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Actinomycetes
University of Udine, Mycology Department
ISSN: 0732-0574
Vol. 4, Num. 3, 1993
Actinomycetes, Vol. 4, Part 3, 65-71, 1993 STRUCTURE OF THE POPULATION OF STREPTOMYCES HYGROSCOPICUS AND CHARACTERISTICS OF ITS VARIANTS

V. GESHEVA and R. GESHEVA

Institute of Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria

Code Number: AC93009
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ABSTRACT. The natural population of Streptomyces hygroscopicus strain 111-81 is heterogeneous. Of the several morphological variants present, the most abundant (main, 88.6%) resembles the parent strain. Following in frequency are white, oligosporic, asporogenic and nocardia-like types. Differences in the morphological, cultural, physiological, biochemical properties and antibiotic production between the original strain and the variants are described.

Variability in microorganisms is related to their evolutionary development, which explains the origin of dissociating forms capable of occupying different ecological niches and thus of securing survival. The study of variability may elucidate some processes connected with species formation, preservation and stabilisation, and strain selection (Kuznetsov, 1987; Kuznetsov et a1.,1990; Rodionova and Danilenko, 1988; Leblond et al., 1989; Ford et a1.,1990; Vesselinova and Gesheva, 1991).

Antibiotics formed by the Streptomyces hygroscopicus group belong to different chemical classes (e.g., aminoglycosides, peptides, polyenic and non-polyenic macrolides, polyethers). S.hygroscopicus, strain 111-81, produces a polyether complex, azalomycin B, hexaene and a non-polyenic macrolide antibiotic complex (Gesheva et a1., 1985), showing antifungal activity in vivo against the phytopathogenic fungi Fusarium graminearum and Botrytis cinerea (Gesheva et a1., 1977).

In this paper results of a comparative study of the biological properties of variants of a natural population of strain 111-81 are reported.

MATERIALS and METHODS

Microorganisms. Strain 111-81 was isolated from Bulgarian soil. Variants were maintained on maize extract agar No. 6 and mineral agar No. 1 (Gauze et a1., 1983).

Growth conditions. Morphological, cultural, physiological and biochemical properties of the variants were studied on media employed in the International Streptomyces Project (ISP) (Shirling and Gottlieb, 1966) and on those described by Gauze et a1., (1983). The colour of aerial and substrate mycelia were determined using Bondartsev's colour scale (1954). Variability was determined on single spore derived colonies according to Kuznetsov's method (1972). Strains were cultivated in 500ml Erlenmeyer flasks containing 50 ml of a glucose-soya complex medium No. 1 or No. 2 (Gesheva et a1.,1986) on a shaker (220 rpm) at 28øC in two stages. Culture broth was processed according to Ivanova et al., (1982). Antibiotics were detected by thin- layer chromatography (Gesheva et a1., 1985). Antifungal activity was assayed by the agar diffusion method against Candida utilis.

>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     
-------------------------------------------------------------------
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          
-------------------------------------------------------------------
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
------------------------------------------------------------------- Table 1 Cultural characteristics of parent and variant strains.

(AM aerial mycelium; SM: substrate mycelium; SP: soluble pigment; - character absent; *: disturbed sporulation)

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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).

REFERENCES

Bondartsev, A. (1954). Scale of Colours. The Academy of Sciences USSR, Moscow

Ford, L., T.Eaton & O.Godfrey (1990) Selection of Streptomyces ambofaciens mutants that produce large quantities of spiramycin and determination of optimal conditions for spiramycin production Appl.Env.Microbiol., 66: 3511-3514

Gauze, G.F., T.P.Preobrazhenskaya, M.A. Svesnikova, L.P.Terekhova & T.S.Maximova (1983). Key to Actinomycetes. Nauka Publ, Moscow

Gesheva, R., P.Panajotov, M.Darakchieva, M.Naumova, H.Tsankov, M.Mladenov & J.Karajova ( 1977). Actinomycetes antagonists of Fusarium graminearum Schw. responsible for fusariosis of wheat Prilojna Mikrobiologiya, 7: 76-81

Gesheva, R., V.Gesheva, M.Darakchieva, V. Ivanova, P.Panajotov, R.Shlegel & H. Thrum (1985). Taxonomy of Streptomyces hygroscopicus 111-81 producing nonpolyenic macrolide antibiotics Compt. Rend.Acad. Bulg. Sci. , 38: 619-622

Gesheva, R., V.Gesheva, V.Ivanova, R.Shlegel & H.Thrum (1986). Study on the biosynthesis of an antifungal antibiotic. In: Antibiotics Farmachim Publ Razgrad, pp 296-299

Ivanova, V7 R.Gesheva, P.Panajotov, D.Belomusova, N.Radonova, V.Gesheva, R. Shlegel, H.Thrum, W.Klainwachter & D.Tresselt ( 1982) Bulgarian Patent No 34842

Kirby, R. & E.Lewis (1981) Unstable genetic elements affecting streptomycin resistance in the streptomycin producing organisms Streptomyces griseus NCIB 8506 and Streptomyces bikiniensis ISP 5235 J.gen.Microbiol., 122: 351-355 Kuznetsov, V.D. (1972). Studies on variation of actinomycetes producing antibiotics and other biologically active substances Antibiotiki, 17: 666471

Kuznetsov, V.D. (1987). Parallelism in hereditary variability and population concept of species in representatives of prokaryotes. J. Gen.Biologii, 48: 466-476

Kuznetsov, V.D. (1991) The population concept in microbiology. Actinomycetes, 1: 63-66

Leblond, P.P., P.Demuyter, L.Moutier, B.Decaris & J.M.Simonet (1989) Hypervariability, a new phenomenon of genetic instability, related to DNA amplification in Streptomyces ambofaciens. J.Bacteriol., 171: 419423

Ogura, M., T.Tanaka, K.Furihata, A.Shimazu & O.Otake (1986) Induction of antibiotic production with ethydium bromide in Streptomyces hygroscopicus.J.Antibiot.,39: 1443-1450

Pronina, M.I., T.P.Efimova, R.A.Zhukova, T.G.Motovilova, I.S.Pechatnikova & I.M.Tereshin (1980). Morphological-biochemical characteristics of Streptomyces hygroscopicus mutants induced by streptomycin and hygromycin B Antibiotiki, 69: 822-827

Rodionova, I.I. & V.N.Danilenko (1988) Factors of streptomycetes genetic instability and their use in selection practice Antibiotiki Chemiotherapiya, 33: 171-179

Roth, M., D.Noack & G Reinhard (1982). Properties of non differentiating derivatives of Streptomyces hygroscopicus. J.gen.Microbiol., 128: 2687-2691

Shirling, E.B. & D.Gottlieb (1966) Methods for characterisation of Streptomyces species. Int.J. Syst.Bacteriol., 16: 313-340

Vesselinova, N. & R.Gesheva (1991). Variability of Streptomyces spectabilis 1000. Actinomycetes, 2: 13-17.

Copyright 1993 C.E.T.A.


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