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1 Instituto de Biotecnología de León, INBIOTEC, Parque Científico de León, Av. Real 1, 24006 León, Spain
2 Área de Microbiología, Fac. CC Biológicas y Ambientales, Universidad de León, Campus de Vegazana s/n, 24071 León, Spain
3 Departamento de Química Orgánica, Universidad Autónoma de Madrid, Cantoblanco, Madrid 28049, Spain
Correspondence
Juan F. Martín
degjmm{at}unileon.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Martín et al., 2000). Some of them serve as quorum-sensing signals or as intercellular cross-talk messengers (Horinouchi & Beppu, 1992; Kaiser & Losick, 1993; Nodwell & Losick, 1998; Federle & Bassler, 2003; Klose, 2006). The antibiotic activity of many of these molecules is well known (Berdy, 2005). Many Streptomyces species and some rare actinomycetes have the ability to produce a class of antifungal compounds, the polyene macrolides (Martín, 1977), that belong to the polyketide family (Omura & Tanaka, 1984; Aparicio et al., 2004). In recent years, clusters of genes encoding enzymes for the biosynthesis of a few polyene macrolides have been reported (Aparicio et al., 1999, 2000, 2003; Brautaset et al., 2000; Caffrey et al., 2001).
Pimaricin is a glycosylated polyene macrolide produced by Streptomyces natalensis. Due to its particularly low toxicity, pimaricin is important for antifungal therapy in animals and it is widely used in the food industry to prevent mould contamination of cheese and other non-sterile foods. The synthesis of pimaricin in S. natalensis requires a complex polyketide synthase (PKS) and additional modification enzymes (Aparicio et al., 2000). The pim gene cluster (85 kb) encodes 13 PKS modules within five multifunctional enzymes, and 12 additional proteins that catalyse post-PKS modifications of the polyketide skeleton (tailoring enzymes), or are involved in export and regulation of gene expression (Mendes et al., 2001, 2005; Antón et al., 2004).
Different types of autoregulatory effectors are known to occur in micro-organisms (Yamada & Nihira, 1999; Klose, 2006). These effectors are synthesized and secreted by the producer strains and internalized by other cells in the population (Robson et al., 1997). The presence of butyrolactones in different species of Streptomyces and their role in triggering secondary metabolite biosynthesis is well documented (Horinouchi & Beppu, 1992). Other autoinducers belong to at least six different structural groups (Yamada & Nihira, 1999; Recio et al., 2004). The biosynthesis of pimaricin is induced by PI-factor, a novel autoinducer produced by the parental wild-type strain S. natalensis ATCC 27448. The structure of PI-factor was determined as 2,3-diamino-2,3-bis-(hydroxymethyl)-1,4-butanediol and is clearly different from that of butyrolactones. The S. natalensis npi287 mutant is unable to synthesize PI-factor and this compound restores pimaricin production in the npi287 mutant when added externally (Recio et al., 2004). The positive response to exogenous PI-factor indicates that this mutant is still able to internalize the PI autoinducer. Very little is known about the secretion and internalization (uptake) of autoinducers. The AI-2 of Salmonella enterica is internalized by the Lsr ACDB complex, an ABC-type transporter, and requires a functional glpD (glycerol-3-phosphate dehydrogenase) gene (Taga et al., 2001; Xavier & Bassler, 2005).
A receptor protein for butyrolactones has been reported recently in S. natalensis (Lee et al., 2005), but it is unknown whether this receptor is specific for classical butyrolactones or whether it may also interact with PI-factor.
During purification of the PI-factor (Recio et al., 2004), we identified a second fraction able to induce production of pimaricin in the PI-factor defective npi287 mutant. This fraction did not contain a butyrolactone, but, as reported in this article, NMR studies showed that it consisted of a mixture of glycerol and lactic acid. It was, therefore, important to study the effect of these compounds and their analogues on the biosynthesis of pimaricin.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) was used to determine the pimaricin-inducing activity of different diols. Candida utilis (syn. Pichia jardinii) CECT 1061 was used as test strain in the bioassay of the antifungal activity of the diverse polyenes.
Culture media and quantification of polyene production in liquid cultures.
The production of different polyenes was studied in NBG [nutrient broth (Oxoid) 13 g l–1, glucose 5 g l–1] medium in liquid cultures. The production of the polyenes was routinely quantified by spectophotometry. A 0.5 ml aliquot of the culture was extracted with 5 ml methanol and further diluted with methanol (if necessary). The UV–visible absorption spectrum was first determined to establish the wavelength of the different absorption peaks of each polyene. The absorption was determined at the wavelength corresponding to the most intense peak for each polyene (319 nm for pimaricin). The absorbance was converted to the respective polyene concentrations using the following molar absorption values in a 1 cm curette: candicidin, 
=1150 l mol–1; nystatin, 
=790 l mol–1; filipin, 
=1360 l mol–1; fungichromin, 
=1231 l mol–1). Since no molar absorption values were known for the other polyenes a unit was defined as the amount of polyene giving an absorption of 0.1 at the peak wavelength of the respective polyene.
Pimaricin was quantified by comparison with a sample of pure pimaricin (Sigma). The different Streptomyces species were grown in NBG medium, except for S. noursei, which was grown in SAO-23 medium (Brautaset et al., 2000). The concentration of extracellular PI-factor was determined in liquid cultures in NBG, YED (yeast extract/dextrose [glucose]) and YEME (yeast extract/malt extract) (Recio et al., 2004).
In addition to the spectrophotometric determination, pimaricin and filipin were also quantified by reverse-phase HPLC using a Waters 600 unit coupled to a PDA 996 detector equipped with a µ-Bondapack C18 column (3.9x300 mm, particle size, 5 µm). Pimaricin eluted at 14.6 min and filipin at 17.8 min using a mobile phase mixture consisting of a linear gradient of methanol/water (from 50 : 50, v/v, at time 0 to 90 : 10, v/v, at 30 min).
Purification of compounds from the second fraction of pimaricin-inducing activity.
A second fraction different from PI-factor that had inducing activity on pimaricin production was isolated, as described previously (Recio et al., 2004). Purification of this fraction was carried out by reverse-phase HPLC using a Waters system equipped with a Polarity C18 column (3.9x150 mm, particle size 5 mm) (Waters). The active compound(s) eluted at 1.9 min using a mobile-phase mixture consisting of a linear gradient of acetonitrile/water (1 : 99, v/v, at time 0 to 70 : 30, v/v, at 15 min).
PI isolation and detection.
The PI-inducing factor was isolated from S. natalensis ATCC 27448 cultures grown in YED medium for 24 h and purified as described previously (Recio et al., 2004). The detection of the PI-factor inducing activity was routinely performed on SPG solid medium cultures of the PI-defective mutant npi287, as described before (Recio et al., 2004). Solid SPG cultures of S. natalensis npi287 produced pimaricin only when supplemented with PI-factor, in a dose-dependent response.
Extraction of intracellular PI-factor.
S. natalensis npi287 was grown in NBG medium with or without supplementation with 100 mM glycerol and the mycelium was collected by centrifugation at 5000 g. The collected mycelium was resuspended in 10 ml 50 mM Tris/HCl buffer pH 7.0 containing 2 % Renex (Imperial Chemical Industries) and disrupted in a French press (Aminco). The disruption of the cells was confirmed by microscopic observation. The debris was removed by centrifugation at 14 000 g and the supernatant was concentrated 10-fold; the presence of PI-factor was tested by bioassay using the inducing test, as described above.
Determination of the pimaricin-inducing activity of different diols.
The biological activity of glycerol and other polyhydroxylated compounds was determined by their ability to induce pimaricin production by the PI-factor-defective mutant S. natalensis npi287, in solid SPG medium and in liquid NBG medium. This activity was further tested in several Streptomyces strains, including wild-type S. natalensis, in liquid and solid medium. In liquid medium, glycerol (or other polyhydroxylated compounds) was added at different concentrations and the production of polyene was compared with that of a control culture without additions. In solid medium tests the strains were grown as lawn cultures on TBO medium for 8 days; then agar plugs were taken from each plate and placed on NAG medium (NBG solidified with 20 g agar l–1) in the vicinity of 0.7 cm diameter wells. The production of polyene was bioassayed after 48 h in the presence or absence of glycerol added to the neighbouring well, and the diameters of inhibition zones, indicative of the production of the antifungals, were measured.
NMR spectroscopy.
The chemical structures of the compounds in the positive inducing samples were determined by NMR spectroscopy, using a combination of one-dimensional NMR (1H-NMR and 13C-NMR) and two-dimensional shift-correlated NMR techniques (HMQC-HSQC and HMBC), for the complete 1H and 13C signal assignments, and mass spectrometry.
NMR spectra were recorded in CD3OD at room temperature using a Bruker WM500 spectrometer [500 MHz (1H-NMR) and 125 MHz (13C-NMR)]. Chemical shifts are given on the 
-scale and were referenced to the solvent and to dioxane as internal signals. The pulse programmes of the two-dimensional experiments were taken from the Bruker software library, and the parameters were as follows: 500/125 MHz gradient-selected HMQC spectra: relaxation delay D1=1.5 s; 500/125 MHz gradient-selected HMBC spectra: relaxation delay D1=1.5 s, evolution delay D2=3.33 ms; delay for evolution of long-range coupling D6=60 ms (Wilker et al., 1993).
Mass spectra were recorded on an LC-TOF (Micromass). Glycerol mass was detected in the positive mode, using formic acid to facilitate ionization. Lactic acid measurement was carried out in the negative mode, using bicarbonate for ionization.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) through a Sephadex G10 column (Fig. 1). The compound(s) purified from the second active fraction (A2) had low molecular mass, lacked amino groups, since it was not derivatized with FMOC (fluorenylmethyl chloroformate), and eluted in reverse-phase HPLC with a retention time of 1.9 min (Fig. 1B).
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) revealed that this fraction consisted of a mixture of glycerol and lactic acid. Exogenously added pure glycerol and lactic acid coeluted with the active fraction in HPLC.
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Glycerol elicits pimaricin biosynthesis in liquid cultures of S. natalensis npi 287 and stimulates production in the wild-type
The results observed in solid medium were compared with those in liquid medium. As shown in Fig. 2, glycerol alone or in combination with lactic acid triggered pimaricin production in the npi287 mutant. Lactic acid (D- or L-) when added alone did not produce any significant stimulating effect on pimaricin production. Addition of lactic acid up to 5 mM had no significant effect on dry weight or pimaricin production; lactic acid at 50 mM was inhibitory.
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). At these concentrations the glycerol had a stimulatory effect on biomass. The best supplementation for pimaricin induction was a mixture of 100 mM glycerol and 100 µM lactic acid. The levels of pimaricin produced by the npi287 mutant when supplemented with glycerol (23 µg pimaricin per mg dry weight or 400 µg per ml) were of the same magnitude as those obtained with the specific inducer PI at 1 µM concentration. However, whereas PI-facto
was effective in the 100 nM to 1 µM range, glycerol was only active at concentrations above 50 mM.
Similar studies were performed with the wild-type strain in NBG medium supplemented with glycerol or with the glycerol/lactic acid mixture. Glycerol alone produced a significant stimulatory effect on pimaricin production (up to 125 µg per mg dry weight) similar to that exerted by the glycerol/lactic acid mixture. The stimulatory effect was higher when glycerol (with or without lactic acid) was added at inoculation time or at 14 h than when added at 24 h after inoculation. The effect of the addition at 24 h was small but still detectable (Fig. 3). The higher effect observed when glycerol is added early to the cultures, as compared to the lower stimulation when added at 24 h, is consistent with an indirect role of glycerol, rather than a nutrient (precursor) effect. Furthermore, when glucose in the NBG medium was replaced by glycerol as carbon source, the pimaricin yield was lower (about 45–50 %), indicating that the glycerol stimulation is not merely a nutrient effect.
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Glycerol lactate esters do not induce pimaricin production
The possible effect of lactate esters of glycerol was tested using glycerol monolactate, glycerol dilactate (not shown) and glycerol trilactate on solid or liquid cultures of the npi287 mutant. None of them induced pimaricin biosynthesis (Fig. 4 and not shown). This result supports the lack of inducing activity observed with glycerol acetate esters described above.
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-hydroxypropionic acid, pyruvic acid, malic acid, tartaric acid, 
-hydroxybutyric acid, succinic acid, -hydroxyvaleric acid, glutaric acid, -hydroxyglutaric acid and the cyclic compounds propiolactone and 
-butyrolactone were tested for their inducing effect.
Of these compounds, only ethylene glycol and 1,2- or 1,3-propanediol, alone or in combination with lactic acid, showed an inducing effect on pimaricin production (Fig. 4). The positive effect was observed both on solid medium (ethylene glycol or glycerol and lactic acid) (Fig. 4A) and in liquid cultures (ethylene glycol, glycerol or propanediol with or without lactic acid) (Fig. 4B) using cultures of either the npi287 mutant or the parental S. natalensis wild-type strain. At the same concentration (100–200 mM), ethylene glycol or propanediol showed a smaller induction effect than glycerol.
Glycerol increases the production of six different polyene macrolides by their producer organisms
To study if the glycerol stimulation of polyene macrolide production was a general phenomenon for other Streptomyces species, we tested the effect of these compounds on the production of nystatin by S. noursei, rimocidin by S. rimosus, candicidin by S. griseus IMRU 3570, filipin by S. filipinensis, tetrafungin by S. albulus, eurocidin by S. eurocidicus and fungichromin by S. cinnamoneus. As an additional control, the effect of glycerol on the production of streptomycin by S. griseus IFO 13350 was also tested. These studies were done on solid medium and in liquid cultures in NBG medium. In all cases the results in liquid cultures agreed with those observed on solid medium.
The production of six out of seven different polyene macrolides tested was stimulated by glycerol (100 mM); the degree of stimulation depended on the producer strain (Fig. 5). A high increase in polyene macrolide yields was observed in the cultures producing nystatin (90 % increase at 96 h), candicidin (270 % increase at 96 h), tetrafungin (60 % at 72 h), filipin (90 % at 72 h) and fungichromin (200 % at 96 h). Moderate increases were obtained in the production of rimocidin (30 % at 72 h) and no increment was observed in eurocidin production by S. eurocidicus at the glycerol concentrations tested. The wild-type isolates of S. albulus and S. rimosus (producers of tetrafungin and rimocidin, respectively) yielded very low levels of the respective polyenes under the conditions used (not optimized) but the stimulatory effect of glycerol was observed in both cases.
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The stimulatory effect of glycerol does not involve intracellular or extracellular PI-factor accumulation in strain npi 287
As described previously, the PI-factor induces pimaricin production both in the npi287 mutant and in the wild-type strain (Recio et al., 2004). To elucidate if the glycerol effect was mediated through the PI signal-transduction cascade, experiments were performed in which the PI-induction system was fully activated (saturated) with high levels of PI (2 µM). Parallel cultures were supplemented with glycerol alone (100 mM) or with glycerol (100 mM) and PI-factor (2 µM). The results (Fig. 6) showed that glycerol produced a higher increase of pimaricin than PI alone and the induction pattern was different. The PI-factor exerted an earlier stimulatory effect (48 h) than glycerol (maximum effect at 72 h) when added separately. Glycerol was still able to increase slightly the production of pimaricin in the PI-saturated system when both PI and glycerol were added simultaneously (Fig. 6).
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The extracellular levels of PI-factor were also determined in cultures of the wild-type strain ATCC 27448 in three different media (NBG, YED and YEME) with or without 100 and 200 mM glycerol. The levels of PI-factor were different in the three media, being about fourfold higher in YED medium than in YEME or NBG, but in all three cases there were only minor differences in cultures with or without glycerol, indicating that glycerol is not a direct precursor of PI-factor.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Fray, 2002; Federle & Bassler, 2003). Secondary metabolism and cell differentiation in actinomycetes are controlled by diffusible low-molecular-mass compounds (Robson et al., 1997; Horinouchi & Beppu, 1992). These belong to at least six different chemical classes (Recio et al., 2004) and there are probably other unknown types of cross-talk signals.
The production of pimaricin by S. natalensis is probably induced by PI-factor through a quorum-sensing-type mechanism. The PI-factor-defective mutant S. natalensis npi287 regained pimaricin biosynthesis ability when supplemented with small amounts of PI-factor (Recio et al., 2004). A gene encoding a butyrolactone-receptor protein has been reported recently in S. natalensis (Lee et al., 2005). However, we have never found a true butyrolactone in S. natalensis. It would be desirable to test if the receptor protein is specific for butyrolactones or whether it may interact with the PI-factor.
In this work a second pimaricin-inducing fraction purified from culture broths of the parental strain was shown by NMR studies to contain glycerol and lactic acid. Indeed, pure commercial glycerol and lactic acid restored pimaricin to strain npi287 in solid medium. When tested in liquid medium, glycerol alone was sufficient to trigger pimaricin biosynthesis. The different requirement for lactic acid in solid and liquid medium is probably related to the distinct physiological conditions in solid and submerged cultures.
The concentration of glycerol required for optimal effect on pimaricin biosynthesis (50–100 mM) might suggest that the triggering effect could be related to a nutritional effect. This does not seem to be the case, since the npi287 mutant does not produce pimaricin when supplemented with other carbon sources (glycerol esters, glucose, acetate or sucrose) that are excellent carbon sources for growth of S. natalensis and for pimaricin production. Furthermore, glycerol, when used instead of glucose as carbon source, decreased the yield of pimaricin, i.e. the inducing effect of glycerol appears to be additional to the effect of biosynthetic precursors provided by glucose.
The high concentration of glycerol required suggests that it may play an indirect role, rather than having a direct effect on pimaricin biosynthesis, e.g. a role in membrane permeability or signal transduction across the membrane. Glycerol is well known to serve as a signal that elicits aggregation and cell differentiation, resulting in fruiting body formation in Myxococcus xanthus (Dworkin & Gibson, 1964; Killeen & Nelson, 1988). A 
glpT M. xanthus mutant defective in the glycerol permease aggregates even in rich medium. Since swarming and aggregation was delayed by glycerol 3-phosphate (G3P), it seems that cell binding and/or catabolism of glycerol is an important step in glycerol regulation of differentiation (Moraleda-Muñoz et al., 2001). Recently glycerol metabolism has been shown to be also involved in regulation of the uptake of the quorum-sensing autoinducer AI-2 in Escherichia coli, an autoinducer that is structurally related to PI-factor (Xavier & Bassler, 2005). A mutation in the glpD gene encoding G3P-dehydrogenase causes a defect in AI-2 internalization.
The effect of glycerol on pimaricin production in both the PI-factor-defective npi287 mutant and the wild-type S. natalensis was also exerted by ethylene glycol and 1,2- or 1,3-propanediol but not by threitol or higher polyalcohols. Neither acetate nor lactate esters of glycerol exerted any effect. These results indicate that the effect of the active compounds is due to the signalling effect of 2-carbon or 3-carbon hydroxylated compounds. Ethylene glycol is known to interact with the cell membranes and it is not catabolized by the glycerol pathway; therefore, a purely nutritional effect of these compounds seems very unlikely.
Addition of glycerol to cultures of strain npi287 did not restore detectable levels of intracellular or extracellular PI-factor in this strain, thus excluding a direct precursor role of glycerol for PI biosynthesis. In addition, the stimulatory effect of glycerol on pimaricin biosynthesis in the wild-type is not accompanied by increased extracellular PI levels in this strain. In summary, glycerol, ethylene glycol and propanediol exert a bypass of the PI inducer in strain npi287.
Glycerol has a stimulatory effect on the biosynthesis of six different polyene macrolides by the respective wild-type producer strains, but no effect on streptomycin production by S. griseus IFO13350 was observed. Glycerol has been reported to stimulate the production of nystatin (Jonsbu et al., 2002) and it is known to be a good carbon source for other Streptomyces species (Kaiser et al., 1994; Borodina et al., 2005). Our results suggest that the effect of glycerol might be specific for polyketides, particularly for polyene macrolides, although this phenomenon should be studied on other antibiotic-producing Streptomyces species.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Aparicio, J. F., Colina, A., Ceballos, E. & Martín, J. F. (1999). The biosynthetic gene cluster for the 26-membered ring polyene macrolide pimaricin. A new polyketide synthase organization encoded by two subclusters separated by functionalization genes. J Biol Chem 274, 10133–10139.
Aparicio, J. F., Fouces, R., Mendes, M. V., Olivera, N. & Martín, J. F. (2000). A complex multienzyme system encoded by five polyketide synthase genes is involved in the biosynthesis of the 26-membered polyene macrolide pimaricin in Streptomyces natalensis. Chem Biol 7, 895–905.[CrossRef][Medline]
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Received 28 February 2006;
revised 30 May 2006;
accepted 7 July 2006.
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, Unsupplemented control; 
, glycerol added at 0 h; 
, glycerol added at 14 h; 
, glycerol added at 24 h.
), 140 mM 1,3-propanediol (
), 150 mM glycerol monolactate (
