FEMS Microbiology Letters 236 (2004) 261–266
www.fems-microbiology.org
Sustained glycolytic oscillations – no need for cyanide
Allan K. Poulsen
a
a,*
, Frants R. Lauritsen
a,b
, Lars Folke Olsen
a
CelCom, Department of Biochemistry and Molecular Biology, Syddansk Universitet, Campusvej 55, DK-5230 Odense M, Denmark
Department of Chemistry – Physical Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen Ø, Denmark
b
Received 15 March 2004; received in revised form 25 May 2004; accepted 27 May 2004
First published online 9 June 2004
Abstract
Using fluorescence spectroscopy we detected long trains of macroscopic oscillations in the glycolytic pathway, in whole cell
suspensions of Saccharomyces cerevisiae, without addition of cyanide. Such oscillations may be induced if argon or another inert gas
is bubbled through the yeast cell suspension. This supports that the synchronizing agent is a volatile compound secreted by the yeast
cells, e.g. CO2 and/or acetaldehyde. Our results show that the rate of acetaldehyde removal is not a crucial parameter to the
synchronization of the yeast cells. The sample cell was connected to a membrane inlet mass spectrometer (MIMS) for online determination of extracellular non-polar compounds. Oscillations in the secretion of CO2 were detected using the MIMS.
Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Glycolytic oscillations; Yeast; CSTR; Carbondioxide; Acetaldehyde; MIMS
1. Introduction
It has been known for several decades that adding a
pulse of glucose to a suspension of yeast cells may result
in oscillations in the glycolytic pathway. In 1957 Duysens and Amesz [1] found, by studying glycolytic intermediates in yeast by fluorescence spectroscopy, that one
of these underwent a damped oscillation with a period
of about 1 min if glucose was added to starved cells. In
1964 Chance et al. [2] continued the experiments with
the damped oscillations initiated by the transition from
aerobic to anaerobic metabolism (the Pasteur effect).
They found by adding glucose to the yeast cells, following addition of sulphide, that the system had to be
shocked into oscillation by inhibition of respiration, and
that blocking of respiration greatly increased both the
amplitude and the number of cycles. Later Betz and
Chance [3] obtained remarkably enhanced oscillations in
yeast suspensions by adding cyanide instead of sulphide.
Further studies by Ghosh et al. [4] of the effect of different inhibitors of respiration showed an increased
*
Corresponding author. Tel.: +45-6550-2478; fax: +45-6550-2467.
E-mail address: [email protected] (A.K. Poulsen).
number of less damped oscillations in the presence of
aldehyde traps such as Girards reagent P, semicarbazide, NADH + acetaldehyde dehydrogenase or cyanide.
If other inhibitors of respiration such as antimycin A
and azide were used instead of cyanide, the oscillations
were more damped [4,5].
Addition of glucose to starved yeast cells and the
inhibition of respiration due to anaerobiosis or inhibition of respiration by any chemical inhibitor is not
sufficient to gain long lasting glycolytic oscillations. The
yeast suspension has further requirements for long
lasting oscillations of intracellular NADH to occur.
Richard et al. [6] found that cyanide must have an additional effect to simulation of anaerobiosis, since other
inhibitors of respiration lead to much shorter trains of
oscillations. Their conclusion was that acetaldehyde is
removed from the extracellular liquid through the reaction of acetaldehyde with cyanide. Furthermore, they
suggested that the acetaldehyde has to be removed at a
certain rate in order to stimulate oscillations. Later
Richard et al. [7] proposed that the subtle balance between secretion of acetaldehyde by the cells and acetaldehyde trapping by reaction with KCN is important for
the coupling to occur and they measured a rate constant
0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2004.05.044
262
A.K. Poulsen et al. / FEMS Microbiology Letters 236 (2004) 261–266
of 1.5 M 1 s 1 for the cyanide/aldehyde reaction forming lactonitrile. This means that free acetaldehyde has a
half-life of approximately 1 min under the conditions
they have stated, and similar to the conditions used in
the experiments described here.
Cyanide is not present in the natural habitats of yeast
and it is difficult to argue for the physiological relevance
of oscillations in the glycolytic pathway, as long as cyanide needs to be added in order to obtain long lasting
oscillations. The oscillations in glycolysis are often referred to as ‘‘essentially sustained’’, but they can only be
considered as sustained in the time scale of glycolysis
and ‘‘essentially’’ serves to indicate that the conditions
limiting the duration of the oscillations are known. It is
well known that the oscillations stop when the cells have
used the glucose in the broth.
The experiments presented in this paper were performed in a set-up combining fluorescence spectroscopy
and membrane inlet mass spectrometry (MIMS, for reviews see [8,9]) allowing for simultaneous measurements
of intracellular NADH and non-polar compounds secreted by the cells. Sustained glycolytic oscillations are
obtained by blowing an inert gas, such as argon,
through the yeast suspension. This ensures that the yeast
undergoes anaerobic metabolism and at the same time
volatiles secreted by the yeast cells are removed at a rate
depending on the gas flow through the suspension. The
experiments show that the non-physiological compound
cyanide is not necessary in order to obtain sustained
oscillations. In the experiments with argon, it was possible to evoke oscillations by addition of ethanol, KOH
or an additional pulse of glucose. It is possible that acetaldehyde couples glycolysis in the cells but the rate at
which acetaldehyde is removed in order to obtain long
lasting glycolytic oscillations is not a crucial parameter,
since removal of acetaldehyde by cyanide happens a lot
faster than removal by argon. Furthermore, truly sustained oscillations in a continuous-flow stirred tank reactor (CSTR), as described by Danø et al. [10,11], were
obtained without addition of cyanide. Instead of an inflow of yeast, glucose, and KCN we used an inflow of
yeast, glucose, and argon.
with 100 mM-potassium phosphate buffer (Merck,
Germany), pH 6.8 (centrifugation, 5 min at 5000 rpm,
GSA, Sorvall), resuspended in the same buffer to a cell
density of 10% by weight and starved for 3 h on a rotary
shaker at 30 °C.
2.2. Photometric measurements
The recording of NADH fluorescence was achieved
with a stabilized mercury lamp (Heraeus, Model St75,
Germany) connected to a stabilized power supply
(DURATEC Analysentechnik GmbH, Germany.
Model NT-HgSt). The lamp was mounted in a lamp
housing made in our own workshop. The exitation filter
(Edmund Industrial Optics, USA, Bandpass UG-1, 355
nm) and the emission filter (Thermo Oriel, USA, Vis/
NIR Long pass filter, 450 nm) were both mounted in the
sample cell. The emission of light was measured with an
end-on photomultiplier tube connected to an IBM
compatible PC through a picoammeter (Keithley,
Model 485, USA) and the data were stored in a program
written in Microsoft Visual Basic 6.0.
The light was guided from the lamp to the reactor and
from the reactor to the photomultiplier by custom made
quartz light guides.
2.3. Mass spectrometry
The sample cell had a volume of 9 ml, and was made
of stainless steel in our workshop; it was stirred with an
impeller connected through a stirring shaft to an electric
motor operating at 1000 rpm. The sample cell was
mounted on a vacuum flange and the only separation
between the yeast suspension in the cell and the vacuum
chamber of a single quadropole mass spectrometer
(Balzers QMG 420) was a 51 lm thick silicone membrane (SIL-TEC Sheeting, Technical Products Inc.,
USA). The principle is shown in Fig. 1. The inlet was
thermostatically controlled at 30 °C by a water bath
pumping water through channels in the sample cell.
With this system it was possible to continuously detect
the volatiles secreted by the yeast cells with response
times less than 2 s.
2. Materials and methods
2.4. The CSTR
2.1. Preparation of yeast
In order to generate truly sustained oscillations we
constructed an experimental set-up similar to the set-up
described by Danø et al. [10,11]. The yeast cells were
kept in a reservoir at 4 °C and stirred to avoid sedimentation. The cells were pumped from the reservoir to
the reactor by a peristaltic pump (Ole Dich Instrumentmakers Aps, Denmark) with a constant rate of
0.50 ml/min. To avoid agglomeration of yeast cells in the
tubes of the peristaltic pump, argon was blown into
the tubes at a constant rate creating small bubbles in the
Saccharomyces cerevisiae diploid strain X2180 was
grown under semiaerobic conditions at 30 °C on a rotary shaker, 180 rpm, in a medium containing 10 g/l
glucose, 6.7 g/l yeast nitrogen base (Bacto) and 100 mMpotassium phthalate (Aldrich, Germany) at pH 5.0. The
yeast was harvested at the point when glucose was depleted as measured with a glucose test strip (MachereyNagel, D€
uren, Germany). The cells were washed twice
A.K. Poulsen et al. / FEMS Microbiology Letters 236 (2004) 261–266
3. Results and discussion
-8.6x10
-8.4x10
-7
1.0
-7
0.8
NADH
-8.2x10
-7
0.6
23 mM glucose
-8.0x10
-7.8x10
-7
m/z 32
0.2
3.9 mM KOH
The usual way to induce oscillations in a suspension
of yeast cells is by the aerobic/anaerobic shock or by the
shock from the addition of cyanide. Only the latter is
capable of inducing sustained oscillations. We found
that another way to induce sustained oscillations is by
introduction of argon or another inert gas to the yeast
suspension. A pulse of glucose was never enough to
initiate a longer train of oscillations. The cells needed an
additional perturbation, either from the introduction of
argon or, if the argon flow was started before the pulse
of glucose, a minimal perturbation, e.g. by addition of
KOH, ethanol (EtOH), or a second pulse of glucose.
However, other compounds might as well-induce oscillations provided that they are added after glucose and
-7.6x10
0.4
-7
m/z 32
Relative abundance
suspension in the tube. An infusion pump (Harvard
Apparatus, Model 22, USA) was mounted with a 5000
ll syringe (Hamilton, Gastight #1005, USA) to provide
a small and constant flow of glucose to the sample cell.
Excess liquid was removed from the sample cell by a
peristaltic pump. It is of great importance that the
pumps are able to give very stable flows. Otherwise, the
base of the oscillations will become unstable.
argon is bubbled through the suspension. Fig. 2 shows a
batch experiment in which oscillations are induced by
addition of KOH after the pulse of glucose and argon is
blown through the suspension. This is the first observation of sustained (limit cycle) macroscopic oscillations
in a yeast cell suspension without addition of cyanide.
The pH of the phosphate buffer was increased from 6.80
to 6.86 by addition of 4 mM KOH. This is similar to the
increase in pH when cyanide is added. Cyanide also
serves to obtain anaerobiosis and in order to ensure that
the experiments were conducted under anaerobic conditions when argon was blown through the broth, the
molecular ion of oxygen at m/z 32 was detected and is
shown in Fig. 2. It is seen in the spectrum, that the intensity is not changed upon addition of glucose to the
yeast cell suspension. The concentration of oxygen in
the broth is less than 100 ppb and addition of sodium
dithionite did not decrease the signal of m/z 32. Flushing
the reactor with argon causes the oxygen signal, as
measured by the MIMS system, to drop by a factor of
100 to a zero-oxygen level caused by leaks in the mass
spectrometer from the surroundings. When the oxygen
level reaches the permanent baseline, we consider the
conditions in the reactor to be anaerobic. The experiment with KOH shows that even compounds that are
not metabolites may induce oscillations in NADH under
anaerobic conditions. No matter how the oscillations
were evoked, with cyanide or with argon flow, they had
the same properties concerning frequency and amplitude. These findings, that the oscillations are easily
NADH Fluorescence a.u.
Fig. 1. Schematic drawing of the membrane inlet system in combination with fluorescence spectroscopy. Sample cell (a); membrane (b); ion
source (c); extraction lens (d); focus lens (e) quadropole analyzer (f);
filament (g); stirrer (h); thermostatted water (i); excitation filter (j);
emission filter (k); quartz window (l); light guide from lamp (m); light
guide to photomultiplier tube (n); inlets for glucose and cells (o); inlet
for argon (p) and outlet for surplus liquid (q). Modified drawing from
[16].
263
-7
0
100
200
300
400
500
600
700
0.0
800
Time [sec.]
Fig. 2. Batch experiment in which glycolytic oscillations are induced by
addition of KOH while argon was blown through the broth. In order
to show that the experiments are conducted under anaerobic conditions the signal from oxygen at m/z 32 has been included in the figure.
The signal corresponds to the zero-baseline of oxygen caused by leaks
in the mass spectrometer from the surroundings. The baseline is
reached after a decrease in oxygen concentration in the broth by a
factor of 100. We consider the conditions in the reactor to be anaerobic. The increase in pH from the addition of KOH is similar to the
change followed by addition of cyanide (a.u.: arbitrary units). Argon
flow 45 ml/min; yeast dry weight 38 mg/ml; NADH oscillating frequency 2.62 min 1 .
264
A.K. Poulsen et al. / FEMS Microbiology Letters 236 (2004) 261–266
NADH Fluorescence
a.u.
induced with non-inhibiting compounds or compounds
that do not react with products of the cells, could indicate that the glycolytic pathway in the cells is oscillating
before macroscopic oscillations occur. The cells probably oscillate with different frequencies or individual
phases before addition of e.g. KOH. The compound
then perturbs the cells and gets them into the same
phase, after which the cells will synchronize.
If the cells were grown, as described in Section 2,
oscillations were easily induced by addition of first
glucose and then cyanide. Induction of the oscillations
without cyanide was not always a triviality. A pronounced behavior by the yeast cells was that the oscillations did not always start by a perturbation after the
first pulse of glucose. If a new pulse of glucose was added after the first pulse was used up by the cells, then a
small perturbation leads to oscillations in the concentration of NADH which did not damp out before the
glucose was used up. Since oscillations do not start
immediately it seems like the cells and the glycolytic
pathway need to be in an activated state gained by the
first pulse of glucose. The concentration of NADH in
the cells increases after the first addition of glucose and
does not return to the initial level after the glucose has
been metabolized (experiments not shown). This suggests that the glycolytic pathway is not completely
emptied before the second pulse of glucose and some of
the enzymatic parameters may have been changed. De
novo synthsis of protein is not a likely explanation since
the cells are starved and washed before the experiments
and no nitrogen source is present.
It is possible to keep the cells in a well-defined oscillating state indefinitely without use of cyanide in a
CSTR. If starved yeast cells and glucose are pumped
into the sample cell with outflow of surplus liquid while
argon is bubbled through the suspension it is in principle
possible to let the oscillations continue forever with
constant amplitude and period. Fig. 3 shows a typical
recording of stable sustained oscillations in the cell
-9.8x10-7
NADH
-9.6x10-7
-9.4x10-7
-9.2x10-7
6500
7000
7500
8000
8500
Time [sec.]
Fig. 3. Sustained oscillations in whole cell yeast suspension, S. cerevisiae diploid strain X2180. An unperturbed part of a CSTR experiment. The retention time was 15.50 min, cell volume 8 ml, yeast dry
weight 37 mg/ml, and the flow concentration of glucose was 37.5 mM.
The argon flow was 48 ml/min. The oscillations persisted for about 6 h
until the yeast cell suspension was used up (a.u.: arbitrary units).
suspension. Only the amount of yeast cells limits the
duration of the run. The experiment shows that cyanide
is not necessary in order to evoke a train of glycolytic
oscillations, and that the oscillations are a part of the
cell metabolism and not only a property provoked by
addition of an inhibitor.
Acetaldehyde is easily detected with MIMS down to
the low-micro-molar range and removal of acetaldehyde
by argon and cyanide from the sample cell was examined. Argon is an inert gas and does not react with any
compound in the suspension. As the gas bubbles
through the suspension, it makes anaerobic conditions
and increases evaporation of volatile compounds. Acetaldehyde is very volatile and evaporates from the
broth when argon is introduced. If acetaldehyde is the
synchronizing agent of the oscillations, the evaporation
results in a decreased concentration of this compound in
the broth. It seems that the yeast cells do not synchronize without a decrease in the concentration of a volatile
compound as, e.g. acetaldehyde, CO2 and/or, a third
compound. Acetaldehyde has often been pointed out as
the synchronizing compound [7,12] and the reduction of
acetaldehyde in the broth is apparently necessary in
order to increase the amplitude of the oscillating secretion of acetaldehyde relative to the acetaldehyde already
present in the broth. Our results show that the rate at
which acetaldehyde is removed from the broth by argon,
is very much slower than removal by reaction with cyanide. It was possible to generate macroscopic oscillations if the argon flow was between 35 and 75 ml/min.
Below 35 ml/min it was not possible to generate oscillations and above 75 ml/min the suspension started to
froth vigorously which rendered both fluorescence and
MIMS measurements impossible. Since Richard et al.
[6,7] have estimated the concentration of free extracellular acetaldehyde to be less than 100 lM after cyanide
is added, experiments were conducted in which removal
of 250 lM acetaldehyde at 30 °C was examined. At flow
rates of argon of 32, 49 and 70 ml/min the half-life period was 1863, 1469 and 1149 s, respectively. These results were compared to the removal of acetaldehyde by
cyanide. When 5 mM cyanide, the standard cyanide
concentration used in order to obtain oscillations, was
added to the sample cell, and the concentration of acetaldehyde was in the micro-molar range (<250 lM), the
reaction followed pseudo-first-order reaction kinetics
and the half-life of acetaldehyde was determined to 53 s
at 30 °C. Cyanide does not react with CO2 or EtOH
(results not shown). A decrease in the concentration of a
volatile compound in the broth is important, but the
rate of removal does not seem to be a crucial parameter
in order for the oscillations to occur. A low content of
acetaldehyde in the broth did not seem important either,
since a perturbation using 250 lM acetaldehyde did not
stop the oscillations (results not shown). Evaporation of
acetaldehyde from the sample cell, when argon is not
A.K. Poulsen et al. / FEMS Microbiology Letters 236 (2004) 261–266
NADH Fluorescence a.u.
-8.6x10-7
23 mM glucose added
0.14
m/z 44
-8.4x10-7
0.12
0.10
-8.2x10-7
0.08
3.9 mMKOH
-8.0x10-7
0.06
-7.8x10-7
0.04
NADH
m/z 44
Relative abundance
0.02
-7.6x10-7
0.00
0
100 200 300 400 500 600 700 800
(a)
Time [sec.]
0.96
Start
0.95
CO2 signal
present was examined and found to be negligible. The
results of the forced evaporation might vary between
different experimental set-ups: design and volume of the
sample cell, stirring of the sample cell, distribution, and
size of the argon bubbles all affect the rate of evaporation. The findings in these experiments, that the acetaldehyde concentration and the rate of removal does not
seem important to the oscillations, are not in agreement
with the finding of Richard et al. [6] who ascribed the
rate of acetaldehyde removal as an important parameter
in order to obtain oscillations. They based their finding
on the fact that the duration of glycolytic oscillations
strongly depended upon the concentration of cyanide.
However, the biochemical system in a cell is complex.
Cyanide inhibits respiration and also reacts with acetaldehyde, but it changes several of the other parameters
too, which again may affect the oscillations. An illustration of the complexity of the system is that addition
of azide, another inhibitor of respiration, after addition
of glucose and cyanide quenched the oscillations. Furthermore, we were not able to induce a long train of
oscillations in the glycolytic pathway if cyanide was
added after azide. Both compounds act as inhibitors of
cytochrome oxidase and cyanide furthermore removes
acetaldehyde. However, oscillations dissappear when
both inhibitors are present, suggesting that the role of
cyanide is more complex than as explained above.
The MIMS technique allows for on-line detection of
volatile compounds secreted by the yeast. The compounds ethanol, CO2 , the fusel oils 3-methyl-1-butanol,
2-methyl-1-butanol, and 2-methyl-1-propanol, were
found in the broth of oscillating yeast cells by this technique. The yeast cells secrete acetaldehyde as well, although practically, it was not possible to detect
acetaldehyde from the oscillating yeast cells since the
fragmentation patterns of ethanol and acetaldehyde are
almost identical and ethanol was secreted in the millimolar range while acetaldehyde was secreted in the micro-molar range. The only compound secreted by yeast
in which we detected oscillations, was CO2 measured at
m/z 44. MIMS has previously been used to detect oscillations in the CO2 transient in glycolysing cell free yeast
extracts [13] but it has never been used in a whole cell
yeast suspension. In Fig. 4(a), oscillations in CO2 and
NADH are shown. The oscillations shown in the figure
are obtained by addition of glucose and KOH while argon was blown through the suspension. Furthermore, we
have recorded CO2 oscillations in yeast cell suspensions
where the train was initiated by a pulse of cyanide and at
varying cell densities ranging from 30 to 40 mg/ml dry
weight. These experiments show that the phase relation
does not depend on the presence of cyanide or changes in
cell density within the range tested in these experiments,
and that NADH and CO2 oscillated with the same frequency. CO2 and acetaldehyde are both products of the
same enzyme, pyruvate decarboxylase and the com-
265
0.94
0.93
End
0.92
0.91
0.90
-9.3x10-7
(b)
-9.3x10-7
-9.2x10-7
NADH Fluorescence
Fig. 4. (a) Oscillations in [NADH] and m/z 44 initiated by addition of
glucose and potassium hydroxide. The peak in the mass spectrum at m/
z 44 is from the molecular ion of CO2 . Acetaldehyde has its molecular
ion at m/z 44 but its intensity is that low compared to CO2 that it has
no effect on the signal (a.u.: arbitrary units). Argon is blown through
the suspension at 45 ml/min. Yeast dry weight 38 mg/ml. (b) The recorded data from MIMS, m/z 44 are plotted against fluorescence. The
traces are formed as distorted spirals since the oscillations were slightly
damped. The angles of the trace indicate a phase difference somewhat
higher than 90°. When the response time from the MIMS is taken into
consideration the phase angle between NADH and CO2 is in a range
from 115° to 130°. The relative phases of CO2 and NADH did not
depend on how the oscillations were evoked. The arrows indicate the
direction of the flow (a.u.: arbitrary units).
pounds are expected to be released simultaneously from
the enzyme. Fig. 4(b) shows m/z 44 plotted against the
fluorescence trace in a phase plane plot, both in arbitrary
units. The phase plot forms a spiral rather than an ellipse, since the average NADH fluorescence and CO2
signal are not constant during their measurement. The
response time for CO2 in the MIMS is less than 2 s. When
taking this into consideration the phase delay between
NADH and CO2 is in the range from 115° to 130°.
NADH and acetaldehyde must have a similar phase relation. Consumption and evaporation of acetaldehyde
and CO2 may have slightly different kinetics, but the low
concentration of the compounds, the size of a yeast cell,
the fact that cell density and the presence or absence of
266
A.K. Poulsen et al. / FEMS Microbiology Letters 236 (2004) 261–266
cyanide does not have any effect on the phase relation
between CO2 and NADH. Therefore, it seems reasonable to expect that the online measurement of CO2 is an
indirect way to detect oscillations in acetaldehyde.
Richard et al. [7] determined a phase delay between acetaldehyde and NADH of around 200° at 20 °C. They
used extraction of suspension from the sample cell and
determination of acetaldehyde by enzymatic assays. If
the relative phases are a property of the experimental
conditions, then the difference in temperature, at which
the experiments were conducted, together with the time
delay in their experiments, could explain the discrepancy
between their results and ours. Our results do not demonstrate that acetaldehyde couples glycolysis in a suspension of yeast cells; neither do they reject this
hypothesis. As found in the work by Danø et al. [11] our
data support that cell synchronization is not a property
of a single chemical species or enzyme but a dynamic
property of the metabolic network, glucose uptake [14],
acetaldehyde, CO2 and heat flux [15], which all contribute to the synchronization.
Acknowledgements
We thank Sune Danø, Preben Graae Sørensen and
Hans Westerhoff for useful discussions. We also thank
the Danish Natural Science Research Council and the
European Science Foundation (REACTOR Programme) for financial support.
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