High performance analysis of the cyanobacterial metabolism via liquid chromatography coupled to a LTQ-Orbitrap mass spectrometer: evidence that glucose reprograms the whole carbon metabolism and triggers oxidative stress

Abstract

Cyanobacteria are environmentally important photosynthetic microorganisms attracting a growing attention in various areas of basic and applied researches. To better understand their metabolism, we presently report on the development of a robust and simple protocol for facile extraction and high throughput analysis of the metabolites of the widely-used strain Synechocystis PCC6803 through liquid chromatography coupled to high resolution mass spectrometry (LC/MS). Our analytical method was developed and tested with 102 reference compounds representative of the chemical diversity of polar cell metabolites, and Synechocystis cell extracts spiked with 37 reference compounds. These samples were analyzed with two chromatographic systems, each coupled to a LTQ-Orbitrap mass spectrometer: a liquid chromatographic system equipped with a pentafluorophenylpropyl column (the PFPP-LC/MS system), and an ultra-high performance liquid chromatographic system with a C18-reversed phase column (the C18-UHPLC/MS system). We showed that the PFPP-LC/MS method performs better than the C18-UHPLC/MS method in terms of retention, separation and detection of metabolites. Consequently, we applied the PFPP-LC/MS method to analyze the metabolome of Synechocystis growing under various conditions of light and glucose, which strongly influence cell growth. We found that glucose increases glucose storage (synthesis of glycogen-like polysaccharide) and catabolism (oxidative pentose phosphate pathway and glycolysis), while it decreases the Calvin–Benson cycle that consumes photosynthetic electrons for CO2 assimilation. Depending on light and glucose availabilities, this global metabolic reprogramming can generate an oxidative stress, likely through the recombination of the glucose-spared electrons with the photosynthetic oxygen thereby producing toxic reactive oxygen species.

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Abbreviations

CE/MS:

Capillary electrophoresis coupled to mass spectrometry

DIMS:

Direct introduction mass spectrometry

ESI:

Electrospray source ionization

GC/MS:

Gas chromatography coupled to mass spectrometry

LC/MS:

Liquid chromatography coupled to mass spectrometry

PFPP:

Pentafluorophenylpropyl

OPP:

Oxidative Pentose phosphate pathway

UHPLC:

Ultra high performance liquid chromatography

References

  1. Abed, R. M., Dobretsov, S., & Sudesh, K. (2009). Applications of cyanobacteria in biotechnology. Journal of Applied Microbiology, 106(1), 1–12.

    PubMed  Article  CAS  Google Scholar 

  2. Anemaet, I. G., Bekker, M., & Hellingwerf, K. J. (2010). Algal photosynthesis as the primary driver for a sustainable development in energy, feed, and food production. NY: Mar Biotechnol.

    Google Scholar 

  3. Atsumi, S., Higashide, W., & Liao, J. C. (2009). Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. Nature Biotechnology, 27(12), 1177–1180.

    PubMed  Article  CAS  Google Scholar 

  4. Capone, D. G., Zehr, J. P., Paerl, H. W., Bergman, B., & Carpenter, E. J. (1997). Trichodesmium, a globally significant marine cyanobacterium. Science, 276, 1221–1229.

    Article  CAS  Google Scholar 

  5. Deusch, O., Landan, G., Roettger, M., Gruenheit, N., Kowallik, K. V., Allen, J. F., et al. (2008). Genes of cyanobacterial origin in plant nuclear genomes point to a heterocyst-forming plastid ancestor. Molecular Biology and Evolution, 25(4), 748–761.

    PubMed  Article  CAS  Google Scholar 

  6. Dismukes, G. C., Carrieri, D., Bennette, N., Ananyev, G. M., & Posewitz, M. C. (2008). Aquatic phototrophs: Efficient alternatives to land-based crops for biofuels. Current Opinion in Biotechnology, 19(3), 235–240.

    PubMed  Article  CAS  Google Scholar 

  7. Dismukes, G. C., Klimov, V. V., Baranov, S. V., Kozlov, Y. N., DasGupta, J., & Tyryshkin, A. (2001). The origin of atmospheric oxygen on Earth: the innovation of oxygenic photosynthesis. Proceedings of the National Academy Science USA, 98(5), 2170–2175.

    Article  CAS  Google Scholar 

  8. Domain, F., Houot, L., Chauvat, F., & Cassier-Chauvat, C. (2004). Function and regulation of the cyanobacterial genes lexA, recA and ruvB: LexA is critical to the survival of cells facing inorganic carbon starvation. Molecular Microbiology, 53(1), 65–80.

    PubMed  Article  CAS  Google Scholar 

  9. Dunnett, C. W. (1955). A multiple comparisons procedure for comparing several treatments with a control. Journal of American Statistical Association, 50, 1096–1121.

    Article  Google Scholar 

  10. Eisenhut, M., Huege, J., Schwarz, D., Bauwe, H., Kopka, J., & Hagemann, M. (2008). Metabolome phenotyping of inorganic carbon limitation in cells of the wild type and photorespiratory mutants of the cyanobacterium Synechocystis sp. strain PCC 6803. Plant Physiology, 148(4), 2109–2120.

    PubMed  Article  CAS  Google Scholar 

  11. Field, C. B., Behrenfeld, M. J., Randerson, J. T., & Falkowski, P. (1998). Primary production of the biosphere: integrating terrestrial and oceanic components. Science, 281(5374), 237–240.

    PubMed  Article  CAS  Google Scholar 

  12. Gangl, E. T., Annan, M. M., Spooner, N., & Vouros, P. (2001). Reduction of signal suppression effects in ESI-MS using a nanosplitting device. Analytical Chemistry, 73(23), 5635–5644.

    PubMed  Article  CAS  Google Scholar 

  13. Ghirardi, M. L., Dubini, A., Yu, J., & Maness, P. C. (2009). Photobiological hydrogen-producing systems. Chemical Society Reviews, 38(1), 52–61.

    PubMed  Article  CAS  Google Scholar 

  14. Grigorieva, G., & Shestakov, S. (1982). Transformation in the cyanobacterium Synechocystis sp 6803. FEMS Microbiology Letters, 13, 367–370.

    Article  CAS  Google Scholar 

  15. Haimovich-Dayan, M., Kahlon, S., Hihara, Y., Hagemann, M., Ogawa, T., Ohad, I., et al. (2011). Cross-talk between photomixotrophic growth and CO(2) -concentrating mechanism in Synechocystis sp. strain PCC 6803. Environmental Microbiology, 13(7), 1767–1777.

    PubMed  Article  CAS  Google Scholar 

  16. Hsieh, Y., Wang, G., Wang, Y., Chackalamannil, S., Brisson, J. M., Ng, K., et al. (2002). Simultaneous determination of a drug candidate and its metabolite in rat plasma samples using ultrafast monolithic column high-performance liquid chromatography/tandem mass spectrometry. Rapid Communications in Mass Spectrometry, 16(10), 944–950.

    PubMed  Article  CAS  Google Scholar 

  17. Junot, C., Madalinski, G., Tabet, J. C., & Ezan, E. (2010). Fourier transform mass spectrometry for metabolome analysis. Analyst, 135(9), 2203–2219.

    PubMed  Article  CAS  Google Scholar 

  18. Kanehisa, M., & Goto, S. (2000). KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Research, 28(1), 27–30.

    PubMed  Article  CAS  Google Scholar 

  19. Kaneko, T., Sato, S., Kotani, H., Tanaka, A., Asamizu, E., Nakamura, Y., et al. (1996). Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Research, 3, 109–136.

    PubMed  Article  CAS  Google Scholar 

  20. Knowles, V. L., & Plaxton, W. C. (2003). From genome to enzyme: analysis of key glycolytic and oxidative pentose-phosphate pathway enzymes in the cyanobacterium Synechocystis sp. PCC 6803. Plant and Cell Physiology, 44(7), 758–763.

    PubMed  Article  CAS  Google Scholar 

  21. Krall, L., Huege, J., Catchpole, G., Steinhauser, D., & Willmitzer, L. (2009). Assessment of sampling strategies for gas chromatography-mass spectrometry (GC-MS) based metabolomics of cyanobacteria. Journal of Chromatography B Analytical Technologies in the Biomedical and Life Sciences, 877(27), 2952–2960.

    Article  CAS  Google Scholar 

  22. Latifi, A., Ruiz, M., & Zhang, C. C. (2009). Oxidative stress in cyanobacteria. FEMS Microbiology Reviews, 33(2), 258–278.

    PubMed  Article  CAS  Google Scholar 

  23. Makarov, A., & Scigelova, M. (2010). Coupling liquid chromatography to Orbitrap mass spectrometry. Journal of Chromatography A, 1217(25), 3938–3945.

    PubMed  Article  CAS  Google Scholar 

  24. Marraccini, P., Bulteau, S., Cassier-Chauvat, C., Mermet-Bouvier, P., & Chauvat, F. (1993). A conjugative plasmid vector for promoter analysis in several cyanobacteria of the genera Synechococcus and Synechocystis. Plant Molecular Biology, 23, 905–909.

    PubMed  Article  CAS  Google Scholar 

  25. Matuszewski, B. K., Constanzer, M. L., & Chavez-Eng, C. M. (2003). Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Analytical Chemistry, 75(13), 3019–3030.

    PubMed  Article  CAS  Google Scholar 

  26. Mazouni, K., Domain, F., Cassier-Chauvat, C., & Chauvat, F. (2004). Molecular analysis of the key cytokinetic components of cyanobacteria: FtsZ, ZipN and MinCDE. Molecular Microbiology, 52(4), 1145–1158.

    PubMed  Article  CAS  Google Scholar 

  27. Mei, H., Hsieh, Y., Nardo, C., Xu, X., Wang, S., Ng, K., et al. (2003). Investigation of matrix effects in bioanalytical high-performance liquid chromatography/tandem mass spectrometric assays: application to drug discovery. Rapid Communications in Mass Spectrometry, 17(1), 97–103.

    PubMed  Article  CAS  Google Scholar 

  28. Mermet-Bouvier, P., & Chauvat, F. (1994). A conditional expression vector for the cyanobacteria Synechocystis sp. PCC6803 and PCC6714 or Synechococcus sp. PCC7942 and PCC6301. Current Microbiology, 28, 145–148.

    PubMed  Article  CAS  Google Scholar 

  29. Partensky, F., Hess, W. R., & Vaulot, D. (1999). Prochlorococcus, a marine photosynthetic prokaryote of global significance. Microbiology and Molecular Biology Reviews, 63, 106–127.

    PubMed  CAS  Google Scholar 

  30. Pelroy, R. A., Rippka, R., & Stanier, R. Y. (1972). Metabolism of glucose by unicellular blue-green algae. Arch Mikrobiol, 87(4), 303–322.

    PubMed  Article  CAS  Google Scholar 

  31. Pereira, S., Zille, A., Micheletti, E., Moradas-Ferreira, P., De Philippis, R., & Tamagnini, P. (2009). Complexity of cyanobacterial exopolysaccharides: Composition, structures, inducing factors and putative genes involved in their biosynthesis and assembly. FEMS Microbiology Reviews, 33(5), 917–941.

    PubMed  Article  CAS  Google Scholar 

  32. Poncelet, M., Cassier-Chauvat, C., Leschelle, X., Bottin, H., & Chauvat, F. (1998). Targeted deletion and mutational analysis of the essential (2Fe-2S) plant-like ferredoxin in Synechocystis PCC6803 by plasmid shuffling. Molecular Microbiology, 28, 813–821.

    PubMed  Article  CAS  Google Scholar 

  33. Richardson, D. L., Reed, R. H., & Stewart, W. D. P. (1983). Synechocystis PCC6803: A euryhaline cyanobacterium. FEMS Microbiology Letters, 18, 99–102.

    Article  CAS  Google Scholar 

  34. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., & Stanier, R. Y. (1979). Generic assignments, strains histories and properties of pure cultures of cyanobacteria. Journal of General Microbiology, 111, 1–61.

    Article  Google Scholar 

  35. Shi, T., & Falkowski, P. G. (2008). Genome evolution in cyanobacteria: The stable core and the variable shell. Proceedings of the National Academy of Sciences USA, 105(7), 2510–2515.

    Article  CAS  Google Scholar 

  36. Smith, C. A., Want, E. J., O’Maille, G., Abagyan, R., & Siuzdak, G. (2006). XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Analytical Chemistry, 78(3), 779–787.

    PubMed  Article  CAS  Google Scholar 

  37. Summerfield, T. C., & Sherman, L. A. (2008). Global transcriptional response of the alkali-tolerant cyanobacterium Synechocystis sp. strain PCC 6803 to a pH 10 environment. Applied and Environmental Microbiology, 74(17), 5276–5284.

    PubMed  Article  CAS  Google Scholar 

  38. Sumner, L. W., Urbanczyk-Wochniak, E., & Broeckling, C. D. (2007). Metabolomics data analysis, visualization, and integration. Methods in Molecular Biology, 406, 409–436.

    PubMed  CAS  Google Scholar 

  39. Takahashi, H., Uchimiya, H., & Hihara, Y. (2008). Difference in metabolite levels between photoautotrophic and photomixotrophic cultures of Synechocystis sp. PCC 6803 examined by capillary electrophoresis electrospray ionization mass spectrometry. Journal of Experimental Botany, 59(11), 3009–3018.

    PubMed  Article  CAS  Google Scholar 

  40. Tiller, P. R., & Romanyshyn, L. A. (2002). Implications of matrix effects in ultra-fast gradient or fast isocratic liquid chromatography with mass spectrometry in drug discovery. Rapid Communications in Mass Spectrometry, 16(2), 92–98.

    PubMed  Article  CAS  Google Scholar 

  41. Williams, P. G. (2009). Panning for chemical gold: marine bacteria as a source of new therapeutics. Trends in Biotechnology, 27(1), 45–52.

    PubMed  Article  CAS  Google Scholar 

  42. Yang, C., Hua, Q., & Shimizu, K. (2002). Integration of the information from gene expression and metabolic fluxes for the analysis of the regulatory mechanisms in Synechocystis. Applied Microbiology and Biotechnology, 58(6), 813–822.

    PubMed  Article  CAS  Google Scholar 

  43. Yang, S., Sadilek, M., & Lidstrom, M. E. (2010). Streamlined pentafluorophenylpropyl column liquid chromatography-tandem quadrupole mass spectrometry and global (13)C-labeled internal standards improve performance for quantitative metabolomics in bacteria. Journal of Chromatography A, 1217(47), 7401–7410.

    PubMed  Article  CAS  Google Scholar 

  44. Yoshida, H., Mizukoshi, T., Hirayama, K., & Miyano, H. (2007). Comprehensive analytical method for the determination of hydrophilic metabolites by high-performance liquid chromatography and mass spectrometry. Journal of Agricultural and Food Chemistry, 55(3), 551–560.

    PubMed  Article  CAS  Google Scholar 

  45. Yoshida, H., Yamazaki, J., Ozawa, S., Mizukoshi, T., & Miyano, H. (2009). Advantage of LC-MS metabolomics methodology targeting hydrophilic compounds in the studies of fermented food samples. Journal of Agricultural and Food Chemistry, 57(4), 1119–1126.

    PubMed  Article  CAS  Google Scholar 

  46. Zehr, J. P., Waterbury, J. B., Turner, P. J., Montoya, J. P., Omoregie, E., Steward, G. F., et al. (2001). Unicellular cyanobacteria fix N2 in the subtropical North Pacific Ocean. Nature, 412, 635–638.

    PubMed  Article  CAS  Google Scholar 

  47. Zhang, C. C., Laurent, S., Sakr, S., Peng, L., & Bedu, S. (2006). Heterocyst differentiation and pattern formation in cyanobacteria: A chorus of signals. Molecular Microbiology, 59(2), 367–375.

    PubMed  Article  CAS  Google Scholar 

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Acknowledgments

We thank our colleagues Geoffrey Madalinski, Aurélie Roux and Ying Xu for their help with the Orbitrap machine, and Eric Ezan and Jean-François Heilier for valuable discussion. This work was supported by French agency for research Grant ANR-09-BIOE-002-01 (EngineeringH2cyano) and CNRS (Centre National recherche scientifique) Programme Interdisciplinaire Energie PIE2 (Reprogramhydrogen). KN was a recipient of PhD thesis fellowship from the CEA-Saclay (Commissariat à l’énergie atomique) France.

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Correspondence to Christophe Junot or Franck Chauvat.

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Christophe Junot and Franck Chauvat contributed equally to this work.

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Narainsamy, K., Cassier-Chauvat, C., Junot, C. et al. High performance analysis of the cyanobacterial metabolism via liquid chromatography coupled to a LTQ-Orbitrap mass spectrometer: evidence that glucose reprograms the whole carbon metabolism and triggers oxidative stress. Metabolomics 9, 21–32 (2013). https://doi.org/10.1007/s11306-011-0382-4

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Keywords

  • Cyanobacterium
  • Synechocystis
  • Central carbon metabolism
  • LC/MS-LTQ-Orbitrap
  • Matrix effect
  • Metabolic reprogramming