Journal of General Microbiology (1990), 136, 2291-2296. Printed in Great Britain 229 1Pigmented antibiotic production by Streptomyces coelicolor A3(2) : kineticsand the influence of nutrientsGLYN M. HOBBS,CATHERINE FRAZER, DAVID J. GARDNER,~ FLETT* C. FIONAand STEPHEN O L I V E R ~ ~ ~ * G.Manchester Biotechnology Centre and Department o Biochemistry and Applied Molecular Biology, fUniversity of Manchester Institute of Science and Technology, PO Box 88, Manchester M60 IQD, UK(Received 23 March 1990; revised 2 July 1990; accepted 17 July 1990) ~ _ _ _ _ _ _ ~ _______~ The production of the pigments actinorhodin and undecylprodigiosin by Stveptomyces coelicolor A3(2) was examined in a chemically defined medium which permits dispersed growth of the organism. The physiological controls on the production of the two pigments were markedly disparate. Actinorhodin production occurred mainly in the stationary phase of batch cultures grown with glucose and sodium nitrate as the principal carbon and nitrogen sources. In the same batch cultures, undecylprodigiosin accumulated during the exponential growth phase. The production of both pigments was sensitive to the levels of ammonium and phosphate in the medium. Actinorhodin production was exquisitely sensitive to ammonium concentration, and was completely inhibited by as little as 1 mM-ammonium chloride, whereas more than 50 mhl-amnonium chloride was required to prevent undecylprodigiosin production. A similar, but less extreme effect was seen with phosphate: actinorhodin pro- duction was completely inhibited by 24 mM-phosphate, whereas undecylprodigiosin was still formed at this high phosphate concentration. The effects of ammonium inhibition of pigmented antibiotic production were relieved by reducing the concentration of phosphate in the medium, but changing the ammonium concentration had no effect on phosphate inhibition. Thus the regulation of pigment production by these two nutrients is interrelated, with phosphate control being epistatic to that of ammonium. The results implicate a phosphorylated intermediate as a major regulator of secondary metabolite synthesis by S. coelicolor.Introduction culture and to determine the effect of medium nutrient composition on product formation.The genetics of Streptomyces coelicolor A3(2) has been S . coelicolor synthesizes two chemically distinctintensively studied using both classical and molecular pigments which are generally regarded as secondaryapproaches (Hopwood, 1988) but the physiology of this metabolites : actinorhodin, a diffusible red-blue pHorganism remains relatively poorly defined. Hodgson indicator, and undecylprodigiosin, a red cell-wall-(1982) used both genetical and physiological techniques associated compound (Rudd & Hopwood, 1980). Genesto investigate glucose uptake and metabolism by which determine the synthesis of these two pigmentsS . coelicolor. Since then, progress has been slow, have been identified (Rudd & Hopwood, 1979, 1980;probably because of the difficulties in culturing Feitelson et al., 1985; Malpartida & Hopwood, 1986)andS . coelicolor in chemically defined growth media. We cloned (Feitelson & Hopwood, 1983; Feitelson et al.,(Hobbs et al., 1989) have recently devised a medium 1986; Malpartida & Hopwood, 1984, 1986). However,which overcomes many of these difficulties and allows the physiological controls which operate on pigmentthe dispersed growth of S . coelicolor under chemically production in S . coelicolor are unknown. Feitelson et al.defined conditions. The medium employs a negatively (1985) reported that the onset of undecylprodigiosincharged polymer (Junlon PW 110) to obtain dispersion production was delayed to mid- or late-exponential phaseand permits the growth of S . coelicolor cultures which are in liquid cultures of S . coelicolor grown on a complex beefboth physiologically homogeneous and readily repro- extract/peptone medium (AM medium; Okanishi et al.,ducible from experiment to experiment. In this paper, we 1974). It was not clear that exponential growth wasreport the use of this culture system to investigate the actually achieved and the complexity of the mediumkinetics of secondary metabolite production in batch precluded identification of a nutritional limitation which0001-6156 O 1990 SGM
2292 G. Hobbs and othersmight have triggered pigment production. We have 4re-examined pigment production in S. coelicolor under 100defined physiological conditions. We demonstrate that d 0the kinetics of accumulation of undecylprodigiosin and 80actinorhodin are markedly disparate and that the w E i? Wcomposition of the growth medium affects the synthesis W 2 2 60 2 u .- M E:of both products. Y 40% 20 2 .gMethods u d Organism. Streptomyces coelicolor A3(2) strain 1147 (Hopwood, 1959) lo4 lo5 lo6 lo7was used. This is a prototrophic strain which contains plasmids SCPl Inoculum (spores ml- )and SCP2. Inoculumpreparation. The inoculum used throughout originated from Fig. 1. Effect of inoculum size on biomass (0)and actinorhodin ( 0 )a single frozen spore stock culture. The spore stock was spread on a production. Biomass and actinorhodin concentrations were deter-plate of sporulation agar (Hobbs et af.,1989)and incubated for 1&14 d mined spectrophotometrically. Spore counts were made with aat 30 "C to allow sporulation. Spores were then streaked by wire loop to haemocytometer. All measurements were made in triplicate and theproduce confluent growth on several plates of sporulation agar. results presented are the means.Distilled water (5 ml) was added to each plate and the surface gentlyscraped to release the spores. Suspensions were harvested bycentrifugation and washed twice with distilled water. Before use asinocula, the spores were dispersed for 10 min in a sonic bath. Inocula medium (HMM) which, through incorporation of awere adjusted to a final concentration of 2 x lo6 spores ml-l. charged polymer, permits growth of the organism as Bacterial growth and its estimation. Basal medium (HMM) was as dispersed filaments (Hobbs et al., 1989). The seconddescribed by Hobbs et al. (1989); Junlon PW 110 was a gift from major factor which influences the reproducibility ofHoneywill & Stein Ltd (Greenfield House, 69/73 Manor Road,Wallington, Surrey, UK). batch culture experiments with this organism is the Cultures were grown in 250 ml flasks containing 100 ml medium at nature of the inoculum. In all experiments reported here,30 "C on an orbital shaker at 200 r.p.m. Biomass concentrations were we used fresh spore inocula to minimize the lag phase.estimated from optical density measurements at 450 nm (Gilford 250 All inocula were derived from a single spore stock.spectrophotometer) and their equivalence in cell dry weight was The medium used in this study (HMM) was ascalculated as described previously (Hobbs et al., 1989). described previously (Hobbs et al., 1989), using glucose Pigment extraction and quuntijication. At each time point, 10-20 ml at a concentration of 2 g 1-l. The effect of inoculum sizeculture samples were taken and biomass estimated as described. Thesample was then divided into two 5-10 ml aliquots for the estimation of on the yield of both biomass and the blue pigment,the pigments. Actinorhodin was extracted by adding an equal volume actinorhodin, was determined. Biomass concentrationof 1 M-sodium hydroxide to one of the culture aliquots. The sample was was determined 116 h after inoculation, by which timethen centrifuged at 1 lOOg for 5 min and the pigment concentration all cultures had entered the stationary phase, irrespectivedetermined by measuring the absorbance of the supernatant at 633 nm of the original inoculum size. Fig. 1 demonstrates thatand applying the formula of Horinouchi & Beppu (1984). Undecyl-prodigiosin was extracted from the cell pellet harvested by centrifu- biomass yield was proportional to inoculum size at sporegation (1 lOOg for 5 min) from the remaining culture aliquot. The concentrations between 3.7 x lo4 and 8-7 x lo5 sporespigment was extracted from the cell pellet using the method of ml-l. Lack of proportionality to biomass yield withWilliams et al. (1956) but modified by incorporating a sonication step larger inocula was not investigated further, but it mayprior to extraction with alkali. Sonication was conducted in an MSE have resulted from the mutual inhibition of sporesonicator at full power using the medium-sized probe. Three 1 min on,30 s off cycles were used to obtain cell breakage. After removing cell germination by high spore densities, or may reflect thedebris by centrifugation, absorbance measurements were made at absolute biomass/substrate yield for this strain. Con-533 nm and the pigment concentration calculated as described by versely, the failure of low spore concentrations toHorinouchi & Beppu (1984). establish a growing culture may indicate that some factor Glucose utilization. Glucose concentrations in the media were which is essential for germination is leached from theestimated using a glucose oxidase test kit (Sigma). spores and is present in insufficient concentration when small inocula are used. Actinorhodin, on the other hand, was only produced at inoculum sizes of > 1 x lo5 sporesResults ml-l (Fig. 1). Cultures grown from small inocula tendedThe nature o the inoculum f to produce pellets rather than dispersed filaments. This response may have resulted in a significant proportion ofTo obtain reproducible batch growth and pigment biomass entering the stationary phase before otherproduction with S. coelicolor, we have devised a defined conditions essential to antibiotic production, either
Pigment product ion by St reptomyces coelicolor 2293 Table 1. Eflects of nitrogen sources on mean culture doubling time and on actinorhodin and undecylprodigiosin production The medium used was H M M supplemented with amino acids or inorganic nitrogen sources at 50 m M with respect to nitrogen. Pigment production was measured after 116 h. Cultures were grown in duplicate and the results are means. Nitrogen Doubling Actinorhodin Undecylprodigiosin source time (h) [pg (mg cells)-] [pg (mg cells)-] ~~ ~ - Ammonium 3 chloride 7.3 0 5 2 Leucine 7.8 0 18 Proline 9.3 46 39 Glycine 8.5 0 35 h Glutamine 7.6 0 19 I Sodium M v nitrate 13.4 50 60 Ammonium nitrate 7.5 0 25 I I I usually displayed by a secondary metabolite, neither can 24 48 72 this red pigment be considered a product of primary Time (h) metabolism. In continuous culture, the titre of undecyl-Fig. 2. (a) Production of biomass, actinorhodin and undecyl- prodigiosin in the steady state is inversely proportional toprodigiosin in batch cultures grown in 250 ml conical flasks containing dilution rate (our unpublished results). In contrast,100 ml HMM. Measurements were made in duplicate in two separate actinorhodin is produced during the period of growthcultures and the results presented are the means. Biomass (0),actinorhodin (0)and undecylprodigiosin ( 0 )were measured spectro- cessation (Fig. 2u) and may therefore be regarded asphotometrically. (b)Glucose concentrationin the medium (H) and COz more typical of a secondary metabolite, since suchproduction (-). compounds are normally produced under sub-optimal growth conditions (Rose, 1979).within or outside the cells, had been satisfied. Alterna- b . e c t of nitrogen source on actinorhodin productiontively, germinating spores may excrete products into themedium whose concentration is critical to the final HMM contains sodium nitrate as the sole nitrogenpigment yield. In all subsequent experiments, an source (Hobbs ut al., 1989). To investigate whetherinoculum size of 2 x lo6 spores ml-l was employed. actinorhodin production is subject to nitrogen catabolite inhibition we grew S . coelicolor on a variety of nitrogenKinetics ojgrowth and antibiotic production sources with glucose always forming the principal source of carbon. Of the nitrogen sources studied, only sodiumThe relationship between growth and pigment pro- nitrate and proline permitted the production ofduction was studied in batch cultures grown in HMM actinorhodin (Table 1). No pigment was produced withcontaining 2 g glucose 1-I. The kinetics of bio- ammonium chloride or ammonium nitrate. S . coelicolormass accumulation, measured spectrophotometrically, is grew more rapidly with ammonia than with nitrate orshown in Fig. 2(a). The similar growth curve (until proline as the sole nitrogen source (Table 1). These dataglucose was exhausted) obtained by monitoring the might indicate that actinorhodin production is related toconcentration of C 0 2 in the exhaust gases (Fig. 26) growth. However, the growth rate on glycine differed bysupports the contention that optical density measure- only 10% from that on proline (Table 1) and noments reliably monitor growth in Junlon-containing actinorhodin was produced in glycine-grown cultures.media (Hobbs et al., 1989). Production of undecyl- Moreover, data from other systems (e.g. Bossinger et al.,prodigiosin paralleled the accumulation of biomass in 1974) have demonstrated that growth rate is unrelatedthe culture (Fig. 2a), indicating that the production of to the severity of repression which nitrogen exerts onthis pigment is growth-associated. While the kinetics of sensitive metabolic pathways. To examine further theundecylprodigiosin production do not conform to those nature of such inhibitory effects in S . coelicolor, we
2294 G . Hobbs and others 20 40 60 80 Ammonium concn (mM)Fig. 3. Effect of ammonium concentration on the production ofactinorhodin ( 0 ) and undecylprodigiosin (0) in the presence of11 mhl-phosphate. The medium used was HMM supplemented withvarious concentrations of ammonium chloride as the sole nitrogen Jsource. Cultures were grown in 250 ml conical flasks containing 100 mlmedium. Pigments were measured after 72 h of growth. Determi-nations were performed on duplicate cultures and the results are the 20 40 60 80means. Ammonium concn (mM) Fig. 5. Effect of ammonium concentration on the production of actinorhodin (e) and undecylprodigiosin (0) in HMM containing 350 1 mM-phosphate. Cultures were grown in 250 ml conical flasks containing 100 ml medium. Pigments were measured after 72 h. Determinations were performed on duplicate cultures and the results are the means. source. The data (Fig. 3) indicate that actinorhodin production is exquisitely sensitive to inhibition by 160 1, 1 ammonium, whereas undecylprodigiosin is not sensitive below an ammonium concentration of 50 mM. Similar data were obtained with ammonium nitrate. Eflect of phosphate concentration on pigment production Phosphate is a major factor in the synthesis of a wide range of antibiotics (Martin, 1977) and it has been 5 10 15 20 25 suggested that phosphorylated metabolites are important Phosphate concn (mM) control elements. Accordingly, we examined the effect ofFig. 4. Effect of phosphate concentration on the production of phosphate concentration on pigment production byactinorhodin (@) and undecylprodigiosin (0). The medium was HMM S . coelicolor in HMM with nitrate as sole nitrogen sourcesupplemented with a range of phosphate concentrations. Pigmentswere measured after 72 h and determinations performed on duplicate (Fig. 4). Phosphate concentrations greater than 24 m Mcultures; the results are the means. completely inhibited actinorhodin production and the yield of actinorhodin increased with decreasing phos- phate concentration, to an optimum at 0-38 mM. The optimum phosphate concentration for the production ofinvestigated the impact of ammonium on the production the two pigments was 1 mM. Higher concentrations ofkinetics of both actinorhodin and undecylprodigiosin. phosphate also inhibited undecylprodigiosin synthesis but, in contrast to actinorhodin, did not completelyAmmonium inhibition o pigment production f prevent it. Since phosphate inhibited pigment pro- duction even on nitrate, a permissive nitrogen source, weS . coelicolor was grown in media containing a range of examined next the interrelationship between inhibitionconcentrations of ammonium chloride as sole nitrogen of pigment production by phosphate and by ammonium.
Pigment production by Streptomyces coelicolor 2295Low phosphate relieues the ammonium repression of true : inhibition of actinorhodin production by highpigment production levels of phosphate is not relieved by reducing the concentration of ammonium.At the phosphate concentration in HMM (11 m ~ ) , This asymmetry in the relationship betweenammonium inhibited production of actinorhodin at ammonium and phosphate control of actinorhodinconcentrations below 1 mM. In contrast, undecyl- production implicates a phosphorylated intermediate asprodigiosin formation was fully inhibited only at a major regulatory element for secondary productammonium concentrations above 75 mM. Reducing the synthesis. Martin (1977) summarized the possible candi-phosphate concentration in HMM to 1 m had no effect M dates: ATP (Silaeva et al., 1965), adenylate energyon the inhibition of undecylprodigiosin production by charge (Atkinson, 1969), polyphosphates (Harold, 1966)high concentrations of ammonium (compare Figs 3 and or highly phosphorylated nucleotides. Ochi (1987)9, but it had a marked effect on actinorhodin pro- emphasized the importance of the level of GTP and itsduction, in that the concentration of ammonium hyperphosphorylated derivative, ppGpp (Gallant, 1979),required for complete inhibition was increased from in controlling synthesis of A-factor, and thereby strepto-1 m to more than 50 m (Fig. 5). This indicated that M M mycin production (Hara & Beppu, 1982),in Streptomycessecondary metabolism in S . coelicolor is impeded by both griseus. Our own studies throw no light on the nature of aammonium and phosphate and that there is some inter- phosphorylated regulatory molecule in S. coelicolor ;relationship between these two control systems. however, recent work by Bibb & Strauch (1990) appears to rule out ppGpp as a candidate. The facility with which both the genetics and the physiology of S . coelicolor mayDiscussion now be controlled suggests that further investigations should definitively answer the question of how secondaryThe results demonstrate that the two pigmented anti- metabolism is regulated in this organism.biotics synthesized by S . coelicolor are differentlycontrolled. Actinorhodin is a secondary metabolite, the This work was supported by the ‘Antibiotics and Recombinantproduction of which is exquisitely sensitive to inhibition DNA’ initiative, which is sponsored by the SERC Biotechnologyor repression by ammonium and is also prevented by Directorate, the Department of Trade and Industry, Beecham Pharmaceuticals, Celltech, Glaxo Group Research and ICI Pharma-high concentrations of phosphate. In the culture con- ceuticals. We are grateful to John Cullum, Paul Broda and, especially,ditions employed in our experiments, undecylprodigiosin Iain Hunter for many useful discussions.is produced during growth. Although it is evident, bothfrom batch culture experiments in rich media (Feitelsonet al., 1985) and from our own unpublished data fromcontinuous cultures, that this red pigment is not a Referencesgrowth-linked primary product, physiological regulation D. ATKINSON, E. (1969). Regulation of enzyme function. Annual Reviewof its production differs markedly from that of actino- o Microbiology 23, 47-68. frhodin. Its synthesis is only moderately affected by BIBB, & STRAUCH, (1990). The stringent response in Streptomyces M. E.ammonium or phosphate concentration whereas syn- coelicolor A3(2). Journal o Cellular Biochemistry 14A, 86. f BOSSINGER, LAWTHER, P. & COOPER, G. (1974). Nitrogen J., R. T.thesis of actinorhodin is severely inhibited by both these repression of allantoin degradative enzymes in Saccharomycesnutrients. The chemically related pigment prodigiosin, cerevisiae. Journal of Bacteriology 118, 821-829.produced by Serratia marcescens, is also sensitive to levels J. D. FEITELSON,S. & HOPWOOD, A. (1983). Cloning of a Streptomyces gene for an o-methyltransferase involved in antibiotic biosynthesis.of phosphate in the medium (Witney et al. 1977).The site Molecular and General Genetics 190, 394-398.of phosphate inhibition, in this instance, was believed to D. FEITELSON, S., MALPARTIDA, & HOPWOOD, A. (1985). Genetic J. F.be enzymes forming the precursors of prodigiosin. and biochemical characterization of the red gene cluster of Streptomyces coelicolor A3(2). Journal o General Microbiology 131, fWe provide no evidence for such a mechanism in 243 1-244 1.S . coelicolor. However, a direct interaction with bio- FEITELSON, S., SINHA, A. M. & Coco, E. A. (1986). Molecular J.synthetic enzymes might explain the differential sensi- genetics of red biosynthesis in Streptomyces. Journal of Natural Products 49, 988-994.tivity of undecylprodigiosin and actinorhodin to GALLANT, A. (1979). Stringent control in Escherichia coli. Annual J.phosphate. Review o Genetics 13, 393-415. f We suggest that ammonium and phosphate are HAROLD, M. (1966). Inorganic polyphosphates in biology; structure, F. metabolism and function. Bacteriological Reviews 30,772-794.both major controllers of secondary metabolism in HARA,0. & BEPPU,T. (1982). Mutants blocked in streptomycinS . coelicolor A3(2) and that their control systems are production in Streptomyces griseus - the role of a-factor. Journal ofinterrelated in some way. A reduction in the phosphate Antibiotics 35, 349-350. HOBBS,G., FRAZER, M., GARDNER, C. J., CULLUM, A. & C. D. J.concentration is able to relieve ammonium inhibition of OLIVER, G. (1989). Dispersed growth of Streptomyces in liquid S.actinorhodin production (Fig. 5). The converse is not culture. Applied Microbiology and Biotechnology 31,272-277.
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