Microbiology (BIOSI 1, Main Building), Cardiff University, PO Box 915, Cardiff CF10 3TL, Wales, UK1
School of Applied Science, University of the South Bank, 103 Borough Road, London SET 0AA, UK2
Author for correspondence: David Lloyd. Tel: +44 29 20874772. Fax: +44 29 20874305. e-mail: Lloydd{at}cf.ac.uk
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ABSTRACT |
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Keywords: mitochondria, redox regulation, NADH, cytochrome, ultradian clock
Abbreviations: CCCP, m-chlorocarbonylcyanide phenylhydrazone; DiBAC4(3), bis(1,3-dibutylbarbituric acid)trimethine oxonol; S-13, 5-chloro-3-t-butyl-2-chloro-41-nitrosalicylanilide; DOT, dissolved O2 tension; SHAM, salicylhydroxamic acid
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INTRODUCTION |
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The oscillatory behaviour of respiration is not dependent on glycolytic oscillations as is evidenced by its occurrence with a similar persistence and temperature-compensated period during growth with ethanol (Keulers et al., 1996a , b
; Murray et al., 2001
). Glycolytic oscillations are different in that they are characterized by shorter periods (about 1 to 5 min), are highly damped and not temperature-compensated (Lloyd & Edwards, 1984
, 1987
).
The aim of the present work is to study changes in mitochondrial structure and function in situ during respiratory oscillations. We conclude that growth is accompanied by in vivo respiratory control (ADP-acceptor control) involving cycles of energization and de-energization of mitochondria (Chance & Williams, 1956 ; Luzikov, 1984
; Lloyd, 1974
) in response to energetic demand.
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METHODS |
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Continuous culture was carried out as described previously (Satroutdinov et al., 1992 ). The fermenter (LH500; Adaptive Biosystems) was operated with a stirrer rate of 900 r.p.m., a working volume of 0·8 l, an airflow rate of 180 ml min-1 and a dilution rate of 0·085 h-1. The temperature was 30 °C and the pH was controlled at 3·4 by the addition of 2·5 M NaOH. Dissolved O2 was monitored continuously using an Ingold O2 electrode. Experiments were conducted in continuous light. On line data acquisition was carried out using an in-house constructed PC-based system. Uncouplers of energy conservation [CCCP (m-chlorocarbonylcyanide phenylhydrazone) and S-13 (5-chloro-3-t-butyl-2-chloro-41-nitrosalicylanilide)] were added as alcoholic solutions.
Cellular respiration.
Organisms in growth medium were transferred directly to a closed O2 electrode system at 30 °C (Rank) for measurements of respiration of the culture samples (2 ml) taken at maxima and minima of dissolved O2 tension (DOT) in the oscillating state. Inhibitors [Na azide, SHAM (salicylhydroxamic acid)], or uncouplers (CCCP, S-13) (Hanstein, 1976 ) were added during the initial linear stage of O2 consumption. Where washed non-proliferating organisms were studied, they were centrifuged at 2000 r.p.m. (average 1000 g) for 2 min at room temperature (22 °C) in a MSE Minor Centrifuge. Resuspension was in 4·5 ml 50 mM citric acid/Na citrate buffer (pH 3·4), followed by rapid recentrifugation in haematocrit tubes and resuspension to a final total volume of 0·5 ml in the same buffer. Dilution with the same buffer (1·5 ml) was followed by supplementation to a final concentration of 10 mM glucose in the reaction mixture in the O2 electrode vessel. Some samples were force-aerated with sterile air at 30 °C (200 cm3 min-1) to purge them of volatile fermentation products (e.g. acetaldehyde, H2S, ethanol, acetic acid): the off-gas was led through a cold-trap kept at 77 K using liquid N2.
Measurement of H2S in the culture off-gas.
Exit gas from the oscillating culture was allowed to impinge on a -impregnated test paper; the absorbance of the black deposit of Ag2S was proportional to the p(H2S) in the mobile gas phase. Calibration (Vogel, 1954
) was with standard solutions of Na2S brought to pH 3·4 with H2SO4 and displacement at 30 °C with an air flow rate of 180 cm3 min-1 (identical to that used for aeration of the continuous culture).
Use of membrane-potential-sensitive fluorophores.
For assessment of plasma-membrane electrochemical potential we used 1 µg ml-1 of an oxonol fluorophore, DiBAC4(3) [bis(1,3-dibutylbarbituric acid)trimethine oxonol] (Dinsdale et al., 1995 ). Incubation with organisms still suspended in their culture medium was for 30 min at room temperature (22 °C). For the inner mitochondrial membrane potential, Rhodamine 123 (1 µg ml-1) was employed under identical conditions (Chen, 1988
). Flow cytometry was performed using a Becton-Dickinson cytometer. Excitation and emission wavelengths were 488 nm and 540 nm, respectively, for both the DiBAC4(3)- and Rhodamine 123-stained yeasts. For direct fluorimetric estimation of the latter, organisms were harvested at intervals from culture samples incubated with 1 µM Rhodamine 123 using excitation at 540 nm, emission at 580 nm.
Redox state measurements.
Samples for cytochrome spectra at high and low DOT (3·8 ml culture) were centrifuged at 1000 g for 2 min in a MSE Minor centrifuge using haematocrit tubes. Resuspension was to a volume of 0·2 ml in 1·0 M mannitol. Reducedoxidized cytochrome spectra were obtained at 77 K in a Unicam SP 1800 spectrophotometer, using Perspex cuvettes with a light path of 5 or 2 mm. The oxidized sample was aerated and the reduced sample was kept anaerobic immediately before freezing. Samples were thawed and then reequilibrated to 77 K. Spectra were obtained at a band-width of 2·2 nm and with a scan rate of 2 nm s-1 over a spectral range of 400 to 620 nm.
The redox state of the cellular NAD(P)H/NAD+ couple was monitored continuously in the culture using the method of Harrison & Chance (1970) using a metabolite fluorimeter (Chance et al., 1975
) directly on the fermentation vessel.
Electron microscopy.
Samples were fixed as rapidly as possible by rapid sedimentation (Eppendorf Centrifuge, 14000 r.p.m., 30 s) followed by mixing equal volumes with 0·1 M cacodylate buffer pH 6·9 containing 1% paraformaldehyde, 2% glutaraldehyde, 4% (v/v) sucrose and 0·02% CaCl2. After 1 h at 4 °C, fixed organisms were sedimented and then resuspended in 1% OsO4 at 4 °C for 1 h. Dehydration by passage through a series of ethanol solutions (50, 70 and 90%, 15 min each; 100%, 2x30 min at room temp.). Infiltration with Spurr resin was overnight, followed by fresh resin for 8 h and polymerized overnight at 60 °C. Sections of 6090 nm thickness were obtained using a LKB Ultramicrotome: staining with uranyl acetate was for 15 min, followed by Reynolds lead citrate for 10 min. The microscope was a JEOL 1210 transmission machine.
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RESULTS |
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Changes in membrane potential
Samples taken at peak and trough DOT values were incubated with 1 µg ml-1 of the anionic membrane-potential-sensitive dye DiBAC4(3). The resulting distribution of fluorescence intensities (Fig. 4ac) indicated an almost homogeneous population (>98% of the total) showing fluorescence emission similar to that due to autofluorescence (not shown). This indicates that the fluorophore had not entered the organisms. The minor, highly fluorescent subpopulation consists of damaged organisms with greatly diminished integrity of the plasma membrane. Organisms from maximal or minimal respiratory phases of the ultradian cycle show identical properties with respect to exclusion of the oxonol dye (median channels of fluorescence intensities at arbitrary value 11 in all three samples).
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Cytochromes
Difference spectra (sodium dithionite reduced minus ammonium persulphate oxidized) indicate that cytochromes c, c1, b and a+a3 are not altered in total amounts between the samples taken at respiratory maxima (Fig. 5a) and minima (Fig. 5b
). However, when culture samples were rapidly quenched to 77 K and difference spectra were obtained directly, the resulting absorption maxima (Fig. 5c
) indicated that the steady-state redox balances were different between the maxima and minima of respiration. In the samples from cultures at low DOT, cytochromes c and a+a3 were more oxidized than in those at high DOT.
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DISCUSSION |
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The results presented here strongly suggest that the ultradian-clock-driven oscillatory response of mitochondrial respiration involves continuous cycles of energization and de-energization. There was at any time little detectable flow of electrons through a SHAM-sensitive alternative terminal oxidase and thus electron apportionment did not detectably change at any stage of the oscillation. Experiments in which organisms and medium from the two extremes of respiratory activity were mixed confirm that the observed changes are intrinsic to the organisms and do not primarily arise from alteration of medium composition.
Thus during the stage of high DOT (low respiratory activity), mitochondrial transmembrane electrochemical potential is increased, and in section in electron micrographs the mitochondria appear to be in the orthodox conformation (Hackenbrock, 1968a , b
). In this state, the organelles are characterized by a relatively large matrix volume and with the inner boundary membrane (the non-cristae component of the inner mitochondrial membrane) closely apposed to the outer membrane, with only a small space between them. In this stage, mitochondrial cytochrome b was more highly reduced, as were the intracellular pools of NAD(P)H and the total flavoprotein.
In the phases of low DOT (high respiratory activity) the mitochondria assume the condensed conformation, in which the inner membrane is pulled away from the outer membrane and the cristae are less clearly defined, the total nicotinamide nucleotide pool is more oxidized as are the cytochromes c and a+a3. Thus the succession of events during respiratory oscillation in yeast is similar to that previously described in the soil amoeba Acanthamoeba castellanii (Edwards & Lloyd, 1978 ; Lloyd et al., 1982a
, b
) where it was concluded that the phenomenon represents in vivo mitochondrial respiratory control (ADP-acceptor control; Chance & Williams, 1956
). The slow kinetics indicates that, as in the amoeba, the cycle of mitochondrial changes in yeast is responding to a slowly changing energetic demand of biosynthetic processes (Edwards & Lloyd, 1980
; Lloyd et al., 1981
), i.e. that it is controlled by an ultradian clock (Lloyd & Edwards, 1984
, 1987
). That the respiratory oscillation in S. cerevisiae has a temperature-compensated period and therefore has a timekeeping function has recently been shown (Murray et al., 2001
) The relaxation time of the oscillating control circuit that determines the 48 min cycle time lies in a slower time domain than that characteristic of mitochondrial energy metabolism (milliseconds to seconds); it belongs rather in the domain that typically includes the processes of transcription and translation.
Other changes (e.g. the oscillatory accumulation of the fermentation products ethanol, acetaldehyde and H2S) may contribute to the respiratory oscillation. During glucose-supported growth, the ethanol concentration varied from 110 to 125 mM, whereas acetaldehyde ranged from 0·4 to 0·85 mM, and H2S varied between 1·5 µM and an undetectable concentration. Thus the respiratory inhibitor H2S reaches its highest concentration in the culture when respiration is at its lowest. However, H2S never exceeded 1·5 µM, and at this concentration, inhibition of yeast respiration was less than 10%. Inhibition by acetaldehyde must also occur, but not at the low concentrations (<0·75 mM) monitored during continuous culture (50% inhibition requires 100 mM and its maximum accumulation corresponds to maximum respiration).
Unlike the oscillatory respiratory metabolism studied in A. castellanii (Edwards & Lloyd, 1978 , 1980
; Lloyd et al., 1982a
) or Schizosaccharomyces pombe (Poole et al., 1973
), that of Sacch. cerevisiae reported here is not directly coupled to the cell division cycle, i.e. does not require cultures in which the growth and division of the population is synchronized for its detection. Yet there must be synchronization of individual organisms to generate coherence in the populations so as to result in an observable oscillatory behaviour. Rather, in this case there is metabolic coupling between individual cells of the culture. It has been proposed that highly diffusible volatile low molecular weight compound(s), e.g. acetaldehyde and H2S, may act as intercellular signalling substances required to maintain coherence of the population (Keulers &Kuriyama, 1998
; Sohn et al., 2000
; Sohn & Kuriyama, 2001
). This must be a rapid process (with a time scale of seconds rather than minutes) to coordinate the oscillatory behaviour with such precision (Klevecz & Murray, 2001
).
Further work is required to determine how the oscillation is controlled, i.e. the extramitochondrial events that produce the slow kinetics of transcriptional switching that generate the time-base of the temperature-compensated ultradian clock (Murray et al., 2001 ; Salgado et al., 2002
). The continuously monitored system described here for study of ultradian clock outputs in S. cerevisiae is more convenient and amenable than other ultradian systems (Lloyd, 1992
) and many circadian systems (Lloyd et al., 1982b
; Lloyd, 1988
) where only discrete sampling is possible (Lloyd & Murray, 2000
), and where the accumulation of useful data requires more extended time intervals.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 15 April 2002;
revised 18 June 2002;
accepted 25 June 2002.
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