Phosphorus (P) is essential to life and its role to sustain ocean productivity is fundamental. The most bioavailable form is dissolved inorganic P (DIP) as orthophosphate, but many compounds of dissolved organic P (DOP) pool can be used as a P source, even under P-sufficient microbial growth. Alkaline phosphatase is one of several potential DOP hydrolyzing enzymes, but it is the best studied and arguably the most important for nutrition since it has the potential to hydrolyze a broad spectrum of DOP compounds (e.g., the phosphomonoesters [P esters]).
The substrate ELF-97 [2-(59-chloro-29-phosphoryloxyphenyl)-6- chloro-4-(3H)-quinazolinone] -phosphate (ELF-P) is used to detect and quantify cell-attached extracellular phosphatase activity. By enzymatic hydrolysis, the water-soluble ELF-P is converted into ELF-alcohol (ELFA) which is insoluble, specific, photostable and highly fluorescent and precipitates at the site of enzyme activity. This enables phosphatase activity to be detected at the single cell level (see figure below).
Before the start of our project, most studies using the ELF labelling technique was on phytoplankton and no protocol was available for marine bacteria.
We thus developed a microscopy approach to measure alkaline phosphatase activity in bacteria using ELF-P :
Van Wambeke F., Nedoma J., Duhamel S., and P. Lebaron, 2008. Alkaline phosphatase activity of marine bacteria studied with ELF97 phosphate: Success and limits in P-limited Mediterranean Sea. Aquatic Microbial Ecology 52, 245–251. DOI: 10.3354/ame01238. Access
Then we developed a flow cytometry approach to detect alkaline phosphatase activity in bacteria using ELF-P:
Duhamel S., Gregori G., Van Wambeke F., and J. Nedoma, 2009. Detection of extracellular phosphatase activity of heterotrophic prokaryotes at the single cell level by flow cytometry. Current Protocols in Cytometry 11.18. DOI: 10.1002/0471142956.cy1118s49. Access
Duhamel S., Gregori G., Van Wambeke F., Mauriac R., and J. Nedoma, 2008. A method for analysing phosphatase activity in aquatic bacteria at the single cell level using flow cytometry. Journal of Microbiological Methods 75, 269–278. DOI:10.1016/j.mimet.2008.1006.1018. Access
We demonstrated the importance of testing ELF-labeling incubation time to properly quantify alkaline phosphatase activity at the single-cell level:
Duhamel S., Gregori G., Van Wambeke F., and J. Nedoma, 2009. Detection of extracellular phosphatase activity at the single cell level by enzyme-labelled fluorescence and flow cytometry: the importance of time kinetics in ELFA labelling. Cytometry: Part A 75A, 163-168. DOI: 10.1002/cyto.a.20686. Open access
We showed that marine and freshwater microbes have similar affinities for the ELF-P substrate than for the more widely used MUF-P substrate which are water soluble (and thus do not allow cell-specific characterization):
Nedoma J., Van Wambeke F., Strojsova A., Strojsova M., and S. Duhamel, 2007. Affinity of extracellular phosphatases for ELF 97 phosphate in aquatic environments. Marine and Freshwater Research 58(5), 454–460. Access
We then successfully applied this method to samples from the North Pacific subtropical gyre at Station ALOHA. First we demonstrated that alkaline phosphatase activity by marine microbes is regulated by phosphate concentration:
Duhamel S., Dyhrman S.T., and D.M. Karl, 2010. Alkaline phosphatase activity and regulation in the North Pacific Subtropical Gyre. Limnology and Oceanography, 55(3), 1414–1425. Open access
A poster can be found here:
Finally, we evaluated the role of alkaline phosphatase activity in the North and South Pacific subtropical gyres:
Duhamel S., Björkman K.M., Van Wambeke F., Moutin T., and D.M. Karl, 2011. Characterization of alkaline phosphatase activity in the North and South Pacific subtropical gyres: implication for the phosphorus cycle. Limnology and Oceanography 56, 1244-1254. Open access
