Letters in Applied Microbiology 2003, 37, 268274

doi:10.1046/j.1472-765X.2003.01394.x

A study of the yeast cell wall composition and structure

in response to growth conditions and mode of cultivation
B. Aguilar-Uscanga and J.M. Francois
Centre de Bioingenierie Gilbert Durand, UMR-CNRS 5504, UMR-INRA 792, Toulouse Cedex, France
2003/0265: received 1 March 2003, revised 18 June 2003 and accepted 19 June 2003

ABSTRACT
B. AGUILAR-USCANGA AND J.M. FRANC
O I S . 2003.

Aim: The polysaccharide composition of the Saccharomyces cerevisiae cell wall was measured under various growth
conditions and was compared with the cell wall structure.
Methods and Results: Chemical and enzymatic methods were used to determine levels of b-1,3-glucan and 1,6glucan, mannan and chitin of the yeast cell wall, whereas the structure/resistance of the wall was qualitatively
assessed by the sensibility to the lytic action by zymolyase. It was found that the dry mass and polysaccharides content
of the cell wall could vary by more than 50% with the nature of the carbon source, nitrogen limitation, pH,
temperature and aeration, and with the mode of cell cultivation (shake flasks vs controlled fermentors). While no
obvious correlation could be found between b-glucan or mannan levels and the susceptibility of whole yeast cells to
zymolyase, increase of b-1,6-glucan levels, albeit modest with respect to the growth conditions investigated, and to a
lesser extent that of chitin, was associated with decreased sensitivity of yeast cells to the lytic action by zymolyase.
Significance and Impact of the Study: Our results indicate that the cell wall structure is merely determined
by cross-linking between cell wall polymers, pointed out the role of b-1,6-glucan in this process. Hence, this study
reinforces the idea that enzymes involved in these cross-linking reactions are potential targets for antifungal drugs.
Keywords: b-1,3-glucan, b-1,6-glucan, chitin, mannan, yeast, zymolyase.

INTRODUCTION
The cell wall of yeast and other fungi determines the cell
shape and integrity of the organism during growth and
cell division. Three main groups of polysaccharides form the
cell wall: polymers of mannose (mannoproteins, ca 40% of
the cell dry mass), polymers of glucose (b-glucan, ca 60% of
the cell wall dry mass) and polymers of N-acetylglucosamine
(chitin, ca 2% of the cell wall dry mass). b-Glucan can be
divided into two subtypes following the mode of glucose
linkages: long chains of ca 1500 b-1,3-glucose units which
represents ca 85% of total cell wall b-glucan, and short
chain of ca 150 b-1,6-glucose units that accounts for ca 15%
of the b-glucan (Klis et al. 2002). The cell wall is a dynamic
structure that can adapt to physiological (i.e. from logarithCorrespondence to: Jean M. Francois, Centre de Bioingenierie Gilbert Durand,
Institut National des Sciences appliquees, Avenue de Rangeuil, F-31077 Toulouse
Cedex 04, France (e-mail: fran_jm@insa-tlse.fr).

mic to stationary phase) and morphological changes (conjugation, sporulation or pseudohyphal growth) (Orlean
1998). Moreover, a cell wall compensatory mechanism is
activated in response to cell wall perturbing agents or cell
wall mutations, which allows remodelling of the cell wall to
combat cell lysis (Orlean, 1998; Klis et al. 2002). One of the
major outcomes of this mechanism is a strong increase of
chitin that can reach up to 20% of the cell wall dry mass
(Popolo et al. 1997; Dallies et al. 1998; Lagorce et al. 2002).
While a high chitin content can contribute to the strength of
the cell wall, it is still a matter of discussion whether such a
modification is necessary for maintenance of the cell wall
integrity (Klis et al. 2002). Another parameter that has not
been carefully investigated is b-1,6-glucan. This short
glucose polymer is implicated in covalent linkages with
b-1,3-glucan, mannoproteins and chitin, and these crosslinkages may also contribute to the modular structure of the
cell wall (Kollar et al. 1995).
2003 The Society for Applied Microbiology

YEAST CELL WALL COMPOSITION AND INTEGRITY

The aim of this work was to investigate the variability of

cell wall composition with respect to several growth
conditions, including the mode of cultivation, the nature
of the carbon source, temperature, pH, aeration, and to
tentatively correlate changes in the levels of b-glucan,
mannan and chitin with those of the cell wall integrity
structure, as assessed by the spheroplast lysis assay developed by Ovalle et al. (1998). This study was motivated not
only by the fact that there are very few reports on effects of
growth conditions on cell wall composition and none of
them have tried to correlate changes in cell wall composition
with cell wall integrity (McMurrough and Rose 1967;
Morris et al. 1993; Kapteyn et al. 2001), but also because
several genome-wide scale transcriptional studies of yeast
cells challenged with various environmental stresses showed
up- and downregulation of many genes implicated in cell
wall biogenesis with no quantitative data of these effects on
cell wall polymers (Gasch et al. 2000; Becerra et al. 2002;
Kwast et al. 2002; Ter Linde and Steensma 2002). Moreover, the control of the cell wall composition could be
relevant for biotechnological purposes, as there is increasing
commercial interest in the production of b-glucan and
mannan for agro-food, pharmaceutical and cosmetic purposes (Donzis 1996; Jozef et al. 1999), and conversely, a willing
to reduce cell wall mass (Daran et al. 1997) to optimize cell
breakage for a more effective and less-time consuming
release of endogenous biomolecules.

MATERIAL AND METHODS

Strains, media and growth conditions
The haploid prototroph CEN.PK133-7D Saccharomyces
cerevisiae strain (Van Dijken et al. 2000) was used in this
work. Unless otherwise stated, a synthetic-mineral medium
(CF medium) was prepared according to Verduyn et al.
(1992), supplemented with 20 g l)1 carbon source. Mineral
medium and sugar solutions were sterilized separately at
120C for 30 min. Vitamins and oligoelements were added
by sterile filtration (using 02 lm sterile nylon filters) to the
sterilized media. Shake flask cultures were carried out in 20-l
flasks containing 04 l of growth medium and agitated on a
gyratory shaker at 200 rev min)1 at 30C. Unless otherwise
stated, the pH was initially adjusted at 50 with HCl 10 M.
Cultivation in batch fermentors was performed in a 2-l
fermentor (Inceltech-SGI, Toulouse, France) with a working volume of 15 l at an impeller speed of 500 rev min)1
and at 30C, unless otherwise stated. The pH was kept
constant at 50 by automatic addition of NaOH 2 M using a
peristaltic pump, and dissolved oxygen was kept above 50%
by an airflow at 05 l min)1. Nitrogen limitation was
controlled by adjusting the initial ammonium sulphate
concentration in the CF medium at 5, 05 or 005 g l)1. The

269

culture media in shake flask and bioreactor were inoculated

with an initial biomass of 05 mg l)1 (or O.D.660  10) with
a preculture that was grown for 24 h at 30C in the same
culture medium.
Growth was monitored by measuring the absorbance of
the culture at 660 nm with an Easyspec IV spectrophotometer (Safas SA, Monaco, France). Appropriate dilution of
the cell suspension in water was made to remain in the
linearity between O.D.660 and dry mass (i.e. from 0 to
04 units at 660 nm). The biomass was determined gravimetrically by filtering 50 ml of culture samples on preweighed nitrocellulose filters (pore size 045 lm; Sartorius
AG, Goettingen, Germany). The filters were washed with
demineralized water and dried in an oven set at 80C. The
dry mass was established by repeatedly weighing the filters
until a constant value was reached (i.e. after ca 1824 h).
Cell wall isolation and quantification
of polysaccharide by HPAEC-Dionex
Cells were collected during the early exponential phase, i.e.
O.D.660  1 (or 04 mg cell dry mass), considering that
yeast cells were under nonlimiting nutritional conditions at
this growth phase. Culture samples (50 ml) were harvested
at 4C by centrifugation (5 min at 4500 g) and washed two
times with ice-cold water. The pellet was resuspended in
05 ml of ice-cold TrisCl 10 mM/EDTA 1 mM at pH 75.
Preparation and acid hydrolysis of the cell wall, and
quantification of glucose and mannose released from
H2SO4 hydrolysis of b-glucan and mannan were performed
by high-performance anionic exchange chromatography
(HPAEC) (Dionex Bio-LC50 system; Sunnyvalle, CA,
USA) as described by Dallies et al. (1998).
Determination of chitin, b-1,3-glucan
and b-1,6-glucan
Chitin was determined using chitinase from Serratia marcescens (purchased from Sigma Chemicals) on whole cells
according to Bulawa et al. (1986). A procedure to estimate
levels of b-1,3-glucan and b-1,6-glucan in yeast cell wall has
been adapted from Hong et al. (1994) and Magnelli et al.
(2002). Briefly, purified cell wall (1020 mg dry mass) was
digested with 5 U zymolyase 20T (purchased from Wako
Chemie, Hamburg, Germany and prepared in 10 mM Tris
buffer, pH 75 containing 50% glycerol) at 37C for 16 h in
10 mM Tris buffer. After heat inactivation, the extract was
centrifuged at 10 000 g for 15 min at 4C. The supernatant
fraction, which contained >90% of mannan, was loaded on a
concanavalin column (05 2 cm) to retain the mannan
fraction, while the b-glucan was eluted with 5 ml water.
This latter fraction and the undigested zymolyase pellet
resuspended in 1 ml of TrisCl buffer were again treated

2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 268274, doi:10.1046/j.1472-765X.2003.01394.x

270 B . A G U I L A R - U S C A N G A A N D J . M . F R A N C O I S

with 5 U zymolyase 20T at 37C, and dialysed using

3500 MW cut-off membranes against 200 volumes of
50 mM acetate buffer, pH 50. The solution in the dialysis
bag was digested with 10 units of recombinant endo-b-1,6glucanase from Trichoderma harzianium for 20 h, and the
products of this enzymatic digestion (glucose, gentobiose
and gentotriose) were quantified by HPAEC-Dionex. The
recombinant b-1,6-glucanase from T. herzanium was purified according to Bom et al. (1998).

change of optical density at 660 nm was followed every

10 min up to 2 h, and then every 30 min, after the addition
of 04 U zymolyase 20T.

RESULTS
Effect of growth medium on cell wall composition
Yeast cells are usually cultured in three different
media, namely a rich Yeast Peptone Dextrose (YPD), a
synthetic medium, Yeast Nitrogen Base (YNB) and a
mineral glucose-containing medium, Cell Factory (CF).
We found that, independently of the culture medium, the
cell wall content was roughly 20% of the cell dry mass
(Table 1). However, the b-glucan and mannan ratio
decreased from 136 in YPD to 082 in YNB and CF
culture. The chitin content was also altered by growth
media, reaching ca 6% of the cell wall mass in the YPD
medium, which is about three times higher than in cells
cultivated in mineral media (Table 1). We also measured
levels of b-1,6-glucan in the b-glucan fraction and found a
modest but reproducible significant change of this type of
polymer, which decreased from 18% in YPD and SD to

Other analytical techniques

Total carbohydrate content of the cell walls was measured
by the phenolsulphuric acid method according to Dubois
et al. (1956) using a mixture of glucose/mannose (60 : 40)
for the calibration curve. Sugars in the growth medium
(galactose, mannose, sucrose and maltose) were determined
by HPAEC using a Dionex Bio-LC50 apparatus using
condition described in Dallies et al. (1998). Susceptibility to
lytic action by zymolyase was carried out following the
procedure of Ovalle et al. (1998) with some modifications.
The assay was performed with 6 107 cells resuspended in
3 ml of TrisCl 10 mM, EDTA 1 mM, pH 75 at 30C. The

Table 1 Effects of growth conditions on cell wall mass, chitin, mannan, b-glucans, b-1,6-glucan and on the sensitivity of whole cells to zymolyase

Growth
condition

Growth
rate
(lmax h)1)

Cell wall*
[dry
mass (%)]

Chitin
[wall
mass (%)]

Mannan
[cell mass
(lg mg)1)]

Total b-glucan
[cell mass
(lg mg)1)]

b-1,6-glucan/
total
glucan (%)

Zymolyase
sensitivity (MLR)
(10)2 min)

YPD
YNB
CF
Glucose
Mannose
Sucrose
Maltose
Galactose
Ethanol
pH 3
pH 4
pH 5
pH 6
22C
30C
37C
pO2  0%
pO2 > 50%

042
035
036
036
033
039
031
023
013
022
032
036
029
019
036
044
033
044

245
212
204
183
142
152
145
164
108
179
189
205
141
124
183
155
142
186

62
30
24
52
43
48
42
47
64
69
71
55
64
52
55
79
14
52

933
895
865
927
597
536
547
453
578
621
576
927
542
416
945
475
948
927

1274
726
714
625
643
627
506
782
387
604
808
665
584
601
685
889
601
625

18
18
15
14
14
13
16
19
21
15
17
14
12
10
14
20
nd
nd

028
032
068
075
066
068
035
015
003
049
062
075
086
085
075
059
nd
nd

25
24
28
20
18
18
20
20
15
30
20
20
16
21
26
20
21
20

055
017
023
032
017
032
009
025
035
04
04
06
14
05
02
09
05
03

32
35
17
27
17
28
32
26
26
21
15
27
34
13
27
13
15
27

32
41
33
29
17
31
18
23
34
26
36
29
32
31
45
49
31
29

30
25
20
25
22
36
27
32
24
20
30
20
15
20
20
20

Yeast cells were cultivated in controlled batch-fermentor in, unless otherwise stated, CF media (cell factory). Other media were YPD (10 g l)1 yeast
extract, 20 g l)1 bactopeptone and 20 g l)1 glucose), YNB (17 g l)1 yeast nitrogen without amino acid and ammonium, 5 g l)1 ammonium sulphate,
20 g l)1 glucose) and CF (see Material and methods). Unless otherwise stated (see column 1), the pH was kept constant at 50, temperature at 30C
and aeration at pO2  50% saturation in the medium. The zymolyase sensitivity was carried out as described in Material and methods. The values
reported are the mean S.E.M. from four independent experiments, and for each experiment, three samples were taken for cell wall analysis.
*Cell wall content was determined by the phenolsulphuric methods as described in Material and methods. MLR, maximal lysis rate; nd, not
determined.
2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 268274, doi:10.1046/j.1472-765X.2003.01394.x

YEAST CELL WALL COMPOSITION AND INTEGRITY

Nutritional effects on cell wall composition

and structure
Yeast can grow on different carbon sources, which are
known to influence their growth behaviour. Thus, one
should expect alteration of cell wall composition and
structure induced by the carbon regimen. However, we also
took into consideration the cultivation mode of yeast cells,
because in shake flasks, growth parameters like pH and
oxygen availability are fixed at the start of the culture and
greatly vary with the progression of growth, while in
controlled batch reactors, these parameters can be kept
constant throughout the fermentation process. Interestingly,
growth of yeast under these two different modes of
cultivation and in the presence of different carbon sources
led to two relevant data with respect to cell wall: (i) the
relative proportion of b-glucan and mannan varied with the
nature of the fermentative carbon source, and these
variations were more noticeable in yeast cells cultivated in
shake flasks than in batch fermentors (Fig. 1); (ii) the cell
wall, as expressed in percentage of the cell dry mass, varied
by almost two times according to the nature of the carbon
source, and these variations were found in both mode of
yeast cultivation. In addition, it can be seen in this table that
the change of cell wall composition was merely an effect of
carbon composition of the medium and not an effect because
of the change of the growth rate (lmax) in the different
media. In quantitative terms, we also found that yeast
cultivated in batch reactor possessed 2035% less cell wall
mass per gram dry mass (Table 1), and contained two- to
threefold more chitin than yeast cultivated in shake flasks
(data not shown), and these differences were independent of
the carbon sources. We then estimated the proportion of
b-1,6-glucan polymer in the total b-glucan fraction of cells
cultivated on these different carbon sources. From four
independent experiments, we found that this proportion
raised from 14 25% in cells cultured in glucose, mannose
and sucrose to 21 3% in cells cultivated on ethanol
(Table 1). Taken together, these data convincingly showed

Sugar (mg per mg dry mass)

160
140
120
100
80
60
40
20
0
Glu Man Gal Suc Mal Etha Glu Man Gal Suc Mal Etha
Shake flask

Batch fermentor

Fig. 1 Effects of the carbon sources on yeast cell wall composition.

Yeast cells were cultivated at 30C in shake flasks or in batch
fermentators containing CF medium in the presence of carbon source
at an initial concentration of 20 g l)1 or ethanol at 2% (v/v). The data
reported are from four independent experiments. Error bars represent
the standard deviation on the histograms. Symbols: open bars are
b-glucan and filled bars represent mannan levels

10

08

06
OD660

15% in CF. We then assessed the susceptibility of yeast cells

to lytic action by zymolyase, as a mean to estimate the cell
wall integrity. For this purpose, we applied the procedure of
Ovalle et al. (1998) to obtain a quantitative value of the rate
of cell lysis [maximal rate lysis (MLR)], e.g. the slope of the
O.D.660 decreases over the time of incubation in the presence
of 04 U zymolyase at 30C and pH 75. As shown in
Table 1, the MLR was very similar between YPD and YNB
media, while it was two times greater with yeast cells
cultivated in a CF medium. It is therefore interesting to
notice that the rate of cell lysis estimated by zymolyase
susceptibility decreased with increased levels of b-1,6-glucan
and chitin in the cell wall.

271

04

02

00
0

100

200

300

400

500

Time (min)

Fig. 2 Effects of the carbon sources on the susceptibility of cell

cultures to lytic action by zymolyase. The susceptibility to zymolyase
digestion was carried out with yeast cells taken from exponentially
growing cells in controlled batch fermentors. Abbreviations and
symbols are: glu, glucose (s); man, mannose (n); suc, sucrose ());
gal, galactose (u); mal, maltose (r) and eth, ethanol (d)

that the polymers composition of the yeast cell wall is

affected by the nature of the carbon source.
We then tested whether the susceptibility of zymolyase
could be altered in yeast cells that have been cultured on
different carbon sources. To this end, cells cultivated in
batch fermentors were harvested early at the exponential
growth phase (O.D.660  1) and then treated with zymolyase 20T, at pH 75. As shown in Fig. 2, the rate of lysis for

2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 268274, doi:10.1046/j.1472-765X.2003.01394.x

272 B . A G U I L A R - U S C A N G A A N D J . M . F R A N C O I S

cells cultured on glucose, mannose and sucrose was in the

same range although their total b-glucan content in the wall
was different (see Fig. 1, Table 1). Yeast cells from maltose
and galactose cultures were found to be less sensitive to
zymolyase, showing a rate of lysis of two- and fourfold,
respectively, lower than cells grown on glucose. More
interestingly, yeast cells from ethanol cultures were almost
completely refractory to zymolyase (Fig. 1, Table 1).
Therefore, it became apparent that the resistance of yeast
cells to this lytic enzyme is not linked to the levels of
b-glucans, but more likely to that of b-1,6-glucan.
We also investigated the nitrogen limitation by measuring
cell wall polymers in nitrogen-depleted yeast cultures
(cultures in 005 g l)1 ammonium sulphate; see Parrou et al.
1999) and compared these values with cells from nitrogen
excess medium (i.e. 5 g l)1 ammonium sulphate). The cell
wall dry mass decreased from 21% in nitrogen-sufficient to
14% in nitrogen-depleted cells, although in both cases, the
b-glucan/mannan ratio remained the same at around 075,
and the chitin content was ca 35% of the cell wall dry mass
(data not shown). These results were somewhat surprising
because of the fact that limitation of nitrogen would rather
reduce protein synthesis and thus, would lower cell wall
proteins. However, genomic data related to nitrogen starvation does not provide any clue about effects of this condition
to alter expression of cell wall related genes (Gasch et al.
2000), which is therefore in accordance with our failure to
detect major differences in the cell wall between cells
cultivated in a nitrogen excess and a nitrogen poor medium.

044 h)1 (Table 1). While levels of mannan and b-glucan

exhibited fluctuations not correlated with temperature
changes, we noticed an increase of the chitin content and a
significant rise in b-1,6-glucan in the b-glucan fraction with
increasing temperature of growth from 22 to 37C. Thus,
one could expect that cells cultivated at elevated temperature
could be more resistant to zymolyase. As a matter of fact, we
found that higher the growth temperature, lower was the
MLR (Table 1). This further argues in favour of a
correlation between the sensibility of yeast cells to zymolyase
and levels of b-1,6-glucan.
We also examined the influence of aeration on the cell
wall composition. To this end, we performed two kinds of
batch fermentor cultures, one in which the pO2 was
maintained well above 50% saturation throughout the
fermentation period, while the second culture was carried
out without aeration and the medium was sparged with
N2 before inoculation. This hypoxic condition led to a
25% reduction of the cell wall mass and to a threefold
decrease in the chitin content, while there was no major
changes in levels of b-glucan and mannans (Table 1). The
lowering of chitin could be because of a reduction of
glutamine amidotransferase activity in hypoxic cells as a
consequence of the repression of GFA1 encoding this
enzyme by type 1 protein phosphatase activity whose
activity is increased under anaerobic conditions (Zheng
et al. 2000; Ter Linde and Steensma 2002). Unfortunately, for technical reason, we could not estimate the
sensitivity of yeast cells cultivated in the absence of
oxygen to zymolyase.

Effect of pH, temperature and aeration

on cell wall composition and structure

DISCUSSION

At 30C and under well-aerated conditions (pO2 > 50%),

the CEN.PK133-7D strain was found to grow optimally at
pH 50 with a maximal growth rate (lmax) of 036 h)1.
Changes of pH caused some fluctuations in levels of
b-glucan, mannan and chitin, but there was no obvious
relation between these fluctuations and pH of the medium
(Table 1). An interesting result was that the relative
proportion of b-1,6-glucan in the b-glucan fraction
decreased by ca 40% as the pH of the culture increased
from 40 to 60 (Table 1). Interestingly, the sensitivity of
cells to zymolyase, as determined by the MLR, of cultures
that were grown at pH 40 was lower than those cultivated at
pH 60. This observation is consistent with the data of
Kapteyn et al. (2001) who showed that yeast cultivated at
pH 35 instead of 55 acquired a stronger resistance to b-1,3glucanase, and this increased resistance was associated with
an induction of a novel type of linkage for glycosylphosphoinositide (GPI)-dependent cell wall proteins.
Regarding effects of temperature, we found that the
CEN.PK strain grew very efficiently at 37C, with a lmax of

This study illustrates a strong influence of the mode and

growth conditions on the cell wall composition of yeast.
These quantitative data, although not spectacular as compared with effects of cell wall mutations on chitin, mannan
and glucan (Dallies et al. 1998; Lagorce et al. 2002) could be
particularly relevant for industrial purposes as yeast cells are
cultivated in large bioreactors, in which many parameters,
such as pH, temperature and aeration, are not homogenous
throughout the whole process. In addition, the carbon
source is rarely pure glucose, but a mixture of several sugars.
As an example, cell wall from ethanol-grown cells is only
1012% of the dry mass, although these cells are more
difficult to break open by mechanical devices and are more
resistant to lytic action by zymolyase.
Another important result of this work was to show that
the cell wall structure, as judged by the lytic action of
zymolyase on whole cells is not directly dependent on
absolute levels of b-glucan and mannan, while a correlation
between the resistance of cells to this b-1,3-glucanase
digestion and levels of b-1,6-glucan was more likely. We

2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 268274, doi:10.1046/j.1472-765X.2003.01394.x

YEAST CELL WALL COMPOSITION AND INTEGRITY

therefore propose that the cell wall integrity is merely

dictated by the degree of b-1,6-glucosidic cross-linking
between b-1,3-glucan, mannoprotein and chitin, although
we do not exclude an important participation of levels of
b-1,3-glucan in this property (i.e. compare levels of b-glucan
and sensitivity with zymolyase of cells grown on ethanol vs
cells cultivated at 37C on glucose). This hypothesis is
supported by the following experimental data: (i) in almost
all growth conditions investigated, the resistance of yeast
cells to zymolyase went together with increased levels of
b-1,6-glucan; (ii) Jamas et al. (1986) showed that purified
glucan matrix to laminarinase degradation was proportional
to the degree of b-1,6-glucosidic cross-linking and (iii) a
genetic manipulation of b-1,6-glucosidic linkage led to cell
wall that are more resistant to laminarinase (Ha et al. 2002).
Taken together, these results support a role of b-1,6-glucan
in the modular organization of the cell wall by assembling
cell wall mannoproteins to the b-1,3-glucan fibrillar network
(Klis et al. 2002). Moreover, the increased polymerization of
the b-1,6-linked branch to b-1,3-glucan matrix could likely
alter the three dimensional conformation of b-glucans (Bohn
and BeMiller 1995). However, the validation of our
hypothesis would require a more refine methodology
combining mild enzymatic digestion of cell wall components
with mass spectrometry analysis and nuclear magnetic
resonance characterization of the b-glucan structure. Nevertheless, our study reinforces the notion that one important
safety guard of the cell to prevent cell lysis is to activate
remodeling enzymes that promote and catalyse crosslinkages between b-glucan, mannoproteins and chitin (Klis
et al. 2002), and thus, these enzymes are potential targets for
antifungal drugs.
ACKNOWLEDGEMENTS
We thank Dr John Chapman from Unilever Research
laboratory (Netherlands) for the gift of Pichia pastoris strain
which expresses recombinant endo-b-1,6-glucanase from
T. harzianium, and P. Escalier for help with the purification
of this enzyme. We also thank our colleagues for critical
discussion and support during this work. This work was
supported in part by a EU grant (QLK3-2000-01537) and by
a grant from Fonds de Recherche Hoechst Marion Roussel
(grant FRHMR2/9922) to J.F. B.A.U. hold a predoctoral
fellowship from the Consejo Nacional de Ciencas y Technologia (CONACYT) of Mexico.
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