A Mimicking-of-DNA-Methylation-Patterns Pipeline for Overcoming the Restriction Barrier of Bacteria

English version České info

Genetic transformation of bacteria harboring multiple Restriction-Modification (R-M) systems is often difficult using conventional methods. Here, we describe a mimicking-of-DNA-methylation-patterns (MoDMP) pipeline to address this problem in three difficult-to-transform bacterial strains. Twenty-four putative DNA methyltransferases (MTases) from these difficult-to-transform strains were cloned and expressed in an Escherichia coli strain lacking all of the known R-M systems and orphan MTases. Thirteen of these MTases exhibited DNA modification activity in Southwestern dot blot or Liquid Chromatography–Mass Spectrometry (LC–MS) assays. The active MTase genes were assembled into three operons using the Saccharomyces cerevisiae DNA assembler and were co-expressed in the E. coli strain lacking known R-M systems and orphan MTases. Thereafter, results from the dot blot and restriction enzyme digestion assays indicated that the DNA methylation patterns of the difficult-to-transform strains are mimicked in these E. coli hosts. The transformation of the Gram-positive Bacillus amyloliquefaciens TA208 and B. cereus ATCC 10987 strains with the shuttle plasmids prepared from MoDMP hosts showed increased efficiencies (up to four orders of magnitude) compared to those using the plasmids prepared from the E. coli strain lacking known R-M systems and orphan MTases or its parental strain. Additionally, the gene coding for uracil phosphoribosyltransferase (upp) was directly inactivated using non-replicative plasmids prepared from the MoDMP host in B. amyloliquefaciens TA208. Moreover, the Gram-negative chemoautotrophic Nitrobacter hamburgensis strain X14 was transformed and expressed Green Fluorescent Protein (GFP). Finally, the sequence specificities of active MTases were identified by restriction enzyme digestion, making the MoDMP system potentially useful for other strains. The effectiveness of the MoDMP pipeline in different bacterial groups suggests a universal potential. This pipeline could facilitate the functional genomics of the strains that are difficult to transform.

Vyšlo v časopise: A Mimicking-of-DNA-Methylation-Patterns Pipeline for Overcoming the Restriction Barrier of Bacteria. PLoS Genet 8(9): e32767. doi:10.1371/journal.pgen.1002987
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1002987

Souhrn

Zdroje

1. BottoneEJ (2010) Bacillus cereus, a volatile human pathogen. Clin Microbiol Rev 23: 382–398.

2. SalterSJ (2011) You cannot B. cereus. Nat Rev Micro 9: 83.

3. HuoY-X, ChoKM, RiveraJGL, MonteE, ShenCR, et al. (2011) Conversion of proteins into biofuels by engineering nitrogen flux. Nat Biotech 29: 346–351.

4. NawyT (2012) Non-model organisms. Nat Meth 9: 37.

5. RobertsRJ, VinczeT, PosfaiJ, MacelisD (2010) REBASE—a database for DNA restriction and modification: enzymes, genes and genomes. Nucleic Acids Res 38: D234–D236.

6. RobertsRJ, BelfortM, BestorT, BhagwatAS, BickleTA, et al. (2003) A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes. Nucleic Acids Res 31: 1805–1812.

7. LabrieSJ, SamsonJE, MoineauS (2010) Bacteriophage resistance mechanisms. Nat Rev Micro 8: 317–327.

8. StarkenburgSR, ArpDJ, BottomleyPJ (2008) d-Lactate metabolism and the obligate requirement for CO2 during growth on nitrite by the facultative lithoautotroph Nitrobacter hamburgensis. Microbiology 154: 2473–2481.

9. ArpDJ, BottomleyPJ (2006) Nitrifiers: more than 100 years from isolation to genome sequences. Microbe 1: 229–234.

10. StarkenburgSR, LarimerFW, SteinLY, KlotzMG, ChainPSG, et al. (2008) Complete genome sequence of Nitrobacter hamburgensis X14 and comparative genomic analysis of species within the genus Nitrobacter. Appl Environ Microbiol 74: 2852–2863.

11. RaskoDA, RavelJ, ØkstadOA, HelgasonE, CerRZ, et al. (2004) The genome sequence of Bacillus cereus ATCC 10987 reveals metabolic adaptations and a large plasmid related to Bacillus anthracis pXO1. Nucleic Acids Res 32: 977–988.

12. ArnaudM, ChastanetA, DébarbouilléM (2004) New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, Gram-positive bacteria. Appl Environ Microbiol 70: 6887–6891.

13. LindbäckT, KolstøA-B (1997) A Bacillus cereus member of the SNF2 family. Microbiology 143: 171–174.

14. LindbäckT, ØkstadOA, RishovdA-L, KolstøA-B (1999) Insertional inactivation of hblC encoding the L2 component of Bacillus cereus ATCC 14579 haemolysin BL strongly reduces enterotoxigenic activity, but not the haemolytic activity against human erythrocytes. Microbiology 145: 3139–3146.

15. XuS-y, NugentRL, KasamkattilJ, FomenkovA, GuptaY, et al. (2011) Characterization of Type II and III restriction-modification systems from Bacillus cereus strains ATCC 10987 and ATCC 14579. J Bacteriol 194: 49–60.

16. ZhangG, DengA, XuQ, LiangY, ChenN, et al. (2011) Complete genome sequence of Bacillus amyloliquefaciens TA208, a strain for industrial production of guanosine and ribavirin. J Bacteriol 193: 3142–3143.

17. ZhangG, BaoP, ZhangY, DengA, ChenN, et al. (2011) Enhancing electro-transformation competency of recalcitrant Bacillus amyloliquefaciens by combining cell-wall weakening and cell-membrane fluidity disturbing. Anal Biochem 409: 130–137.

18. 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.

19. ZhouH, WangY, YuY, BaiT, ChenL, et al. (2012) A non-restricting and non-methylating Escherichia coli strain for DNA cloning and high-throughput conjugation to Streptomyces coelicolor. Curr Microbiol 64: 185–190.

20. MarinusMG (2010) DNA methylation and mutator genes in Escherichia coli K-12. Mutat Res 705: 71–76.

21. LeT, KimK-P, FanG, FaullKF (2011) A sensitive mass spectrometry method for simultaneous quantification of DNA methylation and hydroxymethylation levels in biological samples. Anal Biochem 412: 203–209.

22. BoyeE, Løbner-OlesenA (1990) The role of dam methyltransferase in the control of DNA replication in E. coli. Cell 62: 981–989.

23. HegnaIK, BratlandH, KolstA-B (2001) BceS1, a new addition to the type III restriction and modification family. FEMS Microbiol Lett 202: 189–193.

24. TurgeonN, LaflammeC, HoJ, DuchaineC (2006) Elaboration of an electroporation protocol for Bacillus cereus ATCC 14579. J Microbiol Methods 67: 543–548.

25. FazziniMM, SchuchR, FischettiVA (2010) A novel spore protein, ExsM, regulates formation of the exosporium in Bacillus cereus and Bacillus anthracis and affects spore size and shape. J Bacteriol 192: 4012–4021.

26. FabretC, Dusko EhrlichS, NoirotP (2002) A new mutation delivery system for genome-scale approaches in Bacillus subtilis. Mol Microbiol 46: 25–36.

27. Carsiotis M, Khanna S (1989) Genetic engineering of enhanced microbial nitrification. Cincinnati, OH: US Environmental Protection Agency, Risk Reduction Engineering Laboratory.

28. Sayavedra-SotoLA, SteinLY (2011) Genetic transformation of ammonia-oxidizing bacteria. Methods Enzymol 486: 389–402.

29. 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.

30. DrozdzM, PiekarowiczA, BujnickiJM, RadlinskaM (2012) Novel non-specific DNA adenine methyltransferases. Nucleic Acids Res 40: 2119–2130.

31. SzomolányiÉ, KissA, VenetianerP (1980) Cloning the modification methylase gene of Bacillus sphaericus R in Escherichia coli. Gene 10: 219–225.

32. DonahueJP, IsraelDA, PeekRM, BlaserMJ, MillerGG (2000) Overcoming the restriction barrier to plasmid transformation of Helicobacter pylori. Mol Microbiol 37: 1066–1074.

33. GrootMN, NieboerF, AbeeT (2008) Enhanced transformation efficiency of recalcitrant Bacillus cereus and Bacillus weihenstephanensis isolates upon in vitro methylation of plasmid DNA. Appl Environ Microbiol 74: 7817–7820.

34. Van der RestME, LangeC, MolenaarD (1999) A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA. Appl Microbiol Biotechnol 52: 541–545.

35. DongH, ZhangY, DaiZ, LiY (2010) Engineering Clostridium strain to accept unmethylated DNA. PLoS ONE 5: e9038 doi:10.1371/journal.pone.0009038.

36. VeigaH, PinhoMG (2009) Inactivation of the SauI Type I restriction-modification system is not sufficient to generate Staphylococcus aureus strains capable of efficiently accepting foreign DNA. Appl Environ Microbiol 75: 3034–3038.

37. MarreroR, WelkosSL (1995) The transformation frequency of plasmids into Bacillus anthracis is affected by adenine methylation. Gene 152: 75–78.

38. SitaramanR, LepplaSH (2012) Methylation-dependent DNA restriction in Bacillus anthracis. Gene 494: 44–50.

39. OkibeN, SuzukiN, InuiM, YukawaH (2011) Efficient markerless gene replacement in Corynebacterium glutamicum using a new temperature-sensitive plasmid. J Microbiol Methods 85: 155–163.

40. O'Connell MotherwayM, O'DriscollJ, FitzgeraldGF, Van SinderenD (2009) Overcoming the restriction barrier to plasmid transformation and targeted mutagenesis in Bifidobacterium breve UCC2003. Microbial Biotechnology 2: 321–332.

41. RegoROM, BestorA, RosaPA (2011) Defining the plasmid-borne restriction-modification systems of the lyme disease spirochete Borrelia burgdorferi. J Bacteriol 193: 1161–1171.

42. RaleighEA, MurrayNE, RevelH, BlumenthalRM, WestawayD, et al. (1988) McrA and McrB restriction phenotypes of some E. coli strains and implications for gene cloning. Nucleic Acids Res 16: 1563–1575.

43. Waite-ReesPA, KeatingCJ, MoranLS, SlatkoBE, HornstraLJ, et al. (1991) Characterization and expression of the Escherichia coli Mrr restriction system. J Bacteriol 173: 5207–5219.

44. MorganRD, DwinellEA, BhatiaTK, LangEM, LuytenYA (2009) The MmeI family: type II restriction–modification enzymes that employ single-strand modification for host protection. Nucleic Acids Res 37: 5208–5221.

45. MarinusMG, CasadesusJ (2009) Roles of DNA adenine methylation in host–pathogen interactions: mismatch repair, transcriptional regulation, and more. FEMS Microbiol Rev 33: 488–503.

46. WalkinshawMD, TaylorP, SturrockSS, AtanasiuC, BergeT, et al. (2002) Structure of Ocr from bacteriophage T7, a protein that mimics B-form DNA. Mol Cell 9: 187–194.

47. PatrickS, HoustonS, ThackerZ, BlakelyGW (2009) Mutational analysis of genes implicated in LPS and capsular polysaccharide biosynthesis in the opportunistic pathogen Bacteroides fragilis. Microbiology 155: 1039–1049.

48. XuT, YaoF, ZhouX, DengZ, YouD (2010) A novel host-specific restriction system associated with DNA backbone S-modification in Salmonella. Nucleic Acids Res 38: 7133–7141.

49. ShaoZ, ZhaoH (2009) DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res 37: e16.

50. Daniel GietzR, WoodsRA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350: 87–96.

51. RobzykK, KassirY (1992) A simple and highly efficient procedure for rescuing autonomous plasmids from yeast. Nucleic Acids Res 20: 3790.