Microbiology (1999), 145, 2727–2737
Printed in Great Britain
Candida albicans and Yarrowia lipolytica as
alternative models for analysing budding
patterns and germ tube formation in
dimorphic fungi
Ana B. Herrero, M. Carmen Lo! pez, Luis Ferna! ndez-Lago
and Angel Domı! nguez
Author for correspondence : Angel Domı! nguez. Tel : j34 923 294677. Fax : j34 923 224876.
e-mail : ado!gugu.usal.es
Departamento de
Microbiologı! a y Gene! tica,
Instituto de Microbiologı! a
Bioquı! mica/CSIC,
Universidad de Salamanca,
37071 Salamanca, Spain
The site for bud selection and germ tube emission in two yeasts, Candida
albicans and Yarrowia lipolytica, was analysed. Both dimorphic organisms
display different patterns of budding, which also differ from those described
for Saccharomyces cerevisiae. C. albicans, which is diploid and (until now) lacks
a known sexual cycle, buds in an axial budding pattern. During the
yeast–hypha transition induced by pH, serum, N-acetylglucosamine (GlcNAc) or
temperature, germ tube emergence occurs at approximately 50 % in a polar
manner, while the other 50 % of cells show non-polar germ tube emission. Y.
lipolytica, in which most of the natural isolates are haploid and which has a
well characterized sexual cycle, buds with a polar budding pattern
independently of the degree of ploidy. Germ tube emission during the
yeast–hypha transition in both haploid and diploid cells generally occurs at the
pole distal from the division site (bipolar). The addition of hydroxyurea (HU),
an inhibitor of DNA synthesis, also produces different effects. In its presence,
and therefore in the absence of DNA synthesis, the yeast–hypha transition is
completely abolished in Y. lipolytica. By contrast, in C. albicans germ tube
emission in the presence of HU is similar to that observed in control cultures
for at least 90 min under induction conditions. These results demonstrate that,
rather than a single developmental model, several models of development
should be invoked to account for the processes involved in the morphological
switch in yeasts (the yeast–hypha transition).
Keywords : Yarrowia lipolytica, Candida albicans, budding, yeast–hypha transition,
hydroxyurea
INTRODUCTION
Polarized cell division and spatially ordered cell growth
(polarized growth) occur in a wide variety of species
(both unicellular and multicellular). One of the best
models for exploring both polarized cell growth and
polarized cell division is the unicellular yeast Saccharomyces cerevisiae (Amon, 1996 ; Go$ nczy & Hyman, 1996 ;
Hunter & Plowman, 1997 ; Johnson, 1995 ; Kron, 1997 ;
Kron & Gow, 1995 ; Roemer et al., 1996). S. cerevisiae
cells grow in a polarized manner during vegetative
.................................................................................................................................................
Abbreviations : GlcNAc, N-acetylglucosamine ; HU, hydroxyurea ; DAPI,
4h,6-diamidino-2-phenylindole ; SEM, scanning electron microscopy.
growth by budding from different sites on the cell
surface. In this case, bud sites are defined by some
intracellular landmark and are determined by several
genes whose products are responsible for producing the
defined pattern of bud selection (Chant & Pringle,
1995 ; Chant et al., 1995 ; Lew & Reed, 1995 ; Yang et al.,
1997). S. cerevisiae also polarizes growth towards an
extracellular signal, i.e. the mating pheromones during
conjugation (Jackson & Hartwell, 1990a, b ; Segall,
1993 ; Chenevert et al., 1994). Finally, under starvation
conditions (diploid cells grown on nitrogen-limiting
medium and haploid cells growing on limited glucose) S.
cerevisiae is able to carry out pseudohyphal growth
(Scherr & Weaver, 1953 ; Gimeno et al., 1992,
1993 ; Roberts & Fink, 1994). The switch in S. cerevisiae
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A. B. HERRERO and OTHERS
from yeast to pseudohyphal growth is accompanied by
changes in several cellular processes. One of these is bud
site selection ; a change from the bipolar to the unipolar
mode results in linear filamentous chains of cells (Mo$ sch
& Fink, 1997). This pseudohyphal growth resembles the
dimorphic transition (a reversible change in the growth
mode) that occurs in many pathogenic fungi (Vanden
Bossche et al., 1993). In dimorphic yeasts the yeast–
hypha transition has been analysed using several
approaches (Gow, 1994). Most of them are based on the
assumption that the yeast–filament switch is controlled
by the same morphogenetic pathways in all fungi despite
differences in individual signals and in the shape of the
filaments.
Two yeasts, Yarrowia lipolytica and Candida albicans,
are currently being used in our laboratory as dimorphic
models for analysing the yeast–hypha transition. Y.
lipolytica is a heterothallic yeast, amenable to genetic
analysis, in which most of the natural isolates are
haploid (Barth & Gaillardin, 1996). We have developed
a simple system for inducing the yeast–hypha transition,
replacing the carbon source glucose by N-acetylglucosamine (GlcNAc ; Rodrı! guez & Domı! nguez, 1984)
and have isolated a homeo-gene, HOY1, that is required
for hypha formation and for which no clear counterpart
has been described in either S. cerevisiae or C. albicans
(Torres-Guzma! n & Domı! nguez, 1997).
C. albicans is the most frequently isolated fungal
pathogen in humans and its growth mode is determined
by environmental conditions (high temperature, high
ratio of CO to O , serum, nutrient-poor media, etc.). C.
#
# has no known sexual cycle (Odds,
albicans is diploid,
1988 ; Edwards, 1990) and several pathways regulating
its cell morphology have been described (Saporito-Irwin
et al., 1995 ; Leberer et al., 1996 ; Braun & Johnson,
1997 ; Stoldt et al., 1997 ; Lo et al., 1997). However, data
concerning the budding patterns in both yeasts are
scanty. Chaffin (1984) reported that in C. albicans the
buds emerge primarily at one pole of the mother cell
when cells are grown in the yeast form at 28 mC and
pH 7n4, while bud sites not adjacent to the previous ones
are found in yeast cells growing at 37 mC and pH 4n5.
The same work also describes that the selection of sites
for germ tube formation seems random after the cells
are grown to stationary phase at 28 mC. In a more recent
paper, the isolation of CaRSR1, a gene analogous to the
S. cerevisiae BUD1 gene, has been described (Yaar et al.,
1997). CaRSR1 is required for normal bud site selection,
germ tube emergence (Lee’s broth) and hyphal
elongation. In this study, we show that C. albicans and
Y. lipolytica display different budding patterns, which
also differ from that described for S. cerevisiae. Furthermore, the behaviour of both yeasts also differs with
respect to germ tube emission in the absence of DNA
synthesis. Taken together, all our data suggest that
several experimental models would be necessary if
general conclusions are to be drawn about the developmental pathways involved in the yeast–hypha
transition in fungi.
METHODS
Strains and media. The following strains of Candida albicans
were used in this study : C. albicans ATCC 10261, C. albicans
ATCC 26555, C. albicans SC5314, C. albicans CAF42(∆ura3 : : imm434\∆ura3 : : imm434), C. albicans CAI4
(∆ura3 : : imm434\∆ura3 : : imm434), C. albicans CAI8
(ade2 : : hisG\ade2 : : hisG ∆ura3 : : imm434\∆ura3 : : imm434)
(Fonzi & Irwin, 1993). Yarrowia lipolytica strains used were :
W28 (MatA), E129 (MatA lys11-23 ura3-302 leu2-270 xpr2322), E150 (MatB his-1 ura3-302 leu2-270 xpr2-322 ) (Barth &
Gaillardin, 1996), SA-1 (MatB), ADD1 (MatA\MatB j\his1 lys11-23\j ura3-302\ura3-302 leu2-270\leu2-270 xpr2322\xpr2-322), ADD2 (MatA\MatB lys5-12\j leu2-35\j
ura3-18\j j\his-1) and ADD3 (MatA\MatB lys1-13\j
leu2-35\j j\glcnac-1) (our laboratory). Strains were maintained by periodic transfer to slants of YED medium (1 %
yeast extract, 1 % glucose, 2 % agar). Yeast and hyphal
growth patterns in C. albicans were obtained in Lee’s medium
(Lee et al., 1975), pH 4n5 or 6n8, containing glucose as carbon
source, with or without the addition of 4 % bovine calf serum.
Also, germ tube formation was induced with GlcNac (1 %).
The dimorphic transition was induced as described previously
(Martı! nez et al., 1990). The yeast–hypha transition in Y.
lipolytica was carried out by a modification of a previously
described method (Rodrı! guez & Domı! nguez, 1984). Cells in
early exponential phase were centrifuged and resuspended in
MMWN [0n67 % yeast nitrogen base without amino acids and
ammonium sulphate (Difco), 1 % glucose] for 12 h at 2 i 10(
cells ml−". Cells were then resuspended at 10' cells ml−" in MM
[0n67 % yeast nitrogen base without amino acids (Difco), 1 %
glucose or 1 % GlcNac as carbon sources].
Flow cytometry and light microscopy. Cells were fixed in
70 % ethanol and processed for flow cytometry (Lew et al.,
1992) or with 4h,6-diamidino-2-phenylindole (DAPI) for nuclear staining (Sherman et al., 1986). A Becton–Dickinson
FACScan was used for flow cytometry. Cells were photographed using a phase-contrast Zeiss axiophot photomicroscope equipped with a 35 mm camera using Ilford FP4
Plus film (125 ASA).
Scanning electron microscopy (SEM). Cells were harvested,
washed in 0n1 M sodium phosphate buffer, pH 7n4, prefixed
with glutaraldehyde (5 % glutaraldehyde in 0n1 M sodium
phosphate buffer, pH 7n4) for 1 h, washed twice in buffer and
placed in 1 % osmium tetraoxide for 1 h at 4 mC. The material
was subsequently washed in distilled water and dehydrated in
a graded acetone series. The dehydrated cells, immersed in
absolute acetone, were mounted on specimen holders, airdried, coated with gold in a SEM Coating System (Bio-Rad)
and examined under a Zeiss DSM 940 scanning electron
microscope.
RESULTS
Yeast growth mode and induction of the
yeast–hypha transition
A systematic collection of samples of both yeasts (C.
albicans and Y. lipolytica) and of all the strains described
in Methods growing in the yeast form and during the
yeast–hypha transition was carried out. To determine
budding patterns, cells were observed by staining their
bud scars with Calcofluor white (a fluorescent compound that binds the chitin ring at the mother–daughter
junction) or by SEM.
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Budding patterns and germ tube emission in dimorphic yeasts
Table 1. Distribution of bud scars in C. albicans and Y. lipolytica
.................................................................................................................................................................................................................................................................................................................
Only cells with two or more clearly visible bud scars were scored.
Strain
C. albicans
ATCC 26555
SC5314
ATCC 10261
CAF4-2
CAI4
CAI8
Y. lipolytica
W28
SA-1
E150
E129
ADD1
ADD2
ADD3
(a)
No. of cells
counted
Cells with two or more bud
scars on the same pole
Cells with bud scars
on both poles
Undetermined
200
200
100
50
50
50
187
176
82
40
38
43
4
10
8
4
4
3
9
14
10
6
8
4
200
200
200
50
200
50
50
2
3
2
2
4
1
5
196
190
193
46
191
43
38
2
7
5
2
5
6
7
(b)
.....................................................................................................
Fig. 1. (a and b) Budding pattern of C.
albicans SC5314 growing in the yeast form
(cultures in the exponential phase of
growth) as observed by SEM. All buds are
located on one pole, clearly indicating the
axial budding pattern. Bars, 1 µm.
Bud site selection during the yeast mode of growth
Observations of Calcofluor-stained cells (not shown)
and SEM observations of all the strains tested revealed
that the first bud site in an ellipsoidal cell almost always
(95 % of the cells) formed close to or at the tip of the cell
in both haploid and diploid strains of Y. lipolytica and
in diploid strains of C. albicans. This bud site formation
was similar to what has been reported for S. cerevisiae.
When we examined cells with two or more bud scars, a
different picture emerged. In C. albicans, which is a
diploid organism (Odds, 1988), SEM clearly showed
that most the new bud sites (93 %, Table 1) were
adjacent to the previous ones (axial pattern, Fig. 1a and
b) in agreement with the results described by Chaffin
(1984). In Y. lipolytica (whose cells are more ellipsoid)
haploid (MatA and MatB) or diploid (MatA\MatB)
cells, the second bud was almost invariably (in more
than 95 % of the cells, Table 1) formed opposite the first
bud site (bipolar pattern, Fig. 2a and b). In the case of S.
cerevisiae, it has been described that haploid cells (a cells
and α cells) and diploid cells that are homozygous at the
mating type locus (a\a and α\α cells) bud axially, while
diploid (a\α) cells show a different budding pattern.
Diploid daughters bud opposite the previous bud site
(distal budding) while diploid mother cells mainly bud
bipolarly, i.e. either adjacent to or opposite the previous
bud site (Madden et al., 1992). Thus, our results with Y.
lipolytica and C. albicans show clear differences with
those described for S. cerevisiae.
Bud site selection during the yeast–hypha transition
Cells from all the strains of C. albicans and Y. lipolytica
studied here were transferred to media that induce
hyphal development (see Methods). Fig. 3 shows the
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A. B. HERRERO and OTHERS
(a)
(b)
.....................................................................................................
Fig. 2. Bud scar patterns of Y. lipolytica
cells. (a) Haploid strain E150 ; (b) diploid
strain ADD1 growing in the yeast form. All
cells bud at the distal pole : bipolar budding
pattern. Bars, 1 µm.
(a)
(b)
(c)
.....................................................................................................
Fig. 3. (a–c) Scanning electron micrographs
of germ tube emission in C. albicans. Cells of
C. albicans ATCC 2655 were collected 2 h
after the induction of the yeast–hypha
transition. Polar and lateral germ tube
emission can be observed (arrows). Again,
the axial budding pattern in the yeast cells is
very clear (triangles). Bars, 1 µm.
behaviour of C. albicans ATCC 26555 2 h after induction of the yeast–hypha transition (again, the axial
budding pattern of the yeast mode of growth is clearly
visible ; triangles). The percentage of cells forming germ
tubes was higher than 90 % (Fig. 3a). The orientation of
germ tube emergence with respect to the bud scar(s)
from yeast growth was examined. Germ tubes formed in
positions lateral (approx. 50 % ; i.e. not emerging from
either of the poles but rather from the side of the cell) or
opposite (50 %) to the bud scars (Table 2) in agreement
with the results described by Chaffin (1984). Germ tubes
emerged in a non-adjacent pattern regardless of the
mode of induction (serum, pH, temperature or GlcNac)
or the strain used (some small differences between
strains were observed, Table 2), indicating that this type
of behaviour is general for C. albicans. Our results
clearly show that changes in the selection site for germ
tube emission occur during the dimorphic switch with
respect to the yeast mode of growth. Whether specific
gene products regulate this change remains to be
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Budding patterns and germ tube emission in dimorphic yeasts
Table 2. Bud site selection during the yeast–hypha transition in C. albicans and Y. lipolytica
.................................................................................................................................................................................................................................................................................................................
Only cells with at least one clearly visible bud scar in one pole were scored.
Strain
C. albicans
ATCC 26555
SC5314
Y. lipolytica
SA-1
E150
ADD1
No. of cells
counted
Cells with polar
germ tube emission
Cells with random but never
polar germ tube emission
Undetermined
200
200
96
87
86
72
18
41
200
200
200
187
190
183
3
4
10
10
6
7
(a)
(c)
(b)
(d)
(e)
.....................................................................................................
Fig. 4. Scanning electron micrographs
showing the pattern of bud site selection of
haploid and diploid Y. lipolytica cells during
the yeast–hypha transition. Haploid cells of
strain SA-1 (a) and diploid cells of strain
ADD2 after 4 (b) and 6 h (c, d) in the
presence of GlcNAc. (e) Diploid cells of strain
ADD1 8 h after the induction of the
yeast–hypha transition. Bud scars were
always located on one pole of the cells and
the germ tube emerged in the opposite
position (polar) to the bud scars. Bars, 1 µm.
elucidated. Since we synchronized all our cultures by
starvation (all the cells were in the G1 phase of the cell
cycle, see below), we cannot exclude the possibility that
the lateral-polar pattern of germ tube emission might
have been due to the loss of certain specific signals
during this prolonged starvation period. Another point
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A. B. HERRERO and OTHERS
(a)
affected by refeeding, suggesting that the division site is
marked by a strong signal which always specifies the
location of the new bud on the distal pole. The Y.
lipolytica hypha diameter corresponds to 60–100 % of
that obtained with the yeast cell considered as a sphere
(Fig. 4).
(b)
1
2
Cell synchrony and the effect of hydroxyurea on the
yeast–hypha transition
Cell no.
3
4
5
DNA content
.................................................................................................................................................
Fig. 5. Flow cytometry analysis of the DNA content of C.
albicans SC5314 during germ tube induction in the absence (a)
and presence (b) of 200 mM HU. Lanes : 1, yeast cells growing
exponentially (panel a only) ; 2, synchronized cells ; 3–5, samples
at 90 (3), 105 (4) and 120 min (5) after induction of germ tube
emission. About 10 000 cells were examined in each panel.
meriting comment is the fact that germ tube diameters
were one-quarter of the diameter of the yeast cells
considered as spheres.
However, with Y. lipolytica, a different result was
obtained. The yeast–hypha transition almost always
occurred in a bipolar fashion. Fig. 4 shows that in both
haploid (Fig. 4a) and diploid cells (Fig. 4b and c) germ
tube emission occurred on the pole opposite the previous
bud scar. In cells with bud scars on both poles (Fig. 4d
and e), germ tube emission generally occurred on one
pole, although for the time being we are unable to
specify whether tube formation is adjacent to or opposite
the previous budding site. Thus, with respect to the
budding pattern, the behaviour of Y. lipolytica growing
in the yeast form and during the yeast–hypha transition
(haploid and diploid cells) is always the same ; buds are
formed mostly in a bipolar way (Table 2).
To induce the dimorphic switch, we starved the cells
over a prolonged period of time (see Methods). Again,
all the cells were in the G1 phase of the cell cycle (see Fig.
7). Clearly, in Y. lipolytica the budding pattern was not
In a previous study, we demonstrated that in asynchronous cultures of Y. lipolytica the inhibition of DNA
synthesis by hydroxyurea (HU) treatment prevented the
yeast–hypha transition (Rodrı! guez et al., 1990). To
extend this result and to compare the behaviour of C.
albicans and Y. lipolytica during the dimorphic switch,
we obtained synchronous cultures of both yeast species
(see Methods). Fig. 5 offers a FACS analysis of C.
albicans germ tube emission. Fig. 5(a), lane 1, shows the
DNA content of an exponentially growing culture (yeast
form) of C. albicans SC5314. Two peaks corresponding
to cells in the G1 and G2 phases of the cell cycle can
clearly be seen. Lane 2 represents the DNA content after
starvation. The culture is synchronized and all cells are
in the G1 phase. To address the issue of whether HU
treatment inhibits germ tube emission, the culture was
divided into two aliquots (Fig. 5a, control) and 200 mM
HU was added to one of them (Fig. 5b). In both cultures,
germ tube development was induced by our four
standard conditions (pH, temperature, GlcNAc or
serum) and all of them produced the same result. A clear
increase in fluorescence, indicating normal DNA replication, was observed in Fig. 5(a) up to 120 min (control
culture). Longer incubation times did not produce
reliable results by FACS owing to the size of the hypha
(see Fig. 6). However, as expected, DNA replication did
not occur in the presence of HU (Fig. 5b). At the same
time, the percentage of cells forming germ tubes was
monitored (Fig. 6). Fig. 6(a) (corresponding to Fig. 5a,
lane 1) shows a typical asynchronous yeast culture in the
exponential phase of growth ; both budded and
unbudded cells are present. Fig. 6(b) (corresponding to
Fig. 5, lane 2) shows our synchronous culture. No
budding cells were present, as expected for a culture in
the G1 phase. Figs 6(c) and 6(d) show the behaviour of
the control culture at 120 min after germ tube induction.
Normal germ tube formation occurred in more than
90 % of the cells and DAPI staining revealed normal
DNA replication and the migration of nuclei through
the germ tube.
In the culture carried out in the presence of HU, DNA
synthesis did not occur, as demonstrated by DAPI
staining (Fig. 6f and, in agreement with our FACS
results, Fig. 5b), although more than 85 % of cells were
able to form germ tubes (Fig. 6e). Comparison of both
cultures (Fig. 6c and e) clearly showed that hyphal
growth was reduced in the presence of HU (this effect
was even more evident at prolonged incubation
times ; not shown). However, these experiments did not
show whether the smaller length of the hyphae was a
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Budding patterns and germ tube emission in dimorphic yeasts
(a)
(b)
(c)
(d)
(e)
(f )
.....................................................................................................
Fig. 6. Germ tube formation. C. albicans
cells were grown (a) under conditions
inducing the yeast phase of growth, (b)
synchronized and (c–f) observed 120 min
after transfer to conditions inducing germ
tube formation under normal conditions (c)
(d, DAPI staining) and in the presence of HU
(e) (f, DAPI staining). Bars, 20 µm.
direct effect, due to the lack of DNA replication, or an
indirect effect ; RNA and protein synthesis are known to
be inhibited after 2 h in the presence of HU (Rodrı! guez
et al., 1990). Our results for C. albicans indicate that
germ tube formation occurs in the absence of DNA
synthesis in a similar way to the normal processes up to
times of approximately 90 min.
Similar experiments were carried out with Y. lipolytica.
Fig. 7(a), lane 1 (and Fig. 8a), shows the DNA content of
an exponentially growing culture (yeast form of Y.
lipolytica haploid strain SA-1), with the typical dis-
tribution of cells in the G1 and G2 phases. Cultures
synchronized prior to the induction of germ tube
emission, with all the cells in the G1 phase of the cell
cycle, are represented in Fig. 7(a), lane 2 (an image of the
culture is shown in Fig. 8b). A constant increase in the
DNA content, indicating that all the cells have more
than one nucleus, is evident (Fig. 7a, lanes 4 and 5) in the
control culture, which also developed a normal yeast–
hypha transition (Fig. 8c and d). DNA replication did
not occur in the presence of HU (Fig. 7b) ; in this case the
drug completely blocked the yeast–hypha transition
(Fig. 8e). The observation that some cells were able to
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(a)
(b)
(a)
1
2
3
Cell no.
(b)
4
5
(c)
DNA content
.................................................................................................................................................
Fig. 7. Flow cytometry analysis of the DNA content of Y.
lipolytica SA-1 during germ tube induction in the absence (a)
and presence (b) of 50 mM HU. Lanes : 1, yeast cells growing
exponentially (panel a only) ; 2, synchronized cells ; 3–5, samples
at 4 (3), 6 (4) and 8 h (5) after induction of the yeast–hypha
transition. About 10 000 cells were examined in each panel.
bud is in agreement with the results described for S.
cerevisiae by Slater (1973) and Hartwell (1976), and very
few of them had a more cylindrical shape (Fig. 8e).
(d)
(e)
DISCUSSION
Polarized cell division occurs in a wide variety of
unicellular and multicellular organisms and the elucidation of the mechanisms controlling growth polarity is
essential for understanding cell division and hypha
formation in fungi. The budding yeast S. cerevisiae
undergoes polarized cell growth and polarized cell
division at specific times during its life cycle and during
the developmental processes leading to pseudohyphal
growth. S. cerevisiae cells select bud sites on the basis of
one of two predetermined patterns : MATa and MATα
cells bud in an axial pattern and MATa\MATα cells
bud in a bipolar pattern. It has also been described that
during long incubation times on rich solid media,
haploid S. cerevisiae cells switch from an axial to a
bipolar budding pattern and carry out a process termed
.................................................................................................................................................
Fig. 8. Yeast–hypha transition. Y. lipolytica cells were grown (a)
under conditions inducing the yeast phase of growth, (b)
synchronized and (c–e) observed 6 (c) and 8 h (d) after being
transferred to conditions inducing the yeast–hypha transition
(normal conditions, see Methods) and after 8 h in the presence
of 50 mM HU (e). Bars, 20 µm.
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Budding patterns and germ tube emission in dimorphic yeasts
(a)
(b)
Haploid
(c)
AXIAL
D2
M
Haploid
and
diploid
M
M
Diploid
D1
D1
AXIAL
D2
D1.1
M
Diploid
M
M
D1
M
M
BIPOLAR
BIPOLAR
D2
M
D1
M
M
D1
D1
D1.1
D2
M
(d)
(e)
Diploid
Polar
M
Pseudohyphal growth
M
Diploid
Germ tube emission
polar – lateral
M
M
(f )
Haploid and diploid
Polar
M
Yeast–hypha transition
M
.................................................................................................................................................................................................................................................................................................................
Fig. 9. Budding patterns of vegetatively growing yeast cells of S. cerevisiae (a), C. albicans (b) and Y. lipolytica (c).
Possible models for site selection of pseudohypha formation in S. cerevisiae (d), germ tube emission in C. albicans (e) and
the yeast–hypha transition in Y. lipolytica (f).
invasive growth (Roberts & Fink, 1994). All these
processes are well documented in this model yeast
(Madden et al., 1992 ; Chant & Pringle, 1995 ; Chant et
al., 1995 ; Kron & Gow, 1995 ; Lew & Reed,
1995 ; Roemer et al., 1996 ; Yang et al., 1997) and indepth studies on the role of the genes of the MAPK
pathway in S. cerevisiae and C. albicans morphogenesis
have been carried out (Roberts & Fink, 1994 ;
Herskowitz, 1995 ; Schultz et al., 1995 ; Leberer et al.,
1996 ; Lo et al., 1997 ; Madhani & Fink, 1998a, b).
However, although many yeast species are dimorphic,
the data about budding patterns and germ tube emission
are scanty in non-Saccharomyces yeasts. Here we
compare the budding patterns of C. albicans and Y.
lipolytica, a dimorphic heterothallic yeast that is
genetically closer to filamentous fungi than many other
budding yeasts (Barns et al., 1991).
We analysed several strains of C. albicans and Y.
lipolytica and in the latter case the behaviour of haploid
and diploid strains also. Although some subtle
differences among strains of different backgrounds and
in the same strain under the different conditions of the
yeast–hypha transition were observed, we believe that
the major features of our descriptions can be generalized.
Regarding bud formation during the yeast mode of
growth, in both Y. lipolytica and C. albicans the first
bud generally appeared on one pole of the cell, as has
been described for S. cerevisiae (Madden et al., 1992).
Also, and in agreement with the results obtained for S.
cerevisiae, no cases of overlapping bud sites (overlapping
bud scars) have ever been observed in axially budding or
bipolar budding cells (Chant & Pringle, 1995 ; Figs 1, 2,
3 and 4).
On comparing budding patterns, we observed that
diploid Y. lipolytica cells clearly budded in a bipolar
way (like S. cerevisiae), while C. albicans followed an
axial budding pattern. Surprisingly, haploid cells of Y.
lipolytica also budded with a bipolar pattern, again
differing from the behaviour of S. cerevisiae haploid
cells. Our findings indicate that at least the three
developmental models shown in Fig. 9 are necessary to
understand the budding patterns of vegetatively growing
yeast cells.
The choice of the budding pattern in S. cerevisiae is
controlled by the mating type locus (Chant et al.,
1995 ; Amon, 1996 ; Madden et al., 1992). Possibly, the
observed differences between S. cerevisiae and C.
albicans can be explained in terms of the lack of a sexual
cycle in the latter yeast, although this hypothesis cannot
be applied to Y. lipolytica because its haploid and
diploid strains are stable and its conjugation and
sporulation processes (although with lower efficiency
than in S. cerevisiae) have been described exhaustively
(Ogrydziak et al., 1978 ; Barth & Gaillardin, 1996).
Furthermore, the loci corresponding to both mating
types have been isolated and sequenced (Kurischko et
al., 1992, 1999).
Regarding germ tube emission, again, we observed
similar types of behaviour for all the strains tested under
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A. B. HERRERO and OTHERS
all the conditions assayed. In S. cerevisiae, pseudohyphal
growth requires a polar budding pattern (Gimeno et al.,
1992) and hence haploid filament formation depends on
a switch from an axial to a bipolar mode of bud site
selection (Roberts & Fink, 1994). Our results are
essentially in agreement with this kind of behaviour
although we stress that since Y. lipolytica haploid and
diploid cells show a bipolar budding pattern such a
switch is not necessary. In Y. lipolytica the second bud
site and germ tube emission always occur on the pole
distal to the preceding bud scar, independently of
starvation and refeeding. This implies that nutritionally
starved cells do not lose any specific component involved
in bipolar budding-pattern signalling. For the time being
we can only suggest that either axial genes involved in
bud site selection, such as BUD3, BUD4, BUD10, etc.
(Chant et al., 1995 ; Roemer et al., 1996), are not
functional in Y. lipolytica, or that the gene products
involved in the bipolar pattern act in this yeast in a
dominant way. Experiments to test such possibilities are
currently under way. In C. albicans, germ tube emergence is preceded by a switch from an axial to a different
mode (lateral or polar) of bud site selection, resembling
the behaviour of S. cerevisiae haploid cells during both
invasive and pseudohyphal growth (Roberts & Fink,
1994).
HU inhibited DNA synthesis in C. albicans and Y.
lipolytica (Figs 5 and 7). However, the addition of HU
to cultures undergoing the yeast–hypha transition
produced a different effect in both yeast species : whereas
in C. albicans germ tube emission occurs in an apparently normal way up to 90 min after the addition of
HU (in agreement with the results described by Shepherd
et al., 1980), in Y. lipolytica the morphogenetic switch is
completely abolished (in agreement with our previous
results with asynchronous cultures, Rodrı! guez et al.,
1990). One possible interpretation of this latter result
could be that some preformed mRNA molecules with a
short half life would be degraded during the long
incubation times (4–6 h) necessary for germ tube
induction in synchronized cells of Y. lipolytica in
comparison with the time required by C. albicans (1–2
h).
While it is clear that dimorphism in fungi is controlled
by multiple signalling pathways and that some of these
are conserved among distantly related fungi, this study
and several others suggest that several different models
should be used if we are to understand how fungal cells
integrate the information from different pathways to
effect the dimorphic switch.
ACKNOWLEDGEMENTS
We wish to thank C. Gaillardin for providing some Y.
lipolytica strains, N. Skinner for revising the English version
of this manuscript, and C. Kurischko and E. Fermin4 a! n for
helpful discussions. This work was partially supported by
Grants from the DGICYT (PB94-1384), Junta de Castilla y
Leo! n (SA 46\99) and EU (BMH4-CT96-0310) and by the
Acciones Integradas Hispano-Alemanas 77A and HA1996-
0151. This work was carried out under a UNESCO Chair of
the UNITWIN program. A. B. Herrero was supported by a
fellowship from the Junta de Castilla y Leo! n.
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Received 25 February 1999 ; revised 8 June 1999 ; accepted 28 June 1999.
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