JCC0752

JOURNAL OF CULTURE COLLECTIONS

Volume 5, 2006-2007, pp. 10-15

Taxonomic study of Streptomy= ces sp. strain 34-1

<= o:p>

Stefka Antonova-Nikolova¹*, Va= nya Stefanova¹and Lyubomira Yocheva²
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¹Sofia University, Biological Faculty, Department of General and Industrial Microbiology,
8 Dragan Tzankov st., 1164 Sof= ia, Bulgaria;=

²Institute forCryobiology and = Food Technology, 53 Cherni vrah blvd., 1407 Sofia, Bulgaria<= o:p>

*Corresponding author, e-mai= l: antonova@biofac.uni-sofia.bg

Summary

The present work is= a part of a complex study of Streptomyces sp. strain 34-1 ‑ a producer of trypsin and trypsin-like protease inhibitors with antiviral effect. The taxonomically significant properties of the strain were examined according = to the International Streptomycetes Project (ISP). The identification of the strain was based on Nonomura’s key. The morphological, cultural, physiological and biochemical characteristics of Streptomyces sp. strain 34= -1 were compared with the references for similar streptomycetes species. The s= train was identified as Streptomyces chromofuscus and designated as S. chromofusc= us 34-1.

= Key words:<= /b> streptomycetes, taxon= omy, protease inhibitor.

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Introduction

Microorganisms of the genus = Streptomyces produce a wide spectr= um of bioactive substances (antibiotics, pigments, and enzymes) with application in pharmaceutical and food industries, in biotechnology and laboratory practice [2].

The ability of the streptomy= cetes to synthesize enzyme inhibitors reveals a new aspect of microbial antagonism.<= o:p>

It is known that proper proteolytic enzymes of t= he infected cells play decisive role in the replication of numerous viruses including the influenza viruses. To become infectious, the viruses have to undergo posttranscriptional cleavage of the basic antigen – hemagglutinin. This reaction is carried out by the trypsin-like proteases of the host [7, 13]. The inhibition of these proteases leads to the inhibition of the viral replication.

The proved ability to synthe= size a sub-stance which inhibits the activity of trypsin and trypsin-like proteases and viruses makes Streptomyces = sp. strain 34-1 a perspective producer of the protease inhibitor [1].

This work is a part of a com= plex study of Streptomyces sp. strai= n 34-1 – producer of a protease inhibitor and aims its characterization and taxonomic identification.

Materials and Methods

The soil isolate Streptomyces sp. strain 34-1 from = the collection of the Department of General and Industrial Microbiology at Sofia University was the subject in this study. The taxonomic properties of the strain were deter-mined according to the International Streptomycetes Project (ISP) [9]= and some procedures described by Gause et al. [5].

The strain morphology was ob= served on the 7^th, the 14^th, and the 21^s=
t day for mature cultures. The morphology of the spore-bearing aerial hyphae = was determined by direct microscopic examination of the culture surface. The le= ngth of the spore chain and the spore surface ornamentation were observed by an electron microscope. The electron microscope investigation was made on grids coated with Formvar without shadowing.

The colour of the aerial mas= s, the substrate mycelium and the soluble pigment was determined using the Bondart= sev colour scale [3]. The production of a melanin pigment was observed after 2 = and 4 days.

The ability of the strain to= use different carbon sources was determined according to the ISP recommendation [9]. The starch hydrolysis and the growth on gelatin and skim milk were tes= ted according to Gauze et al. [5].

The sensibility to antibioti= cs was examined by the paper discs method [6]. The following antibiotics (“BulBio”) were used: erythromycin (15 μg/disc), ampicillin (10 μg/disc), streptomycin (10 μg/disc), gentamycin (10 μg/disc), penicillin (10 E/disc), chloramphen= icol (30 μg/disc), tetracyclin (15 μg/disc). The diameters = of the growth inhibition zone were measured after incubation for 48‑72 h= .

The taxonomic identification= of Streptomyces sp. 34-1 was based on Nonomura’s key [8] and the species description according to ISP [10, 11, 12], Gauze et al. [5] and Bergey’s Manual [4].

=

Results and Discussion

Micromorphological characteristics. The substrate mycelium of strain Streptomyces sp. 34-1 did not frag= ment. The aerial mycelium formed monopodially branched spore-bearing hyphae with = the shape of hooks, loops, open or compact spirals with 3-6 curves (Fig. 1= ). The spore chains contained 10‑50 spores. They were ellipsoidal with s= piny surface (Fig. 2).

Fig. 1. Morphology of the spore-bearing aerial hyphae of Stre= ptomyces sp. 34-1 (ISP-3, 800x).

F= ig. 2. Spore chain (A, 13 400x) = and spore surface ornamentation (B, 25 000x) of Streptomyces s= p. 34-1 (ISP-3).

Macromorphological characteristics. The strain was clearly polymorph. It form= ed 7 morphological types of colonies on Gauze I mineral medium. The types of colonies differed in shape (round or ellipsoidal), profile (flat or slightly raised, umbonate or with craters), surface (smooth or wrinkled), edges (undulating or rhizoid). The colonies were completely covered by aerial mycelium or it formed concentric or radial lines (Fig. 3). The dominant type was presented by round, umbona= te colonies, completely cover-ed by pale grey aerial mycelium with slightly undulating edges.

.

F= ig. 3. Morphological types of colonies of Streptomyces sp. 34-1 (Gauze I mineral medium).

Streptomyces sp. 34-1 grew on all of the used media. The abundance and the colour of the aer= ial mycelium depended on the medium composition and the age of the culture. The aerial mass colour varied from white and pale blue to different nuances of = grey (from pale grey to green-grey), therefore it could be assigned to the grey series (Table 1).

The substrate mycelium colou= r varied from pale grey to dark grey depending on the medium composition and the age= of the culture. It could be accepted as not distinctive according to Nonomura’s key [8]. pH changes did not affect the reverse colour.

On most of the media used Streptomyces sp. 34-1 produced a s= oluble pigment whose colour varied from beige-olive (on Gauze I mineral medium, IS= P-3 and ISP-5 media) to dark brown (ISP-2 medium). The strain did not produce a= ny diffusible pigment on ISP-4 medium (Table 1).

Table 1. Cultural characteri= stics of Streptomyces sp. 34-1.

Medium

Growth

Age (days)

Colour of aerial mycelium

Colour of substrate mycelium=

Colour of soluble pigment

ISP-2

abundant

7

white; dark ash-grey<= /p>

dark brown-green

dark brown-green

14

white; mouse-grey

dark ash-grey

brown

21

mouse-grey

brown-yellow

dark brown

ISP-3

abundant

7

pale blue; blue

dark ash-grey

‑

14

pale blue; blue

dark grey

beige-olive

21

green-grey

dark grey

dark beige-olive

ISP-4

abundant

7

white; pale blue

ash-grey

‑

14

white

dark ash-grey

‑

21

mouse-grey

pale grey-violet

‑

ISP-5

moderate

7

white; pale grey-violet

dark grey

‑

14

white; pale grey-violet

dark ash-grey

beige-olive

21

grey-violet

grey-violet

dark beige-olive

Physiological and biochemical proper-ties. The strain produced melanoid pigments on pepton iron agar (ISP-6). Melanin production was not observed on tyrosine a= gar (ISP-7).

Streptomyces sp. 34-1 grew poorly in the presence of D-xylose and i-inositol as a s= ole carbon source. The strain formed abundant mycelium on the media with D-gluc= ose, D-fructose, L-arabinose, L-rhamnose and D-mannitol, but its growth was doub= tful on the media with sucrose and raffinose (Table 2).

Table 2. Carbon utilization = by Streptomyces sp. 34-1.<= /span>

Carbon source

Utilization

No carbon source (negative control)

‑

L-Arabinose

++

D-Xylose

+

i-Inositol

+

D-Mannitol

++

D-Fructose

++

L-Rhamnose

++

Sucrose

±

Raffinose

±

Cellulose

‑

D-Glucose (positive control)

++

Legend: util= ization of the tested carbon is equal to or grater than that of the positive contro= l (++); weaker utilization in comparison to the positive control (+); doubtful utilization (±); no utilization (-).

Strain 34-1 grew on gelatin = without liquefying it. It also grew on skim milk but did not cause coagulation and peptonization. It hydrolysed starch.

The= strain was sensitive to the action of antibiotics such as tetracyclin, gentamycin = and erythromycin (Table 3).

Table 3. Sensibility of Streptomyces sp. 34-1 to different antibiotics.

Antibiotics

Sterile zone [mm]

Tetraciclin

22

Streptomycin

‑

Gentamycin

12

Erythromycin

21

Ampicillin

‑

Penicillin

‑

Chloramphenicol

‑

Identification of Streptomyc= es sp. 34-1. Morphological, cultural, physiological and biochemical properties of the st= rain 34-1 were compared to Streptomyces = species included in Nonomura’s key [8]. Using references given by ISP [10, 11, 12] and Bergey’s Manual [4], the strain was compar= ed with the most similar species (Table 4).

Tab= le 4. Basic taxonomical characteristics of Streptomyces sp. 34-1 and related streptomycetes species.

Species name

Aerial mass colour

Melanoid pigment¹⁼

Reverse side pigment

Soluble pigment

Spore chain²⁼

Spore surface

L-arabinose=

D-xylose

I-inositol<= /span>

D-mannitol<= /span>

D-fructose<= /span>

L-rhamnose<= /span>

Sucrose

Raffinose

D-glucose

Streptomyces sp. 34-1

grey=

+–<= o:p>

–

beige-oli= ve

dark brow= n

SRA<= /o:p>

spiny

+

+

+

+

+

+

±<= o:p>

±<= o:p>

+

S. echinatus

grey=

++

–

–

SRA<= /o:p>

spiny

+

+

+

+

+

+

–

+

+

S. chromo-fuscus

grey=

+–<= o:p>

–

pale yell= ow

SRA³⁼

spiny

+

+

+

+

±<= o:p>

+

–

±<= o:p>

+

S. griseo-chromogenes

grey=

++

–

–

SRA<= /o:p>

spiny

+

+

+

+

+

–

+

+

+

S. ganna-mycicus

grey=

++

–

–

S

spiny

±<= o:p>

+

+

+

+

+

±<= o:p>

+

+

S. flavor-viridis

grey=

++

–

–

SRA<= /o:p>

hairy

+

+

+

+

+

+

–

–

+

<= o:p>

L= egend: ¹ Melanin production on both ISP-6 and ISP-7 media (++); melanin production o= nly on ISP-6 (+–);

² Hooks, loops and open spira= ls (RA); spirals (S);

³ 11 % of the strains be= longing to the species S. chromofuscus = form RA spore-bearing aerial hyphae [6].

Streptomyces sp. 34-1 showed greatest similarity to S. chromofuscus<= /i> [11] in morphology of the sporo= phores (spiral), ornamenttation of the spore surface (spiny), cultural properties (grey aerial mass colour, substrate mycelium without distinctive colour, beige-olive to yellow soluble pigment) and by the absence of melanin produc= tion on the medium ISP-7. The strain 34-1 differed from S. chromofuscus in carbon utilization. It utilized D-fruct= ose and its growth was doubtful in the presence of sucrose. The spore-bearing hyphae of Streptomyces sp. 34-1= formed besides the compact spirals single open spirals, and hooks and loops (RA). It could be concluded on this base that Streptomyces sp. 34-1= is identical or very similar to S.&nbs= p;chromofuscus.

Differences in the “secondary” taxonomic features such as carbon utilization were = the reason to identify the strain as S.= chromofuscus 34-1.

Short characterization of St= reptomyces chromofuscus 34-1. The strain forms monopodial branched sporophores with the shape of hooks, loops, imperfect and perfect spirals with 3‑6 curves (SRA). Sp= ore surface is spiny. The spore chain length is between 10 and 50 spores. The aerial mass colour is of the grey series (from pale grey to grey-violet), a= nd it is white to pale blue on some media and in young cultures. The colour of substrate mycelium is not distinctive (greyish or brownish nuance). The cul= ture is polymorph. It produces olive or brownish soluble pigment on all of the u= sed media with the exception of the ISP-4 medium. It synthesizes melanin on the ISP-6 medium only. It does not possess tyrosinase activity. The strain grow= s on gelatin and liquefies it scarcely perceptibly, inclining to negative. It gr= ows on skim milk without causing its peptonization or coagulation. It hydrolyses starch. The culture utilizes D-glucose, D-fructose, L-arabinose, D-xylose, = L-rhamnose, i-inositol and D-mannitol. The utilization of sucrose and raffinose is doubtful. The strain produces trypsin and trypsin-like proteases inhibitor = [1]. It has antiviral activity. It is sensitive to tetracyclin, gentamycin and erythromycin.

Acknowledgments. This study was supported by the research grant No L-11006/2001 from= The National Science Fund of The Bulgarian Ministry of Education and Science. We express our thanks to Vassilis Kapurdov from the National Center of Hygiene, Sofia for the generous access to electron microscope.

References

1. Angelova, L., E. Ivanova, J. Serkedjieva, I. Ivanova, 2003. Biotechnol. Biotechnol. Eq., 17 (2), 110-114.

2. Antonova-Nikolova, S., I. Ivanova, 1999. Biology of Streptomycetes – producers of antibiotics, Sofia: St. Kl. Ohridski, 107 (in Bulgarian).

3. Bondartsev, A., 1954. Colour Scale, Moskva-Leningrad: Ac= ad. Sci. USSR, 27 (in Russian).

4. Cross, T., H. Lechevalier, 1= 994. Actinomycetes: Group 22-29. In: Ber= gey’s manual of determinative bacteriology, J. Holt, N. Krieg, P. Sneath, J. Staley, S. Williams (Eds), Ninth Edition, Baltimore-Philadelphia-Hong Kong: Williams & Wilkins, 2344-2347. <= /o:p>

5. Gauze, G., T. Preobrazhenska= ja, M. Sveshnikova, L. Terehova, T. Maksimova, 1983. Guide of Actinomycetes, Moskva: Nauka, 248 (in Russian).

6. Gushterov, G., P. Andonov, T= s. Todorov, L. Kominkov, M. Gincheva-Starcheva, 1977. Manual of microbiology and virology, Sofia: Nauka i Izkustvo, 3= 93 (in Bulgarian).

7. Lozitsky, V., A. Fedchuk, L.= Puzis, V. Buiko, I. Girlia, 1987. Voprosi Virusologii, 32, 413-419 (in Russian).

8. Nonomura, H., 1974. J. Ferment. Technol., 52 (7), 78-92.

9. Shirling, E., D. Gottlieb, 1= 966. Intern. J. System. Bacteriol., I, = 16 (3), 313-340.

10. Shirling, E., D. Gottlieb, 1= 968. Intern. J. System. Bacteriol., II,= 18 (2), 106-118.

11. Shirling, E., D. Gottlieb, 1= 968. Intern. J. System. Bacteriol., III= , 18 (4), 307.

12. Shirling, E., D. Gottlieb, 1= 969. Intern. J. System. Bacteriol., V, = 19, 300-303.

13. Zhirnov, O., A. Ovcharenko, = A. Bukrinskaja, 1985. Voprosi Virusolo= gii, 30, 207-214 (in Russian).=

i= 8;аксономичн= 086; изследване на щам Streptomyces sp. 34-1<= /b>

Стефка Антонова-Ни = 82;олова¹*, Ваня Стефанова¹, Любомира Йочева²

=

=

Р&#= 1077;зюме

&= #1053;астоящата работа е час= 090; от комплексно проучване н = 72; щам Streptomyces sp. 34-1, продуцент н = 72; инхибитор н = 72; трипсин и трипсин-под = 86;бни протеази с антивируснk= 2; активност. Представенl= 0; са данни за таксономичl= 5;ите му свойства, получени чрез методите на Международl= 5;ия стрептомицk= 7;тен проект (ISP). При идентифициl= 8;ането му е използван ключът на Nonomura. Морфологичl= 5;ите, културалниm= 0;е и физиолого-б = 80;охимичните характерисm= 0;ики на Streptomyces sp. 34-1 са сравнени с л= 080;тературнит&= #1077; данни за сходни стрептомицk= 7;тни видове. Щамъ= 090; е отнесен къ= 084; вида Streptomyces chromofuscus и е означен кат = 86; S. chromofuscus 34-1.

Medium	Growth	Age (days)	Colour of aerial mycelium	Colour of substrate mycelium=	Colour of soluble pigment
ISP-2	abundant	7	white; dark ash-grey<= /p>	dark brown-green	dark brown-green
14	white; mouse-grey	dark ash-grey	brown
21	mouse-grey	brown-yellow	dark brown
ISP-3	abundant	7	pale blue; blue	dark ash-grey	‑
14	pale blue; blue	dark grey	beige-olive
21	green-grey	dark grey	dark beige-olive
ISP-4	abundant	7	white; pale blue	ash-grey	‑
14	white	dark ash-grey	‑
21	mouse-grey	pale grey-violet	‑
ISP-5	moderate	7	white; pale grey-violet	dark grey	‑
14	white; pale grey-violet	dark ash-grey	beige-olive
21	grey-violet	grey-violet	dark beige-olive

Carbon source	Utilization
No carbon source (negative control)	‑
L-Arabinose	++
D-Xylose	+
i-Inositol	+
D-Mannitol	++
D-Fructose	++
L-Rhamnose	++
Sucrose	±
Raffinose	±
Cellulose	‑
D-Glucose (positive control)	++

Antibiotics	Sterile zone [mm]
Tetraciclin	22
Streptomycin	‑
Gentamycin	12
Erythromycin	21
Ampicillin	‑
Penicillin	‑
Chloramphenicol	‑