1887

Abstract

Nutrient-limited continuous cultures in chemostats have been used to study microbial cell physiology for over 60 years. Genome instability and genetic heterogeneity are possible uncontrolled factors in continuous cultivation experiments. We investigated these issues by using high-throughput (HT) DNA sequencing to characterize samples from different phases of a glucose-limited accelerostat (A-stat) experiment with K-12 MG1655 and a duration regularly used in cell physiology studies (20 generations of continuous cultivation). Seven consensus mutations from the reference sequence and five subpopulations characterized by different mutations were detected in the HT-sequenced samples. This genetic heterogeneity was confirmed to result from the stock culture by Sanger sequencing. All the subpopulations in which allele frequencies increased (, , ) during the experiment were also present at the end of replicate A-stats, indicating that no new subpopulations emerged during our experiments. The fact that ~31 % of the cells in our initial cultures obtained directly from a culture stock centre were mutants raises concerns that even if cultivations are started from single colonies, there is a significant chance of picking a mutant clone with an altered phenotype. Our results show that current HT DNA sequencing technology allows accurate subpopulation analysis and demonstrates that a glucose-limited K-12 MG1655 A-stat experiment with a duration of tens of generations is suitable for studying cell physiology and collecting quantitative data for metabolic modelling without interference from new mutations.

Funding
This study was supported by the:
  • EU (Award EU29994)
  • Estonian targeted and science foundation projects (Award G8165 and SF0140090s08)
  • National Institutes of Health (Award R00 GM087550)
  • National Science Foundation BEACON Center for the Study of Evolution in Action (Award DBI-0939454)
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2011-09-01
2024-03-29
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References

  1. Adams J. ( 2004). Microbial evolution in laboratory environments. Res Microbiol 155:311–318 [View Article][PubMed]
    [Google Scholar]
  2. Barker C. S., Prüss B. M., Matsumura P. ( 2004). Increased motility of Escherichia coli by insertion sequence element integration into the regulatory region of the flhD operon. J Bacteriol 186:7529–7537 [View Article][PubMed]
    [Google Scholar]
  3. Barrick J. E., Lenski R. E. ( 2009). Genome-wide mutational diversity in an evolving population of Escherichia coli. Cold Spring Harb Symp Quant Biol 74:119–129 [View Article][PubMed]
    [Google Scholar]
  4. Barrick J. E., Yu D. S., Yoon S. H., Jeong H., Oh T. K., Schneider D., Lenski R. E., Kim J. F. ( 2009). Genome evolution and adaptation in a long-term experiment with Escherichia coli. Nature 461:1243–1247 [View Article][PubMed]
    [Google Scholar]
  5. Conrad T. M., Joyce A. R., Applebee M. K., Barrett C. L., Xie B., Gao Y., Palsson B. Ø. ( 2009). Whole-genome resequencing of Escherichia coli K-12 MG1655 undergoing short-term laboratory evolution in lactate minimal media reveals flexible selection of adaptive mutations. Genome Biol 10:R118 [View Article][PubMed]
    [Google Scholar]
  6. Ferenci T. ( 2008). Bacterial physiology, regulation and mutational adaptation in a chemostat environment. Adv Microb Physiol 53:169–229 [View Article][PubMed]
    [Google Scholar]
  7. Harris D. R., Pollock S. V., Wood E. A., Goiffon R. J., Klingele A. J., Cabot E. L., Schackwitz W., Martin J., Eggington J. et al. ( 2009). Directed evolution of ionizing radiation resistance in Escherichia coli. J Bacteriol 191:5240–5252 [View Article][PubMed]
    [Google Scholar]
  8. Helling R. B., Vargas C. N., Adams J. ( 1987). Evolution of Escherichia coli during growth in a constant environment. Genetics 116:349–358[PubMed]
    [Google Scholar]
  9. Jishage M., Ishihama A. ( 1997). Variation in RNA polymerase sigma subunit composition within different stocks of Escherichia coli W3110. J Bacteriol 179:959–963[PubMed]
    [Google Scholar]
  10. King T., Ishihama A., Kori A., Ferenci T. ( 2004). A regulatory trade-off as a source of strain variation in the species Escherichia coli. J Bacteriol 186:5614–5620 [View Article][PubMed]
    [Google Scholar]
  11. Kinnersley M. A., Holben W. E., Rosenzweig F. ( 2009). E unibus plurum: genomic analysis of an experimentally evolved polymorphism in Escherichia coli. PLoS Genet 5:e1000713 [View Article][PubMed]
    [Google Scholar]
  12. Lahtvee P.-J., Adamberg K., Arike L., Nahku R., Aller K., Vilu R. ( 2011). Multi-omics approach to study the growth efficiency and amino acid metabolism in Lactococcus lactis at various specific growth rates. Microb Cell Fact 10:12 [View Article][PubMed]
    [Google Scholar]
  13. Lee D.-H., Palsson B. Ø. ( 2010). Adaptive evolution of Escherichia coli K-12 MG1655 during growth on a nonnative carbon source, l-1,2-propanediol. Appl Environ Microbiol 76:4158–4168 [View Article][PubMed]
    [Google Scholar]
  14. Maharjan R., Seeto S., Notley-McRobb L., Ferenci T. ( 2006). Clonal adaptive radiation in a constant environment. Science 313:514–517 [View Article][PubMed]
    [Google Scholar]
  15. Manch K., Notley-McRobb L., Ferenci T. ( 1999). Mutational adaptation of Escherichia coli to glucose limitation involves distinct evolutionary pathways in aerobic and oxygen-limited environments. Genetics 153:5–12[PubMed]
    [Google Scholar]
  16. Mardis E. R. ( 2008). Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet 9:387–402 [View Article][PubMed]
    [Google Scholar]
  17. Monod J. ( 1950). La technique de culture continue, theorie et applications. Ann Inst Pasteur (Paris) 79:390–410
    [Google Scholar]
  18. Naas T., Blot M., Fitch W. M., Arber W. ( 1994). Insertion sequence-related genetic variation in resting Escherichia coli K-12. Genetics 136:721–730[PubMed]
    [Google Scholar]
  19. Naas T., Blot M., Fitch W. M., Arber W. ( 1995). Dynamics of IS-related genetic rearrangements in resting Escherichia coli K-12. Mol Biol Evol 12:198–207[PubMed]
    [Google Scholar]
  20. Notley-McRobb L., Ferenci T. ( 1999a). Adaptive mgl-regulatory mutations and genetic diversity evolving in glucose-limited Escherichia coli populations. Environ Microbiol 1:33–43 [View Article][PubMed]
    [Google Scholar]
  21. Notley-McRobb L., Ferenci T. ( 1999b). The generation of multiple co-existing mal-regulatory mutations through polygenic evolution in glucose-limited populations of Escherichia coli. Environ Microbiol 1:45–52 [View Article][PubMed]
    [Google Scholar]
  22. Notley-McRobb L., Seeto S., Ferenci T. ( 2003). The influence of cellular physiology on the initiation of mutational pathways in Escherichia coli populations. Proc Biol Sci 270:843–848 [View Article][PubMed]
    [Google Scholar]
  23. Novick A., Szilard L. ( 1950a). Description of the chemostat. Science 112:715–716 [View Article][PubMed]
    [Google Scholar]
  24. Novick A., Szilard L. ( 1950b). Experiments with the chemostat on spontaneous mutations of bacteria. Proc Natl Acad Sci U S A 36:708–719 [View Article][PubMed]
    [Google Scholar]
  25. Paalme T., Kahru A., Elken R., Vanatalu K., Tiismaa K., Vilu R. ( 1995). The computer-controlled continuous culture of Escherichia coli with smooth change of dilution rate. J Microbiol Methods 24:145–153 [View Article]
    [Google Scholar]
  26. Soupene E., van Heeswijk W. C., Plumbridge J., Stewart V., Bertenthal D., Lee H., Prasad G., Paliy O., Charernnoppakul P., Kustu S. ( 2003). Physiological studies of Escherichia coli strain MG1655: growth defects and apparent cross-regulation of gene expression. J Bacteriol 185:5611–5626 [View Article][PubMed]
    [Google Scholar]
  27. Valgepea K., Adamberg K., Nahku R., Lahtvee P.-J., Arike L., Vilu R. ( 2010). Systems biology approach reveals that overflow metabolism of acetate in Escherichia coli is triggered by carbon catabolite repression of acetyl-CoA synthetase. BMC Syst Biol 4:166 [View Article][PubMed]
    [Google Scholar]
  28. Wick L. M., Quadroni M., Egli T. ( 2001). Short- and long-term changes in proteome composition and kinetic properties in a culture of Escherichia coli during transition from glucose-excess to glucose-limited growth conditions in continuous culture and vice versa. Environ Microbiol 3:588–599 [View Article][PubMed]
    [Google Scholar]
  29. Wick L. M., Weilenmann H., Egli T. ( 2002). The apparent clock-like evolution of Escherichia coli in glucose-limited chemostats is reproducible at large but not at small population sizes and can be explained with Monod kinetics. Microbiology 148:2889–2902[PubMed]
    [Google Scholar]
  30. Woods R. J., Barrick J. E., Cooper T. F., Shrestha U., Kauth M. R., Lenski R. E. ( 2011). Second-order selection for evolvability in a large Escherichia coli population. Science 331:1433–1436 [View Article][PubMed]
    [Google Scholar]
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