Journal of General Microbiology (1991), 137, 1241-1252.
Printed in Great Britain
1241
Chitosomes and chitin synthetase in the asexual life cycle of Mucor rouxii:
spores, mycelium and yeast cells
TAKASHI
KAMADA,t CHARLES
E. BRACKER~
and SALOMON
BARTNICKI-GARCIA'
*
Department of Plant Pathology, University of California, Riverside, CA 92521, USA
2Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA
(Received 4 February 1991; accepted 19 February 1991)
To help understand the subcellular machinery responsible for cell wall formation in a fungus, we determined the
abundance and subcellular distribution of chitin synthetase (chitin synthase, EC 2.4.1.16) and chitosomes in the
asexual life cycle of Mucor rouxii. Cell-free extracts of ungerminated sporangiospores, hyphaelmycelium in
exponential and stationary phase, and yeast cells were fractionated by isopycnic centrifugationin sucrose density
gradients. The total amount of chitin synthetase per cell increased exponentially during aerobic germination of
spores. In all developmentalstages, the profile of chitin synthetase activity encompassed a broad range of sucrose
density (d = 1.12-1.22) with two distinct zones: a low-density chitosome zone (d = approx. 1.12-1.16) and a highdensity, mixed-membranezone (d = approx. 1.16-1.22). Chitosomes were a major reservoir of chitin synthetase in
all stages of the life cycle, including ungerminated spores. Two kinds of chitin synthetase profiles were recognized
and correlated with the growth state. In nongrowing cells (ungerminated sporangiospores and stationary-phase
mycelium),the profile was skewed toward lower densitieswith a sharp chitosome peak at d = 1.12-1.13. In actively
growing cultures (aerobic mycelium or anaerobic yeast cells), the entire profile of chitin synthetase was displaced
toward higher densities; the average buoyant density of chitosomes was higher (d= 1-14-1.16), and more chitin
synthetase was associated with denser (d= 1.16-1.23) membrane fractions. In all life cycle stages, chitosomal
chitin synthetase was almost completely zymogenic. In contrast to the enzyme from spores or from growing cells,
samples of chitosomal chitin synthetase from stationary-phase mycelium were unstable and contained a high
proportion of larger vesicles in addition to the typical microvesicles. The presence of chitosomes in ungerminated
spores indicates that these cells are poised to begin synthesizing somatic (= vegetative) cell walls at the onset of
germination. The increased buoyant density of chitosomes in actively growing cultures suggests that the
composition of these microvesicles changes significantly as they mobilize chitin synthetase to the cell surface.
Introduction
This investigation is part of a project to determine the
subcellular distribution of chitin synthetase (chitin
synthase, EC 2.4.1.16) during the asexual life cycle of
Mucor rouxii and to determine the role of chitosomes in
cell wall formation. Most of our research on chitosomes
has been done with the yeast form of M . rouxii, whose
chitosomes have a buoyant density of 1.14-1.15 g cm-j
(Ruiz-Herrera et al., 1984). In this study, we compared
spores and somatic (= vegetative) cells (hyphae/mycelium and yeast cells) at different growth phases. This
t Present address: Department of Biology, Faculty of Science,
Okayama University, Okayama 700, Japan
Abbreviations: UDP-GlcNAc, uridine diphosphate N-acetyl-D-glucosamine ; YPG, yeast extract/peptone/glucose medium.
0001-6462 0 1991 SGM
work was made possible by refinements in the techniques for separating chitosomes from other subcellular
organelles by direct isopycnic centrifugation of cell-free
extracts. Because of their low buoyant density, chitosomes of M . rouxii readily separate from other more
abundant subcellular structures during isopycnic sucrose
density gradient centrifugation (Ruiz-Herrera et al.,
1984). Under these conditions, most of the chitin
synthetase in the cell-free extract appears as a single
sharp peak. By using high-performance rotors, such as
the vertical rotor (Flores-Martinez et al., 1990) or the
fixed-angle rotor (Kamada et al., 1987; Leal-Morales et
al., 1988; Lending et al., 1990), we were able to shorten
centrifugation times considerably and run multiple
sucrose density gradient fractionations to compare chitin
synthetase distribution from a variety of cells grown
under different growth conditions.
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1242
T. Kamada, C . E . Bracker and S . Bartnicki-Garcia
Methods
Culture conditions. Mucor rouxii, IM 80 (ATCC 24905), was
maintained on YPG slants [2% (w/v) glucose, 1 % (w/v) peptone, 0.3%
yeast extract, 2.5% (w/v) agar, pM 4-51(Bartnicki-Garcia & Nickerson,
1962). Spore suspensions were harvested from 34-d-old YPG-agar
cultures in 500 ml Koux bottles and washed once with distilled water.
Aerobic hyphal germlings were grown in three 2000 ml Erlenmeyer
flasks each containing 650 ml liquid YPG medium. For stationary
cultures, we used one 250 ml Erlenmeyer flask with 65 ml culture fluid,
a volume sufficient to produce an adequate cell mass. Flasks for aerobic
cultures were fitted with cotton plugs. Cultures were inoculated to a
final density of 2.5 x lo6 spores ml-I. The flasks were shaken in a
reciprocating water bath at 28 "C for different time periods. For yeast
cells, cultures (3 x 650 ml) inoculated to a final density of 5 x 105
spores ml-1 were shaken in a reciprocating water bath at 28 "C for 13 h.
The flasks were fitted with rubber stoppers and glass connectors, and a
stream of N2 / C 0 2(70:30, v/v) was bubbled through them during the
incubation period (Bartnicki-Garcia & Nickerson, 1962).
Preparation of crude cell-fee extracts. ( i ) Hyphal germlings, mycelium,
and yeast cells. Cultures were harvested and filtered on a Millipore unit
with a sintered glass support. Young hyphal cultures ( 4 4 h) and yeast
cultures were filtered on a 5 pm pore membrane; older mycelial cultures
were filtered without a membrane. The cells were washed on the filter
with 0.5 M-sucrose solution in ice-cold phosphate/magnesium buffer
(50 ~ M - K H ~ P O ~ / K , H P
pH
O 6.5;
~ , 0.01 M-MgCl,) and suspended in
10 ml of the same buffered sucrose solution. This cell suspension was
mixed with 10 ml dry glass beads (04-0-50 mm diameter), placed in a
small-capacity (double-bottom) Braun flask, and the cells were broken
with a Braun MSK cell homogenizer for 20 s. During breakage, the
temperature was maintained just above freezing with liquid CO,. In all
subsequent manipulations, samples were maintained at about 1 4 "C.
The cell homogenate was centrifuged in a Beckman 70 Ti rotor at
29500g for 20 min, and the resulting 30k supernatant was applied to
the sucrose gradients.
(ii) Ungerminatedsporangwspores. A large number of sporangiospores
(1.82 x 1Olospores; packed cell volume 2.8 ml) were harvested from 25
Roux bottles with ice-cold phosphate/magnesium buffer. The spore
suspension, filtered through three layers of nylon mesh, was not
contaminated by mycelial fragments. The spores were washed twice
with buffer and once with 0.5 M-sucrose in phosphate/magnesium
buffer by low-speed centrifugations and then suspended in 10 ml of the
same sucrose solution. The total harvesting and washing time was
about 90 min, during which the temperature was maintained at 1 4 "C.
The washed spore suspension was mixed with 10 ml glass beads in a
small Braun bottle and agitated for 30 s. The cell homogenate was
centrifuged at lOOOg for 5 min, and the resulting l k supernatant was
fractionated on a sucrose gradient.
Sucrose gradients. Ultrapure density-grade sucrose (Schwarz-Mann,
Orangeburg, NY, USA) was used. The sucrose solutions were prepared
in phosphate/magnesium buffer. For isopycnic centrifugation, samples
of supernatant were diluted 1 :1 with buffer, 5 ml (spores) or 6 ml (all
other cell types) portions were layered on top of 27 ml (12-65 %, w/v)
linear sucrose gradients in Seton tubes (Sunnyvale, CA, USA), and
centrifuged in a 70 Ti rotor at 90000 g (rav.)for 4.5 h. [Gradients of yeast
cell supernatants received an equivalent centrifugation at 58 000 g
(rav.)for 6.5 h.] Fractions of 1.0 ml were collected from the top with an
ISCO (Lincoln, NE, USA) model 183 density gradient fractionator.
Absorbance values were measured at 280 nm on individual fractions in
a Beckman model 35 spectrophotometer. The relative density (specific
gravity) of individual fractions was calculated from sucrose concentration measured with an Abbe refractometer (Carl Zeiss, Oberkochen,
Germany).
Electron microscopy. Negatively stained specimens were examined by
electron microscopy (Bracker et al., 1976). Droplets of sample were
placed on carbon-coated Formvar films on 300-mesh copper grids. The
carbon films were first treated by glow-discharge to make them
hydrophilic. Aqueous 2.5% (w/v) uranyl acetate was added to each
grid, excess liquid was withdrawn with filter paper, and the grids were
air-dried. Specimens were examined and recorded with a Philips
EM400 at 80 kV. A waffle-type diffraction grating replica (463 nm
spacing) was the magnification standard. Electron micrographs were
selected and trimmed to display, within limitations of the small area
shown, both organelle detail and the distribution of particles in the
sucrose density gradient samples.
Enzyme determinations. Chitin synthetase assays were conducted as
before (Ruiz-Herrera & Bartnicki-Garcia, 1976) but in 0-125 ml
phosphate/magnesium buffer. After incubation at 22 "C, the synthesized [14C]chitin was collected on 2.4 cm Whatman 934-AH glass
microfibre filters, washed with 1 M-acetic acid/95% ethanol (8 : 2, v/v),
and its radioactivity measured in a Beckman model 7500 liquid
scintillation counter, in a cocktail containing 200 mg 1,4-bis[2(4-methyl-5-phenyloxazolyl)]-benzene
and 4 g 2,s-diphenyloxazole in 1
litre of toluene. Zymogenic chitin synthetase was activated during the
assay by addition of a crude acid protease, Rennilase (a gift from Novo
Enzyme Corp., Mamaroneck, NY, USA), at a final concentration of
1 mg m1-I. One unit of chitin synthetase is the amount of enzyme that
catalyses the polymerization of 1 nmol GlcNAc min-' .
Dry weight determinations. Cultures were filtered through sinteredglass crucibles, washed thoroughly with distilled water, and dried at
80 "C overnight before being weighed.
Growth kinetics under aerobic conditions
We determined the growth kinetics of M . rouxii to
compare cellular levels of chitin synthetase at different
stages of aerobic development. Sporangiospores began
germinating promptly, and the cells grew exponentially
for 5-6 h with a doubling time of 1.22 h (Fig. 1). Growth
loooo
*
0
2
8 10 12 14 16 18
Time (h)
Fig. 1. Growth kinetics of M . rouxii under aerobic conditions.
Measurements of dry weight per ml of culture medium were made in
65 ml ( 0 )or 650 ml (0)cultures. ---, Regression line calculated for
values between 3 and 6 h.
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4
6
Chitosomes in the life cycle of Mucor rouxii
2
1
v
%
1243
100
50
Y
5
x
L l l l l l l l l l l l l l l l l l
0
2
4
6
8
10 12 14 16 18
Time (h)
Fig. 3. Total chitin synthetase activity during aerobic germination of
spores and mycelial growth. Values correspond to the total amount of
enzyme recovered, namely the sum of the initial pellet ( 1 k for spores,
30k others) and the total activity in the gradient fractions and in the
gradient pellet. Activity is expressed as units per lo9 spores in the
inoculum. ---, Regression line calculated for values between 4 and
6 h.
did not emerge in an entirely synchronous manner (Fig.
2a, b). The proportion of cells with germ tubes increased
gradually from 2.4%, to 61.5%, to 92.8% after 4 h, 5 h,
and 6 h cultivation, respectively. Subsequently, an
extensively branched mycelium developed and, as the
culture approached stationary phase, numerous arthrospores were formed (Fig. 2c).
Determination of chitin synthetase levels during aerobic
spore germination and vegetative growth
Fig. 2. Appearance of M . rouxii cells at different stages of hyphal/
mycelial development. (a) Sporangiospores germinated for 4 h ; (6)
aerobic germlings at 5.5 h; (c) stationary-phase mycelium at 18 h.
Arrows point to arthrospores. Bar, 50 pm (for all three panels).
decreased gradually after 6 h and reached stationary
phase by 18 h. Similar growth kinetics were obtained in
the 65 ml cultures as in the 650 ml cultures used in most
of our previous work on chitosomes.
Germinating spores produced a germ tube after a
mandatory period of spherical growth (Bartnicki-Garcia
et al., 1968; Bartnicki-Garcia, 1981). Under our culture
conditions, germ tubes appeared after about 4 h, but they
Total chitin synthetase activity in cells was measured at
different cultivation times. Because proteases and
chitinases interfere with the chitin synthetase assays,
measurements in the crude cell-free extract and the 1k or
30k supernatants can grossly underestimate the total
level of chitin synthetase in the cell. We therefore
concluded that the most dependable estimates of the
total chitin synthetase in the homogenate were those
obtained by summing the values for the various final
fractions obtained from each cell-free extract, namely :
(1) activity in the 1k or 30k pellets, and (2) activity in all
sucrose gradient fractions (including the sediment) after
centrifugation of the corresponding l k or 30k supernatants (Table l). In this manner, soluble interfering
enzymes were excluded as they remained in the upper
part of the gradient where no chitin synthetase activity
was present (Kamada et al., 1991).
To compare levels of chitin synthetase between
different cell types, we calculated specific activity in
terms of cell mass (dry weight) or UV absorbance (total
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1244
T. Kamada, C . E . Bracker and S . Bartnicki-Garcia
of the centrifuged cell-free extract) (Table 1). By
either criterion, the exponentially growing aerobic cells
had the highest specific activity. On a dry weight basis,
the chitin synthetase activity of spores and stationaryphase mycelium was 12% and 8 1%, respectively, of that
in exponentially growing cells. When A 2 8 0 was used as a
criterion, the levels of enzyme in spores and stationary
mycelium were even lower, 5.6 and 64.7%, respectively,
than those in exponentially growing cells. By comparison, the level of enzyme in yeast cells was about 20-25%
that in aerobic cells (Table 1).
To follow the kinetics of chitin synthetase production
during development of M . rouxii, we calculated total
enzyme units per lo9 spores in the original inoculum
(Fig. 3). The total amount of chitin synthetase activity in
sporangiospores increased sharply during germination.
Although the data are insufficient to establish precise
kinetics, during the measured span of 4-6 h the increase
in chitin synthetase seems to be exponential with a
doubling time of 1-14h, similar to the doubling time for
cell mass.
A280
Relative density
Fig. 4. Profile of chitin synthetase activity in sporangiospores of M.
rouxii. (a) A 5 ml sample of 1 k supernatant was centrifuged on a (1 265%;27 ml) sucrose gradient in a 70 Ti rotor at 90 100 g (rav,)for 4.5 h.
(6) Recentrifugation of fractions 19-21 from above. Fractions of 0.5 ml
each were combined, diluted with 4.5 ml buffer, applied to a (12-65%;
27 ml) sucrose gradient, and centrifuged in a 70 Ti rotor at 3 1 1 000 g
(rav.) for 15 h. Fraction numbers are shown for selected fractions. 0 ,
Chitin synthetase activity; ---, A z g o .
Subcellular distribution of chitin synthetase in spores,
mycelium, and yeast cells
Ungerminated spores. After centrifugation at 90 100g for
4.5 h, the l k supernatant from ungerminated sporangio-
Table 1. Distribution of chitin synthetase in diflerent morphological stages of M . rouxii
Chitin synthetase
Subcellular distribution (%)#
Activity
Morphological
stage
Spores
Hyphal germlings
Stationary mycelium
Yeast cells
Total*
Cultivation
time
(h)
(units per
lo9 spores)
0
4.3
3.5
181.1
5
5.5
6
18
3.5
13
41 3.6
470.2
507.0
1575.9
24.1
258.3
Specific7
(units per
unit)
,4280
0-23
3.08
3-94
5.19
4.00
2.62
0.99
0.86
(units per
g dry wt)
138.0
1240.3
990.3
904-9
Pellet$
22-0
29.9
29.I
29.9
29.9
31.4
21.5
7.0
Supernatant11
(sucrose gradient)
Fractions
Pellet
66.5
49.4
43.8
48.6
50.3
66.7
69.0
50.3
11.5
20.8
27.1
21-5
19.8
1.9
9.5
42.6
~~
~
* Total chitin synthetase in the cell-free extract was estimated by summing the values from the various fractions: 1 k or 30k
+
~~~
+
pellet all sucrose
sediment in gradient. Total chitin synthetase is expressed in enzymes units per lo9 spores in the original
gradients fractions of supernatant
inoculum.
f Specific activity was calculated by dividing the estimated total activity in the cell-free extract by the dry weight of the harvested cells or by the total
of the cell-free extract (Ik or 30k supernatants).
# The values represent the percentage distribution of chitin synthetase among the 1k or 30k pellet, the sum of activities in the gradient fractions, and
the pellet at the bottom of the sucrose gradient.
$ This pellet was obtained by centrifuging the cell-free extract at 1k (spores) or 30k (all other cells).
)I The resulting 1k or 30k supernatants were fractionated in sucrose density gradients.
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Chitosomes in the life cycle of Mucor rouxii
1245
Fig. 5. Electron microscopy of chitosomes from sporangiospores of M. rouxii. Negatively stained samples from sucrose gradient
fractions. (a) Chitosome peak (fraction 20 from Fig. 40) after the first centrifugation. (6) Sample from UV-rich membrane region
(fraction 24; Fig. 4a). (c) Peak of chitosomes after recentrifugation (fraction 20; Fig. 46). C, chitosome; F, fatty acid synthetase
particle; R, ribosomes; V, large vesicle. Bar, 500 nm (for all three panels).
spores yielded a chitin synthetase profile with a sharp
chitosome peak at a buoyant density of 1.138 (Fig. 4a).
The median relative density for the entire population of
chitin synthetase particles was d = 1.143. Upon recentrifugation of chitosomal peak fractions (19-21) at ultra-
high speed (31 1 OOOg for 15 h), 81 % of the chitin
synthetase sedimented in a sharp peak at the same
buoyant density (1.138) (Fig. 4b). The chitosomal chitin
synthetase from spores was stable; samples kept at 4 "C
for 95 h retained 84% of the initial activity.
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1246
T. Kamada, C. E. Bracker and S . Bartnicki-Garcia
Although the peak of chitosomal chitin synthetase in
the first centrifugation was in a region of the gradient
that was relatively free of UV-absorbing material (Fig.
4a), electron microscopy revealed many ribosomes and
other small contaminating particles together with typical
chitosomal microvesicles (Fig. 5 a). Chitosomes were
abundant in the peak fractions, and there was a rough
correspondence between abundance of microvesicles
and chitin synthetase activity. Deeper in the gradient
(beyond the chitosomal peak), a larger proportion of
ribosomes was observed, and the number of chitosomes
per field fell sharply (Fig. 5b). Microvesicles were
detected in fractions between d = 1.16 and 1.20. Ribosome abundance paralleled the UV absorbance profile
(Fig. 4a). In fractions of about d = 1.175, chitin
synthetase activity was approximately half that in the
peak fraction, but the larger amounts of ribosomes made
it appear as if the chitosome population had fallen more
sharply (Fig. 4a and Fig. 5 b). In fractions with d = 1.20
or higher, we also observed some larger membrane
profiles of unknown identity associated with chitin
synthetase. Recentrifugation of chitosome peak fractions (Fig. 4 b) yielded a substantially purified population
of microvesicles with much less contamination by other
particles (Fig. 5c). It should be noted that the fine
mottling in the background of micrographs, particularly
visible on recentrifuged samples (Figs 5 c and 7d), is not
caused by subcellular particles but represents an artificial staining pattern produced by uranyl acetate.
Hyphalgermlings. Fractionation of cell-free extracts from
hyphal cells in the exponential growth phase (4-6 h
culture) yielded a broad peak of chitin synthetase activity
extending from d = 1.12 to 1-24 in the sucrose density
gradient ; this indicates a mixed population of particles
(vesicles) containing chitin synthetase (Fig. 6a). The
median relative density for the entire population of
chitin synthetase particles was d = 1.179. Although no
discrete chitosomal chitin synthetase peak was detected
after the first centrifugation, the existence of chitosomes
was confirmed by recentrifugation of fractions (19-21) in
the presumed chitosome region of the gradient (Figs 6b,
7 4 . The broad peak shown in Fig. 6(a) appeared to be
formed by at least two overlapping populations of chitin
synthetase particles.
Electron microscopy of fractions in the chitosomal
region (d = 1.12-1-16) of the isopycnic gradient from
hyphal germlings revealed microvesicles and smaller
contaminants such as ribosomes and fatty acid synthetase complexes. The microvesicles in these samples were
not as abundant as in similar fractions from ungerminated spores, even though the chitin synthetase activity per
fraction was significantly higher (Fig. 7a, b). Lower in
the gradient (e.g. at d > 1-16),ribosomes predominated
Loo
1.04
1.08 1.12 1 . 1 6 1.20
Relative density
1.24 "
Fig. 6. Profile of chitin synthetase activity in hyphal germlings (5-6 h
old) of M .rouxii. (a) A 6 ml sample of 30k supernatant from a 6-h-old
aerobic culture was centrifuged on a (1 2 4 5 %; 27 ml) sucrose gradient
in a 70 Ti rotor at 90 lOOg (r,".) for 4.5 h. (b) Profile after
recentrifugationof chitosome fractions (19-2 1) from a gradient similar
to that in (a) but prepared from 5-h-old germlings: individual 0.5 ml
samples from each of the three fractions (19-21) were combined,
diluted with 4.5 ml buffer, applied to a (12-65%; 27 ml) sucrose
gradient, and centrifuged in a 70 Ti rotor at 3 1 1 000 g (rav.)for 16 h. 0 ,
Chitin synthetase activity; ---, A,,,.
in the fields viewed by electron microscopy (Fig. 7c), and
this result again agrees with the high UV absorbance at
d = 1-16-1-20in these gradients (Fig. 6a). After a second
centrifugation at ultrahigh speed (Fig. 6b), the peak
fractions of chitin synthetase activity contained predominantly microvesicles (Fig. 7 d ) resembling the chitosomes we have shown before.
Stationary-phase mycelium. Centrifugation of the 30k
supernatant from mycelium in the stationary phase (18 h
culture) yielded a chitin synthetase profile with a sharp
chitosome peak at a low buoyant density of 1-121 and a
shoulder between d = 1.16 and 1.22 (Fig. 8a). The
median relative density for the entire population of
chitin synthetase particles was d = 1.135. Fractions from
the chitin synthetase peak contained a mixed population
of vesicle sizes together with a variety of other particles
(Fig. 9a). Microvesicles in the size range of chitosomes
were present, but so were larger vesicles exceeding
100 nm diameter. Background contamination included
ribosomes, fatty acid synthetase complexes, and some
small unidentified fragments. Recentrifugation of the
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Chitosomes in the life cycle of Mucor rouxii
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Fig. 7. Electron microscopy of chitosomes from hyphal germlings (5 h old). Negatively stained samples from sucrose gradient fractions.
(a) Sample from chitosome region (equivalent to fraction 18 from Fig. 6a) after the first centrifugation. (6) Sample from chitosome
region (equivalent to fraction 20 from Fig. 6a). (c) Sample from UV-rich membrane region (equivalent to fraction 24; Fig. 6a). Arrows
point to microvesicles. (d) Peak of chitosomes after recentrifugation (fraction 20; Fig. 6b). C, chitosome; F, fatty acid synthetase
particle; R, ribosomes. Bar, 500 nm (for all four panels).
chitosome peak fractions (fractions 18-20) at ultrahigh
speed (31 1 OOOg for 16 h) yielded a sharp peak of chitin
synthetase with a buoyant density of 1.1265 (Fig. 8b).
Electron microscopy of fractions from this peak revealed
that most nonvesicular contaminants had been removed
and the samples consisted mainly of small vesicles (Fig.
9b), some in the size range of typical chitosomes plus
other larger vesicles between 100 and 150 nm.
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T. Kamada, C . E . Bracker and S . Bartnicki-Garcia
particles was d = 1.171. The existence of chitosomes was
confirmed by electron microscopy of fractions (20-22) in
the chitosome region of the gradient.
Stability and zymogenicity of the chitin synthetase
4
3
2
r.00
1.04
1.12 1.16
Relative density
1.08
1.20
IT24
Fig. 8. Profile of chitin synthetase activity in stationary-phase
mycelium (1 8 h old) of M . rouxii. (a)A 6 ml sample of (30k) supernatant
was centrifuged on a (1 2 4 5 %; 27 ml) sucrose gradient in a 70 Ti rotor
at 90 100 g (rav.)for 4.5 h. (b) Recentrifugation of fractions 18-20 from
( a ) :0.5 ml of each fraction was combined, diluted with 4.5 ml buffer,
applied to a ( 1 2 4 5 % ; 27 ml) sucrose gradient, and centrifuged in a
70 Ti rotor at 31 1 OOOg (rav.)for 16 h. 0 , Chitin synthetase activity;
~~~
3
A280.
Deeper in the gradient Of the first centrifugation (Fig'
8a, fraction 25, d = 1.175), fractions contained many
ribosomes, as expected from the UV absorbance profile,
and membrane vesicles of various sizes (Fig. 9c). Most
vesicles exceeded 100 nm in diameter, but a few were
smaller, in the range of chitosomes. Upon recentrifugation of these denser fractions (fractions 24-26;
= 1*165-1*185),
we recovered a sharp peak Of
=
1'183 (profile not shown) that 'Onsynthetase at
tained
large,
1oo-200 nm in
be
and a few microvesicles that
chitosomes (Fig. 9 d ) .
Yeast cells. Since most studies on chitosomes have been
done with yeast cells of M . rouxii, for comparative
purposes, samples of 30k supernatant from 13-h-old
yeast cultures were centrifuged as were the supernatants
from the other morphological stages. As was the case for
the aerobic germlings, yeast cells (3-5 or 13 h culture)
yielded a broad profile of chitin synthetase from d = 1.12
to 1.24 but with a more distinct peak of chitin synthetase
in the chitosome region (Fig. 10). The median relative
density for the entire population of chitin synthetase
Chitin synthetase in the sucrose gradient fractions from
ungerminated sporangiospores and from the hyphal
germlings in the exponential growth phase remained
essentially constant for at least 3-4 d when stored at 4 "C.
By contrast, chitin synthetase from the mycelia in
stationary phase (18 h culture) was unstable, and 86% of
the activity was lost after storage at 4 "C for 64 h. The
loss of activity was greater in the chitosome region.
The chitin synthetase detected in chitosomes from
ungerminated spores or stationary-phase mycelium was
highly zymogenic; about 90-95% of the total chitin
synthetase was expressed only after proteolytic activation with acid protease (Rennilase). These findings
extend those made earlier with growing yeast cells or
mycelium of M . rouxii showing that chitosomal chitin
synthetase of both cell types was highly zymogenic
(Bartnicki-Garcia et al., 1978).
Discussion
Changes in subcellular distribution of chitin synthetase
during development
Some important conclusions can be made from the
profiles of chitin synthetase in the sucrose density
gradients. First, in all developmental stages of M . rouxii
examined (ungerminated spores, young mycelial germlings (4-6 h old), stationary phase mycelium (18 h old),
and anaerobic yeast cells (13 h old), the profile of chitin
synthetase activity encompassed a broad region of the
sucrose gradient from d = 1.12 to 1.22 (Fig. 11). Second,
two
could be recognized in the chitin synthetase
about d = 1.12profile: a low-density &itosome
about
1.16, and a high-density mixed membrane
d = 1.16-1.22. Third, the chitin synthetase profiles fall
into two types which appear to be correlated with growth
state (Fig. 1 1). In nongrowing cultures (sporangiospores
or stationary-phase cultures rich in arthrospores), the
profile is strongly skewed toward the lower-density
region of the gradient, with a sharp peak of chitosomes at
a low buoyant density (d = 1- 12- 1 13)and only a shoulder
of chitin synthetase activity in the zone of the gradient
containing denser membranes. By contrast, in growing
cultures (aerobic hyphae/mycelium or anaerobic yeast
cells), there are no sharp peaks but a broad profile with a
nearly equal abundance of chitin synthetase activity in
the lower and in the higher densities. In yeast cells, a
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Chitosomes in the life cycle of Mucor rouxii
Fig. 9. Electron microscopy of chitosomes from stationary-phase mycelium (18 h old) of M. rouxii. Negatively stained samples from
sucrose gradient fractions. (a) Sample from chitosome peak (fraction 18 from Fig. 8a) after the first centrifugation. (b) Sample from
recentrifuged chitosome peak (fraction 18 from Fig. 8b). (c) Sample from UV-rich membrane region of gradient after the first
centrifugation (fraction 25; Fig. 8a). ( d ) Sample from the peak of chitin synthetase at d = 1.175 (data not shown) obtained after
recentrifugation of pooled fractions 24-26 from Fig. 8(a). C, chitosome; F, fatty acid synthetase particle; R,ribosomes; V, large vesicle.
Bar, 500 nm (for all four panels).
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1249
1250
T. Kamada, C . E . Bracker and S . Bartnicki-Garcia
Relative density
Fig. 10. Profile of chitin synthetase activity in 13-h-old yeast cells of M.
rouxii. A 5 ml sample of 30k supernatant was centrifuged on a (1265%; 27 ml) sucrose gradient in a 70 Ti rotor at 58000g (rav.)for 6.5 h.
0 , Chitin synthetase activity; ---, A,,,.
\
Stationary-phase
mycelium
4
hyphal germlings (d = 1.179) or anaerobic yeast cells
(d = 1.171).
The increased buoyant density of chitosomes in
actively growing cultures suggests that the composition
of these microvesicles changes significantly as they
mobilize chitin synthetase to the cell surface. The overall
increase in the buoyant density of membrane fractions
containing chitin synthetase during active growth may
reflect either changes in the density of chitosomes per se
or associations between chitosomes and membranes of
greater buoyant density.
Two other notable differences between exponentially
growing cells and stationary mycelium were in the
stability of chitin synthetase and the homogeneity of the
vesicle population in the chitosome samples. In growing
cells, the chitosomal peak of chitin synthetase particles
was highly stable, and the samples consisted of microvesicles and rarely contained any large vesicles. In contrast,
the peak of chitosomal chitin synthetase from stationaryphase mycelium was enzymically unstable and the
samples exhibited a mixture of microvesicles with large
vesicles. These two features are probably related.
Presumably, the large vesicles were derived from the
vacuolar system of the fungus, which is known to become
more abundant in older hyphae (Buller, 1933; Grove et
al., 1970). The proteases in these vacuoles (Matile &
Wiemken, 1967; Schwencke et al., 1983) may be
responsible for the marked loss of chitin synthetase
activity from stationary-phase mycelium during sucrose
density gradient fractionation.
Levels of chitin synthetase during development
1.04
I
l
1.08
l
l
l
l
l
l
1.12 1.16 1.20
Relative density
l
l
1-24
l
Fig. 1 1 . Comparative profiles of chitin synthetase at various stages of
development of M. rouxii. Profiles were normalized by assuming a
value of 100% for the fraction with highest chitin synthetase activity.
Each profile has been offset on the ordinate for the sake of clarity.
somewhat greater abundance of chitosomes produces a
more distinct peak at d = 1.15; in aerobic germlings
there was usually little if any evidence of separation
between the two populations on the initial chitin
synthetase profile.
The existence of two different types of chitin
synthetase profiles is also evident in the median values of
relative density for the entire population of chitin
synthetase particles. The median values for sporangiospores (d = 1.143) and stationary-phase mycelium
(d = 1.135) were much lower than those for aerobic
Both the total and the specific chitin synthetase activities
increase during germination. The total amount of chitin
synthetase in the germlings, estimated when most germ
tubes had emerged (6 h), was 147 times that in the spore.
During this time, the dry weight of the cells had
increased only 20-fold. This apparent discrepancy could
be construed as an indication that the amount of enzyme
measured in the sporangiospore was an underestimate of
the total enzyme in these cells. In the cultures from 4 to
6 h of incubation, the increases in enzyme activity closely
paralleled the exponential increases in cell mass.
The exponential increase in chitin synthetase during
this germination period agrees with the earlier finding of
exponential kinetics for the accumulation of cell-wall
aminopolysaccharides in M. rouxii (chitin and chitosan),
which showed exponential kinetics with a doubling time
of 1.3 h (Bartnicki-Garcia & Lippman, 1977). The
kinetics of chitin synthetase would affect not only chitin
but also chitosan synthesis, since the latter is formed by
deacetylation of nascent chitin (Davis & Bartnicki-,
Garcia, 1984).
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Chitosornes in the life cycle of Mucor rouxii
Since the chitin synthetase usually detected in uitro is
essentially all zymogenic (Bartnicki-Garcia & Bracker,
1984; Cabib et al., 1984; Leal-Morales et al., 1988), it
follows that the values reported here and elsewhere for
chitin synthetase represent the potential for chitin
synthesis and not the actual synthetic activity of the cells.
However, the present observation that the total amount
of zymogen in the cells increases in parallel to the amount
of chitin accumulated by the germinating spores (Bartnicki-Garcia & Lippman, 1977), supports the belief that
the levels of zymogenic chitin synthetase in exponentially growing cells reflect the levels of active chitin
synthetase in the cell.
Occurrence of chitosomes in spores
Chitosomes have been found in somatic cells of a variety
of fungi (Bartnicki-Garcia et al., 1978 ; Herrera-Estrella
et al., 1982; Hanseler et al., 1983; Gozalbo et al., 1987),
but their presence in spores was not known previously.
The existence of a chitosome population in spores of M .
rouxii is in harmony with the fact that the sporangiospores of Mucor spp. are ready to germinate as soon as
they are inoculated into nutrient medium (BartnickiGarcia, 198 1 ; Orlowski & Sypherd, 1978). These spores
show only a brief, if any, lag phase (Linz & Orlowski,
1982); in Mucor racernosus, protein synthesis commences
immediately upon addition of nutrient medium (Linz &
Orlowski, 1982). Apparently, sporangiospores of these
species of Mucor are also poised to begin chitin synthesis
immediately at the onset of germination.
Nickerson et al. (1 98 1) have raised the issue that a wetharvested spore of a related fungus, Rhizopus stolonifer, is
not a native spore since metabolic changes occur during
the hydration period. Although we harvested our spores
at 1-4°C to forestall the initiation of germination, we
cannot exclude the possibility that during the long
harvest period (which was needed to collect sufficient
numbers of spores) some internal cellular changes may
have taken place.
Separation and identiJicationof chitin-synthetasecontaining organelles
Because of their uniquely low buoyant density (RuizHerrera et al., 1984; Bartnicki-Garcia et al., 1984; LealMorales et al., 1988), chitosomes from different fungi
were separated readily from other membranous organelles and the microvesicular nature of the low-density
chitin synthetase fractions of the sucrose density gradients could be readily ascertained by electron microscopy.
However, in the high-density region of the gradient, the
complexity and heterogeneity of the membrane fractions
1251
made organelle identification much more difficult and
beyond the scope of this work. From previous studies
mainly on Saccharornyces cereuisiae (Duran et al., 1975 ;
Cabib et al., 1984; Leal-Morales et al., 1988; FloresMartinez & Schwencke, 1988) and other fungi (Gozalbo
et al. 1987; Bartnicki-Garcia et al., 1978, 1984), the likely
containers of chitin synthetase in these fractions are
plasma-membrane vesicles.
The rotor speed and centrifugation time used to
fractionate the cell-free extract (fixed-angle rotor at
90000 g for 4-5 h) provided about 0.4 times the pelleting
efficiency obtained with the SW-27 rotor run at 8 1000 g
for 20 h, as was previously employed by Ruiz-Herrera et
al. (1984). This condition was sufficient to allow most
chitosomes to reach equilibrium. Although higher rotor
speeds in the fixed-angle rotor would produce sharper
and faster separations of vesicular populations, as
demonstrated in the separation of chitin-synthetasecontaining organelles of S . cereuisiae (Leal-Morales et al.,
1988), we did not use these conditions in the initial
centrifugation of cell-free extracts to avoid the serious
problem of soluble proteases moving into the gradient
and destroying or distorting the profile of chitin
synthetase activity (Kamada et al., 1991).
This work was supported in part by grants from the National
Institutes of Health, USA (GM-33513), and the National Science
Foundation, USA (INT-8413728). Journal Paper no. 12629 of the
Purdue University Agricultural Experiment Station.
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