Fluorescence measurements of free [Mg2+] by use of mag-fura 2 in Salmonella enterica.
The Mg2+ fluorescent dye mag-fura 2, entrapped in cells or organelles, has frequently been used for dual excitation ratio-metric determinations of free ionic Mg2+ concentrations in eukaryotic, mostly mammalian cells. Here we report its successful application to measure free Mg2+ concentrations ([Mg2+]i) in Salmonella enterica cells. When kept in nominally Mg2+ free buffer (resting conditions), the [Mg2+]i of wild-type cells has been determined to be 0.9 mM. An increase in the external Mg2+ concentration ([Mg2+]e) resulted in a rapid increase of [Mg2+]i, saturating within a few seconds at about 1.5 mM with [Mg2+]e of 20 mM. In contrast, cells lacking the Mg2+ transport proteins CorA, MgtA, MgtB failed to show this rapid increase. Instead, their [Mg2+]i increased steadily over extended periods of time and saturated at concentrations below those of wild-type cells. Mg2+ uptake rates increased more than 15-fold when corA was overexpressed in these mutant cells. Uptake of Mg2+ into corA expressing cells was strongly stimulated by nigericin, which increased the membrane potential DeltaPsi at the expense of DeltapH, and drastically reduced by valinomycin, which decreased the membrane potential DeltaPsi. These results reveal mag-fura 2 as a useful indicator to measure steady-state [Mg2+]i values in resting bacterial cells and to determine Mg2+ uptake rates. They confirm the role of CorA as the major Mg2+ transport protein and reveal the membrane potential as driving force for Mg2+ uptake into S. enterica cells.
Max F. Perutz Laboratories, Department of Microbiology and Genetics, University of Vienna, Campus Vienna Biocenter, Dr. Bohrgasse 9, A-1030 Vienna, Austria.
Elisabeth M
EM
Froschauer
Martin
M
Kolisek
Frank
F
Dieterich
Monika
M
Schweigel
Rudolf J
RJ
Schweyen
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D027682:Cation Transport Proteins / Q000235:genetics / Q000502:physiology; D002413:Cations, Divalent / Q000032:analysis*; D003593:Cytoplasm / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000494:pharmacology; D016257:Fura-2 / Q000031:analogs & derivatives* / Q000494:pharmacology*; D007476:Ionophores / Q000494:pharmacology; D008274:Magnesium / Q000032:analysis*; D008564:Membrane Potentials; D026901:Membrane Transport Proteins / Q000235:genetics / Q000502:physiology; D009550:Nigericin / Q000494:pharmacology; D018892:Proton-Motive Force; D019779:Salmonella enterica / Q000737:chemistry* / Q000378:metabolism; D014634:Valinomycin / Q000494:pharmacology
D001426:Bacterial Proteins; D027682:Cation Transport Proteins; D002413:Cations, Divalent; C072716:CorA protein, Salmonella; D005456:Fluorescent Dyes; D007476:Ionophores; D026901:Membrane Transport Proteins; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; D014634:Valinomycin; D000251:Adenosine Triphosphatases; C093585:MgtA protein, bacteria; C083836:MgtB protein, Salmonella typhimurium; D008274:Magnesium; D009550:Nigericin; D016257:Fura-2