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PRODIGIOSIN-LIKE AND OTHER METABOLITES PRODUCED BY A STREPTOVERTICILLIUM STRAIN U. Brambilla, G. Nasini, B. Petrolini^*, S. Quaroni^*, M. Saracchi^* and L. Fedeli^*
Centro del CNR per le Sostanze Organiche Naturali, Dipartimento di Chimica del Politecnico, via Mancinelli 7, 20131 Milan and *Istituto di Patologia Vegetale, Universit… di Milano, Via Celoria 2, 20133 Milan, Italy
Code Number: AC95011
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Graphics: Photos (jpg) 74K Abstract. An actinomycete strain, isolated from leek roots and showing a verticillate arrangement of sporophores, was classified by numerical methods as belonging to the cluster Streptoverticillium baldaccii. Relationships with previously described species of the genus are discussed. The organism produces metacycloprodigiosin, undecylprodigiosin, 6,8-dimethoxy-3,methylisocoumarin, 2,5-furanmethanol, Nb- acetyltryptamine and a diketopiperazine compound.
In the course of an investigation on actinomycete populations inhabiting plant roots (Sardi et al., 1992), a red verticillate strain was isolated. Streptoverticillia appear to be typical soil saprophytes, although their isolation from natural habitats is usually problematic (Cardinali et al., 1989; Cardinali and Firrao, 1990). To our knowledge verticillate actinomycetes have never before been isolated from roots of higher plants. Considering the metabolic potential of streptoverticillia, the taxonomy of the strain and the nature of its metabolites were investigated.
MATERIALS AND METHODS
Isolation of the strain. Strain IPV-2793 was isolated from leek (Allium porrum L.) surface- sterilised roots collected in May 1989 in western Lombardy, using a selective procedure previously described (Sardi et al., 1992), on starch-casein agar (Kster and Williams, 1964) with 50 ppm of nystatin and cycloheximide (Williams and Davies, 1965). The verticillate strain was spotted among colonies of other actinomycetes by light microscopy examination of isolation plates because of the typical "barbed-wire" appearance (Locci and Schofield, 1989) of aerial hyphae, emerging from root fragments.
Characteristics of the isolate. Morphological characteristics (sporophore and spore chain morphology) were assessed by light and scanning electron microscopy (Stereoscan 250, Cambridge Sci. Instr. Ltd., UK). Cultural characteristics of the strain were determined on the following media: potato agar (PA), Bacto Czapek solution agar Difco (CA), glycerol asparagine agar Difco = ISP (International Streptomyces Project, Shirling and Gottlieb, 1966) medium 5, glucose asparagine agar (GA = ISP medium 5, with 1% glucose replacing glycerol), inorganic salts-starch agar Difco (ISP medium 4), yeast extract-malt extract agar Difco (ISP medium 2), Bennett's agar (BA) and nutrient agar Oxoid (NA). The media were chosen in order to allow comparison with other species of the genus showing red pigmentation (Locci et al., 1969). Colours were determined according to the Ostwald Color Manual (1939). Carbohydrate utilisation was carried out by the API 50CH system (Bio M‚rieux SA) using as inoculum a 3% agar medium (yeast nitrogen base Difco) neutralized with 20% (v/v) 1% K2HPO4 solution. A similar characterisation was carried out on the type strain of Streptoverticillium pentaticum subsp.jenense, IPV-2002 = KCC (Kaken Chemical Co., Tokyo, Japan) S-0211, for which production of prodigiosin had previously been reported (Locci and Schofield, 1989). Identification of strain IPV-2793. Probabilistic identification of the strain was carried out using a set of 41 characters (Williams et al., 1985; Locci et al., 1986) derived from a numerical taxonomy study of representatives of the genus Streptoverticillium (Locci et al., 1981). Characterisation and purification of metabolites. Strain IPV-2793 was grown in malt-peptone-glucose agar (MPGA; 20:3:20:15 g/l^-1) in Roux flasks (30) incubated at 28 C for 3 weeks. Secondary metabolites were extracted twice with EtOAc- MeOH (100:1) poured over the solid medium, left overnight and the extracts treated with dry Na2SO4. Evaporated extracts (0.65g) were dissolved in CH2Cl2 (15 ml) and subsequently hexane (200 ml) was added; the hexanic layer contains a mixture of prodigiosin pigments 1 and 2 and the residue the more polar metabolites 3-6. Evaporated hexanic-extracts were chromatographed on a column filled with Al2O3 (activity 1) and eluted with hexane- EtOAc (10:1). The mixture of the red-violet metabolites was subjected to PLC (Merck plates-SiO2, 2 mm), using CH2Cl2-MeOH (20:1) as eluent, to give pure compounds 1 (95 mg) and 2 (10 mg). The residue was absorbed onto Si gel, placed on top of a chromatographic column filled with flash Si gel and eluted with EtOAc-MeOH (100:1) to obtain compounds 3 (23 mg), 4 (14 mg), 5b (45 mg) and 6 (25 mg) respectively in order of increasing polarity. Spots were revealed with an oxidative solution containing Ce(SO4)2, 10 g; phosphomolybdic acid, 25 g; H2SO4, 60 ml; water, 940 ml. Metabolites were characterised by UV (Jasco-Uvided-510), IR (Perkin-Elmer 177), MS (Finnigan-MAT-TSQ 70), optical rotation (Jasco-500 DIP-181) and ^1H,^13C NMR (Bruker AC 250). Cultures of Streptoverticillium pentaticum subsp. jenense (IPV-2002) were similarly grown on MPGA in Roux flasks; the EtOAc extract purified as above and metabolites identified as 1 and 2 by TLC, ^1H NMR and MS.
RESULTS
Characteristics of strain IPV-2793. Sporulated hyphae show the typical morphology of genus Streptoverticillium. The aerial mycelium produces verticils of sporophores bearing terminal umbels of straight to flexuous spore chains (fig. 1) showing the typical process of twisting (Locci and Petrolini Baldan, 1970) that can persist even in mature spore chains (fig. 2). The isolate shows a cottony appearance of aerial growth and pale pink spore mass on ISP medium 4. Depending on the substratum, the colony reverse colour ranges from pink to brick orange, red, purple or brown and the aerial growth from pale to strong pink, sometimes with lighter, darker or orange cottony patches. A brown diffusible pigment is formed on NA and ISP medium 2, and a yellow one on BA and PA.
Figure 1 and 2. Verticillate sporophores of 21 day- old culture of IPV-2793 on ISP medium 3. Bar markers equal 4 um in fig. 1 and 2 um in fig. 2.
In Table 1 cultural features of strain IPV-2793 and of Streptoverticillium pentaticum subsp. jenense are compared with those of some species of the genus (Stv.baldaccii, Stv.biverticillatum and Stv.hiroshimense) showing reddish pigmentation of spores and mycelia, as reported by Locci et al. (1969). Strain IPV-2793 differs from the other species in pigmentation, growth on CA and ability to produce soluble pigments. In particular it differs from Stv.pentaticum subsp. jenense in the following characteristics: colony reverse and spore mass generally strongly coloured and aerial growth more cottony, amount of growth and aerial mycelium on CA, yellow diffusible pigment production on BA and PA. The strain utilises only a limited number of the carbon sources tested, in particular: adonitol, D-glucose, D-mannose, inositol, maltose, glycerol, trehalose, starch, glycogen, gluconate and N acetyl glucosamine. In contrast to Stv.pentaticum subsp. jenense, strain IPV-2793 utilises adonitol and inositol, but not ribose. Using the 41 tests suggested for probabilistic identification of the species of the genus Streptoverticillium strain IPV-2793 is placed in the Streptoverticillium baldaccii cluster (Williams et al., 1985; Locci et al., 1986; Locci and Schofield, 1989) with Willcox probability of 0.831, taxonomic distance 0.348 and standard error of taxonomic distance -0.180. Characterisation of metabolites. Pigment 1 was obtained as an orange-brown solid (petroleum ether), m.p. 204-206 C and analysed for C25H33N3O; EIMS spectrum gives a distinct peak at m/z 391(M^+) (100%), 376(10), 348(20) and 334(18); UV exhibits a peak at 472 nm (e 29.800).
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Medium Character IPV-2793 Stv.penta Stv. aldaccii
ticum subsp.
jenense
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G good good abundant
PA R brick orange, brown, pink to salmon,
1X6nc 1X6pe 2X71a
SM pale pink, pale pink, pink to salmon,
1X6la (2IV9ca) 2II8-9ca 2II8ca, 2IV7ea
(withish)
SP yellow none none
G medium poor limited
CA R pink to red, pinkish beige, colourless to
pink traces, pink
1II4ca, 1X6la 1II3ec
SM pink, 2II8ca, traces traces, pink
2IV7ea
G good good good
GA R brick red, pink to orange, red,
1X5-6nc 1IV3ea, 1VIII6ne 1X6nc
SM pink, 1II6ca, pale pink, pale pink,
1IV6gc 2II8ca 2IV8ea (withish)
G good good good
ISP 5 R brick orange, yellow to red, red,
1VIII6ne 1IV3ea,2VIII7ne 1X6nc
SM dark pink, pink, pale pink,
1VI5ga 2IV10ea 2IV7ea
G very good very good good
ISP 4 R purple, brick orange, red,
2X7nc 1VIII6ne 2XII8na
SM pale pink, pale pink, pale pink,
2II8ca(1VI5ga) 2II8ca 2II8-10ca
G very good very good good
ISP 2 R brown, brown, orange red,
1VIII5na 1VIII5na 1X6la
SM pink, 1IV6ea pinkish white, pink, 2IV7-8ea
(1VI5ga) 2II9ca (whitish)
SP brown brown none
G very good good good
R brick orange, brown, orange red,
1X5nc 1VIII5na 1X6la
BA SM dark pink, pink, pale pink,
1IV6ea (1XII5na) 1II5-6ca 2II8ca, 2IV7ea
SP yellow none none
G good good good
R brown,1VI4ng brown,1VI4ng orange-brown
red,1VIII5ea-5lc
NA SM none none pink-violet,
2VIII7ia, 2VI8ga
SP brown brown brown
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Medium Character Stv. bivertici- Stv.hiroshimense
llatum
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G medium moderate
PA R reddish yellow, 1IV6lg brownish, 1VI5pi
SM whitish pink, 1II3ca beige pinkish, 1II3ec
SP none none
G traces traces
CA R colourless off-white, 1II2ea
SM none none
G good good
GA R yellowish reddish, brown red,
1IV2ea, 1XII6pc 1VIII6lc
SM yellowish pink, 1II3ca pinkish beige, 1II3ca
G good good
ISP 5 R yellowish, 1IV2ea dark red, 1VIII6ia
SM yellowish pink, 1II3ca white to pink, 1II5ca
G good good
ISP 4 R yellowish, 1IV2ea raspberry pink,
2VIII8ia
SM yellowish pink, 1II3ca beige pink, 1II3ec-ge
G good good
ISP 2 R reddish brown, brown red, 1VI5-6ng
1VIII5ne
SM pinkish white, 1II3ca beige pink, 1II3-4ge
SP none none
G good good
R brick red, 1X6nc dirty-brown red,
1VIII6pg
BA SM pink, 1II4-5ca pink, 1II4ec
SP none none
G medium poor
R orange yellow, 1X3nc brown, 1VIII4pg
NA S pinkish white(raspberry) light beige, 1II1ca
SP brown traces
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Table 1. Cultural characteristics of strain IPV-2793 and of some Streptoverticillium species with reddish micelia. G = growth; R = reverse colour; SM = secondary mycelium colour; SP = soluble pigment. Code or colour in brackets refer to the colour of SM patches, tufts or overgrowths.
The ^1H NMR spectrum, measured in CDCl3, shows peaks due to NH, aromatic and methoxyl protons, a broad multiplet at d 2.0- 2.3 (3H), a multiplet between d 0.6-1.9 (19H) and more specifically, the peak at d 6.05 is in agreement with the presence of a b proton in a dialkylpyrrole residue, nearly identical to the absorption shown by metacycloprodigiosin (Wasserman et al., 1976). Pigment 2 was isolated as a brown solid and analysed for C25H35N3O and the EIMS spectrum has a molecular ion at m/z 393 and a peak at m/z 252 (loss of C10H21 from the lateral chain); ^1H NMR (CDCl3) spectrum shows absorptions at d 0.9 (t, 3H), 1.15-1.9 (m, 18H) and 2.95 (t, 2H) according to the presence of a n-undecyl side chain; all other NMR data confirm the identity of pigment 2 with the known undecylprodigiosin (Wasserman et al., 1969). On the other hand an extract of Stv.pentaticum subsp. jenense (IPV-2002), known to produce metabolites 1 and 2, showed the same mixture of the two pigments. Metabolite 3 was isolated as crystals m.p. 220-225 C; UV spectrum shows a l[max] 245 and 325 nm (e 36.200 and 4.500) and the IR spectrum (KBr) shows signals at n[max] 1710 (lactone), 1665 and 1600 cm^-1; high resolution mass spectroscopy evidentiates the formula C12H12O4 (found 220.0739, calc. 220.0736); ^1H NMR spectrum (CDCl3) shows two meta-coupled aromatic protons d 6.42 and 6.29, a singlet vinylic signal at d 6.09, two methoxy groups d 3.96 and 3.89 and an aromatic methyl substituent d 2.21, responsible for all the protons of the molecule. Literature data report only a compound with the assigned formula, siderin (4,7-dimethoxy-5-methylcoumarine), isolated from Aspergillus variecolor (Turner and Aldridge, 1983), that normally occurs in Sideritis plants. Moreover the chemical shift values exhibited by compound 3 were not in agreement with NMR data of siderin (Chexal et al.,1975); the structure of 3 was deduced mainly from ^13C NMR analyses [dC(C-1, C-3, C-4a, C-6 and/or C-8; 165.38, 163.27, 159.82, 155.50, 154.30), (C-8a; 142.46), (C-7, C-5 and/or C-4; 103.69, 99.37, 98.13), (C-10 and/or C-11; 56.25, 55.58) and (C-9; 19.50)] and with NOEs measurements as 6,8-dimethoxy,3-methylisocoumarin, a new natural compound. Further support for this structure was the isolation from Stv.mobaraense of 6,8-dihydroxy,3-methylisocoumarin (Turner and Aldridge, 1983). Compound 4 has m.p. 70-72 C; mass spectrum contains peaks at m/z 128 (M^+)(18%) and 110 (M^+-18)(100) and was analysed for C6H8O3; ^1H NMR spectrum (CDCl3) reveals the presence of a singlet of one proton at d 6.24, a singlet at d 4.58 for two protons and a peak corresponding to one aliphatic OH (d 2.35); comparison with an authentic sample of 2,5- furandimethanol (TLC and ^1H NMR spectrum) attributed the symmetrical structure 4 for the metabolite. Metabolite 5b was isolated as an oil and was analysed for C12H14N2O; m/z 202 (M^+) (24%), 143 (100) and 130 (83); UV spectrum shows a typical absorption of an unsubstituted indol moiety (l[max] : 220, 275sh, 282 and 290 nm) and the signal at 1660 cm^-1 in IR spectrum was attributable to an amide function. All this data, together with ^1H and ^13C NMR spectra: two NHs at dH (5,66 and 8,40), two CH2 at dH (3.58 and 2.97) one methyl at dH 1.90 and a CO at dC 170.33, suggest the structure 5b; the synthesis of 5b started from the tryptamine 5a by acetylation with pyridine, Ac2O and DMAP (dimethyl-4-amino pyridine) to obtain the Nb-acetyltryptamine. Metabolite 6 was isolated as a solid, m.p. 149-152 C with an [a]D value of -56.2 (MeOH, c 0.1); IR spectrum (CH2Cl2 solution) shows signals at 3300 (OH) and 1670 (conj.CO) cm^-1; the last band can be attributed to an amide function. The sample when submitted to CIMS (chemical ionization mass spectrometry) shows molecular peak at m/z 261 (MH^+) and was analysed for C14H16N2O3. The ^1H NMR spectrum shows a p-hydroxy-phenyl moiety and a very complex pattern for 10 protons between 1.0-4.4 ppm; the all decoupled ^13C NMR spectrum shows the 14 carbon atoms of the molecule and a DEPT experiment confirms that the signals at dC 22.44, 28.34, 35.95 and 45.43 can be attributed to CH2 groups and the signals at dC 56.29 and 59.14 are due to two aliphatic methine; these data suggested that our metabolite 6 is identical to the diketopiperazine compound isolated from Fusarium nivale (Tatsuno et al., 1971). In fact all spectroscopic measures are in agreement.
DISCUSSION
Following the probabilistic identification of strain IPV- 2793, attributed to the Streptoverticillium baldaccii cluster, a comparison with previous descriptions of culturally similar (red colour) species of the cluster was carried out. In addition Streptoverticillium pentaticum subsp. jenense, described as a producer of prodigiosin (Locci and Schofield, 1989), but belonging to the Streptoverticillium griseocarneum cluster, was subjected to a comprehensive and parallel study. Production of prodigiosin-like pigments by a culture of Stv.rubrireticuli that causes pink staining of vinyl plastic has also been reported (Gerber and Stahly, 1975). However the original strain could not be obtained. IPV-2793 differs from previously described species of Streptoverticillium in cultural and physiological characteristics, and particularly for the production of secondary metabolites. In spite of most Streptoverticillium species having been established on the basis of the production of active secondary metabolites, we do not deem it convenient to propose, at present, a new species to accomodate the strain but prefer to wait for adequate tests for discrimination at the infrageneric level. Verticillate actinomycetes are currently placed in the genus Streptoverticillium (Locci, 1989). In view of the proposed unification for Streptomyces and Streptoverticillium genera (Witt and Stackebrandt, 1990; K„mpfer et al., 1991), we propose for the moment to designate the new isolate as Streptoverticillium sp. IPV-2793 allocated to the Streptoverticillium baldaccii cluster. The lack of effective preferential techniques for the isolation of streptoverticillia is responsable for their irregular and sporadic recovery from natural habitats and consequently not much is known about their ecology (Cardinali et al., 1989). In any case the detection of a Streptoverticillium strain in leek roots appears ecologically interesting. With reference to the metabolites isolated from the strain, metacycloprodigiosin 1 and undecylprogiosin 2 are members of a class of naturally-occurring products, possessing a common characteristic, pyrrolyl- pyrromethane skeleton, which have been shown to possess strong antimicrobial and cytotoxic properties. These compounds are more frequently isolated from species of Streptomyces than of Streptoverticillium. Isocoumarin 3 is a newly isolated product while the prolyltyrosyl anhydride 6 represents a very rare metabolite isolated so far only from Fusarium nivale (Tatsuno et al., 1971). Nb-acetyltryptamine 5b occurs normally in plants (Lee et al., 1988) together with tryptamine and, to our knowledge, this is the first isolation of the compound from a microorganism. REFERENCES
Cardinali, S. & G.Firrao (1990). Problems in the selective isolation of Streptoverticillium species from soil. Actinomycetes, 1: 16-18
Cardinali, S., G.Firrao & R.Locci (1989). Isolamento selettivo di attinomiceti del genere Streptoverticillium dal terreno. Ann.Microbiol., 39: 231-245
Chexal, K.K., C.Fouweather & J.S.E.Holker (1975). Production and biosynthesis of 4,7-dimethoxy-5-methylcoumarin in Aspergillus variecolor. J.Chem.Soc.Perkin, 1: 554-556
Gerber, N.N. & D.P.Stahly (1975). Prodiginine (prodigiosin-like) pigments from Streptoverticillium rubrireticuli, an organism that causes pink staining of polyvinyl chloride. Appl.Microbiol., 30: 807-810
Kampfer, P., R.M. Kroppenstedt & W.Dott (1991). A numerical classification of the genera Streptomyces and Streptoverticillium using miniaturizing physiological tests. J.gen.Microbiol., 137: 1831-1891
Kuster, E. & S.T.Williams (1964). Selection of media for isolation of streptomycetes. Nature (London), 202: 928-929
Lee, S.L., M.Reporter, S.N.Hsu, J.L.Corbin, L.Gregory, P.L.Burkhouse & J.H. Litchfield (1988). The lipid composition and conversion of tryptamine to Nb- acetyltryptamine in a Catharanthus roseus cell line without indole alkaloids. J.Ind.Microb., 3: 293- 297
Locci, R. (1989). Streptomycetes and related genera. In: S.T. Williams, M.E.Sharpe & J.G.Holt (eds.) Bergey's Manual of Systematic Bacteriology. The Williams & Wilkins Co., Baltimore, Vol. 4, pp. 2451-2508
Locci, R. & B.Petrolini Baldan (1970). Morphology and development of Streptoverticillium species as examined by scanning electron microscopy. Giorn.Microbiol., 18: 69-76
Locci, R. & G.M.Schofield (1989). Genus Streptoverticillium Baldacci 1958, 15, emend. mut. char. Baldacci, Farina and Locci 1966, 168^AL. In: S.T.Williams, M.E.Sharpe & J.G.Holt (eds.) Bergey's Manual of Systematic Bacteriology. The Williams & Wilkins Co., Baltimore, Vol. 4, pp. 2492-2504
Locci, R., E.Baldacci & B.Petrolini Baldan (1969). The genus Streptoverticillium. A taxonomic study. Giorn.Microbiol., 17: 1-60
Locci, R., J.Rogers, P.Sardi & G.M.Schofield (1981). A preliminary numerical study on named species of the genus Streptoverticillium. Ann.Microbiol., 31, 115-121
Locci, R., S.T.Williams, G.M.Schofield, J. Vickers, P.H.A.Sneath & A.M.Mortimer (1986). A probabilistic approach to the classification of Streptoverticillium species. In: G.Szabo, S. Biro and M.Goodfellow (eds.) Biological, Biochemical and Biomedical Aspects of Actinomycetes. Akad‚miai Kiad¢, Budapest, pp. 507-516
Ostwald, W. (1939). Die kleine Farbmesstafel, Musterschmidt, Gottingen
Sardi, P., M.Saracchi, S.Quaroni, B.Petrolini, G.Borgonovi & S.Merli (1992). Isolation of endophytic Streptomyces strains from surface-sterilized roots. Appl.Envirom.Microbiol., 58: 2691-2693 Shirling, E.B. & D.Gottlieb (1966). Methods for characterization of Streptomyces species. Int.J. Syst.Bacteriol., 16: 313-340
Tatsuno, T., M.Sato, Y.Kubota & H.Tsunoda (1971). Recherches toxicologiques des sustances metaboliques du Fusarium nivale. Chem.Pharm. Bull., 19: 1498- 1500 Turner, W.B. & D.C.Aldridge (1983). Fungal metabolites II. Academic Press, London
Wasserman, H.H., G.C. Rodgers & D.D. Keith (1969) Metacycloprodigiosin, a tripyrrole pigment from Streptomyces longisporus ruber. J. Amer.Chem.Soc., 91: 1263-1264
Wasserman, H.H., G.C. Rodgers & D.D. Keith (1976) Undecylprodigiosin. Tetrahedron, 32: 1851- 1854
Williams, S.T. & F.L.Davies (1965). Use of antibiotics for selective isolation and enumeration of actinomycetes in soil. J.gen.Microbiol., 38: 251-261
Williams, S.T., R.Locci, J.Vickers, G.M. Schofield, P.H.A.Sneath & M.Mortimer (1985). Probabilistic identification of Streptoverticillium species. J.gen.Microbiol., 131, 1681-1689
Witt, D. & E.Stackebrandt (1990). Unification of the genera Streptoverticillium and Streptomyces, and amendation of Streptomyces Waksman and Henrici 1943, 339^AL. System.Appl.Microbiol., 13: 361-371.
Copyright 1995 C.E.T.A
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