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Random genome deletion methods applicable to prokaryotes


Through their enabling of simultaneous identification of multiple non-essential genes in a genome, large-segment genome deletion methods are an increasingly popular approach to minimize and tailor microbial genomes for specific functions. At present, difficulties in identifying target regions for deletion are a result of inadequate knowledge to define gene essentiality. Furthermore, with the majority of predicted open reading frames of completely sequenced genomes still annotated as putative genes, essential or important genes are found scattered throughout the genomes, limiting the size of non-essential segments that can be safely deleted in a single sweep. Recently described large-segment random genome deletion methods that utilize transposons enable the generation of random deletion strains, analysis of which makes identification of non-essential genes less tedious. Such and other efforts to determine the minimum genome content necessary for cell survival continue to accumulate important information that should help improve our understanding of genome function and evolution. This review presents an assessment of technological advancements of random genome deletion methods in prokaryotes to date.

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  1. Akerley BJ, Rubin EJ, Camilli A, Lampe DJ, Robertson HM, Mekalanos JJ (1998) Systematic identification of essential genes by in vitro mariner mutagenesis. Proc Natl Acad Sci 95:8927–8932

  2. Albert HE, Dale C, Lee E, Ow DW (1995) Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant J 7:649–659

  3. Araki K, Araki M, Yamamura K (1997) Targeted integration of DNA using mutant lox sites in embryonic stem cells. Nucleic Acids Res 25:868–872

  4. Ashour J, Hondalus MK (2003) Phenotypic mutants of the intracellular actinomycete Rhodococcus equi created by in vivo Himar1 transposon mutagenesis. J Bacteriol 185:2644–2652

  5. Austin S, Ziese M, Sternberg N (1981) A novel role for site-specific recombination in maintenance of bacterial replicons. Cell 25:729–736

  6. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:2006.0008

  7. Bethke B, Sauer B (1997) Segmental genomic replacement by Cre-mediated recombination: genotoxic stress activation of the p53 promoter in single copy transformants. Nucleic Acids Res 25:2828–2834

  8. Broach JR, Guarascio VR, Jayaram M (1982) Recombination within the yeast plasmid 2 m circle is site-specific. Cell 29:227–234

  9. Chaconas G Lavoie BD Watson MA (1996) DNA transposition: jumping gene machine, some assembly required. Curr Biol 6:817–820

  10. Chen Y, Narendra U, Iype LE, Cox MM, Rice PA (2000) Crystal structure of a Flp recombinase–Holliday junction complex: assembly of an active oligomer by helix swapping. Mol Cell 6:885–897

  11. Clewell DB, Tomich PK, Gawron-Burke MC, Franke AE, Yagi Y, An FY (1982) Mapping of Streptococcus faecalis plasmids pAD1 and pAD2 and studies relating to transposition of Tn917. J Bacteriol 152:1220–1230

  12. Coulter SN, Schwan WR, Ng EY, Langhorne MH, Ritchie HD, Westbrock-Wadman S, Hufnagle WO, Folger KR, Bayer AS, Stover CK (1998) Staphylococcus aureus genetic loci impacting growth and survival in multiple infection environments. Mol Microbiol 30:393–404

  13. Craig NL (1991) Tn7: a target site-specific transposon. Mol Microbiol 5:2569–2573

  14. Dean D (1981) A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids. Gene 15:99–102

  15. Derbise A, Lesic B, Dacheux D, Ghigo JM, Carniel E (2003) A rapid and simple method for inactivating chromosomal genes in Yersinia. FEMS Immunol Med Microbiol 38:113–116

  16. Fraser CM, Gocayne JD, White O, Adams MD, Clayton RA, Fleischmann RD, Bult CJ, Kerlavage AR, Sutton G, Kelley JM, Fritchman RD, Weidman JF, Small KV, Sandusky M, Fuhrmann J, Nguyen D, Utterback TR, Saudek DM, Phillips CA, Merrick JM, Tomb JF, Dougherty BA, Bott KF, Hu PC, Lucier TS, Peterson SN, Smith HO, Hutchison CA 3rd, Venter JC (1995) The minimal gene complement of Mycoplasma genitalium. Science 270:397–403

  17. Fukiya S, Mizoguchi H, Mori H (2004) An improved method for deleting large regions of Escherichia coli K-12 chromosome using a combination of Cre/loxP and l Red. FEMS Microbiol Lett 234:325–331

  18. Gay P, Le Coq D, Steinmetz M, Berkelman T, Kado CI (1985) Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria. J Bacteriol 164:918–921

  19. Giaever G, Chu AM, Ni L, Connelly C, Riles L, Véronneau S, Dow S, Lucau-Danila A, Anderson K, André B, Arkin AP, Astromoff A, El-Bakkoury M, Bangham R, Benito R, Brachat S, Campanaro S, Curtiss M, Davis K, Deutschbauer A, Entian KD, Flaherty P, Foury F, Garfinkel DJ, Gerstein M, Gotte D, Güldener U, Hegemann JH, Hempel S, Herman Z, Jaramillo DF, Kelly DE, Kelly SL, Kötter P, LaBonte D, Lamb DC, Lan N, Liang H, Liao H, Liu L, Luo C, Lussier M, Mao R, Menard P, Ooi SL, Revuelta JL, Roberts CJ, Rose M, Ross-Macdonald P, Scherens B, Schimmack G, Shafer B, Shoemaker DD, Sookhai-Mahadeo S, Storms RK, Strathern JN, Valle G, Voet M, Volckaert G, Wang CY, Ward TR, Wilhelmy J, Winzeler EA, Yang Y, Yen G, Youngman E, Yu K, Bussey H, Boeke JD, Snyder M, Philippsen P, Davis RW, Johnston M (2002) Functional profiling of the Saccharomyces cerevisiae genome. Nature 418:387–391

  20. Gil R, Silva FJ, Pereto J, Moya A (2004) Determination of the core of a minimal bacterial gene set. Microbiol Mol Biol Rev 68:518–537

  21. Glass JI, Assad-Garcia N, Alperovich N, Yooseph S, Lewis MR, Maruf M, Hutchison CA 3rd, Smith HO, Venter JC (2006) Essential genes of a minimal bacterium. Proc Natl Acad Sci 103:425–430

  22. Goryshin IY, Reznikoff WS (1998a) Tn5 in vitro transposition. J Biol Chem 273:7367–7374

  23. Goryshin IY, Miller JA, Kil YV, Lanzov VA, Reznikoff WS (1998b) Tn5/IS50 target recognition. Proc Natl Acad Sci 95:10716–10721

  24. Goryshin IY, Jendrisak J, Hoffman LM, Meis R, Reznikoff WS (2000) Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes. Nat Biotechnol 18:97–100

  25. Goryshin IY, Naumann TA, Apodaca J, Reznikoff WS (2003) Chromosomal deletion formation system based on Tn5 double transposition: use for making minimal genomes and essential gene analysis. Genome Res 13:644–653

  26. Gutierrez JA, Crowley PJ, Brown DP, Hillman JD, Youngman P, Bleiweis AS (1996) Insertional mutagenesis and recovery of interrupted genes of Streptococcus mutants by using transposon Tn917: preliminary characterization of mutants displaying acid sensitivity and nutritional requirements. J Bacteriol 178:4166–4175

  27. Hashimoto M, Ichimura T, Mizoguchi H, Tanaka K, Fujimitsu K, Keyamura K, Ote T, Yamakawa T, Yamazaki Y, Mori H, Katayama T, Kato J (2005) Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome. Mol Microbiol 55:137–149

  28. Hayes F (2003) Transposon-based strategies for microbial functional genomics and proteomics. Annu Rev Genet 37:3–29

  29. Hoess RH, Ziese M, Sternberg N (1982) P1 site-specific recombination: nucleotide sequence of the recombining sites. Proc Natl Acad Sci 79:3398–3402

  30. Hutchison CA, Peterson SN, Gill SR, Cline RT, White O, Fraser CM, Smith HO, Venter JC (1999) Global transposon mutagenesis and a minimal mycoplasma genome. Science 286:2165–2169

  31. Isberg RR, Lazaar AL, Syvanen M (1982) Regulation of Tn5 by the right-repeat proteins: control at the level of the transposition reaction? Cell 30:883–892

  32. Jacobs MA, Alwood A, Thaipisuttikul I, Spencer D, Haugen E, Ernst S, Will O, Kaul R, Raymond C, Levy R, Chun-Rong L, Guenthner D, Bovee D, Olson MV, Manoil C (2003) Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc Natl Acad Sci 100:14339–14344

  33. Jayaram M (1985) Two-micrometer circle site-specific recombination: the minimal substrate and the possible role of flanking sequences. Proc Natl Acad Sci 82:5875–5879

  34. Ji Y, Zhang B, Van Horn SF, Warren P, Woodnutt G, Burnham MK, Rosenberg M (2001) Identification of critical staphylococcal genes using conditional phenotypes generated by antisense RNA. Science 293:2266–2269

  35. Johnson RC, Yin JC, Reznikoff WS (1982) Control of Tn5 transposition in Escherichia coli is mediated by protein from the right repeat. Cell 30:873–882

  36. Johnson RC, Reznikoff WS (1984) Role of the IS50 R proteins in the promotion and control of Tn5 transposition. J Mol Biol 177:645–661

  37. Kobayashi K, Ehrlich SD, Albertini A, Amati G, Andersen KK, Arnaud M, Asai K, Ashikaga S, Aymerich S, Bessieres P, Boland F, Brignell SC, Bron S, Bunai K, Chapuis J, Christiansen LC, Danchin A, Débarbouille M, Dervyn E, Deuerling E, Devine K, Devine SK, Dreesen O, Errington J, Fillinger S, Foster SJ, Fujita Y, Galizzi A, Gardan R, Eschevins C, Fukushima T, Haga K, Harwood CR, Hecker M, Hosoya D, Hullo MF, Kakeshita H, Karamata D, Kasahara Y, Kawamura F, Koga K, Koski P, Kuwana R, Imamura D, Ishimaru M, Ishikawa S, Ishio I, Le Coq D, Masson A, Mauël C, Meima R, Mellado RP, Moir A, Moriya S, Nagakawa E, Nanamiya H, Nakai S, Nygaard P, Ogura M, Ohanan T, O’Reilly M, O’Rourke M, Pragai Z, Pooley HM, Rapoport G, Rawlins JP, Rivas LA, Rivolta C, Sadaie A, Sadaie Y, Sarvas M, Sato T, Saxild HH, Scanlan E, Schumann W, Seegers JF, Sekiguchi J, Sekowska A, Séror SJ, Simon M, Stragier P, Studer R, Takamatsu H, Tanaka T, Takeuchi M, Thomaides HB, Vagner V, van Dijl JM, Watabe K, Wipat A, Yamamoto H, Yamamoto M, Yamamoto Y, Yamane K, Yata K, Yoshida K, Yoshikawa H, Zuber U, Ogasawara N (2003) Essential Bacillus subtilis genes. Proc Natl Acad Sci 100:4678–4683

  38. Kolisnychenko V, Plunkett G 3rd, Herring CD, Fehér T, Pósfai J, Blattner FR, Pósfai G (2002) Engineering a reduced Escherichia coli genome. Genome Res 12:640–647

  39. Kuhn R, Torres RM (2002) Cre/loxP recombination system and gene targeting. Methods Mol Biol 180:175–204

  40. Lamberg A, Nieminen S, Qiao M, Savilahti H (2002) Efficient insertion mutagenesis strategy for bacterial genomes involving electroporation of in vitro-assembled DNA transposition complexes of bacteriophage Mu. Appl Environ Microbiol 68:705–712

  41. Lee G, Saito I (1998) Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. Gene 216:55–65

  42. Lee L, Sadowski PD (2001) Directional resolution of synthetic Holliday structures by the Cre recombinase. J Biol Chem 276:31092–31098

  43. Mahillon J, Chandler M (1998) Insertion sequences. Microbiol Mol Biol Rev 62:725–774

  44. Mizoguchi H, Mori H, Fujio T (2007) Escherichia coli minimum genome factory. Biotechnol Appl Biochem 46:157–167

  45. Mizuuchi M, Baker TA, Mizuuchi K (1992) Assembly of the active form of the transposase-Mu DNA complex: a critical control point in Mu transposition. Cell 70:303–311

  46. Murphy KC (1998) Use of bacteriophage λ recombination functions to promote gene replacement in Escherichia coli. J Bacteriol 180:2063–2071

  47. Mushegian AR, Koonin EV (1996) A minimal gene set for cellular life derived by comparison of complete bacterial genomes. Proc Natl Acad Sci 93:10268–10273

  48. Nakano M, Odaka K, Ishimura M, Kondo S, Tachikawa N, Chiba J, Kanegae Y, Saito I (2001) Efficient gene activation in cultured mammalian cells mediated by FLP recombinase-expressing recombinant adenovirus. Nucleic Acids Res 29:E40

  49. Naumann TA, Reznikoff WS (2002) Tn5 transposase with an altered specificity for transposon ends. J Bacteriol 184:233–240

  50. Posfai G, Kolisnychenko V, Bereczki Z, Blattner FR (1999) Markerless gene replacement in Escherichia coli stimulated by a double-strand break in the chromosome. Nucleic Acids Res 27:4409–4415

  51. Proteau G, Sidenberg D, Sadowski P (1986) The minimal duplex DNA sequence required for site-specific recombination promoted by the FLP protein of yeast in vitro. Nucleic Acids Res 14:4787–4802

  52. Russell CB, Dahlquist FW (1989) Exchange of chromosomal and plasmid alleles in Escherichia coli by selection for loss of a dominant antibiotic sensitivity marker. J Bacteriol 171:2614–2618

  53. Seibler J, Bode J (1997) double-reciprocal crossover mediated by FLP-recombinase: a concept and an assay. Biochemistry 36:1740–1747

  54. Senecoff JF, Bruckner RC, Cox MM (1985) The FLP recombinase of the yeast 2-micron plasmid: characterization of its recombination site. Proc Natl Acad Sci 82:7270–7274

  55. Serres MH, Gopal S, Nahum LA, Liang P, Gaasterland T, Riley M (2001) A functional update of the Escherichia coli K-12 genome. Genome Biol 2:RESEARCH0035

  56. Steiniger-White M, Rayment I, Reznikoff WS (2004) Structure/function insights into Tn5 transposition. Curr Opin Struct Biol 14:50–57

  57. Suzuki N, Nonaka H, Tsuge Y, Inui M, Yukawa H (2005a) New multiple-deletion method for the Corynebacterium glutamicum genome, using a mutant lox sequence. Appl Environ Microbiol 71:8472–8480

  58. Suzuki N, Okayama S, Nonaka H, Tsuge Y, Inui M, Yukawa H (2005b) Large-scale engineering of the Corynebacterium glutamicum genome. Appl Environ Microbiol 71:3369–3372

  59. Suzuki N, Okai N, Nonaka H, Tsuge Y, Inui M, Yukawa H (2006) High-throughput transposon mutagenesis of Corynebacterium glutamicum and construction of a single-gene disruptant mutant library. Appl Environ Microbiol 72:3750–3755

  60. Taniya T, Mitobe J, Nakayama S, Mingshan Q, Okuda K, Watanabe H (2003) Determination of the InvE binding site required for expression of IpaB of Shigella sonnei virulence plasmid: involvement of a ParB BoxA-like sequence. J Bacteriol 185:5158–5165

  61. Tsuge Y, Suzuki N, Inui M, Yukawa H (2007) Random segment deletion based on IS31831 and Cre/loxP excision system in Corynebacterium glutamicum. Appl Microbiol Biotechnol 74:1333–1341

  62. Uzzau S, Figueroa-Bossi N, Rubino S, Bossi L (2001) Epitome tagging of chromosomal genes in Salmonella. Proc Natl Acad Sci 98:15264–15269

  63. van Kessel JC, Hatfull GF (2007) Recombineering in Mycobacterium tuberculosis. Nat Methods 4:147–152

  64. Vertes AA, Asai Y, Inui M, Kobayashi M, Kurusu Y, Yukawa H (1994a) Transposon mutagenesis of coryneform bacteria. Mol Gen Genet 245:397–405

  65. Vertes AA, Inui M, Kobayashi M, Kurusu Y, Yukawa H (1994b) Isolation and characterization of IS31831, a transposable element from Corynebacterium glutamicum. Mol Microbiol 11:739–746

  66. Westers H, Dorenbos R, van Dijl JM, Kabel J, Flanagan T, Devine KM, Jude F, Seror SJ, Beekman AC, Darmon E, Eschevins C, de Jong A, Bron S, Kuipers OP, Albertini AM, Antelmann H, Hecker M, Zamboni N, Sauer U, Bruand C, Ehrlich DS, Alonso JC, Salas M, Quax WJ (2003) Genome engineering reveals large dispensable regions in Bacillus subtilis. Mol Biol Evol 20:2076–2090

  67. Youngman PJ, Perkins JB, Losick R (1983) Genetic transposition and insertional mutagenesis in Bacillus subtilis with Streptococcus faecalis transposon Tn917. Proc Natl Acad Sci 80:2305–2309

  68. Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci 97:5978–5983

  69. Yu BJ, Sung BH, Koob MD, Lee CH, Lee JH, Lee WS, Kim MS, Kim SC (2002) Minimization of the Escherichia coli genome using a Tn5-targeted Cre/loxP excision system. Nat Biotechnol 20:1018–1023

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We wish to thank Dr. C. Omumasaba (internal) for critical reading of the manuscript.

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Correspondence to Hideaki Yukawa.

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Suzuki, N., Inui, M. & Yukawa, H. Random genome deletion methods applicable to prokaryotes. Appl Microbiol Biotechnol 79, 519–526 (2008). https://doi.org/10.1007/s00253-008-1512-4

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  • Large-segment deletion
  • Essential genes
  • Transposon
  • Minimum bacterial genome