Letters in Applied Microbiology 1999, 28, 199–202
Actinorhodin production by Streptomyces coelicolor A3(2) in iron-restricted media S. Coisne, M. Be´chet and R. Blondeau Laboratoire de Microbiologie, Universite´ des Sciences et Technologies de Lille, Villeneuve d’Ascq, France 1998/98: received 9 December 1998 and accepted 15 December 1998
Production of the polyketide antibiotic actinorhodin by Streptomyces coelicolor A3(2) was investigated using a defined medium with or without iron supplementation. Iron limitation was found to enhance the intracellular production and export of the pigmented antibiotic. The effect of iron deficiency was particularly pronounced when the bacterium was grown with nitrate instead of ammonium. Analysis of the excreted pigment led to the identification of the lactone form of actinorhodin, g-actinorhodin. ´ CH E T A ND R . B LO N DE AU . 1999. S . C OI S NE , M . BE
INTRODUCTION
Actinorhodin, a polyketide antibiotic, is produced by Streptomyces coelicolor A3(2), the best genetically known strain of Streptomyces. Its pigmentation (red at acidic pH and blue at alkaline pH) facilitates visual observation of its produce. Actinorhodin is often used as a model for studying factors regulating the production of antibiotics. Biosynthesis of actinorhodin occurs mainly during the stationary phase in batch cultures, but may be also growthassociated according to the medium used (Ozergin-Ulgen and Mavituna 1993). Numerous factors have been implicated in its synthesis, probably explaining discrepancies between reports. Nitrogen or phosphate depletion generally elicits its production (Doull and Vining 1990), with phosphate control being epistatic to that of ammonium (Hobbs et al. 1990). The presence of sucrose for osmotic balance in a complex medium also leads to actinorhodin production (Elibol and Mavituna 1998). The pH of the culture medium is important as excretion of actinorhodin appears to occur exclusively at pH values above 6·7 (Wright and Hopwood 1976). Moreover, this excretion increases in complex media (Ozergin-Ulgen and Mavituna 1994) but, according to Bystrykh et al. (1996), the excreted pigment is not actinorhodin but its lactone derivative, g-actinorhodin. The aim of the present work was to investigate the effect of iron deficiency on the production of actinorhodin in batch cultures. As polyketide synthesis does not require the presence of iron, iron deficiency may represent a significant regulation factor. Indeed, iron deficiency has been shown to Correspondence to: Dr R. Blondeau, Laboratoire de Microbiologie, Baˆtiment SN2, Universite´ des Sciences et Technologies de Lille, F-59655 Villeneuve d’Ascq-Cedex, France (e-mail:
[email protected]). © 1999 The Society for Applied Microbiology
stimulate the production of tetracyclines in Streptomyces aureofaciens (Be´chet and Blondeau 1998).
MATERIALS AND METHODS Media and cultivation of the strain
The basal culture medium comprised (l−1): K2SO4, 2 g; NaCl, 1 g; K2HPO4, 15 mmol; and NH4Cl or NO3K, 40 mmol. Any contaminating Fe ions were removed by passage through Chelex 100 resin (Bio-Rad). The medium was then supplemented with: MgSO4.7H2O, 80 mg; ZnSO4.7H2O, 2 mg; and 0·1 ml of trace element solutions containing (l−1): CuSO4.5H2O, 0·5 g; MnSO4.H2O, 5 g; H3BO3, 4 g; CoCl2.6H2O, 0·5 g; NiCl2.6H2O, 2 g; and Na2MoO4.2H2O, 3 g. The final pH was adjusted to 7·0 with (i) HCl or (ii) KOH when the medium was buffered with 50 mmol l−1 TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulphonic acid). Medium (100 ml) was distributed into 500 ml serum flasks, previously washed with 10% nitric acid to remove traces of iron. After autoclaving, the flasks were further supplemented with CaCl2.2H2O, 0·1 g l−1 and glucose, 5 g l−1 (both autoclaved separately), and filter-sterilized yeast extract, 50 mg l−1 (previously deferrated by Chelex 100). Where appropriate, filter-sterilized FeSO4.7H2O was added (20 mmol l−1 final concentration) to the flasks. Streptomyces coelicolor A3(2) 1147, provided by Prof. D.A. Hopwood (John Innes Institute, Norwich, UK), was kept as spore suspensions in 20% glycerol at – 20 °C. Each culture flask was inoculated with 0·2 ml diluted suspension containing about 3·0 × 105 spores. After 12 h incubation in still culture
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at 30 °C, the cultures were transferred to an orbital shaker (100 rev min−1) and grown in the dark. The iron deficiency level of the cultures was checked by measuring the production of siderophores, using the reagent of Schwyn and Neilands (1987).
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Intracellular and extracellular levels of the actinorhodinrelated pigment concentrations were determined according to Bystrykh et al. (1996). For further TLC characterization, the pigment was extracted from the culture filtrates, after acidification to pH 2·0 with HCl, either by passage through XAD-8 resin (Merck) followed by elution with methanol, or by treatment with chloroform. Thin layer chromatography assays were conducted on silica gel 60 F254 plates (Merck) with benzene-acetic acid (9:1; v/v) as solvent.
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RESULTS
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Pigment synthesis with ammonium as nitrogen source
Added at low concentration to the medium containing ammonium chloride or potassium nitrate as sole nitrogen source, yeast extract served mainly to stimulate germination of the spores and to produce more homogeneous mycelial pellets. Whether or not the culture medium was buffered with TES, the results observed when ammonium was the nitrogen source were very similar. However, bacterial growth, as dry weight of washed pellets, was lower in non-buffered medium because the pH dropped. Figure 1 shows the results from non-buffered cultures. In iron-deprived medium, the production of siderophores started as early as the second day of incubation and the stationary phase occurred more quickly. Intracellular pigment was produced only during the stationary phase but synthesis began earlier in the presence of iron; the final concentration of the intracellular pigment was similar. The biggest difference concerned colorization of the culture medium due to excretion of the pigment, which arose as soon as it was intracellularly synthesized in the medium devoid of iron. Here, extracellular pigment concentration was much higher than in iron-containing cultures. In the latter cultures, colorization of the medium appeared a long time after intracellular synthesis of the pigment, and may have resulted partly from cell lysis. Pigment production with nitrate as nitrogen source
Substitution of ammonium by nitrate led to very different results according to the presence of iron in the culture medium; no actinorhodin-related pigment synthesis occurred in the presence of iron, while its production occurred immedi-
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Fig. 1 Growth and pigment production by Streptomyces coelicolor A3(2) in non-buffered medium containing ammonium as nitrogen source, without (a) or with iron (b). (R) Mycelium dry weight from 100 ml culture; (Ž) intracellular pigment; (ž) pigment excreted in the culture medium. Measurements were made in duplicate from two separate flasks and the results are the means
ately followed by excretion into the iron-deficient medium. The results obtained in buffered medium are shown in Fig. 2. In non-buffered medium, pigment synthesis was much less significant in the absence of iron, and the pH dropped below 5·0. In the experiments carried out with 50 mmol l−1 TESbuffered medium, the pH dropped slightly to 6·7 and 6·5 in the absence or presence of iron, respectively. This variation, however, could not explain the difference in pigment production because similar results were obtained when this slight acidification was avoided by a stronger buffering of the medium with 100 mmol l−1 TES. In addition, increasing the glucose concentration in the medium had no effect on actinorhodin-related pigment synthesis, whereas a lower concentration led to a lower biomass and less marked results.
© 1999 The Society for Applied Microbiology, Letters in Applied Microbiology 28, 199–202
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1998), produced early in a defined culture medium under conditions of iron deficiency. It is worth noting that, like tetracyclines and probably other polyketides with structures possessing close ketone functions and hydroxyl groups, actinorhodin and its derivatives behave as chelators of iron. In the media used in our experiments, the concentrations of phosphate and nitrogen were relatively high as they allowed iron deficiency to be followed more easily by favouring maximal growth. These concentrations have been found to inhibit actinorhodin synthesis in other media (Hobbs et al. 1990; Bystrykh et al. 1996). The different behaviour of S. coelicolor A3(2) according to the nature of mineral nitrogen in the medium has already been reported for actinorhodin production (Hobbs et al. 1990) and for spore formation (Karandikar et al. 1997). This may result from the different assimilation pathways of these two forms of nitrogen, or their effects on other metabolic functions. In other respects, the excretion of g-actinorhodin in the media used appears to be closely related to iron deficiency. In S. coelicolor A3(2), genes involved in the synthesis and excretion of g-actinorhodin have been identified (Caballero et al. 1991; Ferna´ndez-Moreno et al. 1991; Bystrykh et al. 1996). However, the effects of iron on genetic regulation of the synthesis of actinorhodin remain to be investigated.
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Fig. 2 Growth and pigment production by Streptomyces coelicolor
A3(2) in TES-buffered medium containing nitrate as nitrogen source, without (a) or with iron (b). (R) Mycelium dry weight from 100 ml culture; (Ž) intracellular pigment; (ž) pigment excreted in the culture medium. Measurements were made in duplicate from two separate flasks and the results are the means
Pigment characterization
The absorption maximum of the pigment in chloroform extracts of the culture filtrates, measured in acidic methanol, was at 535 nm, but measured in 1 mol l−1 KOH, it was at 638 nm. The pigment was insoluble in dioxane. Thin layer chromatography assays on silica-gel 60 F254 plates yielded a single spot with an Rf of 0·28. According to Bystrykh et al. (1996), this value indicates the presence of the lactone derivative of actinorhodin in the culture filtrates, i.e. g-actinorhodin. The solubility characteristics of g-actinorhodin are totally different from those of actinorhodin, which is insoluble in chloroform and methanol but soluble in dioxane. DISCUSSION
The results presented here show that g-actinorhodin is a further polyketide, after tetracyclines (Be´chet and Blondeau
REFERENCES Be´chet, M. and Blondeau, R. (1998) Iron deficiency-induced tetracycline production in submerged cultures by Streptomyces aureofaciens. Journal of Applied Microbiology 84, 889–894. Bystrykh, L.V., Ferna´ndez-Moreno, M.A., Herrema, J.K., Malpartida, F., Hopwood, D.A. and Dijkhuizen, L. (1996) Production of actinorhodin-related ‘blue pigments’ by Streptomyces coelicolor A3(2). Journal of Bacteriology 178, 2238–2244. Caballero, J.L., Malpartida, F. and Hopwood, D.A. (1991) Transcriptional organization and regulation of an antibiotic export complex in the producing Streptomyces culture. Molecular and General Genetics 228, 372–380. Doull, J.L. and Vining, L.C. (1990) Nutritional control of actinorhodin production by Streptomyces coelicolor A3(2): suppressive effects of nitrogen and phosphate. Applied Microbiology and Biotechnology 32, 449–454. Elibol, M. and Mavituna, F. (1998) Effect of sucrose on actinorhodin production by Streptomyces coelicolor A3(2). Process Biochemistry 33, 307–311. Ferna´ndez-Moreno, M.A., Caballero, J.L., Hopwood, D.A. and Malpartida, F. (1991) The act cluster contains regulatory and antibiotic export genes, direct targets for translational control by the bldA transfer RNA gene of Streptomyces. Cell 66, 769–780. Hobbs, G., Frazer, C.M., Gardner, D.C.J., Flett, F. and Oliver, S.G. (1990) Pigmented antibiotic production by Streptomyces coelicolor A3(2): kinetics and the influence of nutrients. Journal of General Microbiology 136, 2291–2296. Karandikar, A., Sharples, G.P. and Hobbs, G. (1997) Differentiation
© 1999 The Society for Applied Microbiology, Letters in Applied Microbiology 28, 199–202
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of Streptomyces coelicolor A3(2) under nitrate-limited conditions. Microbiology 143, 3581–3590. Ozergin-Ulgen, K. and Mavituna, F. (1993) Actinorhodin production by Streptomyces coelicolor A3(2): kinetic parameters related to growth, substrate uptake and production. Applied Microbiology and Biotechnology 40, 457–462. Ozergin-Ulgen, K. and Mavituna, F. (1994) Comparison of the activity of immobilised and freely suspended Streptomyces coel-
icolor A3(2). Applied Microbiology and Biotechnology 41, 197– 202. Schwyn, B. and Neilands, J.B. (1987) Universal chemical assay for the detection and determination of siderophores. Analytical Biochemistry 160, 47–56. Wright, L.H. and Hopwood, D.A. (1976) Actinorhodin is a chromosomally-determined antibiotic in Streptomyces coelicolor A3(2). Journal of General Microbiology 96, 289–297.
© 1999 The Society for Applied Microbiology, Letters in Applied Microbiology 28, 199–202
