A Spectrofluorophotometrical Method Based on Fura-2-AM Probe to Determine Cytosolic Ca2+ Level in Pseudomonas syringae Complex Bacterial Cells
Simone Trabalza,
Roberto Buonaurio,
Alberto Del Pino,
Carlo Palmerini,
Harrold van den burg,
Chiaraluce Moretti
Affiliations
Simone Trabalza
Department of Agricultural, Food and Environmental Science, University of Perugia, Perugia, ItalyMolecular Plant Pathology, Swammerdam Institute for Life Sciences (SILS), University of Amsterdam, Amsterdam, the Netherlands
Roberto Buonaurio
Department of Agricultural, Food and Environmental Science, University of Perugia, Perugia, Italy
Alberto Del Pino
Department of Agricultural, Food and Environmental Science, University of Perugia, Perugia, Italy
Carlo Palmerini
Department of Agricultural, Food and Environmental Science, University of Perugia, Perugia, Italy
Harrold van den burg
Molecular Plant Pathology, Swammerdam Institute for Life Sciences (SILS), University of Amsterdam, Amsterdam, the Netherlands
Chiaraluce Moretti
Department of Agricultural, Food and Environmental Science, University of Perugia, Perugia, Italy
Calcium signaling is an emerging mechanism by which bacteria respond to environmental cues. To measure the intracellular free-calcium concentration in bacterial cells, [Ca2+]i, a simple spectrofluorometric method based on the chemical probe Fura 2-acetoxy methyl ester (Fura 2-AM) is here presented using Pseudomonad bacterial cells. This is an alternative and quantitative method that can be completed in a short period of time with low costs, and it does not require the induction of heterologously expressed protein-based probes like Aequorin. Furthermore, it is possible to verify the properties of membrane channels involved in Ca2+ entry from the extracellular matrix. This method is in particular valuable for measuring [Ca2+]i in the range of 0.1-39.8 µM in small cells like those of prokaryotes.
