Separation of pimaricin-inducing fractions from the culture broth of S. natalensis ATCC 27448. (A) Fractionation of the lyophilized solvent extract dissolved in water through a Sephadex G10 column. The two active fractions A1 and A2 are shaded. Size markers: sucrose (solid line), glucose (dotted line) and NaCl (dashed line). The initial peak (fractions 14–15) corresponds to the dextran blue marker. Note that the active fraction A2 eluted between glucose ( M r 180) and NaCl ( M r 58 ? 5). The elution peak of NaCl was followed by precipitation with a silver nitrate solution. (B) HPLC analysis in a C 18 column 

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... 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; Anto ́ n et al. , 2004). Different types of autoregulatory effectors are known to occur in micro-organisms (Yamada & Nihira, 1999; Klose, 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. 2006). During A receptor These purification protein effectors of for the are butyrolactones PI-factor synthesized (Recio and has et secreted been al. , 2004), reported by the we producer identified recently in a strains S. second natalensis and fraction internalized (Lee et able al. , to 2005), by induce other but production it cells is unknown in the of population whether pimaricin this in receptor (Robson the PI-factor is et specific al. defective , for 1997). classical npi The 287 butyrolactones mutant. presence This of butyrolactones or fraction whether did it not may in contain also different interact a butyrolactone, species with PI-factor. of but, Streptomyces as reported and in their this article, role in triggering NMR studies secondary showed metabolite that it consisted biosynthesis of is a well mixture documented of glycerol (Horinouchi and lactic & acid. Beppu, It was, 1992). therefore, Other autoinducers important to study belong the to effect at of least these six compounds different structural and their groups analogues (Yamada on the biosynthesis & Nihira, 1999; of pimaricin. 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 npi 287 mutant is unable to synthesize PI-factor and this compound restores pimaricin production in the npi 287 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). 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 npi 287 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. Unexpectedly, the mass spectra (molecular masses of 93 ? 2 and 89 ? 3) and NMR spectra of the purified active fraction (Table 1) 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. During Unexpectedly, The pimaricin-inducing purification the mass of the activity spectra PI inducer of (molecular the using mixture an masses induction of these of 93 two test ? 2 based and chemicals 89 on ? 3) the was and restoration confirmed NMR spectra using of of pimaricin pure the purified compounds. production active Addition fraction to the inducer-defective (Table 1) of glycerol revealed together strain that with this npi lactic fraction 287, a acid second consisted to SPG active of solid fraction a mixture medium was of isolated glycerol cultures after and of fractionation lactic the non-producer acid. Exogenously of the lyophilized npi 287 added strain solvent pure triggered glycerol extract of and pimaricin the lactic culture acid production. broth coeluted (Recio Such with et al. the an , 2004) active effect through fraction was not a in Sephadex observed HPLC. G10 when column each compound (Fig. 1). The was compound(s) tested separately purified on from solid the second medium active cultures, fraction but (A2) glycerol had alone low molecular was sufficient mass, to lacked elicit amino pimaricin groups, production since it in was liquid not cultures derivatized (see with below). FMOC A (fluorenylmethyl chloroformate), and eluted in reverse- phase HPLC with a retention time of 1 ? 9 min (Fig. 1B). The pimaricin-inducing activity of the mixture of these two chemicals was confirmed using pure compounds. Addition of glycerol together with lactic acid to SPG solid medium cultures of the non-producer npi 287 strain triggered pimaricin production. Such an effect was not observed when each compound was tested separately on solid medium cultures, but glycerol alone was sufficient to elicit pimaricin production in liquid cultures (see below). A stimulation of the production of pimaricin in the wild-type S. natalensis by the mixture of both compounds was also observed in solid medium. Triacetylated glycerol, in contrast to pure glycerol, did not exert any inducing effect, although it was efficiently used as carbon source (when supplied instead of glucose). These results suggested that free alcohol groups are required to produce the inducing effect. Glycerol alone triggered pimaricin biosynthesis in liquid cultures of npi 287 in NBG medium at concentrations in the range 70–120 mM. At concentrations higher than 100 mM the stimulatory effect was smaller (Fig. 2). 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 m M lactic acid. The levels of pimaricin produced by the npi 287 mutant when supplemented with glycerol (23 m g pimaricin per mg dry weight or 400 m g per ml) were of the same magnitude as those obtained with the specific inducer PI at 1 m M concentration. However, whereas PI-facto ` was effective in the 100 nM to 1 m M 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 m g 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. The The Similar Glycerol results lack studies alone of observed requirement were triggered performed in solid pimaricin of medium lactic with biosynthesis the acid were wild-type for compared the in strain glycerol liquid with in those stimulatory NBG cultures in medium of liquid npi effect 287 medium. supplemented in observed NBG As medium in shown liquid with in at glycerol cultures concentrations Fig. 2, glycerol suggests or with in alone that the the or the glycerol/lactic range in different 70–120 combination physiological mM. acid At mixture. with concentrations conditions lactic Glycerol acid in higher triggered submerged alone than produced pimaricin 100 cultures mM a production makes significant the stimulatory lactic stimulatory in acid the unnecessary. effect npi 287 effect was mutant. on smaller pimaricin Lactic (Fig. 2). acid production ( D At - or these (up L -) to when concentrations 125 m added g per mg alone the dry glycerol weight) did not had similar produce a stimulatory to that any exerted significant effect by the on glycerol/lactic stimulating biomass. The effect best acid supplementation on mixture. pimaricin The production. for stimulatory pimaricin Addition effect induction was of higher lactic was a mixture acid when up glycerol of to 100 5 mM (with mM had or glycerol without no significant and lactic 100 acid) m M effect lactic was on added acid. dry at The weight inoculation levels or pimaricin of time pimaricin or production; at 14 produced h than lactic when by acid the added npi at 287 at 50 24 mM mutant h after was inoculation. when inhibitory. supplemented The effect with of the glycerol addition (23 at m g 24 pimaricin h was small per but mg still dry weight detectable or 400 (Fig. 3). m g per ml) The were higher of the effect same observed magnitude when as glycerol those obtained is added with early the to the specific cultures, inducer as compared PI at to 1 m the M lower concentration. stimulation ...