter marinus IMCC12053

Celeribacter marinus IMCC12053의 외향고리 GpC DNA 메틸트랜스퍼라아제

Kim, Junghee;Oh, Hyun-Myung
김정희;오현명

  • Received : 2019.01.09
  • Accepted : 2019.04.10
  • Published : 2019.06.30

Abstract

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 $N^6$-adenine or $N^4$-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 $N^6$-methyladenosine and conversion of $N^4$-cytosine of 5'-GpC-3' to $N^4$-methylcytosine. Exocyclic DNA methyltransferase from the genome of strain IMCC12053 was chosen using phylogenetic analysis and $N^4$-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

References

  1. Adhikari S and Curtis PD. 2016. DNA methyltransferases and epigenetic regulation in bacteria. FEMS Microbiol. Rev. 40, 575-591. https://doi.org/10.1093/femsre/fuw023
  2. Baek K, Choi A, Kang I, and Cho JC. 2014. Celeribacter marinus sp. nov., isolated from coastal seawater. Int. J. Syst. Evol. Microbiol. 64, 1323-1327. https://doi.org/10.1099/ijs.0.060673-0
  3. Choi DH, Kwon YM, Kwon KK, and Kim SJ. 2015. Complete genome sequence of Novosphingobium pentaromativorans US6-$1^T$. Stand. Genomic Sci. 10, 107.
  4. Eberhard J, Oza J, and Reich NO. 2001. Cloning, sequence analysis and heterologous expression of the DNA adenine-($N^6$) methyltransferase from the human pathogen Actinobacillus actinomycetemcomitans. FEMS Microbiol. Lett. 195, 223-229. https://doi.org/10.1111/j.1574-6968.2001.tb10525.x
  5. Eddy SR. 2011. Accelerated profile HMM searches. PLoS Comp. Biol. 7, e1002195. https://doi.org/10.1371/journal.pcbi.1002195
  6. Eid J, Fehr A, Gray J, Luong K, Lyle J, Otto G, Peluso P, Rank D, Baybayan P, Bettman B, et al. 2009. Real-time DNA sequencing from single polymerase molecules. Science 323, 133-138. https://doi.org/10.1126/science.1162986
  7. Gonzalez D, Kozdon JB, McAdams HH, Shapiro L, and Collier J. 2014. The functions of DNA methylation by CcrM in Caulobacter crescentus: a global approach. Nucleic Acids Res. 42, 3720-3735. https://doi.org/10.1093/nar/gkt1352
  8. Harrison A and Parle-McDermott A. 2011. DNA methylation: A timeline of methods and applications. Front. Genet. 2, 74.
  9. Herring JL, Rogstad DK, and Sowers LC. 2009. Enzymatic methylation of DNA in cultured human cells studied by stable isotope incorporation and mass spectrometry. Chem. Res. Toxicol. 22, 1060-1068.
  10. Jang HS, Shin WJ, Lee JE, and Do JT. 2017. CpG and Non-CpG methylation in epigenetic gene regulation and brain function. Genes 8, 148. https://doi.org/10.3390/genes8060148
  11. Jeltsch A, Christ F, Fatemi M, and Roth M. 1999. On the substrate specificity of DNA methyltransferases. adenine-$N^6$ DNA methyltransferases also modify cytosine residues at position $N^4$. J. Biol. Chem. 274, 19538-19544. https://doi.org/10.1074/jbc.274.28.19538
  12. Jurkowska RZ and Jeltsch A. 2016. Mechanisms and biological roles of DNA methyltransferases and DNA methylation: From past achievements to future challenges. Adv. Exp. Med. Biol. 945, 1-17.
  13. Kang I, Jang H, Oh HM, and Cho JC. 2012. Complete genome sequence of Celeribacter bacteriophage P12053L. J. Virol. 86, 8339-8340. https://doi.org/10.1128/JVI.01153-12
  14. Kozdon JB, Melfi MD, Luong K, Clark TA, Boitano M, Wang S, Zhou B, Gonzalez D, Collier J, Turner SW, et al. 2013. Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle. Proc. Natl. Acad. Sci. USA 110, E4658-E4667. https://doi.org/10.1073/pnas.1319315110
  15. Kumar S, Stecher G, and Tamura K. 2016. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870-1874. https://doi.org/10.1093/molbev/msw054
  16. Lawrence CE, Altschul SF, Boguski MS, Liu JS, Neuwald AF, and Wootton JC. 1993. Detecting subtle sequence signals: a Gibbs sampling strategy for multiple alignment. Science 262, 208-214. https://doi.org/10.1126/science.8211139
  17. Li Y, Zou X, Ma F, Tang B, and Zhang CY. 2017. Development of fluorescent methods for DNA methyltransferase assay. Methods Appl. Fluoresc. 5, 012002. https://doi.org/10.1088/2050-6120/aa6127
  18. Loenen WA and Raleigh EA. 2014. The other face of restriction: modification-dependent enzymes. Nucleic Acids Res. 42, 56-69.
  19. Luo YR, Kang SG, Kim SJ, Kim MR, Li N, Lee JH, and Kwon KK. 2012. Genome sequence of benzo(a)pyrene-degrading bacterium Novosphingobium pentaromativorans US6-1. J. Bacteriol. 194, 907. https://doi.org/10.1128/JB.06476-11
  20. Maier JA, Albu RF, Jurkowski TP, and Jeltsch A. 2015. Investigation of the C-terminal domain of the bacterial DNA-(adenine $N^6$)-methyltransferase CcrM. Biochimie 119, 60-67. https://doi.org/10.1016/j.biochi.2015.10.011
  21. Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, Geer RC, He J, Gwadz M, Hurwitz DI, et al. 2015. CDD: NCBI's conserved domain database. Nucleic Acids Res. 43, D222-D226. https://doi.org/10.1093/nar/gku1221
  22. Mohapatra SS, Fioravanti A, and Biondi EG. 2014. DNA methylation in Caulobacter and other Alphaproteobacteria during cell cycle progression. Trends Microbiol. 22, 528-535. https://doi.org/10.1016/j.tim.2014.05.003
  23. Perez A, Castellazzi Chiara L, Battistini F, Collinet K, Flores O, Deniz O, Ruiz Maria L, Torrents D, Eritja R, Soler-Lopez M, et al. 2012. Impact of methylation on the physical properties of DNA. Biophys. J. 102, 2140-2148. https://doi.org/10.1016/j.bpj.2012.03.056
  24. Renbaum P, Abrahamove D, Fainsod A, Wilson GG, Rottem S, and Razin A. 1990. Cloning, characterization, and expression in Escherichia coli of the gene coding for the CpG DNA methylase from Spiroplasma sp. strain MQ1(M. SssI). Nucleic Acids Res. 18, 1145-1152. https://doi.org/10.1093/nar/18.5.1145
  25. Roberts RJ, Vincze T, Posfai J, and Macelis D. 2015. REBASE-a database for DNA restriction and modification: enzymes, genes and genomes. Nucleic Acids Res. 43, D298-D299. https://doi.org/10.1093/nar/gku1046
  26. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard MA, and Huelsenbeck JP. 2012. MrBayes 3.2: Efficient bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539-542. https://doi.org/10.1093/sysbio/sys029
  27. Sohn JH, Kwon KK, Kang JH, Jung HB, and Kim SJ. 2004. Novosphingobium pentaromativorans sp. nov., a high-molecularmass polycyclic aromatic hydrocarbon-degrading bacterium isolated from estuarine sediment. Int. J. Syst. Evol. Microbiol. 54, 1483-1487. https://doi.org/10.1099/ijs.0.02945-0
  28. Stamatakis A. 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22, 2688-2690. https://doi.org/10.1093/bioinformatics/btl446
  29. van der Wijst MGP, van Tilburg AY, Ruiters MHJ, and Rots MG. 2017. Experimental mitochondria-targeted DNA methylation identifies GpC methylation, not CpG methylation, as potential regulator of mitochondrial gene expression. Sci. Rep. 7, 177. https://doi.org/10.1038/s41598-017-00263-z
  30. Vincze T, Posfai J, and Roberts RJ. 2003. NEBcutter: a program to cleave DNA with restriction enzymes. Nucleic Acids Res. 31, 3688-3691. https://doi.org/10.1093/nar/gkg526
  31. Xu M, Kladde MP, Van Etten JL, and Simpson RT. 1998. Cloning, characterization and expression of the gene coding for a cytosine-5-DNA methyltransferase recognizing GpC. Nucleic Acids Res. 26, 3961-3966. https://doi.org/10.1093/nar/26.17.3961
  32. Yang JA, Kang I, Moon M, Ryu UC, Kwon KK, Cho JC, and Oh HM. 2016. Complete genome sequence of Celeribacter marinus $IMCC12053^T$, the host strain of marine bacteriophage P12053L. Mar. Genomics 26, 5-7. https://doi.org/10.1016/j.margen.2015.11.012
  33. Yang JA, Kwon KK, and Oh HM. 2017. Complete genome sequence of Flavobacteriales bacterium strain UJ101 isolated from a xanthid crab. Genome Announc. 5, e01551-16.

Acknowledgement

Supported by : Korea National Research Fund