Applied Microbiology and Biotechnology

, Volume 77, Issue 2, pp 489–496 | Cite as

Development of a novel continuous culture device for experimental evolution of bacterial populations

  • E. de Crécy
  • D. Metzgar
  • C. Allen
  • M. Pénicaud
  • B. Lyons
  • C. J. Hansen
  • V. de Crécy-Lagard
Methods

Abstract

The availability of a robust and reliable continuous culture apparatus that eliminates wall growth problems would lead to many applications in the microbial field, including allowing genetically engineered strains to recover high fitness, improving biodegradation strains, and predicting likely antibiotic resistance mechanisms. We describe the design and implementation of a novel automated continuous culture machine that can be used both in time-dependent mode (similar to a chemostat) and turbidostat modes, in which wall growth is circumvented through the use of a long, variably divisible tube of growth medium. This tube can be restricted with clamps to create a mobile growth chamber region in which static portions of the tube and the associated medium are replaced together at equal rates. To functionally test the device as a tool for re-adaptation of engineered strains, we evolved a strain carrying a highly deleterious deletion of Elongation Factor P, a gene involved in translation. In 200 generations over 2 weeks of dilution cycles, the evolved strain improved in generation time by a factor of three, with no contaminations and easy manipulation.

Keywords

Experimental evolution Continuous culture Turbidostat Natural selection Adaptation Metabolic engineering Biodegradation 

Notes

Acknowledgements

We thank Phillippe Marlière for inspiration, Paul Schimmel for encouragements in the initial stages of the project, and Daniel Dykhuizen for critical reading of the manuscript. V d C-L work was funded in part by grant MCB-0128901 from the National Science Foundation.

References

  1. Aoki H, Dekany K, Adams SL, Ganoza MC (1997) The gene encoding the elongation factor P protein is essential for viability and is required for protein synthesis. J Biol Chem 272:32254–32259CrossRefGoogle Scholar
  2. Bonomo J, Warnecke T, Hume P, Marizcurrena A, Gill RT (2006) A comparative study of metabolic engineering anti-metabolite tolerance in Escherichia coli. Metab Eng 8:227–239CrossRefGoogle Scholar
  3. Bryson V, Szybakski W (1952) Microbial selection. Science 116:45–51CrossRefGoogle Scholar
  4. de Crécy E (2005) Continuous culture apparatus with mobile vessel allowing selection of fitter cell variants. WO/2005/083052Google Scholar
  5. de Crécy-Lagard VA, Bellalou J, Mutzel R, Marlière P (2001) Long term adaptation of a microbial population to a permanent metabolic constraint: overcoming thymineless death by experimental evolution of Escherichia coli. BMC Biotechnol 1:10CrossRefGoogle Scholar
  6. Dykhuizen DE (1993) Chemostats used for studying natural selection and adaptive evolution. Methods Enzymol 224:613–631CrossRefGoogle Scholar
  7. Dykhuizen DE, Hartl DL (1983) Selection in chemostats. Microbiol Rev 47:150–168Google Scholar
  8. Fong SS, Palsson BO (2004) Metabolic gene-deletion strains of Escherichia coli evolve to computationally predicted growth phenotypes. Nat Genet 36:1056–1058CrossRefGoogle Scholar
  9. Fong SS, Burgard AP, Herring CD, Knight EM, Blattner FR, Maranas CD, Palsson BO (2005) In silico design and adaptive evolution of Escherichia coli for production of lactic acid. Biotechnol Bioeng 91:643–648CrossRefGoogle Scholar
  10. Fong SS, Nanchen A, Palsson BO, Sauer U (2006) Latent pathway activation and increased pathway capacity enable Escherichia coli adaptation to loss of key metabolic enzymes. J Biol Chem 281:8024–8033CrossRefGoogle Scholar
  11. Ganoza MC, Aoki H (2000) Peptide bond synthesis: function of the efp gene product. Biol Chem 381:553–559CrossRefGoogle Scholar
  12. Helling RB, Kinney T, Adams J (1981) The maintenance of plasmid-containing organisms in populations of Escherichia coli. J Gen Microbiol 123:129–141Google Scholar
  13. Larsen DH, Dimmick RL (1964) Attachment and growth of bacteria on surfaces of continuous culture vessels. J Bacteriol 88:1380–1387Google Scholar
  14. Lenski RE (1993) Evaluating the fate of genetically modified microorganisms in the environment: are they inherently less fit? Experientia 49:201–209CrossRefGoogle Scholar
  15. Lenski RE, Travisano M (1994) Dynamics of adaptation and diversification: a 10,000-generation experiment with bacterial populations. Proc Natl Acad Sci USA 91:6808–6814CrossRefGoogle Scholar
  16. Metzgar D, Bacher JM, Pezo V, Reader J, Döring V, Schimmel P, Marlière P, de Crécy-Lagard V (2004) Acinetobacter sp. ADP1: an ideal model organism for genetic analysis and genome engineering. Nucleic Acids Res 32:5780–5790CrossRefGoogle Scholar
  17. Meynial-Salles I, Forchhammer N, Croux C, Girbal L, Soucaille P (2007) Evolution of a Saccharomyces cerevisiae metabolic pathway in Escherichia coli. Metab Eng 9:152–159CrossRefGoogle Scholar
  18. Monod J (1950) La technique de culture continue. Théorie et applications. Ann Inst Pasteur 19:390–410Google Scholar
  19. Mutzel R, Mazel D, Marlière P (2003) Method for obtaining cells with new properties WO 03/004656Google Scholar
  20. Notley-McRobb L, Ferenci T (1999) The generation of multiple co-existing mal-regulatory mutations through polygenic evolution in glucose-limited populations of Escherichia coli. Environ Microbiol 1:45–52CrossRefGoogle Scholar
  21. Novick A, Szilard L (1950a) Description of the chemostat. Science 112:715–716CrossRefGoogle Scholar
  22. Novick A, Szilard L (1950b) Experiments with the chemostat on spontaneous mutations of bacteria. Proc Natl Acad Sci USA 36:708–719CrossRefGoogle Scholar
  23. Spratt BG (1994) Resistance to antibiotics mediated by target alterations. Science 264:388–393CrossRefGoogle Scholar
  24. Swaney S, McCroskey M, Shinabarger D, Wang Z, Turner BA, Parker CN (2006) Characterization of a high-throughput screening assay for inhibitors of elongation factor p and ribosomal peptidyl transferase activity. J Biomol Screen 11:736–742CrossRefGoogle Scholar
  25. Vidal O, Longin R, Prigent-Combaret C, Dorel C, Hooreman M, Lejeune P (1998) Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression. J Bacteriol 180:2442–2449Google Scholar
  26. Wahl LM, Gerrish PJ, Saika-Voivod I (2002) Evaluating the impact of population bottlenecks in experimental evolution. Genetics 162:961Google Scholar
  27. Zhou S, Yomano LP, Shanmugam KT, Ingram LO (2005) Fermentation of 10% (w/v) sugar to D: (−)-lactate by engineered Escherichia coli B. Biotech Lett 27:1891–1896CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • E. de Crécy
    • 1
  • D. Metzgar
    • 1
  • C. Allen
    • 2
  • M. Pénicaud
    • 1
  • B. Lyons
    • 2
  • C. J. Hansen
    • 1
  • V. de Crécy-Lagard
    • 2
  1. 1.GainesvilleUSA
  2. 2.Department of Microbiology and Cell ScienceUniversity of FloridaGainesvilleUSA

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