Neurospora crassa conidia incubating in buffer at low water activities (a(w)) release a germination-essential component as well as 260-nm absorbing and ninhydrin-positive materials, regardless of whether an electrolyte or non-electrolyte is used to reduce a(w). Chloroform and antibiotics known to increase cell-membrane permeability have a similar effect. This suggests that membrane damage occurs in media of low a(w) and that an increase in permeability is responsible for the release of cellular components. The damage caused in media of low a(w) is nonlethal in most cases, and the conidia recover when transferred to nutrient medium

Charlang, Gisela

Horowitz, N. H.

PubMed

Neurospora crassa conidia incubating in buffer at low water activities (aw) release a germination-essential component as well as 260-nm absorbing and ninhydrin-positive materials, regardless of whether an electrolyte or non-electrolyte is used to reduce aw. Chloroform and antibiotics known to increase cell-membrane permeability have a similar effect. This suggests that membrane damage occurs in media of low aw and that an increase in permeability is responsible for the release of cellular components. The damage caused in media of low aw is nonlethal in most cases, and the conidia recover when transferred to nutrient medium

Caltech Authors - Main

JOURNAL OF BACTERIOLOGY, Jan. 1974, p. 261-264
Copyright 0 1974 American Society for Microbiology
Vol. 117, No. 1
Printed in U.S.A.
Membrane Permeability and the Loss of Germination Factor
from Neurospora crassa at Low Water Activities
GISELA CHARLANG AND N. H. HOROWITZ
Division of Biology, California Institute of Technology, Pasadena, California 91109
Received for publication 23 July 1973
Neurospora crassa conidia incubating in buffer at low water activities (a,)
release a germination-essential component as well as 260-nm absorbing and
ninhydrin-positive materials, regardless of whether an electrolyte or non-elec-
trolyte is used to reduce a,. Chloroform and antibiotics known to increase
cell-membrane permeability have a similar effect. This suggests that membrane
damage occurs in media of low a, and that an increase in permeability is
responsible for the release of cellular components. The damage caused in media
of low a, is nonlethal in most cases, and the conidia recover when transferred to
nutrient medium.
Living organisms have a high and, within
narrow limits, apparently irreducible require-
ment for water. Most microorganisms are una-
ble to grow in media whose water activity (a,) is
below 0.9. A few unusual bacteria, the extreme
halophiles (7, 18), grow at a, 0.75. Growth of
some fungi has been reported at a, 0.62 (18),
but the growth is so slow even under ideal
conditions of temperature and nutrition that its
biological significance is doubtful. Some very
dry natural environments, such as locales in the
dry valleys of Antarctica where the prevailing
aw is 0.45 or less at 0 C, are abiotic (6). In many
other regions of the world, also, water is the
life-limiting factor. Yet little is known about
what determines the water requirements of
microorganisms, or what cellular responses re-
sult when these requirements are not met.
Charlang and Horowitz (3) have presented
evidence for the existence of a substance in
conidia of Neurospora crassa which is essential
for their germination and which is lost from the
cells in media of low aw. The substance, called
"germination factor" (GF), can be extracted
from conidia or mycelium. When added to
media of low aw, it shortens the long germina-
tion lag that is observed in these media. GF
activity is found in extracts of other fungi, as
well-e.g., Aspergillus nidulans and Penicil-
lium chrysogenum -but not yeast; Escherichia
coli contains a trace (unpublished data from
this laboratory). GF is not present in complete
medium (see composition under Materials and
Methods).
GF loss occurs in media of low aw regardless of
whether an electrolyte or non-electrolyte is used
to reduce a,. In this paper, we present evidence
that a nonlethal alteration of the permeability
of the cell membrane in such media is responsi-
ble for the loss of GF, along with other mole-
cules. A similar, but usually lethal, effect is
obtained when antibiotics known to damage the
membrane specifically are used.
MATERIALS AND METHODS
Wild-type N. crassa, strain 74A, was grown on
slants or in 125-ml flasks of complete medium [Vogel
minimal medium N (19) supplemented with 0.5%
yeast extract, 0.25% casein hydrolysate, and 1.5%
agar] in the manner previously described (3).
Experimental procedures. Conidia from 4-day-old
cultures were harvested by suspension in distilled
water and filtered through four layers of cheesecloth
to remove mycelial debris. They were washed once
and suspended in 0.067 M sodium phosphate buffer,
pH 6.0, before being counted in a haemocytometer. A
total of 106 conidia/ml were incubated in buffer at low
aw or in buffer with added antibiotics or chloroform,
at 30 C for up to 48 h with shaking. At various time
intervals, samples were taken, and the conidia were
removed by centrifugation and Millipore filtration.
The cell-free solutions were tested for GF activity,
260-nm absorbance, and ninhydrin reaction.
The aw of the buffer (initially 0.998) was lowered to
0.938, 0.908, or 0.843 by adding NaCl or glycerol. The
required amounts of solutes were calculated from the
tables of Robinson and Stokes (17), Scott (18), and
Ingram (7).
Certain antibiotics have been shown to damage the
cell membrane with a resulting increase in permeabil-
ity (5). Of these, we used nystatin (8, 9, 10) at 5 1g/ml,
polymyxin B (5, 8, 15) at 50 g/ml, and tyrocidine (12)
at 20 jug/ml. Nystatin was decomposed photochemi-
cally (10) before GF assays were carried out. Nystatin
and polymyxin were obtained from Sigma Chemical
261
CHARLANG AND HOROWITZ
Co., and tyrocidine was obtained from Nutritional
Biochemicals Corp.
Assays. The GF assay was essentially the one
previously described (3). Samples to be tested were
added to 50 ml of complete medium plus NaCl at a,
0.934. Inoculation was with 102 conidia/ml, and the
flasks were incubated with shaking at 30 C for 90 h.
The maximum absorbance in the 250- to 260-nm
range was read from spectral curves obtained on a
recording Cary 15 spectrophotometer. The method of
Moore and Stein (13) was used for determinations of
ninhydrin-positive material.
RESULTS
Experiments at low a,. Loss of GF from
conidia is correlated with loss of 260-nm absorb-
ing and ninhydrin-positive materials. This is
shown in Table 1 for solutions of lowered aw and
in Table 2 for substances known to damage the
cell membrane. Germination does not occur
during 48-h exposure to any of the solutions
listed in these tables, except for the tyrocidine
case discussed below.
When conidia are incubated in buffer at a,
0.998, there is some loss of 260-nm absorbing
and ninhydrin-positive materials into the me-
dium, but there is no detectable loss of GF. At
low aw, however, GF is released along with
larger amounts of 260-nm absorbing and ninhy-
drin-positive substances (Table 1). It should be
noted that the loss of materials from the cells is
both aw- and time-dependent.
All of the solutions (except buffer) listed in
TABLE 1. Amounts of GF activity, 260-nm absorbing and ninhydrin-positive materials released from conidia at
different levels of a,
Molal
concen- a, of sus- GF actiVitya OD at 260 nm Ninhydrin-positive
Suspending medum of solute pending
(NaCl or medium 6hd 24h 48h 6h 24h 48h 6h 24h 48h
glycerol)
Buffer 0.998 0.0 0.0 0.0 0.13 0.23 0.33 0.014 0.014 0.025
Buffer plus NaCl 1.78 0.938 1.5 5.0 0.20 0.24 0.068 0.12
2.57 0.908 6.3 10.5 18.3 0.195 0.30 0.74 0.075 0.164 0.242
4.15 0.843 6.3 9.4 15.4 0.190 0.33 0.84 0.06 0.151 0.259
Buffer plus glycerol 3.32 0.938 0.0 2.7 2.2 0.12 0.275 0.68 0.039 0.045 0.05
5.0 0.908 1.3 5.0 10.8 0.17 0.33 1.06 0.05 0.078 0.156
8.75 0.843 1.9 8.6 47.3 0.16 0.77 3.30 0.092 0.28 0.99
a GF bioassay of 0.25 ml of incubation mixture. Amounts are expressed in mg of mycelial dry weight, average
of two flasks.
b Peak absorbance in 250 to 260 nm range.
c Expressed in mM amino acid equivalents based on a leucine standard.
d Time of exposure.
TABLE 2. Amounts of GF activity, 260-nm absorbing and ninhydrin-positive materials released from conidia by
antibiotics or chloroform
Concentra- GF activitya OD at 260 nmb Ninhydrin-positive
Suspending medium tion ofmaterialt
(jg/ml) 3hd 6h 24h 3h 6h 24h 3h 6h 24h
Buffer 0.0 0.0 0.0 0.13 0.13 0.23 0.012 0.014 0.014
Buffer plus nystatin 5 trace 4.0 57.7 1.43 2.2 4.9 2.26 2.28 3.21
Buffer plus polymyxin B 50 45.0 69.5 4.3 11.1 2.9 6.5
Buffer plus tyrocidine 20 46.4 1.8 1.2 2.83 1.43 0.58 1.35 0.36 0.067
Buffer plus chloroform saturated 72.2e 6.5e 3.05e
a GF bioassay of 0.25 ml of incubation mixture. Amounts expressed in mg dry weight (average of 2 flasks).
Peak absorbance in 250 to 260 nm range.
c Expressed in mM amino acid equivalents based on a leucine standard.
d Time of exposure.
e Exposure was for 1 h.
262 J. BACTERIOL.
MEMBRANE PERMEABILITY OF N. CRASSA
Table 1 cause extensive plasmolysis of conidia.
No, or at best only partial, recovery from
plasmolysis occurs during a 48-h exposure to the
NaCl solutions. It thus appears that the altera-
tion in permeability that is induced by these
solutions is not so extensive as to permit free
entry of NaCl into the cells. Although plasmo-
lyzed, the cells are nevertheless fully viable, as
shown by subsequent plate counts on sorbose
medium (2). In the case of glycerol solutions,
there is complete recovery from plasmolysis
within 48 h at a, 0.938 and a, 0.908, and the
cells show full viability when plated out. In
glycerol at a, 0.843, however, viability is re-
duced by about 40%, and recovery from plas-
molysis is abnormal. The cells are enlarged and
have a transparent center with the protoplasm
distributed around the periphery. In contrast,
normal conidia tend to be uniformly dense.
The data of Table 1 parallel these observa-
tions in that they show that glycerol is more
damaging than NaCl at high concentrations. At
low concentrations (high a,), the reverse is true.
Our previous growth studies showed that glyc-
erol is less toxic than NaCl at water activities in
the neighborhood of 0.9 or higher (3). For
example, N. crassa 74A will grow very slowly in
complete medium plus glycerol at a, 0.898, but
it does not grow at all at the same water activity
with NaCl.
Experiments with antibiotics and
chloroform. The experiments summarized in
Table 2 were performed to test the hypothesis
that loss of GF from conidia in media of low aw
results from altered permeability of the cell
membrane. A number of antibiotics are known
to be specifically active against membranes.
Nystatin and tyrocidine have been shown to
inhibit germination and growth of N. crassa by
irreversibly damaging the cell membrane (8, 10,
12). Polymyxin B is known to breach the
osmotic barrier, especially in gram-negative
bacteria (15), probably by inducing a reorienta-
tion of membrane lipids (5). The effect of
polymyxin B on N. crassa cell membranes has
not been studied in detail, but Kinsky (8) has
shown that it does inhibit both germination and
growth. Chloroform causes extensive damage to
plasma membranes (5). It was used in our
experiments to induce maximum leakage.
No conidia survive after a 1-h exposure to
chloroform-saturated buffer, and values for re-
leased GF, 250-nm absorbing, and ninhydrin-
positive material are high (Table 2). In ny-
statin-supplemented buffer, viability of conidia
decreases gradually to nearly zero in 24 h. The
opposite trend is shown in amounts of material
released. Polymyxin B has a similar effect, but
there were some survivors after 24 h, and they
had germinated in the incubation mixture. It
appears that the damage caused to the sensitive
fraction of the population was extens,ive, judg-
ing from the large quantities of material re-
leased.
The reaction of tyrocidine is unusual. At a
concentration of 20 ug and 107 conidia/ml, the
antibiotic is lethal, viability being reduced to
less than 1% after a 2-h exposure. With 108
conidia/ml, the same tyrocidine concentration
causes an immediate and rapid loss of 260-nm
absorbing and ninhydrin-positive material, as
well as GF. But after 1 h, the reaction is
reversed, and the released material begins to
disappear from the medium. After 3 h, the
conidia germinate. At 6 h, only traces of GF
activity, about half of the 260-nm absorbing and
20% of the ninhydrin-positive materials, remain
in the medium; at the same time, germination
is essentially complete. After 24 h, no further
growth has occurred, but the quantities of
intracellular material in the medium have been
further reduced. Because germination does not
occur in unsupplemented buffer, it appears that
tyrocidine-or, more likely, an impurity in the
commercial preparation-provides nutrient to
the conidia.
Dialysis results. Mach and Slayman (12)
found that 44% of the 260-nm absorbing mate-
rial released by Neurospora mycelium during a
TABLE 3. Amounts of nondialyzable 260-nm
absorbing material released from conidia at different
levels of a, or by chloroform
Nondi-
Time ODa ODa alyzableSuspending medium (h) before after material(h) dialysis dialysis (% of
9 total)
Buffer only at 6 0.192 0.036 18.2
a, 0.998 24 0.194 0.032 16.5
48 0.230 0.043 18.7
Buffer plus NaCl at 6 0.159 0.023 14.5
aW 0.843 24 0.250 0.035 14.2
48 0.415 0.051 12.3
Buffer plus glycerol 6 0.338 0.076 22.5
at aW 0.843 24 0.791 0.135 17.1
48 4.69 0.350 7.47
Buffer plus excess 1 7.86 2.49 31.8
chloroform
a Peak absorbance in 250 to 260 nm range.
VOL. 117, 1974 263
CHARLANG AND HOROWITZ
2-min exposure to an inactivating concentration
of tyrocidine was nondialyzable. We dialyzed
260-nm absorbing material released from co-
nidia exposed to low a, or chloroform for 24 h at
4 C against four changes of water. The results,
presented in Table 3, show that increasing
absolute amounts of nondialyzable material are
correlated with increased length of exposure to
low a,. The fraction of nondialyzable material
in the total 260-nm absorbing material de-
creases with time. It thus appears that, as an
index of damage, the absolute amount rather
than the percentage of nondialyzable material is
more revealing. On the other hand, with a short
exposure to a highly damaging compound like
chloroform, one observes the release of a large
percentage of nondialyzable material, as Mach
and Slayman did with tyrocidine.
DISCUSSION
Exposure of Neurospora conidia to media of
low a, results in the release of a germination-
essential component, together with 260-nm ab-
sorbing and ninhydrin-positive materials. Simi-
lar results are obtained regardless of whether an
electrolyte or a non-electrolyte is used to reduce
aw. We conclude that reduction of aw, and not
specific solute toxicity, is responsible. Because
membrane-specific antibiotics also induce a
release of GF and other materials from conidia,
it is likely that the loss of intracellular sub-
stances at low aw results from alteration of the
cell membrane.
Membrane damage at low aw may explain the
water requirements of many microorganisms,
and resistance to such damage may explain
xerotolerance. Hypertonic solutions of NaCl
appear to cause membrane damage in blue-
green algae (1), and high sucrose concentrations
make E. coli permeable to nucleoside triphos-
phates (4). The loss of putrescine from E. coli
growing at moderately elevated osmolarities,
however, is not the result of membrane damage
(14). Preliminary experiments with A. nidulans
and P. chrysogenum in our laboratory have
shown that these species are much more xerotol-
erant than N. crassa: their conidia retain GF
and germinate at aw <0.9. Since their GF is
readily diffusible-as shown by its rapid loss
from conidia in chloroform-saturated buffer-
there is an evident correlation between ability
to germinate at low aw and resistance to
permeability changes.
Specific toxicities may also be manifested at
high solute concentrations, superimposed on
the effects of low aw. The results presented in
this paper show that at the lowest water activi-
ties tested, glycerol begins to be lethal, but
NaCl does not. In our earlier experiments on the
effects of reduced a, on germination and
growth, sucrose was more inhibitory than NaCl,
glycerol, or glucose (3). Specific solute toxicities
have also been observed in yeast (11, 16).
ACKNOWLEDGMENTS
This work was supported by a grant from the National
Aeronautics and Space Administration (NGR 05-002-121).
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264 J. BACTERIOL.


Membrane Permeability and the Loss of Germination Factor from Neurospora crassa at Low Water Activities

English

A modified ninhydrin reagent for the photometric determination of amino acids and related compounds.

Alterations in the permeability of Neurospora crassa due to polyene antibiotics.

Antimicrobial agents and membrane functions.

Decryptification of RNA polymerase in whole cells of Escherichia coli.

Der osmotische Wert als begrenzender Faktor fUr das Wachstum and die Gsrung von Hefen.

Distribution of lysine pathways among fungi: evolutionary implications.

Effect of polyene antibiotics on protoplasts of Neurospora crassa.

Electrolyte solutions.

Germination and growth of Neurospora at low water activities.

Growth responses of blue-green algae to sodium chloride concentration.

Micro-organisms resisting high concentrations of sugars or salts.

Mode of action of tyrocidine on

Nystatin binding by protoplasts and a particulate fraction of Neurospora crassa, and a basis for the selective toxicity of polyene antifungal antibiotics.

Sorbose toxicity&quot;

Studies on marine occurring yeasts: respiration, fermentation, and salt tolerance.

the dry valleys of Antarctica.

The properties and mode of action of the polymyxins.

the putrescine content of Escherichia coli on the osmotic strength of the medium.

Water relations of food spoilage microorganisms.

http://authors.library.caltech.edu/2826/1/CHAjbact74.pdf

Membrane Permeability and the Loss of Germination Factor from Neurospora crassa at Low Water Activities

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