Analytical and Bioanalytical Chemistry

, Volume 407, Issue 8, pp 2273–2282 | Cite as

Monitoring metabolites from Schizophyllum commune interacting with Hypholoma fasciculare combining LESA–HR mass spectrometry and Raman microscopy

  • Riya C. Menezes
  • Marco Kai
  • Katrin Krause
  • Christian Matthäus
  • Aleš SvatošEmail author
  • Jürgen Popp
  • Erika Kothe
Research Paper
Part of the following topical collections:
  1. Mass Spectrometry Imaging


Microbial competition for territory and resources is inevitable in habitats with overlap between niches of different species or strains. In fungi, competition is brought about by antagonistic mycelial interactions which alter mycelial morphology, metabolic processes, secondary metabolite release, and extracellular enzyme patterns. Until now, we were not able study in vivo chemical interactions of different colonies growing on the same plate. In this report, we developed a fast and least invasive approach to identify, quantify, and visualize co culture-induced metabolites and their location of release within Schizophyllum commune. The pigments indigo, indirubin, and isatin were used as examples to show secondary metabolite production in the interaction zone with Hypholoma fasciculare. Using a combinatory approach of Raman spectroscopy imaging, liquid extraction surface analysis (LESA), and high-resolution mass spectrometry, we identified, quantified, and visualized the presence of indigo and indirubin in the interaction zone. This approach allows the investigation of metabolite patterns between wood degrading species in competition to gain insight in community interactions, but could also be applied to other microorganisms. This method advances analysis of living, still developing colonies and are in part not destructive as Raman spectroscopy imaging is implemented.


Raman spectroscopy LESA–HRMS Mass spectrometry pigment production Indigo Wood-decaying fungi Basidiomycetes 



We gratefully acknowledge financial support from the Deutsche Forschungsgemeinschaft (Jena School of Microbial Communication—MikroInter). Thanks to Imam Hardiman for conceiving the idea of the co-cultures and Elke-Martina Jung for help with Fig. 1. We thank Dr. Matthias Gube for providing strains and Petra Mitscherlich for general technical help.

Supplementary material

216_2014_8383_MOESM1_ESM.pdf (273 kb)
ESM 1 (PDF 272 kb)


  1. 1.
    Moree WJ, Phelan VV, Wu C-H, Bandeira N, Cornett DS, Duggan BM, Dorrestein PC (2012) Interkingdom metabolic transformations captured by microbial imaging mass spectrometry. Proc Natl Acad Sci U S A 109(34):13811–13816CrossRefGoogle Scholar
  2. 2.
    Watrous J, Roach P, Heath B, Alexandrov T, Laskin J, Dorrestein PC (2013) Metabolic profiling directly from the petri dish using nanospray desorption electrospray ionization imaging mass spectrometry. Anal Chem 85(21):10385–10391CrossRefGoogle Scholar
  3. 3.
    Walter A, Erdmann S, Bocklitz T, Jung EM, Vogler N, Akimov D, Dietzek B, Rosch P, Kothe E, Popp J (2010) Analysis of the cytochrome distribution via linear and nonlinear Raman spectroscopy. Analyst 135(5):908–917CrossRefGoogle Scholar
  4. 4.
    Popp J, Tuchin VV, Chiou A, Heinemann SH (2011) Volume 1: basics and techniques. Handbook of biophotonics. Chiou A HS, Popp J, Tučin VV (eds). Wiley-VCHGoogle Scholar
  5. 5.
    Kertesz V, Van Berkel GJ (2010) Fully automated liquid extraction-based surface sampling and ionization using a chip-based robotic nanoelectrospray platform. J Mass Spectrom 45(3):252–260CrossRefGoogle Scholar
  6. 6.
    Harz M, Rosch P, Popp J (2009) Vibrational spectroscopy—a powerful tool for the rapid identification of microbial cells at the single-cell level. Cytometry A 75(2):104–113CrossRefGoogle Scholar
  7. 7.
    Krafft C, Dietzek B, Popp J (2009) Raman and CARS microspectroscopy of cells and tissues. Analyst 134(6):1046–1057CrossRefGoogle Scholar
  8. 8.
    Delhaye M, Dhamelincourt P (1975) Raman microprobe and microscope with laser excitation. J Raman Spectrosc 3(1):33–43CrossRefGoogle Scholar
  9. 9.
    Tague T (2009) Infrared and Raman microscopy: pushing the limits of spatial resolution. Microsc Microanal 15(Supplement S2):562–563CrossRefGoogle Scholar
  10. 10.
    Eikel D, Vavrek M, Smith S, Bason C, Yeh S, Korfmacher WA, Henion JD (2011) Liquid extraction surface analysis mass spectrometry (LESA-MS) as a novel profiling tool for drug distribution and metabolism analysis: the terfenadine example. Rapid Commun Mass Spectrom 25(23):3587–3596CrossRefGoogle Scholar
  11. 11.
    Johnson D, Krsek M, Wellington EMH, Stott AW, Cole L, Bardgett RD, Read DJ, Leake JR (2005) Soil invertebrates disrupt carbon flow through fungal networks. Science 309(5737):1047CrossRefGoogle Scholar
  12. 12.
    Lundell TK, Mäkelä MR, Hildén K (2010) Lignin-modifying enzymes in filamentous basidiomycetes—ecological, functional and phylogenetic review. J Basic Microbiol 50(1):5–20CrossRefGoogle Scholar
  13. 13.
    Kirk TK, Farrell RL (1987) Enzymatic “combustion”: the microbial degradation of lignin. Annu Rev Microbiol 41:465–505CrossRefGoogle Scholar
  14. 14.
    Eriksson KEL, Blanchette RA, Ander P. (2012) Microbial and enzymatic degradation of wood and wood components. Springer London, LimitedGoogle Scholar
  15. 15.
    Hatakka A (1994) Lignin-modifying enzymes from selected white-rot fungi: production and role from in lignin degradation. FEMS Microbiol Rev 13(2–3):125–135CrossRefGoogle Scholar
  16. 16.
    Rayner ADM, Griffith GS, Howard GW (1994) Induction of metabolic and morphogenetic changed during mycelial interactions among species of higher fungi. Biochem Soc Trans 22:389–394Google Scholar
  17. 17.
    Rayner ADM, Webber JF (1984) Interspecific mycelial interactions—an overview. In: Jennings DH, Rayner ADM (eds) The ecology and physiology of the fungal mycelium. Cambridge University Press, Cambridge, pp 383–417Google Scholar
  18. 18.
    White NA, Boddy L (1992) Extracellular enzyme localization during interspecific fungal interactions. FEMS Microbiol Lett 98(1–3):75–79CrossRefGoogle Scholar
  19. 19.
    Boddy L (2000) Interspecific combative interactions between wood-decaying basidiomycetes. FEMS Microbiol Ecol 31(3):185–194CrossRefGoogle Scholar
  20. 20.
    Rayner ADM (1991) The challenge of the individualistic mycelium. Mycologia 83(1):48–71CrossRefGoogle Scholar
  21. 21.
    Raudaskoski M, Kothe E (2010) Basidiomycete mating type genes and pheromone signaling. Eukaryot Cell 9(6):847–859CrossRefGoogle Scholar
  22. 22.
    Ohm RA, de Jong JF, Lugones LG, Aerts A, Kothe E, Stajich JE, de Vries RP, Record E, Levasseur A, Baker SE, Bartholomew KA, Coutinho PM, Erdmann S, Fowler TJ, Gathman AC, Lombard V, Henrissat B, Knabe N, Kues U, Lilly WW, Lindquist E, Lucas S, Magnuson JK, Piumi F, Raudaskoski M, Salamov A, Schmutz J, Schwarze FW, vanKuyk PA, Horton JS, Grigoriev IV, Wosten HA (2010) Genome sequence of the model mushroom Schizophyllum commune. Nat Biotechnol 28(9):957–963CrossRefGoogle Scholar
  23. 23.
    Papazian HP (1950) The physiology of the incompatibility factors in Schizophyllum commune. Bot Gaz 112:143–163CrossRefGoogle Scholar
  24. 24.
    Miles PG, Lund H, Raper JR (1956) The identification of indigo as a pigment produced by a mutant culture of Schizophyllum commune. Arch Biochem Biophys 62(1):1–5CrossRefGoogle Scholar
  25. 25.
    Willstaedt H (1935) Über die Farbstoffe des echten Reizkers (Lactarius deliciosus L.) (I. Mitteil.). Ber Dtsch Chem Ges A B Ser 68(2):333–340CrossRefGoogle Scholar
  26. 26.
    Willstaedt H (1936) Über die Farbstoffe des echten Reizkers (Lactarius deliciosus L.) (II. Mitteil.). Ber Dtsch Chem Ges A B Ser 69(5):997–1001CrossRefGoogle Scholar
  27. 27.
    Kögl F, Erxleben H, Jänecke L (1930) Untersuchungen über Pilzfarbstoffe. IX. Die Konstitution der Thelephorsäure. Justus Liebigs Ann Chem 482(1):105–119CrossRefGoogle Scholar
  28. 28.
    Kögl F, Deijs WB (1935) Untersuchungen über Pilzfarbstoffe. XI. Über Boletol, den Farbstoff der blau anlaufenden Boleten. Justus Liebigs Ann Chem 515(1):10–23CrossRefGoogle Scholar
  29. 29.
    Cartwright KSG, Findlay WPK (1946) Decay of timber and its prevention. Her Majesty’s Stationary Office, LondonGoogle Scholar
  30. 30.
    Ujor VC, Monti M, Peiris DG, Clements MO, Hedger JN (2012) The mycelial response of the white-rot fungus, Schizophyllum commune to the biocontrol agent, Trichoderma viride. Fungal Biol 116(2):332–341CrossRefGoogle Scholar
  31. 31.
    Schwalb MN, Miles PG (1967) Morphogenesis of Schizophyllum commune. II. Effect of microaerobic growth. Mycologia 59:610–622CrossRefGoogle Scholar
  32. 32.
    Miljkovic M, Chernenko T, Romeo MJ, Bird B, Matthaus C, Diem M (2010) Label-free imaging of human cells: algorithms for image reconstruction of Raman hyperspectral datasets. Analyst 135(8):2002–2013CrossRefGoogle Scholar
  33. 33.
    Hedegaard M, Matthäus C, Hassing S, Krafft C, Diem M, Popp J (2011) Spectral unmixing and clustering algorithms for assessment of single cells by Raman microscopic imaging. Theor Chem Accounts 130:1249–1260CrossRefGoogle Scholar
  34. 34.
    Xia J, Mandal R, Sinelnikov IV, Broadhurst D, Wishart DS (2012) MetaboAnalyst 2.0—a comprehensive server for metabolomic data analysis. Nucleic Acids Res 40:W127–133, Web Server issueCrossRefGoogle Scholar
  35. 35.
    Wang CS, Miles PG (1966) Studies of the cell walls of Schizophyllum commune. Am J Bot 53(8):792–800CrossRefGoogle Scholar
  36. 36.
    Tuma R (2005) Raman spectroscopy of proteins: from peptides to large assemblies. J Raman Spectrosc 36(4):307–319CrossRefGoogle Scholar
  37. 37.
    Zhang K, Geissler A, Fischer S, Brendler E, Bäucker E (2012) Solid-state spectroscopic characterization of α-chitins deacetylated in homogeneous solutions. J Phys Chem B 116(15):4584–4592CrossRefGoogle Scholar
  38. 38.
    Lee CM, Cho E-M, Yang SI, Ganbold E-O, Jun J, Cho K-H (2013) Raman spectroscopy and density functional theory calculations of β-glucans and chitins in fungal cell walls. Bull Korean Chem Soc 34(3):943–945CrossRefGoogle Scholar
  39. 39.
    Bauer H, Kowski K, Kuhn H, Lüttke W, Rademacher P (1998) Photoelectron spectra and electronic structures of some indigo dyes. J Mol Struct 445(1–3):277–286CrossRefGoogle Scholar
  40. 40.
    Baran A, Fiedler A, Schulz H, Baranska M (2010) In situ Raman and IR spectroscopic analysis of indigo dye. Anal Methods 2(9):1372CrossRefGoogle Scholar
  41. 41.
    Falconer RE, Bown JL, White NA, Crawford JW (2008) Modelling interactions in fungi. J R Soc Interface 5(23):603–615CrossRefGoogle Scholar
  42. 42.
    Swack NS, Miles PG (1960) Conditions affecting growth and indigotin production by strain 130 of Schizophyllum commune. Mycologia 52(4):574–583CrossRefGoogle Scholar
  43. 43.
    Abadulla E, Robra K-H, Gübitz GM, Silva LM, Cavaco-Paulo A (2000) Enzymatic decolorization of textile dyeing effluents. Textile Res J 70(5):409–414CrossRefGoogle Scholar
  44. 44.
    Medvedev A, Buneeva O, Glover V (2007) Biological targets for isatin and its analogues: implications for therapy. Biologics 1(2):151–162Google Scholar
  45. 45.
    Sriram D, Bal TR, Yogeeswari P (2004) Design, synthesis and biological evaluation of novel non-nucleoside HIV-1 reverse transcriptase inhibitors with broad-spectrum chemotherapeutic properties. Bioorg Med Chem 12(22):5865–5873CrossRefGoogle Scholar
  46. 46.
    Chohan ZH, Pervez H, Rauf A, Khan KM, Supuran CT (2004) Isatin-derived antibacterial and antifungal compounds and their transition metal complexes. J Enzyme Inhib Med Chem 19(5):417–423CrossRefGoogle Scholar
  47. 47.
    Velišek J, Cejpek K (2011) Pigments of higher fungi—a review. Czech J Food Sci 29:87–102Google Scholar
  48. 48.
    Epstein E, Miles P (1966) Identification of indirubin as a pigment produced by mutant cultures of the fungus Schizophyllum commune. J Plant Res 79:566–571Google Scholar
  49. 49.
    Hosoe T, Nozawa K, Kawahara N, Fukushima K, Nishimura K, Miyaji M, Kawai K (1999) Isolation of a new potent cytotoxic pigment along with indigotin from the pathogenic basidiomycetous fungus Schizophyllum commune. Mycopathologia 146(1):9–12CrossRefGoogle Scholar
  50. 50.
    Henion J, Eikel D, Linehan SL, Heller D, Murphy K, Rudewicz PJ & Prosser SJ (2011) Liquid extraction surface analysis mass spectrometry (LESA MS) - drug distribution and metabolism of diclofenac in the mouse. in 59th Conference of the American Society for Mass Spectrometry. DenverGoogle Scholar
  51. 51.
    Agilent Technologies I. Considerations for selecting GC/MS or LC/MS for metabolomics. 2007; Available from:
  52. 52.
    Schubert D, Raudaskoski M, Knabe N, Kothe E (2006) Ras GTPase-activating protein gap1 of the homobasidiomycete Schizophyllum commune regulates hyphal growth orientation and sexual development. Eukaryot Cell 5(4):683–695CrossRefGoogle Scholar
  53. 53.
    Kai M, Gonzalez I, Genilloud O, Singh SB, Svatos A (2012) Direct mass spectrometric screening of antibiotics from bacterial surfaces using liquid extraction surface analysis. Rapid Commun Mass Spectrom 26(20):2477–2482CrossRefGoogle Scholar
  54. 54.
    Porta T, Varesio E, Hopfgartner G (2013) Gas-phase separation of drugs and metabolites using modifier-assisted differential ion mobility spectrometry hyphenated to liquid extraction surface analysis and mass spectrometry. Anal Chem 85(24):11771–11779CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Riya C. Menezes
    • 1
    • 2
  • Marco Kai
    • 4
  • Katrin Krause
    • 1
  • Christian Matthäus
    • 2
    • 3
  • Aleš Svatoš
    • 4
    Email author
  • Jürgen Popp
    • 2
    • 3
  • Erika Kothe
    • 1
  1. 1.Department of Microbial Communication, Institute of MicrobiologyFriedrich Schiller UniversityJenaGermany
  2. 2.Leibniz Institute of Photonic Technology e.V.JenaGermany
  3. 3.Institute of Physical Chemistry and Abbe Center of PhotonicsFriedrich Schiller UniversityJenaGermany
  4. 4.Research Group Mass SpectrometryMax Planck Institute for Chemical EcologyJenaGermany

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