Aspartate metabolism and pyruvate homeostasis triggered by oxidative stress in Pseudomonas fluorescens: a functional metabolomic study

Abstract

There is mounting evidence that metabolic reprogramming is critical for the survival of organisms exposed to changing and stressed environments. Using the soil microbe Pseudomonas fluorescens as a model system, we demonstrate that the metabolic networks aimed at the conversion of aspartate into pyruvate are enhanced in the presence of hydrogen peroxide (H2O2). The metabolites pyruvate, oxaloacetate and acetate were increased in the treated cultures as measured by HPLC. Enzymes such as aspartate transaminase and phosphoenolpyruvate carboxylase (PEPC) that mediate the conversion of aspartate to phosphoenolpyruvate (PEP) were up-regulated. This high-energy phosphate was readily converted into ATP, a process facilitated by the increased activity of pyruvate orthophosphate dikinase (PPDK) and phosphoenolpyruvate synthase (PEPS) as oxidative phosphorylation was severely compromised. The ensuing formation of pyruvate readily detoxified reactive oxygen species with the concomitant formation of acetate. This H2O2-induced metabolic reconfiguration not only helps generate the antioxidants necessary to thwart oxidative stress but also powers the formation of energy.

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Abbreviations

AL:

Aspartate lyase

AT:

Aspartate transaminase

AK:

Adenylate kinase

CFE:

Cell free extract

G6PDH:

Glucose 6-phosphate dehydrogenase

H2O2 :

Hydrogen peroxide

ICDH:

Isocitrate dehydrogenase

MDH:

Malate dehydrogenase

ME:

Malic enzyme

OP:

Oxidative phosphorylation

PEPC:

Phosphoenolpyruvate carboxylase

PEPS:

Phosphoenolpyruvate synthase

PPDK:

Pyruvate orthophosphate dikinase

ROS:

Reactive oxygen species

NADPH:

Reduced nicotinamide adenine dinucleotide phosphate

TCA:

Tricarboxylic acid

References

  1. Alhasawi, A., Auger, C., Appanna, V. P., Chahma, M., & Appanna, V. D. (2014). Zinc toxicity and ATP production in Pseudomonas fluorescens. Journal of Applied Microbilogy, 117, 65–73.

    CAS  Article  Google Scholar 

  2. Anderson, S., Appanna, V. D., Huang, J., & Viswanatha, T. (1992). A novel role for calcite in calcium homeostasis. FEBS Letters, 308, 94–96.

    CAS  Article  PubMed  Google Scholar 

  3. Appanna, V. D., Gazso, L. G., & Pierre, M. S. (1996). Multiple-metal tolerance in Pseudomonas fluorescens and its biotechnological significance. Journal of Biotechnology, 52, 75–80.

    CAS  Article  Google Scholar 

  4. Appanna, V. D., & Preston, C. M. (1987). Manganese elicits the synthesis of a novel exopolysaccharide in an artic Rhizobium. FEBS Letters, 215, 79–82.

    CAS  Article  Google Scholar 

  5. Arora, A., Sairam, R. K., & Srivastava, G. C. (2002). Oxidative stress and antioxidative system in plants. Current Science, 82, 1227–1238.

    CAS  Google Scholar 

  6. Auger, C., & Appanna, V. (2014). A novel ATP-generating machinery to counter nitrosative stress is mediated by substrate-level phosphorylation. Biochimica et Biophysica Acta, 1850, 43–50.

    Article  PubMed  Google Scholar 

  7. Auger, C., Appanna, V., Castonguay, Z., Han, S., & Appanna, V. D. (2012). A facile electrophoretic technique to monitor phosphoenolpyruvate-dependent kinases. Electrophoresis, 33, 1095–1101.

    CAS  Article  PubMed  Google Scholar 

  8. Auger, C., Lemire, J., Cecchini, D., Bignucolo, A., & Appanna, V. D. (2011). The metabolic reprogramming evoked by nitrosative stress triggers the anaerobic utilization of citrate in Pseudomonas fluorescens. PLoS One, 6, e28469.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. Aydin, S., Yargicoglu, P., Derin, N., Aliciguzel, Y., Abidin, İ., & Agar, A. (2005). The effect of chronic restraint stress and sulfite on visual evoked potentials (VEPs): Relation to lipid peroxidation. Food and Chemical Toxicology, 43, 1093–1101.

    CAS  Article  PubMed  Google Scholar 

  10. Beriault, R., Hamel, R., Chenier, D., Mailloux, R. J., Joly, H., & Appanna, V. D. (2007). The overexpression of NADPH-producing enzymes counters the oxidative stress evoked by gallium, an iron mimetic. BioMetals, 20, 165–176.

    CAS  Article  PubMed  Google Scholar 

  11. Bignucolo, A., Appanna, V. P., Thomas, S. C., Auger, C., Han, S., Omri, A., & Appanna, V. D. (2013). Hydrogen peroxide stress provokes a metabolic reprogramming in Pseudomonas fluorescens: Enhanced production of pyruvate. Journal of Biotechnology, 167, 309–315.

    CAS  Article  PubMed  Google Scholar 

  12. Bradford, M. M. (1976). A Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.

    CAS  Article  PubMed  Google Scholar 

  13. Bruno-Bárcena, J. M., Azcárate-Peril, M. A., & Hassan, H. M. (2010). Role of antioxidant enzymes in bacterial resistance to organic acids. Applied and Environment Microbiology, 76, 2747–2753.

    Article  Google Scholar 

  14. Chenier, D., Beriault, R., Mailloux, R., Baquie, M., Abramia, G., Lemire, J., & Appanna, V. D. (2008). Involvement of fumarase C and NADH oxidase in metabolic adaptation of Pseudomonas fluorescens cells invoked by aluminum and gallium toxicity. Applied and Environment Microbiology, 74, 3977–3984.

    CAS  Article  Google Scholar 

  15. Chubukov, V., Gerosa, L., Kochanowski, K., & Sauer, U. (2014). Coordination of microbial metabolism. Nature Reviews Microbiology, 12, 327–340.

    CAS  Article  PubMed  Google Scholar 

  16. Coustou, V., Besteiro, S., Biran, M., Diolez, P., Bouchaud, V., Voisin, P., et al. (2003). ATP generation in the Trypanosoma brucei procyclic form: Cytosolic substrate level is essential, but not oxidative phosphorylation. Journal of Biological Chemistry, 278, 49625–49635.

    CAS  Article  PubMed  Google Scholar 

  17. Esposito, E., Capasso, M., di Tomasso, N., Corona, C., Pellegrini, F., Uncini, A., & Sensi, S. L. (2007). Antioxidant strategies based on tomato-enriched food or pyruvate do not affect disease onset and survival in an animal model of amyotrophic lateral sclerosis. Brain Research, 1168, 90–96.

    CAS  Article  PubMed  Google Scholar 

  18. Filomeni, G., De Zio, D., & Cecconi, F. (2014). Oxidative stress and autophagy: The clash between damage and metabolic needs. Cell Death and Differentiation, 2014, 1–12.

    Google Scholar 

  19. Forrester, M. T., & Foster, M. W. (2012). Protection from nitrosative stress: A central role for microbial flavohemoglobin. Free Radical Biology and Medicine, 52, 1620–1633.

    CAS  Article  PubMed  Google Scholar 

  20. Friedberg, E. C. (2003). DNA damage and repair. Nature, 421, 436–440.

    Article  PubMed  Google Scholar 

  21. Ganesan, B., Seefeldt, K., & Weimer, B. C. (2004). Fatty acid production from amino acids and α-keto acids by Brevibacterium linens BL2. Applied and Environment Microbiology, 70, 6385–6393.

    CAS  Article  Google Scholar 

  22. Graf, E., & Penniston, J. T. (1980). Method for determination of hydrogen peroxide, with its application illustrated by glucose assay. Clinical Chemistry, 26, 658–660.

    CAS  PubMed  Google Scholar 

  23. Han, S., Auger, C., Appanna, V., Lemire, J., Castonguay, Z., & Appanna, V. D. (2012). A blue native polyacrylamide gel electrophoretic technology to probe the functional proteomics mediating nitrogen homeostasis in Pseudomonas fluorescens. Journal of Microbiol Methods, 90, 206–210.

    CAS  Article  Google Scholar 

  24. Han, G. D., Zhang, S., Mashall, D. J., Ke, C. H., & Dong, Y. W. (2013). Metabolic energy sensors (AMPK and SIRT1), protein carbonylation and cardiac failure as biomarkers of thermal stress in an intertidal limpet: Linking energetic allocation with environmental temperature during aerial emersion. The Journal of Experimental Biology, 216, 3273–3282.

    CAS  Article  PubMed  Google Scholar 

  25. Kim, J. Y., Lee, Y. A., Wittmann, C., & Park, J. B. (2013). Production of non-proteinogenic amino acids from α-keto acid precursors with recombinant Corynebacterium glutamicum. Biotechnology and Bioengineering, 110, 2846–2855.

    CAS  Article  PubMed  Google Scholar 

  26. Lemire, J., Auger, C., Mailloux, R., & Appanna, V. D. (2014). Mitochondrial lactate metabolism is involved in antioxidative defense in human astrocytoma cells. Journal of Neuroscience Research, 92, 464–475.

    CAS  Article  PubMed  Google Scholar 

  27. Lemire, J., Kumar, P., Mailloux, R., Cossar, K., & Appanna, V. D. (2008). Metabolic adaptation and oxaloacetate homeostasis in P. fluorescens exposed to aluminum toxicity. Journal of Basic Microbiology, 48, 252–259.

    CAS  Article  PubMed  Google Scholar 

  28. Lemire, J., Milandu, Y., Auger, C., Bignucolo, A., Appanna, V. P., & Appanna, V. D. (2010). Histidine is a source of the anti-oxidant a-ketoglutarate in Pseudomonas fluorescens challenged by oxidative stress. FEMS Microbiology Letters, 309, 170–177.

    CAS  PubMed  Google Scholar 

  29. Li, S. F., Liu, H. X., Zhang, Y. B., Yan, Y. C., & Li, Y. P. (2010). The protective effects of α-ketoacids against oxidative stress on rat spermatozoa in vitro. Asian Journal of Andrology, 12, 247–256.

    CAS  Article  PubMed  Google Scholar 

  30. Liu, H., Sun, Y., Ramos, K. R., Nisola, G. M., Valdehuesa, K. N., Lee, W. K., et al. (2013). Combination of Entner-Doudoroff pathway with MEP increases isoprene production in engineered Escherichia coli. PLoS One, 8, e83290.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Mailloux, R. J., Bériault, R., Lemire, J., Singh, R., Chénier, D. R., Hamel, R. D., & Appanna, V. D. (2007). The tricarboxylic acid cycle, an ancient metabolic network with a novel twist. PLoS One, 2, e690.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Mailloux, R. J., Darwich, R., Lemire, J., & Appanna, V. (2008). The monitoring of nucleotide diphosphate kinase activity by blue native polyacrylamide gel electrophoresis. Electrophoresis, 29, 1484–1489.

    CAS  Article  PubMed  Google Scholar 

  33. Mailloux, R. J., Singh, R., Brewer, G., Auger, C., Lemire, J., & Appanna, V. D. (2009). α-ketoglutarate dehydrogenase and glutamate dehydrogenase work in tandem to modulate the antioxidant α-ketoglutarate during oxidative stress in Pseudomonas fluorescens. Journal of Bacteriology, 191, 3804–3810.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. Masip, L., Veeravalli, K., & Georgiou, G. (2006). The many faces of glutathione in bacteria. Antioxidants and Redox Signaling, 8, 753–762.

    CAS  Article  PubMed  Google Scholar 

  35. Navari-Izzo, F., Quartacci, M. F., & Sgherri, C. (2002). Lipoic acid: A unique antioxidant in the detoxification of activated oxygen species. Plant Physiology and Biochemistry, 40, 463–470.

    CAS  Article  Google Scholar 

  36. Noctor, G. (2006). Metabolic signalling in defence and stress: The central roles of soluble redox couples. Plant, Cell and Environment, 29, 409–425.

    CAS  Article  PubMed  Google Scholar 

  37. Puntel, R. L., Roos, D. H., Grotto, D., Garcia, S. C., Nogueira, C. W., & Batista Teixeira Rocha, J. (2007). Antioxidant properties of krebs cycle intermediates against malonate pro-oxidant activity in vitro: A comparative study using the colorimetric method and HPLC analysis to determine malondialdehyde in rat brain homogenates. Life Sciences, 81, 51–62.

    CAS  Article  PubMed  Google Scholar 

  38. Ravasz, E., Somera, A. L., Mongru, D. A., Oltvai, Z. N., & Barabási, A. L. (2002). Hierarchical organization of modularity in metabolic networks. Science, 297, 1551–1555.

    CAS  Article  PubMed  Google Scholar 

  39. Sauer, U., & Eikmanns, B. J. (2005). The PEP–pyruvate–oxaloacetate node as the switch point for carbon flux distribution in bacteria. FEMS Microbiology Reviews, 29, 765–794.

    CAS  Article  PubMed  Google Scholar 

  40. Schägger, H., & von Jagow, G. (1991). Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Analytical Biochemistry, 199, 223–231.

    Article  PubMed  Google Scholar 

  41. Singh, R., Mailloux, R. J., Puiseux-Dao, S., & Appanna, V. D. (2007). Oxidative stress evokes a metabolic adaptation that favors increased NADPH synthesis and decreased NADH production in Pseudomonas fluorescens. Journal of Bacteriology, 189, 6665–6675.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. Singh, R. B., Middaugh, R., Hamel, J., Chenier, R., Appanna, D., & Kalyuzhnyi, V. D. S. (2005). Aluminum-tolerant Pseudomonas fluorescens: ROS toxicity and enhanced NADPH production. Extremophiles, 9, 367–373.

    CAS  Article  PubMed  Google Scholar 

  43. Smirnov, S. V., Sokolov, P. M., Kodera, T., Sugiyama, M., Hibi, M., Shimizu, S., & Ogawa, J. (2012). A novel family of bacterial dioxygenases that catalyse the hydroxylation of free l-amino acids. FEMS Microbiology Letters, 331, 97–104.

    CAS  Article  PubMed  Google Scholar 

  44. Spura, J., Reimer, L. C., Wieloch, P., Schreiber, K., Buchinger, S., & Schomburg, D. (2009). A method for enzyme quenching in microbial metabolome analysis successfully applied to gram-positive and gram-negative bacteria and yeast. Analytical Biochemistry, 394, 192–201.

    CAS  Article  PubMed  Google Scholar 

  45. Takao, M., Aburatani, H., Kobayashi, K., & Yasui, A. (1998). Mitochondrial targeting of human DNA glycosylases for repair of oxidative DNA damage. Nucleic Acids Research, 26, 2917–2922.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. Zeller, T., & Klug, G. (2004). Detoxification of hydrogen peroxide and expression of catalase genes in Rhodobacter. Microbiology, 150, 3451–3462.

    CAS  Article  PubMed  Google Scholar 

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Acknowledgment

This study was funded by Laurentian University and the Northern Ontario Heritage Fund. Azhar Alhasawi is a recipient of funding from the Ministry of Higher Education of Saudi Arabia.

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Correspondence to Vasu D. Appanna.

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Alhasawi, A., Leblanc, M., Appanna, N.D. et al. Aspartate metabolism and pyruvate homeostasis triggered by oxidative stress in Pseudomonas fluorescens: a functional metabolomic study. Metabolomics 11, 1792–1801 (2015). https://doi.org/10.1007/s11306-015-0841-4

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Keywords

  • Pyruvate
  • Antioxidant
  • Metabolic networks
  • Pyruvate synthase
  • Energy