Detection of Extracellular Phosphatase Activity of Heterotrophic Prokaryotes at the Single‐Cell Level by Flow Cytometry

Solange Duhamel1, Gerald Gregori1, France Van Wambeke1, Jiří Nedoma2

1 Université de la Méditerranée, CNRS, Marseille, France, 2 Academy of Sciences of the Czech Republic, České Budějovice, Czech Republic
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.18
DOI:  10.1002/0471142956.cy1118s49
Online Posting Date:  July, 2009
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Monitoring cell activity using substrates, which turn fluorescent due to biological activity, allows observing the presence and dynamics of sub‐populations, and provides a very valuable insight in ecological studies. The phosphatase substrate ELF97 phosphate (ELF‐P) is a useful tool to detect and quantify phosphatase activity (PA) of microorganisms at the single‐cell level. Most of the studies dealing with PA at the single‐cell level focus on autotrophic cells and only few concern heterotrophic prokaryotes (referred as bacteria in the text). While flow cytometry is a promising tool to assess the single‐cell analysis, only microscopy tools have been used until now to measure the ELF labeling associated with bacteria expressing PA. Therefore, we have developed a new protocol that enables the detection of ELF alcohol (ELFA), the product of ELF‐P hydrolysis, making possible the specific identification of bacteria showing PA using flow cytometry. Curr. Protoc. Cytom. 49:11.18.1‐11.18.8. © 2009 by John Wiley & Sons, Inc.

Keywords: aquatic heterotrophic bacteria; phosphatase activity; ectoenzyme; flow cytometry; cell concentration; functional measurement

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • Fresh marine or freshwater sample (i.e., non‐preserved)
  • Tween 80 diluted to 100 mg/liter (polyoxyethylenesorbitan monooleate; Sigma)
  • ELF‐P solution (see recipe)
  • PBS/formalin solution (see recipe)
  • 0.2 µm‐filtered sample
  • Internal standard microsphere mixture: 2 µm‐beads (Fluoresbrite, Polysciences) in sheath fluid
  • Sheath fluid made of 0.2 µm‐filtered distilled water
  • DAPI solution (see recipe)
  • 15‐ml Beckman polyallomer centrifuge tubes
  • Centrifuge (capable of generating acceleration ≥20,000 × g) with rotor adapted to 15‐ml Beckman polyallomer centrifuge tubes
  • Sterile pipets
  • Vortex
  • Syringe filters, 0.2‐µm pore (Sartorius)
  • A flow cytometer equipped with:
    • UV light source (351 nm)
    • 440‐nm long‐pass dichroic mirror to separate DAPI fluorescence from ELFA fluorescence
    • 375‐ to 435‐nm band‐pass filter for isolating DAPI fluorescence
    • 490‐ to 570‐nm band‐pass filter for isolating ELFA fluorescence
    • 620‐nm long‐pass filter for the detection of Chlorophyll a fluorescence
    • Flow cytometry software to acquire and analyze the data
  • 12 × 75–mm flow cytometry test tubes
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Literature Cited

   Dignum, M., Hoogveld, H.L., Floris, V., Gons, H.J., Matthijs, H.C.P., and Pel, R. 2004a. Flow cytometric detection of phosphatase activity combined with 13C‐CO2 tracer‐based growth rate assessment in phytoplankton populations from a shallow lake. Aquat. Microb. Ecol. 37:159‐169.
   Dignum, M., Hoogveld, H.L., Matthijs, H.C.P., Laanbroek, H.J., and Pel, R. 2004b. Detecting the phosphate status of phytoplankton by enzyme‐labelled fluorescence and flow cytometry. FEMS Microbiol. Ecol. 48:29‐38.
   Duhamel, S., Gregori, G., Van Wambeke, F., Mauriac, R., and Nedoma, J. 2008. A method for analysing phosphatase activity in aquatic bacteria at the single‐cell level using flow cytometry. J. Microbiol. Methods 75:269‐278. doi:10.1016/j.mimet.2008.1006.1018.
   Duhamel, S., Gregori, G., Van Wambeke, F., Nedoma, J. 2009. Detection of extracellular phosphatase activity at the single‐cell level by enzyme‐labeled fluorescence and flow cytometry: The importance of time kinetics in ELFA labeling. Cytometry A 75:163‐168.
   Dyhrman, S.T. and Palenik, B. 1999. Phosphate stress in cultures and field populations of the dinoflagellate Prorocentrum minimum detected by a single‐cell alkaline phosphatase assay. Appl. Environ. Microbiol. 65:3205‐3212.
   Elser, J. and Kimmel, B. 1986. Alteration of phytoplankton phosphorus status during enrichment experiments: Implications for interpreting nutrient enrichment bioassay results. Hydrobiologia 133:217‐222.
   Gonzalez‐Gil, S., Keafer, B.A., Jovine, R.V.M., Aguilera, A., Lu, S., and Anderson, D.M. 1998. Detection and quantification of alkaline phosphatase in single cells of phosphorus‐starved marine phytoplankton. Mar. Ecol. Prog. Ser. 164:21‐35.
   Hoppe, H.‐G. 2003. Phosphatase activity in the sea. Hydrobiologia 493:187‐200.
   Huang, Z., You, W., Haugland, R.P., Paragas, V.B., Olson, N.A., and Haugland, R.P. 1993. A novel fluorogenic substrate for detecting alkaline phosphatase activity in situ. J. Histochem. Cytochem. 41:313‐317.
   Karl, D.M. 2000. Phosphorus, the staff of life. Nature 406:31‐32.
   Labry, C., Delmas, D., and Herbland, A. 2005. Phytoplankton and bacterial alkaline phosphatase activities in relation to phosphate and DOP availability within the Gironde plume waters (Bay of Biscay). J. Exp. Mar. Biol. Ecol. 318:213‐225.
   Mackey, K.R.M., Labiosa, R.G., Calhoun, M., Street, J.H., Post, A.F., and Paytan, A. 2007. Phosphorus availability, phytoplankton community dynamics, and taxon‐specific phosphorus status in the Gulf of Aqaba, Red Sea. Limnol. Oceanogr. 52:873‐885.
   Marie, D., Simon, N., Guillou, L., Partensky, F., and Vaulot, D. 2000. Enumeration of phytoplankton, bacteria, and viruses in marine samples. Curr. Protoc. Cytom. 10:11.11.1‐11.11.15.
   Nedoma, J., štrojsová, A., Vrba, J., Komárková, J., and šimek, K. 2003. Extracellular phosphatase activity of natural plankton studied with ELF97 phosphate: Fluorescence quantification and labelling kinetics. Environ. Microbiol. 5:462‐472.
   Nicholson, D., Dyhrman, S., Chavez, F., and Paytan, A. 2006. Alkaline phosphatase activity in the phytoplankton communities of Monterey Bay and San Francisco Bay. Limnol. Oceanogr. 51:874‐883.
   Telford, W.G., Cox, W.G., Stiner, D., Singer, V.L., and Doty, S.B. 1999. Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF‐97 phosphatase substrate. Cytometry 37:314‐319.
   Van Wambeke, F., Nedoma, J., Duhamel, S., and Lebaron, P. 2008. Alkaline phosphatase activity of marine bacteria studied with ELF97 phosphate: success and limits in the P‐limited Mediterranean Sea. Aquat. Microb. Ecol. 52:245‐251. doi: 10.3354/ame01238.
Key References
   Duhamel et al., 2008. See above.
   Duhamel et al., 2009. See above.
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