Antonie van Leeuwenhoek

, Volume 106, Issue 3, pp 431–438 | Cite as

Fumarate metabolism and ATP production in Pseudomonas fluorescens exposed to nitrosative stress

  • Varun P. Appanna
  • Christopher Auger
  • Sean C. Thomas
  • Abdelwahab OmriEmail author
Original Paper


Although nitrosative stress is known to severely impede the ability of living systems to generate adenosine triphosphate (ATP) via oxidative phosphorylation, there is limited information on how microorganisms fulfill their energy needs in order to survive reactive nitrogen species (RNS). In this study we demonstrate an elaborate strategy involving substrate-level phosphorylation that enables the soil microbe Pseudomonas fluorescens to synthesize ATP in a defined medium with fumarate as the sole carbon source. The enhanced activities of such enzymes as phosphoenolpyruvate carboxylase and pyruvate phosphate dikinase coupled with the increased activities of phospho-transfer enzymes like adenylate kinase and nucleoside diphophate kinase provide an effective strategy to produce high energy nucleosides in an O2-independent manner. The alternate ATP producing machinery is fuelled by the precursors derived from fumarate with the aid of fumarase C and fumarate reductase. This metabolic reconfiguration is key to the survival of P. fluorescens and reveals potential targets against RNS-resistant organisms.


Energy production Fumarate Reactive nitrogen species (RNS) Pseudomonas fluorescens 


  1. Adam RD (2001) Biology of Giardia lamblia. Clin Microbiol Rev 14:447–475PubMedCentralPubMedCrossRefGoogle Scholar
  2. al-Aoukaty A, Appanna VD, Falter H (1992) Gallium toxicity and adaptation in Pseudomonas fluorescnes. FEMS Microbiol Lett 71:265–272PubMedCrossRefGoogle Scholar
  3. Auger C, Lemire J, Cecchini D, Bignucolo A, Appanna VD (2011) The metabolic reprogramming evoked by nitrosative stress triggers the anaerobic utilization of citrate in Pseudomonas fluorescens. PLoS ONE 6:e28469PubMedCentralPubMedCrossRefGoogle Scholar
  4. Auger C, Appanna V, Castonguay Z, Han S, Appanna VD (2012) A facile electrophoretic technique to monitor phosphoenolpyruvate-dependent kinases. Electrophoresis 33:1095–1101PubMedCrossRefGoogle Scholar
  5. Auger C, Han S, Appanna VP, Thomas SC, Ulibarri G, Appanna VD (2013) Metabolic reengineering invoked by microbial systems to decontaminate aluminum: implications in bioremediation technologies. Biotechnol Adv 31:266–273PubMedCrossRefGoogle Scholar
  6. Bignucolo A, Appanna VP, Thomas SC, Auger C, Han S, Omri A, Appanna VD (2013) Hydrogen peroxide stress a metabolic reprogramming in Pseudomonas fluorescens: enhanced production of puyruvate. J Biotechnol 167:309–315PubMedCrossRefGoogle Scholar
  7. Bradford MB (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  8. Charoo NA, Shamsher AAA, Lian LY, Abrahamsson B, Cristofoletti R, Groot DW, Kopp S, Langguth P, Polli J, Shah VP, Dressman J (2014) Biowaiver monograph for immediate-release solid oral dosage forms: bisoprolol fumarate. J Pharma Sci 103:378–391CrossRefGoogle Scholar
  9. Chénier D, Bériault R, Mailloux R, Baquie M, Abramia G, Lemire J, Appanna VD (2008) Metabolic adaptation in Pseudomonas fluorescens evoked by aluminum and gallium toxicity: involvement of fumarase C and NADH oxidase. Appl Environ Microbiol 74:3977–3984PubMedCentralPubMedCrossRefGoogle Scholar
  10. Couston V, Besterio S, Biran M, Diolez P, Bouchaud V, Voisin P, Michels PAM, Canioni P, Baltz T, Bringaud F (2003) ATP generation in the Trypanosoma brucei procyclic form cytosolic substrate level phosphorylation is essential, but not oxidative phosphorylation. J Biol Chem 278:49625–49635CrossRefGoogle Scholar
  11. Hall JW, Ji Y (2013) Sensing and adapting to anaerobic conditions by Staphylococcus aureus. Adv Appl Microbiol 84:1–25PubMedCrossRefGoogle Scholar
  12. Han S, Auger C, Castonguay Z, Appanna VP, Thomas SC, Appanna VD (2012) The unravelling of metabolic dysfunctions linked to metal-associated diseases by blue native polyacrylamide gel electrophoresis. Anal Bioanal Chem 405(6):1821-1831 Google Scholar
  13. Han S, Auger C, Thomas SC, Beites CL, Appanna VD (2013) Mitochondrial biogenesis and energy production in differentiating stem cells: a functional metabolic study. Cell Reprogram 16:84–90PubMedCrossRefGoogle Scholar
  14. Hunt KA, Flynn JM, Naranjo B, Shikhare ID, Gralnick JA (2010) Substrate-level phosphorylation is the primary source of energy conservation during anaerobic respiration of Shewanella oneidensis strain MR-1. J Bacteriol 192:3345–3351PubMedCentralPubMedCrossRefGoogle Scholar
  15. Ingram-Smith C, Martin SR, Smith KS (2006) Acetate kinase: not just a bacterial enzyme. Trends Microbiol 14:249–253PubMedCrossRefGoogle Scholar
  16. Kim D, Yu BJ, Kim JA, Lee Y, Choi S, Kang S (2013) The acetylproteome of Gram-positive model bacterium Bacillus subtilis. Proteomics 13:1726–1736PubMedCrossRefGoogle Scholar
  17. Lemire J, Auger C, Bignucolo A, Appanna VP, Appanna VD (2012) Metabolic strategies deployed by Pseudomonas fluorescens to combat metal pollutants: biotechnological prospects in current research, technology and education topics in applied microbiology and microbial biotechnology. In: Mendez-vilas A (ed). Formalex Publisher, pp 177–187Google Scholar
  18. Li K, Pidatala RV, Shaik R, Datta R, Ramakrishna W (2014) Integrated metabolomic and proteomic approaches dissect the effect of metal-resistant bacteria on maize biomass and copper uptake. Environ Sci Technol 48:1184–1193PubMedCrossRefGoogle Scholar
  19. Lushchak OV, Piroddi M, Galli F, Lushchak VI (2014) Aconitase post-translational modification as a key in linkage between Krebs cycle, iron homeostasis, redox signaling, and metabolism of reactive oxygen species. Redox Rep 19:8–15PubMedCrossRefGoogle Scholar
  20. Ma Y, Guo C, Li H, Peng X (2013) Low abundance of respiratory nitrate reductase is essential for Escherichia coli in resistance to aminoglycoside and cephalosporin. J Proteom 87:78–88CrossRefGoogle Scholar
  21. Mailloux RJ, Darwich R, Lemire J, Appanna V (2008) The monitoring of nucleotide diphosphate kinase activity by blue native polyacrylamide gel electrophoresis. Electrophor 29:1484–1489Google Scholar
  22. Mailloux RJ, Lemire J, Appanna VD (2011) Metabolic networks to combat oxidative stress in Pseuodomonas fluorescens. Antonie Van Leeuwenhoek 99:433–442PubMedCrossRefGoogle Scholar
  23. Miranda KM, Espey MG, Wink DA (2001) A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide 5:62–71PubMedCrossRefGoogle Scholar
  24. Poole RK (2005) Nitric oxide and nitrosative stress tolerance in bacteria. Biochem Soc Trans 33:176–180PubMedCrossRefGoogle Scholar
  25. Quijano C, Alvarez B, Gatti RM, Augusto O, Radi R (1997) Pathways of peroxynitrite oxidation of thiol groups. Biochem J 322:167–173PubMedCentralPubMedGoogle Scholar
  26. Schagger H, von Jagow G (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199:223–231PubMedCrossRefGoogle Scholar
  27. Singh R, Chenier D, Beriault R, Mailloux R, Hamel RD, Appanna VD (2005) Blue native polyacrylamide gel electrophoresis and the monitoring of malate- and oxaloacetate-producing enzymes. J Biochem Biophys Methods 64:189–199PubMedCrossRefGoogle Scholar
  28. Singh R, Lemire J, Mailloux RJ, Chénier D, Hamel R, Appanna VD (2009) An ATP and oxalate generating variant tricarboxylic acid cycle counters aluminum toxicity in Pseudomonas fluorescens. PloS One 4:e7344 Google Scholar
  29. Watanabe S, Zimmerman M, Goodwin MB, Sauer U, Barry CE, Boshoff HI (2011) Fumarate reductase activity maintains an energized membrane in anerobic Mycobacterium tuberculosis. PLoS ONE 7:1–15Google Scholar
  30. Zielonka J, Zielonka M, Sikora A, Adamus J, Joseph J, Hardy M, Ouari O, Dranka BP, Kalyanarama B (2012) Global profiling of reactive oxygen and nitrogen species in biological systems: high-throughput real-time analyses. J Biol Chem 287:2984–2995PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2014

Authors and Affiliations

  • Varun P. Appanna
    • 1
  • Christopher Auger
    • 1
  • Sean C. Thomas
    • 1
  • Abdelwahab Omri
    • 1
    Email author
  1. 1.Department of BiologyLaurentian UniversitySudburyCanada

Personalised recommendations