JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1980, p. 291-293 Vol. 12, No. 2 0095-1 137/80/08-0291/03$02.00/0 Production of p-Hydroxyhydrocinnamic Acid from Tyrosine by Peptostreptococcus anaerobius MARY ANN LAMBERT AND C. WAYNE MOSS* Analytical Bacteriology Branch, Center for Disease Control, Atlanta, Georgia 30333 Peptostreptococcus anaerobius was found to metabolize tyrosine to p-hydroxy, hydrocinnamic acid [3-(p-hydroxyphenyl)propionic acid]. This acid was detected in spent growth media by gas-liquid chromatography, and its identity was confirmed by mass spectrometry. A recent report by Babcock (1) showed that cultures of Peptostreptococcus anaerobius could be differentiated from other anaerobic gram-positive cocci by their abiity to degrade crystals of L-tyrosine which were dispersed throughout a modified Schaedler agar medium. Use of this new medium provides a simple and specific test for the presumptive identification of P. anaerobius in the clinical laboratory. Most bacteria metabolize tyrosine through the homogentisate (2, 4, 6, 9) or homoprotocatechuate (4, 6, 13) pathways to various hydroxyphenolic compounds. In aerobic species, the benzene ring is further hydroxylated, split, and degraded to short-chain carbon compounds which can then enter the Krebs cycle or other metabolic pathways (2, 4, 6, 9, 13, 14). However, in anaerobic bacteria, the benzene ring usually remains intact, and only the aliphatic side chains of these phenolic compounds are metabolized (2). This report describes the identification of a major acid metabolite produced when L-tyrosine is degraded by P. anaerobius. Cultures of P. anaerobius 17642 (VPI 4329), 19112, and 17790 were obtained from the stock culture collection of the Center for Disease Control Anaerobe Section. They were inoculated into thioglycolate broth (135-C; BBL Microbiology Systems, Cockeysville, Md.) and incubated anaerobically for 24 h at 35°C. These cultures were used to inoculate Schaedler agar plates containing 0.3% L-tyrosine (1), Schaedler broth containing 0.05% L-tyrosine, and Schaedler agar and broth without added tyrosine. The agar plates were prepared, stored, and inoculated as described by Babcock (1), except that only one strain was inoculated per plate, and the inoculum was spread over a 40-mmdiameter area in the center of the plate. The broth was prepared according to the manufacturer's directions (BBL Microbiology Systems), dispensed in 100-ml volumes, and inoculated with 0.3 ml of the thioglycolate culture. The inoculated media and uninoculated media controls were incubated under anaerobic conditions for 48 h at 35°C. The cultures of P. anaerobius grew well in all media; disappearance of the tyrosine crystals in the agar plates beneath the area of growth was observed as previously described (1). The cells were removed from the agar plates and discarded. A 0.3-ml amount of 25% H2SO4 was distributed onto the surface of each plate and allowed to stand for 15 to 30 min. The agar was cut into small pieces with a spatula, transferred to 50-ml screw-capped centrifuge tubes, and melted in a water bath at 80 to 85°C. After cooling to room temperature, the agar was extracted with 20 ml of diethyl ether; the ether layer was transferred to a small beaker, concentrated, and transferred to a screw-capped test tube. The butyl ester or trifluoroacetyl butyl esters of the acids were prepared as described previously (8, 10). The broth cultures were centrifuged, and 5-ml volumes of the spent growth medium were acidified to pH 2 with 6 N HCL. The acids were extracted with two 5-ml volumes of diethyl ether, and the ether layers were combined in a small beaker, concentrated, and derivatized (8, 10). The butyl ester or trifluoroacetyl butyl ester was analyzed with a Perkin-Elmer model 990 gas chromatograph (The Perkin-Elmer Corp., Norwalk, Conn.) equipped with a flame ionization detector and a coiled glass column (3.6 m by 4 mm [inside diameter]) packed with 5% OV1 coated on 80/100-mesh, acid-washed, dimethylchlorosilane-treated Chromosorb W (Analabs, North Haven, Conn.) The instrument conditions and column temperatures were the same as those used in an earlier report (10). Combined gas-liquid chromatography-mass spectrometry was done on a model 21-491B mass spectrometer (Du Pont Instruments, Wilmington, Del.) interfaced with a Varian 2700 gas chromatograph (Varian Instruments, Walnut Park, Calif.). The 291 292 J. CLIN. MICROBIOL. NOTES 2700 instrument contained a glass column (2 m valeric acids were produced regardless of the by 2 mm [inside diameter]) packed with 3% 101 medium used. The identity of these acids was on 100/200-mesh Gas-Chrom Q. The mass spec- established by gas-liquid chromatography retentrometer was equipped for both electron impact tion time comparison with standards and by ionization and chemical ionization. The reagent mass spectrometry. The large peak eluting at 17.5 min in both chromatograms was identified gas for chemical ionization was isobutane. Chromatograms of the esterified acids pro- by gas-liquid chromatography-mass spectromeduced by P. anaerobius in Schaedler broth with try as hydrocinnamic acid or 3-phenylpropionic and without added tyrosine are shown in Fig. 1 acid. The electron impact ionization spectrum of and 2, respectively. Large to moderate amounts the butyl ester showed a molecular ion (M+) of of acetic, isocaproic, butyric, and benzoic acids 206, major ions at m/e 91 and m/e 104, and and small amounts of isobutyric, lactic, and iso- relatively large fragment ions at m/e 151 (M+55) and m/e 133 (M+-73). The chemical ionization spectrum showed a large M+1 ion at m/e 207. These data are identical to those reported previously for the butyl ester derivative of a standard of hydrocinnamic acid (3). The peak at retention time of 19.6 min in Fig. 1 was identified asp-hydroxyhydrocinnamic acid or 3-(p-hydroxyphenyl)propionic acid. The mass spectrum of the butyl ester showed an M+ at 222 in the electron impact mode and an M+1 ion at 223 in the chemical mode. In the electron impact ionization spectrum, the base peak ion was at m/e 107, and characteristic fragment ions were at m/e 120 (M+-102), m/e 149 (M+-73), and m/e 166 (M+-56). Identical mass spectra and retention time data were observed for a standard of FIG. 1. Gas-liquid chromatogram of esterified 3-(p-hydroxyphenyl)propionic acid (Aldrich short-chain acids produced by P. anaerobius 17642 Chemical Co., Milwaukee, Wis.) derivatized and after growth in Schaedler broth containing 0.05% L analyzed under the same conditions. When the tyrosine. Abbreviations: C2, acetic acid; R, reagent; butyl ester derivatives of the culture extracts or iC4, isobutyric acid; C4, butyric acid; L, lactic acid; thep-hydroxyhydrocinnamic acid standard were iC5, isovaleric acid; iCe,, isocaproic acid; B> benzoic treated with trifluoroacetic anhydride (Pierce acid; HCn, hydrocinnamic acid; and OH-HCn, hydroxyhydrocinnamic acid. Analysis was made on a Chemical Co., Rockford, Ill.), the retention time decreased from 23.1 to 19.6 min, indicating that 5% OV-I column. the hydroxyl group on the benzene ring had reacted to form a more volatile compound. The electron impact ionization mass spectrum of the trifluoroacetyl butyl ester derivative had an M+ ce 9 c4 CI at 318, confirming the addition of -COCF3 to the butyl ester. A small amount of hydroxyhydrocinnamic was produced by all cultures grown in acid MICa Schaedler medium without added tyrosine (Fig. 2), indicating that low concentrations of this amino acid were present in unsupplemented met4 ItF dia. The substantial increase in the amount of this acid produced by all cultures of P. anaerobius grown in the tyrosine-enriched medium (Fig. 1) clearly shows that hydroxyhydrocinnamic acid is a major product when tyrosine is i i n i à2 à4 à metabolized by this species. _sb These data indicate that the homogentisate FIG. 2. Gas-liquid chromatogram of esterified or homoprotocatechuate pathways are not inshort-chain acids produced by P. anaerobius 17642 after growth in Schaedler broth. Analysis was made volved in the degradation of tyrosine by P. anaerobius since the expected intermediates of on a 5% OV-I column. Abbreviations are as in the legend to Fig. 1. these pathways (p-hydroxyphenylpyruvic,p-hy- 1 ai 1 S NOTES VOL. 12, 1980 droxyphenyllactic, or p-hydroxyphenylacetic acid) were not detected (2, 4, 9, 13). This organism may metabolize tyrosine by initial deamination to p-hydroxycinnamic acid and subsequent reduction of this acid to p-hydroxyhydrocinnamic acid. The deamination reaction has been demonstrated in several fungi (4) and is catalyzed by either a tyrosine or phenylalanine ammonia-lyase (EC 184.108.40.206). The latter enzymatic reaction could be similar to that reported for Lactobacillus pastorianus subsp. quinicus with various phenolic compounds (14). Similar reactions may have been observed previously in Clostridium sporogenes when phenylalanine, an aromatic amino acid structurally related to tyrosine, was first metabolized to cinnamic acid and then to hydrocinnamic acid (11). An analogous enzyme system may be responsible for the degradation of tyrosine and phenylalanine in P. anaerobius because p-hydroxyhydrocinnamic and hydrocinnamic acids were both major metabolites of this organism. Obviously, other studies are needed to firmly establish the metabolic pathway(s) involved in these reactions. Other microorganisms, including the pathogenic aerobic actinomycetes (5), nonfermentative gram-negative bacilli (7), and members of the Enterobacteriaceae (12), can also actively degrade L-tyrosine. To our knowledge, the metabolites produced from tyrosine by these organisms have not been determined. We thank Ann Y. Armfield, Anaerobe Section, Center for Disease Control for providing the cultures used in this study. LITERATURE CITED 1. Babcock, J. B. 1979. Tyrosine degradation in presumptive identification of Peptostreptococcus anaerobius. J. 293 Clin. Microbiol. 9:358-361. 2. Barker, H. A. 1961. Fermentations of nitrogenous organic compounds, p. 179-181. In I. C. Gunsalus and R. Y. Stanier (ed.), The bacteria, vol II. Academic Press, Inc., New York. 3. Choudhary, G., and C. W. Moss. 1976. Gas chromatography-mass spectrometry of some biologically important short chain acid butyl esters. J. Chromatogr. 128: 261-270. 4. Dagley, S. 1978. Pathways for the utilization of organic growth substrates, p. 353-355. In L. N. Ornston and J. R. Sokatch (ed.), The bacteria, vol. VI. Academic Press, Inc., New York. 5. Gordon, M. A. 1974. Aerobic pathogenic Actinomycetaceae, p. 175-184. In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, 2nd ed., American Society for Microbiology, Washington, D.C. 6. Gottschalk, G. 1979. Catabolic activities of aerobic heterotrophs, p. 126-128. In M. P. Starr (ed.), Bacterial metabolism. Springer-Verlag New York, Inc., New York. 7. Kurup, V. P., and J. B. Babcock. 1979. Use of casein, tyrosine, and hypoxanthine in the identification of nonfermentative gram-negative bacilli. Med. Microbiol. Immunol. 167:71-75. 8. Lambert, M. A., and C. W. Moss. 1972. Gas-liquid chromatography of short chain fatty acids on Dexsil 300 GC. J. Chromatogr. 74:335-338. 9. Meister, A. 1965. Biochemistry of the amino acids, vol. II, 2nd ed., p. 922-927. Academic Press, Inc., New York. 10. Mose, C. W., C. L Hatheway, M. A. Lambert, and L. M. McCroskey. 1980. Production of phenylacetic and hydroxyphenylacetic acids by Clostridium botulinum type G. J. Clin. Microbiol. 11:743-745. 11. Moss, C. W., M. A. Lambert, and D. J. Goldsmith. 1970. Production of hydrocinnamic acid by Clostridia. Appl. Microbiol. 19:375-378. 12. Sheth, N. K., and V. P. Kurup. 1975. Evaluation of tyrosine medium for the identification of Enterobacteriaceae. J. Clin. Microbiol. 1:483-485. 13. Sparnins, V. L., and P. J. Chapman. 1976. Catabolism of L-tyrosine by the homoprotocatechuate pathway in gram-positive bacteria. J. Bacteriol. 127:362-366. 14. Whiting, G. C., and J. G. Carr. 1959. Metabolism of cinnamic acid and hydroxy-cinnamic acids by Lactobacillus pastorianus var. quinicus. Nature (London) 184:1427-1428.
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