Detection and identification of Bacteriovorax stolpii UKi2 sphingophosphonolipid molecular species

  • Koka Jayasimhulu
  • Shannon M. Hunt
  • Edna S. Kaneshiro
  • Yoko Watanabe
  • José-Luis Giner


Bacteriovorax stolpii is a predator of larger gram-negative bacteria and lives as a parasite in the intraperiplasmic space of the host cell. This bacterium is unusual among prokaryotes in that sphingolipids comprise a large proportion of its lipids. We here report the presence of 18 molecular species of B. stolpii UKi2 sphingophosphonolipids (SPNLs). 31P NMR spectroscopy and analysis of Pi released by a differential hydrolysis protocol confirmed the phosphonyl nature of these lipids. The SPNLs were dominated by those with 1-hydroxy-2-aminoethane phosphonate (hydroxy-aminoethylphosphonate) polar head groups; aminoethylphosphonate was also detected in minor SPNL components. The long-chain bases (LCBs) were dominated by C17 iso-branched phytosphingosine; C17 iso-branched dihydrosphingosine was also present in some SPNLs. The N-linked fatty acids were predominantly iso-branched and most contained an α-hydroxy group (C15 α-hydroxy fatty acid was the major fatty acid). Minor molecular species containing nonhydroxy fatty acids were also detected. The definitive iso-structures of the predominant fatty acids and LCBs present in the B. stolpii SPNLs were established using 13C and 3H nuclear magnetic resonance spectroscopy; less than 20% were unbranched. Detection and analyses of intact compounds by MS-MS were performed by a hybrid quadrupole time-of-flight (Q-TOF-II) MS equipped with an electrospray ionization source. Analyses of peracetylated derivatives verified the structural assignments of these lipids.


Nuclear Magnetic Resonance Ceramide Head Group Nuclear Magnetic Resonance Spectroscopy Hydroxy Fatty Acid 
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  1. 1.
    Baer, M. L.; Ravel, J.; Chun, J.; Hill, R. T.; Williams, H. N. A Proposal for the Reclassification of Bdellovibrio stolpii and Bdellovibrio starrii into a New Genus, Bacteriovorax. gen. nov. as Bacteriovorax stolpii comb. nov. and Bacteriovorax starrii comb. nov., respectively. Int. J. Syst. Evol. Microbiol. 2000, 50, 219–224.CrossRefGoogle Scholar
  2. 2.
    Hespell, R. B.; Miozzari, G. F.; Rittenberg, S. C. Ribonucleic Acid Destruction and Synthesis during Intraperiplasmic Growth of Bdellovibrio bacteriovorus. J. Bacteriol. 1975, 123, 481–491.Google Scholar
  3. 3.
    Kuenen, J. G.; Rittenberg, S. C. Incorporation of Long-Chain Fatty Acids of the Substrate Organism by Bdellovibrio bacteriovorax during Intraperiplasmic Growth. J. Bacteriol. 1975, 121, 1145–1157.Google Scholar
  4. 4.
    Steiner, S.; Conti, S. F.; Lester, R. L. Occurrence of Phosphonosphingolipids in Bdellovibrio bacteriovorus Strain Uki2. J. Bacteriol. 1973, 116, 1199–1211.Google Scholar
  5. 5.
    Stoffel, W. Sphingolipids. Ann. Rev. Biochem. 1971, 40, 57–82.CrossRefGoogle Scholar
  6. 6.
    Morrel, P.; Braun, B. Biosynthesis and Metabolic Degradation of Sphingolipids Not Containing Sialic Acid. J. Lipid Res. 1972, 13, 293–309.Google Scholar
  7. 7.
    Ferguson, K. A.; Conner, R. L.; Mallory, F. B.; Mallory, C. W. α-Hydroxy Fatty Acids in Sphingolipids of Tetrahymena. Biochim. Biophys. Acta. 1972, 270, 111–116.CrossRefGoogle Scholar
  8. 8.
    Viswanathan, C. V.; Rosenberg, H. Isolation of Ceramide Monomethylaminoethylphosphonate from the Lipids of Tetrahymena pyriformis W. J. Lipid Res. 1973, 14, 327–330.Google Scholar
  9. 9.
    Ferguson, K. A.; Davis, F. M.; Conner, R. L.; Landrey, J. R.; Mallory, F. B. Effect of Sterol Replacement in Vivo on the Fatty Acid Composition of Tetrahymena. J. Biol. Chem. 1975, 250, 6998–7005.Google Scholar
  10. 10.
    Sugita, M.; Fukunaga, Y.; Ohkawa, K.; Nozawa, Y.; Hori, T. Structural Components of Sphingophosphonolipids from the Ciliated Protozoan, Tetrahymena pyriformis WH-14. J. Biochem. (Tokyo). 1979, 86, 281–288.Google Scholar
  11. 11.
    Rhoads, D. E.; Kaneshiro, E. S. Characterizations of Phospholipids from Paramecium tetraurelia Cells and Cilia. J. Protozool. 1979, 26, 329–338.CrossRefGoogle Scholar
  12. 12.
    Kaneshiro, E. S.; Matesic, D. F.; Jayasimhulu, K. Characterizations of Six Ethanolamine Sphingophospholipids from Paramecium Cells and Cilia. J. Lipid Res. 1984, 25, 369–377.Google Scholar
  13. 13.
    Kaneshiro, E. S.; Jayasimhulu, K.; Lester, R. L. Characterizations of Inositol Lipids from Leishmania donovani Promastigotes: Identification of an Inositol Sphingolipid. J. Lipid Res. 1986, 27, 1294–1303.Google Scholar
  14. 14.
    Kaneshiro, E. S.; Jayasimhulu, K.; Sul, D.; Erwin, J. A. Identification and Initial Characterizations of Free, Glycosylated, and Phosphorylated Ceramides of Paramecium. J. Lipid Res. 1997, 38, 2399–2410.Google Scholar
  15. 15.
    Kariotoglou, D. M.; Mastronicolis, S. K. Sphingophosphonolipid Molecular Species from Edible Mollusks and a Jellyfish. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2003, 136, 27–44.CrossRefGoogle Scholar
  16. 16.
    Liu, G.; Kleine, L.; Hebert, R. L. Advances in the Signal Transduction of Ceramide and Related Sphingolipids. Crit. Rev. Clin. Lab. Sci. 1999, 36, 511–573.CrossRefGoogle Scholar
  17. 17.
    Hannun, Y. A.; Luberto, C. Ceramide in the Eukaryotic Stress Response. Trends Cell. Biol. 2000, 10, 73–80.CrossRefGoogle Scholar
  18. 18.
    Futerman, A. H.; Hannun, Y. A. The Complex Life of Simple Sphingolipids. EMBO Rep. 2004, 5, 777–782.CrossRefGoogle Scholar
  19. 19.
    Pettus, B. J.; Chalfant, C. E.; Hannun, Y. A. Sphingolipids in Inflammation: Roles and Implications. Curr. Mol. Med. 2004, 4, 405–418.CrossRefGoogle Scholar
  20. 20.
    Brown, D. A.; London, E. Structure and Function of Sphingolipid- and Cholesterol-Rich Membrane Rafts. J. Biol. Chem. 2000, 275, 17221–17224.CrossRefGoogle Scholar
  21. 21.
    London, E.; Brown, D. A. Insolubility of Lipids in Triton X-100: Physical Origin and Relationship to Sphingolipid/Cholesterol Membrane Domains (Rafts). Biochim. Biophys. Acta. 2000, 1508L, 182–195.CrossRefGoogle Scholar
  22. 22.
    Chazal, N.; Gerlier, D. Virus Entry, Assembly, Budding, and Membrane Rafts. Microbiol. Mol. Biol. Rev. 2003, 67, 226–237.CrossRefGoogle Scholar
  23. 23.
    Gulbins, E.; Dreschers, S.; Wilder, B.; Grassme, H. Ceramide, Membrane Rafts, and Infections. J. Mol. Med. 2004, 82, 357–363.CrossRefGoogle Scholar
  24. 24.
    Martin, S. W.; Konopka, J. B. Lipid Raft Polarization Contributes to Hyphal Growth in Candida albicans. Eukaryot. Cell. 2004, 3, 675–684.CrossRefGoogle Scholar
  25. 25.
    Seveau, S.; Bierne, H.; Giroux, S.; Prevost, M. C.; Cossart, P. Role of Lipid Rafts in E-Cadherin- and HGF-R/Met-Mediated Entry of Listeria monocytogenes into Host Cells. J. Cell Biol. 2004, 166, 743–753.CrossRefGoogle Scholar
  26. 26.
    van Meer, G.; Sprong, H. Membrane Lipids and Vesicular Traffic. Curr. Opin. Cell Biol. 2004, 16, 373–378.CrossRefGoogle Scholar
  27. 27.
    Kawahara, K.; Seydel, U.; Matsuura, M.; Danbara, H.; Rietschel, E. T.; Zahringer, U. Chemical Structure of Glycosphingolipids Isolated from Sphingomonas paucimobilis. FEBS Lett. 1991, 292, 107–110.CrossRefGoogle Scholar
  28. 28.
    LaBach, J. P.; White, D. C. Identification of Phosphorylethanolamine and Ceramide Phosphorylglycerol in the Lipids of an Anaerobic Bacterium. J. Lipid Res. 1969, 10, 528–534.Google Scholar
  29. 29.
    Watanabe, Y.; Nakajima, M.; Hoshino, T.; Jayasimhulu, K.; Brooks, E. E.; Kaneshiro, E. S. A Novel Sphingophosphonolipid Head Group 1-Hydroxy-2-Aminoethyl Phosphonate in Bdellovibrio stolpii. Lipids 2001, 36, 513–519.CrossRefGoogle Scholar
  30. 30.
    Sugita, M.; Hori, T. Isolation of Diacylglycerol-2-Aminoethylphosphonate from Tetrahymena pyriformis. J. Biochem. (Tokyo) 1971, 69, 1149–1150.Google Scholar
  31. 31.
    Matesic, D. F.; Kaneshiro, E. S. Incorporation of Serine into Paramecium Ethanolamine Phospholipid and Phosphonolipid Head Groups. Biochem. J. 1984, 222, 229–233.Google Scholar
  32. 32.
    Kaneshiro, E. S. Lipids of Paramecium. J. Lipid Res. 1987, 28, 1241–1258.Google Scholar
  33. 33.
    Liebisch, G.; Drobnik, W.; Reil, M.; Trumbach, B.; Arnecke, R.; Olgemoller, B.; Roscher, A.; Schmitz, G. Quantitative Measurement of Different Ceramide Species from Crude Cellular Extracts by Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS). J. Lipid Res. 1999, 40, 1539–1546.Google Scholar
  34. 34.
    Costello, C. E.; Beach, D. H.; Singh, B. N. Acidic Glycerol Lipids of Trichomonas vaginalis and Tritrichomonas foetus. Biol. Chem. 2001, 382, 275–282.CrossRefGoogle Scholar
  35. 35.
    Jensen, N. J.; Tomer, K. B.; Gross, M. L. FAB MS/MS for Phosphatidylinositol, -glycerol, -ethanolamine and Other Complex Phospholipids. Lipids 1987, 22, 480–409.CrossRefGoogle Scholar
  36. 36.
    Bligh, E. G.; Dyer, W. J. A Rapid Method of Total Lipid Extraction and Purification. Can. Biochem. Physiol. 1959, 37, 911–917.CrossRefGoogle Scholar
  37. 37.
    Dittmer, J. C.; Lester, R. L. A Simple, Specific Spray for the Detection of Phospholipids on Thin-Layer Chromatograms. J Lipid Res. 1964, 15, 126–127.Google Scholar
  38. 38.
    Harrison, K. A.; Davies, S. S.; Marathe, G. K.; McIntyre, T.; Prescott, S.; Reddy, K. M.; Falck, J. R.; Murphy, R. C. Analysis of Oxidized Glycerophosphocholine Lipids Using Electrospray Ionization Mass Spectrometry and Microderivatization Techniques. J. Mass Spectrom. 2000, 35, 224–236.CrossRefGoogle Scholar
  39. 39.
    Glonek, T.; Henderson, T. O.; Hilderbrand, R. L.; Myers, T. C. Biological Phosphonates: Determination by Phosphorus-31 Nuclear Magnetic Resonance. Nature 1970, 169, 192–194.Google Scholar
  40. 40.
    Meneses, P.; Glonek, T. High Resolution 31P NMR of Extracted Phospholipids. J. Lipid Res. 1988, 29, 679–687.Google Scholar
  41. 41.
    Liang, C. R.; Rosenberg, H. The Biosynthesis of the Carbon-Phosphorus Bond in Tetrahymena. Biochim. Biophys. Acta. 1968, 156, 437–439.CrossRefGoogle Scholar
  42. 42.
    Rosenberg, H. Phosphonolipids. In Form and Function of Phospholipids; Ansell, G. B.; Dawson, R. M. C.; Hawthorne, J. N., Eds.; Elsevier Publishing: Amsterdam, 1973; pp 333–344.Google Scholar
  43. 43.
    Hori, T.; Nozawa, Y. Phosphonolipids. In Phospholipids; Hawthorne, J. N.; Ansell, E. G., Eds.; Elsevier Biomedical Press: New York, 1982; pp 95–128.Google Scholar

Copyright information

© American Society for Mass Spectrometry 2007

Authors and Affiliations

  • Koka Jayasimhulu
    • 1
  • Shannon M. Hunt
    • 1
  • Edna S. Kaneshiro
    • 1
  • Yoko Watanabe
    • 2
  • José-Luis Giner
    • 3
  1. 1.Department of Biological SciencesUniversity of CincinnatiCincinnatiUSA
  2. 2.Department of Medical Technology, School of Health Sciences, Faculty of MedicineNiigata UniversityNiigataJapan
  3. 3.Department of ChemistryState University of New York-ESFSyracuseUSA

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