Metabolomics

, Volume 10, Issue 2, pp 241–249 | Cite as

Global metabolomics characterization of bacteria: pre-analytical treatments and profiling

Original Article

Abstract

Pre-analytical treatments of bacteria are crucial steps in bacterial metabolomics studies. In order to achieve reliable samples that can best represent the global metabolic profile in vivo both qualitatively and quantitatively, many sample treatment procedures have been developed. The use of different methods makes it difficult to compare the results among different groups. In this work, E. coli samples were tested by using NMR spectroscopy. Both liquid N2 and cold methanol quenching procedures reduce the cell membrane integrity and cause metabolites leakage. However, liquid N2 quenching affected the cell viability and the NMR metabolites’ profile less than cold methanol procedure. Samples obtained by metabolite extraction were significantly superior over cell suspensions and cell lysates, with a higher number of detectable metabolites. Methanol/chloroform extraction proved most efficient at extraction of intracellular metabolites from both qualitative and quantitative points of view. Finally, standard operating procedures of bacterial sample treatments for NMR metabolomics study are presented.

Keywords

NMR Metabolomics Bacteria Pre-analytical treatments Standard operating procedure 

Supplementary material

11306_2013_571_MOESM1_ESM.doc (1.9 mb)
Supplementary material 1 (DOC 1995 kb)

References

  1. Arita, M. (2004). The metabolic world of Escherichia coli is not small. Proceedings of the National Academy of Sciences United States of America, 101, 1543–1547.CrossRefGoogle Scholar
  2. Bingol, K., & Bruschweiler, R. (2011). Deconvolution of chemical mixtures with high complexity by NMR consensus trace clustering. Analytical Chemistry, 83, 7412–7417.CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bolten, C. J., Kiefer, P., Letisse, F., Portais, J. C., & Wittmann, C. (2007). Sampling for metabolome analysis of microorganisms. Analytical Chemistry, 79, 3843–3849.CrossRefPubMedGoogle Scholar
  4. Boroujerdi, A. F., Jones, S. S., & Bearden, D. W. (2012). NMR analysis of metabolic responses to extreme conditions of the temperature-dependent coral pathogen Vibrio coralliilyticus. Letters in Applied Microbiology, 54, 209–216.CrossRefPubMedGoogle Scholar
  5. Cano, K. E., Li, L., Bhatia, S., Bhatia, R., Forman, S. J., & Chen, Y. (2011). NMR-based metabolomic analysis of the molecular pathogenesis of therapy-related myelodysplasia/acute myeloid leukemia. Journal of Proteome Research, 10, 2873–2881.CrossRefPubMedPubMedCentralGoogle Scholar
  6. Cuperlovic-Culf, M., Barnett, D. A., Culf, A. S., & Chute, I. (2010). Cell culture metabolomics: Applications and future directions. Drug Discovery Today, 15, 610–621.CrossRefPubMedGoogle Scholar
  7. Dietmair, S., Timmins, N. E., Gray, P. P., Nielsen, L. K., & Kromer, J. O. (2010). Towards quantitative metabolomics of mammalian cells: Development of a metabolite extraction protocol. Analytical Biochemistry, 404, 155–164.CrossRefPubMedGoogle Scholar
  8. Duarte, I. F., Marques, J., Ladeirinha, A. F., Rocha, C., Lamego, I., Calheiros, R., et al. (2009). Analytical approaches toward successful human cell metabolome studies by NMR spectroscopy. Analytical Chemistry, 81, 5023–5032.CrossRefPubMedGoogle Scholar
  9. Ganzera, M., Vrabl, P., Worle, E., Burgstaller, W., & Stuppner, H. (2006). Determination of adenine and pyridine nucleotides in glucose-limited chemostat cultures of Penicillium simplicissimum by one-step ethanol extraction and ion-pairing liquid chromatography. Analytical Biochemistry, 359, 132–140.CrossRefPubMedGoogle Scholar
  10. Govindaraju, V., Young, K., & Maudsley, A. A. (2000). Proton NMR chemical shifts and coupling constants for brain metabolites. NMR in Biomedicine, 13, 129–153.CrossRefPubMedGoogle Scholar
  11. Grob, M. K., O’Brien, K., Chu, J. J., & Chen, D. D. (2003). Optimization of cellular nucleotide extraction and sample preparation for nucleotide pool analyses using capillary electrophoresis. Journal of Chromatography B Analytical Technologies in the Biomedical Life Sciences, 788, 103–111.CrossRefPubMedGoogle Scholar
  12. Gromova, M., & Roby, C. (2010). Toward Arabidopsis thaliana hydrophilic metabolome: Assessment of extraction methods and quantitative 1H NMR. Physiologia Plantarum, 140, 111–127.CrossRefPubMedGoogle Scholar
  13. Hoerr, V., Zbytnuik, L., Leger, C., Tam, P. P., Kubes, P., & Vogel, H. J. (2012). Gram-negative and gram-positive bacterial infections give rise to a different metabolic response in a mouse model. Journal of Proteome Research, 11, 3231–3245.Google Scholar
  14. Ishii, N., Nakahigashi, K., Baba, T., Robert, M., Soga, T., Kanai, A., et al. (2007). Multiple high-throughput analyses monitor the response of E.coli to perturbations. Science, 316, 593–597.CrossRefPubMedGoogle Scholar
  15. Jozefczuk, S., Klie, S., Catchpole, G., Szymanski, J., Cuadros-Inostroza, A., Steinhauser, D., et al. (2010). Metabolomic and transcriptomic stress response of Escherichia coli. Molecular Systems Biology, 6, 364.CrossRefPubMedPubMedCentralGoogle Scholar
  16. Le Belle, J. E., Harris, N. G., Williams, S. R., & Bhakoo, K. K. (2002). A comparison of cell and tissue extraction techniques using high-resolution 1H NMR spectroscopy. NMR in Biomedicine, 15, 37–44.CrossRefPubMedGoogle Scholar
  17. Lee, S. Y. (1996). High cell-density culture of Escherichia coli. Trends in Biotechnology, 14, 98–105.CrossRefPubMedGoogle Scholar
  18. Lin, C. Y., Wu, H., Tjeerdema, R. S., & Viant, M. R. (2007). Evaluation of metabolite extraction strategies from tissue samples using NMR metabolomics. Metabolomics, 3, 55–67.CrossRefGoogle Scholar
  19. Lindon, J. C., Nicholson, J. K., & Holmes, E. (2007). Handbook of metabonomics and metabolomics. Amsterdam: Elsevier.Google Scholar
  20. Lodi, A., & Ronen, S. M. (2011). Magnetic resonance spectroscopy detectable metabolomic fingerprint of response to antineoplastic treatment. PLoS One, 6, e26155.CrossRefPubMedPubMedCentralGoogle Scholar
  21. Maharjan, R. P., & Ferenci, T. (2003). Global metabolite analysis: The influence of extraction methodology on metabolome profiles of Escherichia coli. Analytical Biochemistry, 313, 145–154.CrossRefPubMedGoogle Scholar
  22. Niles, A. L., Moravec, R. A., & Riss, T. L. (2009). In vitro viability and cytotoxicity testing and same-well multi-parametric combinations for high throughput screening. Current Chemical Genomics, 3, 33–41.CrossRefPubMedPubMedCentralGoogle Scholar
  23. Oostendorp, M., Engelke, U. F., Willemsen, M. A., & Wevers, R. A. (2006). Diagnosing inborn errors of lipid metabolism with proton nuclear magnetic resonance spectroscopy. Clinical Chemistry, 52, 1395–1405.CrossRefPubMedGoogle Scholar
  24. Rabinowitz, J. D. (2007). Cellular metabolomics of Escherichia coli. Expert Review of Proteomics, 4, 187–198.CrossRefPubMedGoogle Scholar
  25. Samuelsson, L. M., Forlin, L., Karlsson, G., Adolfsson-Erici, M., & Larsson, D. G. (2006). Using NMR metabolomics to identify responses of an environmental estrogen in blood plasma of fish. Aquatic Toxicology, 78, 341–349.CrossRefPubMedGoogle Scholar
  26. Sellick, C. A., Hansen, R., Maqsood, A. R., Dunn, W. B., Stephens, G. M., Goodacre, R., et al. (2009). Effective quenching processes for physiologically valid metabolite profiling of suspension cultured Mammalian cells. Analytical Chemistry, 81, 174–183.CrossRefPubMedGoogle Scholar
  27. Tang, J. (2011). Microbial metabolomics. Current Genomics, 12, 391–403.CrossRefPubMedPubMedCentralGoogle Scholar
  28. Tomiya, N., Ailor, E., Lawrence, S. M., Betenbaugh, M. J., & Lee, Y. C. (2001). Determination of nucleotides and sugar nucleotides involved in protein glycosylation by high-performance anion-exchange chromatography: Sugar nucleotide contents in cultured insect cells and mammalian cells. Analytical Biochemistry, 293, 129–137.CrossRefPubMedGoogle Scholar
  29. Viant, M. R., Bundy, J. G., Pincetich, C. A., de Ropp, J. S., & Tjeerdema, R. S. (2005). NMR-derived developmental metabolic trajectories: An approach for visualizing the toxic actions of trichloroethylene during embryogenesis. Metabolomics, 1, 149–158.CrossRefGoogle Scholar
  30. Villas-Boas, S. G., Moxley, J. F., Akesson, M., Stephanopoulos, G., & Nielsen, J. (2005). High-throughput metabolic state analysis: The missing link in integrated functional genomics of yeasts. Biochemical Journal, 388, 669–677.CrossRefPubMedPubMedCentralGoogle Scholar
  31. Villas-Boas, S. G., Roessner, U., Hansen, M. A. E., Smedsgaard, J., & Nielsen, J. (2007). Metabolome analysis:An introduction. New york: Wiley.CrossRefGoogle Scholar
  32. Winder, C. L., Dunn, W. B., Schuler, S., Broadhurst, D., Jarvis, R., Stephens, G. M., et al. (2008). Global metabolic profiling of Escherichia coli cultures: An evaluation of methods for quenching and extraction of intracellular metabolites. Analytical Chemistry, 80, 2939–2948.CrossRefPubMedGoogle Scholar
  33. Wishart, D. S., Knox, C., Guo, A. C., Eisner, R., Young, N., Gautam, B., et al. (2009). HMDB: A knowledgebase for the human metabolome. Nucleic Acids Research, 37, D603–D610.CrossRefPubMedGoogle Scholar
  34. Wittmann, C., Kromer, J. O., Kiefer, P., Binz, T., & Heinzle, E. (2004). Impact of the cold shock phenomenon on quantification of intracellular metabolites in bacteria. Analytical Biochemistry, 327, 135–139.CrossRefPubMedGoogle Scholar
  35. Ye, Y., Wang, X., Zhang, L., Lu, Z., & Yan, X. (2012a). Unraveling the concentration-dependent metabolic response of Pseudomonas sp. HF-1 to nicotine stress by 1H NMR-based metabolomics. Ecotoxicology, 21, 1314–1324.CrossRefPubMedGoogle Scholar
  36. Ye, Y., Zhang, L., Hao, F., Zhang, J., Wang, Y., & Tang, H. (2012b). Global metabolomic responses of Escherichia coli to heat stress. Journal of Proteome Research, 11, 2559–2566.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Magnetic Resonance Center (CERM)University of FlorenceSesto FiorentinoItaly
  2. 2.Department of ChemistryUniversity of FlorenceSesto FiorentinoItaly
  3. 3.FiorGen FoundationSesto FiorentinoItaly

Personalised recommendations