Journal of General Microbiology ( r g p ) , 71,301-309
Printed in Great Britain
Inhibition of Mucor rouxii by Polyoxin D: Effects on Chitin
Synthetase and Morphological Development
By S . B A R T N I C K I - G A R C I A A N D E L E A N O R L I P P M A N
Department of Plant Pathology, University of Califorliia, Riverside,
California 92502, U.S.A.
(Acceptedfor publication 29 January 1972)
SUMMARY
Polyoxin D is a strong inhibitor of growth and spore germination of Mucor
rouxii. This antibiotic inhibited the growth of both the yeast and mycelial forms of
M . rouxii. It caused some minor morphological alterations but did not decisively
affect the pattern of dimorphic development. Polyoxin D is a powerful competitive
.
growing at inhibitory coninhibitor of chitin synthetase ( K , = 0.6 p ~ )Organisms
centrations of the antibiotic exhibited weakened walls that were susceptible to
bursting.
INTRODUCTION
Polyoxins are a new group of antifungal antibiotics isolated from Streptomyces cacaoi
which inhibit the growth of a number of mycelial fungi (Isono, Nagatsu, Kawashima &
Suzuki, 1965; Isono, Nagatsu, Kobinata, Sasaki & Suzuki, 1967) by interfering with chitin
synthesis (Endo, Kakiki & Misato, 1970; Ohta, Kakiki & Misato, 1970). Polyoxin D is a
strong competitive inhibitor of chitin synthetase in Neurospora crassa (Endo et a/. 1970).
Polyoxin D shares a gross structural similarity with uridine diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) in agreement with its role as acompetitive inhibitor (Fig. I). Polyoxin
has also been reported to cause some marked morphological alterations (Endo et a/. I 970).
Because of the potential use of polyoxins as experimental tools to manipulate chitin
synthesis as well as fungal morphology, we decided to investigate the action of this antibiotic
on Mucor rouxii, an organism whose dimorphism and wall biosynthesis have been examined
in our laboratory (Bartnicki-Garcia, I 963 ; Bartnicki-Garcia & Lippman, 1969; McMurrough & Bartnicki-Garcia, 197I ; McMurrough, Flores-Carreon & Bartnicki-Garcia, 197I).
Mucor racemosus was reported to be insensitive to polyoxins (Isono et al. 1965; Isono et
al. I 967). Since Mucor spp. have chitinous walls (Bartnicki-Garcia & Nickerson, 1962;
Jones, Bacon, Farmer & Webley, 1968) and M . rouxii synthesizes chitin by the same pathway
(McMurrough et a/. 1971) as in polyoxin-sensitive fungi (Endo et a/. 1970) we were also
interested in resolving a possible discrepancy in the relationship between chitin synthetase
and polyoxin inhibition in different fungi.
METHODS
Chemicals. Polyoxin D was the generous gift of M. Sakamaki of the Kaken Chemical
Company, Ltd, Tokyo, Japan. [I-14C]Glucosamine (New England Nuclear Corporation)
was used to prepare uridine diph~spho-N-acetyl-~~C-~-glucosamine
(UDP-14C-GlcNAc)
according to the method of McMurrough et al. (1971).
Microbiological. Mucor rouxii strain ~ ~ - was
8 0 used. The basic culture medium was
MIC
20
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71
S. B A R T N I C K I - G A R C I A A N D E. L I P P M A N
0
302
COOH
€1
H-C
I
I
OH H
I
l
H
NH,
I
l
-.C--C--C-C--N-
I I I I I
HZNCO FI
II
OHH
0
0
OH
OH
Polyoxin D
HOH,C
OH
!
O-P-O-
11
0
C=O
I
c' H .,
Fig.
I.
I
t
OH OH
U rid 1iir: dipliosph o-N - ace t y I-u -sl~cosiim1ne
Molecular structures of uridine diphospho N-acetyl-D-glucosamine and polyoxin D
(Isono, Asahi & Suzuki, 1969).
YPG: 0.3yo yeast extract, I yo peptone, 2 glucose (pH 4.5). Stock cultures were maintained on YPG agar (2.5 %) slants.
Both anaerobic and aerobic liquid cultures were used to evaluate the morphological
effects of the antibiotic on the fungus. The culture media were inoculated to a final concentration of 104 spores per ml. To produce anaerobic hyphae, the spores were germinated
at 28 "C in liquid YPG medium containing 0.1 or 0.2 yoglucose; purified nitrogen was continuously bubbled through the cultures (Bartnicki-Garcia, 1968a). Yeast cells were grown under
similar conditions except that N, was substituted by a mixture of 30 yo CO, + 70 yopurified
N,. Aerobic hyphae were grown in Erlenmeyer flasks containing liquid YPG medium with
0.1% glucose. The flasks were agitated on a reciprocating shaker at 28 "C.
Colonies of Mucor rouxii on agar pIates were used to examine the effects of polyoxin on
hyphal tips. The plates were prepared with YPG medium diluted 16-fold and adjusted to
pH 5-5. Plates were inoculated with a loop of M . rouxii spore suspension placed on the
centre of the plate. The fungus was grown for 16 h at 28 "C.Several drops of 480 pM-polyoxin
(PH 5-35) were applied to a small area on the periphery of the colony.
Crude chitin synthetase preparation. A flask with 500 ml of liquid YPG medium was
inoculated with 10' spores and incubated at 28 "C on a reciprocating shaker for 8 h. The
resulting young hyphae were harvested by filtration on a coarse sintered glass filter and
washed with 0.05 M-potassium phosphate buffer, pH 6.5, containing 0-01M-M~CI,.The
slurry was transferred to a centrifuge tube and sedimented at 20008 for 10min. The pellet
was disrupted by freeze-thawing (McMurrough et al. 1971) and the preparation was centrifuged at 2000 g for 10min at 4 "C.The supernatant was discarded and the pellet washed with
buffer until a clear supernatant was obtained. A suspension of the resulting pellet in buffer
constituted the crude chitin synthetase preparation.
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Polyoxin inhibition of Mucor rouxii
303
Chitin synthetase assay. The assay system contained 20 mM-N-acetyl-D-glucosamine,
0.4 mM-ATP, 0.2 mM or 0.4 ~ M - U D P - ~ ~ C - G ~ C
(0.5
NA
xC
105d.p.m.) and 0.2 ml of crude
chitin synthetase in a total volume of 0.5 ml. Polyoxin D in water was included in the concentrations shown. After 10 min at 22 "C, the reaction was stopped by acidification with
20 ,ul of glacial acetic acid and the mixture filtered through a millipore filter (type HA;
0.45 pm). After washing with ethanolic-alkali (aqueous I N-NaOH : 95 yo ethanol, I :2, vlv),
the filter with the residue was suspended in 'Aquasol' (New England Nuclear Corporation)
and counted in a Packard Tricarb liquid scintillation spectrometer. The radioactivity in the
residue was a measure of chitin synthesis (McMurrough et al. 1971).
RESULTS
Eflects on growth and germination. The sporangiospores of Mucor rouxii germinate in
two stages (Bartnicki-Garcia, Nelson & Cota-Robles, 1968): in Stage I, the ellipsoidal spore
grows into a large sphere; in Stage 11, a germ tube (or a spherical bud in yeast cultures) is
emitted. Polyoxin D affected both stages of germination and at 19 to 190 ,UM (10 to IOO pg/
ml) inhibited the germination and growth of M . rouxii. Inhibition was greater in anaerobic
cultures, thus I g pM-polyoxin D caused severe retardation of germ tube emission in cultures
grown under N, (Fig. 2c, d ) ; in aerobic cultures, there was only a slight reduction in germ
tube length (Fig. 2 a, b). Eventually growth inhibition by this concentration of polyoxin was
overcome and the amount of growth under both aerobic and anaerobic conditions approached
that of the control cultures. In aerobic cultures with 19 pM-polyoxin D, essentially all spores
germinated, but in anaerobic cultures (N2 or CO,) many spores failed to reach stages I or
I1 of germination (Fig. 2d).
At 190 to 480,~M-polyoxinD, growth was almost completely inhibited under either
aerobic or anaerobic incubation, most spores failed to germinate, the few that did developing
no further than Stage I germination (large spheres). In aerobic cultures, an occasional
spherical cell emitted atypical, highly branched hyphae (Fig. 3a). On transfer from this
concentration of polyoxin to antibiotic-free YPG agar plates, growth was not resumed.
~ , majority of the spores
At high concentrations of polyoxin D, particularly 4 8 0 , ~the
incubated under aerobic conditions rounded slightly and exhibited a small protuberance
as if they were about to go into stage I1 of germination, by-passing Stage I (Fig. 4). However,
these cells had died as they failed to recover when transferred to polyoxin-free medium.
Eflect on dimorphism. To test if polyoxin D had any effect on the mycelial-yeast dimorphism
of Mucor rouxii, we examined cultures incubated under an atmosphere of N, or 30% CO,
within the inhibitory range of the antibiotic (38,76, I 14, 152 and 190 p ~ )These
.
atmospheres
would normally lead to the formation of hyphae or yeast cells respectively.
Despite severe growth inhibition, polyoxin D did not alter the usual pattern of hyphal
morphology obtained in cultures incubated under N,. In cultures incubated under 30 yoCO,,
most of the spores, which were capable of germination in the presence of polyoxin D,
developed typical yeast buds. Occasionally, the buds were elongated, rather than spherical,
and on prolonged incubation some developed into hyphae. However, hyphae were not
formed in polyoxin-inhibited cultures when the glucose concentration of the medium was
raised to I o/o.
Incompletely germinated spores (Stage I spherical cells) showed a tendency to aggregate
to one another and to ungerminated spores, sometimes giving the false impression that they
were budding yeast cells. These inhibited cells also showed a ragged external appearance
(Fig. 3b).
20-2
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304
S . B A R T N I C K I - G A R C I A AND E. L I P P M A N
Fig. 2. Effect of polyoxin D on germination and growth of Mucor rouxii under aerobic and anaerobic
conditions. Bar equals IOO ,urn. (a) Control incubated under air for 8 h; (b) incubated with 19 p ~ polyoxinunder air for 8 h; (c) control incubatedunder N, for I I h; (d)incubated with 19pwpolyoxin
under N, for I I h. Arrow points at ungerminated spore.
Interference with apical growth. Application of rgo to 480 pM-pOlyOXin to the margin of
a colony of Mucor rouxii grown for 16 h on dilute YPG agar caused the growing tips to
burst (Fig. 5 a). Bursting time and frequency depended on concentration; the earliest bursts
occurred about I min after addition of 480 pM-polyoxin or two to three min for 190 ,UMpolyoxin. The effect was not due to simple osmotic shock since addition of distilled water
caused no bursting of the hyphal tips of M . rouxii grown on dilute YPG.
Bursting of organisms in liquid cultures. Bursting was also commonly observed (Fig. 5 b)
in liquid cultures incubated with polyoxin D at concentrations that were partially growth
inhibitory (19 to 76 p ~ )Bursting
.
occurred regardless of the atmosphere of incubation; i.e.
both yeast and hyphal forms were capable of disintegration. In the presence of 0.5 M-mannitOl,
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Polyoxin inhibition of Mucor rouxii
305
Fig. 3. Abnormal cells produced with 190,uM-polyoxin D. (a) Aerobic culture incubated for
Bar equals IOO ,urn; (6) culture incubated under N2 for 2 2 h. Bar equals 10pm.
Fig. 4. Abortive incipient germination of sporangiospores of Mucor rouxii. Bar equals
Inoculum spores; (b) after I I h aerobic incubation with 480 ,uM-polyoxin D.
20
h.
1 0pm. (a)
bursting was drastically reduced. The combination of antibiotic plus osmoticant did not
markedly affect hyphal morphology. However, it caused the appearance of giant yeast cells
in cultures containing 76 pu-polyoxin D incubated under 30 yo C o g . Some of these cells
were three to four times larger in diameter than those obtained in polyoxin-free cultures;
these giant cells discharged their cytoplasm upon dilution of the medium with distilled water.
Inhibition of chitin synthetase. Polyoxin D was a powerful inhibitor of chitin synthetase
of Mucor rouxii. As little as 1-25pM-polyoxin caused 50 yo inhibition of chitin synthesis
under the assay conditions employed (Fig. 6).
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S. B A R T N I C K I - G A R C I A A N D E. L I P P M A N
306
Fig. 5. Cell bursting (arrows) caused by polyoxin D. (a) On the margin of a colony of Mucor vouxii
treated with 480~~-polyoxin
D. Bar equals ~ o o p m(b)
; in liquid culture under N, for 8 h with
I 9 pwpolyoxin D. Bar equals 20 pm.
r
70
300
.-
c
f
U
50
30
1
3
#
Pol>oxin Ipxr)
Fig. 6
-K,
0
1.0
Pol!,o.c in
2.0
(,mi)
Fig. 7
Fig. 6. Inhibition of chitin synthesis by different concentrations of polyoxin D in the presence of
0.2 mM UDP[14C]GlcNAc.Percentage inhibition calculated relative to a control without polyoxin
D. The rate of incorporation of UDP-GlcNAc in the control was 2-32nm mol/min/ml.
Fig. 7. Kinetics of polyoxin D inhibition of chitin synthetase. Reaction velocity in the presence of
polyoxin (6)measured as described in Methods. Values plotted according to Dixon (1953).
Regression lines were calculated for each set of data.
The kinetics of inhibition of Mucor rouxii by polyoxin D were determined at various
concentrations of polyoxin and at two different substrate concentrations. The kinetic data
(Fig. 7) plotted according to Dixon (I953), indicate inhibition of a competitive type and a
K , value of 0.6 ,UM. The K, for UDP-GlcNAc measured earlier was 500 p~ (McMurrough
et al. 1971).
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Polyoxin inhibition of Mucor rouxii
Table
I.
307
Kinetic constants for chitin synthetase and its inhibition by polyoxins
Fungus
Mucor rouxii"
Neurospora crassat
Saccharomyces cerevisiael
Km
(UDP-GlcNAc)
Polyoxin
D
D
A
Ki
(Polyoxin)
(PM)
0.5
1-43
0.6 to 0.9
0.6
1.4
0.5
* McMurrough et al. (1971) and results herein.
t
Endo et al. (1970).
$ Keller & Cabib (1971).
DISCUSSION
Growth inhibition. Polyoxin D is a strong inhibitor of the growth of Mucor rouxii in a
concentration range comparable to that found for Neurospora crassa and other fungi (Endo
et al. 1970; Isono et al. 1967).
Only higher fungi, particularly phytopathogenic species, have hitherto been listed as
organisms susceptible to polyoxins (Isono et al. 1967; Endo et al. 1970); our findings
demonstrate that Zygomycetes should also be included among the polyoxin-sensitive fungi.
Moreover, the mode of action of this antibiotic in Mucor rouxii appears to be the same
proposed for Neurospora crassa (Endo et al. 1970), namely, a powerful competitive
inhibitor of UDP-GlcNAc in chitin synthesis. The degree of inhibition of chitin synthetase
of M . rouxii was comparable to that found for other fungi. In all cases, the K, values were
about 1000 times smaller than the corresponding K, for UDP-GlcNAc (Table I).
Cell wall efects. Endo et al. (1970) found that spherical cells formed in cultures of Cochliobolus miyabeanus treated with polyoxin D were osmotically fragile - an indication of a
weakened cell wall. The frequent finding of burst cells in liquid cultures of Mucor rouxii is
evidence that polyoxin D also causes a weakening of its cell walls. This conclusion is supported by the bursting of growing hyphal tips of M . rouxii during treatment with polyoxin
D. The bursting occurs at the precise region of chitin synthesis, i.e. the hyphal apex
(Bartnicki-Garcia & Lippman, 1969, 1971), and the brief delay following application of the
antibiotic probably represents the time necessary to produce a sufficiently weak, chitindeficient, apical wall.
Morphological efects. Even though polyoxin D appears to act by the same basic mechanism in different fungi, i.e. chitin synthetase inhibition, the exact morphological effects
may vary. In Neurospora crassa and Cochliobolus miyabeanus, Endo et al. (1970) reported
no inhibition of germination rate at ~ o o p ~ - p o l y o x D,
i n but the germ tubes were greatly
distorted. The germ tubes of N . crassa eventually ceased growing, but those of C . rniyabeanus
continued to grow until they ruptured. However, when the latter were osmotically protected
they formed large protoplast-like structures. In contrast, in Mucor rouxii, germ tube morphology was not markedly affected whereas the frequency and rate of germination were greatly
diminished by comparable concentrations of polyoxin D.
In view of the finding that polyoxin D caused marked changes in the morphology of
Cochliobolus iniyabeanus (Endo et al. 1970), we tested the possibility that polyoxin, via
inhibition of chitin synthesis, could also affect the patterns of dimorphic development of
Mucor rouxii; e.g. induce the formation of yeast forms under conditions which would
normally favour hyphal morphogenesis or vice versa. This was not the case. Within the
growth inhibitory range, there was no evidence of yeast formation under either N 2 or air.
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S. B A R T N I C K I - G A R C I A A N D E. L I P P M A N
Some spherical forms observed were spores which had failed to develop beyond an initial
obligatory spherical stage in the germination process. Furthermore, those few spherical
forms which were capable of subsequent development did not produce yeast buds but
hyphal tubes. Conversely, under cultivation conditions which normally lead to yeast development of M . rouxii, few if any hyphal tubes were developed as a result of polyoxin addition.
Moreover, these hyphae appeared under conditions which may be regarded as marginal
for yeast development (0.1yo glucose and 30 7; C 0 2 in the atmosphere) (Bartnicki-Garcia,
I 968 a) and disappeared when a higher glucose concentration was used.
Role of chitin synthesis in wall structure and morphogenesis. In view of the suspected correlation between wall structure and morphogenesis of fungi (Bartnicki-Garcia, 1968b), one may
pose the question as to the role of chitin in wall structure and form development of Mucor
rouxii. The extensive bursting caused by polyoxin D suggests that chitin is an essential component of the wall of the vegetative form of Mucor rouxii. Since polyoxin D does not bring
about a decisive shift in the pattern of vegetative morphogenesis, we can also conclude that
the overall rate or extent of chitin synthesis is not a decisive factor in dimorphic development.
This conclusion is also supported by the earlier finding that the yeast and hyphal forms of
M . rouxii contain similar proportions of chitin in their walls (Bartnicki-Garcia & Nickerson,
I 962). These observations, however, do not necessarily exclude a role for chitin synthesis
in morphogenesis ; they merely indicate that morphogenetic shifts are not accomplished via
scalar changes in chitin synthesis and, hence, they emphasize the previous contention that
vectorial changes in the pattern of chitin synthesis (i.e. polarized versus non-polarized
synthesis) play a key role in dimorphism (Bartnicki-Garcia & Lippman, 1969).
This investigation was supported in part by research grant AI-05540 from the National
Institutes of Health, United States Public Health Service, U.S.A.
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