THE AMINO ACID REQUIREMENTS OF STREPTOCOCCUS FAECALIS AND THE USE OF THIS ORGANISM FOR THE DETERMINATION OF THREONINE IN NATURAL PRODUCTS*

THE AMINO ACID REQUIREMENTS OF STREPTOCOCCUS FAECALIS AND THE USE OF THIS ORGANISM FOR THE DETERMINATION OF THREONINE IN NATURAL PRODUCTS* BY IRENE TATMAN GREENHUT, B. S. SCHWEIGERT, AND C. A. ELVEHJEM (From the Department of Biochemistry, College of Agriculture, University of Wisconsin, Madison) (Received for publication, August 0, 95) Many workers (l6) have successfully used microbiological methods for the assay of several amino acids in natural materials. Lactobacillus arabinosus has been used as the test organism in many of these studies. Stokes and Gunness (7) have shown that Lactobacillus arabinosus is not satisfactory for the assay of threonine. It was necessary therefore to find another organism which would give a quantitative response to threonine under all conditions. Since Mitchell and Snell (8), Luckey et al. (9), and Teply and Elvehjem (0) have used Xtreptococcusfaecalis, American Type Culture Collection, No. 80 (formerly () referred to as X. lactis R),l for the assay of folic acid and Snell and Guirard () have reported the qualitative amino acid requirements of this organism, the use of X. faecalis for the assay of the threonine content of natural materials was investigated. The amino acid requirements of this organism were studied in detail and suitable techniques were devised for determining the threonine content of various protein materials. EXPERIMENTAL The constituents of the medium used in these studies, other than the amino acids, are identical to those reported by Teply and Elvehjem (0). The amino acid composition of the medium used for the preliminary work was identical to that used in earlier work with Lactobacillus arabinosus (Schweigert et al. ()), with the exception of glycine and serine which were added at 0. and.0 mg. levels per tube, respectively. Snell and Guirard () showed that these amino acids were also required for Streptococcus faecalis. The complete composition of the medium used is given in Table I. The method of preparing the vitamin and the xanthine solutions was Downloaded from http://www.jbc.org/ by guest on November 7, 08 * Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported in part by a grant from the National Live Stock and Meat Board. Niven and associates have shown that Streptococcus Zactis R is an enterococcus, specifically Streptococcus faecalis. 69

70 THREONINE CONTENT OF NATURAL PRODUCTS the same as described by Luckey et al. (9). At the end of the 7 hour incubation period, the lactic acid was titrated with 0. N alkali, thymol blue being used as the indicator. All the other techniques were the same as described by Schweigert et al. (). The requirement of Streptococcus faecalis for each of the amino acids listed in Table I was tested by omitting the appropriate amino acid from Constituent Glucose... Sodium citrate.... KzHPOd.... Salts B MgS0.7HzO.... NaCl... FeSOa.7Hz0... MnS0.H?O.... Adenine.... Guanine.... Uracil.... Xanthine.... Thiamine... Ca pantothenate... Pyridoxine.... Riboflavin.... Nicotinic acid... Biotin... Folic acid concentrate?. TABLE Basal Medium for Assay &dies with Streptococcus faecalis* Amount 00 50 50 mg. per tube 0. 0. 0. 0. 0. 0. 0. Y 6 0.00 0.0 (B,) I Constituent I(+)Glutamic acid.. IAsparagine. I(+)Lysine monohydrochloride... clthreonine. dlvaline dlisoleucine. dlaalanine... I()Cystine. _, I(+)Leucine. dlmethionine. dlphenylalanine. I(+)arginine hydrochloride. E(+)Histidine monohydrochloride... I()Tyrosine.. _. _. l()tryptophane.. Glycine... dlserine... Amount per tube mg. * In adapt,ing this medium for the assay of threonine the folloiving changes in amino acid concentration were made in accordance with the results obtained: arginine, 0.5 mg.; serine, 0.5 mg. ; and leucine,.0 mg. per tube. t Prepared according to the method of Hutchings et al. (). We are indebted to Mr. Keith McCall for supplying this preparation. 0.5 0.8 0. Downloaded from http://www.jbc.org/ by guest on November 7, 08 the basal medium. Several levels of each amino acid were then added and from the curves obtained by plotting the number of cc. of alkali used against the amino acid concentration the amount, of amino acid required for maximum growth was comput,ed. These results and the titration obtained when none of the amino acid was added are given in Table II. Lack of growth as indicated by low acid production when the amino acid was omitted from the medium was t,aken as evidence for the essential character

GREENHUT, SCHWEIGERT,.ND ELVEHJEM 7 of this amino acid. A summary of several assays indicates that alanine, tyrosine, phenylalanine, and glycine are stimulatory in their action. TABLE II Amino Acid Requirement of Streptococcus faecalis I(+)Glutamic IAsparagine I(+)Lysine.. dlaalanine. dlvaline. I (+) Leucine. dlisoleucine dlphenylalanine dlmethionine.. Amino acid * Stimulatory. acid.. CC.. 6.6 5. 9..6.. 7.6.6 Amiio acid per tube ref luired fol I naximur growth %. 0.50 0.0 0.60 * 0.50 0.5 0.50 * 0.5 Amino acid I(+)Arginine.. I(+)Histidine.. I()Tyrosine. dlserine.. Glycine. I () Tryptophane I()Cystine.. dlthreonine.. All present. _. Amino Blank acid per :itration, tube re 0. N quired for NaOH maximum growth I L cc. ms..0 0.0.8 0.0 8. * 5. 0.5 7.9 *. 0.075.7 0.90.5 0.5.5 Downloaded from http://www.jbc.org/ by guest on November 7, 08 I 0 50 00 50 00 MICROGRAM5 dlthreonine FIG.. Typical dlthreonine standard curve When dlthreonine was used as the standard, it was found to be 50 per cent as active as the naturally occurring isomer (d()threonine). In We are indebted to Dr. R. T. Major of Merck and Company, Inc., for supplying some of the samples of threonine used in this work.

7 THREONINE CONTENT OF NATURAL PRODUCTS confirmabion of the work of Stokes and Gunness () the unnatural isomer (Z(f)threonine) was found to be completely inactive. Standard curves were obtained by using dlthreonine over a range of 0 to 50 y per tube. A typical standard curve is shown in Fig.. Samples of muscle and organ tissues, casein, lactalbumin, soy bean oil meal, and ground corn were analyzed for threonine. In all cases the percentage of total nitrogen was determined in duplicate by the Kjeldahl method. The meat samples were also analyzed for moisture and fat. Veal Pork Lamb Beef liver I kidney brain tongue Proximate Tissue Sample No. 5 Ana/ TABLE III :is of Animal Tissues Moisture Fat Protein per cent 7.5 7. 7.0 7.0 5.8 5.8 55.7 9.8 58. 67. 67.7 6. 6. 75. 67. 69.5 66.8 79. 79. 66.6 per cent.7.9 5..5 8.5.8 5..0... 0.0 6.9 6....0.0 S.8 6.9 ger cent. 0.6 9..0 6..0 8.8 6.0 6. 8.6 8.6 6.6 7.5 7.7 9.8 0.0..5.6. 0. 6.0 Downloaded from http://www.jbc.org/ by guest on November 7, 08.0 N hydrochloric acid was tried as the hydrolytic agent for liberating the threonine from the protein, since this reagent was found to be satisfactory for liberating leucine, valine, and isoleucine from natural materials (, ). Maximum liberation of threonine occurred when a gm. sample was autoclaved with 5 cc. of reagent for to 5 hours.,n autoclaving time of 5 hours was used for all subsequent assays. The stability of threonine to this procedure of hydrolysis was studied by adding dlthreonine to several different samples of animal tissue prior to acid treatment. In

GREENHUT, SCHWEIGERT, AND ELVEHJEM 7 twelve different trials the recovery ranged from 89 to 5 per cent, with seven of the values falling between 9 and 0 per cent. Veal Pork Lamb Foodstuff Beef liver kidney brain tongue Ground corn Soy bean oil meal Casein* La&albumin* TABLE Threonine Content of Natural Foodstuffs e Threonine protein in Total N per cm!. per cent per cent 5 I 0.99 0.78 0.76 0.96 IV..8.0..5.0.07.5 0.78.8.56 0.6.8.08 0.66.5.0 0.7.6.56 0.75.5.6 0.80..98 0.80..98 0.70..66 0.68.9.80 0.65.7.8 0.9.6.8 0.90.5.0.0..67 0.6.. 0.7.9. 0.5.6.80 0.8.7.6 0.76.7.56 0.0.7.5.70.9 7.0.5.7 (.8).6.75 5.0 (5.) 5. Threom.sNN of pe7 cent..8.9.. (Average).5.5.6.. (Average).....9. (Average).7.5....6...5.5.9.7.7 Downloaded from http://www.jbc.org/ by guest on November 7, 08 * All values are calculated to 6 per cent N. The values in parentheses are calculated to 5.6 per cent nitrogen for casein (Chibnall et al. (5)) and 5. per cent nitrogen for lactslbumin (Jones and Johns (6)). The proximate analyses of the various animal tissues are given in Table III. In Table IV are summarized the threonine data expressed as per cent of the sample as analyzed, per cent threonine in the protein (calculated to 6 per cent N), and the per cent threonine N of the total N.

7 THREONINE CONTENT OF NdTUR.L PRODUCTS DISCUSSION It is evident from Table II that glutamic acid, asparagine, lysine, valine, leucine, isoleucine, methionine, arginine, histidine, serine, tryptophane, cystine, and threonine give standard responses with Streptococcus faecalis. Alanine, tyrosine, phenylalanine, and glycine seem to have only a stimulatory action under the conditions of these experiments. This work suggests that Streptococcus faecalis can also be used for the estimation of methionine and histidine. The qualitative requirements for the amino acids as determined in the present work differ somewhat from those reported by Snell and Guirard (). They reported that no decrease in the amount of growth resulted when methionine, valine, histidine, and isoleucine were omitted from the basal medium. They also reported that alanine was needed for the growth of Streptococcus faecalis but in the present work only a slight increase in growth resulted when levels of alanine up to 0.5 to.0 mg. per tube were tested; consequently mg. per tube was used in all subsequent work. Undoubtedly the purity of the amino acids and the different assay techniques employed were largely responsible for the differences noted. The reliability of Streptococcus faecalis for the threonine assay has been shown by excellent recoveries and excellent checks obtained with different levels of the test samples. Autoclaving samples of animal tissues for as long as 8 hours with N HCI did not result in an increase or a decrease in the values obtained. The adequacy of the method of hydrolysis was further established by the fact that no increase in values was obtained after autoclaving for hours with N HCl. In the first half hour as much as 75 per cent of the threonine was liberated. The standard was shown to be reliable, since the naturally occurring isomer gave the same growth response to Streptococcus faecalis as twice that amount of dlthreonine. There are no values in the literature for the threonine content of biological material determined by the microbiological method. However, the values obtained in this study compare reasonably well with those obtained by the chemical methods summarized by Block and Bolling (7, 8). The following comparisons can be made when both values are expressed as per cent threonine N of the total N and the microbiological values are given first: casein.7 and.9, lactalbumin.7 and.9, soy bean meal.8 and.9, ground corn.5 and.6. It is interesting from a nutritional point of view that most muscle meats contain about 0.7 to 0.8 per cent threonine or approximately.0 per cent on a protein basis. When the results are expressed as per cent threonine nitrogen of total nitrogen, the values are remarkably constant for muscle meats. The values for the other tissues tend to be slightly higher but Downloaded from http://www.jbc.org/ by guest on November 7, 08

GREENHUT, SCHWEIGERT, AND ELVEHJEM 75 many more values are needed before any significance can be attached to these small differences. It is clearly evident that lactalbumin contains more threonine than casein, because values of 5. and.8 are obtained when the calculations are made on the true nitrogen content. The values for the natural products are minimum when expressed as per cent threonine N of the total N, because some of the total N of these samples is nonprotein N (Beach et al. (9)). It would be valuable to know the exact quantity of protein and nonprotein nitrogen in each sample but many difficulties are involved in such determinations. With the improved methods for the estimation of amino acids now becoming available it may soon be possible to determine directly all of the amino acids in biological materials with considerable accuracy. When this. can be done, the best procedure may be to calculate the N in each amino acid as per cent of total N and the nonamino acid nitrogen can then be estimated by difference. SUMMARY The amino acid requirements of Streptococcus faecalis have been investigated. Leucine, threonine, glutamic acid, asparagine, lysine, valine, isoleucine, methionine, arginine, histidine, serine, tryptophane, and cystine were required and alanine, tyrosine, phenylalanine, and glycine were found to be stimulatory. Xtreptococcus faecalis has been used for the assay of threonine in natural materials. Suitable recovery experiments and a study of methods of hydrolysis established the validity of the assay. The following values were obtained: animal tissue protein. to.7, casein.7, lactalbumin 5.0, soy bean oil meal.9, and whole corn. per cent threonine in the protein (calculated to 6 per cent N). The values are also expressed for each sample on the basis of the per cent threonine N of the total N. Downloaded from http://www.jbc.org/ by guest on November 7, 08 BIBLIOGRAPHY. Kuiken, K. A., Norman, W. H., Lyman, C. M., Hale, F., and Blotter, L., J. Biol. Chem., 6, 65 (9).. Hegsted, D. M., J. Biol. Chem., 6,9 (9).. Schweigert,, B. S., McIntire, J. M., Elvehjem, C. A., and Strong, F. M., J. Biol. Chem., 66, 8 (9).. Schweigert, B. S., Tatman, I. E., and Elvehjem, C. A., Arch. Biochem., 6, 77 (95). 5. Dunn, M. S., Camien, M. Pj., Shankman, S., Frankl, W., and Rockland, L. B., J. Biol. Chem., 66, 75 (9). 6. Stokes, J. L., and Gunness, M., J. Biol. Chem., 6 7,65 (95). 7. Stokes, J. L., and Gunness, M., Science, 0, (95). 8. Mitchell, I. K., and Snell, E. E., CTniv. l exas Pub., No..97, 6 (9).

76 THREONINE CONTENT OF NATURAL PRODUCTS 9. Luckey, T. D., Briggs, G. M., Jr., and Elvehjem, C. A., J. Biol. Chem., 6, 57 (9). 0. Teply, L. J., and Elvehjem, C. A., J. Biol. Chem., 67, 0 (95).. Niven, C. F., Jr., and Sherman, J. M., J. Bact., 7, 5 (9). Niven, C. F., Jr., J. Bat., 7, (9). Gunsalus, I. C., Niven, C. F., Jr., and Sherman, J.M., J. Bact., 8,6 (9).. Snell, E. E., and Guirard, B. M., Proc. Nut. Ad. Xc., 9, 66 (9).. Hutchings, B. L., Bohonos, N., and Peterson, W. H., J. Biol. Chem.,, 5 (9).. Stokes, J. L., and Gunness, M., J. Biol. Chem., 6,75 (9). 5. Chibnall, A. C., Rees, M. W., and Williams, E. F., Biochem. J., 7, 5 (9). 6. Jones, D. B., and Johns, C. O., J. Biol. Chem., 8, 7 (9). 7. Block, R. J., and Bolling, D., J. Am. Dietet. Aesn., 0, 69 (9). 8. Block, R. J., and Bolling, D., The amino acid composition of proteins and foods, Springfield ( 95). 9. Beach, E. F., Munks, B., and Robinson, A., J. Biol. Chem., 8, (9). Downloaded from http://www.jbc.org/ by guest on November 7, 08

THE AMINO ACID REQUIREMENTS OF STREPTOCOCCUS FAECALIS AND THE USE OF THIS ORGANISM FOR THE DETERMINATION OF THREONINE IN NATURAL PRODUCTS Irene Tatman Greenhut, B. S. Schweigert and C. A. Elvehjem J. Biol. Chem. 96, 6:6976. Access the most updated version of this article at http://www.jbc.org/content/6//69.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's email alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/6//69.citation.full.ht ml#reflist Downloaded from http://www.jbc.org/ by guest on November 7, 08

CORRECTIONS On page 79, Vol. 6, No., April, 96, line 5, read 660 mg. for 66 mg. On page 590, Vol. 6, No., June, lq6, footnote, line, read C/(A + Z?) X 00 for C(A + ) X 00. On page 7, Vol. 6, No., January, 96, Table IV, under the heading Threonine in protein, read casein.6 and lactalbumin.8 for 6.8 and 6.6 respectively. On page 0, Vol. 65, No., September, 96, Table III, under the heading Tryptophane in protein, read lactalbumin 6.67 and casein.60 for.88 and.68 respectively.