, Volume 21, Issue 5, pp 1314–1324 | Cite as

Unraveling the concentration-dependent metabolic response of Pseudomonas sp. HF-1 to nicotine stress by 1H NMR-based metabolomics

  • Yangfang Ye
  • Xin Wang
  • Limin Zhang
  • Zhenmei Lu
  • Xiaojun Yan


Nicotine can cause oxidative damage to organisms; however, some bacteria, for example Pseudomonas sp. HF-1, are resistant to such oxidative stress. In the present study, we analyzed the concentration-dependent metabolic response of Pseudomonas sp. HF-1 to nicotine stress using 1H NMR spectroscopy coupled with multivariate data analysis. We found that the dominant metabolites in Pseudomonas sp. HF-1 were eight aliphatic organic acids, six amino acids, three sugars and 11 nucleotides. After 18 h of cultivation, 1 g/L nicotine caused significant elevation of sugar (glucose, trehalose and maltose), succinate and nucleic acid metabolites (cytidine, 5′-CMP, guanine 2′,3′-cyclic phosphate and adenosine 2′,3′-cyclic phosphate), but decrease of glutamate, putrescine, pyrimidine, 2-propanol, diethyl ether and acetamide levels. Similar metabolomic changes were induced by 2 g/L nicotine, except that no significant change in trehalose, 5′-UMP levels and diethyl ether were found. However, 3 g/L nicotine led to a significant elevation in the two sugars (trehalose and maltose) levels and decrease in the levels of glutamate, putrescine, pyrimidine and 2-propanol. Our findings indicated that nicotine resulted in the enhanced nucleotide biosynthesis, decreased glucose catabolism, elevated succinate accumulation, severe disturbance in osmoregulation and complex antioxidant strategy. And a further increase of nicotine level was a critical threshold value that triggered the change of metabolic flow in Pseudomonas sp. HF-1. These findings revealed the comprehensive insights into the metabolic response of nicotine-degrading bacteria to nicotine-induced oxidative toxicity.


Nicotine Oxidative stress Metabolomics NMR spectroscopy 



We thank supports from National Natural Science Foundation of China (No. 31100032, 31170115), Zhejiang Provincial Natural Science Foundation of China (No. Y3090046), University National Oceanographic Public Welfare Project (201205029), K. C. Wong Magna Fund in Ningbo and Academic Discipline Project of Ningbo University (xkl11089). We also acknowledge that all the NMR detection was performed at the Analysis and Test Laboratory, Wenzhou Medical College, in the form of paid service.

Supplementary material

10646_2012_885_MOESM1_ESM.doc (419 kb)
Supplementary material 1 (DOC 419 kb)


  1. Allen J, Davey HM, Broadhurst D, Heald JK, Rowland JJ, Oliver SG, Kell DB (2003) High-throughput classification of yeast mutants for functional genomics using metabolic footprinting. Nat Biotechnol 21(6):692–696CrossRefGoogle Scholar
  2. Al-Naama M, Ewaze JO, Green BJ, Scott JA (2009) Trehalose accumulation in Baudoinia compniacensis following abiotic stress. Int Biodeterior Biodegrad 63(6):765–768CrossRefGoogle Scholar
  3. Arguelles JC (2000) Physiological role of trehalose in bacteria and yeasts, a comparative analysis. Arch Microbiol 174(4):217–224CrossRefGoogle Scholar
  4. Benaroudj N, Lee NH, Goldberg AL (2001) Trehalose accumulation during cellular stress protects cells and cellular proteins from damage by oxygen radicals. J Biol Chem 276(26):24261–24267CrossRefGoogle Scholar
  5. Benowitz NL, Hall SM, Herning RI, Jacob P, Jones RT, Osman AL (1983) Smokers of low-yield cigarettes do not consume less nicotine. N Engl J Med 309:139–142CrossRefGoogle Scholar
  6. Boroujerdi AFB, Vizcaino MI, Meyers A, Pollock EC, Huynh SL, Schock TB, Morris PJ, Bearden DW (2009) NMR-based microbial metabolomics and the temperature-dependent coral pathogen Vibrio coralliilyticus. Environ Sci Technol 43(20):7658–7664CrossRefGoogle Scholar
  7. Brown SAE, McKelvie JR, Simpson AJ, Simpson MJ (2010) 1H NMR metabolomics of earthworm exposure to sub-lethal concentrations of phenanthrene in soil. Environ Pollut 158(6):2117–2123CrossRefGoogle Scholar
  8. Bundy JG, Willey TL, Castell RS, Ellar DJ, Bindle KM (2005) Discrimination of pathogenic clinical isolates and laboratory strains of Bacillus cereus by NMR-based metabolomic profiling. FEMS Microbiol Lett 242(1):127–136CrossRefGoogle Scholar
  9. Campain JA (2004) Nicotine, potentially a multifunctional carcinogen. Toxic Sci 79(1):1–3CrossRefGoogle Scholar
  10. Cánovas D, Fletcher SA, Hayashi M, Csonka LN (2001) Role of trehalose in growth at high temperature of Salmonella enterica serovar Typhimurium. J Bacteriol 183(11):3365–3371CrossRefGoogle Scholar
  11. Chen FF, Zhang JT, Song XS, Yang J, Li HP, Tang HR, Liao YC (2011) Combined metabonomic and quantitative real-time PCR analyses reveal systems metabolic changes of Fusarium graminearum induced by Tri5 gene deletion. J Proteome Res 10(5):2273–2285CrossRefGoogle Scholar
  12. Civilini M, Domenis C, Sebastianutto N, de Bertoldi M (1997) Nicotine decontamination of tobacco agro-industrial waste and its degradation by micro-organisms. Waste Manag Res 15(4):349–358Google Scholar
  13. Cloarec O, Dumas ME, Trygg J, Craig A, Barton RH, Lindon JC, Nicholson JK, Holmes E (2005) Evaluation of the orthogonal projection on latent structure model limitations caused by chemical shift variability and improved visualization of biomarker changes in 1H NMR spectroscopic metabonomic studies. Anal Chem 77(2):517–526CrossRefGoogle Scholar
  14. Coen M, Homes E, Lindon JC, Nicholson JK (2008) NMR-based metabolic profiling and metabonomic approaches to problems in molecular toxicology. Chem Res Toxicol 21(1):9–27CrossRefGoogle Scholar
  15. Dai H, Xiao CN, Liu HB, Tang HR (2010) Combined NMR and LC-MS analysis reveals the metabonomic changes in Salvia miltiorrhiza Bunge induced by water depletion. J Proteome Res 9(3):1460–1475CrossRefGoogle Scholar
  16. Ding LN, Hao FH, Shi ZM, Wang YL, Zhang HX, Tang HR, Dai JY (2009) Systems biological responses to chronic perfluorododecanoic acid exposure by integrated metabonomic and transcriptomic studies. J Proteome Res 8(6):2882–2891CrossRefGoogle Scholar
  17. Duman F, Aksoy A, Aydin Z, Temizgul R (2010) Effects of exogenous glycinebetaine and trehalose on cadmium accumulation and biological responses of an aquatic plant (Lemna gibba L). Water Air Soil Pollut 217(1–4):545–556Google Scholar
  18. Fan TW-M (1996) Metabolite profiling by one- and two-dimensional NMR analysis of complex mixtures. Prog Nucl Magn Reson Spectrosc 28:161–219Google Scholar
  19. Fan TWM, Lane AN (2008) Structure-based profiling of metabolites and isotopomers by NMR. Prog Nucl Magn Reson Spectrosc 52:69–117CrossRefGoogle Scholar
  20. Fang MA, Frost PJ, Lida-Klein A, Hahn TJ (1991) Effects of nicotine on cellular function in UMR 106–01 osteoblast-like cells. Bone 12(4):283–286CrossRefGoogle Scholar
  21. Ganas P, Sachelaru P, Mihasan M, Igloi GL, Brandsch R (2008) Two closely related pathways of nicotine catabolism in Arthrobacter nicotinovorans and Nocardioides sp. strain JS614. Arch Microbiol 189(5):511–517CrossRefGoogle Scholar
  22. Gasch AP, Spellman PT, Kao CM, Carmel-Harel O, Eisen MB, Storz G, Botstein D, Brown PO (2000) Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell 11(12):4241–4257Google Scholar
  23. Gavaghan CL, Li JV, Hadfield ST, Hole S, Nicholson JK, Wilson ID, Howe PWA, Stanley PD, Holmes E (2010) Application of NMR-based metabolomics to the investigation of salt stress in maize (Zea mays). Phytochem Anal 22(3):214–224CrossRefGoogle Scholar
  24. Gjersing EL, Herberg JL, Horn J, Schaldach CM, Maxwell RS (2007) NMR metabolomics of planktonic and biofilm modes of growth in Pseudomonas aeruginosa. Anal Chem 79(21):8037–8045CrossRefGoogle Scholar
  25. Gunasekera TS, Csonka LN, Paliy O (2008) Genome-wide transcriptional responses of Escherichia coli K-12 to continuous osmotic and heat stresses. J Bacteriol 190(10):3712–3720CrossRefGoogle Scholar
  26. Gutierrez-Ríos RM, Freyre-Gonzalez JA, Resendis O, Collado-Vides J, Saier M, Gosset G (2007) Identification of regulatory network topological units coordinating the genome-wide transcriptional response to glucose in Escherichia coli. BMC Microbiol 7:53–71CrossRefGoogle Scholar
  27. Halouska S, Chacon O, Fenton RJ, Zinniel DK, Barletta RG, Powers R (2007) Use of NMR metabolomics to analyze the targets of D-cycloserine in Mycobacteria: role of d-alanine racemase. J Proteome Res 6(12):4608–4614CrossRefGoogle Scholar
  28. Himmelreich U, Somorjai RL, Dolenko B, Lee OC, Daniel HM, Murray R, Mountford CM, Sorrell TC (2003) Rapid identification of Candida species by using nuclear magnetic resonance spectroscopy and a statistical classification strategy. Appl Environ Microbiol 69(8):4566–4574CrossRefGoogle Scholar
  29. Holmes E, Loo RL, Stamler J, Bictash M, Yap IKS, Chan Q, Ebbels T, De Iorio M, Brown IJ, Veselkov KA, Daviglus ML, Kesteloot H, Ueshima H, Zhao L, Nicholson JK, Elliott P (2008) Human metabolic phenotype diversity and its association with diet and blood pressure. Nature 453(7193):396–400CrossRefGoogle Scholar
  30. Jozefczuk S, Klie S, Catchpole G, Szymanshi J, Cuadros-Inostroza A, Steinhauser D, Selbig J, Willmitzer L (2010) Metabolomic and transcriptomic stress response of Escherichia coli. Mol Syst Biol 6:364–380CrossRefGoogle Scholar
  31. Kaplan F, Guy CL (2004) β-amylase induction and the protective role of maltose during temperature shock. Plant Physol 135(3):1674–1684CrossRefGoogle Scholar
  32. Konno S, Oronsky BT, Semproni AR, Wu JM (1991) The effect of nicotine on cell proliferation and synthesis of secreted proteins in BALB/C3T3 cells. Biochem Int 25(1):7–17Google Scholar
  33. Lenz EM, Weeks JM, Lindon JC, Osborn D, Nicholson JK (2005) Qualitative high field 1H-NMR spectroscopy for the characterization of endogenous metabolites in earthworms with biochemical biomarker potential. Metabolomics 1(2):123–136CrossRefGoogle Scholar
  34. Li HJ, Li XM, Duan YQ, Zhang K-Q, Yang JK (2010) Biotransformation of nicotine by microorganism, the case of Pseudomonas spp. Appl Microbiol Biotechnol 86(1):11–17CrossRefGoogle Scholar
  35. Lin CY, Viant MR, Tjeerdema RS (2006) Metabolomics, methodologies and applications in the environmental sciences. J Pestic Sci 31(3):245–251CrossRefGoogle Scholar
  36. Lindon JC, Holmes E, Nicholson JK (2004) Toxicological applications of magnetic resonance. Prog Nucl Magn Reson Spectrosc 45(1–2):109–143CrossRefGoogle Scholar
  37. Liu XL, Zhang LB, You LP, Yu JB, Zhao JM, Li LZ, Wang Q, Li F, Li CH, Liu DY, Wu HF (2011) Differential toxicological effects induced by mercury in gills from three pedigrees of Manila clam Ruditapes philippinarum by NMR-based metabolomics. Ecotoxicology 20(1):177–186CrossRefGoogle Scholar
  38. Malmendal A, Overgaard J, Bundy JG, Sørensen JG, Nielsen NC, Loeschche V, Holmstrup M (2006) Metabolomic profiling of heat stress: hardening and recovery of homeostasis in Drosophila. Am J Physiol Regul Integr Comp Physiol 291(1):R205–R212CrossRefGoogle Scholar
  39. Mandal AK, Samaddar S, Banerjee R, Lahiri S, Bhattacharyya A, Roy S (2003) Glutamate counteracts the denaturing effect of urea through its effect on the denatured state. J Biol Chem 278(38):36077–36084CrossRefGoogle Scholar
  40. Marles-Wright J, Lewis RJ (2007) Stress responses of bacteria. Curr Opin Struct Biol 17(6):755–760CrossRefGoogle Scholar
  41. McKelvie JR, Yuk J, Xu YP, Simpson AJ, Simpson MJ (2009) 1H NMR and GC/MS metabolomics of earthworm responses to sub-lethal DDT and endosulfan exposure. Metabolomics 5(1):84–94CrossRefGoogle Scholar
  42. McKelvie JR, Wolfe DM, Celejewski M, Simpson AJ, Simpson MJ (2010) Correlations of Eisenia fetida metabolic responses to extractable phenanthrene concentrations through time. Environ Pollut 158(6):2150–2157CrossRefGoogle Scholar
  43. Nicholson JK, Lindon JC, Holmes E (1999) ‘Metabonomics’, understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica 29(11):1181–1189CrossRefGoogle Scholar
  44. Pereira CS, Hünenberger PH (2006) Interaction of the sugars trehalose, maltose and glucose with a phosphollipid bilayer: a comparative molecular dynamics study. J Phys Chem B 110(31):15572–15581CrossRefGoogle Scholar
  45. Pirintsos SA, Kotzabasis K, Loppi S (2004) Polyamine production in lichens under metal pollution stress. J Atmos Chem 49(1–3):303–315CrossRefGoogle Scholar
  46. Robert M, Soga T, Tomita M (2007) E. coli metabolomics: capturing the complexity of a “simple” model. Metabolomics 18:189–234CrossRefGoogle Scholar
  47. Ruan AD, Min H, Peng XH, Huang Z (2005) Isolation and characterization of Pseudomonas sp. strain HF-1, capable of degrading nicotine. Res Microbiol 156(5–6):700–706CrossRefGoogle Scholar
  48. Saum SH, Müller V (2008) Regulation of osmoadaptation in the moderate halophile Halobacillus halophilus: chloride, glutamate and switching osmolyte strategies. Saline Systems 4(1):4–18CrossRefGoogle Scholar
  49. Shao TJ, Yuan HP, Yan B, Lu ZM, Min H (2009) Antioxidant enzyme activity in bacterial resistance to nicotine toxicity by reactive oxygen species. Arch Environ Contam Toxicol 57(3):456–462CrossRefGoogle Scholar
  50. Shao TJ, Yang GQ, Wang MZ, Lu ZM, Min H, Zhao L (2010) Reduction of oxidative stress by bioaugmented strain Pseudomonas sp. HF-1 and selection of potential biomarkers in sequencing batch reactor treating tobacco wastewater. Ecotoxicology 19(6):1117–1123CrossRefGoogle Scholar
  51. Singer MA, Lindquist S (1998) Multiple effects of trehalose on protein folding in vitro and in vivo. Mol Cell 1(5):639–648CrossRefGoogle Scholar
  52. Slupsky CM, Rankin KN, Wagner J, Fu H, Chang D, Wdljie AM, Saude EJ, Lix B, Adamko DJ, Shah S, Greiner R, Sykes BD, Marrie TJ (2007) Investigations of the effects of gender, diurnal variation and age in human urinary metabolomic profiles. Anal Chem 79(18):6995–7004CrossRefGoogle Scholar
  53. Son HS, Hwang GS, Kim KM, Kim EY, Van Den Berg F, Park WM, Lee C-H, Hong YS (2009) 1H NMR-based metabolomic approach for understanding the fermentation behaviors of wine yeast strains. Anal Chem 81(3):1137–1145CrossRefGoogle Scholar
  54. Tabor CW, Tabor H (1985) Polyamines in Microorganisms. Microbiol Rev 49(1):81–99Google Scholar
  55. Tang HR, Wang YL (2006) Metabonomics-a revolution in progress. Prog Biochem Biophys 33(5):401–417Google Scholar
  56. Tang HZ, Wang SN, Ma LY, Meng XZ, Deng ZX, Zhang D, Ma CQ, Xu P (2008) A novel gene encoding 6-hydroxy-3-succinoylpyridine hydroxylase, involved in nicotine degradation by Pseudomonas putida strain S16. Appl Environ Microbiol 74(3):1567–1574CrossRefGoogle Scholar
  57. Tang HZ, Wang LJ, Meng XZ, Ma LY, Wang SN, He XF, Wu G, Xu P (2009) Novel nicotine oxidoreductase-encoding gene involved in nicotine degradation by Pseudomonas putida strain S16. Appl Environ Microbiol 75(3):772–778CrossRefGoogle Scholar
  58. Tjeerdema RS (2008) Application of NMR-based techniques in aquatic toxicology: brief examples. Mar Pollut Bull 57(6–12):275–279CrossRefGoogle Scholar
  59. Tkachenko AG, Nesterova LY, Pshenichnov MP (2001) Role of putrescine in the regulation of the expression of the oxidative stress defense genes of Escherichia coli. Microbiology 70(2):133–137CrossRefGoogle Scholar
  60. Tremaroli V, Workentine ML, Weljie AM, Vogel HJ, Ceri H, Viti C, Tatti E, Zhang P, Hynes AP, Turner RJ, Zannoni D (2009) Metabolomic investigation of the bacterial response to a metal challenge. Appl Environ Microbiol 75(3):719–728CrossRefGoogle Scholar
  61. Trygg J, Wold S (2002) Orthogonal projections to latent structures (O-PLS). J Chemom 16(3):119–128CrossRefGoogle Scholar
  62. Trygg J, Holmes E, Lundstedt T (2007) Chemometrics in metabonomics. J Proteome Res 6(2):469–479CrossRefGoogle Scholar
  63. van den Berg RA, Hoefsloot HC, Westerhuis JA, Smilde AK, van der Werf MJ (2006) Centering, Scaling, and transformations, improving the biological information content of metabolomics data. BMC Genomics 7:142–156CrossRefGoogle Scholar
  64. Wang YL, Tang HR, Nicholson JK, Hylands PJ, Sampson J, Whitcombe I, Stewart CG, Caiger S, Oru I, Holmes E (2004) Metabolomic strategy for the classification and quality control of phytomedicine: a case study of chamomile flower (Matricaria recutita L). Planta Med 70(3):250–255CrossRefGoogle Scholar
  65. Wang YL, Tang HR, Nicholson JK, Hylands PJ, Sampson J, Holmes E (2005) A metabonomic strategy for the detection of the metabolic effects of chamomile (Matricaria recutita L) ingestion. J Agric Food Chem 53(2):191–196CrossRefGoogle Scholar
  66. Wang YL, Holmes E, Tang HR, Lindon JC, Sprenger N, Turini ME, Bergonzelli G, Fay LB, Kochhar S, Nicholson JK (2006) Experimental metabonomic model of dietary variation and stress interactions. J Proteome Res 5(7):1535–1542CrossRefGoogle Scholar
  67. Wang SN, Liu Z, Tang HZ, Meng J, Xu P (2007) Characterization of environmentally friendly nicotine degradation by Pseudomonas putida biotype A strain S16. Microbiology 153(5):1556–1565CrossRefGoogle Scholar
  68. Wang MZ, Yang GQ, Min H, Lu ZM (2009a) A novel nicotine catabolic plasmid pMH1 in Pseudomonas sp. strain HF-1. Can J Microbiol 55(3):228–233CrossRefGoogle Scholar
  69. Wang MZ, Yang GQ, Min H, Lu ZM, Jia XY (2009b) Bioaugmentation with the nicotine-degrading bacterium Pseudomonas sp. HF-1 in a sequencing batch reactor treating tobacco wastewater, degradation study and analysis of its mechanisms. Water Res 43(17):4187–4196CrossRefGoogle Scholar
  70. Welsh DT (2000) Ecological significance of compatible solute accumulation by micro-organisms: from single cells to global climate. FEMS Microbiol Rev 24(3):263–290CrossRefGoogle Scholar
  71. Westerhuis JA, Hoefsloot HJC, Smit S, Vis DJ, Smilde AK, van Velzen EJJ, van Duijnhoven JPM, van Dorsten FA (2008) Assessment of PLSDA cross validation. Metabolomics 4(1):81–89CrossRefGoogle Scholar
  72. Xiao CN, Dai H, Liu HB, Wang YL, Tang HR (2008) Revealing the metabonomic variation of rosemary extracts using 1H NMR spectroscopy and multivariate data analysis. J Agric Food Chem 56(21):10142–10153CrossRefGoogle Scholar
  73. Xiao CN, Hao FH, Qin XR, Wang YL, Tang HR (2009) An optimized buffer system for NMR-based urinary metabonomics with effective pH control, chemical shift consistency and dilution minimization. Analyst 134(5):916–925CrossRefGoogle Scholar
  74. Xu J, Li M, Mak NK, Chen F, Jiang Y (2011) Triphenyltin induced growth inhibition and antioxidative responses in the green microalga Scenedesmus quadricauda. Ecotoxicology 20(1):73–80CrossRefGoogle Scholar
  75. Yap IKS, Clayton TA, Tang H, Everett JR, Hanton G, Provost J-P, Le Net J-L, Charuel C, Lindon JC, Nicholson JK (2006) An integrated metabonomic approach to describe temporal metabolic disregulation induced in the rat by the model hepatotoxin allyl formate. J Proteome Res 5(10):2675–2684CrossRefGoogle Scholar
  76. Ye YF, Zhang LM, An YP, Hao FH, Tang HR (2011) Nuclear magnetic resonance for analysis of metabolite composition of Escherichia coli. Chin J Anal Chem 39(8):1186–1194CrossRefGoogle Scholar
  77. Yildiz D, Ercal N, Armstrong DW (1998) Nicotine enantiomers and oxidative stress. Toxicology 130(2–3):155–165CrossRefGoogle Scholar
  78. Yu H, Tang H, Wang L, Yao Y, Wu G, Xu P (2011) Complete genome sequence of the nicotine-degrading Pseudomonas putida strain S16. J Bacteriol 193(19):5541–5542CrossRefGoogle Scholar
  79. Zhang LM, Ye YF, An YP, Tian Y, Wang YL, Tang HR (2011) Systems responses of rats to aflatoxin B1 exposure revealed with metabonomic changes in multiple biological matrices. J Proteome Res 10:614–623CrossRefGoogle Scholar
  80. Zhao XJ, Huang CY, Lei HH, Nie X, Tang HR, Wang YL (2011) Dynamic metabolic response of mice to acute mequindox exposure. J Proteome Res 10:5183–5190CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Yangfang Ye
    • 1
  • Xin Wang
    • 2
  • Limin Zhang
    • 3
  • Zhenmei Lu
    • 2
  • Xiaojun Yan
    • 1
  1. 1.School of Marine ScienceNingbo UniversityNingboChina
  2. 2.College of Life ScienceZhejiang UniversityHangzhouChina
  3. 3.State Key Laboratory of Magnetic Resonance and Atomic and Molecular PhysicsWuhan Institute of Physics and Mathematics, Chinese Academy of SciencesWuhanChina

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