Document 6wRkERzLmQ6K47BRydvpNjMjE

:* I ^ Applied MrcROfiioLOcv, Nov 1975, p. o Copyright ^ 1975 Amoricim Snnotv fm Mi ri*hn|ogy V..l :ut. . I*rinIt'll in C S A Prodiginine (Prodigiosin-Like) Pigments from Streptoverticillium. rubrircticuli, an Organism That Causes Pink Staining of Polyvinyl Chloride NANCY N. GKItHKR and DONALD P STAHI.Y' Institute of Microbiology. Hutgers Unieersity. The Stole University New Jersey. New Urnnsteirlt. New Jersey (Will. and Deportment o/ Microbiology, College of Medicine. University <1/ lomi. lotm1 City, town 52242* Received for publication 1 April 1975 Red pigments were extracted from Streptoverticilliiim rnhrireticitli strain 10019, an organism frequently incriminated in pink staining of polyvinyl chloride These pigments were identified sis undocvlprodiginine and hutvlcycloheptylprodiginine. Pink discoloration of polyvinyl chloride due to microbial growth has frequently plagued the plastics industry K>. IHi. Such discoloration oc curs on vinyl-coveted furniture, vinyl wall cov ering. and vinyl doors. In an attempt to induce artificially the pink discoloration of vinyl plastic. Yeager il7) bur ied vinyl in soil and observed production of pink spots on the vinyl. Of the many organisms iso lated. only one. Streptoverticilliiim rnhnreticuli (formerly Strcptomyces ri/brircticiili). pro duced the pink discoloration of vinyl. Because of the lack of information in the literature on the nature of the pink pigments produced by S. rubrireticuh. a study was initi ated to characterize these substances and to determine cultural conditions suitable for their formation. The evidence presented indicates tat the pink pigment is actually a mixture of two prodiginine pigments, undocvlprodiginine and butvlcvcloheptylprodiginine. MATERIALS AND METHODS Organism. S. rubrireticuh strain 100-19. an orga nism kindly provided by G Tirpak. Tenneco Chemi cals. Inc., was used in this study. It was originally isolated from pink-stained vinyl. Solid media. Five different media solidified with 15 g of agar per liter (USP no. 1. Meer Corp.. New York. N Y ' were used. These included <i 1 Bennett's medium '5': <ii) yeast-Czapek medium (5a); (iii) Pablum medium containing 60 g of Pablum mixed cereal per liter (Canadian Pablum, Mead Johnson Co., Toronto, Ont.) in tap water, no pH adjustments; (iv) malt medium containing 10 g of malt extract per liter 'Fisher Chemical Co., Pittsburg, Pa.) in tap water; and fv) nutrient glycerol medium contain ing 60 mi of glycerol, 5 g of Wilson's peptone 851, 5 g of NaCl, and 3 g of meat extract iDifco Labora tories, Detroit, Mich. I per liter of tap water. Liquid media. The five liquid media used were (i) Bennett's, 1 ii yeast-Czapek. ` iii' modified yeast-Cza pek in which thi' phosphate salt was replaced by calcium carbonate 1 Ml gliteri. iiv> yeast-dextrose 'Si. and v 1 soybean media tat. The soybean meal in the latter medium differs from that used previously tot in that it contains 49") solvent-extracted soybean meal from Central Soya (Fort Wavne. Ind.i. It con tains not less than 49'; crude protein; the ingredi ents are solvent-extracted dehullod soybean meal with added kaolin. Cultural conditions. Slant cultures on solid me dia were maintained at 2X ('. Growth in liquid me dia was in 50-ml batches in 25(l-ml Erlenmeyer flasks shaken on a New Brunswick model V rotary shaker at 180 to 220 rpm. When the organism was grown for extraction and purification of the pig ments. a slant culture was inoculated into soybean medium or Bennett's broth, shaken 3 days at 28 C. and then transferred into 10 flasks of soybean me dium. After 3 days, the cells were harvested hy filtration. Extraction and purification of the prodiginine pigments. The following steps were taken 'ii The cells were shaken overnight with acetone. Spectrophotometric assay at 530 nm (5). the wavelength at which maximum absorbance occurred, indicated about 4 mg of prodiginine pigments in the acetone extract, iii) This acetone extract was concentrated and poured into water, and the mixture was exracted twice with chloroform. Before the second extraction the aqueous lavtr w as made strongly acid and the shaken mixture was allowed to stand over night before the lower, pigment-containing chloro form layer was withdrawn. This procedure was known from previous work with other microorga nisms to result in higher yields of chloroform-extractable pigments. <iii) The pigment mixture was further purified and resolved into two fractions hy column chromatography on alumina and silica gel *5i. Techniques for characterization of the pig ments. Procedures for spectrophotometric analysis thin-layer chromatography 1TLC1, and mass spec troscopy were described previously i5t. Mass spectra were obtained at 2 kV with an inlet temperature of 240 C 807 21490001 KKSUI.TS I'lgmentution and cultural conditions. .S' rubnntu uli grown on five solid media, previ ously found useful for eliciting prodiginine pigments. showed strong red nondilTusing pigmen tation on nutrient-glycerol agar only. Weak red pigmentation was observed in the growth on Bennett's agar, but it was nearly obscured by the intense dark diffusing pigment that was also produced. Only the dark diffusing pigment was observed when this organism was grown on mall. Pablum, or yeast-Czapek agar. During the past 2 years one of us iN.N.G.) has studied 12 Strcpiomyces or Strvptoverticillium strains that produce prodiginine pigments.S. rubrireti- cuii strain 100-19 is the first such strain that failed to show red pigment on yeast-Czapek agar. In shaken broth cultures, strain 100-19 grew well in yeast dextrose and soybean media, less well in Bennett's broth, and poorly in the two types of yeast-Czapek broth. Red pigmentation was most noticeable in cells grown in soybean medium. Some pigmentation was apparent in cells from Bennett's and yeast-dextrose media, but it was absent from cells in the two yeastCzapek media. More pink pigment was produced at 28 to 31 C than at 22 C. At 38 C pigment production was slight to none. Maximum pigment accumu lation occurred during the early stationary phase of growth. Characterization of the prodiginine pig ments. The pigments extracted from cells were purified and resolved into two fractions lA and B) as described in Materials and Methods. The purified pigments were compared with authen tic samples of the nine known, fully character ized, naturally occurring prodiginines. Figment A exhibited visible absorption max ima at 528 nm in acid-CHCl3 and at 525 nm in acid-ethanol. These maxima as well as the ob served TLC behavior (color, fluorescence, and Hf) were identical with those of authentic sam ples of undecylprodiginine and nonylprodiginine (4). Mass spectroscopy indicated that pig ment A was undecylprodiginine (Fig. IA; Fig. 2). The molecular ion was clearly 393 mass units, and the spectrum closely resembled that of authentic undecylprodiginine (H). Nonylprodi- ginine has a molecular ion at 30f> mass units. Figment B was identical wil.li authentic bu- tvlcycloheptylprodiginine (Fig IB) in visible absorption maxima 1542 nm m ai id-CHCl3 and 536 nm in acid-ethanol) and TLC behavior (5a). On the TLC plate, spots of piement B were slightly behind those of authentic metacyclo- prodigiosin (15) and pinker (lee; orange). Spots of pigment B were the same ('"'or as those of authentic prodigiosin (10, but slightly ahead. The structure was verified by mass spectroscopy (Fig. 2). The mass spectrum was similar to that obtained previously for butvl- cycloheptylprodiginine (5a) and clearly different from that of metacycloprodiginsin, which does have the same molecular formula but a differ ent structure. DISCUSSION Two prodiginine pigments have been identi fied as products of S. rubrireticuli, undecyl prodiginine and butylcycloheptylprodiginine. These pigments were recently found by Gerber (5a) to also be produced by Streptomyces sp. Y-42, a strain isolated from leaf and grass com post. Several other members of the order Actinomycetales have been found to be capable of producing prodiginine pigments (l-5a, 7-9, 11-15). It is probable that the "pink staining" of poly vinyl chloride caused by S. rubrireticuli is due to the prodiginine pigments produced by the organism. Yeager U8) reported that "the pres ence of the organism in the vinyl system is not necessary to produce the stain. Instead, the discoloration is caused by a migration of the stain from a nearby substrate on which the organism is growing." The prodiginine pig ments ofS. rubrireticuli are soluble in diisodecyl adipate, one of the plasticizers used commer cially by some vinyl manufacturers. Exposure of the plasticizer to red mycelia resulted in ex traction of the pigment (Stahly, unpublished data). Thus, the plasticizer is the probable vehi- } I ____ ,004, n__nf xi HH AB Fic. 1. Prodiginine pigments of S- rubrireticuh. (A) Undecylprodiginine; (B) butylcycloheptylprodiginine. 21490002 BFG14404 T Vol. 30. 1976 . IO0 *> PKODK'ilNINK PICMKNTS FROM S. RURRlRETtCUU 809 A) Pigment A *v JJL 41 100 200 tuimy rtw , * 300 350 B) Pigment 8 I <1i I I % T 50 100 IT* .- T T_......... TT-- 150 200 MASS UNITS 300 IM m see _i___ 350 400 Fig. 2. Mdgt/^pectra of prodiginine pigments of S. ruhrireticuli. (A) Pigment A. undecylprodiginine; (B) ftpigment B, 1-lftfJeycloheptyiprodiginine. de through wjuch the pigment is transported from S. rubrtiiiticuli throughout the vinyl sys tem. The mass, spectrum of pigment A with its strong molecular ion at 393 mass units is dearly different from that of nonylprodiginine. the moleculaf ibn of which is 28 mass units less. Both show th<jlj252 mass unit peak caused by fi cleavage of tB? aliphatic side chain. Although both metacydlftprodigiosin and butylcycloheptylprodiginipBi.have strong molecular ions at 391 mass unfb), the next largest peaks are 307 and 320 for former and 348 and 334 for the latter ITEBATURE CITED 1 Arcamone. A. Di Marco. M. Ghione. and T. Scotii. 195? Stu 1 pigmento simile alia prodigiosina prodotto da ali ii actinomiceti. G. Microbiol. 4:77--8S 2 Dietsel. E. 1!^ Uber das Vorkommen von prodigiosinahnlichnif rbstoiTen bei Actinomyceten Naturals- senschaften 35:345. 3. Dietzel. E. 1949 Uber prodigiosinahnliche Farbatoffe bei Actinomyceten. Hoppe-Seyler's 2. Physiol. Chem. 284:262-271. 4. Gerber. N. N 1969 Prodigiosin-like pigments from Acli* nomadura Nocardia\ pelletieri and Actinomadura madurae. Appl. Microbiol. 18:1-3. 5. Gerber, N N. 1973 Minor prodiginine pigments from Actinomadura madurae and Actinomadura peilefieri. J. Heterocycl. Chem. 10:925-929. 5a. Gerber, N. N. 1975 A new prodiginine (prodigiosin- like) pigment from Streptomycea. Antimalerial activ ity of several prodiginines. J. Antibiot. 28:194-199. 6 Girard. T. A-, and C F. Koda. 1959 Pink discoloration of vinyls. Mod. Plast. 36:148. 7. Harashima, K.. N. Tsuchida, and J. Nagatau. 1966. Prodigiosin-25 C. A new prodigiosin-like pigment. Agric. Biol. Chem. 30:309-310. 8. Harashima. K.. N. Tsuchida, T. Tanaka, and J. Na gstsu. 1967. Prodigiosin-25 C Isolation and chemical structure. Agric. Biol. Chem. 31:481-489. 9. Khoklova, I. M.. A. V. Puchnina. and 0 I. Artamova. 1964. Chemical study of the chief component# of vitamycin. Biokhimiya 29:841-845. T ; " - "If:.-.`--A*T- &* <VM*5I ,4f1".';}/x^: ;.' i -i- s-v-^r/ 9 .-. SI, 810 IIKKHKK AND STABLY Appl. Microbiol. 10. Morgan. fc N.. and K. M. Tanner. 1955. Prodigiosin. J. Chem Sue. 1955:3.105. 11. Perry. *1 4 1961 Prodigiosin in an nctmomycete. Na ture i Londoru 191:77-78. 12. Wasserman. H. H . D. J. Friedland. and D. A. Mornson 1968 A novel dipyrrulydpyrromethane prodig iosin analog from Serratfa marcesvens. Tetrahedron Lett. 1968:641-644. 13. Wasserman. H. H.. J. Keggi. F Bohlmann. and W. Ludors. 1960. Struktur eines prodigiosin-ahnlichen Pilzfarbetoffs aus Streptomyves longisporus ruber. An* gew. Chem. 72:779, 14. Wasserman. H. H.. G. C. Rodgers. Jr., and D. D. Keith1966. The structure and synthesis of undecylprodig* iosin. A prodigiosin analogue from Strtptomyces. Chem. Commun. 1966:825. 15. Wasserman, H. H., G. C. Rodgers, and Keith. 1969. Metacycloprodigiosin, a tripyrfl^w pigment from Streptomyces longisporus ruber. J A*n. Chem. Soc. 91:1263-1264. 16. Williams. R. P.. and W. R. Hearn. 1967. P^^igiosin. p. 410-432. 449. In D. Gottlieb and P. D. Shaw ted.), Antibiotics, vol. 2. Biosynthesis. Springer-Verlag. Inc.. New York. 17. Yeager. C. C. 1962. Pink staining in polyvinyl chloride. Plast. World 20:14-15. 18. Yeager, C. C. 1968. Deterioration of vinyTvesin ays- tems-special considerations, p. 222-225 'IH Develop ments in industrial microbiology, vol. (1- Am. Inst. Biol. Sci.. Washington. D.C. | f A C A Ft s i of ! | tha (24) iter beei repx U bee I it e: lept tion the (ND ble I tion f lidhi : preh CO who 10 C elecl i. stric s BFG14406 T