>Figure 1. Colonies of S.hygroscopicus; a: parent
culture, b: main, c: oligosporic, d: white, e:
asporogenic and f: nocardia-like types.
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Parent strain Variant strains
Medium
No.111-81 Main No.13
-------------------------------------------------------------------
Yeast-malt AM whitish to pale gray whitish to mouse gray
extract agar SM pale yellow to brown honey yellow
(ISP2) SP
Oatmeal AM dark to violet gray violet to dark gray
agar SM brown hazelnut to brown
(ISP3) SP
Inorganic AM dark to violet gray whitish to mouse gray
salt-starch SM brown pale sandy
agar SP
Glycerol- AM pale to ash gray chalk white
asparagine SM yellowish-brown straw yellow
agar (ISP 6) SP
Tyrosine AM pale gray (poor) pale gray (poor)
agar SM dark brown brown-yellow
(ISP7) SP dark brown brown
Glucose AM dark gray white
nitrate SM yellow-brown yellow ochre
agar SP
Mineral AM dark gray pale to dark gray
agar SM brown brown
No.1 SP
Maize AM gray to ash gray mouse to dark gray
extract agar SM yellow-brown brown-yellow
No.6 SP
Bennett's AM pale to dark gray whitish to pale gray
agar SM pale sandy sandy
SP
Czapek's AM ash gray mouse to dark gray
agar SM yellow-brown pale sandy
SP
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Table contd.
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Medium Variant strains
White Oligosporic
No.l No.353
-------------------------------------------------------------------
---
Yeast-malt AM whitish smoke gray
extract agar SM pale to dark sandy pale sandy
(ISP2) SP
Oatmeal AM whitish to smoky white
agar SM pale sandy brown-yellow
(ISP3) SP
Inorganic AM smoke gray smoke gray
salt-starch SM honey yellow straw yellow
agar SP
Glycerol- AM chalk white whitish
asparagine SM pale sandy pale sandy
agar (ISP 6) SP
Tyrosine AM whitish to smoky smoke gray
agar SM dark chestnut brown
(ISP7) SP brown brown
Glucose AM whitish ash gray
nitrate SM straw yellow sandy
agar SP
Mineral AM whitish chestnut*
agar SM pale sandy sandy
No.1 SP
Maize AM whitish whitish to ash
gray
extract agar SM pale sandy brown-yellow
No.6 SP
Bennett's AM whitish ash gray
agar SM pale sandy pale sandy
SP
Czapek's AM smoke gray mouse gray
agar SM dark sandy pale gray violet
SP
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Table contd.
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---
Variant strains
Medium
Asporogenic Nocardia-like
No.3 No.230
-------------------------------------------------------------------
---
Yeast-malt AM
extract agar SM pale sandy ochre
(ISP2) SP
Oatmeal AM
agar SM ochre sulphur yellow
(ISP3) SP
Inorganic AM
salt-starch SM pale sandy whitish
agar SP
Glycerol AM
asparagine SM ash gray ochre
agar (ISP 6) SP
Tyrosine AM whitish
agar SM brown-yellow colourless
(ISP7) SP brown-yellow
Glucose AM
nitrate SM pale sandy pale sandy
agar SP
Mineral AM pinkish
yellow
agar SM pale sandy yellow-brown
No.1 SP
Maize AM
extract agar SM pale sandy sandy
No.6 SP
Bennett's AM
agar SM sandy pale yellow
SP
Czapek's AM
agar SM whitish pale gray
SP
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Table 1 Cultural characteristics of parent and variant
strains.
(AM aerial mycelium; SM: substrate mycelium; SP: soluble pigment; -
character absent; *: disturbed sporulation)
-------------------------------------------------------------------
Resistance was studied on maize extract agar No. 6 using the
following antibiotics: ampicillin, bacitracin, carbenicillin,
chloramphenicol, erythromycin, gentamicin, kanamycin, nalidixic
acid, oxacillin, penicillin, streptomycin and tetracycline.
RESULTS and DISCUSSION
Composition of strain 111-81 population. Observations
of S.hygroscopicus 111-81 population on nine media show the
great influence of medium composition on the expression of the
distinct features of the variants. The highest dissociation is
obtained on glucose nitrate agar No.1.
The population is heterogeneous and consists of five
morphological variants: main, white, oligosporic, asporogenic
and nocardia-like (Fig. 1).
The main type prevails (88.6%) in the population. The
colonies protrude radially, are concentrically folded and often
have a cleft in the centre. White variants (6.33%) are
distinguished by white aerial mycelium which in some colony regions
is sterile. The colonies are round with a slightly protruding,
folded centre. Oligosporic variants (4.55%) are
characterised by an aerial mycelium which does not cover the whole
surface uniformly. The colonies are flat with a well-outlined
periphery. Asporogenic variants, of limited frequency
(0.53%), usually lack spores. The colonies are folded and
convoluted in structure. Nocardia-like variants are also
rare (0.02%). The colonies have irregular shape and soft
consistency. During the course of five years no reversions to the
parent strain of white, asporogenic and nocardia-like
variants have been found.
Morphological, cultural and physiological properties of the
variants. Main type variants show abundant sporulation and
tight spiral sporophores. White and oligosporic
variants form primitive spirals (Retinaculum apertum).
On some media a few cultures of asporogenic variants
produce poor aerial mycelium with coremia. The spore surface, of
the variants which form spores, is rough, as is that of the parent
strain.
Cultural properties of representative strains of each
morphological type are listed in Table 1. The colour of aerial
mycelium of the main variant shows different nuances of
grey-pale grey, dark grey, dark ash grey, mouse grey and, after
lysis, turns black on some culture media. The substrate mycelium
varies from sandy to yellow or brown. White variants differ
from the main strain in their whitish aerial mycelium and
poor sporulation. The substrate mycelium changes from pale to dark
sandy. The aerial mycelium in nocardia-like variants is more
scarce, it does not cover the whole surface of the colonies or it
is absent. The substrate mycelium is fragmented and pale sandy,
ochre or sulphur yellow in colour.
Physiological features, utilisation of carbon sources and
biosynthesis of antibiotics are summarised in Table 2. Differences
are noted in melanin formation, utilisation of carbon sources and
antibiotic production. Asporogenic and nocardia-like
variants respectively utilise inositol or sucrose, but do not
form melanin and azalomycin B. It is suggested that the loss of
these features (azalomycin, melanin and aerial mycelium formation)
is due to a pleiotropic effect and it is perhaps connected with
changes in the genotype. Similar results were reported for non
differentiating derivatives of the mutant S.hygroscopicus JA
6599, producer of the macrolide antibiotic turimycin, which were
not able to form turimycin, aerial mycelium and spores (Roth et
al., l982).
-------------------------------------------------------------------
Feature Parent Variant strains
strain
111-81 13 1 353 3
230
----------------------------------------------------------------
------
Melanin formation + + + + - -
Milk coagulation + + + + + -
Nitrate reduction + + + + + -
Starch hydrolysis + + + + + -
Sucrose inversion + + + tr + +
Utilisation of sucrose - - - - - +
Utilisation of inositol - - - - + -
Biosynthesis of antibiotics:
Polyether complex + + + + + tr
Non-polyenic macrolide
complex + + + + + tr
Azalomycin B + + + - - -
Hexaene + + + - - -
----------------------------------------------------------------
---
Table 2 Physiological and biochemical characters of the
parent strain 111-81 and of the variants (+ = positive, - =
negative, tr = traces Gelatine liquefaction, milk peptonisation and
utilisation of arabinose, fructose, glucose, maltose, raffinose and
xylose are positive for all strains
----------------------------------------------------------------
---
--------------------------------------------------------------
Antibiotic Parent Variant strains
strain
(ug/ml) 111-81 Main White
-------------------------------------------------------------------
Bacitracin (10) 0 0 0
Nalidixic acid (30) 0 0 0(a)
Ampicillin ( 10) 0 0 0
Penicillin ( 10) 0 0 0 (a)
Oxacillin (10) 16 0 0 (a)
Carbenicillin (100) 11 25.5+/-9.8 15.7+/-3.7
Kanamycin (30) 48 52.2+/-9.0 50.3+/-6.4
Gentamicin (30) 64 62.5+/-7.4 56 5+/-6.5
Tetracycline (30) 11 19.7+/-3.7 18.3+/-4.9
Chloramphenicol (30) 17 19.6+/-2.7 17.5+/-6.0
Streptomycin (30) 23 31.4+/-3.5 28.5+/-6.5
Erythromycin (15) 15 23.7+/-2.5 26.7+/-2 5
----------------------------------------------------------------
--
Table 3 contd.
-------------------------------------------------------------------
Antibiotic Variant type
(ug/ml) Oligo- Asporo- Nocardia
sporic genic -like
-------------------------------------------------------------------
Bacitracin (10) 0(a) 0(a) 0
Nalidixic acid (30) 0(a) 0)a) 0
Ampicillin ( 10) 0 0 0
Penicillin ( 10) 0 0 0
Oxacillin (10) 0(a) 0(a) 0
Carbenicillin (100) 26.0+/-3.2 27.8+/-6.3 0
Kanamycin (30) 49.0+/-2.9 57.1+/-8.8 22.5+/-7.3
Gentamicin (30) 46.3+/-8.7 61.0+/-6.3 35.2+/-4.7
Tetracycline (30) 21.8+/-8.1 21.9+/-8.1 25.2+/-3.5
Chloramphenicol (30) 19.0+/-7.8 16.6+/-5.7 19.5+/-5.3
Streptomycin (30) 31.8+/-4.9 31.5+/-6.1 25.2+/-4.9
Erythromycin (15) 23.4+/-6.5 26.0+/-7.4 17.0+/-2.3
----------------------------------------------------------------
---
Table 3. Inhibition ( x+/-Sx in mm) by antibiotics of parent
strain and of variants
0 = resistant;
o(a) = some ( 1-3) representatives of the group are sensitive)
----------------------------------------------------------------
---
Antibiotic resistance and antibiotic productivity.
Results of antibiotic resistance of the variants are shown in
Table 3.
The main cultures possess the same spectrum of
sensitivity and resistance to observed antibiotics as the parent
strain. Resistance to ampicillin, oxacillin, nalidixic acid,
penicillin, bacitracin is genetically unstable in the population of
S. hygroscopicus 111-81. The occurrence of the single
sensitive representatives in these cases was noted in variants with
poor aerial mycelium. Possibly impaired regulation of aerial
mycelium formation and sensitivity are connected events.
Similarly in S.bikiniensis changes from streptomycin
resistance to sensitivity was related to variations in aerial
mycelium production and auxotrophy induction (Kirby and Lewis,
1981).
Asporogenic and nocardia-like cultures do not
grow on agar supplemented with 50 ug/ml of the non-polyenic
macrolide antibiotic complex, while other groups of variants are
resistant to the same concentration, as previously described for
non producing derivatives of S.hygroscopicus JA 6599 (Roth
et a1., 1982). Interestingly enough, nocardia-like
forms show resistance to carbenicillin and are more sensitive
to the aminoglycosides kanamycin and gentamicin.
Data on antibiotic productivity are summarised in Table 4.
-----------------------------------------------------------
--------
Antifungal activity
Strains _ _ _
Relative _ _ x-tSx-x+tSx
activity x+/-Sx P=0.05
----------------------------------------------------------------
------
Main 163 1206.0+/-174.7 865.0-1547.0
White 93 687.0+/-87.0 463.4-850.0
Oligosporic 52 381.0+/-41.3 300.0-461.9
Asporogenic 0.9 6 7+/-1.2 1.2-12.2
Nocardia-like tr
Parent culture 100
----------------------------------------------------------------
---
Table 4. Antifungal activity (ug/ml) of the variants of
S.hygroscopicus 111-81
(x = mean, Sx = standard error, x-tSx - x+tSx = confidence
intervals).
----------------------------------------------------------------
---
Variants differ in their biosynthetic abilities. Cultures of
the main type possess a higher antifungal activity than the
parent strain. This represents a potential that could be exploited
by selection. In the case of the white strains activity is
lower. These results suggest that a close relationship exists
between differentiation and biosynthesis of the antifungal
antibiotic complex. Variants forming poor aerial mycelium
(oligosporic, asporogenic and nocardia-like types)
possess low productivity. Similar results were obtained with
mutants derived from S.hygroscopicus strains producing
turimycin (Roth et a1., 1982), hygromycin (Pronina et
a1., 1980) and carriomycin (Ogura et al., 1986).
Possibly the lower antifungal activity of these S.hygroscopicus
111-81 variants is due to the reduced number of components of
the non-polyenic macrolide complex (Fig. 2).
Results clearly indicate that S.hygroscopicus is a
polymorphic species. The population consists of variants differing
from the ancestor in their ability to form aerial mycelium, spores
and melanin, antibiosis and resistance to antibiotics. It is
suggested that distinct features of variants represent a mechanism
for strain survival in nature.
Figure 2. Thin-layer chromatography of antibiotics of
S.hygroscopicus. Lanes: A, parent strain 111-81; B, main
variant 13; C, white variant 1; D, oligosporic
variant 353; E, asporogenic variant 3; F,
nocardia-like variant 230 (1-6: components of the
non-polyenic macrolide antibiotic complex; 7: azalomycin B; 8:
polyether antibiotic complex).
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Copyright 1993 C.E.T.A.
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