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Prodigiosin synthesis in Serratia marcescens: isolation of a pyrrole-containing precursor
578
SHORT COMMUNICATIONS, PRELIMINARY NOTES
VOL.
19 (1956)
1 E. E. SNELL, N. S. RADIN AND M. IKA\VA, J. Biol. Chem., 217 (1955) 803. 2 H. A. KREBS, in J. B. SUMNER AND 1~. MYRBACK, The Enzymes, Academic Press, New" York, Voh I I (1951), p. 505 • a M. S. DUNN, M. N. CAMIEN, S. SHANKMAN AND H. BLOCK, J. Biol. Chem., I68 (I947) 43. 4 p. BOULANGER AND R. OSTEUX, Biochim. Biophys..4cta, 5 (195 °) 416C. H. W. HIRS, S. MOORE AND W. H. STEIN, J . . 4 m . Chem. Soc., 76 (z954) 6o63. H. E. SAUBERLICrI AND C. A. BAUMANN, dr. Biol. Chem., 177 (19491 545. 7 B. F. STEELE, H. E. SAUBERLICH, M. S. REYNOLDS AND C. A. BAUMANN, J. Biol. Chem., 177 (19491 533. 8 l,. A. ELSON AND W. T. J. MORGAN, Biochem. J., 27 (19331 1824. 9 M. R. J. SALTON, Biochim. Biophys. ~4cta, 8 (19.52) 5IO. 10 C. S. CUMMINS AND H. HARRIS, .[. Gen. Microbiol., 13 (t9551 iii. 11 j . T. PARK, J. Biol. Chem., 194 (19521 897. 12 V. BRUCKNER AND G. IVANOVICS,Z. physiol. Chem., 247 (19351 281 ; Z. Immuuitats/orsch., 90 (19371 304; M. BOVARNICK, J . Biol. Chem., 145 (19421 415 . 13 A. GAUHE, P. GYORGY, J. R. E. HOOVER, R. KUHN, C. S. ROSE, R. W. RUELIUS AND F. ZILLIKAN .~rch. Biochem. Biophys., 48 (19541 214. Received December 23rd, 1955
Prodigiosin synthesis in S e r r a t i a m a r c e s c e n s : isolation of a pyrrole-containing precursor* .q'erratia marcescens produces a distinctive red pigment, prodigiosin, which has been characterized 1 as a t r i p y r r y l m e t h e n e . Evidence has recently been obtained by RIZKI 2 and b y WILLIAMS AND GREEN a t h a t some stable prodigiosin-deficient m u t a n t s of this species accumulate substances capable of causing the appearance Of p i g m e n t in certain other m u t a n t s impaired in prodigiosin synthesis. A pair of such m u t a n t s , not previously reported to exhibit cross-feeding, has been examined in some detail, since one of the two m u t a n t s (9-3-3) was found to excrete a relatively stable substance t h a t permitted p i g m e n t formation in the other (W-l). The excreted substance has now" been isolated in pure crystalline form; the progress of the isolation was followed by bioassay with strain \Y-I. This assay detects a b o u t o.1 t*g of the pure materiah For the isolation of the excreted substance, strain 9-3-3 was grown at 3 ° for four days in aerated peptone-glycerol m e d i u m 4. The culture was centrifuged, and the s u p e r n a t a n t liquid was extracted with chloroform. The chloroform layer was washed successively with i N hydrochloric acid, 1 N sodium hydroxide, and distilled water, and was then taken to dryness in vacuo. The residue was taken up in hexane (in which the active substance is sparingly soluble) and transferred to an alumina (Fisher Scientific Co., for c h r o m a t o g r a p h i c analysis) colunln. The latter was washed successively with benzene, and benzene-ether, and the active substance was then eluted with ether and ether-chloroform. After recrystallization from ethanol, the substance was obtained as colorless needles t h a t do not melt at 250', b u t decompose on f u r t h e r heating. The yield was a b o u t 1 mg per liter of culture. E l e m e n t a r y analysis agrees with the formula C10H1002N,~ (calculated: C, 63.2; H, 5.3; O, i6.8; N, 14.7; CHaO, I6.3; found: C, 63.0; H, 5.3; O, 17.o, N, 14.7; CHaO, 16.6)**. The isolated substance has neither pronounced basic nor p r o n o u n c e d acidic properties, i t is sparingly soluble in such solvents as ethanol, chloroform, ethyl ether, benzene, or t e t r a h y d r o f u r a n ; it is very sparingly soluble in water. The substance absorbs strongly in the ultra-violet; in ethanol solution it gives absorption m a x i m a at 363 and 254 mt, with molar extinction coefficients of a b o u t 3.5" IO4 and i. 3 • IO4, respectively. I t rapidly forms a red color at 25 with p - d i m e t h y l a m i n o b e n z a l d e h y d e in ethanolic hydrochloric acid; therefore, it p r e s u m a b l y contains a pyrrole ring with a free alpha-position. The excretion of the isolated material by one m u t a n t strain and its utilization by a n o t h e r indicate t h a t this c o m p o u n d is an intermediate in the synthesis of prodigiosin. This indication was s u p p o r t e d by tracer experiments. Strain 9-3-3 was cultivated in the usual m e d i u m supplemented with glycine-2-14C (c/. HUBBARD AND RIMINGTONS). Crystalline material was isolated as • This investigation was s u p p o r t e d in p a r t by the Atomic E n e r g y Commission, Contract No. AT-(3o-i)-ioi7 . • * The value for oxygen was obtained by direct analysis. Molecular weight d e t e r m i n a t i o n s are complicated by the low solubility of the substance in c o m m o n solvents; the formula given agrees with one m e t h o x y l group per formula weight. This formula weight was used in the c o m p u t a t i o n of the molar extinction coefficients and the specific activity of the substance. Microanalyses were performed by the Schwarzkopf Microanalytical Laboratory, by the Microchemical L a b o r a t o r y of New York University, and by Mr. STANLEY MILLS.
VOL.
19 (1956)
SHORT COMMUNICATIONS, PRELIMINARY NOTES
579
described above and found to be radioactive. I t was supplied to strain W - I , and the red p i g m e n t c o n s e q u e n t l y formed b y the organisms was extracted b y a modification of the m e t h o d of WREDE AND HETTCHE6 and was t h e n c h r o m a t o g r a p h e d on paper. The p a p e r was p r e t r e a t e d with propylene glycol, and the p i g m e n t was applied in toluene solution; chloroform was used as the mobile phase (C[. ZAFFARONI AND BURTON2). The pigment, which had the same RF as authentic prodigiosin, was eluted w i t h 5 o~o aqueous m e t h a n o l and r e c h r o m a t o g r a p h e d on p a p e r with hexane-glacial acetic acid (9:1). U p o n elution, the red p i g m e n t showed the same absorption s p e c t r u m in the visible region as did authentic prodigiosin. These results indicate t h a t the m a j o r p i g m e n t produced b y strain W - I in the presence of the isolated substance is indeed prodigiosin. Moreover, the prodigiosin t h u s formed was found to be labeled, its specific activity being equal, within experim e n t a l error, to t h a t of the labeled material supplied to strain W - I . I t is therefore concluded t h a t the substance isolated f r o m cultures of strain 9-3-3 is in fact a precursor of prodigiosin. Strain 9-3-3 was generously supplied by Dr. M. I. BUNTING and strain W - I b y Dr. M. T. M. RIZKI. A sample of authentic prodigiosin was kindly provided by Mr. J. E. McKEON. Valuable discussions with Drs. D. M. BONNER, M. I. BUNTING, and M. T. M. RIZKI are gratefully acknowledged.
Department o/Microbiology, Yale University, New Haven, Conn. (U.S.A.)
URSULAV. SANTER* HENRY J. VOGEL
1 F. WREDE AND A. ROTHHAAS, Z. Physiol. Chem., 226 (1934) 952 M. T. M. RIZKI, Proc. Natl. Acad. Sci., 4 ° (1954) lO57. 3 R . P . WILLIAMS AND J . A . GREEN, Bacteriological Proc., (1955) 55. 4 E . L. LABRUM AND M. I. BUNTING, J. Bacteriol., 65 (1953) 394.
5 R. HUBBARD AND C. RIMINGTON, Bioehem. J., 46 (195 o) 220. 6 F. WREDE AND O. HETTCHE, Ber. Deutsch. Chem. Ges., 62 (1929) 2678. 7 A. ZAFFARONI AND R . BURTON, ft. Biol. Chem., 193 (1951) 749. Received J a n u a r y 3rd, 1956 * National Science F o u n d a t i o n Predoctoral Fellow, 1954-1956. This work is p a r t of a dissertation to be presented by U. V. SANTER for the degree of Doctor of Philosophy in Yale University.
The adenosine deaminase of crustaceans* DUCHATEAU, FLORKIN AND FRAPPEZ 1-4 have reported t h a t invertebrates contain adenase b u t no adenosine deaminase and t h a t vertebrates contain adenosine deaminase b u t no adenase. FLORKIN4 has concluded " T h e appearance of an enzymatic s y s t e m for the deamination of nucleosides is a biochemical characteristic of vertebrates in c o n t r a s t to i n v e r t e b r a t e s " . On the other hand, WAGNER AND MITCHELL~ have reported adenosine deaminase in the larva of Drosophila melanogaster and LENNOX~ has found adenosine deaminase and guanase in blowfly larva. The p r e s e n t r e p o r t deals with the d e m o n s t r a t i o n of adenosine deaminase in two crustaceans, a lobster (Homarus americanus) and a local fresh w a t e r crayfish (unidentified species). We have been unable to detect adenase in e x t r a c t s of the h e p a t o p a n c r e a s e s of either of these crustaceans. The lobster hepatopancreas is a convenient source of adenosine deaminase t h a t is relatively free of other enzymes of purine metabolism. The animals were killed by decapitation, the h e p a t o p a n c r e a s e s r e m o v e d immediately and homogenized with five volume of o.i ionic s t r e n g t h p H 7.o p h o s p h a t e buffer in a glass homogenizer. The extraction and all s u b s e q u e n t steps were carried out near o ° C. The h o m o g e n a t e s werf centrifuged for an h o u r at 20,000 × g and the clear s u p e r n a t a n t s were used in tests for the various enzyme activities. An e x t r a c t of lobster h e p a t o p a n c r e a s was fractionated with a m m o n i u m sulfate and m o s t of the adenosine deaminase precipitated between o. 5 and o. 7 saturation. Acetone p o w d e r s of the h e p a t o p a n c r e a s e s were prepared; these and the fresh tissues were extracted with p H 9.5 borate buffer for 2o min at 4 °0 C 2 to determine if uricase could be removed. E n z y m e activities were m e a s u r e d spectrophotometrically with the following s u b s t r a t e s and b y m e t h o d s indicated in the references: adenosine deaminase, adenine deoxyriboside deaminase, adenosine3 ' - p h o s p h a t e and a d e n o s i n e - J - p h o s p h a t e deaminases, x a n t h i n e oxidase, nucleoside phosphorylase, and uricaseS; adenase~; guanase, guanosine and guanine deoxyriboside deaminasesl0; cytosine deaminase::; purine-purine transribosidase and purine-purine transdeoxyribosidase 12. The positive findings are listed in Table I. Adenosine deaminase and a v e r y weak guanase activity were found in the crayfish h e p a t o p a n c r e a s extracts; adenosine deaminase, guanase, and
* This investigation was s u p p o r t e d by a research g r a n t Ci73o from the National Cancer Institute, of the :National I n s t i t u t e s of Health, U.S. Public H e a l t h Service.
38
SHORT COMMUNICATIONS, PRELIMINARY NOTES
VOL.
19 (1956)
1 E. E. SNELL, N. S. RADIN AND M. IKA\VA, J. Biol. Chem., 217 (1955) 803. 2 H. A. KREBS, in J. B. SUMNER AND 1~. MYRBACK, The Enzymes, Academic Press, New" York, Voh I I (1951), p. 505 • a M. S. DUNN, M. N. CAMIEN, S. SHANKMAN AND H. BLOCK, J. Biol. Chem., I68 (I947) 43. 4 p. BOULANGER AND R. OSTEUX, Biochim. Biophys..4cta, 5 (195 °) 416C. H. W. HIRS, S. MOORE AND W. H. STEIN, J . . 4 m . Chem. Soc., 76 (z954) 6o63. H. E. SAUBERLICrI AND C. A. BAUMANN, dr. Biol. Chem., 177 (19491 545. 7 B. F. STEELE, H. E. SAUBERLICH, M. S. REYNOLDS AND C. A. BAUMANN, J. Biol. Chem., 177 (19491 533. 8 l,. A. ELSON AND W. T. J. MORGAN, Biochem. J., 27 (19331 1824. 9 M. R. J. SALTON, Biochim. Biophys. ~4cta, 8 (19.52) 5IO. 10 C. S. CUMMINS AND H. HARRIS, .[. Gen. Microbiol., 13 (t9551 iii. 11 j . T. PARK, J. Biol. Chem., 194 (19521 897. 12 V. BRUCKNER AND G. IVANOVICS,Z. physiol. Chem., 247 (19351 281 ; Z. Immuuitats/orsch., 90 (19371 304; M. BOVARNICK, J . Biol. Chem., 145 (19421 415 . 13 A. GAUHE, P. GYORGY, J. R. E. HOOVER, R. KUHN, C. S. ROSE, R. W. RUELIUS AND F. ZILLIKAN .~rch. Biochem. Biophys., 48 (19541 214. Received December 23rd, 1955
Prodigiosin synthesis in S e r r a t i a m a r c e s c e n s : isolation of a pyrrole-containing precursor* .q'erratia marcescens produces a distinctive red pigment, prodigiosin, which has been characterized 1 as a t r i p y r r y l m e t h e n e . Evidence has recently been obtained by RIZKI 2 and b y WILLIAMS AND GREEN a t h a t some stable prodigiosin-deficient m u t a n t s of this species accumulate substances capable of causing the appearance Of p i g m e n t in certain other m u t a n t s impaired in prodigiosin synthesis. A pair of such m u t a n t s , not previously reported to exhibit cross-feeding, has been examined in some detail, since one of the two m u t a n t s (9-3-3) was found to excrete a relatively stable substance t h a t permitted p i g m e n t formation in the other (W-l). The excreted substance has now" been isolated in pure crystalline form; the progress of the isolation was followed by bioassay with strain \Y-I. This assay detects a b o u t o.1 t*g of the pure materiah For the isolation of the excreted substance, strain 9-3-3 was grown at 3 ° for four days in aerated peptone-glycerol m e d i u m 4. The culture was centrifuged, and the s u p e r n a t a n t liquid was extracted with chloroform. The chloroform layer was washed successively with i N hydrochloric acid, 1 N sodium hydroxide, and distilled water, and was then taken to dryness in vacuo. The residue was taken up in hexane (in which the active substance is sparingly soluble) and transferred to an alumina (Fisher Scientific Co., for c h r o m a t o g r a p h i c analysis) colunln. The latter was washed successively with benzene, and benzene-ether, and the active substance was then eluted with ether and ether-chloroform. After recrystallization from ethanol, the substance was obtained as colorless needles t h a t do not melt at 250', b u t decompose on f u r t h e r heating. The yield was a b o u t 1 mg per liter of culture. E l e m e n t a r y analysis agrees with the formula C10H1002N,~ (calculated: C, 63.2; H, 5.3; O, i6.8; N, 14.7; CHaO, I6.3; found: C, 63.0; H, 5.3; O, 17.o, N, 14.7; CHaO, 16.6)**. The isolated substance has neither pronounced basic nor p r o n o u n c e d acidic properties, i t is sparingly soluble in such solvents as ethanol, chloroform, ethyl ether, benzene, or t e t r a h y d r o f u r a n ; it is very sparingly soluble in water. The substance absorbs strongly in the ultra-violet; in ethanol solution it gives absorption m a x i m a at 363 and 254 mt, with molar extinction coefficients of a b o u t 3.5" IO4 and i. 3 • IO4, respectively. I t rapidly forms a red color at 25 with p - d i m e t h y l a m i n o b e n z a l d e h y d e in ethanolic hydrochloric acid; therefore, it p r e s u m a b l y contains a pyrrole ring with a free alpha-position. The excretion of the isolated material by one m u t a n t strain and its utilization by a n o t h e r indicate t h a t this c o m p o u n d is an intermediate in the synthesis of prodigiosin. This indication was s u p p o r t e d by tracer experiments. Strain 9-3-3 was cultivated in the usual m e d i u m supplemented with glycine-2-14C (c/. HUBBARD AND RIMINGTONS). Crystalline material was isolated as • This investigation was s u p p o r t e d in p a r t by the Atomic E n e r g y Commission, Contract No. AT-(3o-i)-ioi7 . • * The value for oxygen was obtained by direct analysis. Molecular weight d e t e r m i n a t i o n s are complicated by the low solubility of the substance in c o m m o n solvents; the formula given agrees with one m e t h o x y l group per formula weight. This formula weight was used in the c o m p u t a t i o n of the molar extinction coefficients and the specific activity of the substance. Microanalyses were performed by the Schwarzkopf Microanalytical Laboratory, by the Microchemical L a b o r a t o r y of New York University, and by Mr. STANLEY MILLS.
VOL.
19 (1956)
SHORT COMMUNICATIONS, PRELIMINARY NOTES
579
described above and found to be radioactive. I t was supplied to strain W - I , and the red p i g m e n t c o n s e q u e n t l y formed b y the organisms was extracted b y a modification of the m e t h o d of WREDE AND HETTCHE6 and was t h e n c h r o m a t o g r a p h e d on paper. The p a p e r was p r e t r e a t e d with propylene glycol, and the p i g m e n t was applied in toluene solution; chloroform was used as the mobile phase (C[. ZAFFARONI AND BURTON2). The pigment, which had the same RF as authentic prodigiosin, was eluted w i t h 5 o~o aqueous m e t h a n o l and r e c h r o m a t o g r a p h e d on p a p e r with hexane-glacial acetic acid (9:1). U p o n elution, the red p i g m e n t showed the same absorption s p e c t r u m in the visible region as did authentic prodigiosin. These results indicate t h a t the m a j o r p i g m e n t produced b y strain W - I in the presence of the isolated substance is indeed prodigiosin. Moreover, the prodigiosin t h u s formed was found to be labeled, its specific activity being equal, within experim e n t a l error, to t h a t of the labeled material supplied to strain W - I . I t is therefore concluded t h a t the substance isolated f r o m cultures of strain 9-3-3 is in fact a precursor of prodigiosin. Strain 9-3-3 was generously supplied by Dr. M. I. BUNTING and strain W - I b y Dr. M. T. M. RIZKI. A sample of authentic prodigiosin was kindly provided by Mr. J. E. McKEON. Valuable discussions with Drs. D. M. BONNER, M. I. BUNTING, and M. T. M. RIZKI are gratefully acknowledged.
Department o/Microbiology, Yale University, New Haven, Conn. (U.S.A.)
URSULAV. SANTER* HENRY J. VOGEL
1 F. WREDE AND A. ROTHHAAS, Z. Physiol. Chem., 226 (1934) 952 M. T. M. RIZKI, Proc. Natl. Acad. Sci., 4 ° (1954) lO57. 3 R . P . WILLIAMS AND J . A . GREEN, Bacteriological Proc., (1955) 55. 4 E . L. LABRUM AND M. I. BUNTING, J. Bacteriol., 65 (1953) 394.
5 R. HUBBARD AND C. RIMINGTON, Bioehem. J., 46 (195 o) 220. 6 F. WREDE AND O. HETTCHE, Ber. Deutsch. Chem. Ges., 62 (1929) 2678. 7 A. ZAFFARONI AND R . BURTON, ft. Biol. Chem., 193 (1951) 749. Received J a n u a r y 3rd, 1956 * National Science F o u n d a t i o n Predoctoral Fellow, 1954-1956. This work is p a r t of a dissertation to be presented by U. V. SANTER for the degree of Doctor of Philosophy in Yale University.
The adenosine deaminase of crustaceans* DUCHATEAU, FLORKIN AND FRAPPEZ 1-4 have reported t h a t invertebrates contain adenase b u t no adenosine deaminase and t h a t vertebrates contain adenosine deaminase b u t no adenase. FLORKIN4 has concluded " T h e appearance of an enzymatic s y s t e m for the deamination of nucleosides is a biochemical characteristic of vertebrates in c o n t r a s t to i n v e r t e b r a t e s " . On the other hand, WAGNER AND MITCHELL~ have reported adenosine deaminase in the larva of Drosophila melanogaster and LENNOX~ has found adenosine deaminase and guanase in blowfly larva. The p r e s e n t r e p o r t deals with the d e m o n s t r a t i o n of adenosine deaminase in two crustaceans, a lobster (Homarus americanus) and a local fresh w a t e r crayfish (unidentified species). We have been unable to detect adenase in e x t r a c t s of the h e p a t o p a n c r e a s e s of either of these crustaceans. The lobster hepatopancreas is a convenient source of adenosine deaminase t h a t is relatively free of other enzymes of purine metabolism. The animals were killed by decapitation, the h e p a t o p a n c r e a s e s r e m o v e d immediately and homogenized with five volume of o.i ionic s t r e n g t h p H 7.o p h o s p h a t e buffer in a glass homogenizer. The extraction and all s u b s e q u e n t steps were carried out near o ° C. The h o m o g e n a t e s werf centrifuged for an h o u r at 20,000 × g and the clear s u p e r n a t a n t s were used in tests for the various enzyme activities. An e x t r a c t of lobster h e p a t o p a n c r e a s was fractionated with a m m o n i u m sulfate and m o s t of the adenosine deaminase precipitated between o. 5 and o. 7 saturation. Acetone p o w d e r s of the h e p a t o p a n c r e a s e s were prepared; these and the fresh tissues were extracted with p H 9.5 borate buffer for 2o min at 4 °0 C 2 to determine if uricase could be removed. E n z y m e activities were m e a s u r e d spectrophotometrically with the following s u b s t r a t e s and b y m e t h o d s indicated in the references: adenosine deaminase, adenine deoxyriboside deaminase, adenosine3 ' - p h o s p h a t e and a d e n o s i n e - J - p h o s p h a t e deaminases, x a n t h i n e oxidase, nucleoside phosphorylase, and uricaseS; adenase~; guanase, guanosine and guanine deoxyriboside deaminasesl0; cytosine deaminase::; purine-purine transribosidase and purine-purine transdeoxyribosidase 12. The positive findings are listed in Table I. Adenosine deaminase and a v e r y weak guanase activity were found in the crayfish h e p a t o p a n c r e a s extracts; adenosine deaminase, guanase, and
* This investigation was s u p p o r t e d by a research g r a n t Ci73o from the National Cancer Institute, of the :National I n s t i t u t e s of Health, U.S. Public H e a l t h Service.
38