Báo cáo khoa học: Training of yeast cell dynamics ppt

The extracellular dynamics changed the intracel-lular dynamics – which was monitored through NADH fluorescence – from steady to equally dynamic; the latter followed the extracellular dyna

Karin A Reijenga1,*, Barbara M Bakker1, Coen C van der Weijden1 and Hans V Westerhoff1,2,3

1 Department of Molecular Cell Physiology, CRbCS, BioCentrum Amsterdam, Faculty of Earth and Life Sciences, Vrije Universiteit,

Amsterdam, the Netherlands

2 Department of Mathematical Biochemistry, BioCentrum Amsterdam, Swammerdam Institute for Life Sciences, the Netherlands

3 Stellenbosch Institute for Advanced Study, University of Stellenbosch, South Africa

Physiological and biochemical experiments are usually

designed in a way that conditions are ideal and

homo-geneous For example, cell densities are low, substrate

concentrations are well defined and constant, and

product concentrations are kept low to reduce product

inhibition However, in their natural environment cells

often encounter less ideal conditions For example, in

industrial fermenters the discontinuous feeding of

sub-strate and the high cell density can cause mixing

prob-lems and inhomogeneous cultures [1] The organism in

the fermenter experiences a fluctuating extracellular

environment, e.g in terms of concentrations of

sub-strates [2] and products as well as with respect to pH

and oxygen tension Glucose fluctuations encountered

by microorganisms in an industrial bioreactor were

found to be in the subminute to minute timescale [3]

In this paper we set out to mimic one aspect of these nonideal conditions and studied whether the temporal dynamics of the extracellular glucose concentration could induce a dynamic response inside the yeast cells This issue of whether extracellular dynamics can cause intracellular dynamics should not be confused with the issue of whether static extracellular or intracel-lular conditions can influence autonomous intracelintracel-lular dynamics The latter issue was addressed by Reijenga

et al [4], for so-called ‘autonomous’ glycolytic oscilla-tions arising under static extracellular condioscilla-tions, i.e limit-cycle oscillations The glucose transporter and therewith the effective concentrations of extracellular glucose and of inhibitors of glucose transport had sub-stantial control on the frequency of the oscillations Subsequently, it was shown that the glucose transporter

Keywords

dynamics; glycolysis; oscillations; training;

yeast

Correspondence

H V Westerhoff, Department of Molecular

Cell Physiology, CRbCS, BioCentrum

Amsterdam, Faculty of Earth and Life

Sciences, Vrije Universiteit, Amsterdam,

the Netherlands

Fax: +31 20 444 7229

Tel: +31 20 444 7230

E-mail: hans.westerhoff@falw.vu.nl

*Present address

DSM Anti-Infectives, PO 425, NL-2600 MA,

Delft, the Netherlands

(Received 28 September 2004, revised 18

January 2005, accepted 24 January 2005)

doi:10.1111/j.1742-4658.2005.04582.x

In both industrial fermenters and in their natural habitats, microorganisms often experience an inhomogeneous and fluctuating environment In this paper we mimicked one aspect of this nonideal behaviour by imposing a low and oscillating extracellular glucose concentration on nonoscillating suspensions of yeast cells The extracellular dynamics changed the intracel-lular dynamics – which was monitored through NADH fluorescence – from steady to equally dynamic; the latter followed the extracellular dynamics at the frequency of glucose pulsing Interestingly, the amplitude of the oscilla-tion of the NADH fluorescence increased with time This increase in ampli-tude was sensitive to inhibition of protein synthesis, and was due to a change in the cells rather than in the medium; the cell population was

‘trained’ to respond to the extracellular dynamics To examine the mechan-ism behind this ‘training’, we subjected the cells to a low and constant extracellular glucose concentration Seventy-five minutes of adaptation to a low and constant glucose concentration induced the same increase of the amplitude of the forced NADH oscillations as did the train of glucose pul-ses Furthermore, 75 min of adaptation to a low (oscillating or continuous) glucose concentration decreased the KM of the glucose transporter from

26 mm to 3.5 mm When subsequently the apparent KM was increased by addition of maltose, the amplitude of the forced oscillations dropped to its original value This demonstrated that the increased affinity of glucose transport was essential for the training of the cells’ dynamics

and several of the glycolytic enzymes exerted

substan-tial control over the eigenvalues of the glycolytic

path-way, thereby codetermining whether yeast glycolysis

oscillates or is steady [5] These findings confirmed that

static intra- and extracellular conditions can affect

intracellular dynamics Here we shall address how

dynamic extracellular conditions affect otherwise steady

intracellular processes

In previous studies on glycolytic oscillations there was

an aspect of adaptation Alterations in the make-up of

the yeast cells made them prone to limit cycle

oscilla-tions [6,7] For the cells to engage in sustained rather

than transient oscillations, they had to be harvested at

the diauxic shift (i.e the shift from using glucose to

using ethanol as a carbon source), and subsequently

starved for 2 h The essential nature of the pretreatment

has remained unclear, but it is known that the growth

conditions affect the affinity of the glucose transporters

over a wide range [8] Moreover, starvation induces the

(partial) degradation of glucose transporters [9–11]

possibly bringing yeast glycolysis into the ‘oscillatory

window’ [5] Indeed, glucose transport offers a wealth

of possibilities for Saccharomyces cerevisiae to exhibit

adaptation The organism has a large family of hexose

transporters, six of which confer growth on glucose

These are HXT1–4 and HXT6–7 [12] An hxt1–7

dele-tion mutant does not grow on glucose anymore [12]

HXT1 and HXT3 are transporters with low affinity

kin-etics, whereas HXT4 has a moderate affinity for

extra-cellular glucose and HXT6 and HXT7 display a high

affinity [13] HXT2 exhibits different affinities

depend-ing on the growth conditions [13,14]

The effects of external periodic events on oscillations,

and their possible advantages, have been studied

previ-ously, however, mostly by means of mathematical

mod-els [15–17,18] Here we perform in vivo experiments to

study these effects In this study we address cells that

should themselves settle for a steady-state, were it not

for an entrainment by extracellular dynamics We

har-vested the cells during exponential growth on glucose

and immediately put them on ice, without starvation [6]

Subsequently the cells were subjected to the repetitive

addition of aliquots (‘pulses’) of glucose As the cells

consumed (part of) the glucose during the intervals, this

led to an oscillating extracellular glucose concentration

Previously [19], we combined modelling and experiments

to demonstrate resonance of the intracellular dynamics

with an extracellular glucose oscillation under these

con-ditions It was shown that the amplitude of the

intra-cellular oscillation was considerably higher when the

frequency of the extracellular oscillations was close to

the eigenfrequency of the cells, than at other frequencies

(the eigenfrequency of the cells was taken to be the

frequency of the autonomous oscillations they engaged

in under slightly different conditions) Here, the same experimental system is used, but the focus is not on resonance, but on the question of whether the cellular make-up can change such that their dynamic response increases We report that the cells’ response to the dynamic extracellular glucose increased with prolonged exposure to the extracellular glucose oscillations, and identify a possible mechanism that may be responsible for this increase

Results

Response of intracellular dynamics to the pulsing

of glucose

S cerevisiae X2180 was grown on glucose and harves-ted during exponential phase After washing, the cells were resuspended to a protein concentration of approximately 13 gÆl)1 The cell suspension was incu-bated in a thermostated cuvette (25
C), and aliquots

of 0.8 mm glucose were administered at a frequency of 1.5 min)1 This frequency is in the range that is observed in industrial fermenters due to incomplete mixing [3] As the cells consumed the glucose, this should lead to an oscillating extracellular glucose con-centration It was observed, using glucose indicators that the extracellular glucose concentration at the end

of the experiment was below 2.8 mm (results not shown) This indicated that glucose did not accumulate during the experiment Intracellular NADH fluores-cence was measured continuously in the suspension The cell suspension responded to the glucose pulses by

an increase in NADH fluorescence, followed by a decrease (Fig 1) The insert to Fig 1 focuses on a part

of the experiment and indicates the time points where the glucose pulses were given The cells were forced into an oscillation with a frequency that was equal to the frequency of pulsing Glucose was given in series

of eight pulses, with 3 min and 20 s without glucose addition between the series For the later pulses the NADH fluorescence increased before the glucose was added (c.f the insert to Fig 1) When the pulsing was stopped, the oscillations continued for a few periods These trailing oscillations were damped and had a slightly higher frequency than the forced oscillations (c.f the insert to Fig 1) Clearly, extracellular dynam-ics induced intracellular dynamdynam-ics

Training Interestingly, the number of trailing oscillations increased with the time of exposure to the glucose

pulsing regime At first, no damped oscillations were

observed, whereas later on, two or three periods were

observed (Fig 1) Moreover, the amplitude of the

enforced oscillations increased strongly in time,

ulti-mately by a factor of four (Figs 1 and 2, Table 1)

During the first two series of pulses the NADH

oscilla-tions had a low amplitude From the third series

onward its amplitude started to increase until it

reached a plateau after eight series at t¼ 60 min

(Figs 1 and 2) We will refer to this increase in

ampli-tude as ‘training’, and to the cells that have reached

the maximum amplitude as ‘trained’ cells When a

trained cell suspension was mixed with a fresh cell

sus-pension, the amplitude was close to the average

ampli-tude of the two independent cultures (minimum 0.04,

maximum 0.14, average 0.09, after mixing 0.10),

indi-cating that the fresh cells were not entrained by the

trained cells (Table 1) To distinguish between the

increase of the amplitude of the NADH oscillation

being due to a change of the cells or to a change in

their environment, the cell suspension was centrifuged

and the cells were separated from the supernatant

First, trained cells were resuspended in fresh medium

and pulses of glucose were given to the suspension as

described before In this case the amplitude was similar

to the maximum amplitude of trained cells (0.145)

Subsequently, fresh cells were resuspended in the

supernatant of trained cells In the latter case the

amplitude was similar to the minimum amplitude at

t¼ 6 min (0.021) (Table 1) These results indicated that the increase in amplitude as seen in Figs 1 and 2

0

0.2

0.4

0.6

0.8

1

Time (min)

0.4

0.6

0.8

Time (min)

A

B

Fig 1 Intracellular response of yeast cells

to extracellular dynamics The NADH fluor-escence (solid line) was measured in a sus-pension of intact yeast cells Cells were grown on glucose, harvested during expo-nential phase, washed and resuspended to

a protein concentration of
13 gÆL)1 At

t ¼ 1 min, glucose was added to a final concentration of 3.2 m M From t ¼ 6 min, glucose was added in a pulsatile manner in the following regime: eight times 0.8 m M at 40-s intervals, followed by a pause of 200 s, then again eight times 0.8 m M at 40-s inter-vals, etc (Inset) one series of eight pulses The solid line represents the NADH fluores-cence The triangles indicate when the glucose pulses were given Each glucose pulse amounted to a glucose concentration

of 0.8 m M and the period of pulsing was

40 s The bold line represents the six periods that were used to calculate the average amplitude of the series.

0 0.05 0.1 0.15 0.2

0 20 40 60 80 100 120

Time (min)

Fig 2 Amplitude of the intracellular NADH oscillation The amplitude was determined from experiments like the one shown in Fig 1 The average amplitude of NADH fluorescence during one series was determined by averaging the amplitudes of all the individual periods

of that series The first two periods of each series were not taken into account, as, especially towards the end of the experiment, the frequency and amplitude of these periods were substantially differ-ent from the six subsequdiffer-ent periods The damped oscillations that were observed after the periodic glucose addition had been stopped were not taken into account either Average amplitudes were deter-mined for all 12 subsequent series of pulses Here, the average val-ues of the amplitudes are plotted in time, for five independent experiments, carried out with cells from independent cultures.

was due to a change in the make-up of the cells rather

than to some extracellular product in the supernatant;

the cells appeared to have been trained

Inhibition of protein synthesis

To test whether the synthesis of new proteins was

required for the increase in amplitude, protein

synthe-sis was blocked by cycloheximide Inhibiting

eukaryo-tic peptidyl transferase, this compound prevents the

formation of new peptide bonds No increase in

ampli-tude was observed when cycloheximide had been

added to the cell suspension before glucose (average

amplitude 0.032) (Fig 3) Two different concentrations

were used (5 lgÆmL)1, 50 lgÆmL)1) and the results

were similar This indicated that protein synthesis was essential for the training of the cells and that the proteins involved have a net positive control on the amplitude

Continuous low glucose concentration The increase in amplitude could be caused by two dif-ferent properties of the extracellular glucose signal: either (a) its pulsatile character, or (b) its low concen-tration The former hypothesis should require that the cells have a memory for dynamics The second hypo-thesis would involve regulation of protein synhypo-thesis and breakdown, after the shift of the cells from a high glucose concentration during exponential growth to a low concentration in the cuvette To distinguish between these possibilities, cells were subjected to a continuous glucose feed for 75 min On average, the rate of glucose addition was the same as in the pulse experiments described above After the incubation, cells were subjected to pulses of glucose in the cuvette,

as described before, and NADH fluorescence was measured The amplitude of the NADH oscillations after a continuous and low glucose feed (0.17 of our arbitrary units) was similar to the maximum amplitude cells that were trained by a pulsatile and low glucose feed (0.15) (Fig 4) This indicated that the low glucose concentration, rather than its pulsatile addition, led to the expression of proteins that have a pronounced effect on the amplitude of the intracellular NADH oscillation

Glucose transport The above-described results raised the question of which proteins were expressed that caused the increase

of the amplitude of the NADH oscillations during incubation at low glucose concentration High affinity

Table 1 Amplitude training of yeast cells Summary of the results of the different experiments described The second and third column reflect the average amplitude at t ¼ 10 min and t ¼ 90 min, respectively The fourth column reflects the average amplitude after different treatments of the cell suspension The first experiment (no additions) corresponds to Figs 1 and 2.

Amplitude (a.u.)

Average amplitude after different treatments

a Amplitude after mixing or resuspending of cells b Amplitude after 75 min of continuous feeding of glucose to the yeast cells a.u., Arbitrary units of NADH fluorescence.

0

0.05

0.1

0.15

0.2

0 20 40 60 80 100

Time (min)

Fig 3 Effect of inhibition of protein synthesis on the amplitude of

the intracellular NADH oscillation The amplitude of the intracellular

NADH oscillation was determined in the presence of 0 (d), 5 (–)

and 50 (·) mgÆL)1cycloheximide The stock solution of

cyclohexi-mide (1 gÆL)1) had been dissolved in phosphate buffer (100 m M ,

pH 6.8) The average values of the amplitudes were determined as

described in the legend to Fig 2 Cycloheximide was added 10 min

before the first addition of glucose A duplicate experiment gave

essentially the same result.

glucose transporters were obvious candidates for two

reasons First, it is known that after a shift from high

to low glucose concentration, as was the case in these

experiments, high affinity glucose transporters are

induced [8] It had to be investigated, however,

whe-ther this occurred even over the short time scale of

these experiments Secondly, glucose transport exerts

a substantial control on the frequency of autonomous

oscillations [4] We wondered if it also controlled the

amplitude of these forced oscillations To test the

hypothesis that high affinity transporters were

respon-sible for the training of the cells, we first investigated

whether the affinity of glucose transport did change

during the pulsing of glucose Glucose transport

kinet-ics were measured both in cells harvested during

expo-nential phase, and in cells that had been subjected to

a pulsatile glucose concentration for 75 min From

Fig 5 it becomes clear that during the experiment the

kinetics of the glucose transporter changed

substan-tially For cells harvested during exponential phase,

the Vmax and the KM of the transporter were

496 ± 23 nmolÆmin)1 per mg protein and 26.2 ±

0.3 mm, respectively For the cells after pulsatile

addi-tion of glucose, the Vmax and the KMof the

transpor-ter were 518 ± 11 nmolÆmin)1 per mg protein and

3.5 ± 0.1 mm, respectively We concluded that the

Vmax of the transport system hardly changed, whereas

the affinity of transport for glucose changed from low

to high

Subsequently, we asked the question whether the

observed decrease of the KMof glucose transport was

required for the increase of the amplitude of the NADH oscillation To this end we increased the KMof glucose transport in trained cells artificially to approxi-mately its value in fresh cells, by addition of maltose,

a competitive inhibitor [20] As the Kiof glucose trans-port for maltose varies with strains and conditions [20] (presumably depending on which transporters are expressed), we determined its value in the trained cells The Kiwas then 32 mm (results not shown) It was cal-culated that a final concentration of 210 mm of malt-ose was needed to increase the apparent KM for glucose transport from 3.5 mm (trained cells) back to

26 mm (exponentially grown cells) At t¼ 100 min maltose was added to various final concentrations Fig 6 shows a substantial decrease in the amplitude of the forced oscillations, after the addition of maltose The drop in amplitude increased with the final concen-tration of maltose as can be seen in Fig 7 The minor increase in amplitude upon addition of phosphate buf-fer may reflect an effect on the fluorescence signal due

to dilution The values for the amplitude after addition

of maltose were corrected for this dilution From inter-polation, it was concluded that a final concentration of

210 mm of maltose, which should increase the KM of the transporter back to its original value, was sufficient

to abolish the increase of the amplitude due to training

by 90% (Fig 7) These results indicated that, within experimental error, the difference in KM of glucose

0

0.05

0.1

0.15

0.2

0 20 40 60 80 100 120

Time (min)

Fig 4 Effect of low continuous and low pulsatile glucose

concen-trations on the intracellular dynamics of yeast The amplitude of the

intracellular NADH oscillation was determined for cells that were

subjected to a pulsatile glucose concentration (s) and for cells that

were first subjected to a low continuous glucose concentration for

75 min and subsequently subjected to a pulsatile glucose

concen-tration (d) The average values of the amplitudes were determined

as described in the legend to Fig 2 A duplicate experiment gave

essentially the same result.

0 200 400 600 800

0 20 40 60 80 100 120 140 160

V/S (nmol.min –1 .mg protein –1 .mM –1 )

–1 mg pr

–1 )

Fig 5 Glucose transport kinetics Eadie–Hofstee plot of zero-trans influx kinetics of glucose transport in exponentially grown cells (d), and in exponentially grown cells subjected to a pulsatile glucose concentration for 75 min (s) The glucose uptake experiments were performed twice on independent cultures The average values

of Vmax and KM are given here with their standard deviations For cells harvested during exponential phase, the Vmaxand the KMof the transporter were 496 ± 23 nmolÆmin)1 per mg protein and 26.2 ± 0.3 m M , respectively For the cells subjected to a pulsatile glucose concentration, the Vmax and the KM of the transporter were 518 ± 11 nmolÆmin)1 per mg protein and 3.5 ± 0.1 m M , respectively.

transport was sufficient to explain the difference in

amplitudes of the forced oscillation between trained

and untrained cells

Discussion

In this paper we studied the dynamic response of

other-wise steady yeast glycolysis to an oscillating

extra-cellular substrate concentration Cells were harvested

during exponential growth phase, as these cells do not

engage in limit cycle oscillations, when subjected to glucose [6] Indeed, when given steady extracellular glucose (data not shown), or after the first single addi-tion of 3.2 mm glucose (Fig 1) the cell populaaddi-tions in our experiments exhibited a fairly steady, nonoscillatory level of NADH fluorescence, but when glucose was added in a pulsatile fashion, the NADH fluorescence followed dynamically This showed either that extracel-lular dynamics can induce dynamics of intracelextracel-lular NADH, hence presumably of intracellular glycolysis,

or that the pulsatile addition of glucose synchronized pre-existing oscillations of the individual cells

The latter explanation assumes that the individual cells oscillated anyway but out of phase We consider this explanation unlikely for the following reasons: (a)

it has been demonstrated that the exponentially grown cells used here could not be synchronized by partly trapping acetaldehyde, the synchronizing agent [6]; (b) mixing trained with nontrained cells led to oscillations

of average amplitude, whereas synchronization should have led rapidly to the full amplitude (compare with [21]); (c) cycloheximide prevented the training, suggest-ing that at least the amplitude increase due to trainsuggest-ing was not due to synchronization; (d) added maltose reduced the amplitude immediately of cells that should already have been synchronized, suggesting again that

at least the amplitude increase due to training is not

an effect of synchronization

The dynamics of the intracellular response was quasi-sinusoidal at the same frequency as the extracel-lular perturbation It was not chaotic, as it might have been if the cells engaged in limit cycle oscillations themselves (compare with [22,23]) There was an active aspect to the cellular response, i.e after a few glucose pulses, the response seemed to run slightly ahead of the extracellular pulsing and the intracellular oscilla-tions persisted for some time after the extracellular pulsing had been stopped, and at this somewhat higher frequency This behaviour suggests that the cells were not in a stable node but in a stable focus, and had a dominant eigenfrequency that was slightly higher than the frequency of extracellular pulsing This was the fastest response of the cells

There were two slower responses The first of these was the increase in average NADH fluorescence observed during the first three series of pulses (Fig 1) From the data on the presumed extracellular glucose concentration and the measured KM and Vmax values

of glucose transport, we estimated the glucose trans-port rate at the beginning and at the end of the experi-ment At the beginning of the experiment the glucose transport activity should have been too low to com-pletely consume the extracellular glucose within 40 s,

Fig 6 Effect of inhibition of glucose transport on the amplitude of

the intracellular NADH oscillation The amplitude of the intracellular

NADH oscillation was determined before and after the addition of

maltose, a competitive inhibitor of glucose transport, to the cell

suspension At t ¼ 100 min, maltose [to final concentrations of

22 m M (s), 111 m M (·) or 223 m M (–)] or phosphate buffer

[100 m M , pH 6.8 (d)] was added to the cell suspension The

aver-age values of the amplitudes were determined as described in the

legend to Fig 2.

0%

20%

40%

60%

80%

100%

120%

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

[Maltose] (M)

Fig 7 Effect of addition of maltose on decrease in amplitude of

the intracellular NADH oscillation After addition of maltose to

trained cells, a decrease in amplitude was seen The amplitudes

after addition of maltose were corrected for dilution of the cell

sus-pension, as the addition of phosphate buffer caused the measured

amplitude to increase The decrease in amplitude was calculated,

relative to the maximum amplitude.

and therefore extracellular glucose may have

accumu-lated Two effects will cause the glucose transport rate

to increase during the experiment: (a) the presumed

accumulation of extracellular glucose; and (b) the

decrease of the KM of the transporter These two

effects should have led to an increased glucose

trans-port activity and therefore they should eventually have

stabilized the glucose concentration averaged over a

pulse and therewith the NADH fluorescence averaged

over a pulse This may explain the initial increase in

average NADH fluorescence

The cellular response at the slowest time scale was

the gradual and more than fourfold increase of the

amplitude of intracellular NADH oscillations, a

phe-nomenon we attributed to ‘training’ We observed a

decrease of the KM of glucose transport from 26 to

3.5 mm during the training period and proved that

increasing the KMback to its original value abolished

the effect of the training on the amplitude Strictly

speaking we cannot conclude from this result that the

change in KM of glucose transport is sufficient to

achieve the training effect and that no other change in

the cellular make-up is involved This conclusion

would require the opposite experiment of decreasing

the KM of untrained cells, without changing anything

else in the cells, which is a more complicated if not

impossible task Considering, however, that increasing

the KM of untrained cells decreased the amplitude

back to its original value, we may conclude that the

KM change is necessary And although we have not

proven it definitively, we find it at least likely that the

KM change is the major if not the only change

required to achieve the training effect Which of the

Hxt proteins are involved, remains open for further

investigation

Previously, we have determined the control of

glu-cose transport on the frequency of autonomous

oscilla-tions [4] Glucose transport had substantial control

over the frequency of these oscillations, i.e on this

dynamic behaviour Additionally, we have studied

forced oscillations in terms of resonance phenomena in

the same, exponentially grown yeast cells that were

used in this study [19] As resonance occurs only in

systems around a stable focus, we can conclude that

these yeast cell populations are stable but do have a

dynamic component We might hypothesize that

through the change in the kinetics of the glucose

transporter the training has an effect on the dynamic

component of the stable cells, i.e on their

eigenfre-quency The required change of eigenfrequency would

be very small, since a sharp resonance peak was

observed in the untrained cells at approximately

1.7 min)1, indicating that the untrained cells have an

eigenfrequency of 1.7 min)1 [19] (as compared to a glucose pulsing frequency of 1.5 min)1 in this study) However, the decrease of the KM of the transporter in time should lead to an increase of the rate of the trans-porter and according to the positive control of the transporter on the frequency [4] this should increase the eigenfrequency of the system and thereby move the eigenfrequency even further away from the extracellu-lar frequency Moreover, the previously observed reso-nance increased the amplitude by less than a factor of two, while we observe an increase of a factor of four

in this study Therefore we consider it more likely that the mechanism of training merely reflects the positive control exerted by glucose transport on the amplitude

of enforced oscillations After all, enhanced influx of glucose should be expected to enhance the reduction of NAD(P)H

This study effectively also explored consequences of fluctuating extracellular conditions in industrial fer-menters What do the results mean then for yeast cells in an industrial environment? During industrial production of yeast, the average sugar concentration

in the fermenter is low, the cell density is high and high affinity glucose transporters are expected to be expressed, as is the case in glucose-limited chemostat experiments [8] This situation resembles the condi-tions described in this paper, particularly as the time scale of sugar fluctuations in an industrial reactor may be similar to the frequency of pulsing applied here [3] Consequently, we might speculate that the intracellular metabolism of yeast cells in industrial fermenters also reacts to extracellular dynamics by engaging itself in those dynamics And, it is conceiv-able that the response itself is subject to training Of course the regular glucose pulses that were applied in the present study are a only a first approximation of the more complex glucose profile that the cells encounter in a bioreactor when they pass the medium inlet repeatedly Due to the complex time dependence

of extracellular glucose concentration for individual cells in such a bioreactor, the time scales involved might have subtle or drastic implications for the metabolic performance of those cells and for their gene expression pattern

This paper illustrates that metabolism can only be understood by including gene expression and protein synthesis, as these processes partly determine the activity of the enzymes involved As described earlier [24], the different levels of regulation and control can-not be treated separately and therefore a hierarchical approach should be taken to explain the overall cellu-lar behaviour Furthermore, the ability of cells to adapt to their environment and to anticipate to certain

changes therein may enhance their chances of survival,

by tightly regulating the use of their available free

energy

Experimental procedures

Chemicals

Yeast nitrogen base without amino acids was from BD

(Franklin Lakes, NJ, USA) Glucose was from Boom

(Meppel, the Netherlands) (when used as carbon and

energy source in the medium) or from Sigma (St Louis,

MO, USA) (when used in the glucose transport assay)

d-[U-14C]glucose was from GE Healthcare (St Giles, UK),

glass microfibre filters (GF⁄ C) from Whatman (Brentford,

Middlesex, UK) and liquid scintillation fluid from

Perkin-Elmer (Boston, MA, USA) All other reagents were

obtained from Merck (Whitehouse Station, NJ, USA),

Sigma or Fluka (St Louis, MO, USA), and were of

ana-lytical grade or higher

Strain, growth conditions and preparation

of the cell suspensions

The yeast strain S cerevisiae X2180 was used in all

experi-ments (grown in-house) Cells were grown semiaerobically

on yeast nitrogen base, containing 1% glucose and 100 mm

phthalic acid (pH 5.0, KOH) at 30
C and harvested at an

D600of
1.0 Cells were washed twice with 100 mm

phos-phate buffer (pH 6.8, KOH) and resuspended in the same

phosphate buffer to an D600of 80, corresponding to a

pro-tein concentration of 13 gÆL)1 Protein concentrations were

determined according to Lowry [25] When large amounts

were needed, cells were grown in a 2-L batch fermenter at a

working volume of 1.5 L, a stirrer speed of 800 r.p.m., an

air flow of 45 LÆh)1, and at 30
C and pH 5.0 The medium

was yeast nitrogen base containing 1% glucose and 100 mm

phthalic acid, set to an initial pH of 5.0 by addition of

KOH, but not pH controlled

Forced oscillations

Cells were incubated in a thermostated cuvette (25
C, 300–

600 lL) and NADH fluorescence was measured on-line

(excitation 338 nm, emission 456 nm) Fluorescence

inten-sity is given in arbitrary units (a.u.), as the value depends on

instrument settings and cell density The latter parameters

were standardized, in order to be able to compare

experi-ments between each other The D600of the suspension was

80 (13 gÆL)1 protein) and conditions in the cuvette were

semianaerobic, i.e the dense suspension was stirred in the

absence of an additional air supply Glucose was added

to the suspension either manually or by means of an

automated pump This computer controlled pump (KD

Scientific 200 two-syringe pump, Holliston, MA, USA) was operated with a 100-lL Hamilton syringe (#1710 with Tef-lon luer lock; ؼ 1.46 mm) The syringe was connected to the cuvette through Teflon tubing (
40 cm, Ø ¼
1 mm) using two needles (one with a luer lock for the Hamilton syringe) It was checked that both methods gave similar results (results not shown) At t¼ 1.0 min, glucose was added to a final concentration of 3.2 mm Starting at t¼ 6.0 min, eight aliquots (‘pulses’) of glucose were given, each corresponding to an increase in concentration of 0.80 mm,

at a frequency of 1.5 min)1(T¼ 40 s) Pulsing was stopped for 200 s and at t¼ 14 min the pulsing was repeated Dur-ing a typical experiment, a total of 12 series of eight pulses were given (Fig 1) The average amplitude of NADH fluor-escence during one series was determined by calculating the amplitudes of all the individual periods, and averaging them over that series The first two periods of each series were not taken into account, as, especially at the end of the experi-ment, the frequency and amplitude of these periods were substantially different from the six following periods The damped oscillations that were observed after the pulsing was stopped were not taken into account either Average amplitudes were determined for all 12 subsequent series of pulses

Continuous glucose feed For a continuous feed of glucose, a thermostated (25
C) and stirred 15-mL vessel was used Ten millilitres of cells with an D600of 95 were incubated, and glucose was added continuously using a masterflex pump (microprocessor pump drive; Cole-Palmer Instrument Company (Vernon Hills, IL, USA); flow 43.5 lLÆmin)1; glucose stock 0.22 m)

In terms of its time average, the rate of glucose addition was the same as in the pulse experiments After 75 min, cells were taken out of the vessel and incubated in a cuvette, under the same conditions Subsequently, glucose pulses were given, as described above, and the cellular response was again read in terms of NADH fluorescence

Glucose transport Before and after 75 min of pulsing of glucose, as described above, the kinetic characteristics of glucose transport were measured according to Walsh et al [26] Cells that had been subjected to a pulsatile glucose concentration were washed and resuspended in phosphate buffer (100 mm,

pH 6.8) The transport assay was performed at 25
C

Acknowledgements

This work was supported financially by the Nether-lands Organization of Scientific Research (NWO) and the Technology Foundation (STW)

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