Convenient fluorescence-based methods to measure membrane potential and intracellular pH in the Archaeon Methanobacterium thermoautotrophicum

New and improved methods to determine the membrane potential (Delta psi) an d the Delta pH in methanogenic archaea were developed and tested in Methano bacterium thermoautotrophicum strain DeltaH. The Delta pH measurements took advantage of the pH-dependent fluorescence properties of coenzyme F-420, t he major intracellular electron carrier in the organism. The protonophore p -nitrophenol did not show any interference with the F-420 fluorescence spec tra and was therefore suitable to equalize internal and external pH. The me thod developed allowed the determination of the intracellular pH with an er ror of less than 0.05 pH units. Membrane potentials could easily be assessed using the fluorescent probe bi s-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC(4)(3)) with an accur acy of approximately 10 mV. Both methods were tested with cell suspensions of M. thermoautrophicum incu bated at medium pH values between 5.5 and 8. It was found that Delta psi an d Delta pH values remained constant under these conditions. Membrane potent ials were about - 160 mV and Delta pH was kept at 0.35 pH units (inside min us outside) resulting in a total proton motive force of about - 180 mV (ins ide negative). (C) 2001 Elsevier Science B.V. All rights reserved.