Actinomycetes University of Udine, Mycology Department ISSN: 0732-0574 Vol. 7, Num. 1, 1996

STREPTOMYCES SCOPIFORMIS SP. NOV.

Z. LIU and Z. YAMEI

Institute of Microbiology, Academia Sinica, Beijing, P.R. China

Code Number: AC96005
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ABSTRACT. On the basis of morphological and biochemical characteristics an actinomycete isolate is proposed as the type strain of the new species Streptomyces scopiformis sp. nov.

From a soil sample, collected in China, an unusual actinomycete strain was isolated.

In the present note the organism is characterised both for its morphological and biochemical properties.

MATERIALS and METHODS

Organism. Strain A25 was isolated from the rhizosphere soil of the Chinese hemlock (Tsuga chinensis) in Lushan, Jiangxi province, China. The sample was plated on glycerol-asparagine medium (ISP, International Streptomyces Project, medium 5, Shirling and Gottlieb, 1966) for 2 weeks at 28 C.

Morphological and cultural characterisation. Morphology of the strain was carried out by light and electron microscopy. Cultural characteristics were determined after 2 weeks incubation on media suggested by Shirling and Gottlieb (1966) and by Gordon et al. (1974).

Physiological characters. Media and tests previously recommended were used (Shirling and Gottlieb, 1966; Kampfer and Kroppenstedt, 1991).

Biochemical characterisation. Biomass for chemotaxonomic analysis was grown in submerged culture on modified Sauton medium (Lechevalier and Lechevalier, 1980) at 28 C. Characteristics determined were cell wall (Becker et al., 1965; Lechevalier and Lechevalier, 1980), fatty acid (Kroppenstedt, 1985; O'Donnell, 1993), phospholipid (Lechevalier and Lechevalier, 1980), menaquinone (Collins, 1985; Wu et al., 1989) and mycolic acid (Minnikin et al., 1980; Liu et al., 1990) composition.

DNA was extracted using a modified procedure (Hopwood et al., 1975; Liu et al., 1992). The thermal denaturation method of Marmur and Doty (1962) was used to determine the guanine (G) plus cytosine (C) content. DNA from E.coli was used as a control.

RESULTS

Phenotypic characterisation. The non fragmenting substrate mycelium consists of septate and swollen elements (Fig. 1 c). Aerial spore chains arise directly from the substrate mycelium (Fig. l a, b). Spore chains are arranged in a broom-like structure. Spores are roundish (0.7-0.8um in diameter) and show a spiny surface (Fig. l d)

Strain A25 grows well on organic media, but poorly on inorganic salts-starch agar. The isolate does not grow on MacConkey medium. Substrate mycelium colour ranges from light blue grey to brown blue. The colour of aerial mycelium is blue grey or light blue grey (Table 1). No soluble pigment is produced.

Physiological test are shown in Table 2.

Biochemical characters. Strain A25 has a type I cell wall composition, with no characteristic sugars. The phospholipid pattern is of type PII (containing PE, DPG) and the main menaquinone component is MK9(H4,6). The fatty acid composition is shown in Table 3. The iso-C15:0, anteiso-C15:0, anteiso-C16:0 and C16:0 predominant composition is differ- ent from that of other actinomycetes. No mycolic acids were detected. The DNA composition is 69.7 mol% (Tm 89.3 C).

DISCUSSION

On the basis of morphological and chemotaxonomic characteristics strain A25 belongs to the genus Streptomyces. A comparison with previously published species of the genus shows the uniqueness of the organism, with reference to the characteristics of the spore bearing apparatus and fatty acid composition (O'Donnnell, 1993). In particular anteiso-C16:0 fatty acid is rare in Streptomyces species. Though different criteria have been used for the characterisation of Streptomyces species (Gilson et al., 1994; Kampfer and Kroppenstedt, 1991), in our opinion the strain represents a new species for which the name Streptomyces scopiformis sp. nov. is proposed.

Description of Streptomyces scopiformis sp. nov.

Streptomyces scopiformis (sco.pi.for'mis. L.n.f. scopa, a broom; L.n.f. forma, form. scopiformis, broom-like pattern, referring to the structure of the spore chains).

Aerobic mesophilic, Gram positive organism. Substrate mycelium non fragmenting, septate and swollen. Aerial mycelium light blue- grey to blue grey. Spore chains arranged in a broom-like pattern arising directly from the substrate mycelium.

Type I cell wall, type II phospholipids and MK9(H4,6) as dominant menaquinone. Predominant fatty acids: iso-C15:0, anteiso-C15:0, anteiso-C16:0 and C16:0. No mycolic acids. DNA G+C content 69.7% (Tm 89.3 C).

Isolated from soil.

Strain A25, deposited at the China Committee Culture Collection of Microorganisms (CCCCM) as AS 4.1331, is the type strain of the species.

ACKNOWLEDGEMENTS. The authors thank Dr.D.E.Minnikin, Newcastle University, UK for the determination of fatty acid and the Technological Department of the Microbiology Institute, Chinese Academy of Sciences, for the electron microscopy.

REFERENCES

Becker, B., M.P.Lechevalier & H.A.Lechevalier (1965). Chemical composition of cell-wall preparation from strains of various form genera of aerobic actinomycetes. Appl.Microbiol., 13: 236-243

Collins, M.D. (1985). Isoprenoid quinone analyses in bacterial classification and identification. In: M.Goodfellow & D.E.Minnikin (eds.) Bacterial Systematics. Academic Press, London, pp. 267-287

Gilson, P.M., J.Zakrzewska-Czerwinska, E. Alalan & M.Goodfellow (1994). Towards minimal standards for the description of Streptomyces species. Abstr. 9th International Symposium on the Biology of Actinomycetes, Moscow

Gordon, R.E., D.A.Barnett, J.B.Handerhan & C.Hor-nay Pang (1974). Nocardia coeliaca, Nocardia autotrophica, and the Nocardia strain. Int.J.Syst.Bacteriol., 24: 54-63

Hopwood, D.A., M.J.Bibb, K.F.Chater, T.Kieser, C.J.Bruton, H.M.Kieser, D.J.Lydiate, C.P.Smith & H.Schrempf (1985). Genetic manipulation of Streptomyces: a Laboratory Manual. John Innes Foundation, Norwich

Kampfer, P. & R.M.Kroppenstedt (l991). Probabilistic identification of streptomycetes using miniaturized physiological tests. J.gen.Microbiol., 137: 1893-1902

Kroppenstedt, R.M. (1985). Fatty acid and menaquinone analysis of actinomycetes and related organisms. In: M.Goodfellow & D.E.Minnikin (eds.) Bacterial Systematics. Aca- demic Press, London, pp. 173-199

Lechevalier, M.P. & H.A.Lechevalier (1980). The chemotaxonomy of actinomycetes. In: A.Dietz & D.W.Thayer (eds.) Actinomycete Taxonomy. SIM Special Publ., No.. 6, Arlington, Virginia, pp. 277-284

Liu, Z.H., J.Su & J.S.Ruan (l990) Determination of mycolates by gas chromatography. Microbiology (China), 17: 307- 310

Liu, Z.H., J.Ruan, J.Zakrzewska-Czerwinska & M.Mordarski (1992). Analyses of DNA homology and rDNA restriction patterns of some species in genus Nocardiopsis. Actinomycetes, 3: 51-54

Marmur, J. & P.Doty (1962). Determination of basic- composition of deoxyribonucleic acid from its denaturation temperature. J.Mol.Biol., 5: 109-118

Minnikin, D.E., I.G.Hutchinson, A.B.Caldicot & M.Goodfellow (1980). Thin-layer chromatography of methanolysates of mycolic acid-containing bacteria. J.Chrom., 188: 221-233

O'Donnell, A.G. (1993). Quantitative and qualitative analysis of fatty acids in the classification and identification of microorganisms. In: D.L.Hawksworth (ed.) Identification and Characterization of Pest Organisms. CAB International, Wallingford, UK, pp. 323-335

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

Wu, C., X.Lu, M.Gin & J.S.Ruan (1989). The analysis of menaquinone compositions in microbial cell wall by HPLC. Microbiology (China), 18: 176-178.

Figure 1. Morphology of strain A25: aerial mycelium (a), broom-like spore chains (b) and substrate growth (c); magnification 1200x. Spore morphology (d), 7000x.

Medium                    Mycelium colour    
----------------------------------------------------
                      Substrate       Aerial

Gauze starch      li ght blue grey   blue grey
Czapek sucrose    light blue grey    light blue grey
Czapek glycerol   brown    
Czapek peptone    light blue grey    light blue grey
Inorganic salts 
- starch (ISP 4)  light brown    
Potato extract    light blue grey    light blue grey
Glycerol calcium 
malate            blue grey    
Oatmeal (ISP 3)   blue grey          blue grey
Glycerol asparagine 
(ISP 5)           light blue    
Emerson           yellow brown  

Table 1. Cultural characteristics of strain A25. Growth is poor on ISP 4 and moderate on ISP 7 media.

Character            Reaction    Character         Reaction
------------------------------------------------------------
Acid fastness           -        
------------------------------
Decomposition of:               Hydrolysis of:   

Adenine                 +       Esculin            +
Casein                  +       Hippurate          -
Hypoxanthine            +       Starch             +
Urea                    -       Cellulose          -
------------------------------------------------------------
Growth at:                      Acid production
from:    
10 C                    -       Adonitol           -
50 C                    -       L (+) arabinose    +
Nitrate reduction       +       Dulcitol           -
Utilisation of:                 i-Erythrol         -
Benzoate                -       D (+) galactose    +
Citrate                 -       Glucose            +
Lactate                 +       Inositol           +
Malate                  +       D (+) lactose      +
Mucate                 (+)      D (+) maltose      + 
Oxalate                 -       D (+) mannose      +
Succinate               +       D (+) melibiose    +
Resistance to:                  D (+) raffinose    -
Bacitracin              -       D (+) rhamnose     +
Lysozyme                -       D (+) xylose       +
Penicillin              +       Trehalose          +
Methyl violet           -       D sorbitol         -
Pyrinin B               -       alpha-Methyl glycoside +
Biomycin (5 ug)         +       Glucose oxidation      +
Gelatine liquefaction   +       Glucose fermentation   -

Table 2. Physiological characteristics of strain A25. +: positive, -: negative, (+): weak reaction.

Fatty acid    Retention time  Amount (%)
                  (min)
----------------------------------------
ai-C14:0         8.21         3.507
C14:0            8.64         1.371
iso-C15:0        9.57        21.519
ai-C15:0         9.66        13.211
C15:0            9.94         4.576
unknown         10.42         1.926
ai-16:0         10.79        18.953
C16:1           10.91         4.364
C16:0           11.21        13.516
unknown         11.60         2.808
unknown         11.71         1.289
iso-C17:0       11.92         4.399
ai-C17:0        12.04         6.160
C17:0           12.31         1.374

Table 3. Fatty acid composition of strain A25. The amount is the percentage of methyl esters as determined by gas chromatography.

Copyright 1996 C.E.T.A., The International Centre for Theoretical and Applied Ecology, Gorizia

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