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) and the Delta pH in methanogenic archaea were developed and tested in Methanobacterium thermoautotrophicum strain Delta H. The Delta pH measurements took advantage of the pH-dependent fluorescence properties of coenzyme F(420), the major intracellular electron carrier in the organism. The protonophore p-nitrophenol did not show any interference with the F(420) fluorescence spectra and was therefore suitable to equalize internal and external pH. The method developed allowed the determination of the intracellular pH with an error of less than 0.05 pH units.Membrane potentials could easily be assessed using the fluorescent probe bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC(4)(3)) with an accuracy of approximately 10 mV. Both methods were tested with cell suspensions of M. thermoautrophicum incubated at medium pH values between 5.5 and 8. It was found that Delta Psi and Delta pH values remained constant under these conditions. Membrane potentials were about -160 mV and Delta pH was kept at 0.35 pH units (inside minus outside) resulting in a total proton motive force of about -180 mV (inside negative).
de Poorter
L M
LM
Department of Microbiology, Faculty of Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED, Nijmegen, The Netherlands.
Keltjens
J T
JT
D001463:Barbiturates / Q000378:metabolism; D002138:Calibration; D003470:Culture Media; D003593:Cytoplasm / Q000378:metabolism; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007555:Isoxazoles / Q000378:metabolism; D008564:Membrane Potentials; D017014:Methanobacterium / Q000166:cytology / Q000502:physiology*; D018892:Proton-Motive Force / Q000502:physiology; D012256:Riboflavin / Q000031:analogs & derivatives* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D001463:Barbiturates; D003470:Culture Media; D007555:Isoxazoles; C052795:bis(1,3-dibutylbarbiturate)trimethine oxonol; C015084:coenzyme F420; D012256:Riboflavin