ALTERATIONS IN THE PERMEABILITY OF NEUROSPORA CRASSA

DUE TO POLYENE ANTIBIOTICS

STEPHEN C. KINSKY
Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri

Received for publication June 13, 1961

MATERIALS AND METHODS

KINSKY, STEPHEN C. (Washington University,

St. Louis, Mo.). Alterations in the permeability
of Neurospora crassa due to polyene antibiotics.
J. Bacteriol. 82:889-897. 1961. -Thirty-three
antibiotics and synthetic fungicides were examined for their effect on the growth and morphology of Neurospora crassa. Only the polyene
antibiotics (nystatin, amphotericin B, and filipin)
caused a decrease in the dry weight of mycelial
mats, which was accompanied by the appearance
of cytoplasmic constituents in the medium. The
influence of polyene concentration, incubation
time, mycelial age, various metabolic inhibitors,
pH, and media composition on this phenomenon
are described. The present evidence indicates
that the polyenes exert their primarily fungicidal
effect by an alteration of permeability, probably
due to direct action on the membrane of sensitive

Chemicals. The antibiotics and fungicides which

were examined in this study were generously
provided by the individuals and pharmaceutical
companies cited in the footnote to Table 1.
Dimethylformamide was obtained from Eastman
Organic Chemicals, Rochester, N. Y., and 3,3'dimethylglutaric acid was purchased from the
Aldrich Chemical Company, Inc., Milwaukee,
Wis. All chemicals were used without further

purification.

Growth and antibiotic sensitivity. N. crassa

strain 5297a was employed throughout this
investigation. Antibiotic sensitivity was determined by two methods, using either a conidial
suspension or mycelial mat as inoculum. In the
first procedure, appropriate quantities of antibiotic (see Results) were added to 25 ml of sterile
growth medium (Nason and Evans, 1953) in
125-ml Erlenmeyer flasks, which were subseorganisms.
quently inoculated with approximately 0.2 ml
of a dense spore suspension of Neurospora (zero-hr
During the course of an investigation on in- inoculum). After 48 hr growth at 25 C without
duced enzyme synthesis in Neurospora crassa, shaking (stationary cultures), the mycelial mats
an attempt was made to prepare protoplasts of were harvested by filtration in a Buchner funnel,
this organism by means of antibiotics and syn- carefully folded into small rectangles, and dried
thetic fungicides. It was thought that some of over phosphorous pentoxide for 12 hr in an
these compounds might interfere with fungal evacuated desiccator. Dry weight, which was
cell wall formation in a manner analogous to the used as the index of growth, was determined
action of penicillin on Escherichia coli and Staphy- with a Mettler Type H16 balance (Mettler Inlococcus aureus (Lederberg, 1956; Park and strument Corp., Hightstown, N. J.). In the second method devised for assaying response to antiStrominger, 1957). Although no gross morpholog- biotic, a 48-hr inoculum (i.e., mycelial mat) was
ical changes which could be interpreted as pro- employed. Mycelial mats grown as above in
toplast production were apparent, the action of standing cultures, but in the absence of antiseveral polyene antibiotics led to the conclusion biotic, were transferred to 125-ml Erlenmeyer
that these agents altered the permeability of flasks containing 25 ml of fresh medium suppleNeurospora. The present paper, which amplifies a mented with the antibiotic. These flasks were
preliminary note (Kinsky, 1961), provides addi- then shaken at 25 C in a Gyrotory Shaker (New
tional evidence in support of this idea, and also Brunswick Scientific Company, New Brunswick,
includes some observations obtained with other N. J.). The mats were harvested after 2 and 5 hr
antibiotics and fungicides.
incubation by Biichner filtration, and dried an(l
889

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ABSTRACT

[VOL. 82

KINSKY

890

weighed as described previously. The increase

in dry weight between 2 and 5 hr was used as a
measure of growth. As indicated below, mycelial
mats in control flasks, which did not receive any
antibiotic, generally gained approximately 25%
of the initial dry weight under these conditions.
Variations of this procedure were routinely
employed in experiments with the polyene antibiotics, and are detailed in the Results.

Stock solutions of water-insoluble antibiotics

and fungicides were prepared with either absolute
ethanol or dimethylformamide immediately
before use. No attempt was made to sterilize
these solutions, since periodic examination of the
medium containing these agents did not reveal
any bacterial contamination after completion
of the experiment. Control flasks contained a
comparable amount of solvent when necessary.

Antibiotics

Synthetic fungicides

Inhibitiont with 100 pg/ml

Antibacterial

A. None

Antifungal

Chlortetracyclinee
Chloramphenicolh
Cycloserinef
Erythromycinf
Neomycink
Penicillinf
Puromycine
Streptomycink
Vancomycinf
Viridogreseinh

B. Partial

C. Complete

Bacitracinf
Gramicidini
Novobiocink
Psicofuraninek

Gladiolic acidd

Polymixin Bb

Amphotericin Bi
C-275f
Cycloheximidek

Mycophenolic acidd
Griseofulvini

Eulicing
Filipink
Nystatini
Pimaricine
Viridind

a-Dichloroacetamido-43hydroxy-p-nitropropiophenoneh

Acrizanea
BenzethoniuMh
Chlorodimorinea
Furasporc
NF-6Ac

Tridecylpyridine oxideh
U-587c

* The following companies and individuals geiserously provided the antibiotics and fungicides which
made this investigation possible:
a Abbott Laboratories, North Chicago, Ill. (W. E. Grundy).
b Burroughs Wellcome and Co., Tuckahoe, N. Y. (G. H. Hitchings).
c Eaton Laboratories, Norwich, Conn. (A. B. Neill).
d Imperial Chemical Industries, Ltd., Welwyin, Herts., England (P. W. Brian).
e Lederle Laboratories, Pearl River, N. Y. (B. L. Hutchings).
f Lilly Research Laboratories, Indianapolis, Ind. (E. R. Shephard).
9 Merck Sharp and Dohme, Rahway, N. J. (F. J. Wolff).
h Parke, Davis and Co., Detroit, Mich. (A. L. Rawlins).
Schering Corporation, Bloomfield, N. J. (E. J. Foley).
i Squibb Institute for Medical Research, New Brunswick, N. J. (R. E. Bennett).
k The Upjohn Company, Kalamazoo, Mich. (G. M. Savage).
t Inhibition was noted as "none" when mats from flasks containing the antibiotic had a dry weight
within 5% of the weight of control mycelial mats grown in the absence of antibiotic. "Complete" inhibition means that no growth was observed after 48 hr (see text).

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TABLE 1. Effect of antibiotics and synthetic fungicides on growth of Neurospora crassa*

POLYENE ANTIBIOTICS ON NEUROSPORA

1961]

Optical density was determined with either a

Zeiss or Cary Model 14 recording spectrophotometer.
RESULTS

16

891

A.

01)
4

I
C,

Id

C.,

Compound

50% Inhibition, 0 hr

pg/mi

Eulicin .......
Cycloheximide..
Amphotericin B.
Acrizane........
C-275...........
Viridin .........
Filipin .........
Nystatin..
Benzethonium.
Pimaricin.......
Tridecylpyridine oxide....
U-587...........
NF-6A .........
Polymixin B....
Chlorodimorine .
Furaspor .......

0.020
0.042
0.054
0.072
0.11
0.16
0.20
0.27
0.63
0.71

1.65
2.70
3.65
10.0
18.0
18.0

100% Inhibition (MIC)

0 hr

48 hr

pg/ml

pg/mi

4.1 X 10-8
1.5
1.5 X 10-7 1.5
0.3
5.6 X 10-8 0.3
3.0
1.9 X 10-7 0.5
6.8 X 10-8 1.0 >20
3-30
4.5 X 10-7 3.0
1.0
3.5 X 10-7 1.0
2.0
2.9 X 10-7 2.0
1.4 X 10-6 1.6 20.0
3-6
1.0 X 10-6 3.0
6.0 X 10-6 5.0 >5
1.1
19.0
10-' 6.0
12-24
2.3 X 10-' 12
>50
8.7 X 10-6 50
>60
4.9 X 10-5 60
1.2 X 10-4 70
>70
X

were harvested, dried, and weighed.

actual amount required for inhibition may be

less than the values cited in Table 2, since data
concerning the purity of the antibiotics and
fungicides were generally not obtainable. For
this reason, the final molar concentration in this
and succeeding experiments are only approximations made on the assumption of 100% purity.
Table 2 also indicates the least amount of each
compound which will produce complete inhibition, as estimated from the growth-concentration
curve (zero-hr inoculum). These minimum
inhibitory concentrations (MIC) were then
tested for their effect on 48-hr mycelial mats
grown in the absence of antibiotic by the procedure described above. Significantly higher levels
of acrizane, C-275, benzethonium chloride,

tridecyl-pyridine oxide, U-587, polymixin B,

chlorodimorine, and Furaspor, were (or would
be) required to give complete growth inhibition

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Response of Neurospora to various antibiotics 3' (i) 8

and fungicides. During preliminary screening of
antibiotics and fungicides, these compounds were ( 16
arbitrarily classified according to the extent of
growth inhibition produced at a concentration of
2
-0.4 2.0
20
CONC. NYSTATIN- lml
100 j,g per ml of medium (complete, partial, or
20
(20.0
NYS.
B.
5/ml)
no inhibition, using a zero-hr inoculum as de- (2
AMPHO. B.(0.06 5/ml)
6
scribed under Methods). Table 1, rows A and B,
indicates that the majority of common anti- 0 12
NYS. (0.4l/mI)
bacterial antibiotics had no effect. Complete
growth inhibition was obtained with the com- LIdU,) 8pounds listed in row C. These included most of 4Lcr
4the antifungal antibiotics and synthetic fungicides
ad
relative
which were tested. To compare the
IC
3
2
potencies of these agents, the concentration
INCUBATION TIME-HOURS
required to produce 50% inhibition after 48 hr
FIG. 1. Effect of polyene concentration and ingrowth was determined (zero-hr inoculum), and cubation
time on dry weight of mycelial mats. A.
in
Table
2.
The
antiare
presented
the results
The 48-hr-old mycelial mats were transferred to
fungal antibiotics were effective at extremely low fresh medium, buffered with 0.02 M dimethylglutaconcentrations (10-6 to 10-8 M), whereas higher rate, pH 4.5, containing different concentrations of
levels of the synthetic fungicides were required nystatin, as indicated on the abscissa. After 3 hr
to produce the same degree of inhibition. The shaking, the mats were harvested, dried, and weighed
(-y denotes ,.g). B. The 48-hr-old mycelial mats were
TABLE 2. Inhibitory concentrations of various transferred to fresh medium, buffered with 0.02 ir
dimethylglutarate, pH 4.5, containing the concenantibiotics and synthetic fungicides on
trations of mystatin and amphotericin B indicated
Neurospora crassa
in the figure. After 0.5 and 3 hr shaking, the mats

892

[VOL. 82

KINSKY

20AMPHOTERICIN

and

FLIPIN (F)

(A)

260

mA

remained

essentially

constant

throughout the experiment (Fig. 3C). Material

18
which gave a positive reaction with the Folin
260(+ F) /
protein reagent was also detected in the medium
24
D.W. (-A) "
16
this material is
appearance
(Fig. 3D).
14
A ,,'
"protein"
as The
(using ofcrystalline
plotted
Y. 14 -o
bovine
o * - 1616
//o
a
-J 12 _
8 g serum albumin as a standard), although a
-. "/
O significant, but variable, fraction was dialyzable.
, f
/
- DW(-F) 0
This may be due to tryptophan, tyrosine, small
DW260(+A)
E 10
0
(0
(+A)
peptides,
guanine, and other bases (see below),
D.W.
D.W.(+F)
8 =
//
81
/ > ss
which are known to interfere with this method
Cs
of protein determination (Lowry et al., 1951).
61 6
'------__*
.0 6
After 4 to 5 hr, the mycelial mats no longer
6(-)o/
260 (-F)
(-A)
4 -260
24
lost weight, but began growing (Fig. 3A). Under
identical conditions, the disappearance of the
0
/
,

INCUBATION TIME-HOURS
FIG. 2. Effect of amphotericin B and filipin. The
43 -hr-old mycelial mats were transferred to fresh
edium, buffered with 0.02 M dimethylglutarate,
i 4.7, containing amphotericin B, 0.3 lAg/ml
(c(a. 3.2 X 10-7 M), or filipin, 3.0 ,Ag/ml (ca. 5.3 X
10--7 M). Control flasks did not contain antibiotic.
fter shaking for varying intervals, the mats from
plicate flasks were harvested, dried, and weighed.
Tike average changes in dry weight (increase or decriease) relative to the weight of mats at the beginning
Of the experiments are shown on the right ordinate.
T- ze average increase in absorption of the medium
at 260 m,u (expressed as optical density units per
25 ml medium) is shown on the left ordinate. These
vailues were essentially nil in zero-time flasks.

characteristic absorption spectrum of nystatin

could be demonstrated in growth medium alone.
These results suggested that regrowth of the
mycelium was probably a consequence of antimycedestruction
s proby
ofantiby photo-oxidation (Vandebiotic
putte, Washtel,' and Stiller, 1956) and not caused
by the development of resistance (e.g. induction
of an enzyme which inactivated nystatin). This
conclusion was supported by the experiments
represented in Fig. 4. Continued addition of

ahonsequen

nystatin after 4 hr prevented subsequent growth

of the mycelial mats and, in fact, induced further
loss in dry weight (Fig. 4A). Figure 4B shows

o indte insthe preceding experin

(as
of additional nystatin,
that, in the absence

renewed growth occurred after the initial polyeneinduced weight loss. However, the mycelia were

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off 48-hr mycelia. These results could be explained the presence of excess antibiotic. This value
bi y lack of penetration into older mycelia or represented a 25% decrease in dry weight of
m ietabolic transformation (inactivation).
48-hr mycelial mats (range: 70 to 85 mg).
Changes accompanying mycelial atrophy. The
Effects of polyene antibiotics. Among the most
a(ctive agents tested were the polyene antibiotics, polyene-induced weight loss was accompanied by
niystatin, amphotericin B, and filipin. These the appearance in the medium of compounds
aintibiotics were examined in some detail because
absorbing at 260 m,u. This is shown in Fig. 2 for
it was observed that addition of the MIC not amphotericin B and filipin. Both the rate and
01 nly produced complete growth cessation when
extent of optical density increase in control
te sted on 48-hr mycelial mats, but actually flasks was very much smaller than in experimental
re sulted in a significant decrease in dry weight. flasks supplemented with the polyene antibiotics.
F 'igure 1A shows the loss of dry weight pro- It should be noted that appreciable growth
diuced by increasing concentrations of nystatin (i.e., increase in dry weight) was observed in the
af fter 3 hr incubation. The time course described absence of either amphotericin B or filipin after
in Fig. 1B indicates that this weight loss began a lag period of 1 to 2 hr.
innmediately and, at low levels of nystatin (also
The action of nystatin was examined over a
armphotericin B), proceeded linearly. The average longer period of time (Fig. 3). In addition to 260
WIeight loss, obtained in 15 experiments, was 20
m,i absorbing material (Fig. 3B), there also
mLg (range: 19 to 31 mg) after 5 hr incubation in appeared in the medium compound(s) absorbing
at 280 m,. The ratio of optical densities at 280

19B1 ]

POLYENE ANTIBIOTICS ON NEUROSPORA

893

20

0E

ui

U0

300

00

Y.

104

0 l-

o0 -,,
2

12
INCUBATION TIME- HOURS

24

FIG. 3. Changes accompanying nystatin-induced weight loss. The 48-hr-old mycelial mats were transferred to fresh medium, buffered with 0.02 M dimethylglutarate, pH 4.5, containing nystatin, 0.8
,ug/ml (ca. 8.5 X 10-v M). After shaking for varying intervals, the mats were harvested, dried, and
weighed. The filtrate was analyzed for 260 and 280 m,u absorption and protein. See text for additional details.

still sensitive to the antibiotic. This is shown by

the marked decrease in weight produced by
nystatin after 22 hr, at which time the mats
actually weighed more than at the start of the

experiment.
Requirements for weight loss induced by polyene
antiobiotics. Mycelial mats were transferred to
fresh medium at the beginning (36 hr), middle
(59 hr), and end (84 hr) of the exponential growth
phase of stationary cultures. Table 3 indicates
that the effect of the polyene (nystatin in this
experiment) was relatively independent of the age
of the mycelium used as "inoculum", since the
antibiotic caused essentially the same percentage
weight loss after 3 hr in all cases. Because younger
mats might be expected to grow more rapidly
than older ones, this would suggest that the
polyene-induced weight loss was not a function
of the rate of growth.
To decide whether mycelial growth was
essential, the effect of several metabolic inhibitors
on the action of nystatin was determined. Figure
5 shows that addition of nystatin to mats, whose

growth had been completely inhibited by

iodoacetamide, p-fluorophenylalanine, sodium
azide, sucrose omission, cycloheximide, or viridin,
produced a weight loss which was qualitatively
and quantitatively identical to the decrease
induced by the antibiotic initially in the absence
of any inhibitor. It must also be emphasized
that, in this experiment, only growth inhibition
due to the polyene antibiotics was attended by a
decrease in dry weight, and that none of the
other agents or antibiotics inhibitory for Neurospora produced this effect.
Influence of pH and media composition. Subsequent experiments have shown that the polyene-

induced weight loss occurred at nearly identical

rates at pH 4.5 and 7.1. Mycelial atrophy could
be demonstrated not only in the minimal medium
employed in the present investigation, but also
when 48-hr-old mats from stationary cultures
were transferred either to 20% sucrose, used as
a stabilizing medium for the preparation of
Neurospora protoplasts (Bachmann and Bonner,
1959), or distilled water.

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894

[VOL. 82

KINSKY

TABLE 3. Effect of age of Neurospora

myceliumiti on nystatin-induced
atrophy
Age of culture (hr)
36

Initial weight (ing)....... 46.77

Final weight* (mg)...... . 37.99
Weight loss (img) ....... 8.78
Weight loss (%) .......... 18.8

59

84

89.50

107.88
93.24

79.53
9.97
11.1

14.64
13.6

* After 3 hoturs shaking in fresh medium, buffered at pH 4.5 with 0.02 M dimethylglutarate and
containing 0.42 ,ug nystatin per ml (ca. 4.5 X
10-7 M). Weights recorded are the average of
mycelial mats harvested from duplicate flasks.

purine or pyrimidine bases-ultraviolet albsorl)tion.

It should bc noted that similar compounds,
although in much smaller amounts, have been
detected in zero-time control flasks and flasks
shaken for 30 min in the absence of any antibiotics. These results probably reflect the inability to wash 48-hr mycelial mats completely
free of bound material before transfer to fresh
medium. This explanation may also account for
the slow increase in 260 m,u absorption observed
during growth in the absence of antibiotic

(Fig. 2).
m
(D
ui

DISCUSSION

(,t"

3.1

Before discussing the effects of the polyene

an examination of the results obantibiotics,
6 (2
with
other coml)ounds might be al)l)rotained
E
.0
priate. The absence of growth inhibition by
penicillin and cycloserine was not surprising, in
viewv of the exvidence that these agents interfere
with the synthesis of a specific component of the
bacterial cell wall (Strominger, 1960). However,
INCUBATION TIME- HOURS
the ineffectiveness of chloramphenicol and purFIG. 4. Effect of nystatin addition at different
times. Sixteen mycelial mats were grown for 36 omycin can not be readily explained, since these
hr as stationary cultures and then transferred to antibiotics are generally regarded as inhibifresh mediunm, buffered with 0.02 .1 dimethylgluta- tors of )rotein synthesis. Recent evidence sugrate, pH 4.5. A single mycelial nmat was harvested gests that this inhibition is caused by interafter shaking for varying intervals, as indicated on ference with the transfer of amino acids from
the abscisa. The arrows denote the times at which soluble ribonucleic acid to microsomes (Yar0.02 nml of stock nystatin solution (1 ntg per ml of
molinsky andl De La Haba, 1959; Lacks and
dimethylformamide) was added to the renmaining
flasks (final concentration: 0.8 Ag/Inl, ca. 8.5 X 10-7 Gros, 1959). Although the results obtained with
M). The changes in dry weight (increase or decrease) Neurospora may reflect either lack of penetration
relative to the weight of nmats at the beginning of the or metabolic inactivation of chloramphenicol and
experiment are indicated on the ordinate. See text puromycin, the possibility that this mold possesses a mode of protein synthesis which is
for- additional details.
>-

cr
0

0
2

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Additional characterization of mlaterials appearing in the medium. The occurrence of the weight
loss in distilled wvater at neutral pH has facilitated
accumulation of materials "liberated" in the
presence of polyene antibiotics. Preliminary
chromatographic experiments have demonstrated
the following in the medium after only 30 min
incubation in the presence of saturating levels of
nystatin: nucleotides, amnino acids or peptides,
phosphorylated sugars, free sugars, inorganic
phosphate, and purine or pyrimidine bases. Thus
far, insufficient quantities have been isolated to
permit definitive chemical characterization of
these components. Tentative evidence for the
existence of these materials in the medium was
based on the following criteria: nueleotidescorrespondence of ultraviolet-absorbing and
acid-labile phosphate-containing areas in several
solvent systems; amino acids or small l)el)tidesspots detected with ninhydrin; phosphorylated
sugars-correspondence of areas containing
acid-labile phosphate, and revealed with silver
nitrate or acid aniline-diphenylamine spray in
several solvent systems; reducing sugars-silver
nitrate and acid aniline-diphenylamine spray;

POLYENE ANTIBIOTICS ON NEU'ROSPORA

19611]

-NAA

80
70
60
tI-

85

*N
_
I<
_
N

+ IODOACETAMIDE

N"'---l

-N_.

70
-

(2%)

+ CYCLOHEXIMIDE

'.

3 75

+ p-F0ALANINE

(IO-5 M)

( 10-3M)

-- p-FOlALANINE

60

-I

+ No

50

+N

-NoN3 '

+N

~~~+NN

40

8C

(10-3M)

70
6C

5
4
3
2
INCUBATION TIME HOURS
-

(9

N3

+N

-+

F(x105M)
VIRIDIN

-VIR
IIDIN
I
5
4
2
3
INCUBATION TIME- HOURS

5. Effect of various inhibitors on nystatin-induced weight loss. The 48-hr-old mycelial mats (except
experiment C) were transferred to fresh medium, buffered with 0.02 M dimethylglutarate, pH 4.5, containing
no inhibitor (solid curve) or inhibitor (dashed curve) at the concentrations cited in the figure. In experiment
C, 39-hr-old mats were employed; dimethylglutarate was omitted from the medium, and the pH was maintained at 6.9 with 0.02 potassium phosphate. In experiment D, the mats were washed extensively with distilled water and sucrose-free mediutm, and the "inhibited" mats were added to flasks which did not contain
sucrose normally present in the growth medium. Nystatin (+N) was added to flasks at the times indicated
by the arrows (final concentration: 0.8 ,ug/ml, ca. 8.5 X 10-7 Mi-). After shaking for varying intervals, the
average dry weight of mycelial mats from duplicate flasks was determined.
FIG.

insensitive to these antibiotics must still be against fungi containing chitin (Brian, 1960).
Neurospora is known to contain chitinous material
considered.
in
in
its cell wall, and an enzyme system which
hr
growth
48
after
1\Iycelial mats formed
the presence of novobiocin (100 ,ig per ml synthesizes chitin has been described in this
medium) not only weighed less than control organism (Glaser and Brown, 1957). Lack of
mats, but were also characterized by a profuse inhibition was probably not due to inactivation
formation of aerial hyphae and conidia. (The of the antibiotic, since preliminary experiments
mycelial mats in control flasks after 48 hr sta- have not shown any change in the absorption
tionary incubation were still submerged, with no spectrum of griseofulvin when it was incubated
indication of sporulation.) Novobiocin-induced with 72-hr mycelial mats.
Polyene antibiotics. This investigation has
filamentation and hyphal production has been
previously described in bacteria; magnesium ion shown that all of the polyene antibiotics tested
(nystatin, amphotericin B, filipin) caused a
was reported to antagonize this effect (Brock,
1956). In the present investigation, an attempt marked decrease in dry weight of Neurospora
mycelial mats, which, at extremely low antiwas made to obtain complete growth inhibition
of Aleurospora by lowering the magnesium sulfate biotic concentration, began immediately, proconcentration (0.05%) of the basal growth ceeded at a constant rate, and was accompanied
medium. Instead, it was observed that decreasing by the appearance of various cytoplasmic
levels of MgSO4 actually reduced the degree of constituents in the medium. Furthermore, dry
weight loss was specifically observed with the
inhibition produced by novobiocin.
The poor inhibition caused by griseofulvin polyenes and was not obtained with any other
(20 to 50% with 100 Mug per ml of medium) was agent or antibiotic (cycloheximide, viridin)
unexpected, because earlier studies have sug- inhibitory for Neurospora. Such results suggest
gested that this agent was primarily active that these clinically important agents produce

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65[

p-F-N LAN

S UCROSE

N\-.,,-

+
+SUCROSE

t+

60

(10-3 M)

-N*N
- IODOACETAMIDE
/ ----- ----

801

895'

896

KINSKY

glycolysis.
The structural unit p)rincipally responsible for
maintenance of selective permeability is the cell
membrane, and it would be plausible to assume
that this might be the l)rincipal site of polyene
action. (Recent evidence indicates that the
antibacterial antibiotics polymixin B (Newton,
1956) and streptomycin (Anand and Davis, 1960)
also act on the cell membrane and alter cellular
permeability.) The selective toxicity of the
polyenes for fungi and some algae would conse-

quently be due to a unique component in the

membrane of these organisms which specifically
combines with the polyene antibiotics. In the
present investigation, growth of Neurospora
could be comp)letely inhibited by a variety of
agents without altering the response of imycelial
mats to nystatin. This indicates that de novo
membrane synthesis is probably not inhibited by
the l)olyene antibiotics. Direct action of nvstatiii
on the membrane is further suggested by experiments which show a rapid enlargement of
iNeurospora lprotoplasts upon the addition of the
antibiotic (Kinsky, in press).
ACKNOWLEDGMENT

This research was supported by grant Ino.

RG-6941 from the U. S. Public Health Service.
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loss of material not necessary to maintain

[voi.. 82

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