STUDIES OP THE NITROGEN METABOLISM OP SELECTED MYCOBACTERIA By WILLIAM S . BONIECE A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OP PHILOSOPHY Department of. Bacteriology and Public Health ACKNOWLEDGEMENT The writer wishes to express his sincere appreciation, to Dr. W. L. Mallraann for his assis­ tance and guidance in conducting this investiga­ tion, and to Dr.‘s Sell, Benne, and Leucke of the Department of Agricultural Chemistry for their generosity in permitting the use of certain facil­ ities of their respective laboratories. TABLE OF CONTENTS INTRODUCTION.......................................... 1 EXPERIMENTAL A. Growth and Preparation ofthe B. Microbiological Assays TestOrganisms...... 4 forAmino Acids.......... 5 DISCUSSION........................................... 26 SUMMARY.......................................... 29 LITERATURE CITED............... ..................... ?0 INTRODUCTION. If chemical and physical changes could be detected in a microor­ ganism as a result of its adaptation to a specific substrate under de­ fined conditions, a knowledge of those changes conceivably would pro­ vide important clues as to the nature of the mechanisms, involved in metabolizing the specific substrate. Such studies coupled with an at­ tempt at an integration of the facts learned from the usual enzyme and metabolism studies might clarify the interrelationships among the var­ ious cellular processes, and thereby increase our understanding of the overall function, of the cell as a biological unit. It is a well established fact (1 ,2 ) that within the range of the potential enzymic constitution of an organism, wide variations in the actual enzymic, constitution may occur in response to alterations in the external environment. Such variations in the enzymic, constitution conceivably would be reflected in quantitative changes in the total pro­ tein: of the cell as well as in the amino acid composition of that protein. Hence, the most obvious line of investigation would involve analyses of the amino acid and protein content of microorganisms after growth upon a: variety of media of known composition, under defined conditions. Numerous such investigations have been conducted in the past, the results of which have been carefully reviewed by Camien, Salle, and Dunn (3 )* In general, the results obtained by the earlier workers tended to support the prevalent assumption that the amino acid composition of bac­ terial proteins is fixed and characteristic of the organism concerned, regardless of the conditions of growth. However, the data obtained by these earlier workers could not be highly accurate because of the limi­ tations of the analytical methods employed. With this in mind, the above authors assayed microbiologically four lactobacilli and Escherichia coli after growth upon two different media* They found no significant dif­ ferences in the total nitrogen or in the quantitative content of the five to seven amino acids; assayed for comparison, and concluded that their data strongly support the original hypothesis. Freeland and Gale (4 ) have analyzed a number of bacteria and yeasts for amino acid content, employing-the manometries specific decarboxylase method (5 ) • Two of the organisms studied, E. coli and Aerobacter aerogenes were grown on. various buffered media, and no significant quantitative changes were noted in content of the five amino acids (arginine, lysine, histidine, tyrosine, and glutamic acid) which may be assayed for by the decarboxylase technique. However, in. the most comprehensive study of its nature to date, Stokes and Gunness (6) found evidence that while the amino acid composi­ tion of an organism is quantitatively a stable and characteristic; property Under fixed conditions of growth, it nay vary significantly with changes in. the substrate and other environmental conditions. These authors as­ sayed for ten amino acids by microbiological procedures estimated to be accurate within 10 per cent. The data obtained for the two fungi, Streptomyces griseus and Penioillium notaturn were more striking than those obtained for Bacillus subtilis. Although analyses of the latter organ­ ism showed that significantly more leucine waB present in the cells after growth under one of the four environmental conditions tried, it was felt that most of the small quantitative differences in the other amino acids were within.the limits of error of the analytical method. More recently, Dunlop (7 )> although mainly interested in ascertain­ ing whether E. coli synthesizes amounts of amino acids in excess of its own cellular requirements, also assayed that organism microbiologically for ten amino acids after growth upon two different media. Although he found little difference in the amino acid content of the cells grown on the two types of media? Dunlop concluded that his results varied suf­ ficiently from those of other investigators, who employed different media, to suggest that the composition of the medium may affect the amino acid composition of the organism. In view of the seeming.; contradictions and inconclusiveness of these data regarding the amino acid composition, of microorganisms, particularily those relating to bacteria, it was felt that another, more exhaustive study might prove of value. In the present investigation;two closely related acid-fast bacilli of very simple nutritional requirements were studied. These bacteria have absolutely no vitamin requirements, and are able to synthesize all of their constituent amino acids from ammonium salts in the presence of a suitable carbon source and certain inorganic ions. It seemed logical that the greatest quantitative changes in the amino acid and protein con­ tent of the organisms, if such changes actually occurred, would result from variations in. the source of nitrogen. Hence the organisms were grown on three media which differ, as exclusively as possible, only in the type of substrate nitrogen. After growth the organisms were assayed microbiologically for fifteen amino acids. To procure a more accurate estimate of what would constitute a ’’significant difference” in the anal­ ytical values obtained for any amino acid, in each instance duplicate samples were weighed, hydrolyzed, and analyzed simultaneously throughout every amino acid determination. EXPERIMENTAL A* Growth and Preparation of the Test Organisms The two organisms selected for study were the aerobic, acid-fast bacilli, Mycobacterium avium and Mycobacterium phlei , both of which can be grown in large quantities on simple, liquid media. Growth of these aerobic bacilli on such media appears in the form of a heavy, surface pell­ icle, and the organisms can be harvested by ordinary filtration. The compositions of the three media employed are given in Table I. Medium "A", in which the sole source of nitrogen is ammonium citrate, is an adaptation of the medium recommended by Long and Seibert (8) for the production of tuberculin in large quantities. The substrate nitrogen for medium "B" consists of Bacto-Peptone, a partially hydrolyzed (enzym­ atically) protein, and for medium "0 " it consists of "vitamin free" BactoOasamino Acids (acid hydrolyzed casein) supplemented with tryptophane and cystine. In order to minimize the formation of precipitates in the lat­ ter two media, decreases in the amounts of phosphates and magnesium sul­ fate added were necessary. After pH adjustments, the media were filtered and dispensed into florence flasks of 1 liter capacity, 500 ml to each flask. plugged and autoclaved at 15 lbs pressure for 15 minutes. The flasks were The two myco­ bacteria were cultured on two to six liters of each test medium. In every instance care was taken to float the inoculum on the surface of the broth. The cultures were incubated at 57 °0 for three weeks. Before harves­ ting, the gross cultural characteristics of each organism on the three types of media were studied, and smears were prepared, stained (acid-fast) American Type Culture No.'s 7992 and respectively. and examined microscopically* No significant differences were observed in- the cultural characteristics (macroscopic; or microscopic) of either of the two organisms grown on the three different media. The organisms were harvested by ordinary filtration, washed five to six times on the filter paper with distilled water, placed in an Arnold sterilizer for JO minutes, quick-frozen and dried by sublimation (lyophilization)• The dried bacilli were extracted (continuous Soxhlet extractors) with “peroxide free" ether for 18 - 24 hours, and then dried over anhy­ drous calcium chloride in. a vacuum desiccator for 24 - 48 hours. All nitrogen determinations were performed in triplicate. The 2^0 mg; samples of the dried, defatted bacilli were weighed on. an analytical bal­ ance and digested by the accepted, Kjeldahl-Gunning procedure, employing: conc. H2SO4. in the presence of K2SO4 and CUSO4. Upon liberation the NH^ was collected in known aliquots of 0 .2 N HgSO^, which were then backtitrated with 0.1 N NaOH in the presence of methyl red indicator. B. Microbiological Assays for Amino Acids Stock cultures of the assay organisms, Lactobacillus plantarum 17-J, Leuconostoc mesenteroides P-60, and Streptococcus faecalis were carried as stabs on a solid medium of the following composition per 100 ml: Agar Peptone Yeast extract Na acetate (anhyd.) Glucose K2HPO4 KH2PO4 MgS04*7 HOH NaOl FeS04*7 HOH MnS04«H0H 1.0 0.8 0.1 0.1 1.0 0.05 0.05 0.02 0.001 0.001 0.001 gm Fresh stock transfers were made weekly, incubated for 12 - 18 hours at 57 °C and stored in the refrigerator until needed. Cultures for seeding the assay tubes were prepared from the stock stabs by transfers to tubes of the above medium with the agar omitted. I 0 incubated for 12 - 18 hours at 57 These broth subcultures were the cells were then centrifuged asep- tically, washed once with sterile saline, and resuspended in. saline for seeding the assay tubes, one drop to each tube. The compositions of the basal media employed in the assays are given in Table II. These media were prepared from stock solutions of the various amino acids, salts, purines and pyrimidines, and vitamins. The basal media and organisms used for the various specific determinations are listed in Table III. For each determination ^ wl of the appropriate double strength basal medium, minus the amino acid in question, were added per tube, so that the final volume of each culture was 10 ml. Pyrex culture tubes, 18 x l^O mm, and glass caps were used throughout, and all sterilization was by autoclaving at 15 lbs pressure for 10 minutes. For the standard curve determinations each level was run in tripli­ cate. In every case graded aliquots of the pure amino acid solution were added to the standard tubes, which contained sufficient amounts of dis­ tilled water to make each final volume 5 basal medium. before the addition of the The L-amino acid increment in micrograms per tube of the ascending series for each standard rack is indicated in Table III. Duplicate hydrolysates were prepared for each sample of dried, de­ fatted organisms, and analyzed concurrently throughout every amino acid determination. The enzymatic hydrolysates for the assay of tryptophane were prepared by the method of Wooley and Sebrell (9 )> employing 200 mg samples and digestion with pepsin and trypsin. The acid hydrolysates 7 used throughout the other amino acid analyses were prepared by autoclaving 1 gm samples with 6 N HOI at 15 lbs pressure for 8 hours. Prom the results of preliminary assays it was possible to estimate what dilutions of the hydrolysates, and aliquots thereof, were necessary for the sample values to fall within the best range of each standard curve. In every final determination, the hydrolysates were assayed in duplicate at three different levels of the standard curve. As in the preparation of the standard tubes, the final volume of each sample tube was adjusted to 5 id. with distilled water before adding the basal medium. After inoculation and incubation, at 57°0 for J2 hours, the relative acid production; in each assay tube was determined by transferring the con­ tents of the tubes to beakers with distilled water and titrating to neu­ trality with N/lO NaOH, employing a:.continuous reading Beckman pH meter. Representative standard curves, one for each amino acid determination, are illustrated on. the following pages. In every assay, the final analytical value recorded for each sample of dried, defatted bacilli was calculated by averaging, the results obtained for the duplicate hydrolysates. Each hydrolysate value was determined by averaging the results obtained at the three levels. The amino acid con­ tents of the two test organisms, calculated to 16 % nitrogen, are recorded in Tables IV and V. TABLE I. Growth Media for the Test Organisms Medium (per liter) Constituent Di-ammonium citrate A 10.0 gm B c - - Bacto-Peptone (Difco) - Casamino Acids * - - L-(_)_Gy stine - - 0.05 DL-Tryptophane - - 0.10 8.0 gm 18.0 gm Na2C0^ (anhyd.) 5.0 - - KHgPO^ 5.0 1.0 1.0 k 2h p o 4 - 1.0 1.0 NaCl 2.0 2.0 - MgS04*7 HOH 1.0 0.20 0.20 Ferric ammon. citrate 0.05 0.05 0.05 Glycerol pH (before autoclav.) 50.0 50.0 50.0 6.9 6.6 6 .9 * Difco "Vitamin Free" TABLE II. Media for Microbiological Assay of Amino Acids Medium (per 500 ml) 1(10) Constituent 11(11) 111(12) IV(15 ) V(14 ) 7*5 gm Hg02 treated peptone 5 .0 gm Casein hydrolysate DL -®C -Alanine 200 mg 100 mg 200 mg L (+)-Arginine *HC1 50 5° 100 L-Asparagine 200 200 200 L(-)-Cystine 100 200 200 L(+)-Glutamic acid 4oo 4 oo 400 Glycine 20 20 100 50 50 100 DL-Isoleucine 200 200 200 DL-Leucine 200 200 200 L(+)-Lysine*HC1 *H0H 200 200 200 DL-Methionine 100 100 200 DL-Fhenylalanine 100 100 100 L(-)-Proline 50 50 50 DL-Serine 50 50 200 DL-Threonine 200 200 200 DL-Tryptophane 50 100 100 100 L(-)-Tyrosine 50 100 100 100 DL-Valine 200 200 200 Glucose 20 gm 20 gm 20 gm 20 gm 20 gm Na Acetate (anhyd.) 20 20 20 12 L (+)-Histidine»HC1 «H0H NOTE: 200 mg ( ) after Roman numerals indicate references 100 mg 100 TABLE II. (concluded) Medium (per 500 ml) Constituent I II III 17 V 25 gm Na Citrate‘HOH 6 gm NH4CI 500 mg 500 mg 500 mg 5 500 500 500 200 200 mg 200 200 200 PeS04 *7 HOH 10 10 10 10 10 MnSO^ ’4 HOH 10 10 10 10 10 NaCl 10 10 10 10 10 Adenine sulfate*2 HOH 10 10 10 10 10 Guanine *HG1 *2 HOH 10 10 10 10 10 Uracil 10 10 10 10 10 Xanthine 10 10 10 Thiamine*HC1 0.50 0.50 0.50 0.10 1.0 Pyridoxine *HC1 1.0 1.0 1.0 0.10 2.0 DL-Ca Pantothenate 0.50 O.5O 0.50 0.10 2.0 Riboflavin 0.50 0.50 0.50 2.0 2.0 Nicotinic acid 1.0 1.0 1.0 o.4 c 2.0 0.10 0.10 0.10 0.10 0.01 Biotin 0.001 0.001 0.001 0.2 ^Ag 0.005 Folic acid 0.01 0.01 0.01 6.6-6.8 6.6-6.8 6.6-6.8 k h 2p o ^ 500 mg K2HP04 500 MgS04 *7 HOH .PABA pH (before autoclav.) 0.0015 6.6-6.8 6.9-7*0 TABLE III. Organisms and Media Used for Determining Specific Amino Acids Std. Curve Increment ^ig/tube Organism Glutamic acid L. plantarum 10 I 0.6 - 2.5 Leucine n 5 I 0.3 - 1.U Isoleucine n 5 I -iB** 0.3 - 3.5 Valine tt 5 I *BBf- 0.3 - 1.9 Phenylalanine it 5 I 0.8 - 2.0 II 0.8 - 3.3 II 0.1 - 0.7 Threonine Arginine S. faecalis tt 10 Medium Range of Disagreement* Dupl. Samples Amino Acid * Per cent of the mean value ## + 5 mg NH^Cl and 20 ^ig glutamine per tube (l5»l6) + 20ml tomato eluate (17) per 500 ml medium TABLE III. (concluded) Organisms and Media Used for Determining Specific Amino Acids Amino Acid Organism Histidine S. faecalis Aspartic acid L. mesenteroides Std. Curve Increment ^ig/tube Medium II 10 III, phosphates incr. lix’s Range of Disagreement* Dupl. Samples 0,0 - 1 . 8 0.0 - 3.8 lysine ti 25 Proline ii 5 Tyrosine n 5 III 0.0 - 2.6 2.2 - 8.3 III 0.0 - 3.5 L. plantarum 1 TJ Methionine L. mesenteroides 5 V n l V 0.0 - 6.0 Cystine * Per cent of the mean value . 0 1 ro Tryptophane o . III, + lO^ig Proline/tube 0.0 - 2.7 Representative Standard Curves L(-)-PROLINE 0 10 20 50 Micrograms L(-)-TYROSINE 0 10 20 50 Micrograms 14* Representative Standard Ourves 10 8 6 DL-METHIONINE 4 2 0 20 40 60 80 100 Micrograms 4 5 2 L(-)-CYSTINE 1 0 2 4 6 Micrograms 8 10 15 Representative Standard Curves 10 8 6 DL-ISOLEUCINE 4 2 0 20 60 80 100 Micrograms £ DL-VALINE 100 Micrograms Representative Standard Curves DL-ASPARTIC ACID 80 160 120 200 Micrograms L(+)-GLUTAMIC ACID 0 20 ho 60 Micrograms • 80 - 100 Representative Standard Curves 6 DL-LEUCINE 4 Ml N/lO NaOH 8 2 0 20 40 60 Micrograms 80 100 12 10 NaOH 8 6 N/10 DL-PHENYL ALANINE 4 2 0 20 4o 60 Micrograms 80 100 Representative Standard Curves 80 120 160 200 Micrograms DL-THREONINE 20 60 Micrograms 80 100 Representative H* o Standard M Curves Micrograms M oo Q\ 03 o NaOH NAO Ml NaOH NAO Ml 'O Representative Standard Curves L(-)-TRYPTOPHANE 0 2 4 6 Micrograms 8 10 TABLE IV. Amino Acid Content of Mycobacterium ohlei (calculated to 16 % nitrogen) Substrate Nitrogen Constituent Ammonium Citrate Peptone Casamino Acids Total nitrogen 1 1 .5 6 % 10.64 % Protein (N x 6 .25) 7 1 .0 0 6 6 .5 0 54.06 Arginine 7.42 6 .8 5 5.78 Aspartic acid 4.92 5 .7 8 4.59 Cystine 0 .2 5 1 0.221 0 .2 5 1 8.65 % 10.62 12.54 9.81 Histidine 1 .6 7 1.72 1.52 Isoleucine 4 .5 5 5 .8 1 5.98 Leucine 7 .7 1 6.59 6.54 Lysine 5 .5 0 5.66 2.48 Methionine 1.47' 1.27 1.49 Phenylalanine 5.58 5-59 2.97 Proline 4.80 4.08 4.42 Threonine 4.96 4.56 4.68 Tryptophane 0 .5 8 7 0.145 0 .1 5 8 Tyrosine 2 .5 4 1.86 1.88 Valine 6.95 5 .8 0 5 .8 6 6 5 .5 2 8 6 1 .8 5 6 5 6 .0 0 9 Glutamic acid Total * * Per cent of Total Protein accounted for. TABLE V. Amino Acid Content of Mycobacterium avium (calculated to 1 6 % nitrogen) Substrate Nitrogen Constituent Ammonium Citrate Peptone Casamino Acids 11.54 % Total nitrogen 11.55 % Protein (N x 6 .25) 12*59 58.44 7 0 .8 8 1.62 6.82 7.09 Aspartic acid '4.67 4.04 5.05 Cystine 0 .2 1 5 0.248 0.175 1 0 .2 5 9.11 9.45 Histidine 1.95 1 .8 0 1.98 Isoleucine .4.48 6 .8 5 4.55 Leucine 7.56 7 .0 1 7 .0 8 Lysine 5.95 4.84 2.97 Methionine 1.57 1.40 1.69 Phenylalanine 5.46 5.45 5.48 Proline 4.75 4.26 4.87 Threonine 4.98 4.84 5.25 Tryptophane 0 .2 9 7 0.465 0 .5 5 6 Tyrosine 2.24 1.86 2 .5 0 Valine 6 .8 5 6.45 7 .0 5 64.420 6 5 .4 0 5 65.461 Arginine Glutamic acid Total * 9.55 % * Per cent of Total Protein accounted for. TABLE VI. Variation in Amino Acid Content of Bacterial Proteins With Substrate Nitrogen Bacto-Peptone Amino Acid M. phlei M. avium Oasamino Acids M. phlei M. avium Arginine - 7-95 -1 0 .5 -2 2 .1 - 6 .9 8 Aspartic acid -2 5 .2 -15-5 -1 0 .8 + 7.70 Cystine -11.9 +16.4 0 .0 Glutamic acid +16.2 -ll.l - 7-65 - 8.00 Histidine + 2.95 - 7.70 - 8 .9 8 + 1.54 Isoleucine +27.7 +5 5 .0 -12.5 + 1.12 L eucine -14.5 - 4 .7 6 -1 7 .8 - 5 .8 1 Lysine + 4.57 +2 2 .5 -22.7 -24.8 Methionine -1 5 .6 + 2 .1 9 + 1.58 +23.4 Phenylalanine - 5.51 - 0 .8 7 -17.0 + O .5 8 Proline -1 5 .0 -1 0 .5 - 7.92 + 2.55 Threonine -12.1 - 2.81 -5.65 + 5 ‘02 Tryptophane -62.6 +5 8 .6 -59-2 +8 7 .5 Tyrosine -26.8 -17.0 -26.0 + 2.68 Valine -16.6 - 5*86 -15-7 + 5.22 -1 7 .8 * Expressed as per cent increase or decrease from the analytical values for the organisms grown on ammonium citrate. 2iu FIGURE 1. Variation in Amino Acid Content of Mycobacterium phlei Cell Proteins with Substrate Nitrogen 80 Per cent of Ammonium Citrate Value 60 1*0 Peptone 20 0 \ Casein ^ Hydrolysate -20 -Uo -6 0 -80 o < o •rl bO -P & «=< oR "O •H O «< O p (Q -P P H O 0) a •H T3 •H •P W •H « w a 0 p 0) H O (0 M I o o o a t a ! p -i § a o o Q) sE-t & •P a> a 10 o ■3 > Per Cent of Amnonitm Citrate Value o d o to o o CO o Arginine Aspartic Acid Cell Cystine K O Histidine Proteins Glutamic Acid Isoleucine with Leucine Substrate lysine Methionine Proline Threonine Tryptophane Tyrosine Valine Nitrogen Phenylalanine DISCUSSION The results obtained clearly indicate a variation in the amino acid and protein content of each test organism in response to changes in the substrate nitrogen. To illustrate more Btrikingly the scope of the quantitative changes involved, the variation in content of each amino acid after growth of the organisms upon peptone and casein hydrol­ ysate, respectively, was calculated as per cent increase or decrease from the analytical value found after growth upon ammonium citrate. These per cent variations are recorded in Table VI, and illustrated graphically in Figures 1 and 2. With a few exceptions, each variation is much greater than could be reasonably attributed to the inaccuracy of the particular analytical method. Reference is made to Table III, in which are recorded the ranges of disagreement between duplicate samples, compiled from all the data obtained for each assay procedure. The protein of a bacterial cell is a complex mixture of a large var­ iety of simple and conjugated proteins. Amino acid analysis of the total cell protein is a procedure which disregards changes in the various pro­ teins unless they are accompanied by quantitative changes in the amino acids. Conceivably, other important changes in the characteristics of the cellular proteins, such as the sizes of the molecules and arrange­ ments or sequences of the amino acids contained therein, could occur while the amino acid and/or protein content of the cell remained osten­ sibly the same. However, since significant quantitative differences in the amino acid and protein content were obtained by varying the type of substrate nitrogen, it may be considered that these reflect important modifications in the amount, character and distribution of the cellular proteins. These modifications may be in the structural proteins of the cell as well as the enzymes, although it seems likely that changes in the enzymic con­ stitution were of the greatest importance in the present study. On medium "A” the organisms had to evolve the enzymes necessary for the syn­ thesis of all their constituent amino acids, with an ammonium salt sup­ plying the nitrogen. On medium mB" the organisms presumably evolved pro­ teolytic enzymes to break down the peptone to free amino acids and small peptides, which were then absorbed and utilized in the synthesis of cell­ ular proteins. Oasein hydrolysate, a source high in content of free amino acids and small peptides, supplied those substances for immediate absorption: when the organisms v/ere grown on medium "0” . Since the organisms would have to produce intracellular enzymes for the synthesis of all their constituent amino acids when grown on the ammonium citrate medium, they presumably would have a higher enzymic, and hence total protein, content than when grown on media complete in all the amino acids. This hypothesis is supported by the observation that the total protein of each organism was significantly less (refer to Tables IV and V) when it was cultured on the peptone medium. In the case of M. phlei the total protein decreased still further when that organism was grown on the casein hydrolysate medium. However, on the same medium the total protein of M. avium was observed to increase significantly to a value only slightly less than that found for the organism grown on ammonium citrate. From the data presented in Table VI and illustrated graphically in Figures 1 and 2 it is evident that the observed variations in amino acid content do not necessarily parallel one another, even though the organ­ isms are of closely related species. This is true for those changes which occurred when the two organisms were cultured on the same medium, as well as for those changes observed in the same organism grown on two media which differ primarily in the availability of the amino acids sup­ plied . Of the eleven significant variations (10 % or more for tryptophane, 5 % or more for all others) observed for M. avium and thirteen for M. phlei after growth on the peptone medium, only six were parallel; the arginine, aspartic acid, proline, tyrosine, and valine content de­ creased, while the isoleucine content increased in both cases. eight significant variations observedfor M. avium Of the and thirteenfor M. phlei after growth on the casein hydrolysate medium, only three were parallel; the arginine, glutamic acid, and lysine content decreased in both cases. The concurrent, large increases in content of tryptophane observed for M. avium after growth on the peptone and casein hydrolysate media are of interest, although that amino acid was detected in only small amounts in both organisms throughout the study. The data obtained for M. phlei after growth on the same twomedia reveal concurrent, cant decreases in content of tyrosine, valine, and tryptophane, signifi­ which are especially large in case of the latter. These pronounced differences in the variations of the amino acid content of the two bacilli with the type of substrate nitrogen suggest that fundamental differences may exist between these organisms in the nature of the enzymes and mechanisms involved in their nitrogen metab­ olism. SUMMARY 1. Significant quantitative changes in the amino acid and protein content of two related acid-fast bacilli were induced by varying the type of substrate nitrogen. 2. It may be considered that these changes reflect important modi­ fications in the character and distribution of the enzymes involved in the nitrogen metabolism of the two organisms. The pronounced differences observed in the variations of the amino acid content of the two bacilli suggest that fundamental differ­ ences may exist between these organisms in the nature of the enzymes and mechanisms involved in their nitrogen metabolism. 50. LITERATURE CITED 1. Gale, E. P. Factors influencing the enzymic activities of bacteria. Bact. Rev. ]t 159 (1945) 2. Spiegelman, S. Nuclear and cytoplasmic factors controlling enzymic; constitution. Cold Spring Harbor Symposia Quant. Biol. 1 1 : 256 (1946) 5. Camien, M. N., Salle, A. J., and Dunn, M. S. Investigations of amino acids, peptides, and proteins XXXII. Percentages of some amino acids in lactobacilli. Arch. Biochem. 8 : 67 (1945) 4. Preeland, J. C., and Gale, E. F. The amino acid composition of cer­ tain bacteria and yeast. Biochem. J. 4l: 155 (1947) 5 . Gale, E. P. Studies on bacterial amino acid decarboxylases 5* The use of specific decarboxylase preparations in the estima­ tion of amino acids and in protein analysis. Biochem. J. 22: 46 (1945) 6. Stokes, J. L., and Gunness, M. The amino acid composition of micro­ organisms. J. Bact. ^2: 195 (1946) 7. Dunlop, S. G. The synthesis of amino acids by Escherichia coli in pure culture. J . Bact. £8: 457 (19495 8. Long, E. R., and Seibert, P. B. I. A non-protein medium suitable for the production of tuberculin in large quantity. Am. Rev. Tuberc. 1^5: 595 (1926) 9. Wooley, J. G., and Sebrell, W. H. Two microbiological methods for the determination of L(-)-tryptophane in proteins and other complex substances. J. Biol. Ohem. 157j l4l (1945) 10. Schweigert, B. S., McIntyre, J. M., Elvehjem, 0. A., and Strong, P. M. The direct determination of valine and leucine in fresh ani­ mal tissues. J. Biol. Ohem. 155? I85 (1944-) 11. Greenhut, I. T., Schweigert, B. S., and Elvehjem, 0. A. The amino acid requirements of Streptococcus faecalis and the use of this organism for the determination of threonine in. natural products. J. Biol. Ohem. 162: 69 (1946) 12. McMahan, J. R., and Snell, E. E. The microbiological determination of amino acids I. Yaline and arginine. J. Biol. Ohem. 152: 85 (1944) 15. Krehl, W. A., Strong, F. M., and Elvehjem, 0. A. Determination of nicotinic acid. Ind. Eng. Ohem.(anal, ed.) 1^: 471 (1945) 51. LITERATURE CITED (concluded) 14. Lyman, C. M., Moseley, 0., Butler, B., Wood, S., and Hale, F. The microbiological determination of amino acids III. Methionine. J. Biol. Chem 166: 161 (1946) 15. Lyman, C. M., Kuiken, K. A., Blotter, L., and Hale, F. The microbio­ logical determination of amino acids II. Glutamic acid. J. Biol. Chem. l ^ s 595 (1945) 16. Hac, L. R., Snell, E. E., and Williams, R. J. The microbiological determination of amino acids II. Assay and utilisation of glutamic.; acid and glutamine by Lactobacillus arabinosus. J. Biol. Chem. l^g: 275 (19451 17* Kuiken, K. A., Norman, V/. H., Lyman, C. M., Hale, F., and Blotter, L. The microbiological determination of amino acids I. Valine, leucine, and isoleucine. J. Biol. Chem. 151: 615 (194-5) 4