Vol. 126, No. 1 Printed in USA.
JOURNAL OF BACTERIOLOGY, Apr. 1976, p. 544-546 Copyright © 1976 American Society for Microbiology
Formation of Indole-3-Carboxylic Acid by Chromobacterium violaceum PATRICK J. DAVIS, MARK E. GUSTAFSON, AND JOHN P. ROSAZZA* Division of Medicinal Chemistry-Natural Products, College of Pharmacy, The University of Iowa, Iowa City, Iowa 52242
Received for publication 19 January 1976
L-Tryptophan is converted to indole-3-carboxylic acid by growing cultures and resting cell suspensions of Chromobacterium violaceum
Chromobacterium violaceum yields a number of metabolites of the aromatic amino acid Ltryptophan. These include 5-hydroxy- (11, 12) and 6-hydroxytryptophan (2); indole (15), and the products of further metabolism of indole, violacein, and indigo (1, 4, 5, 15); and transamination products, indole-3-pyruvic acid and indole-3-acetic acid (R. D. Moss and N. R. Evans, Bacteriol. Proc., p. 117, 1957). The presence of the pigments renders the bacterium a deep violet color. We reported on the unusual ability of this microorganism to introduce a double bond into the side chain of N-carbobenzoxyl-L-tryptophan (3). To obtain this metabolite, the culture medium normally contained L-tryptophan, which served as an inducer for the enzyme, causing the side-chain dehydrogenation reaction to occur. In the course of this work, an unknown tryptophan metabolite consistently appeared on thin-layer chromatograms run on extracts of culture media. The metabolite reacted with strongly acidic reagents to yield a bright red color. The mobility of this compound in any of our commonly used solvent systems (3, 14) was not consistent with any of the known tryptophan metabolite standards on hand. This report describes the isolation and characterization of the metabolite as indole-3-carboxylic acid, as well as preliminary experiments indi. cating how the compound is formed by C. violaceum. C. violaceum (ATCC 12472) was used throughout the study. The basal medium employed and the fermentation procedure were described by Davis et al. (3). The metabolite was first isolated as a side product of a fermentation that was conducted as follows. C. violaceum was grown in a stirred fermentor operating at 350 rpm, 27 C, with air being sparged in at 0.4 volumes per liter of medium per min, in 10 liters of medium containing 0.5 mg of L-tryptophan per ml and 0.5 mg of
N-carbobenzoxyl-i,tryptophan per ml. Samples (4 ml) of the fermentation were withdrawn at various time intervals. These were acidified to pH 2 (5 N HCl) and extracted with ethyl acetate (1 ml). Then 30 u1l of the extract was examined by thin-layer chromatography on one or more of the following solvent systems: (i) ether-hexane-90% formic acid (75:50:1); (ii) ethyl acetateisopropanol-25% NH,OH (9:5:1); (iii) benzene95% ethanol (4:1); (iv) chloroform-glacial acetic acid (95:5). Developed chromatograms were visualized by spraying with a p-anisaldehyde spray reagent (3). Indole-3-carboxylic acid gave a bright red color on chromatograms with this reagent, and could be detected in amounts as low as 1 jig/ml of culture medium by this assay procedure. After 72 h, the fermentation was harvested, adjusted to pH 2 with 5 N HCl, and exhaustively extracted with ethyl acetate. The combined ethyl acetate extracts were evaporated to yield a black tar (14.3 g). The tar was dissolved in a minimum volume of acetone, adsorbed onto 15 g of silica gel (Baker, 3405), and added to the top of a silica gel column (300 g, 5.5 by 50 cm). Fractions of 6 ml were eluted at a flow rate of 3 ml/min with chloroform-95% ethanol-glacial acetic acid (40:2:1), and fractions 83 to 119 gave 300 mg of the unknown metabolite. The metabolite was further purified by thick-layer chromatography on silica gel GF2-,4 using solvent system (i). Recrystallization of the metabolite from chloroform gave an analytical sample with the following physical properties: melting point, 210 to 212C (literature value for indole-3carboxylic acid, 213C l17]); mass spectrum, mle (percent relative intensity) 161 (100), 144 (87), 116 (27); nuclear magnetic resonance, dimethyl sulfoxide-D6 (8) 7.33 (m, 3H), 8.01 (m, 3H), 11.77 (s, 1H, COOH); infrared (KBr disk), cm-', 2,500-3,000, 1,740, 1,410, 1,305, 1,230, 1,218, 1,128, 1,105; ultraviolet, 95% ethanol, nm, Xm,. (e), 215 (3.12 x 104, 282 (9.95 x
544
VOL. 126, 1976
103); thin-layer chromatography, silica gel
NOTES
545
33%, respectively, from these substrates.
GF2,4, solvent system: (i), Rf 0.45; (ii), Rf 0.4; Traces of indole-3-carboxaldehyde and indole-3carboxylic acid were obtained with indole-3(iii), Rf 0.11; (iv), Rf 0.45. The methyl ester of the metabolite was prepared by dissolving 21.6 mg in 10 ml of absolute methanol and adding 1.4 ml (0.28 mmol) of an ethereal solution of diazomethane. After 3 h, the solvent was removed under vacuum, and the resulting solids were recrystallized from ethanol-water (1:1) to give 17 mg of the methyl ester: melting point, 148.5 to 149.5; mass spectrum, mle (percent relative intensity) 175 (40), 144 (100), 116 (29); nuclear magnetic resonance, dimethyl sulfoxide-D6 3.85 (s, 3H, -OCH3), 7.33 (m, 3H), 8.01 (m, 2H), 9.97 (broad s, 1H). The metabolite and its methyl ester were identical in all respects to authentic indole-3-carboxylic acid (K & K Laboratories Inc., Plainview, N.Y.) and its methyl ester. An experiment was conducted to verify that the isolated metabolite was not an artifact of the previous fermentation. Fermentations were conducted in 125-ml Erlenmeyer flasks in 25 ml of medium on a gyratory shaker (New Brunswick G-25) at 200 rpm and 27 C. Controls consisted of sterile medium (no culture) with and without L-tryptophan, while the organism was also grown with and without L-tryptophan. Indole-3-carboxylic acid could only be detected in flasks containing C. violaceum and L-tryptophan. The ability of resting cells (3) of C. violaceum to convert a variety of substrates into indole-3carboxylate was examined. Resting cell incubations were conducted in 10 ml of 0.1 M phosphate buffer, pH 6.0, in 125-ml Erlenmeyer flasks shaken at 180 rpm. The cell suspensions represented a 10-fold concentration of cells relative to fermentation media (3). Such resting cell suspensions rapidly converted L-tryptophan to indole-3-carboxylic acid and other metabolites within 1 h, and all of the added L-tryptophan was consumed within 24 h. Indole-3-acetic acid, indole-3-acetaldehyde, indole-3-propionic acid, indole-3-pyruvic acid, indole-3-carboxaldehyde, and L-tryptophan were added as substrates to such cell suspensions in concentrations of 0.5 mg/ml. Controls consisted of cell suspensions containing no substrates, as well as each of the substrates dissolved in phosphate buffer alone. Samples were taken at 1 and 24 h, and were examined by thin-layer chromatography using solvent systems (i), (ii), and (iii). Of the substrates used, only L-tryptophan and indole-3-carboxaldehyde served as efficient precursors for indole-3-carboxylic acid. Estimated yields of the metabolite were 25% and
acetic acid as substrate. Results with indole-3pyruvic acid were inconclusive because this compound decomposes readily in aqueous medium to several products (14). The mechanism by which L-tryptophan is converted into indole-3-carboxylic acid in microorganisms remains to be elucidated. It has been suggested that indole-3-acetaldehyde may serve as a precursor for both indole-3-acetic acid and indole-3-carboxylic acid (9). An alternative suggestion involves an indole-3-acetic acid oxidase/peroxidase enzyme system similar to that found in plants (13). The low yields of products obtained in our experiments with indole-3acetic acid may have been due to the inability of this substrate to enter whole cells of C. violaceum. This substrate may also serve as an inhibitor of indole-3-carboxylic acid synthesis. Although a womplete understanding of the path by which indole-3-carboxylic acid is formed in C. violaceum will require the application of cell-free systems, it appears that a reasonable reaction sequence is: L-tryptophan -* indole-3-acetic acid indole-3-carboxaldehyde indole-3-carboxylic acid
Indole-3-carboxylic acid has been detected in several types of microorganisms including Azotobacter species (16), Nectria galligena (10), actinomycetes (8), yeasts (6), various other bacteria including other members of the Rhizobiaceae (7, 8), and Corydyceps militaris (7). In all of the above studies, indole-3-carboxylic acid was identified only by thin-layer or paper chromatographic procedures. This report appears to be the first one in which indole-3-carboxylic acid has been identified by chemical methods. LITERATURE CITED 1. Ballentine, J. A., C. B. Barrett, R. J. S. Beer, S. Eardley, A. Robinson, B. L. Shaw, and T. H. Simpson. 1958. Chemistry of the bacteria. VII. The structure of violacein. J. Chem. Soc. p. 755-760. 2. Contractor, S. F., M. Sandier and J. Wragg. 1964. 6-
Hydroxytryptophan formation from Chromobacterium violaceum. Life Sci. 3:996-1006. 3 Davis, P. J., M. E. Gustafson, and J. P. Rosazza. 1975. Metabolism of N-carbobenzoxyl-L-tryptophan by Chromobacterium violaceum. Biochim. Biophys. Acta 385:133-144. 4. DeMoss, R. D., and N. R. Evans. 1959. Physiological aspects of violacein biosynthesis in non-proliferating cells. J. Bacteriol. 78:583-588. 5. DeMoss, R. D., and N. R. Evans. 1960. Incorporation of '4C-labeled substrates into violacein. J. Bacteriol. 79:729-733.
546
J. BACTERIOL.
NOTES
6. Glombitza, K. W., and T. Hartmann. 1966. Der Tryptophan-abbau bei Endomycopsis vernalis und anderen Hefen. Planta 69:135-149. 7. Horegott, V. H. 1973. Zum Tryptophanabbau bei Corydyceps militaris-Stammen in saprophytischen Kulturen. Biochem. Physiol. Pflanzen 164:500-508. 8. Kaunat, H. 1969. Bildung von Indol-derivaten durch rhizospharenspezifische Bakterien und Aktinomyceten. Zentralkl. Bakteriol. Parasitenk. Infektionskr. Hyg., Abt. 2 123:501-515. 9. Libbert, E., R. Schroder, A. Drawert, and E. Fischer. 1970. Pathway of IAA production from tryptophan by plants and their epiphytic bacteria; a comparison. III. Metabolism of tryptamine, indole-3-acetaldehyde, tryptophol and indoleacetamide, effects of a native inhibitor. Physiol. Plant. 23:287-293. 10. Marchal, P., and J. Rigaud. 1972. Sur La degradation de i'acide indolyl-3-ac6tique par Nectria galligena Bres var Major Wr. Arch. Mikrobiol. 85:1-5. 11. Mitoma, C., H. Weissbach, and S. Udenfriend. 1955. Formation of 5-hydroxytryptophan from tryptophan
12.
13.
14. 15. 16. 17.
by Chromobacterium violaceum. Nature (London) 175:994-995. Mitoma, C., H. Weissbach, and S. Udenfriend. 1966. 5Hydroxytryptophan formation and tryptophan metabolism in Chromobacterium violaceum. Arch. Biochem. Biophys. 63:122-130. Rigaud, J., and C. Bulard. 1965. Sur la presence d'indolyl-3-aldehyde et d'acide indolyl-3-carboxylique dans les milieux de culture de Rhizobium. C. R. Acad. Sci. Paris 261:784-786. Rosazza, J. P., R. Juhl, and P. J. Davis. 1973. Tryptophol formation by Zygosaccharomyces priorianus. Appl. Microbiol. 26:98-105. Sebek, 0. K., and H. Jiger. 1962. Divergent pathways of indole metabolism in Chromobacterium violaceum. Nature (London) 196:793-795. Vancura, V., and J. Macura. 1960. Indole derivatives in Azotobacter cultures. Folia Microbiolog. (Prague) 5:293-297. Ward, F. W. 1923. The absorption spectra of some indole derivatives. Biochem. J. 17:891.
JOURNAL OF BACTERIOLOGY, Apr. 1976, p. 544-546 Copyright © 1976 American Society for Microbiology
Formation of Indole-3-Carboxylic Acid by Chromobacterium violaceum PATRICK J. DAVIS, MARK E. GUSTAFSON, AND JOHN P. ROSAZZA* Division of Medicinal Chemistry-Natural Products, College of Pharmacy, The University of Iowa, Iowa City, Iowa 52242
Received for publication 19 January 1976
L-Tryptophan is converted to indole-3-carboxylic acid by growing cultures and resting cell suspensions of Chromobacterium violaceum
Chromobacterium violaceum yields a number of metabolites of the aromatic amino acid Ltryptophan. These include 5-hydroxy- (11, 12) and 6-hydroxytryptophan (2); indole (15), and the products of further metabolism of indole, violacein, and indigo (1, 4, 5, 15); and transamination products, indole-3-pyruvic acid and indole-3-acetic acid (R. D. Moss and N. R. Evans, Bacteriol. Proc., p. 117, 1957). The presence of the pigments renders the bacterium a deep violet color. We reported on the unusual ability of this microorganism to introduce a double bond into the side chain of N-carbobenzoxyl-L-tryptophan (3). To obtain this metabolite, the culture medium normally contained L-tryptophan, which served as an inducer for the enzyme, causing the side-chain dehydrogenation reaction to occur. In the course of this work, an unknown tryptophan metabolite consistently appeared on thin-layer chromatograms run on extracts of culture media. The metabolite reacted with strongly acidic reagents to yield a bright red color. The mobility of this compound in any of our commonly used solvent systems (3, 14) was not consistent with any of the known tryptophan metabolite standards on hand. This report describes the isolation and characterization of the metabolite as indole-3-carboxylic acid, as well as preliminary experiments indi. cating how the compound is formed by C. violaceum. C. violaceum (ATCC 12472) was used throughout the study. The basal medium employed and the fermentation procedure were described by Davis et al. (3). The metabolite was first isolated as a side product of a fermentation that was conducted as follows. C. violaceum was grown in a stirred fermentor operating at 350 rpm, 27 C, with air being sparged in at 0.4 volumes per liter of medium per min, in 10 liters of medium containing 0.5 mg of L-tryptophan per ml and 0.5 mg of
N-carbobenzoxyl-i,tryptophan per ml. Samples (4 ml) of the fermentation were withdrawn at various time intervals. These were acidified to pH 2 (5 N HCl) and extracted with ethyl acetate (1 ml). Then 30 u1l of the extract was examined by thin-layer chromatography on one or more of the following solvent systems: (i) ether-hexane-90% formic acid (75:50:1); (ii) ethyl acetateisopropanol-25% NH,OH (9:5:1); (iii) benzene95% ethanol (4:1); (iv) chloroform-glacial acetic acid (95:5). Developed chromatograms were visualized by spraying with a p-anisaldehyde spray reagent (3). Indole-3-carboxylic acid gave a bright red color on chromatograms with this reagent, and could be detected in amounts as low as 1 jig/ml of culture medium by this assay procedure. After 72 h, the fermentation was harvested, adjusted to pH 2 with 5 N HCl, and exhaustively extracted with ethyl acetate. The combined ethyl acetate extracts were evaporated to yield a black tar (14.3 g). The tar was dissolved in a minimum volume of acetone, adsorbed onto 15 g of silica gel (Baker, 3405), and added to the top of a silica gel column (300 g, 5.5 by 50 cm). Fractions of 6 ml were eluted at a flow rate of 3 ml/min with chloroform-95% ethanol-glacial acetic acid (40:2:1), and fractions 83 to 119 gave 300 mg of the unknown metabolite. The metabolite was further purified by thick-layer chromatography on silica gel GF2-,4 using solvent system (i). Recrystallization of the metabolite from chloroform gave an analytical sample with the following physical properties: melting point, 210 to 212C (literature value for indole-3carboxylic acid, 213C l17]); mass spectrum, mle (percent relative intensity) 161 (100), 144 (87), 116 (27); nuclear magnetic resonance, dimethyl sulfoxide-D6 (8) 7.33 (m, 3H), 8.01 (m, 3H), 11.77 (s, 1H, COOH); infrared (KBr disk), cm-', 2,500-3,000, 1,740, 1,410, 1,305, 1,230, 1,218, 1,128, 1,105; ultraviolet, 95% ethanol, nm, Xm,. (e), 215 (3.12 x 104, 282 (9.95 x
544
VOL. 126, 1976
103); thin-layer chromatography, silica gel
NOTES
545
33%, respectively, from these substrates.
GF2,4, solvent system: (i), Rf 0.45; (ii), Rf 0.4; Traces of indole-3-carboxaldehyde and indole-3carboxylic acid were obtained with indole-3(iii), Rf 0.11; (iv), Rf 0.45. The methyl ester of the metabolite was prepared by dissolving 21.6 mg in 10 ml of absolute methanol and adding 1.4 ml (0.28 mmol) of an ethereal solution of diazomethane. After 3 h, the solvent was removed under vacuum, and the resulting solids were recrystallized from ethanol-water (1:1) to give 17 mg of the methyl ester: melting point, 148.5 to 149.5; mass spectrum, mle (percent relative intensity) 175 (40), 144 (100), 116 (29); nuclear magnetic resonance, dimethyl sulfoxide-D6 3.85 (s, 3H, -OCH3), 7.33 (m, 3H), 8.01 (m, 2H), 9.97 (broad s, 1H). The metabolite and its methyl ester were identical in all respects to authentic indole-3-carboxylic acid (K & K Laboratories Inc., Plainview, N.Y.) and its methyl ester. An experiment was conducted to verify that the isolated metabolite was not an artifact of the previous fermentation. Fermentations were conducted in 125-ml Erlenmeyer flasks in 25 ml of medium on a gyratory shaker (New Brunswick G-25) at 200 rpm and 27 C. Controls consisted of sterile medium (no culture) with and without L-tryptophan, while the organism was also grown with and without L-tryptophan. Indole-3-carboxylic acid could only be detected in flasks containing C. violaceum and L-tryptophan. The ability of resting cells (3) of C. violaceum to convert a variety of substrates into indole-3carboxylate was examined. Resting cell incubations were conducted in 10 ml of 0.1 M phosphate buffer, pH 6.0, in 125-ml Erlenmeyer flasks shaken at 180 rpm. The cell suspensions represented a 10-fold concentration of cells relative to fermentation media (3). Such resting cell suspensions rapidly converted L-tryptophan to indole-3-carboxylic acid and other metabolites within 1 h, and all of the added L-tryptophan was consumed within 24 h. Indole-3-acetic acid, indole-3-acetaldehyde, indole-3-propionic acid, indole-3-pyruvic acid, indole-3-carboxaldehyde, and L-tryptophan were added as substrates to such cell suspensions in concentrations of 0.5 mg/ml. Controls consisted of cell suspensions containing no substrates, as well as each of the substrates dissolved in phosphate buffer alone. Samples were taken at 1 and 24 h, and were examined by thin-layer chromatography using solvent systems (i), (ii), and (iii). Of the substrates used, only L-tryptophan and indole-3-carboxaldehyde served as efficient precursors for indole-3-carboxylic acid. Estimated yields of the metabolite were 25% and
acetic acid as substrate. Results with indole-3pyruvic acid were inconclusive because this compound decomposes readily in aqueous medium to several products (14). The mechanism by which L-tryptophan is converted into indole-3-carboxylic acid in microorganisms remains to be elucidated. It has been suggested that indole-3-acetaldehyde may serve as a precursor for both indole-3-acetic acid and indole-3-carboxylic acid (9). An alternative suggestion involves an indole-3-acetic acid oxidase/peroxidase enzyme system similar to that found in plants (13). The low yields of products obtained in our experiments with indole-3acetic acid may have been due to the inability of this substrate to enter whole cells of C. violaceum. This substrate may also serve as an inhibitor of indole-3-carboxylic acid synthesis. Although a womplete understanding of the path by which indole-3-carboxylic acid is formed in C. violaceum will require the application of cell-free systems, it appears that a reasonable reaction sequence is: L-tryptophan -* indole-3-acetic acid indole-3-carboxaldehyde indole-3-carboxylic acid
Indole-3-carboxylic acid has been detected in several types of microorganisms including Azotobacter species (16), Nectria galligena (10), actinomycetes (8), yeasts (6), various other bacteria including other members of the Rhizobiaceae (7, 8), and Corydyceps militaris (7). In all of the above studies, indole-3-carboxylic acid was identified only by thin-layer or paper chromatographic procedures. This report appears to be the first one in which indole-3-carboxylic acid has been identified by chemical methods. LITERATURE CITED 1. Ballentine, J. A., C. B. Barrett, R. J. S. Beer, S. Eardley, A. Robinson, B. L. Shaw, and T. H. Simpson. 1958. Chemistry of the bacteria. VII. The structure of violacein. J. Chem. Soc. p. 755-760. 2. Contractor, S. F., M. Sandier and J. Wragg. 1964. 6-
Hydroxytryptophan formation from Chromobacterium violaceum. Life Sci. 3:996-1006. 3 Davis, P. J., M. E. Gustafson, and J. P. Rosazza. 1975. Metabolism of N-carbobenzoxyl-L-tryptophan by Chromobacterium violaceum. Biochim. Biophys. Acta 385:133-144. 4. DeMoss, R. D., and N. R. Evans. 1959. Physiological aspects of violacein biosynthesis in non-proliferating cells. J. Bacteriol. 78:583-588. 5. DeMoss, R. D., and N. R. Evans. 1960. Incorporation of '4C-labeled substrates into violacein. J. Bacteriol. 79:729-733.
546
J. BACTERIOL.
NOTES
6. Glombitza, K. W., and T. Hartmann. 1966. Der Tryptophan-abbau bei Endomycopsis vernalis und anderen Hefen. Planta 69:135-149. 7. Horegott, V. H. 1973. Zum Tryptophanabbau bei Corydyceps militaris-Stammen in saprophytischen Kulturen. Biochem. Physiol. Pflanzen 164:500-508. 8. Kaunat, H. 1969. Bildung von Indol-derivaten durch rhizospharenspezifische Bakterien und Aktinomyceten. Zentralkl. Bakteriol. Parasitenk. Infektionskr. Hyg., Abt. 2 123:501-515. 9. Libbert, E., R. Schroder, A. Drawert, and E. Fischer. 1970. Pathway of IAA production from tryptophan by plants and their epiphytic bacteria; a comparison. III. Metabolism of tryptamine, indole-3-acetaldehyde, tryptophol and indoleacetamide, effects of a native inhibitor. Physiol. Plant. 23:287-293. 10. Marchal, P., and J. Rigaud. 1972. Sur La degradation de i'acide indolyl-3-ac6tique par Nectria galligena Bres var Major Wr. Arch. Mikrobiol. 85:1-5. 11. Mitoma, C., H. Weissbach, and S. Udenfriend. 1955. Formation of 5-hydroxytryptophan from tryptophan
12.
13.
14. 15. 16. 17.
by Chromobacterium violaceum. Nature (London) 175:994-995. Mitoma, C., H. Weissbach, and S. Udenfriend. 1966. 5Hydroxytryptophan formation and tryptophan metabolism in Chromobacterium violaceum. Arch. Biochem. Biophys. 63:122-130. Rigaud, J., and C. Bulard. 1965. Sur la presence d'indolyl-3-aldehyde et d'acide indolyl-3-carboxylique dans les milieux de culture de Rhizobium. C. R. Acad. Sci. Paris 261:784-786. Rosazza, J. P., R. Juhl, and P. J. Davis. 1973. Tryptophol formation by Zygosaccharomyces priorianus. Appl. Microbiol. 26:98-105. Sebek, 0. K., and H. Jiger. 1962. Divergent pathways of indole metabolism in Chromobacterium violaceum. Nature (London) 196:793-795. Vancura, V., and J. Macura. 1960. Indole derivatives in Azotobacter cultures. Folia Microbiolog. (Prague) 5:293-297. Ward, F. W. 1923. The absorption spectra of some indole derivatives. Biochem. J. 17:891.
