Abstract
The fluorogenic substrate Enzyme-Labeled Fluorescence 97 (ELF-P) is hydrolyzed by the P-cleaving enzyme phosphatase, producing ELF 97 alcohol (ELFA), a fluorescent-insoluble product. This reaction is used for monitoring phosphatase activity at the single-cell level. Most frequently, ELF-P has been used to determine the P-limitation status of microphytoplankton, but rarely of heterotrophic bacteria. We incubated ELF-P on filters to monitor marine bacterial cultures and oligotrophic Mediterranean Sea samples. Results were compared to classical measurements of bulk alkaline phosphatase activity using the fluorogenic substrate 4-methylumbelliferyl phosphate (MUF-P). A high percentage of the cultured cells were labeled with ELFA (the ratio of ELFA spots to total DAPI counts in P-limited cultures ranged from 26 to 100%, depending on the strain). In contrast, this ratio never exceeded 0.01 % in Mediterranean samples, even when P was demonstrated to be a significant limiting factor. This protocol is useful for application on cruises and with cultures, but was not sufficiently sensitive to detect P-stressed bacterial cells in oligotrophic marine environments.
Highlights
Developing new techniques for detecting phosphorus (P)-stress in bacteria at the cell level from natural environments is a challenge in microbial ecology
Culture populations and field communities of heterotrophic bacteria exhibited different phosphatase activities as seen from the specific activities found with methylumbelliferyl phosphate (MUF-P) substrate and the varying percentages of ELF labeling
A minimum threshold of extracellular phosphatase activity of 10 fmol cell–1 h–1 for phytoplankton (Nedoma et al 2003) and 0.17 fmol cell–1 h–1 for bacteria in acidified mountain lakes (Nedoma & Vrba 2006) is necessary to observe significant ELF alcohol (ELFA) spot formation and to allow quantitative per cell estimates
Summary
Developing new techniques for detecting phosphorus (P)-stress in bacteria at the cell level from natural environments is a challenge in microbial ecology. The molecule 2-(5’-chloro2’-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (Enzyme-Labeled-Fluorescence 97 phosphatase substrate [ELF-P], Molecular Probes) is a soluble substrate, which, when cleaved by the cell’s enzyme (phosphatase), produces ELF 97 alcohol (ELFA), a bright fluorescent yellow-green precipitate labeling the site of enzymatic activity (Gonzalez-Gil et al 1998) In aquatic systems, this technique has mainly been used as a tool for identifying P-limited phytoplankton cells. Only 4 published studies have focused on natural bacterial communities This includes studies on activated sludge (Van Ommen Kloeke & Geesey 1999), an acidified lake (Nedoma & Vrba 2006), and bacterial colonies and biofilms (Huang et al 1998, Espeland & Wetzel 2001). In most of these studies, the abundance and activity of bacteria were fairly high
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