In the bacterial world, methylation is most commonly associated with restriction-modification systems that provide a defense mechanism against invading foreign genomes. In addition, it is known that methylation plays functionally important roles, including timing of DNA replication, chromosome partitioning, DNA repair, and regulation of gene expression. However, full DNA methylome analyses are scarce due to a lack of a simple methodology for rapid and sensitive detection of common epigenetic marks (ie N6-methyladenine (6 mA) and N4-methylcytosine (4 mC)), in these organisms. Here, we use Single-Molecule Real-Time (SMRT) sequencing to determine the methylomes of two related human pathogen species, Mycoplasma genitalium G-37 and Mycoplasma pneumoniae M129, with single-base resolution. Our analysis identified two new methylation motifs not previously described in bacteria: a widespread 6 mA methylation motif common to both bacteria (5′-CTAT-3′), as well as a more complex Type I m6A sequence motif in M. pneumoniae (5′-GAN7TAY-3′/3′-CTN7ATR-5′). We identify the methyltransferase responsible for the common motif and suggest the one involved in M. pneumoniae only. Analysis of the distribution of methylation sites across the genome of M. pneumoniae suggests a potential role for methylation in regulating the cell cycle, as well as in regulation of gene expression. To our knowledge, this is one of the first direct methylome profiling studies with single-base resolution from a bacterial organism.
Vyšlo v časopise: Comprehensive Methylome Characterization of and at Single-Base Resolution. PLoS Genet 9(1): e32767. doi:10.1371/journal.pgen.1003191 Kategorie: Research Article prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003191
1. CasadesusJ, D'AriR (2002) Memory in bacteria and phage. Bioessays 24 : 512–518.
2. JorgensenHF, BirdA (2002) MeCP2 and other methyl-CpG binding proteins. Ment Retard Dev Disabil Res Rev 8 : 87–93.
3. KloseRJ, BirdAP (2006) Genomic DNA methylation: the mark and its mediators. Trends Biochem Sci 31 : 89–97.
4. LewisJD, MeehanRR, HenzelWJ, Maurer-FogyI, JeppesenP, et al. (1992) Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA. Cell 69 : 905–914.
5. NanX, CrossS, BirdA (1998) Gene silencing by methyl-CpG-binding proteins. Novartis Found Symp 214 : 6–16 discussion 16–21, 46–50.
6. RobertsRJ, VinczeT, PosfaiJ, MacelisD (2010) REBASE–a database for DNA restriction and modification: enzymes, genes and genomes. Nucleic Acids Res 38: D234–236.
7. Lobner-OlesenA, SkovgaardO, MarinusMG (2005) Dam methylation: coordinating cellular processes. Curr Opin Microbiol 8 : 154–160.
8. MarinusMG, MorrisNR (1973) Isolation of deoxyribonucleic acid methylase mutants of Escherichia coli K-12. J Bacteriol 114 : 1143–1150.
9. MayMS, HattmanS (1975) Analysis of bacteriophage deoxyribonucleic acid sequences methylated by host - and R-factor-controlled enzymes. J Bacteriol 123 : 768–770.
10. BarrasF, MarinusMG (1989) The great GATC: DNA methylation in E. coli. Trends Genet 5 : 139–143.
11. ModrichP (1989) Methyl-directed DNA mismatch correction. J Biol Chem 264 : 6597–6600.
12. PalmerBR, MarinusMG (1994) The dam and dcm strains of Escherichia coli–a review. Gene 143 : 1–12.
13. WionD, CasadesusJ (2006) N6-methyl-adenine: an epigenetic signal for DNA-protein interactions. Nat Rev Microbiol 4 : 183–192.
14. CasadesusJ, LowD (2006) Epigenetic gene regulation in the bacterial world. Microbiol Mol Biol Rev 70 : 830–856.
15. SohanpalBK, El-LabanyS, LahootiM, PlumbridgeJA, BlomfieldIC (2004) Integrated regulatory responses of fimB to N-acetylneuraminic (sialic) acid and GlcNAc in Escherichia coli K-12. Proc Natl Acad Sci U S A 101 : 16322–16327.
16. BlynLB, BraatenBA, LowDA (1990) Regulation of pap pilin phase variation by a mechanism involving differential dam methylation states. EMBO J 9 : 4045–4054.
17. PolaczekP, KwanK, CampbellJL (1998) GATC motifs may alter the conformation of DNA depending on sequence context and N6-adenine methylation status: possible implications for DNA-protein recognition. Mol Gen Genet 258 : 488–493.
18. PolaczekP, KwanK, LiberiesDA, CampbellJL (1997) Role of architectural elements in combinatorial regulation of initiation of DNA replication in Escherichia coli. Mol Microbiol 26 : 261–275.
19. HerndayA, BraatenB, LowD (2004) The intricate workings of a bacterial epigenetic switch. Adv Exp Med Biol 547 : 83–89.
20. HerndayA, KrabbeM, BraatenB, LowD (2002) Self-perpetuating epigenetic pili switches in bacteria. Proc Natl Acad Sci U S A 99 Suppl 4 : 16470–16476.
21. BanasJA, BiswasS, ZhuM (2011) DNA Methylation Affects Virulence Gene Expression in Streptococcus mutans. Appl Environ Microbiol 77 : 7236–7242.
22. BrunetYR, BernardCS, GavioliM, LloubesR, CascalesE (2011) An epigenetic switch involving overlapping fur and DNA methylation optimizes expression of a type VI secretion gene cluster. PLoS Genet 7: e1002205 doi:10.1371/journal.pgen.1002205.
23. MannarelliBM, BalganeshTS, GreenbergB, SpringhornSS, LacksSA (1985) Nucleotide sequence of the Dpn II DNA methylase gene of Streptococcus pneumoniae and its relationship to the dam gene of Escherichia coli. Proc Natl Acad Sci U S A 82 : 4468–4472.
24. TaghbaloutA, LandoulsiA, KernR, YamazoeM, HiragaS, et al. (2000) Competition between the replication initiator DnaA and the sequestration factor SeqA for binding to the hemimethylated chromosomal origin of E. coli in vitro. Genes Cells 5 : 873–884.
25. MolinaF, SkarstadK (2004) Replication fork and SeqA focus distributions in Escherichia coli suggest a replication hyperstructure dependent on nucleotide metabolism. Mol Microbiol 52 : 1597–1612.
26. KangS, LeeH, HanJS, HwangDS (1999) Interaction of SeqA and Dam methylase on the hemimethylated origin of Escherichia coli chromosomal DNA replication. J Biol Chem 274 : 11463–11468.
27. CampbellJL, KlecknerN (1990) E. coli oriC and the dnaA gene promoter are sequestered from dam methyltransferase following the passage of the chromosomal replication fork. Cell 62 : 967–979.
28. KaguniJM (2006) DnaA: controlling the initiation of bacterial DNA replication and more. Annu Rev Microbiol 60 : 351–375.
29. CokusSJ, FengS, ZhangX, ChenZ, MerrimanB, et al. (2008) Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452 : 215–219.
30. ListerR, O'MalleyRC, Tonti-FilippiniJ, GregoryBD, BerryCC, et al. (2008) Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133 : 523–536.
31. FlusbergBA, WebsterDR, LeeJH, TraversKJ, OlivaresEC, et al. (2010) Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat Methods 7 : 461–465.
32. ClarkTA, MurrayIA, MorganRD, KislyukAO, SpittleKE, et al. (2012) Characterization of DNA methyltransferase specificities using single-molecule, real-time DNA sequencing. Nucleic Acids Res 40: e29.
33. ChinerE, Signes-CostaJ, AndreuAL, AndreuL (2003) [Mycoplasma pneumoniae pneumonia: and uncommon cause of adult respiratory distress syndrome]. An Med Interna 20 : 597–598.
34. JensenJS (2004) Mycoplasma genitalium: the aetiological agent of urethritis and other sexually transmitted diseases. J Eur Acad Dermatol Venereol 18 : 1–11.
35. DandekarT, HuynenM, RegulaJT, UeberleB, ZimmermannCU, et al. (2000) Re-annotating the Mycoplasma pneumoniae genome sequence: adding value, function and reading frames. Nucleic Acids Res 28 : 3278–3288.
36. PetersonSN, HuPC, BottKF, HutchisonCA3rd (1993) A survey of the Mycoplasma genitalium genome by using random sequencing. J Bacteriol 175 : 7918–7930.
37. FraserCM, GocayneJD, WhiteO, AdamsMD, ClaytonRA, et al. (1995) The minimal gene complement of Mycoplasma genitalium. Science 270 : 397–403.
38. YusE, MaierT, MichalodimitrakisK, van NoortV, YamadaT, et al. (2009) Impact of genome reduction on bacterial metabolism and its regulation. Science 326 : 1263–1268.
39. WilsonGG, MurrayNE (1991) Restriction and modification systems. Annu Rev Genet 25 : 585–627.
40. SmithDW, CrowderSW, ReichNO (1992) In vivo specificity of EcoRI DNA methyltransferase. Nucleic Acids Res 20 : 6091–6096.
41. ReichNO, OlsenC, OstiF, MurphyJ (1992) In vitro specificity of EcoRI DNA methyltransferase. J Biol Chem 267 : 15802–15807.
42. YusE, GuellM, VivancosAP, ChenWH, Lluch-SenarM, et al. (2012) Transcription start site associated RNAs in bacteria. Mol Syst Biol 8 : 585.
43. GuellM, van NoortV, YusE, ChenWH, Leigh-BellJ, et al. (2009) Transcriptome complexity in a genome-reduced bacterium. Science 326 : 1268–1271.
44. MaierT, GuellM, SerranoL (2009) Correlation of mRNA and protein in complex biological samples. FEBS Lett 583 : 3966–3973.
45. MurrayNE (2000) Type I restriction systems: sophisticated molecular machines (a legacy of Bertani and Weigle). Microbiol Mol Biol Rev 64 : 412–434.
46. KongH, LinLF, PorterN, StickelS, ByrdD, et al. (2000) Functional analysis of putative restriction-modification system genes in the Helicobacter pylori J99 genome. Nucleic Acids Res 28 : 3216–3223.
47. MottML, BergerJM (2007) DNA replication initiation: mechanisms and regulation in bacteria. Nat Rev Microbiol 5 : 343–354.
48. RazinA, RazinS (1980) Methylated bases in mycoplasmal DNA. Nucleic Acids Res 8 : 1383–1390.
49. VoelkerLL, DybvigK (1996) Gene transfer in Mycoplasma arthritidis: transformation, conjugal transfer of Tn916, and evidence for a restriction system recognizing AGCT. J Bacteriol 178 : 6078–6081.
50. LuoW, TuAH, CaoZ, YuH, DybvigK (2009) Identification of an isoschizomer of the HhaI DNA methyltransferase in Mycoplasma arthritidis. FEMS Microbiol Lett 290 : 195–198.
51. GautamA, BastiaD (2001) A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. J Biol Chem 276 : 8771–8777.
52. SkarstadK, TorheimN, WoldS, LurzR, MesserW, et al. (2001) The Escherichia coli SeqA protein binds specifically to two sites in fully and hemimethylated oriC and has the capacity to inhibit DNA replication and affect chromosome topology. Biochimie 83 : 49–51.
53. SkarstadK, LuederG, LurzR, SpeckC, MesserW (2000) The Escherichia coli SeqA protein binds specifically and co-operatively to two sites in hemimethylated and fully methylated oriC. Mol Microbiol 36 : 1319–1326.
54. HiragaS, IchinoseC, OnogiT, NikiH, YamazoeM (2000) Bidirectional migration of SeqA-bound hemimethylated DNA clusters and pairing of oriC copies in Escherichia coli. Genes Cells 5 : 327–341.
55. Castilla-LlorenteV, Munoz-EspinD, VillarL, SalasM, MeijerWJ (2006) Spo0A, the key transcriptional regulator for entrance into sporulation, is an inhibitor of DNA replication. EMBO J 25 : 3890–3899.
56. Marinus MG (1996) Methylation of DNA; al. Ne, editor. Washington, D.C.: ASM Press. 782–791 p.
57. Lobner-OlesenA, MarinusMG, HansenFG (2003) Role of SeqA and Dam in Escherichia coli gene expression: a global/microarray analysis. Proc Natl Acad Sci U S A 100 : 4672–4677.
58. WangMX, ChurchGM (1992) A whole genome approach to in vivo DNA-protein interactions in E. coli. Nature 360 : 606–610.
59. CharlierD, GigotD, HuysveldN, RooversM, PierardA, et al. (1995) Pyrimidine regulation of the Escherichia coli and Salmonella typhimurium carAB operons: CarP and integration host factor (IHF) modulate the methylation status of a GATC site present in the control region. J Mol Biol 250 : 383–391.
60. WallechaA, MunsterV, CorrentiJ, ChanT, van der WoudeM (2002) Dam - and OxyR-dependent phase variation of agn43: essential elements and evidence for a new role of DNA methylation. J Bacteriol 184 : 3338–3347.
61. CorrentiJ, MunsterV, ChanT, WoudeM (2002) Dam-dependent phase variation of Ag43 in Escherichia coli is altered in a seqA mutant. Mol Microbiol 44 : 521–532.
62. TullyJG, RoseDL, WhitcombRF, WenzelRP (1979) Enhanced isolation of Mycoplasma pneumoniae from throat washings with a newly-modified culture medium. J Infect Dis 139 : 478–482.
63. TraversKJ, ChinCS, RankDR, EidJS, TurnerSW (2010) A flexible and efficient template format for circular consensus sequencing and SNP detection. Nucleic Acids Res 38: e159.
64. KrzywinskiM, ScheinJ, BirolI, ConnorsJ, GascoyneR, et al. (2009) Circos: an information aesthetic for comparative genomics. Genome Res 19 : 1639–1645.
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