, Volume 8, Issue 4, pp 566–578 | Cite as

Deuterium-exchange metabolomics identifies N-methyl lyso phosphatidylethanolamines as abundant lipids in acidophilic mixed microbial communities

  • Curt R. Fischer
  • Paul Wilmes
  • Benjamin P. Bowen
  • Trent R. Northen
  • Jillian F. BanfieldEmail author
Original Article


Natural microbial communities are extremely diverse and contain uncharacterized but functionally important small molecules. By coupling a deuterium (D) labeling technique to high mass accuracy untargeted liquid chromatography-electrospray ionization-mass spectrometry (LC–ESI–MS) metabolomic analysis, we found that natural acidophilic microbial biofilms dominated by bacteria of the genus Leptospirillum contained unusual lyso phosphatidylethanolamine (PE) lipids in high abundance (more than 10 nmol/mg of dry biomass). The unusual polar head group structure of these lipids is similar to lipids found in phylogenetically unrelated acidophilic chemoautolithotrophs and may be related to the affinity of these lipids for iron and calcium ions. Correlations of lyso phospholipid and proteome abundance patterns suggest a link between the lyso phospholipids and the UBA-type substrain of Leptospirillum group II. By combining untargeted metabolomics with D exchange we demonstrate the ability to identify cryptic but biologically functional small molecules in mixed microbial communities.


Deuterium exchange Untargeted metabolomics Microbial community Lyso lipid 



The authors thank Mr. T. Arman for access to the Richmond Mine, Mr. R. Caver for on-site assistance, the Banfield lab members for assistance with biofilm sampling in the field, Susan Spaulding for assistance with bioreactor operation and sample preparation, and John Hayes for valuable technical discussions. This research was funded by the US Department of Energy, Office of Biological and Environmental Research Carbon-Cycling Program (DE-SC0004665), the DOE Genomics:GTL Program grant number DE-FG02-05ER64134. PWs recent involvement was supported by a Luxembourg National Research Fund ATTRACT grant (FNR/A09/03).

Conflicts of interests

The authors declare no competing interests.

Supplementary material

11306_2011_344_MOESM1_ESM.docx (1.3 mb)
Supplementary material 1 (DOCX 1381 kb)


  1. Agate, A. D., & Vishniac, W. (1972). Iron transport by phospholipids in a two phase system containing water and n-pentanol. Chemistry and Physics of Lipids, 9(3), 247–254.PubMedCrossRefGoogle Scholar
  2. Agate, A. D., & Vishniac, W. (1973). Changes in phospholipid composition of Thiobacillus neapolitanus during growth. Archives of Microbiology, 89(3), 247–255.CrossRefGoogle Scholar
  3. Allen, E. E., Tyson, G. W., Whitaker, R. J., Detter, J. C., Richardson, P. M., & Banfield, J. F. (2007). Genome dynamics in a natural archaeal population. Proceedings of the National Academy of Sciences, 104(6), 1883.CrossRefGoogle Scholar
  4. Ames, G. F. (1968). Lipids of Salmonella typhimurium and Escherichia coli: Structure and metabolism. Journal of Bacteriology, 95(3), 833.PubMedGoogle Scholar
  5. Aston, J. E., Apel, W. A., Lee, B. D., & Peyton, B. M. (2009). Toxicity of select organic acids to the slightly thermophilic acidophile Acidithiobacillus caldus. Environmental Toxicology and Chemistry, 28(2), 279–286.PubMedCrossRefGoogle Scholar
  6. Baker, B. J., Comolli, L. R., Dick, G. J., Hauser, L. J., Hyatt, D., Dill, B. D., et al. (2010). Enigmatic, ultrasmall, uncultivated Archaea. Proceedings of the National Academy of Sciences, 107(19), 8806.CrossRefGoogle Scholar
  7. Baran, R., Bowen, B. P., Bouskill, N. J., Brodie, E. L., Yannone, S. M., & Northen, T. R. (2010). Metabolite identification in Synechococcus sp. PCC 7002 using untargeted stable isotope assisted metabolite profiling. Analytical Chemistry, 82(21), 9034–9042.Google Scholar
  8. Belnap, C. P., Pan, C., VerBerkmoes, N. C., Power, M. E., Samatova, N. F., Carver, R. L., et al. (2010). Cultivation and quantitative proteomic analyses of acidophilic microbial communities. ISME Journal, 4(4), 520–530.PubMedCrossRefGoogle Scholar
  9. Buist, P. H. (2007). Exotic biomodification of fatty acids. Natural Product Reports, 24(5), 1110–1127.PubMedCrossRefGoogle Scholar
  10. Chang, Y. Y., & Cronan, J. E. (2002). Membrane cyclopropane fatty acid content is a major factor in acid resistance of Escherichia coli. Molecular Microbiology, 33(2), 249–259.CrossRefGoogle Scholar
  11. Cowie, B. R., Slater, G. F., Bernier, L., & Warren, L. A. (2009). Carbon isotope fractionation in phospholipid fatty acid biomarkers of bacteria and fungi native to an acid mine drainage lake. Organic Geochemistry, 40(9), 956–962.Google Scholar
  12. Denef, V. J., Kalnejais, L. H., Mueller, R. S., Wilmes, P., Baker, B. J., Thomas, B. C., et al. (2010). Proteogenomic basis for ecological divergence of closely related bacteria in natural acidophilic microbial communities. Proceedings of the National Academy of Sciences of USA, 107(6), 2383–2390.CrossRefGoogle Scholar
  13. Goldfine, H. (1962). The characterization and biosynthesis of an N-methylethanolamine phospholipid from Clostridium butyricum. Biochimica et Biophysica Acta, 59(2), 504–506.PubMedCrossRefGoogle Scholar
  14. Goldfine, H., & Ellis, M. E. (1964). N-methyl groups in bacterial lipids. Journal of Bacteriology, 87(1), 8–15.PubMedGoogle Scholar
  15. Goldfine, H., & Hagen, P.-O. (1968). N-methyl groups in bacterial lipids III. Phospholipids of hypomicrobia. Journal of Bacteriology, 95(2), 367–375.PubMedGoogle Scholar
  16. Goltsman, D. S. A., Denef, V. J., Singer, S. W., VerBerkmoes, N. C., Lefsrud, M., Mueller, R. S., et al. (2009). Community genomic and proteomic analyses of chemoautotrophic iron-oxidizing “Leptospirillum rubarum” (Group II) and “Leptospirillum ferrodiazotrophum” (Group III) bacteria in acid mine drainage biofilms. Applied and Environmental Microbiology, 75(13), 4599.PubMedCrossRefGoogle Scholar
  17. Han, X., & Gross, R. W. (1996). Structural determination of lysophospholipid regioisomers by electrospray ionization tandem mass spectrometry. Journal of the American Chemical Society, 118(2), 451–457.CrossRefGoogle Scholar
  18. Han, X., & Gross, R. W. (2005). Shotgun lipidomics: Electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samples. Bioorganic Chemistry, 24, 367–412.Google Scholar
  19. Jeans, C., Singer, S. W., Chan, C. S., VerBerkmoes, N. C., Shah, M., Hettich, R. L., et al. (2008). Cytochrome 572 is a conspicuous membrane protein with iron oxidation activity purified directly from a natural acidophilic microbial community. The ISME Journal, 2(5), 542–550.PubMedCrossRefGoogle Scholar
  20. Kind, T., & Fiehn, O. (2006). Metabolomic database annotations via query of elemental compositions: mass accuracy is insufficient even at less than 1 ppm. BMC Bioinformatics, 7, 234.PubMedCrossRefGoogle Scholar
  21. Kind, T., & Fiehn, O. (2007). Seven Golden Rules for heuristic filtering of molecular formulas obtained by accurate mass spectrometry. BMC Bioinformatics, 8, 105.PubMedCrossRefGoogle Scholar
  22. Lam, W., & Ramanathan, R. (2002). In electrospray ionization source hydrogen/deuterium exchange LC-MS and LC-MS/MS for characterization of metabolites. Journal of the American Society for Mass Spectrometry, 13(4), 345–353.PubMedCrossRefGoogle Scholar
  23. Liu, D. Q., Hop, C. E. C. A., Beconi, M. G., Mao, A., & Chiu, S. H. L. (2001). Use of on-line hydrogen/deuterium exchange to facilitate metabolite identification. Rapid Communications in Mass Spectrometry, 15(19), 1832–1839.PubMedCrossRefGoogle Scholar
  24. Liu, D. Q., Wu, L., Sun, M., & MacGregor, P. A. (2007). On-line H/D exchange LC-MS strategy for structural elucidation of pharmaceutical impurities. Journal of Pharmaceutical and Biomedical Analysis, 44(2), 320–329.PubMedCrossRefGoogle Scholar
  25. Lo, I., Denef, V. J., Verberkmoes, N. C., Shah, M. B., Goltsman, D., DiBartolo, G., et al. (2007). Strain-resolved community proteomics reveals recombining genomes of acidophilic bacteria. Nature, 446(7135), 537–541.PubMedCrossRefGoogle Scholar
  26. Mueller, R. S., Denef, V. J., Kalnejais, L. H., Suttle, K. B., Thomas, B. C., Wilmes, P., Smith, R. L., Nordstrom, D. K., McCleskey, R. B., Shah, M. B., VerBerkmoes, N. C., Hettich, R. L., & Banfield, J. F. (2010). Ecological distribution and population physiology defined by proteomics in a natural microbial community. Molecular Systems Biology, 6, 314.Google Scholar
  27. Neufeld, J. D., Wagner, M., & Murrell, J. C. (2007). Who eats what, where and when? Isotope-labelling experiments are coming of age. The ISME Journal, 1(2), 103–110.PubMedCrossRefGoogle Scholar
  28. Pulfer, M., & Murphy, R. C. (2003). Electrospray mass spectrometry of phospholipids. Mass Spectrometry Reviews, 22(5), 332–364.PubMedCrossRefGoogle Scholar
  29. Ram, R. J., Verberkmoes, N. C., Thelen, M. P., Tyson, G. W., Baker, B. J., Blake, R. C., et al. (2005). Community proteomics of a natural microbial biofilm. Science, 308(5730), 1915–1920.PubMedCrossRefGoogle Scholar
  30. Shively, J. M., & Benson, A. A. (1967). Phospholipids of Thiobacillus thiooxidans. Journal of Bacteriology, 94(5), 1679.PubMedGoogle Scholar
  31. Short, S. A., White, D. C., & Aleem, M. I. H. (1969). Phospholipid metabolism in Ferrobacillus ferrooxidans. Journal of Bacteriology, 99(1), 142.PubMedGoogle Scholar
  32. Singer, S. W., Chan, C. S., Zemla, A., VerBerkmoes, N. C., Hwang, M., Hettich, R. L., et al. (2008). Characterization of cytochrome 579, an unusual cytochrome isolated from an iron-oxidizing microbial community. Applied and Environmental Microbiology, 74(14), 4454.PubMedCrossRefGoogle Scholar
  33. Sohlenkamp, C., López-Lara, I. M., & Geiger, O. (2003). Biosynthesis of phosphatidylcholine in bacteria. Progress in Lipid Research, 42(2), 115–162.PubMedCrossRefGoogle Scholar
  34. Tyson, G. W., Chapman, J., Hugenholtz, P., Allen, E. E., Ram, R. J., Richardson, P. M., et al. (2004). Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature, 428(6978), 37–43.PubMedCrossRefGoogle Scholar
  35. Wilmes, P., Remis, J. P., Hwang, M., Auer, M., Thelen, M. P., & Banfield, J. F. (2009). Natural acidophilic biofilm communities reflect distinct organismal and functional organization. The ISME Journal, 3(2), 266–270.PubMedCrossRefGoogle Scholar
  36. Wilmes, P., Bowen, B. P., Thomas, B. C., Mueller, R. S., Denef, V. J., VerBerkmoes, N. C., et al. (2010). Metabolome-proteome differentiation coupled to microbial divergence. mBio, 1(5), e00246.PubMedCrossRefGoogle Scholar
  37. Zhang, X., Borda, M. J., Schoonen, M. A. A., & Strongin, D. R. (2003). Adsorption of phospholipids on pyrite and their effect on surface oxidation. Langmuir, 19(21), 8787–8792.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Curt R. Fischer
    • 1
    • 4
  • Paul Wilmes
    • 1
    • 5
  • Benjamin P. Bowen
    • 3
  • Trent R. Northen
    • 3
  • Jillian F. Banfield
    • 1
    • 2
    Email author
  1. 1.Department of Earth and Planetary ScienceUniversity of California, BerkeleyBerkeleyUSA
  2. 2.Department of Environmental Science, Policy and ManagementUniversity of CaliforniaBerkeleyUSA
  3. 3.Life Sciences DivisionLawrence Berkeley National LaboratoryBerkeleyUSA
  4. 4.Ginkgo BioworksBostonUSA
  5. 5.Department of Environment and Agro-biotechnologies, Public Research Centre—Gabriel Lippmann, and Luxembourg Centre for Systems Biomedicine (LCSB)University of LuxembourgBelvauxGrand-Duchy of Luxembourg

Personalised recommendations