Institute of Plant Biology, University of Zürich, Zollikerstraße 107, CH-8008 Zürich, Switzerland1
Author for correspondence: Reinhard Bachofen. Tel: +41 1 634 82 80. Fax: +41 1 634 82 04. e-mail: bachofen{at}botinst.unizh.ch
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ABSTRACT |
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Keywords: cytoplasm, pH gradient, intracellular pH, protonmotive force, methanogenic bacteria
Abbreviations: BESA, bromoethanesulfonic acid; CF, carboxyfluorescein; TCS, tetrachlorosalicylanilide
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INTRODUCTION |
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In suspension cultures of micro-organisms, the external pH is easily followed by using electrodes. In contrast, the determination of cytoplasmic pH in cells as small as bacteria is more difficult. Certain methods are based on the distribution of radiolabelled weak acids or bases whereby the cells (after incubation) are separated from the medium by rapid filtration or centrifugation. As sampling is periodic, the data from these methods are not continuous (Padan et al., 1981 ). Spectrometric methods, however, either absorption or fluorescence spectrometry, use specific pH indicators to monitor the cytoplasmic pH continuously.
The first descriptions of pH estimations using dyes (fluorogenic esters) were given by Thomas et al. (1979 , 1982
) for tumour cells and bacteria. The prerequisites of the method are as follows: (1) the cells must be permeable to these colourless and non-fluorescent esters; (2) the indicators must be concentrated in the cells; (3) an intracellular esterase must cleave off an absorbing or fluorescing species; (4) the membrane must be impermeable to the negatively charged species formed by the hydrolysis (so that the indicator remains in the cells, at least for the duration of the experiment); (5) a calibration curve must be obtained; and (6) no other cellular compounds should interfere (by absorbance or fluorescence) with the marker compound. Generally, not all of these prerequisites are fulfilled. To prevent leakage of the pH-indicator, a dye forming covalent bonds with cytoplasmic compounds has been developed (Breeuwer et al., 1996
). However, the question as to whether or not indicator dyes interact with cytoplasmic proteins and cause erroneous results remains open to debate (Yassine et al., 1997
). To compensate for unequal uptake of the dye and varying esterase activity, fluorescence ratios from two wavelengths were analysed to follow the pH (Aono et al., 1997
). Recently, fluorescence ratio microscopy imaging even made it possible to follow the pH of single bacterial cells in a mixed culture (Siegumfeldt et al., 1999
). These studies demonstrate the versatility of spectroscopic techniques in the investigation of pH homeostasis and the dynamics of pH changes during energy transduction.
The metabolism (including energy transduction) of methanogenic bacteria has been studied intensively in recent decades, as reviewed, for example, by Deppenmeier et al. (1996) and Schäfer et al. (1999)
. In the process of the stepwise reduction of CO2 to CH4 by H2, protons are extruded, giving rise to a pH gradient, which, along with a Na+ gradient and the membrane potential, is an important component of the driving force for ATP synthesis in a chemiosmotic mechanism. Changes in membrane potential upon the energization of whole cells of Methanobacterium thermoautotrophicum have been measured by Butsch & Bachofen (1984)
. Using the dye carboxyfluorescein (CF), Bachofen & Butsch (1986)
demonstrated, qualitatively, the formation of a pH gradient upon cell energization. The length of the signal correlated with the partial pressure of hydrogen gas introduced, whereas its magnitude was independent of the partial pressure over the range 2080% (v/v) hydrogen in the gas mixture.
In the present work, the endogenous factor F420 was used as an intrinsic pH indicator. It fulfils most of the requirements cited above for a cytoplasmic pH indicator.
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METHODS |
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Organism, growth medium and growth conditions.
M. thermoautotrophicum strain Hveragerdi (DSM 3590) was isolated earlier in our laboratory (Butsch & Bachofen, 1984 ). Stock cultures were kept at -80 °C. The medium was based on that of Schönheit et al. (1979)
, as modified by Butsch & Bachofen (1984)
, and contained the following: NH4Cl (40 mM), MgCl2.6H2O (1·5 mM), nitrilotriacetate (0·15 mM), NaCl (10 mM), KH2PO4 (10 mM), CoCl2.6H2O (1 µM), Na2MoO4.2H2O (1 µM), NiCl2.6H2O (1 µM) and FeCl2.4H2O (25 µM). Cells were grown in chemostat mode in a 2 l bioreactor equipped with controls for temperature, pH and redox potential, as described by Jud et al. (1997)
. The temperature was held at 58 °C and the pH at 6·9. The culture was supplied with Na2S (310 mM) at intervals, producing a final concentration of 0·5 mM sulfide in the reactor. The gas supplied was H2/CO2, 80%:20% (v/v); the rate was controlled electronically and kept at 220 ml min-1 (equal to 0·12 vol. per vol. per min). Traces of oxygen were removed by a BASF catalyst, R020, sealed in an iron tube in the gas supply line. The sterile media were kept under nitrogen.
Spectroscopic investigations.
The absorption spectra of solutions were obtained with a Uvicon 810 spectrophotometer. Optical measurements of cell suspensions were obtained with an Aminco DW-2 dual-wavelength spectrophotometer using either specially made anaerobic cuvettes with rubber septa or a 30 ml minibioreactor built in our workshop and coupled to the DW-2 optics by light pipes. The spectrophotometer was connected to a computer with an ADALAB A/D converter (Interactive Microware).
Gas analysis.
Hydrogen, methane, oxygen, carbon monoxide and carbon dioxide were quantified by gas chromatography [using a Shimadzu GC-R1A with integrator RPR-G1 and a CSS column Carbosieve S 120/140 (Supelco)] with a TCD detector. The gas was supplied reproducibly to the cuvettes and the minibioreactor through a stainless steel needle as pulses of 10 s at a flow rate of 240 ml min-1 by a computer-controlled valve.
Cell preparations.
Cells were harvested by centrifugation (15 min at 1800 g, 4 °C) and washed twice with growth medium. All steps were performed under anoxic conditions using bottles flushed nine times with nitrogen (cycling between 0·5 and 2 bar). For the experiments, the cell concentration, measured as OD660, was set between 1·5 and 2, which is equivalent to 0·81·1 g cells (dry weight) l-1. The suspension was transferred either into an anaerobic cuvette equipped with a valve for gas pulses or into the minibioreactor in an anaerobic box (Forma Scientific 1024). To ensure the complete absence of oxygen, all glassware was kept in the anaerobic box for 24 h prior to the experiments. All manipulations were done under strictly anaerobic conditions, in closed vessels under purified nitrogen or in the anaerobic glove box. If these precautions for anoxic conditions were observed, the cells could be used reproducibly in experiments over a period of at least 4 h.
Measurement of the pHin.
The cuvette or the minibioreactor was stirred and kept at constant temperature under strictly anaerobic conditions during the measurements. The Aminco DW-2 spectrophotometer was used in the dual-wavelength mode, which allows the quantification of small absorption changes in the presence of a large optical background signal (cell scatter). The determination of the relevant wavelengths and the calibration of the intracellular pH (pHin) are described in Results.
Isolation of F420.
F420 was isolated, according to Cheeseman et al. (1972) and Schönheit et al. (1981)
, by extracting the compound using 50% (v/v) acetone followed by chromatography twice on QAE-Sephadex A-25.
Other determinations.
The minibioreactor was equipped with electrodes for the continuous measurement of pH, redox potential and Na+ ions (Ingold). The OD600 of the cell suspension was calibrated by using dry-weight determination.
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RESULTS AND DISCUSSION |
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The experiment presented in Fig. 2 is an example showing the kinetics of the changes in pHin as calculated from the calibration curve. The rapid alkalinization of the cytoplasm upon the substrate pulse cannot be visualized because bubbles produced during gas injection make optical measurements impossible. Under defined conditions, the duration of the alkalinization until the pHin returned to the initial pH of the medium was highly reproducible within the same cell preparation but it could vary between cell batches. The redox potential in the medium did not change after a hydrogen pulse (not shown). The size of the pH increase in the cytoplasm was dependent on the pHout: it is larger at acid values of the medium pH and becomes smaller towards neutrality. Around neutrality, Schönheit & Beimborn (1985)
found, using M. thermoautotrophicum, that they could not measure a
pH under metabolically active conditions; in a more acidic environment (pH 5), however, the cytoplasm was found to be more alkaline, resulting in a pH gradient of 11·3 pH units under energized conditions. This is in close agreement with our observations. In contrast, the
pH reported by Dybas & Konisky (1992)
for Methanococcus voltae under growing conditions was only a few millivolts.
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It has been suggested that F420 is not homogeneously distributed within the cell but is, rather (for functional reasons) concentrated near the membrane at the site of the hydrogenase (Muth, 1988 ). Thus, an averaging method such as the distribution of weak acids and bases (Schönheit & Beimborn, 1985
) or measurement with homogeneously distributed indicator dyes may not give the same values as a more localized pH indicator (Kotyk & Slavik, 1989
). The pHin measured by F420 probably does not indicate the mean pH within the cell but, rather, represents the pH found close to the membrane. This would support the suggestion that the pumped protons are held along the membrane by anionic lipids and are not in equilibrium with the cytoplasm (Haines, 1983
).
The concentration of Na+ ions in the medium, important for the formation of the in methanogens, was measured simultaneously. It increases when hydrogen is supplied, and the return to the original value is delayed relative to the decay of the
pH. The decay clearly accelerates after the
pH has dropped to below approximately 50% of the original size (Fig. 4
). At the cell concentration chosen for the experiments, the ratio between the volume of medium and the volume of cells has been estimated to be approximately 600. Thus, an Na+ increase of a few millimoles per litre in the medium upon an H2 pulse would represent a drastic decrease in the ion concentration in the cell. Although most of the Na+ ions may have been bound to cell components, it indicates the formation of a noticeable membrane potential. Indeed, it has been suggested that Na+ and K+ ions are strongly complexed with specific lipids of the cell membrane (Kramer et al., 1988
). The Na+ efflux is probably the result of a directly coupled Na+ pump, whereas the Na+/H+ antiporter driven by H+ extrusion during methanogenesis acts as a mechanism for regulating the pHin (Deppenmeier et al., 1996
; Schäfer et al., 1999
). Because of the presence of CO2 in the gas pulse, the pH of the medium drops slightly and returns slowly (within 3040 s) to the original value before the pulse (Fig. 4
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 22 May 2000;
revised 30 August 2000;
accepted 12 September 2000.
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