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A novel metabolic network leads to enhanced citrate biogenesis in Pseudomonas fluorescens exposed to aluminum toxicity

Extremophiles volume 12, pages 451–459 (2008)Cite this article

Abstract

Aluminum (Al), an environmental toxin, is known to have a negative impact on various biological systems. However, some microbes have devised intricate mechanisms to combat the toxic influence of this trivalent metal. In this study, Pseudomonas fluorescens grown in malate invoked a unique metabolic shift to promote the synthesis of citrate, a metabolite involved in the sequestration of Al. Electrophoretic and spectrophotometric assays revealed several malate-metabolizing enzymes including malate dehydrogenase (MDH) and malic enzyme (ME) displayed increases in activity and expression in the Al-treated cells. Whereas pyruvate dehydrogenase (PDH) also showed increased activity and expression in the Al-stressed cultures, phosphoenolpyruvate carboxykinase (PEPCK) displayed a marked diminution in the Al-treated cells. The upregulation of citrate synthase (CS) coupled with the diminished activities of aconitase (ACN) and NAD-isocitrate dehydrogenase (NAD-ICDH) appeared to be instrumental in the accumulation of citrate. HPLC experiments revealed high levels of citrate in the Al-stressed cultures. Thus, an Al-enriched environment provoked a metabolic shift in P. fluorescens dedicated to the conversion of malate to citrate.

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Abbreviations

ME:

Malic enzyme

MDH:

Malate dehydrogenase

PEPCK:

Phosphoenol pyruvate carboxykinase

ACN:

Aconitase

PDH:

Pyruvate dehydrogenase

CS:

Citrate synthase

NAD-ICDH:

NAD-dependent isocitrate dehydrogenase

Al:

Aluminum

References

  • Aich S, Imabayashi F, Delbaere LT (2003) Expression, purification, and characterization of a bacterial GTP-dependent PEP carboxykinase. Protein Expr Purif 31(2):298–304

    PubMed  Article  CAS  Google Scholar 

  • Alvarez G, Ramos M, Ruiz F, Satrustegui J, Bogonez E (2003) Pyruvate protection against beta-amyloid-induced neuronal death: role of mitochondrial redox state. J Neurosci Res 73(2):260–269

    PubMed  Article  CAS  Google Scholar 

  • Anderson S, Appanna VD, Huang J, Viswanatha T (1992) A novel role for calcite in calcium homeostasis. FEBS Lett 308(1):94–96

    PubMed  Article  CAS  Google Scholar 

  • Anoop VM, Basu U, McCammon MT, McAlister-Henn L, Taylor GJ (2003) Modulation of citrate metabolism alters aluminum tolerance in yeast and transgenic canola overexpressing a mitochondrial citrate synthase. Plant Physiol 132(4):2205–2217

    PubMed  Article  CAS  Google Scholar 

  • Appanna VD, Hamel RD, Levasseur R (2003) The metabolism of aluminum citrate and biosynthesis of oxalic acid in Pseudomonas fluorescens. Curr Microbiol 47(1):32–39

    PubMed  Article  CAS  Google Scholar 

  • Becaria A, Campbell A, Bondy SC (2002) Aluminum as a toxicant. Toxicol Ind Health 18(7):309–320

    PubMed  CAS  Article  Google Scholar 

  • Beriault R, Chenier D, Singh R, Middaugh J, Mailloux R, Appanna V (2005) Detection and purification of glucose 6-phosphate dehydrogenase, malic enzyme, and NADP-dependent isocitrate dehydrogenase by blue native polyacrylamide gel electrophoresis. Electrophoresis 26(15):2892–2897

    PubMed  Article  CAS  Google Scholar 

  • Cabiscol E, Piulats E, Echave P, Herrero E, Ros J (2000) Oxidative stress promotes specific protein damage in Saccharomyces cerevisiae. J Biol Chem 275(35):27393–27398

    PubMed  CAS  Google Scholar 

  • Crichton RR, Wilmet S, Legssyer R, Ward RJ (2002) Molecular and cellular mechanisms of iron homeostasis and toxicity in mammalian cells. J Inorg Biochem 91(1):9–18

    PubMed  Article  CAS  Google Scholar 

  • Farrell SO, Fiol CJ, Reddy JK, Bieber LL (1984) Properties of purified carnitine acyltransferases of mouse liver peroxisomes. J Biol Chem 259(21):13089–13095

    PubMed  CAS  Google Scholar 

  • Fedotcheva NI, Sokolov AP, Kondrashova MN (2006) Nonezymatic formation of succinate in mitochondria under oxidative stress. Free Radic Biol Med 41(1):56–64

    PubMed  Article  CAS  Google Scholar 

  • Hansford RG (1976) Studies on the effects of coenzyme A-SH: acetyl coenzyme A, nicotinamide adenine dinucleotide: reduced nicotinamide adenine dinucleotide, and adenosine diphosphate: adenosine triphosphate ratios on the interconversion of active and inactive pyruvate dehydrogenase in isolated rat heart mitochondria. J Biol Chem 251(18):5483–5489

    PubMed  CAS  Google Scholar 

  • Imlay JA (2002) How oxygen damages microbes: oxygen tolerance and obligate anaerobiosis. Adv Microb Physiol 46:111–153

    PubMed  Article  CAS  Google Scholar 

  • Kawano T, Kadono T, Furuichi T, Muto S, Lapeyrie F (2003) Aluminum-induced distortion in calcium signaling involving oxidative bursts and channel regulation in tobacco BY-2 cells. Biochem Biophys Res Commun 308(1):35–42

    PubMed  Article  CAS  Google Scholar 

  • Komuniecki R, Rhee R, Bhat D, Duran E, Sidawy E, Song H (1992) The pyruvate dehydrogenase complex from the parasitic nematode Ascaris suum: novel subunit composition and domain structure of the dihydrolipoyl transacetylase component. Arch Biochem Biophys 296(1):115–121

    PubMed  Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685

    PubMed  Article  CAS  Google Scholar 

  • Lemire J, Mailloux R, Appanna VD (2007) Zinc toxicity alters mitochondrial metabolism and leads to decreased ATP production in hepatocytes. J Appl Toxicol (in press)

  • Ligaba A, Shen H, Shibata K, Yamamoto Y, Tanakamaru S, Matsumoto H (2004) The role of phosphorus in aluminium-induced citrate and malate exudation from rape (Brassica napus). Physiol Plant 120(4):575–584

    PubMed  Article  CAS  Google Scholar 

  • MacDiarmid CW, Gardner RC (1998) Overexpression of the Saccharomyces cerevisiae magnesium transport system confers resistance to aluminum ion. J Biol Chem 273(3):1727–1732

    PubMed  Article  CAS  Google Scholar 

  • Mailloux R, Lemire J, Appanna V (2007a) Aluminum-induced mitochondrial dysfunction leads to lipid accumulation in human hepatocytes: a link to obesity. Cell Physiol Biochem 20(5):627–638

    PubMed  Article  CAS  Google Scholar 

  • Mailloux RJ, Beriault R, Lemire J, Singh R, Chenier DR, Hamel RD, Appanna VD (2007b) The tricarboxylic acid cycle, an ancient metabolic network with a novel twist. PLoS ONE 2(1):e690

    PubMed  Article  CAS  Google Scholar 

  • Mailloux RJ, Hamel R, Appanna VD (2006a) Aluminum toxicity elicits a dysfunctional TCA cycle and succinate accumulation in hepatocytes. J Biochem Mol Toxicol 20(4):198–208

    PubMed  Article  CAS  Google Scholar 

  • Mailloux RJ, Singh R, Appanna VD (2006b) In-gel activity staining of oxidized nicotinamide adenine dinucleotide kinase by blue native polyacrylamide gel electrophoresis. Anal Biochem 359(2):210–215

    PubMed  Article  CAS  Google Scholar 

  • Marino D, Gonzalez EM, Frendo P, Puppo A, Arrese-Igor C (2007) NADPH recycling systems in oxidative stressed pea nodules: a key role for the NADP+ -dependent isocitrate dehydrogenase. Planta 225(2):413–421

    PubMed  Article  CAS  Google Scholar 

  • Middaugh J, Hamel R, Jean-Baptiste G, Beriault R, Chenier D, Appanna VD (2005) Aluminum triggers decreased aconitase activity via Fe-S cluster disruption and the overexpression of isocitrate dehydrogenase and isocitrate lyase: a metabolic network mediating cellular survival. J Biol Chem 280(5):3159–3165

    PubMed  Article  CAS  Google Scholar 

  • Mottram JC, Coombs GH (1985) Purification of particulate malate dehydrogenase and phosphoenolpyruvate carboxykinase from Leishmania mexicana mexicana. Biochim Biophys Acta 827(3):310–319

    PubMed  CAS  Google Scholar 

  • Mukhopadhyay B, Concar EM, Wolfe RS (2001) A GTP-dependent vertebrate-type phosphoenolpyruvate carboxykinase from Mycobacterium smegmatis. J Biol Chem 276(19):16137–16145

    PubMed  Article  CAS  Google Scholar 

  • Nayak P (2002) Aluminum: impacts and disease. Environ Res 89(2):101–115

    PubMed  Article  CAS  Google Scholar 

  • Oshiro S, Kawahara M, Mika S, Muramoto K, Kobayashi K, Ishige R, Nozawa K, Hori M, Yung C, Kitajima S, Kuroda Y (1998) Aluminum taken up by transferrin-independent iron uptake affects the iron metabolism in rat cortical cells. J Biochem (Tokyo) 123(1):42–46

    CAS  Google Scholar 

  • Pina RG, Cervantes C (1996) Microbial interactions with aluminium. Biometals 9(3):311–316

    PubMed  Article  CAS  Google Scholar 

  • Romanov V, Merski MT, Hausinger RP (1999) Assays for allantoinase. Anal Biochem 268(1):49–53

    PubMed  Article  CAS  Google Scholar 

  • Sapan CV, Lundblad RL, Price NC (1999) Colorimetric protein assay techniques. Biotechnol Appl Biochem 29(Pt 2):99–108

    PubMed  CAS  Google Scholar 

  • Singh R, Beriault R, Middaugh J, Hamel R, Chenier D, Appanna VD, Kalyuzhnyi S (2005a) Aluminum-tolerant Pseudomonas fluorescens: ROS toxicity and enhanced NADPH production. Extremophiles 9(5):367–373

    PubMed  Article  CAS  Google Scholar 

  • Singh R, Chenier D, Beriault R, Mailloux R, Hamel RD, Appanna VD (2005b) Blue native polyacrylamide gel electrophoresis and the monitoring of malate- and oxaloacetate-producing enzymes. J Biochem Biophys Methods 64(3):189–199

    PubMed  Article  CAS  Google Scholar 

  • Singh R, Mailloux RJ, Puiseux-Dao S, Appanna VD (2007) Oxidative stress evokes a metabolic adaptation that favors increased NADPH synthesis and decreased NADH production in Pseudomonas fluorescens. J Bacteriol 189(18):6665–6675

    PubMed  Article  CAS  Google Scholar 

  • Valderrama R, Corpas FJ, Carreras A, Gomez-Rodriguez MV, Chaki M, Pedrajas JR, Fernandez-Ocana A, Del Rio LA, Barroso JB (2006) The dehydrogenase-mediated recycling of NADPH is a key antioxidant system against salt-induced oxidative stress in olive plants. Plant Cell Environ 29(7):1449–1459

    PubMed  Article  CAS  Google Scholar 

  • Yoshino M, Ito M, Haneda M, Tsubouchi R, Murakami K (1999) Prooxidant action of aluminum ion–stimulation of iron-mediated lipid peroxidation by aluminum. Biometals 12(3):237–240

    PubMed  Article  CAS  Google Scholar 

  • Zhao WN, McAlister-Henn L (1996) Assembly and function of a cytosolic form of NADH-specific isocitrate dehydrogenase in yeast. J Biol Chem 271(17):10347–10352

    PubMed  Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was funded by Ontario Center of Excellence and the Northern Ontario Heritage Fund.

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Affiliations

  1. Department of Chemistry and Biochemistry, Laurentian University, Sudbury, ON, Canada, P3E 2C6

    Ryan J. Mailloux, Joseph Lemire & Vasu Appanna

  2. Department of Chemical Enzymology, Moscow State University, 119992, Moscow, Russia

    Sergey Kalyuzhnyi

Authors
  1. Ryan J. Mailloux
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  2. Joseph Lemire
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  3. Sergey Kalyuzhnyi
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  4. Vasu Appanna
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Corresponding author

Correspondence to Vasu Appanna.

Additional information

Communicated by G. Antranikian.

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Mailloux, R.J., Lemire, J., Kalyuzhnyi, S. et al. A novel metabolic network leads to enhanced citrate biogenesis in Pseudomonas fluorescens exposed to aluminum toxicity. Extremophiles 12, 451–459 (2008). https://doi.org/10.1007/s00792-008-0150-1

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  • DOI: https://doi.org/10.1007/s00792-008-0150-1

Keywords

  • Aluminum
  • Malate
  • Citrate
  • Metabolic adaptation
  • Malic enzyme
  • Citrate synthase