Engineering Gram-Negative Microbial Cell Factories Using Transposon Vectors

  • Esteban Martínez-García
  • Tomás Aparicio
  • Víctor de Lorenzo
  • Pablo I. Nikel
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1498)

Abstract

The construction of microbial cell factories à la carte largely depends on specialized molecular biology and synthetic biology tools needed to reprogram bacteria for modifying their existing functions or for bestowing them with new-to-Nature tasks. In this chapter, we document the use of a series of broad-host-range mini-Tn5 vectors for the delivery of gene(s) into the chromosome of Gram-negative bacteria and for the generation of saturated, random mutagenesis libraries for studies of gene function. The application of these tailored mini-transposon vectors, which could also be used for chromosomal engineering of a wide variety of Gram-negative microorganisms, is demonstrated in the platform environmental bacterium Pseudomonas putida KT2440.

Key words

Mini-transposon Tn5 transposon Pseudomonas putida Escherichia coli Synthetic biology Metabolic engineering Microbial cell factory Genome editing 

Notes

Acknowledgments

The work described in this protocol was supported by the CAMBIOS Project of the Spanish Ministry of Economy and Competitiveness (RTC-2014-1777-3), the ST-FLOW (FP7-KBBE-2011-5-289326), EVOPROG (FP7-ICT-610730), ARISYS (ERC-2012-ADG-322797), and EmPowerPutida (EU-H2020-BIOTEC-2014-2015-6335536) Contracts of the European Union, and the PROMPT Project of the Autonomous Community of Madrid (CAM-S2010/BMD-2414). The authors declare that there is no conflict of interest. All the bacterial strains and plasmids described in the text are available upon request.

References

  1. 1.
    de Lorenzo V, Herrero M, Jakubzik U, Timmis KN (1990) Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in Gram-negative eubacteria. J Bacteriol 172:6568–6572PubMedPubMedCentralGoogle Scholar
  2. 2.
    de Lorenzo V, Timmis KN (1994) Analysis and construction of stable phenotypes in Gram-negative bacteria with Tn5- and Tn10-derived minitransposons. Methods Enzymol 235:386–405CrossRefPubMedGoogle Scholar
  3. 3.
    Reznikoff WS (2008) Transposon Tn5. Annu Rev Genet 42:269–286CrossRefPubMedGoogle Scholar
  4. 4.
    Berg DE (1989) Transposon Tn5. In: Berg DE, Howe MM (eds) Mobile DNA. American Society for Microbiology Press, Washington, D.C., pp 185–210Google Scholar
  5. 5.
    Reznikoff WS (2006) Tn5 transposition: a molecular tool for studying protein structure-function. Biochem Soc Trans 34:320–323CrossRefPubMedGoogle Scholar
  6. 6.
    Phadnis SH, Sasakawa C, Berg DE (1986) Localization of action of the IS50-encoded transposase protein. Genetics 112:421–427PubMedPubMedCentralGoogle Scholar
  7. 7.
    Martínez-García E, Calles B, Arévalo-Rodríguez M, de Lorenzo V (2011) pBAM1: an all-synthetic genetic tool for analysis and construction of complex bacterial phenotypes. BMC Microbiol 11:38CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    de Lorenzo V, Herrero M, Sánchez JM, Timmis KN (1998) Mini-transposons in microbial ecology and environmental biotechnology. FEMS Microbiol Ecol 27:211–224CrossRefGoogle Scholar
  9. 9.
    Martínez-García E, Aparicio T, de Lorenzo V, Nikel PI (2014) New transposon tools tailored for metabolic engineering of Gram-negative microbial cell factories. Front Bioeng Biotechnol 2:46PubMedPubMedCentralGoogle Scholar
  10. 10.
    Nikel PI, de Lorenzo V (2013) Implantation of unmarked regulatory and metabolic modules in Gram-negative bacteria with specialised mini-transposon delivery vectors. J Biotechnol 163:143–154CrossRefPubMedGoogle Scholar
  11. 11.
    Schweizer HP (2003) Applications of the Saccharomyces cerevisiae Flp-FRT system in bacterial genetics. J Mol Microbiol Biotechnol 5:67–77CrossRefPubMedGoogle Scholar
  12. 12.
    Martínez-García E, Aparicio T, Goñi-Moreno A, Fraile S, de Lorenzo V (2014) SEVA 2.0: an update of the Standard European Vector Architecture for de-/re-construction of bacterial functionalities. Nucleic Acids Res 43:D1183–D1189CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Silva-Rocha R, Martínez-García E, Calles B, Chavarría M, Arce-Rodríguez A et al (2012) The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Res 41:D666–D675CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Nikel PI, Martínez-García E, de Lorenzo V (2014) Biotechnological domestication of pseudomonads using synthetic biology. Nat Rev Microbiol 12:368–379CrossRefPubMedGoogle Scholar
  15. 15.
    Nikel PI, de Lorenzo V (2014) Robustness of Pseudomonas putida KT2440 as a host for ethanol biosynthesis. New Biotechnol 31:562–571CrossRefGoogle Scholar
  16. 16.
    Benedetti I, de Lorenzo V, Nikel PI (2016) Genetic programming of catalytic Pseudomonas putida biofilms for boosting biodegradation of haloalkanes. Metab Eng 33:109–118CrossRefPubMedGoogle Scholar
  17. 17.
    Timmis KN (2002) Pseudomonas putida: a cosmopolitan opportunist par excellence. Environ Microbiol 4:779–781CrossRefPubMedGoogle Scholar
  18. 18.
    Nikel PI, Chavarría M, Fuhrer T, Sauer U, de Lorenzo V (2015) Pseudomonas putida KT2440 strain metabolizes glucose through a cycle formed by enzymes of the Entner-Doudoroff, Embden-Meyerhof-Parnas, and pentose phosphate pathways. J Biol Chem 290:25920–25932CrossRefPubMedGoogle Scholar
  19. 19.
    Nikel PI, Kim J, de Lorenzo V (2014) Metabolic and regulatory rearrangements underlying glycerol metabolism in Pseudomonas putida KT2440. Environ Microbiol 16:239–254CrossRefPubMedGoogle Scholar
  20. 20.
    Nikel PI, Romero-Campero FJ, Zeidman JA, Goñi-Moreno A, de Lorenzo V (2015) The glycerol-dependent metabolic persistence of Pseudomonas putida KT2440 reflects the regulatory logic of the GlpR repressor. mBio 6:e00340-00315CrossRefGoogle Scholar
  21. 21.
    Nikel PI, Silva-Rocha R, Benedetti I, de Lorenzo V (2014) The private life of environmental bacteria: pollutant biodegradation at the single cell level. Environ Microbiol 16:628–642CrossRefPubMedGoogle Scholar
  22. 22.
    Makovets S (2013) Basic DNA electrophoresis in molecular cloning: a comprehensive guide for beginners. Methods Mol Biol 1054:11–43CrossRefPubMedGoogle Scholar
  23. 23.
    Pratt LA, Kolter R (1998) Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili. Mol Microbiol 30:285–293CrossRefPubMedGoogle Scholar
  24. 24.
    Martínez-García E, de Lorenzo V (2012) Transposon-based and plasmid-based genetic tools for editing genomes of Gram-negative bacteria. Methods Mol Biol 813:267–283CrossRefPubMedGoogle Scholar
  25. 25.
    Zechner EL, Lang S, Schildbach JF (2012) Assembly and mechanisms of bacterial type IV secretion machines. Philos Trans R Soc Lond B Biol Sci 367:1073–1087CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Ilangovan A, Connery S, Waksman G (2015) Structural biology of the Gram-negative bacterial conjugation systems. Trends Microbiol 23:301–310CrossRefPubMedGoogle Scholar
  27. 27.
    Álvarez-Martínez CE, Christie PJ (2009) Biological diversity of prokaryotic type IV secretion systems. Microbiol Mol Biol Rev 73:775–808CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Babic A, Guérout AM, Mazel D (2008) Construction of an improved RP4 (RK2)-based conjugative system. Res Microbiol 159:545–549CrossRefPubMedGoogle Scholar
  29. 29.
    Iwasaki K, Uchiyama H, Yagi O, Kurabayashi T, Ishizuka K et al (1994) Transformation of Pseudomonas putida by electroporation. Biosci Biotechnol Biochem 58:851–854CrossRefPubMedGoogle Scholar
  30. 30.
    Choi KH, Kumar A, Schweizer HP (2006) A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J Microbiol Methods 64:391–397CrossRefPubMedGoogle Scholar
  31. 31.
    Das S, Noe JC, Paik S, Kitten T (2005) An improved arbitrary primed PCR method for rapid characterization of transposon insertion sites. J Microbiol Methods 63:89–94CrossRefPubMedGoogle Scholar
  32. 32.
    Zimmermann J, Voss H, Schwager C, Stegemann J, Ansorge W (1988) Automated Sanger dideoxy sequencing reaction protocol. FEBS Lett 233:432–436CrossRefPubMedGoogle Scholar
  33. 33.
    Shendure JA, Porreca GJ, Church GM, Gardner AF, Hendrickson CL et al (2011) Overview of DNA sequencing strategies. Curr Prot Mol Biol 96:7.1.1–7.1.23Google Scholar
  34. 34.
    Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410CrossRefPubMedGoogle Scholar
  35. 35.
    Winsor GL, Lam DK, Fleming L, Lo R, Whiteside MD et al (2011) Pseudomonas Genome Database: improved comparative analysis and population genomics capability for Pseudomonas genomes. Nucleic Acids Res 39:D596–D600CrossRefPubMedGoogle Scholar
  36. 36.
    Berg DE, Weiss A, Crossland L (1980) Polarity of Tn5 insertion mutations in Escherichia coli. J Bacteriol 142:439–446PubMedPubMedCentralGoogle Scholar
  37. 37.
    Liberati NT, Urbach JM, Miyata S, Lee DG, Drenkard E et al (2006) An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants. Proc Natl Acad Sci U S A 103:2833–2838CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Cherepanov PP, Wackernagel W (1995) Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158:9–14CrossRefPubMedGoogle Scholar
  39. 39.
    Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP (1998) A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86CrossRefPubMedGoogle Scholar
  40. 40.
    Bachmann BJ (1996) Derivations and genotypes of some mutant derivatives of Escherichia coli K-12. In: Neidhardt FC, Curtiss R III, Ingraham JL, Lin ECC, Low KB Jr et al (eds) EcoSalEscherichia coli and Salmonella: cellular and molecular biology. American Society for Microbiology Press, Washington, DC, pp 2460–2488Google Scholar
  41. 41.
    de las Heras A, Carreño CA, de Lorenzo V (2008) Stable implantation of orthogonal sensor circuits in Gram-negative bacteria for environmental release. Environ Microbiol 10:3305–3316CrossRefPubMedGoogle Scholar
  42. 42.
    Herrero M, de Lorenzo V, Timmis KN (1990) Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in Gram-negative bacteria. J Bacteriol 172:6557–6567PubMedPubMedCentralGoogle Scholar
  43. 43.
    Miller VL, Mekalanos JJ (1988) A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol 170:2575–2583PubMedPubMedCentralGoogle Scholar
  44. 44.
    de Lorenzo V, Cases I, Herrero M, Timmis KN (1993) Early and late responses of TOL promoters to pathway inducers: identification of postexponential promoters in Pseudomonas putida with lacZ-tet bicistronic reporters. J Bacteriol 175:6902–6907PubMedPubMedCentralGoogle Scholar
  45. 45.
    Ferrières L, Hémery G, Nham T, Guérout AM, Mazel D et al (2010) Silent mischief: bacteriophage Mu insertions contaminate products of Escherichia coli random mutagenesis performed using suicidal transposon delivery plasmids mobilized by broad-host-range RP4 conjugative machinery. J Bacteriol 192:6418–6427CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–472CrossRefPubMedGoogle Scholar
  47. 47.
    Worsey MJ, Williams PA (1975) Metabolism of toluene and xylenes by Pseudomonas putida (arvilla) mt-2: evidence for a new function of the TOL plasmid. J Bacteriol 124:7–13PubMedPubMedCentralGoogle Scholar
  48. 48.
    Bagdasarian M, Lurz R, Rückert B, Franklin FC, Bagdasarian MM et al (1981) Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene 16:237–247CrossRefPubMedGoogle Scholar
  49. 49.
    Ditta G, Stanfield S, Corbin D, Helinski DR (1980) Broad host range DNA cloning system for Gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A 77:7347–7351CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Kessler B, de Lorenzo V, Timmis KN (1992) A general system to integrate lacZ fusions into the chromosomes of Gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy. Mol Gen Genet 233:293–301CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Esteban Martínez-García
    • 1
  • Tomás Aparicio
    • 1
  • Víctor de Lorenzo
    • 1
  • Pablo I. Nikel
    • 1
  1. 1.Systems and Synthetic Biology ProgramCentro Nacional de Biotecnología (CNB-CSIC)MadridSpain

Personalised recommendations