flow). (.3... hr... .«fiz r/Zh... v. J..l rpflrh/ inn..." kn?» .1 OF UM ... .a..» bi m4. 4.». .. . ..~..w.o .. .5... WEI cf 91': NE reg I. «9...:- 29...}: .3 . 1.7... / xrdwmvflrrnaf .4... , s? .m .mz, .4. 2 ND: 1% menu's t the: Arm I' IN ”ST HAN afi CATABOUSM A 5,, 4 CHIGAN E. m I._. T . . .4: Ir- T . . “Govt.“r/ . vrflv.I1/fl..el.xl I... ., . .. . franc... «wafakmitfi. . . . . .. .arfi'nrwuvc.r Erwin. . (“(!r.:.‘a I .u. . . . . j . . 21f... 7..-. .. . u s .. . f a. . ,. 5...... _ p . .... inc... Hangar”... .. 4...“ :5. 9a:l..rt.1:<.l..7 5.... 5... .2! This is to certify that the thesis entitled The Catabolism and Transport of Tryptophan in Bacillus megaterium presented by Reynard R. Bouknight has been accepted towards fulfillment of the requirements for Ph- D- degreein Microbiology AM!) 1% A/;/ Major professor Date 13114544913— 0-7639 -. y’a’m‘cnu *4, I 4‘ -. 3‘ l W’ ‘ a ' a . ¢ 3 ABSTRACT THE CATABOLISM AND TRANSPORT OF TRYPTOPHAN IN BACILLUS MEGATERIUM BY Reynard R. Bouknight Bacillus megaterium was able to grow and sporulate in a medium which contained L-tryptophan as the sole car- bon, nitrogen, and energy source. This growth was accompanied by the production of a brown pigment which was not necessary for sporulation and which may have been catechol or a product thereof. The identification of intermediates such as kynurenine, anthranilic acid, and catechol indicated that this organism used the anthranilic acid pathway for tryptophan degradation. A mutant which was unable to grow on tryptophan accumulated anthranilic acid when exposed to the former, thus providing additional proof that this pathway was used. Also, cells which were grown on L-tryptophan oxidized the intermediates kynurenine, alanine, anthranilic acid, and to a lesser extent, catechol. The presence of enzymes such as tryptophan oxygenase (E.C. 1.13.1.12), kynureninase (E.C. 3.7.13.3), and catechol oxygenase Reynard R. Bouknight ( E.C. 1.13.1.1) in cell-free extracts provided addi- tional evidence that E; megaterium oxidized tryptophan via the anthranilic acid pathway. Bacillus tryptOphan oxygenase was inhibited by sodium azide, potassium cyanide, and hydroxylamine indicating that the enzyme had a functional heme group. There was always a lag before maximal activity was attained during tryptophan oxygenase assays, similar to that seen in assays of this enzyme from other sources. Although L-tryptophan was oxidized, D-tryptOphan was not a substrate for trypto- phan oxygenase and the D-isomer did not inhibit this enzyme. Preliminary evidence was obtained which indi- cated that Eagillu§_kynureninase may attack N—formyl- kynurenine in addition to kynurenine which would re— sult in the alternate pathway: L-tryptophan + N-formyl- L-kynurenine + N-formylanthranilic acid + catechol ++ succinate + acetate. Formamidase (E.C. 3.5.1.9) and anthranilate hydroxylase were not present in extracts. Tryptophan catabolism was inducible in E. megaterium and was subject to catabolite repression by sucrose, glucose, and glutamate. Alanine, aspartic acid, asparagine, and arginine did not cause repression. Both kynurenine and anthranilic acid seemed to act as inducers, indicating that part of the pathway was controlled by sequential induction. Kynurenine in- duced both tryptophan oxygenase and kynureninase. Reynard R. Bouknight Anthranilate apparently induced anthranilate hydroxylase. The synthesis of tryptOphan oxygenase and kynureninase was not coordinate. Tryptophan-grown cells of E; megaterium also formed a permease system which transported both D- and L-tryptophan. Arginine-grown cells contained little tryptophan permease activity, showing that the system was inducible. At least a portion of this permease was specific for tryptophan and would not transport leucine, arginine, nor phenylalanine. Sodium azide effectively prevented tryptophan transport. Arginine repressed tryptophan permease as well as leucine per- mease and phenylalanine permease. Kynurenine was a more effective inducer of the tryptOphan transport system than D- or L-tryptophan. THE CATABOLISM AND TRANSPORT OF TRYPTOPHAN IN BACILLUS MEGATERIUM BY Reynard R. Bouknight A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1974 To LaClaire - my lover, colleague, and friend ii ACKNOWLEDGEMENTS i1. Harold L. Sadoff has been a source of intel- lectual stimulation throughout these studies. I thank him for his guidance, understanding, and patience. I am also grateful to Dr. Costilow for his helpful sugges- tions during Dr. Sadoff's sabbatical and to Dr. Brubaker for our thought-provoking discussions during that period. I wish to thank Dr. Velicer for his encouragement and Dr. Bieber for his challenging biochemistry courses through which I learned many of the techniques used in these studies. I am truly indebted to Barbara Shimei who prepared the figures in this thesis and assisted in the characterization of the mutants and the prepara- tion of the manuscript. I am also grateful to Dorothy Okazaki and Hazel Green for assistance in the preparation of the manuscript and to Jerry Stelma who has been a true fidcmul. iii TABLE OF CONTENTS DEDICATION . . . . . . . . . . . . . . ACKNOWLEDGEMENTS . . . . . . . . . . . TABLE OF CONTENTS . . . . . . . . . . LIST OF TABLES . . . . . . . . . . . . LIST OF FIGURES O O O O O O O O O O I INTRODUCTION . . . . . . . . . . . . . LITERATURE SURVEY . . . . . . . . . . Pathways of Tryptophan Catabolism Indole Pathway . . . . . . . . . Kynurenic Acid Pathway . . . . . Anthranilic Acid Pathway . . . . Enzymes of the Anthranilic Acid Pathway Tryptophan Oxygenase . . . . . . Kynureninase . . . . . . . . . . Anthranilate Hydroxylase . . . Products of Tryptophan Metabolism . Identification of Intermediates . Pigment Production . . . . . . . Nicotinamide Adenine Dinucleotide Tryptophan Products and Disease Tryptophan Transport . . . . . . . MATERIALS AND METHODS . . . . . . . . Organisms and Cultivation . . . . . Organisms . . . . . . . . . . . . Media . . . . . . . . . . . . . . Inoculation and Growth . . . . . Radioisotopes and Sources . . . . Scintillation Counting . . . Chemicals . . . . . . . . . . . Protein Concentration . . . . . . iv Page ii iii iv vii viii 29 29 29 29 3O 31 31 31 32 Growth, TryptOphan Utilization, and Pigmentation on Trp-S . . . . . . . . . . . . . . . . . . Isolation of "Late" Pigment . . . . . . . . . Isolation of Mutants . . . . . . . . . . . . . D-Cycloserine-Resistant Mutants . . . . . . Tryptophan Mutants . . . . . . . . . . . . . Chromatography . . . . . . . . . . . . . . . . Paper Chromatography . . . . . . . . . . . . Thin-Layer Chromatography . . . . . . . . . Manometry . . . . . . . . . . . . . . . . . . Preparation of Whole Cells . . . . . . . . . Warburg Constant-Volume Respirometry . . . . Enzyme Assays . . . . . . . . . . . . . . . . Preparation of Cell-Free Extracts . . . . . Assays . . . . . . . . . . . . . . . . . . . Tryptophan-2,3-Oxygenase . . . . . . . . . . Kynureninase . . . . . . . . . . . . . . . . Catechol Oxygenase . . . . . . . . . . . . . Other Enzymes . . . . . . . . . . . . . Time Course of Tryptophan Oxygenase and Kynureninase Synthesis . . . . . . . . . . . Amino Acid Transport . . . . . . . . . . . . . Competition Experiments . . . . . . . . . . RESULTS . . . . . . . . . . . . . . . . . . . . . Pigment Production . . . . . . . . . . . . . . Tryptophan Oxidation . . . . . . . . . . . . . Pathway Intermediates . . . . . . . . . . . TryptOphan Mutants . . . . . . . . Oxidation of Tryptophan and Intermediates by Whole Cells . . . . . . . . . . . . . . . Characterization of the Pathway . . . . . . Co-Metabolism of Kynurenine and Anthranilate Enzymes of the Anthranilate Pathway . . . . . Tryptophan Oxygenase . . . . . . . . . . . . Kynureninase . . . . . . . . . . . . . . . . Catechol Oxygenase . . . . . . . . . . . . . Other Enzymes . . . . . . . . . . . . . . . Catabolite Repression . . . . . . Induction and Synthesis of Tryptophan Oxygenase and Kynureninase . . . . . . . . . Induction of Tryptophan Oxygenase . . . . Kynurenine as an Inducer . . . . . . . . . . Non-Coordinate Induction of TryptOphan Oxygenase and Kynureninase . . . . . . . V Page 32 33 34 34 35 37 37 38 38 38 39 4O 40 40 41 41 42 42 42 43 44 45 45 56 56 61 73 74 89 95 95 103 108 108 108 110 110 113 115 Tryptophan Permease . . . . . . . . . . . . Induction of Tryptophan Permease . . . . Transport of D- and L-Tryptophan . . . . Arginine, Leucine and Phenylalanine Competition . . . . . . . . . . . . . . Transport of Phenylalanine, Repression by Arginine . . . . . . . . . . . . . . Product Induction of Tryptophan Permease DISCUSSION . . . . . . . . . . . . . . . . . . The Anthranilic Acid Pathway of B. megaterium . . . . . . . . . . . . . . . Alternate Pathway . . . . . . . . . . . Selective Advantage of the Anthranilate Pathway . . . . . . . . . . . . . . . . Pathway Controls . . . . . . . . . . . . Post-Translational Controls . . . . . . . Metabolite Repression . . . . . . . . . . Pigment Production . . . . . . . . . . . Bacillus Tryptophan Permease . . . . . LIST OF REFERENCES I O O O O I O O I O O I O 0 vi Page 119 119 122 122 126 130 138 140 142 143 145 147 148 149 151 153 LI ST OF TABLES Table Page 1. Energy sources and pigmentation . . . . . . . 49 2. Identification of intermediates . . . . . . . 59 3. Accumulation of anthranilate by Brs-S . . . . 65 4. Mutants of B; megaterium unable to grow on tryptophan . . . . . . . . . . . . . . . . 68 5. Induction and repression of tryptophan oxygenase and kynureninase . . . . . . . . 109 6. Induction of tryptophan oxygenase and kynureninase by metabolic products . . . . 114 7. Non-coordinate induction of tryptophan oxygenase and kynureninase . . . . . . . . 118 vii Figure 1. 10. 11. 12. LIST OF FIGURES Page Pathways for tryptophan catabolism in bacteria . . . . . . . . . . . . . . . . . 6 Growth and pigment production of B. megaterium in "Pig" broth . . . . . . . . 46 Growth, tryptophan utilization, and pigmentation of B; megaterium in Trp-S beth 0 o o o o o o o o o o o o o o 51 Isolation of "late" pigment . . . . . . . . 54 Production by B: megaterium of N- formylkynurenine and kynurenine . . . . . 57 UV spectra of tryptophan catabolites . . . . 62 Growth of B; megaterium ATCC 19213, Brs-S, and Brs-12 on arginine . . . . . . . . . . 66 Oxidation of intermediates of the anthranilic acid pathway by B. megaterium ATCC 19213, Brs-8, Brs-lO, Brs-12, and Brs-15 . . . . . . . . 7O Oxidation of L-tryptophan and intermediates of the anthranilic acid pathway by whole cells of B; megaterium grown on L— tryptophan . . . . . . . . . . . . . . . . 75 Oxidation of L-alanine and other inter- mediates of the anthranilic acid pathway by whole cells of B; megaterium grown on tryptophan . . . . . . . . . . . . . . 77 Oxidation of anthranilic acid and kynurenic acid by stationary whole cells of B. megaterium grown on L-tryptophan . . . . . 79 Induction of tryptophan oxidation . . . . . 82 viii Figure Page 13. Oxidation of L-tryptophan by cells grown on tryptophan plus L-alanine and tryp- tophan plus L-aspartic acid . . . . . . . . 84 14. Oxidation of L-tryptOphan, anthranilic acid, and L-kynurenine by cells grown on L-tryptOphan plus L-arginine . . . . . . 87 15. Growth of B. megaterium on L- -tryptophan, L- kynuren1ne, or anthranilic acid . . . . . 9O l6. Oxidation of L-tryptophan, anthranilic acid, and L-kynurenine by cells of B. megaterium grown with sucrose as the sole carbon source. . . . . . . . . . . . . 93 17. Tryptophan oxygenase, kynureninase, and anthranilate hydroxylase in cell-free extracts of B; megaterium . . . . . . . . . 96 18. UV spectrum of the product of trytophan oxygenase activity. . . . . . . . . . . . 100 19. Inhibition of tryptophan oxygenase by sodium azide, potassium cyanide, and hydroxylamine . . . . . . . . . . . . . . . 101 20. The effect of D-tryptophan on tryptophan oxygenase activity . . . . . . . . . . . . 104 21. UV spectrum of the product of kynureninase activity . . . . . . . . . . . . . . . . . 106 22. Synthesis of tryptophan oxygenase . . . . . . 111 23. Synthesis of tryptophan oxygenase and kynureninase . . . . . . . . . . . . . . . 116 24. Induction of tryptophan permease . . . . . . 120 25. Competition of unlabeled D— and L- tryptophan for tryptophan permease A O O O O O I O O O O O O I O O O O O O O 124 B . . . . . . . . . . . . . . . . . . . . 125 ix Figure 26. 27. 28. 29. Competition of unlabeled L-arginine, L- leucine, and L-phenylalanine for tryptophan permease A C O O I O I O I O O O O O O O O B I I O O O O O O I O O O O O O O O Repression of tryptophan permease and phenylalanine permease by arginine . Repression of leucine permease and phenylalanine permease by arginine . Induction of tryptOphan permease by L-kynurenine . . . . . . . . . . . . Page 128 129 131 133 136 INTRODUCTION The biochemical aspects of cellular differentiation and regulation persist as intriguing areas of research because they pose problems involving temporal gene expression, in addition to transcriptional, translational, and post- translational controls. The recent rapid advances in genetics, biochemistry, and molecular biology have depended to a large extent on the use of bacteria as experimental systems. These have the following advantages: (1) ethical issues are avoided, (2) bacteria are maintained inexpen- sively, (3) bacteria can be easily grown and in massive numbers in a matter of hours, (4) specific mutants and recombinants can be selected and studied by manipulating nutrients or growth conditions—-conditional mutants are especially valuable, (5) the simple bacterial chromosome allows analysis and mapping through the techniques of transduction, transformation, and conjugation, and (6) information gained through the use of these simpler procaryotes can be applied to the more complex eucaryotes due to the unity of biology at the molecular level. The metabolism of lactose in Escherichia coli is probably the most extensively studied bacterial system of control. Studies on lactose utilization in B; coli 1 have led to the development of the basic concepts of molecular biology and to our understanding of information transfer in cells. Lactose can be used by B;_gg££ as its sole source of carbon. The bacterium makes two proteins which are essential for lactose metabolism. One is a galactoside permease which is found in the bacterial membrane and directs the transport and accumulation of lactose within the cell. The other is B-galactosidase, an enzyme inside the cell which catalyzes the hydrolysis of lactose into its two component monosaccharides, glucose and galactose. When £1.2211 is grown on most carbon sources, other than lactose, these two proteins are pre- sent in amounts of the order of ten or so molecules per cell. However, in the presence of lactose or of other B-galactosides, the rate of synthesis of these proteins is increased by as much as one thousand—fold. A third protein which is induced, thiogalactoside transacetylase, is not essential for lactose utilization. Its role is unknown. The genes which determine the ability of £1.2211 to make and to regulate the synthesis of these proteins are clustered in a small region of the §;.EQ££ chromosome. The structures of the three proteins are determined by the "2" gene (B-galactosidase), the "y" gene (permease) and the "a" gene (thiogalactoside transacetylase). The transcription of these three genes is initiated at a single site, the lactose promoter. A group of genes thus transcribed into a single polygenic messenger ribonucleic acid species is termed an operon. In the absence of an inducer of the lactose operon, the transcription of the genes is prevented by the presence of the repressor gene. The repressor most probably acts to stop transcription by binding to the Operator deoxyribonucleic acid (DNA) which lies between the promoter and the structural genes. The inducer allows transcription of the operon by binding to the repressor and causing its release from the operator. Although the lactose operon is the prototype of bacterial regulatory mechanisms other systems exist and are sometimes more complex. Regulation of DNA synthesis, ribonucleic acid (RNA) synthesis, and protein synthesis are a few examples. Also, the breakdown of many energy sources is not necessarily controlled by a regulatory unit such as the operon. Other energy sources may introduce complicating factors which are not involved in the degradation of sugars. For instance, how does the cell handle a substrate which has functions other than energy generation? More specifically, how does a cell degrade amino acids for energy and at the same time conserve an amino acid pool for protein synthesis? It is precisely the answer to this question which was the basis of the study reported in this dissertation. During sporulation, the cell uses amino acids as an energy source since carbohydrates such as glucose are depleted before sporulation begins. In Bacillus cereus, tryptophan catabolism may be involved in sporu- lation since the enzymes for tryptophan utilization were not formed until the cell began to differentiate into a spore and glucose apparently did not repress enzyme formation (90). Also, tryptophan catabolism leads to the production of pigments and pigmentation is associated with sporulation in Bacillus subtilis (8, 31, 101). An increased understanding of the breakdown of tryptophan could aid in our knowledge of how the cell uses, yet conserves, this building block and how tryptophan degrad- ation is related to sporulation. This thesis attempts to provide new insight in these areas. The pathway for tryptophan oxidation and the tryptophan transport system of Bacillus megaterium are described. These are compared to tryptophan pathways and transport systems in other bacteria for an overall View of adaptation, selection and mechanisms of control. Pigment production is also described. LITERATURE SURVEY Pathways of Tryptophan Catabolism Bacteria degrade tryptophan by one of three path- ways. If the indole pathway is used the indole ring is conserved (127). If the kynurenic acid or anthranilic acid pathways are used the indole ring is cleaved (110), (see Figure 1). All three of these pathways may be operative in Bacillus (46, 90, 100). Indole Pathway Hopkins and Cole (47) first described the pro- duction of indole from tryptophan in 1903. Wood SE 31° (127) later found that the pathway consists of essentially one step in which the enzyme, tryptophanase, cleaves the alanine moiety from tryptophan with indole, pyruvate, ammonia, and water as the resulting products. The tryptophanase reaction was originally thought to be irreversible, but recent data show that the reaction is reversible in the presence of high concentrations of pyruvate and ammonium chloride (123). Both forward and reverse reactions probably proceed through an enzyme- bound a-aminoacrylic acid (75). 5 Figure l.--Pathways for tryptophan catabolism in bacteria. 7 INDOLE INDOLE PATHWAY u «coon mil”: N u TRYPTOPHAN ocuzencoon NH, ANTHRANILIC ”5° ACID FORMYLKYNURENINE PATHWAY 8 OOH OCch’CHCOOH (I (I NH, "2 ANTHRANILIC KYNURE NINE ACID II 0:: CATEC HOL 8 ¢>toou kvxoon CIS.CIS‘MUCONIC ACID 8 COOH I cu, I CO I"2 CH2 I COOH B—KETOADIPIC ACID coon I coon ca, '5 gm: + é"? N"; + ”2f‘\?° COOH H00 COOH KYNURENIC ACID PATHWAY KYNURENI C Tryptophanase is found in Escherichia coli from which it has been crystallized (78). The enzyme is a tetramer with a molecular weight of 223,000 and is made up of four polypeptide chains (63). Each of these sub- units has a molecular weight of 55,000. Quantitative end group determinations and examination of cyanogen bromide cleavage products showed that the subunit contains a single peptide chain with methionine and valine, respec- tively, as the amino and carboxyl terminal residues, and one binding site for pyridoxal phosphate. Edman degrada- tions showed the amino terminal sequence to be Met-Glu- Asn-Phe-Lys (54). B:_ggl£ tryptophanase is inducible and control mutations (ESE) affect its synthesis. The addition of adenosine 3', 5'-phosphate (cAMP) by itself to SEE mutants does not induce tryptophanse, nor does the addition of tryptophan alone. A combination of cAMP plus tryptophan does induce tryptophanase. It has been postu- lated that cAMP is necessary for the translation of the tryptophanase messenger ribonucleic acid. Results from .225 mutants support this hypothesis but also indicate that the control of the indole pathway is more complex and cannot be fully explained by a simple model (24). Bacillus alvei also oxidizes tryptophan via the indole pathway. Tryptophanase has been isolated from this organism and exhibits the expected spectrum of pyridoxal- 5'-phosphate dependent reactions. Tryptophanases from B:_ggl£ and B; Blygi_are similar in that both enzymes are tetramers held together by non-covalent bonds and both enzymes have four pyridoxal phosphate cofactors per molecule. However, pyridoxal phosphate is required for the integrity of B; 3123; tryptophanase but not for B; ggli tryptophanase. B;_B£ygi tryptophanase also requires ammonium or potassium ions whereas B; coli tryptophanase does not (46). gynurenic Acid Pathway Both the kynurenic acid and anthranilic acid pathways involve cleavage of the indole ring. The kynurenic acid pathway was originally called the "quinoline" pathway by Stanier BB 31° (111). However, this name is misleading since quinolinic acid is not produced via this route. The more descriptive term "kynurenic acid pathway" will be used. The initial step is the cleavage of the pyrrole ring between carbons two and three by tryptophan—2, 3- dioxygenase (E.C. 1.13.1.12). The product, N-formyl- kynurenine, is attacked by formylkynurenine formamidase (E.C. 3.5.1.9) with the elimination of the formyl group and the formation of kynurenine. Transamination of the amino group of the alanine moiety occurs via kynurenine transaminase (E.C. 2.6.1.7) with subsequent ring closure to form kynurenic acid (48). 10 Stanier and Tsuchida (108) were the first to study the kynurenic acid pathway in 1949. Using an unidentified Pseudomonas sp. and adaptive manometry experiments, they demonstrated that kynurenic acid was oxidized by cells which were grown on D- or L-tryptophan. They coined the phrase "simultaneous adaptation" to explain this phenomenon. Later, a survey of twenty- seven strains of Pseudomonas showed that five used the kynurenic acid pathway (110). Each strain using this pathway oxidized both D- and L-tryptophan. Tashiro SE _£. (118) suggested that D-kynurenic acid is formed from D-kynurenine through a transaminase reaction. Miller BE B1. (69) were the first to demonstrate kynurenine transaminase activity in bacterial extracts. Kynurenic acid formation was shown to depend on the presence of d-Ketoglutarate. Mason (66) later found a similar enzyme in rat kidney. This enzyme also converted kynurenine to kynurenic acid and required pyridoxal phosphate and d-ketoglutarate or oxalacetate. Hayaishi g: 31. (41) studied the oxidation of kynurenic acid by extracts of Pseudomonas fluorescens and found that the products were L-glutamic acid, D— and L-alanine, and acetic acid. Radioisotope studies indicated that the carbon skeleton of glutamic acid may be provided directly from the benzene moiety of kynurenic acid. The pyridine ring of kynurenate is the source of the carbon atoms of acetic acid and alanine (48). ll Kynurenate hydroxylase (kynurenate NAD(P) H:02 oxidoreductase (hydroxylating E.C. 1.14.1.3) oxidizes kynurenic acid to form 7,8-dihydrokynurenic acid-7,8-diol. This enzyme was purified from Pseudomonas and requires ferrous ion and flavin nucleotide for maximum activity. It is inhibited by gfphenanthroline and d,d'-dipyridyl (72). 7,8-Dihydrokynurenic acid-7,8-diol is dehydrogenated to form 7,8-dihydroxykynurenic acid by NAB-dependent 7,8- dihydrokynurenic acid-7,8-diol dehydrogenase (5, 116). 7,8-Dihydroxykynurenic acid is converted to 5-(Y-carboxy- y-oxopropenyl) 4,6-dihydroxypicolinic acid. The latter compound is reduced by a NADP-linked dehydrogenase to S-Y-carboxy-y-oxopropyl)-4,6-dihydroxypicolinic acid which is then decarboxylated to 5-(B—formylethyl)-4,6,- dihydroxypicolinic acid. Further oxidation of the latter compound yields 5-(B-carboxyethyl)—4,6-dihydroxypicolinic acid (61). Tremblay 3E Bl. (119) showed that the first four enzymes of the kynurenic acid pathway of B; fluorescens were induced in a sequential pattern in order of their position along the pathway. The non-metabolizable analogue, d-methyl-D,L-tryptophan, caused a measurable elevation in the levels of the first three enzymes. It was believed that this induction was due to accumulation of endogenous tryptophan. However, Rosenfeld and Feigelson (95) found that oxidation of tryptophan through the kynurenic acid 12 pathway was product-induced by kynurenine. The ratio of igyyiyg induced activities of tryptophan oxygenase and kynurenine formamidase was alterable under various circumstances, indicating that these two enzymes were either non-coordinately induced at the transcriptional level or were each subject to separate translational regulation. 7-Azotryptophan, which lacked inducing ability per se, when added to media containing trypto- phan caused an augmented inducing efficiency of tryp- tophan oxygenase. The authors called this "synergistic induction". The possibility that 7-azotryptophan may increase the specific activity of tryptophan oxygenase by binding to and stabilizing the enzyme was not considered. Bacillus subtilis and mammals also produce kynurenic acid by oxidizing tryptophan (110, 55). Mammals are unable to further oxidize the kynurenic acid. Oxidation of this compound has not been studied in B; subtilis. Anthranilic Acid Pathway This pathway was originally referred to as the "aromatic pathway" by Stanier SE BB. (111). The more descriptive term, "anthranilic acid pathway", will be used to avoid the confusion which results from usage of the former term. The first two steps in the anthranilic acid pathway are exactly the same as in the kynurenic l3 acid pathway with cleavage of the indole nucleus between carbons two and three and elimination of carbon two. Therefore, N-formylkynurenine and kynurenine are common intermediary metabolites in the two pathways. As was previously mentioned, in the kynurenic acid pathway kynurenine is transformed into kynurenic acid by kynurenine transaminase. In the anthranilic acid pathway kynurenine is transformed into anthranilic acid by kynureninase (E.C. 3.7.1.3) (110). Anthranilic acid is oxidized by anthranilic acid hydroxylase to form catechol. At this point the anthranilic acid pathway merges with the oxidative pathways of mandelic acid, phenol, and benzoic acid. The common central inter- mediary metabolite for all of these pathways is catechol (105). Its benzene ring is cleaved to form cis,cis- muconic acid which is transformed into B-ketoadipic acid (109). The end products of B-ketoadipic acid oxidation are succinic acid and acetic acid. In 1949, Suda EE.§£° (113) studied the adaptive patterns caused by exposure of an unidentified Pseudomonas to tryptophan. They deduced the following metabolic sequence: L-tryptophan+L-kynurenine+anthranilic acid+ catechol. Using cell-free extracts in 1951, Hayaishi and Stanier (43) confirmed the results of Suda SE BL. and also found that catechol was oxidized to B-ketoadipic acid via cis,cis-muconate. The enzyme system which 14 converted anthranilate to catechol was very unstable. Twenty out of the twenty-seven strains of Pseudomonas studied by Stanier EE.E$° used the anthranilic acid path- way (110). Although D-tryptophan is not typically metabolized by strains of bacteria which use this path- way, Behrman and Cullen (6) found one Pseudomonas species which oxidized D-tryptophan via tryptophan racemase (E.C. 5.1.1.9). The D-isomer was transformed first into the L-isomer before oxidation occurred. More recently Alcaligenes eutrophus and Bacillus cereus have been shown to use the anthranilic acid pathway for tryptophan oxidation. After growth at the expense of L-tryptophan cells of B: eutrophus respire L-tryptophan, L- kynurenine and anthranilate at high and identical rates but are unable to respire catechol or kynurenate at significant rates. The fate of anthranilate was not determined (51, 52). Cis,cis-muconate, cis,trans- muconate, and trans,trans-muconate can also be utilized by this organism. Prasad and Srinivasan (90) isolated two intermediates from a sporulating culture of B; cereus and identified them as kynurenine and anthranilic acid by their spectral properties, paper chromatography and staining reactions. Tryptophan oxygenase, formamidase, and kynureninase were detected in extracts of cells exposed to L—tryptophan. Attempts to determine patterns of enzyme production were hampered due to the use of a 15 complex medium containing energy sources such as glucose and glutamate which have been shown to repress certain enzyme systems, and in particular, the system of tryp- tophan catabolism. Earlier studies indicated a functional anthranilic acid pathway in B; subtilis (100) although Yanofsky was unable to confirm this (128). Other species of microorganisms which use the anthranilic acid path- way are among the genera Xanthomonas, Neurospora, Nocardia, Streptomyces, and Saccharomyces (62). Of the three pathways for tryptophan catabolism, the anthranilic acid pathway is the most common. It is also found in man, mammals, birds, and insects. In 1924 Supniewski (114) showed that a strain of Pseudomonas pyocaneus formed carbon dioxide, water, and ammonia from the breakdown of anthranilic acid. It is now known that the first step in the oxidation of anthra- nilate involves hydroxylation to form catechol (117). Many strains of Pseudomonas have been isolated which are capable of performing this reaction (71) but certain strains which oxidize tryptophan by the anthranilate pathway are unable to oxidize anthranilate due to an enzymatic defect (112). Conversion of anthranilic acid to catechol is not well understood. It has been shown that benzoic acid is converted to catechol via 1,2-di- hydro-l,2-dihydroxybenzoic acid. Apparently the same mode of catechol production does not occur in the 16 anthranilate system (91). Anthranilate oxidation has also been studied in Nocardia opaca. The pathway is the same as in Pseudomonas (16, l7, l8). Evans and Smith were the first to show that bacteria oxidize catechol to form cis,cis-muconic acid (27). The enzyme responsible for this reaction is catechol-1,2-oxygenase (E.C. 1.13.1.1, catechol: oxygen 1,2-oxido-reductase). In Pseudomonas the synthesis of catechol oxygenase is elicited by its product, cic,cis- muconic acid. Although catechol has not been unequivocally excluded, it probably is not an inducer. It is thought that low levels of catechol oxygenase in uninduced cells must be sufficient to permit an effective endogenous generation of inducer. Also, cic,cis-muconate elicits coordinate synthesis of the two enzymes that catalyze its conversion to B-ketoadipate enol-lactone (81). The catechol oxidation enzymes of Acinetobacter are likewise induced by cis,cis-muconic acid (51,52). In contrast, cis,cis-muconate does not induce catechol oxygenase in A; eutrophus. The catechol oxygenase of this organism is temperature sensitive and its inducer has not been identified (81). Palleroni and Stanier (84) did an extensive study on the regulation of tryptophan oxidation in a strain of P. fluorescens that used the anthranilic acid pathway. They found that kynurenine induced tryptophan oxygenase, 17 formamidase, and kynureninase. Tryptophan had no induc- tive preperties. The first two enzymes were induced coordinately. Kynureninase and anthranilate hydroxylase were induced sequentially. Mutants were used to deter- mine inducer identity. High levels of constitutive tryptophan oxygenase were attributed to a leaky repressor. It was hypothesized that tryptophan oxygenase had a higher Km than tryptophan aminoacyl synthetase which allowed maintenance of internal pools of tryptophan (84). In Xanthomonas pruni, the first three enzymes in the anthranilic acid pathway are induced coordinately by L-tryptophan. Gratuitous inducers of these enzymes include D—tryptophan, d-methyl-D,L-tryptophan, and 4- methyl-D,L-tryptophan. N-formyl-L-kynurenine and L- kynurenine are not effective as inducers (12). Enzymes of the Anthranilic Acid Pathway Tryptophan Oxygenase Knox and Mehler (58) were the first to describe the enzymatic breakdown of tryptophan. They used a soluble system from rat liver. Later Tanaka and Knox (115) compared a Pseudomonas tryptophan oxygenase to the hepatic tryptophan oxygenase and found them to be the same in every respect. Both enzymes were inhibited by potassium cyanide, sodium azide, carbon monoxide, and catalase. 18 Diamond 3E Bl. (26) purified tryptophan oxygenase from B; acidovorans using Sephadex and ion exchange chro- matography. The enzyme was eluted in two distinct bands. When present in low concentrations, cadmium stimulated enzymatic activity. Feigelson's group (87) purified tryptophan oxygenase to a state of homogeneity from substrate- induced B; acidovorans. The sedimentation and diffusion -13 coefficients were 820,w, 6.26 x 10 sec and D 4.78 x 10.7 cm2 sec"l 20,w, , respectively. From equilibrium sedimentation data, the molecular weight was 120,000. Amino acid analysis revealed no sulfur-containing amino acid residues other than methionine. Quantitative analytical data showed one mole of ferriprotoporphyrin IX and only trace amounts of copper and non-heme iron per mole of holoenzyme. The major absorption bands in the optical spectrum of the native holoenzyme occurred at 280 and 405 nm. Treatment with guanidinium chloride, alkaline pH, or sodium dodecyl sulfate resulted in dissociation of the native protein into four subunits. The molecular weights of the subunits were calculated as 31,300 to 35,800 depending on the method. Turnover number (moles of tryptophan oxidized per minute per mole of enzyme) was 950. Antibodies against the hepatic tryptophan oxygenase did not cross-react to precipitate the bacterial enzyme (103). l9 Koike and Feigelson (14, 65) have recently shown that the heme prosthetic group of tryptophan oxygenase oscillates in valence during catalysis. The enzyme possesses regulatory as well as catalytic sites. The substrate, tryptophan, is capable of binding to both. The tryptOphan analog, a-methyltryptOphan, binds only to the regulatory site(s) under appropriate conditions (104). S-Fluorotryptophan binds exclusively to the catalytic site and is a competitive inhibitor. Cyanide binds to the heme group of the enzyme. This binding is affected by the presence of an effector (tryptophan or S-fluorotryptophan) at the catalytic site but no effect on cyanide binding results from interactions at the regulatory site (30, 59). By using the two enzymes superoxide dismutase and catalase, reductive activation by sodium borohydride, hydroperoxide, and dithiothreitol of tryptophan oxygenase was shown to involve superoxide hydroperoxide as well as direct transfer of electrons from the reducing agent (9). Brady and Feigelson (10) recently studied photoactivation of tryptophan oxygenase and proposed a mechanism by which electrons enter tryptophan oxygenase via "electron ejection" from a photoexcited L-tryptOphan bound at the catalytic site. Tryptophan oxygenase has also been studied in partially purified extracts of B; pruni. The dialyzed 20 enzyme required both heme and ascorbate for maximal activity. Other reducing agents were able to substitute for ascorbate. The enzyme exhibited sigmoid saturation kinetics. Reduced nicotinamide adenine dinucleotide, (NADH), reduced nicotinamide adenine dinucleotide phOSphate, (NADPH), nicotinic acid mononucleotide, and anthranilic acid enhanced the sigmoid kinetics and presumably bound to allosteric sites on the enzyme. The sigmoid kinetics were diminished in the presence of a-methyl-tryptophan (122). Kynureninase In 1953 Miller and Adelberg (70) elucidated the mechanism of kynureninase. They identified anthranilic acid and alanine as the products of the reaction. More recently, Mariguchi SE 91' (73) crystallized and charac- terized kynureninase from B; marginalis. The enzyme was homogeneous by the criteria of ultracentrifugation and disc gel electrophoresis. The molecular weight was 100,000 and one mole of pyridoxal-5'—phosphate was bound per mole of enzyme. Kynureninase was inactivated by incubation with alanine. The reactivity was restored by addition of pyridoxal-5'-phosphate (74). Neurospora crassa has two different "kynureninases". The noninducible enzyme, hydroxykynureninase, preferentially catalyzes L-3-hydroxykynurenine to 3-hydroxyanthranilate and appears to be mainly present in uninduced cells for 21 the indispensable synthesis of nicotinamide adenine dinucleotide. The inducible enzyme, kynureninase, is induced by tryptophan to a concentration far in excess of that needed to meet the requirements of the cells for nicotinamide adenine dinucleotide, resulting in the excretion of anthranilate into the medium (33). Anthranilate Hydroxylase Anthranilate hydroxylase is very unstable in cellular extracts. However, this enzyme has been purified from B; fluorescens (117) and characterized. Anthranilate hydroxylase required NADH or NADPH. Ferrous ions had no effect on the stimulation of the enzymatic activity. Both mercuric chloride and pfchloromercuricbenzoate were inhibitors. The Optimum pH was 7.5. Catechol was identified as the product by chromatography, spectroscopy and radioisotope studies. Catechol Oxygenase Catechol oxygenase has been purified from Pseudomonas and crystallized (42). It is distinctly red, resistant to oxidizing agents, and readily inacti- vated by reducing agents. Hayaishi BE a1. (39) found that when catechol oxygenase acted on catechol in the presence of H2018 there was no incorporation of label into cis,cis-muconic acid. However, when the reaction was carried out in the presence of 1802 the product, 22 cis,cis-muconic acid was heavily labeled. Information concerning the redox state of iron in catechol oxygenase was obtained with electron spin resonance (77). The proposed reaction mechanism for this enzyme is as follows: the iron is initially in the trivalent state. Upon combination with catechol and then oxygen the iron is transiently reduced (42). Products of Tryptophan Metabolism Identification of Intermediates Paper chromatography, thin layer chromatography, column chromatography, and ultraviolet fluorescence have been used to identify and characterize the various tryptophan metabolites. Early studies involved the use of paper chromatography and ultraviolet fluorescence was used to detect the intermediates (67, 86). Mason and Berg (68) studied kynurenine and anthranilate quantitatively by subjecting these compounds to diazo- tization and coupling with N-(l-naphthyl)-ethylene diamine. The reaction products were measured colorime- trically. More recently, Haworth and Walmsley (37) used a two dimensional thin layer chromatographic procedure with thin layers of Silica Gel GF to unambiguously 254, separate thirty-two tryptophan metabolites and related compounds. This procedure was used to identify compounds from serotonin, indican, and indole—3—acetic acid to 23 kynurenine, anthranilate kynurenate, and xanthurenate to quinolinate and nicotinamide (37). Ion exchange column chromatography has also been used to isolate tryptOphan metabolites (4, 20). Pigment Production Species of Nocardia and Pseudomonas are able to grow on anthranilic acid as sole carbon, nitrogen, and energy source. In almost all strains an intense reddish- brown pigment results from the metabolism of this substrate. The pigment is thought to result from condensation of nitroquinones which are formed by the oxidation of nitrocatechols (19). The Emmochrome pigments of insects are also end- products of tryptophan metabolism. Xanthommatin is the product of the oxidative condensation of two molecules of 3-hydroxykynurenine, one of which undergoes quinoline ring formation in the process. Ten eye-color mutants of the housefly were studied for tryptophan metabolism. It was shown that the eye—color mutations were due to the loss of tryptophan oxygenase, formamidase, and kynurenine hydroxylase. The enzyme deficiencies apparently caused eye color variants due to the lack of tryptophan metabolites (44). 24 Nicotinamide Adenine Dinucleotide One of the most important products of tryptophan metabolism is NAD. Some of the microorganisms which synthesize NAD from tryptophan are among the genera Xanthomonas, Neurospora, Streptomyces, and Saccharomyces (62). Kynurenine, 3-hydroxykynurenine, and 3—hydroxyan- thranilic acid have been established as intermediates in the synthesis of NAD. Nishizuka and Hayaishi (79) found that 3-hydroxyanthranilic acid was converted to nicotinamide ribonucleotide in the presence of 5- phosphoribosyl-l-pyrophosphate. In addition, 2-amino- 3-acroleyl-fumaric and quinolinic acids were identified as intermediates in this conversion. Free nicotinamide was not involved. The nicotinamide ribonucleotide produced from 3-hydroxyanthranilate was converted to deamido NAD in the presence of glutamine and adenosine triphosphate (ATP) by NAD synthetase. The activity leading to niacin ribonucleotide synthesis seemed to be inversely related to picolinic carboxylase activity. This enzyme removed 2-amino-3-acroleyl-fumaric acid rapidly and limited quinolinic acid available for niacin ribonucleotide biosynthesis. This is a possible control mechanism for the regulation of NAD synthesis. Tryptophan Products and Disease In man, the inability to synthesize nicotinic acid ribonucleotide from tryptophan catabolites accounts 25 for the pellagra-like rash which occurs in three inborn errors of tryptophan metabolism: Hartnup disease, hypertryptOphanemia, and 3-hydroxykynureninuria. Also in man, impaired synthesis of pyridoxal phosphate results in an elevated urinary excretion of kynurenine, 3-hydroxykynurenine, and xanthurenic acid because the kynureninase which is responsible for conversion of 3-hydroxykynurenine to 3-hydroxyanthranilic acid is more sensitive to a lack of available coenzyme than kynurenine transaminase (94). Elevation of urinary tryptophan metabolites has been associated with bladder cancer, renal cancer, breast cancer, prostatic cancer, and Hodgkin's disease. The cause of abnormal tryptophan metabolism in breast cancer may reflect a disturbed estrogen-androgen balance due to impaired androgen production with consequent estrogenic overstimulation Tryptophan Transport There are no detailed studies on tryptophan trans- port in Bacillus. Konings and Freese (60) studied amino acid transport in B; subtilis using membrane vesicles. They observed energized transport for all of the natural amino acids with the exception of L-tryptophan. However, competition studies did show that tryptophan could be transported by a general aromatic amino acid permease /‘_\___ 26 which had affinity for tryptophan, tyrosine, and phenylalanine. There was no involvement of a phos- phorylated intermediate for any of the amino acids that were transported. B; 2913 has at least three systems for tryptophan transport (7, l3, 14). The general aromatic transport system is constitutive and has an affinity for tryptophan, phenylalanine, and tyrosine. There is also a specific constitutive transport system which transports only tryptophan. The third system is inducible and is thought to function in tryptophan catabolism. This permease is induced by tryptophan in the presence of casamino acids and is repressed by glucose. Its Km 5 is 7 x 10' M. Salmonella typhimurium has at least two systems that transport tryptophan (l, 2). The general aromatic permease transports tryptophan, tyrosine, phenylalanine, and histidine. A specific permease transports tryptophan only. A study of the specificity of the general aromatic permease demonstrated that the nature of the side chain can vary considerably although some aromatic character is necessary. Data indicated that carboxyl activation is not involved in transporting any of the aromatic amino acids. 27 The tryptophan permease of B; acidovorans is inducible and sensitive to both sodium azide and 2,4- dinitrophenol. The inducer of this permease is kynurenine and not tryptophan. This is the first reported example of the product induction of a permease. Regardless of whether B; acidovorans is grown in the presence of D- or L-tryptophan, the resulting induced tryptophan per- mease is specific for the L-isomer. Leucine, phenyla- lanine, and D,L-hydroxytryptophan are not transported. However, both D,L-hydroxytryptophan and D,L-fluorotryp- tophan competitively inhibit tryptophan uptake. The synthesis of tryptophan permease is not coordinate with that of tryptophan oxygenase. Tryptophan transport is strongly inhibited by formylkynurenine and kynurenine which seem to have separate distinct permeases (96). Tryptophan transport in N; crassa is mediated by a distinct stereospecific system which is chemically specific for a family of neutral amino acids. Leucine and phenylalane competitively inhibit the rate of tryptophan transport. It is believed that an uncharged side chain and an a-amino group, next to a carboxyl group, represent three attachment points for the uptake site (124). The rate of tryptophan transport is regulated by the intracellular pool of tryptophan. Cknutinued protein synthesis is required for the main- terLance of the system (125). Mutants which are resistant 28 to 4-methyl-tryptophan and Effluorophenylalanine are defective in the uptake of tryptophan. Both unlinked and linked supressor mutants have been isolated (11). The binding protein of the Neurospora tryptOphan trans- port system has been isolated from germinated conidia using cold, osmotic shock. The dissociation constant for binding is approximately equal to the Km for tryp- tophan transport (126). In Claviceps species strain SD-58, tryptophan uptake is mediated by an energy-requiring, unidirectional transport system which is specific for neutral aromatic and aliphatic amino acids. The optimal temperature and pH for uptake are 30 C and 5.0, respectively. Trypto— phan transport is markedly influenced by the intracellular concentration of free tryptophan (92). MATERIALS AND METHODS Organisms and Cultivation Organisms The parent strain in these studies was Bacillus megaterium ATCC 19213. This aerobic spore-former grows well in simple defined media requiring only a sugar, salts, and trace elements. It is therefore excellent for studying physiological and biochemical systems. A spontaneous D-cycloserine resistant mutant, DCSr-S, isolated from the wild type, is resistant to 500 ug/ml of D-cycloserine. B; megaterium ATCC 19213 is inhibited by 50 ug/ml of D-cycloserine. DCSr-S was the parent strain for mutants Brs-3, Brs-S, Brs-8, Brs-lO, Brs-12, Brs-lS, Brs-22, and Brs-23, all of which are unable to use L-tryptOphan as a sole energy source. Media For most experiments, a modified SS medium (106) was used. This defined medium contains the following in grams per liter: 2g sucrose, 5g KH2P04, 1g (NH4)2HPO4,0.2g MgSO4, 1g NaCl, 0.005g CaClZ, 0.007g bhuSO4. H20, 0.01g ZnSO4, and 0.01g FeSO4. When sucrose twas replaced by tryptophan the medium was called Trp-S. 29 30 When sucrose was replaced by arginine the medium was called Arg-S, and so on. The KH2P04 and (NH4)2HPO4 were combined to form SS buffer. This buffer, solu- tions of energy source, MgSO4 plus NaCl and trace ele- ments were autoclaved separately and combined in appropriate amounts after sterilization. Energy sources were sometimes filter-sterilized with a Nalgene filter unit (0.2um, plain membrane; Nalge Sybron Corporation). In early experiments "Pig" medium was used and contains the following in grams per liter: 1.0g K2HPO4, 4.0g (NH 1.0g casamino acids, 0.1g MnSO 'H O, 1.64g 4’2504' 4 2 MgSO '7H 0, 4.0g sodium acetate, 0.1g CaClz, 0.001g 4 2 FeSO '7H 0, 0.01g CuSO '5H O, 0.0177g ZnSO '7H 0, and 4 2 4 2 4 2 2g L-tryptOphan. In mutant studies, D—cycloserine medium was used and consists of SS medium + varying amounts of D-cycloserine. Organisms were occasionally grown in nutrient broth (Difco) or Bacto-penassay broth. When a semi-solid medium was desired Bacto-agar was added to a final concentration of ng/liter. Inoculation and Growth All organisms were maintained at -20 C or 4 C in washed Spore suspensions. Spores were routinely heat shocked in one milliliter amounts at 70 C for thirty minutes. A portion of the heat-shocked spore ENiSpension was used to inoculate a 500 m1 Erlenmeyer I l i I. "IA; 31 flask containing 100 ml of fresh, sterile medium. The culture was aerated at 30 C with a New Brunswick rotary platform shaker at 180 rpm. Starter cultures contained inosine and alanine (100 ug/ml) to initiate germination. RadioisotOpes and Sources The following isotopes were utilized in this study: l4C-D,L-tryptophan (Calatomic, 29.1 mCi/mmole), l4C-L-phenylalanine (New England Nuclear, 47 mCi/mmole), l4C-L-leucine (New England Nuclear, 312 mCi/mmole), l4C-L-tyrosine (International Chemical and Nuclear, 360 mCi/mmole). Scintillation Counting Radioisotopes were assayed by liquid scintillation counting with samples adsorbed to an inert support (Millipore or Whatman filter). The scintillation fluid contained 6 grams of 2,5-diphenyloxazole (PPO) and 0.01 gram of 1,4-bis-2-(S—phenyloxazolyl)-benzene (POPOP) per liter of toluene (15). Ten milliliters of scintilla- tion fluid was added to glass vials containing dried samples. Radioactivity was assayed with a Model 3320 Packard Tri-Carb Scintillation Spectrometer. Chemicals All chemicals were analytical or reagent grade and were purchased from standard commercial sources. 32 Protein Concentration Protein concentration was measured spectrOpho- tometrically by the procedure of Lowry SE 51’ (64). The standard was bovine serum albumin. Growth,_Tryptophan Utilizatigg and Pigmentation on Trp-S In this and all other experiments turbidity was an index of growth and was measured as the optical density of a culture at 620 nm. A Gilford spectropho- tometer was used for optical density measurements. Pigment production was monitored by measuring the absorbance of culture supernatants at 400 or 415 nm with a Perkin-Elmer Model 124, double-beam diffraction grating spectrophotometer coupled to a Sargent Model SL recorder. Culture supernatants were obtained by centri- fugation at 15,000 x g. TryptOphan concentration was measured by the method of Udenfriend and Peterson (120) which detects the indole ring with pfdimethylaminobenzaldehyde, NaNO and concentrated HCl. Results were recorded 2: in terms of umoles of tryptophan used per 100 ml of culture. Cellular protein is also an index of growth. The procedure of Slapikoff, 2E 31' (107) was used to isolate the crude bulk protein from the growing culture. Cells were sedimented by centrifugation at 10,000 x g. 33 The pellet was suspended in 2 ml 5% trichloroacetic acid (TCA). The precipitate was washed once with cold 5% TCA, twice with ethanolether (3:1, v/v) at 37 C for 10 minutes and once with 5% TCA heated at 90 C for 15 minutes. The residue was dissolved in 1.0 ml or 2.0 ml of 0.1 N NaOH. Protein concentration was measured spectrophotometrically and was recorded as mg of protein /ml of culture. Isolation of "Late" Pigment The medium contained 0.1% sucrose and 0.05% l-tryptophan. A starter culture of the same medium was the source of the inoculum. After inocultaion, the culture was allowed to develop to the stationary phase. At this time l4C-D,L-tryptophan was added to a final concentration of 100 nCi/ml. The addition of the labelled tryptophan after exponential growth decreased its chances of being incorporated into protein since there is no net protein synthesis during the stationary phase. The culture was allowed to develop for a total of forty-eight hours and then cleared by centrifugation at 13,000 rpm for thirty minutes in a refrigerated RCZ-B centrifuge. The supernatant solution was clear and orange. An equal amount of 95% ethanol was added to the solution which was left in the cold overnight. The precipitate was removed by filtration 34 and the pigment was concentrated by flash evaporation at 40 C. The remaining procedures were carried out at 25 C. The concentrated pigment was applied to a column of DEAE cellulose which was 1.5 x 14 cm and had been equilibrated with 0.1 M Tris-HCl buffer,pH 7.3. The pigment stuck to the top of the column. The column was washed with 60 m1 of the same buffer. Formylkyn- urenine, kynurenine, formic acid, and unreacted l4-C- D,L-tryptophan were eluted with 40 m1 of 0.5 M NaCl in 0.1 M Tris-HCl, pH 7.3 and fractions of these pro- ducts and reactants were collected. The column was washed with 40 ml of 2 M NaCl in tris buffer (pH 7.3), and 20 ml of tris buffer (pH 10). The pigment remained on the column during all of this procedure. The pigment (or its hydrolysis products) was finally eluted with concentrated hydrochloric acid. The UV spectrum of the late pigment was determined using a Perkin-Elmer Model 124 double—beam diffraction grating spectrophotometer coupled to a Sargent Model SL recorder. Radioactivity was assayed by liquid scintilla- tion spectrometry. Isolation of Mutants D-Cycloserine-Resistant Mutants At concentrations of 50 ug/ml D-cycloserine completely inhibits colony formation of B; megaterium 35 ATCC 19213 on semi-solid media. Spontaneous D-cycloserine resistant mutants were obtained by the following procedure: Heat-shocked spores (2 x 107) were spread on penassay agar containing 100 ug of D-cycloserine/ml. Resistant colonies appeared after 30 hours of incubation at 30 C. This was the only procedure in which spontaneous D- cycloserine-resistant mutants were obtained. Spreading vegetative cells was unsuccessful. The resistant mutants were labelled DCSr-l through DCSr—ll. The mutation rate 6 7 was 10- to 10- . All mutants were identified as B; megaterium based on colonial morphology, cellular and spore morphology, and growth characteristics. Mutants were re-streaked on SS agar containing 100 ug of D- cycloserine/ml and were sequentially cycled by re- streaking on 200 ug/ml, 300 ug/ml, 400 ug/ml, and finally 500 pg of D-cycloserine/ml in SS agar. Thus the final group of mutants was ten times more resistant to D-cycloserine than the wild type. DCSr-S was the most resistant and was used as the parent strain for tryptophan mutants. D-cycloserine-resistance served as a genetic marker. Tryptophan Mutants A spore suspension of DCSr-S was heat-shocked at 70 C for thirty minutes and used to inoculate 100 ml of Penassay broth. After growth overnight, the cells were sub-cultured with 50 ml of fresh Penassay broth. 36 The culture was in the mid—exponential phase after six hours of growth. Cells were harvested, resuspended in 50 m1 of Penassay broth, and treated with 30 pg of N-methyl-N'-nitro-N-nitrosoguanidine per m1 (NTG; Aldrich Chemical Co.) for thirty minutes at 30 C. The cells were washed twice in 0.02 M phosphate buffer, pH 7.3 and resuspended in 5 ml of the same buffer. One milliliter of this suspension was transferred to SS broth (0.4% sucrose) and incubated overnight to allow segregation of mutant and non—mutant nuclei from multi- nucleated cells and to permit the phenotypic expression of the induced mutations. This step also tends to reduce the proportion of mutants with nutritional require- ments since they are unable to grow in SS broth which is a minimal medium. Survivors (0.1 ml) were transferred to 10 m1 of Trp-S containing 300 units of penicillin/ ml. The suspension was aerated on a platform shaker at 30 C for twelve hours. During this time interval, cells which could utilize tryptophan were killed off while tryptophan mutants survived. Appropriate dilu- tions of the penicillin culture were spread on minimal media containing 0.01% sucrose and 0.2% tryptophan. Presumptive tryptOphan mutants formed pinpoint colonies on this medium and large colonies on 0.4% sucrose agar. Restreaking on tryptophan agar and D-cycloserine agar confirmed the tryptophan mutants. Through this 37 selection procedure mutants of B; megaterium were obtained which did not completely degrade tryptophan but grew and sporulated without extreme nutritional requirements. The eight mutants are Brs-3, Brs-S, Brs-8, Brs-lO, Brs-12, Brs-lS, Brs-22, and Brs-23. Chromatography Paper Chromatography Pigmented supernatant from a one-liter culture of B; megaterium ATCC 19213 grown on Trp-S was lyophilized and redissolved in distilled water. This was followed by dialysis against distilled water. Ten microliters of the dialyzable material was spotted 3 cm from the bottom of Whatman No. 1 paper, 22 x 31 cm. The dialyzable material was also extracted with diethyl ether. The ether extract was concentrated by evaporation and 10 microliters were applied to the chromatogram. Standards of kynurenine, kynurenic acid, and anthranilic acid were also applied (0.1 micromole). The chromatogram was placed in a chamber 31 X 39 cm and developed by the ascending method for 3-5 hours with butanol-glacial acetic acid—water (4:1:1, based on volume). The solvent front was marked with a pencil and the chromatogram was air dried in a hood. Ultraviolet fluorescent and absorbant compounds were detected with a UV lamp and circled. Ninhydrin and 38 ferric chloride sprays were used to detect primary amino groups and phenols, respectively. Two-dimen- sional chromatography was carried out with water as the second solvent. Thin Layer Chromatograpgy The standards, samples, and spotting procedures were the same. The solvents were butanol-acetic acid- water (12:3:5) (solvent A) and chloroform-acetic acid- methanol-water (65:20:10:5) (solvent B). Chambers (22 cm X 22 cm X 10 cm. DESAGA, Germany) were equili- brated overnight. Prior to development, the old solvent was discarded and replaced with fresh solvent. Two- demensional chromatograms were developed first with solvent A and then with solvent B. The thin layer plates were coated with silica gel (Uniplate, pre- coated with Silica Gel G, 250 microns thick, Analtech, Inc.). All plates were developed by the ascending method (37). Manometry Preparation of Whole Cells Cells were grown in 100 ml batches to the late-exponential phase (O.D.620¥1.0) and harvested by centrifugation (15,000 x g, 15 min.). The pellet was washed in 100 ml and resuspended in 5 ml of .IM potas- sium phosphate buffer, pH 7.0. Oxidation of various 39 substrates by 0.1 m1 of this cell suspension was determined by the interval uptake method (121). Warburg Constant-Volume Respirometry Oxygen uptake studies were conducted with whole cells in a Warburg respirometer at 30 C. The flasks contained 0.1 ml of the cell suspension and 2.2 ml of 0.1 M potassium phosphate buffer (pH 7.0) in the main compartment, 0.2.ml 20% KOH in the center well, and 5 umoles (0.5 ml) of substrate in the side arm. Respi- rometers were equilibrated for five minutes before t tipping the substrate into the major compartment. Readings were generally made every five minutes. Re- sults were recorded in terms of microliters of oxygen consumed according to the formula: x = hk where x = amount of gas exchanged h = alternation in reading on the Open arm of the manometer k = flask constant The flask constants were determined by the method of Umbreit SE 31. (121). Flasks and manometers were numbered and were always used in the same combination. The manometer fluid (Krebs formulation) had a density of 1.033 g/ml. Oxygen uptake was linear. 40 Enzyme Assays Preparation of Cell- Free Extracts Packed cells from a liter culture were washed in .033M potassium phosphate buffer (pH 7.0) and resuspended in ten m1 of .1M phosphate buffer (pH 7.0) containing .01 mM l-tryptophan to stabilize trypto- phan oxygenase. Cells were disrupted in a sonic oscillator (Measuring and Scientific Equipment, Ltd) for a total of two minutes with four thirty-second bursts and one minute rests. The suspension was cooled with an ice bath throughout this procedure. The sonicated preparation was clarified by centrifugation (55,000 xg for one hour). The resulting extract was assayed directly for enzyme activity. Assays All enzyme assays were monitored spectrophoto- metrically using a Perkin—Elmer double beam spectrophoto- meter equipped with a Sargent recorder, model SR. Assay volumes were 1.0 m1 and the temperature was 25 C. Unless otherwise specified, the reaction blanks contained all of the assay components except the substrate. For each enzyme, one unit of enzymatic activity was defined as the amount necessary to use 1 n mole of substrate or form 1 n mole of product per minute at 25 C. Specific 41 activity was eXpressed as units per milligram of pro- tein. Readings were made at 30 second intervals. Tryptgphan-2,3-Oxygenase Activity of tryptophan-2,3-oxygenase was assayed by a modification of the method described by Feigelson SE 31' (28). The formation of N-formyl-L-kynurenine was followed continuously by measuring increased absorbance at 321 nm. The extinction coefficient of 3 cm2/ N-formyl-L-kynurenine at 321 nm is 3.75 x 10 mmole (115). The reaction mixture contained one micromole of L-tryptophan, 0.2 ml of enzyme, .Sml potassium phosphate buffer (0.1 M, pH 7.0) and water to give a final volume of 1.0 ml. Kynureninase Activity of kynureninase was assayed by a modification of the method of Knox (57). The dis- appearance of L-kynurenine was followed continuously by measuring the decreased absorbance at 360 nm. The extinction coefficient of kynurenine at 360 nm is 4.53 x 103 cmZ/mmole (115). The reaction mixture contained 0.2 umoles of L-kynurenine, 0.2 m1 of enzyme, 0.5 ml of potassium phosphate buffer (0.1 M, pH 7.0) and water to give a final volume of 1.0 ml. 42 Catechol Oxygenase Activity of catechol 1,2-oxygenase was assayed by the method of Hayaishi EE.31° (40). The formation of cis,cis-muconic acid was followed continuously by measuring increased absorbance at 260 nm. At this wavelength the difference in extinction coefficients of catechol and cis,cis-muconic acid is 1.6 x 104 cmz/mmole. The reaction mixture contained 0.2 umoles of catechol, 0.1 ml enzyme, 0.5 ml potassium phosphate buffer (0.1 M, pH 7.0) and water to give a final volume of 1.0 ml. Other Enzymes Attempts were made to detect other enzymes in cell-free extracts. Formamidase was assayed by the method of Brown and Wagner (12). Kynurenine trans- aminase was assayed by the method of Tremblay SE 31' (119). Anthranilic acid hydroxylase was assayed by the method of Tanuichi SE al. (117). Time Course of Tryptophan Oxygenase and Kynureninase Synthesis B; megaterium ATCC 19213 was grown in Arg-S in three-liter batches. The cells were harvested, washed in SS buffer, and resuspended in a medium con- taining 0.4% arginine. Appropriate flasks contained 0.1% L—tryptophan and chloramphenicol (50 ug/ml). The 43 flasks were incubated at 30 C on a platform shaker for specific time intervals. Incubation was terminated by chilling to 4 C and adding chloramphenicol to a final concentration of 50 ug/ml. Each sample was clarified by centrifugation (15,000 x g), washed in 0.033 M potassium phOSphate buffer (pH 7.0), and resuspended in 5 or 10 ml of 0.1 M potassium phosphate buffer (pH 7.0) + 0.01 mM L-tryptophan. The extracts from these resuspended samples were assayed for tryp- tophan oxygenase and kynureninase. Amino Acid Transport The procedure used for monitoring the transport of l4C-D,L-tryptophan, l4C-L-phenylalanine, and 14C- L-leucine was a modification of the procedure used by Schlesinger and Magasanik (102). Bacteria were har- vested from exponential phase cultures, washed with SS buffer, and resuspended to an O.D. of 0.2 620 nm (2.0 x 107 cells/ml). The incorporation medium (20 ml) contained approximately 4 x 108 cells, 0.05 uCi/ m1 of radioactive amino acid, and 100 ug/ml of chlor- amphenicol. Appropriate flasks contained 100 umole/ ml of sodium azide. Reaction mixtures were incubated at 30 C on a New Brunswick rotary shaker at 180 rpm. Radioactive compounds were added following three minutes of incubation. After specific time intervals, 1 m1 44 samples were withdrawn and passed through a membrane filter (Gelman Instrument Co., Metricel GN-6, pore size 0.45 pm 25 mm diameter). The filter was washed with cold SS buffer (addition of unlabeled tryptophan did not alter the results), dried in an oven, and placed in a glass vial for liquid scintillation spectrometry. Competition Experiments The ability of unlabelled amino acids to compete with the uptake of l4C-D,L-tryptophan was measured. Unlabelled D-tryptOphan, L-tryptophan, L-leucine, L- arginine, and L-phenylalanine were added to reaction mixtures in two different concentrations, 20 nmoles/ ml and l mmole/ml. Radioactive D,L—tryptophan was always present in a concentration of 1.72 nmoles/ml. 14 Uptake of C-D,L-tryptophan was determined as des- cribed above. RESULTS Pigment Production Many investigators have studied the early biochemical events of sporulation to gain an under- standing of cellular differentiation in procaryotes (98, 36). Enzymes such as exoprotease are found in Bacillus cultures only after exponential growth has ceased. Sadoff SE Bl. (97) reported that one function of this exoprotease is to modify vegetative aldolase, which is smaller and more heat resistant. Products such as antibiotics are also produced only after the cessation of exponential growth (98). In B; megaterium, pigment production (or release) followed the same pattern (Figure 2). When this organism grew in a medium which was rich in amino acids, large amounts of brown pigment were produced. This pigment was excreted into the medium, but only during the station- ary phase. In B. subtilis, the production of brown pigments has been associated with sporulation, which also occurs during the stationary phase. In all of these post-eXponential events there must be a control rmechanism which turns a system off during the exponential grtnvth phase and turns it on during the stationary 45 46 Figure 2.--Growth and pigment production of B; megaterium in "Pig" broth. Symbols: (0) OD6 0 nm’ (I) OD400nm of culture supernatant. Experimental details are described in Materials and Methods. 47 .19 .18 17 .16 .15 .14 .13 .12 .10 .09 08 w02HHMHuHCH mp3 m uomm .A>\>\>\>v Amuoauomumwv Hmum3laosmzumelpflom OHumUMIEH0moHOH£o mm3 ucm>H0m mne .Hmm mueaflm mo mummma Ceca so Umnmmwmoumfionco mumB memepwEHmucH m>HumESmmnm map cam Hocowumo .A>\>v AHuHuvv umuMBIpflom oeumUMIHOGMpSQ ca momma co Umnmmnmosmfionno wwmz mmuMHUmEHmucH m>HumEdmmHm map was .mumaflcmunucm .mcflcmuscmx .mmpMHmeHmucH mo GOHpMUHMHucmpHII.N magma 60 .susfisnsHOm swaps How Umummp mumz mpcsomEOU .pmcefiumpoo #02 .1 ® + + + + I I *nmnum ® Es mum Es cam Es cam ® Es omm .xmz >2 + + I I I I msomm I I I I + + cflnpmscHz I I umaofl>Im5Hn pmHoH>Imch snap mama mean mama .uosam >D sh. as. as. mm. mm. mm. msam> as m uomw Hocomumo o womm mumaflcmunuc¢ v pomm mcflcmuscmx .mmumameHmucH mo COAHMUAMHucmpHII.N wanes 61 its pale-blue UV fluorescence and positive ninhydrin reaction. In addition to its Rf value, spot 5 was identified as catechol by its positive FeCl3 reaction, ether solubility, and lack of UV fluorescence. In addition to its Rf value, spot 6 was identified as anthranilate by its blue-violet fluorescence, ether solubility, and UV maximum at 310 nm. Chromatography revealed that at least seven intermediates were excreted when B; megaterium oxidized tryptophan. Three could be extracted with ether and had UV maxima at 310 nm, 295 nm, and 280 nm. The 310 nm compound was probably anthranilic acid (Figure 6) and the 280 nm compound was probably catechol. It was believed that the 295 nm compound was N-formylanthranilic acid for reasons which will be discussed later. The presence of anth— ranilate and catechol in the ether extract was confirmed by paper and thin-layer chromatography. Tryptophan Mutants The identification of kynurenine, anthranilate, and catechol indicated that B; megaterium used the anthranilic acid pathway for tryptophan oxidation. Additional data was obtained which supported this when it was found that Brs-5, a mutant which was unable to grow on tryptophan, accumulated anthranilic acid vfllen grown on a medium containing tryptophan and 62 Figure 6.--UV spectra of tryptophan catabolites. Symbols: (0) commercial anthranilic acid, (0) ether- soluble tryptOphan catabolites produced by B. megaterium. Experimental details are described in Materials and Methods. ABSORBANCE 280 63 300 320 340 WhAVELEHVGTFIInm) 360 64 arginine (Table 3). The medium did not show the presence of significant amounts of other intermediates and catechol was not detected even after the culture supernatant solu- tion was concentrated. Both Brs-S and Brs-12 (another mutant which was unable to grow on tryptophan) were compared to the wild type for their abilities to grow on arginine. The mutants had growth rates which were equal to or nearly equal to the wild type growth rate (Figure 7). Since these and other B£B_mutants had no nutritional growth requirements, it was concluded that B; megaterium did not synthesize NAD via tryptophan catabolism as does X; pruni. The B£§_mutants were also tested for their abilities to sporulate on sucrose and arginine. Some sporulated well while others were oligosporogenous (Table 4). Brs-S had a presumptive block in the path— way at anthranilate but sporulated well when grown on both sucrose and arginine. Brs-8 also had a presumptive block in the pathway at anthranilate (Figure 8) but was oligosporogenous when grown on sucrose or agrinine. Brs-lO had a presumptive block in the pathway at either anthranilate or catechol (Figure 8) and was oligosporo- genous when grown on sucrose or arginine. Brs-12 had a presumptive block in the pathway at catechol (Figure 8) but sporulated well when grown on both sucrose and arginine. Therefore, although oligosporogeny was 65 .Hmcum cuHB Umuomuuxm mm3 ucmumcummsm one .A>\>\>\>v a. ,E “i i... 7... mo ibii §-Vllyll~ I-I‘IIs.a'. ® Amuoauomummv nmuszHoamsumsIeHom owumUMIEH0moHOH£o cufi3 pmmoaw>mp mums mamumoumEouso HmmImoHHHm Hmmmalcflse % + + muflasnsHom umsum uoaofl> uoaofl> mocmommuosam .> .5 Es cam Es cam Ezeflxmz .> .3 mm. mm. msam> mm ucmumcummsm mlmum musaflcmucuc¢ @ .mlmum an wumaflcmucucm mo coflumHsEsoodII.m «wanes 66 Figure 7.--Growth of B; megaterium ATCC 19213 (I), Brs-S (A), and Brs-12 (I) on arginine (4 mg/ml). Experimental details are described in Materials and Methods. 67 EcOmo 00 HOURS 68 .ooom um sofluoa moss IoQSOCH unocINH o Hopwo cu3oum oHQHmH> Mom cocHono ouoB mouoam .ocfluomoHomoIo mo HE\mn ooa mcflcfloucoo Homo mm co samuuo nooo mcflxmouum ma pocHEHopop mos ocflwomoHomoIQ 0p oocoumflmom .GOHuonsocH mo musoc «N Houwo omme UHHB one was qumoo neon sow em>smmno was suzosm xosas .ooom um cofluonsocfl mo mason pcmflolmuuom Hopmo SpBOHm oHQHmH> MOM mcflxooa Ugo Homo mImHB co ucouDE sumo mcflxoouum we cocHE Iuopop mo3 coceoummquq co cuzouw .Uoom no cofipocsocfl mo muse: mHINH uoumo poucsoo oHoB moflcoHoo mcfluasmon ocp was Homo ucofluusc co pouoam oHoB mouomm ucoumfloou Iuoom .Uooh um mouscflE hpnflsu How monomm ooum mcflcfloacoo onsuaso o mcflxoochuooc ma posflsuouop ommz HE\moHomm .cocmoummwan :0 30mm ou oanoco Esfluouomofi .m mo mucouSSII.e oHQoB 69 I - 12.9 .I II Imp. + moa m.m moa x v.a I mmImHm + moa o.w boa x w.v I NNImHm + boa v.H moa x H.o I mHIowm + moa m.m moa x m.H I NHIoum + MOH m.m moa x n.m I oaImnm + moa m.m moa x v.m I mImwm + noa m.m boa x o.v I mImwm + sea m.> woa x o.a I MImwm + noa H.m moa x m.v + qumOo I boa v.m moa x m.m + mamma Dose A.HE\mn ooav oocoumwmom ocflcflmudIA co omouosm co cocooudmuqu ocfluomoHomuIQ .HE\mou0Qm .HE\mouomm co cuzouw cflouum .cocdoummquq so 30mm Op oanocs Esfluouomofi 4m we mucousle.v oHQoB 70 Figure 8.--Oxidation of intermediates of the anthranilic acid pathway by B; megaterium ATCC 19213, Brs-8 (A), Brs-lO (B), Brs-lZ (C), and Brs-lS (D). Each strain was tested for its ability to oxidize tryptophan (T), kynurenine (K), anthranilic acid (A), and catechol (C) by Warburg respirometry. The oxidation of the particular intermediate was recorded as ul of oxygen consumed/min/mg of cells (dry weight)+ The rate of oxygen consumed by the wild-type (w ) was set at 100% for each interme- diate. The mutant oxygen consumption rates were compared to the wild type oxygen consumption rates in terms of percentage. Organisms were grown in a medium containing tryptophan (2 mg/ml) and arginine (4 mg/ml). 71 Brs-10———sl K——Brs-8——->I W+ 72 thomwQ thOEwa W" Ie—Brs-IS—H a1 k—Brs-12 w‘. 73 associated with some of the mutants which were deficient in the oxidation of anthranilate, and catechol, the re- sults did not unequivocally relate tryptophan cata- bolism to sporulation, since the oligosporogeny may have been due to double mutations and since some of the mutants sporulated well. The mutants which were defective in the oxidation of anthranilic acid accumu- lated this compound and did not produce the brown pigment. The mutants which had defects in the oxida- tion of catechol apparently accumulated this compound (according to UV Spectra of culture supernatants) and did produce the brown pigment. Oxidation of Tryptophan and Intermediates by Whole Cells Stanier (108) and Suda (113) simultaneously discovered that the intermediates of a pathway were oxidized by cells which had been exposed to the initial substrate. This technique was used to confirm the pathway for tryptophan catabolism in B; megaterium. Cells were grown on L-tryptophan to late exponential phase (0 =0.9). They were harvested, washed, .D.620 and incubated with L-tryptophan, D-tryptophan, L- kynurenine, anthranilic acid, and kynurenic acid in a Warburg respirometer. A compound was identified as an intermediate if it significantly stimulated oxygen uptake over the endogenous rate. L-tryptophan, L- 74 kynurenine, and anthranilic acid were all oxidized immediately with high rates of oxygen consumption (Figure 9). Alanine was also found to be a pathway intermediate (Figure 10). Kynurenic acid and D- tryptophan were not oxidized. These data confirm the conclusions drawn from the chromatography results. B; megaterium ATCC 19213 oxidized tryptophan via the anthranilic acid pathway instead of the kynurenic acid pathway or the indole pathway. D-Tryptophan is typically oxidized only by organisms which use the kynurenic acid pathway (110). The possibility remained that the kynurenic acid pathway was operative in the stationary phase but not in the exponential phase. Figure 11 shows that stationary whole cells oxidized anthranilate at high rates but kynurenic acid was inert. Cell-free extracts were also unable to oxidize kynurenic acid. Therefore, it seems safe to conclude that B; megaterium ATCC 19213 oxidized tryptophan only by the anthranilic acid pathway or a modification thereof. Characterization of the Pathway When bacteria were grown on an energy source other than tryptOphan, this amino acid did not stimu- late oxygen uptake by cells in a respirometer. The inductive nature of tryptophan oxidation was investigated. 75 .mpocpoz pco maoflwouoz CH ponflnomop one maflouop Houcoefluomxm .cocmovmmnqu Adv .msocomOpco ADV .pfloo owcousc>x ADV .ocflcowscwaq AIV .paoo OHHflcmunuco AIV .cocmosmwqua hmv "mHOQEmm .cocmoumhuulq :0 CBOHm Esflwowomofi .m we AucmfloB hue m8 m.mv mHHoU oHOQB we manwom pfloo OHHHcoHcpco onu mo mouoflpofiuoecfl Ugo connowmwuulq mo coauoeflxOII.m ousqflm 76 MINUTES 77 Figure lO.--Oxidation of L-alanine and other intermediates of the anthranilic acid pathway by whole cells of B; megaterium grown on L-tryptophan. ( ) 30 mg of cells (dry weight), (—-n—) 12 mg of cells (dry weight). Endogenous values were subtracted. Symbols: (I) L-tryptophan, (I) L- alanine, (A) anthranilic acid, (*) L—kynurenine. Experimental details are described in Materials and Methods. 78 400 350 300 25 0 200 OwEDWZOU 2w0>x0 m0 150 wmwh.40m0.§ _ 0 m 50 60 50 40 30 20 10 MINUTES 79 Figure ll.--Oxidation of anthranilic acid and kynurenic acid by stationary whole cells of B. mega- terium grown on L-tryptophan. Endagenous values were subtracted. Symbols: (I) anth- ranilic acid, (0) kynurenic acid. Experi- mental details are described in Materials and Methods. 80 350 300 250 _ ONZDmZOO 2w0>x0 uO mam..._..0m0_s_ MINUTES 81 Cells were grown on sucrose, and washed. Their oxygen consumption rates were measured in the presence and absence of L-tryptOphan. After ninety minutes the oxygen consumption rate of the cells which were exposed to L-tryptophan began to increase and continued to increase until it was well above the endogenous rate (Figure 12). This shows that tryptophan oxidation was inducible in this organism. In addition, when cells were grown in the presence of sucrose and tryptophan, there was no in- duction of tryptophan oxidation. The system was pre- sumed to be subject to catabolite repression. Gluta- mate also repressed tryptophan oxidation. Other energy sources were tested to determine their effects. Neither alanine nor aspartic acid caused the repression of tryptOphan oxygenase (Figure 13A and 13B). This was advantageous because patterns of enzyme synthesis could be studied using these compounds as energy sources. However, alanine was unsuitable because it could cause feedback inhibition of kynureninase and aspartate was unsuitable because it was oxidized at very low rates. It was found that arginine was an excellent energy source and did not repress the oxidation of L-trypto- phan, L-kynurenine, or anthranilate (Figure 14). There- fore, arginine was chosen as the energy source for studies on the synthesis of enzymes of tryptophan catabolism. 82 Figure 12.--Induction of tryptophan oxidation. Cells (3.9 mg dry weight) which had been grown on sucrose were tested for their oxygen consumption rates in the presence (I) and absence (A) of L-tryptophan. Experimental details are described in Materials and Methods. 83 100 O 0 0 8 6 4 OwEDwZOO Zm0>x0 uO wmeSOEHZE 20 400 300 200 100 MINUTES 84 Figure l3.--Oxidation of L-tryptophan by cells grown on tryptOphan plus alanine (A) and tryptophan plus aspartic acid (B). Symbols for A: (o) L-tryptophan, (I) endogenous. Symbols for B: (I) L-tryptophan, (I) endogenous. Experimental details are described in Materials and Methods. 85 140 60L.— _ _ 120 IOO__ DwZDmZOO Zw0>x0 no mmwh30m0_2 20__ 60 50 40 30 20 10 MINUTES 86 400 CC; 200 m OwEDmZOO 7w0>xO “.0 wmthOmUZ). 60 50 40 30 20 10 MINUTES 87 Figure l4.--Oxidation of L-tryptophan (I), anthranilic acid (A), and L-kynurenine (I) by cells grown on L-tryptophan plus L-arginine. (o) endogenous. Dry weight of cells was 2.6 mg. Experimental details are described in Materials and Methods. 88 _ _ _ o 0 8 6 100 DmfiDmZOU z m0>xO do OIL _ _ 0 0 4 2 0mm»_40m0.§ 60 50 40 30 20 10 MINUTES 89 Co-metabolism of Kynurenine and Anthranilate The term, "co-metabolism" refers to any oxida- tion of substrates without the utilization of the energy derived from the oxidation to support micro- bial growth (49). Kynurenine and anthranilate were co-metabolized by B; megaterium. As previous results have shown, these compounds were oxidized at high rates without a lag. However, neither supported growth (Figure 15). There were four possible explanations: (l) Tryptophan synthesis was repressed or inhibited by anthranilate and kynurenine (2) The compounds were toxic in the concentrations used (3) The compounds did not induce the enzymes for their utilization whereas tryptophan did induce these enzymes (4) Enough of the compound was transported to give sig- nificant oxygen uptake rates, but not enough was transported to support growth. There was no evidence for the first explanation. Kynurenine would not be expected to exert any control over tryptophan synthesis because it is not an intermediate in the pathway. The control of tryptophan synthesis seems to be at the level of anthranilate synthetase which forms anthrani- .Late from chorismate. In some systems this enzyme is iffllibited by tryptophan. In others it is repressed by ‘tryptophan. The second explanation was tested by 90 Figure 15.--Growth of B; megaterium on L—tryptophan, 2 mg/ml (I); L-kynurenine, 2 mg/ml (A); or anthranilic acid, 2 mg/ml, (I). In each case the culture was inoculated with cells grown on L-tryptophan. Experimental details are described in Materials and Methods. 91 , I); P i v.» II _. A i. In 16 20 I 2 HOURS 92 inoculating cells into a medium containing arginine and kynurenine and into a medium containing arginine and anthranilate. Growth occurred in the presence kynurenine, but not in the presence of anthranilate. These results suggest the toxicity of the latter. The mode of action is unknown. The toxicity of anthranilate was somewhat unexpected since both Pseudomonas and Nocardia grow well on this compound (17). However, anthranilate inhibits the growth of Mycobacterium tuberculosis, B; coli and N; crassa (32, 99, 129). In M; tubercu- lggig its site of inhibition is catalase (80). The third explanation was tested by determining the ability of cells to adapt to kynurenine and anthranilate. The oxygen consumption rates of cells in the presence of L-tryptophan, anthranilate, and L-kynurenine began to increase after 90 minutes, 170 minutes, and 260 minutes respectively (Figure 16). Thus, cells were able to adapt to all three compounds but adaptation to kynurenine was poor. The reason that cells did not grow on kynurenine was probably the inability of sufficient amounts of kynurenine to get into the cell. Later results confirmed this compound as the inducer Of kynureninase. The ability of cells to adapt to énTthranilate indicated that its toxicity was not due 93 Figure 16.--Oxidation of L-tryptophan (I), anthranilic acid (I), and L-kynurenine (A) by cells of B; megaterium grown on sucrose as the sole carbon source. Experimental details are described in Materials and Methods. 94 7O mil-17.. . . I . £1.14": j A.» I “II . . II III.» :lil.‘ II I!. . 0 0 0 0 6 5 4 3 DMEDmZOO ZmO>xO m0 wmwh_40m0_§ 20 10 500 400 300 200 100 MINUTES 95 to inhibition of protein synthesis because new enzymes must have been formed in its presence. It can be concluded from these experiments that at least part of the control of the anthranilic acid pathway in B; megaterium ATCC 19213 was due to sequential induction. Enzymes of the Anthranilate Pathway» Warburg respirometry can be used successfully for determining the general character of a pathway, but it is not sensitive enough to provide quantitative estimates of low enzyme concentrations. Therefore, direct measurements of specific enzyme activities were made in cell-free extracts. Tryptophan Oxygenase When cells were exposed to L-tryptophan in the absence of catabolite repression, their extracts contained increased levels of tryptophan oxygenase. This enzyme was assayed spectrophotometrically by measuring the increased absorbance at 321 nm (28) ciue to the cleavage of the tryptophan indole ring ‘Mith the concomitant formation of N-formyl-L- ikynurenine (Figure 17). During each assay there was a typical lag befkare maximum rates were attained. This lag was not (due to the warming of the reaction mixture in 96 Figure l7.-—Tryptophan oxygenase, kynureninase, and anthranilate hydroxylase in cell-free extracts of B; megaterium. Cells were grown on L-tryptophan, harvested at the late exponential growth phase, washed, and sonicated. The extract was clarified by centrifugation and the resulting solution was assayed directly for trypto- phan oxygenase (OD 1 n I), kynureninase (ADD360 nm I), an anxtlhranilate hydroxylase (AOD3 0 nm’ A ). Experimental details are descr16ed in Materials and Methods. 97 EC eon DOQ .0 EC 0; 004 .0 EC an DO 12 10 MINUTES 98 the spectrOphotometer because the mixture was thermally equilibrated before assays were initiated. Kynureninase assays (Figure 17) were not subject to the same lag. This characteristic has also been demonstrated in enzyme preparations from Pseudomonas, Xanthomonas, and mammalian liver (115, 122). It is due to the allosteric nature of tryptophan oxygenase which is substrate activated. The lag in tryptophan oxygenase activity from the aforementioned three sources is abolished by ascorbate, but ascorbate had no effect on enzyme acti- vity in B: megaterium extracts. The lag time interval seemed to be inversely proportional to activity and protein concentration. The product of B; megaterium tryptophan oxy- genase activity was N-formylkynurenine as shown by its UV spectrum with a well-defined peak at 315-325 nm (Figure 18). In some assays, an additional product was formed which had a peak at 295 nm. It was believed that this compound was formylanthranilic acid based on its absorption characteristics, its fluorescence, its ether solubility, and the conditions under which it was formed. Bacillus tryptophan oxygenase was inhibited b)? sodium azide, potassium cyanide, and hydroxylamine (Ffiigure 19). These results indicated that this enzyme iS a heme protein, as are all tryptophan oxygenases. LII .3 99 Figure l8.--UV spectrum of the product of tryptophan oxygenase activity (12.5 units/mg protein). Experimental details are described in Materials and Methods. 100 wOZfluoa osmsomdm co sufl>uuoa osoflomsm smasouasns oumnumnsm ruzoso .omocwcoudcmx pco ommcomwxo condoummue mo coflmmoumom Uco COAUUSpCHII.m oHQoB 110 kynureninase was somewhat less affected. When arginine was used as substrate there occurred a total induction of tryptophan oxygenase and substantial induction of kynureninase. Overall, alanine and asparagine were less effective as substrates for the growth of E; megaterium but they did not repress the induction of either enzyme. Induction and Synthesis of Tryptophan Oxygenase and Kynureninase Since arginine did not repress tryptophan oxygenase or kynureninase, it was used as a sole carbon source to study the synthesis of these two enzymes. Induction of Tryptophan Oxygenase Cells were grown in Arg-S, harvested in the exponential growth phase, washed with SS buffer, and resuspended. Equal portions of the cell suspension were added to flasks containing arginine only; arginine and tryptophan; and arginine, tryptophan, and chloramphenicol. At specific time intervals, a given flask (which constitutes a sample) was chilled and treated with chloramphenicol. The extract of each sample was assayed for tryptophan oxygenase. The basal activity ("zero" sample) was subtracted. Fisgure 22 shows that incubating cells with tryptophan re5111ted in an almost immediate increase in specific '0 A '. J‘l “.'~'—S§l‘ 111 Figure 22.--Synthesis of tryptophan oxygenase. B. megaterium was grown in Arg-S in thrEe- 1iter batches. The cells were harvested in the late exponential growth phase, washed in SS buffer, and resuspended in media containing 0.4 % L-arginine (A), 0.1 % L-tryptophan + 0.4 % L-arginine (I), and 0.1 % L-tryptophan + 0.4 % L- arginine + 50 ug/ml chloramphenicol (O). The flasks were incubated at 30 C on a platform shaker for 5, 10, 30, 40, 50, and 60 minutes. Incubation was terminated by chilling to 4 C and adding chloramphenicol to a final concentration of 50 ug/ml. Cells were harvested and crude extracts were pre- pared by sonication. Extracts were assayed directly for tryptophan oxygenase after the removal of cells and debris. Specific activ— ity was recorded in terms of units/mg pro- tein. Basal values for tryptophan oxygenase activity (tryptophan oxygenase in cells in- cubated with arginine only) were subtracted. 112 .12 6 . 4 o. o. .08 >._._>_._.O< O_u _Owam .02 MINUTES 113 activity. Induction of tryptophan oxygenase continued throughout the experiment. Chloramphenicol effectively inhibited enzyme synthesis. This shows that tryptOphan oxygenase was made d3 pgyg. Induction did not occur in the absence of tryptophan. Figure 22 and Table 5 demonstrate some of the advantages of using the sensitive spectrophotometric assay for tryptOphan oxygenase. In the respirometer studies, there was a lag of ninety minutes before enzyme induction was observed. The carbon source, sucrose, may have influenced these results. The spectrophotometric assays revealed very low levels of tryptophan oxygenase even in the presence of catabolite repression. Kynurenine as an Inducer Although the inductive properties of tryptophan were readily obvious, the levels of enzyme activity were not as high as expected. This raised the possi- bility that tryptophan either was not the inducer of tryptophan oxygenase or was not the only inducer. A survey of tryptophan-related compounds uncovered L- kynurenine as the true inducer of both tryptophan oxygenase and kynureninase (Table 6). Addition of L-tryptophan did not increase induction. Anthranilate did not increase the specific activities of either enzyme. This shows that the 114 July". .smumaa 1HE\SENV Hcflmuo co czowv oHoB mHHoOI muoospcfl osu mo oocomonm ocu cs AHE\mEvV oc mumHAcmsrusa nvo. mva. mm.m m.m smeaosassqu poo ocflcoHSC%MIq m.m mo.m ocflcoHSCSMIA mas. ems. gaseoussssIo mam. mo.a gaseoussusIa no. ma. II .mE\muasa .mE\mUHcD omocommxo condoumhue «uoosccH mo sus>suo< oflusomsm .muosooum oflaonouo: omocflcouschx mo suflssuoa Usesoosm pco omocomhxo cosmovmxue mo coflUoSchII.w oanoe we omocflsouscwx 115 inducer is kynurenine and not its metabolite. The D-isomer of tryptophan was not an inducer of either enzyme. By way of explanation, D—tryptophan was not attacked by tryptophan oxygenase, kynurenine was not formed and induction could not occur. In X; pruni, the inducer of tryptOphan oxygenase is L-tryptophan instead of kynurenine but D-tryptophan causes gratuitous induction (12). Non-Coordinate Induction of Tryptophan Oxygenase and Kynureninase Since kynurenine induced both tryptophan oxygenase and kynureninase, it was possible that the synthesis of these enzymes was coordinate, iyg; con- trolled by the same "operon". Comparison of the syn- thesis of these two enzymes indicated that they were probably not coordinately induced. The rates of syn- thesis were quite different (Figure 23). Tryptophan reached its maximum specific activity after four hours whereas kynureninase had not reached its maximum at eight hours. In other words, kynureninase was being synthesized while the synthesis of tryptophan oxygenase liad ceased. Enzymes which are coordinately induced Imaintain a constant ratio of specific activities because they'are synthesized in a constant ratio. Table 7 stunvs that Bacillus kynureninase and tryptophan oxy- genasne did not have a constant ratio of specific 'il‘ 0' 116 Figure 23.--Synthesis of tryptophan oxygenase and kynureninase. B. megaterium was grown in Arg-S in thrEE-liter batches. The cells were harvested in the late expo- nential growth phase, washed in SS buffer, and resuspended in media con- taining 0.4 % L-arginine or 0.1 % L- tryptophan + 0.4 % L-arginine. Cell- free extracts were measured for trypto- phan oxygenase or kynureninase activity. Specific activity was recorded as units/ mg protein. Experimental details are described in Materials and Methods. Symbols: (I) cells were incubated with L-tryptophan and L—arginine, extracts were assayed for kynureninase activity; (I) cells were incubated with L-tryptophan and L-arginine, extracts were assayed for tryptophan oxygenase activity; (0) cells were incubated with L-arginine, extracts were assayed for typtophan oxygenase activ- ity; (0) cells were incubated with L- arginine, extracts were assayed for kynu— reninase activity. The eight-hour samples showed that 95 % of the L-tryptophan was not used. ACTIVITY SPECIFIC 117 1.4 .._ 1.2 I... 2 4 I F I 5 L "i .T —‘.':,a I L -, I I .I‘ 118 .Acflououm .mE\mUHcsv omocommxo cosmoumwue .Acflououm .mE\mpHcsv omocflcouscmm 1 A W. IMP-l: .. Ill“— . I n IIINQIISIIENF|Fn II II ME—I ¥ h©.m vm.H mmH. m mN.m oN.H oma. o H.@ mH.H NmH. v mm.oa mv. Hvo. N mm.m mNH. NNO. H cflououm .mE\mUHca cflououm .mE\muHco musom «B\M omocflcouscwm omocomxxo soSQOUquB oHdEom we oEHB .omocflcoudcxm pco omocomxxo cosmoummue mo cofluosch ouocflpuoooICOZII.h oHnoB 119 activities and therefore are probably not synthesized coordinately. Previous results showed that these enzymes are also subject to different degrees of re- pression and induction. Therefore, it was concluded that tryptophan oxygenase and kynureninase were not coordinately induced. Unequivocal evidence will await genetic studies. Tryptophan Permease Before a cell can utilize any compound as an eriergy source, that compound must be transported across ‘tlie cell's membrane barrier. Proteins which facilitate ‘tfiaea transport of amino acids and carbohydrates are gnaruerally called permeases (21, 83, 85). A series cxf experiments were undertaken to demonstrate the {pxrezsence and properties of Bacillus tryptophan per- mease. .Eflfillction of Tryptophan Permease When grown on arginine, bacteria had low per- Ineéissee activity. However, when bacteria were grown on L“"tjrjyptophan, high levels of tryptophan permease were iruflquzed (Figure 24). Sodium azide effectively prevented trEVIDtuophan transport, implying that the generation of AT“? VVas necessary for the process. Therefore, the acctlnnulation of 4C-tryptophan in cells was not due tc’ Eslilnple or facilitated diffusion. Rather, tryptophan - .m.»!..\.~.\17 V I 5' Nil—3.. 120 Figure 24.--Induction of tryptophan permease. Bacteria were grown on Trp-S broth (I) or Arg-S broth (A) and harvested in the exponential phase. Cells were washed with SS buffer7and resus- pended to approximately 2.0 x 10 cells/ml (viable count). The reaction mixturg (20 m1) contained approximately 4.0 x 10 cells, 0.05 uCi/ml of C—D,L-tryptophan, and 100 pg/ml of sodium azide (I). The uptake of C-D,L-tryptophan by cells was monitored by the procedure given in Materials and Methods. 121 I 0.0 9.0 8.0 7.0 6.0 CPM 5.0 10 4.0 1‘ “A 3.0 1 I‘- 2.0 I. :7 1 I 5 10 15 20 MINUTES 122 was transported by an active process which required energy and probably specific membrane proteins. Transport of D- and L-Tryptophan In some experiments cells transported all of the radioactive tryptophan which had been added. Since 14C-D,L-tryptophan was being used, it was suspected that Bacillus tryptophan permease transported both isomers. Competition experiments with unlabeled D- and L-tryptOphan indicated that this was the case. When either isomer was added to the reaction mixture at a concentration of 10 X the labelled tryptophan, uptake of the label was inhibited (Figure 25 A). That this was true competition was demonstrated when raising the concentration of either unlabeled isomer to 500 X completely prevented the transport of the radioactive amino acid (Figure 25 B). Arginine, Leucine, and PhenylalanIfie Competition Other amino acids were tested to determine their affinity for tryptophan permease. Arginine, leucine and phenylalanine did not markedly inhibit the uptake of l4C-D,L-tryptophan by cells of B; megaterium capable of tryptophan transport. However, L-leucine and L-phenyla- lariine did cause minor inhibition. The effects of L- leLicine on tryptOphan transport were not considered conqoetitive because there was little change in the 123 Figure 25.--Competition of unlabeled D- and L-tryptophan for tryptophan permease. Unlabeled D- and L-tryptophan were added to reaction mixtures in two different concentrations, 20 n mole/ml (A) and l mmole/ml (B). Radioactive D,L- tryptophan was present in a concentration of 1.72 n moles/ml. Symbols for (A): (I) No addition of unlabeled tryptophan; (A) addition of unlabeled D-trypto- phan, 20 n mole/m1; (I) addition of unlapfiled L-tryptophan, 20 n mole/m1. Values for - tryptophan transport in the presence of sodium azide were subtracted. Symbols for (B): (I) No addition of unlabeled tryptophan; (A) addition of unlabeled D-trypto- phan, l umole/ml; (0) addition of unlabeled L-tryptophan, 1 umole/ml; (0) addition of sodium azide, 100 umole/ml, no unlabeled trypto- phan. 124 MINUTES CPM IoJ 125 MINUTES F’s} 126 pattern of uptake when this unlabeled amino acid was raised from a concentration of 10 X to 500 X (Figure 26 B). Since there were very high levels of leucine permease in tryptophan-grown cells (Figure 28), the inhibition of tryptophan transport by L—leucine was probably due to non-specific competition for the general transport requirements such as ATP. Arginine did not cause the same inhibition because it was readily used for the generation of ATP whereas leucine and phenylalanine were not. However, when the concentra- tion of L-arginine was raised from 10 X to 500 X, the same non-competitive inhibition of tryptophan trans- port was observed (Figure 26 B). Transport of Phenylalanine, Repression by Arginine A similar argument could be evoked for explaining the minor inhibition of tryptophan transport by pheny- lalanine. On the other hand, many investigators have reported the existence of general aromatic permeases which transport phenylalanine, tyrosine, and tryptophan (l, 2, 7, l3, 14, 85). Therefore, the Bacillus permease which was induced may have been specific for all three aromatic amino acids and not just tryptophan. This question was investigated by determining the rates at 14 which cells transported C-L-phenylalanine when trypto- phan permease was induced or repressed. Arginine g? 127 Figure 26.--Competition of unlabeled L—arginine, L-leucine, and L—phenylalanine for tryptophan permease. Unlabeled amino acids were added to reaction mixtures in two different concentrations, 20 n mole/m1 (A) and l umole/ml (B). Radioactive D,L-tryptophan was present in a concentration of 1.72 n mole/ml. Symbols for (A): (I) No addition of unlabeled amino acids; (I) addition of unlabeled L-argin- ine, 20 n mole/m1; (0) addition of unlabeled phenylalanine, 20 n mole/ml; (A) addition of unlabeled L-leucine, 20 n mole/ml. Symbols for (B): (I) No addition of unlabeled amino acids; (A) addition of unlabeled L- arginine, l mmole/ml; (D) addition of unlabeled L-leucine, 1 umole/ml; (0) addition of unlabeled D—tryptophan, 1 umole/ml. 10 CPM 3.0 2.0 1.0 128 I.____.I____I__I_. 5 10 15 MINUTES 129 4.0 ._. 3.0 _. I: 1: 2.0 _. 1.0 30 2O 10 MINUTES 130 repressed both tryptophan permease and phenylalanine permease (Figure 27). Phenylalanine permease was derepressed along with tryptophan permease but not to the same extent. The possibility remains that these two permeases are the same. However, general aromatic permeases in B; coli and B; typhimurium have almost equal affinity for all three amino acids (1, 85). Therefore, if phenylalanine and tryptophan were transported only by a general aromatic permease, phenylalanine should have markedly inhibited trypto- phan transport and cells should have accumulated at least equal amounts of l4C—phenylalanine and 14C- tryptophan. Arginine may be a general permease re- pressor since it also repressed leucine permease (Figure 28). The increased phenylalanine transport was probably due to derepression of phenylalanine permease rather than transport by tryptophan permease. It was concluded that the inducible D,L-trypto- phan permease was specific for tryptophan and did not transport other amino acids. A general aromatic amino acid permease may exist, but it is not the same as the inducible tryptophan permease. Product Induction of Tryptophan Permease Kynurenine was tested for its effects on trypto- phan permease with arginine added as the energy source. 131 Figure 27.--Repression of tryptophan permease and phenyl- alanine permease by arginine. Bacteria were grown in the presence of tryptophan or trypto— phan plus arginine. Cells were washed with SS buffer and resuspended to approximately 2.0 x 107 cells/ml (viable count). The ability of these cells to transport l4C-D,L-tryptophan and l4C-L-phenylalanine was determined as described in Materials and Methods. Symbols: (I) uptake of l4C-D,L—tryptophan, cells were grown on L-tryptophan; (o) uptake of l4C-D,L-tryptophan, cells were grown on tryptophan plus arginine; (I) uptake of 14C- L-phenylalanine, cells were grown on trypto- phan; (o) uptake of l4C-L-phenylalanine, cells were grown on tryptophan plus arginine. CPM 103 132 MINUTES 133 Figure 28.-—Repression of leucine permease and phenyla- lanine permease by arginine. Bacteria were grown in the presence of tryptophan or tryp- tophan plus arginine. Cells were washed with SS buffer and resuspended to approximately 2.0 x 107 cells/m1 (viable count). he ability of thise cells to transport 4C-L- leucine and C-L-phenylalanine was determined as described in Materials and Methods. Symbols: (I) uptake of l4C-L-leucine, celli were grown on L-tryptophan; (o) uptake of l C- L-leucine, cells were grown on -tryptophan plus L-arginine; (A) uptake of C-L-phenyla- lanine, cells were grown on L-tryptophan; (I) uptake of C-L-phenylalanine, cells were grown on L-tryptophan + L-arginine. CPM 10’ 12.0 1 0.0 8.0 6.0 4.0 2.0 134 MINUTES 20 30 135 Kynurenine was a more effective inducer of tryptophan permease (Figure 29) than was tryptophan. Therefore, kynurenine appears to be the true inducer of trypto- phan oxygenase, kynureninase, and also tryptophan permease. There is only one other reported case of product induction of a transport system (96). 136 Figure 29.--Induction of tryptophan permease by L-kyn- urenine. Bacteria were grown in the presence of L-arginine (p), L-arginine + L-tryptophan (I), or L-arginine + L—kynurenine (A). The ability of these cells to transport l4C-D,L- tryptophan was determined as described in Materials and Methods. Values for tryptophan transport in the presence of sodium azide were subtracted. 137 1.4 1.2 __ 1.0 CPM Io3 “_ .4._________ -———————-—— 5 10 MINUTES 15 20 DISCUSSION There are few reports on tryptOphan catabolism in Bacillus (46, 90). This genus oxidizes tryptOphan via the anthranilic acid and indole pathways. The kynurenic acid pathway may be used also, because B. subtilis produces kynurenic acid when grown on trypto- phan (100). Few definitive data have been presented on the control of these pathways. Prasad and Srinivasan (90) reported that tryptophan catabolism in B; cereus is not subject to catabolite repression by glucose. They reached these conclusions after finding that the delayed pattern of enzyme synthesis was not changed when cells were grown in a defined medium in the absence of glucose. However, their medium contained large amounts of glutamate (76) and these investigators failed to recognize the repressive effects of this amino acid (35). The results in this thesis clearly show that B; megaterium uses the same pathway as B; cereus and that B; megaterium is subject to catabolite repression by glucose (Table 5). Definitive data could be obtained with this organism due to its ability to grow on minimal media containing only glucose and salts. Prasad and Srinivasan also suggested a role for tryptophan catabolism 138 139 in sporulation. Some of the B; megaterium Brs mutants which lacked an intact pathway for tryptophan catabolism also had a reduced ability to sporulate. This apparent relationship may have been due to double mutations or due to a mutation in a regulator gene which affected more than one cellular function. This would be similar to the SEE mutations in B;_ggl£ (24). The oxidation of anthranilate and catechol does not seem to be re- quired for sporulation because mutants which oxidized these substrates at significantly lower rates than the wild type were not impaired with respect to their abilities to form heat-resistant spores. These results do not necessarily contradict the conclusions reached by Prasad and Srinivasan because they linked sporulation with the oxidation of L-tryptophan, N-formyl—L-kynurenine, and L-kynurenine and not anthranilate and catechol. With respect to NAD synthesis in B; subtilis, Yanofsky reached the right conclusions but possibly for the wrong reasons (128). He found NAD-requiring mutants could not use kynurenine as a substitute for NAD. B; megaterium appeared to be somewhat impermeable to kynurenine and the same factors may have accounted for Yanofsky's results. Mutants which were deficient in tryptophan catabolism were used to study tryptophan catabolism as a requirement for NAD synthesis. Brs-5, 140 Brs-8, and Brs-10 grew well on defined media and re- quired no nutritional supplements. All three seemed to have reduced anthranilate hydroxylase activity when compared to the wild type. Since anthranilate must be oxidized to form NAD via the tryptophan pathway, these results imply that B; megaterium synthesizes NAD by another route. The Anthranilic Acid Pathway of B. megaterium B; megaterium was able to grow and sporulate on media containing L-tryptophan as the sole carbon, nitrogen, and energy source. The pathway involved the cleavage of the indole ring of tryptophan, removal of a formyl group, and hydrolysis of kynurenine to form anthranilic acid. This sequence has been referred to as the aromatic or anthranilic acid pathway. Inter- mediates of the pathway, kynurenine, anthranilate and catechol, were excreted into culture media and were identified by thin layer chromatography, paper chromatography, and UV spectra. The presence of the various intermediates in Trp-S cultures implied that B; megaterium was capable of oxidizing these compounds and the oxidation of L-kynurenine, L-alanine, and anthranilic acid by cells grown on L-tryptophan was found to occur at very high rates. Thus, B; megaterium 141 was capable of synthesizing the enzymes of the anthranilic acid pathway. It is also possible that kynurenic acid was a product of tryptophan catabolism. Culture supernatant solutions consistently contained compounds whose UV spectra matched kynurenate's with maxima at 332 nm and 345 nm. Kynurenine transaminase was not detected in cell-free extracts, but this may have been partially due to the high levels of kyn- ureninase which were present. Mutants which were unable to grow on trypto- phan accumulated compounds in the media which had the UV spectra of anthranilate and catechol. The accumu- lation of anthranilate by Brs-5 was confirmed by thin- layer chromatography. Other mutants were not able to oxidize significant amounts of anthranilate and/or catechol. These strains had been treated with a mutagen (NTG) which must have caused a genetic alteration which was phenotypically expressed as the inability to oxidize specific intermediates in the anthranilic acid pathway. These mutants provided additional evidence that this particular pathway was used for tryptophan catabolism in B. megaterium. Sucrose, glucose, and glutamic acid caused catabolite repression of the pathway but alanine, arginine, asparagine, and aspartic acid, did not. At 142 least part of the pathway was controlled by sequential induction because cells that were exposed to kynurenine or anthranilate adapted to these substrates as indicated by the increased rates that such cells consumed oxygen. These results implied that the cells synthesized the enzymes necessary for the oxidation of kynurenine and anthranilate after being exposed to these substrates. Tryptophan oxygenase, kynureninase, and catechol oxygenase were found in cell-free extracts of cells grown on L—tryptOphan. Anthranilate hydroxylase activity was detected and assayed manometrically but was too unstable to be measured spectrophotometrically. The presence of these four enzymes in B; megaterium grown on tryptophan was additional proof that the anthranilate pathway was the route for tryptophan oxidation. For- mamidase was not detected in extracts, but the presence of kynurenine in culture supernatants indicated that it must have been active in whole cells. Alternate Pathway In addition to the oxidation of tryptophan via kynurenine, B; megaterium may use an alternate pathway. The proposed sequence of intermediates is: L-trypto- phan + N-formyl-L—kynurenine + N-formylanthranilic acid + anthranilic acid + catechol ++ succinate + acetate. The presence of an alternate pathway was 143 suspected when it was found that a compound with a UV maximum at 295 nm was formed after tryptophan was added to extracts with tryptophan oxygenase activity. Since formamidase was absent from these extracts, N-formyl- L—kynurenine presumably accumulated. The only other enzyme which can use formylkynurenine as a substrate is kynureninase which was present in these extracts. This enzyme promotes the formation of N-formylanthranilic acid from N-formylkynurenine (50). The 295 nm absorbing compound was similar to N-formylanthranilic acid in its UV fluorescence, Rf value, ether solubility, and UV spectrum. If the prOposed alternate pathway exists, it is probably functional under normal conditions since the 295 nm absorbing compound is excreted into the medium during bacterial growth on tryptophan. However, it would be a minor route since kynurenine is excreted into the medium in micromolar amounts and since this intermediate plays an important role in tryptophan catabolism. Selective Advantage of the Anthranilate Pathway The selective advantage of oxidation of tryp- tophan via the anthranilic acid pathway is its convergence with the E-ketoadipic acid pathway. The latter is common to the oxidation of compounds such as phenol, 144 benzoate, and mandelate. This provides an efficient means for the degradation of aromatic structures. Additional regulatory or structural genes and proteins are not required beyond the step of catechol because the pathway and its regulation are established. The kynurenic acid pathway requires a number of different enzymes and protein repressors due to the lack of its early convergence. One selective advantage of the kynurenic acid pathway is that it avoids the production of toxic products such as anthranilic acid and catechol. An- other advantage is that D-tryptophan is typically oxidized by this route. Organisms which use the anthranilic acid pathway do not degrade the D—isomer. B; 391$ oxidizes tryptophan efficiently by the indole pathway. This organism cleaves the alanine moiety and obtains pyruvate in one step. Therefore, fewer enzymes are required for energy production in the indole pathway than are required in the anthranilic acid or kynurenic acid pathways. Since B; coli is generally found in the intestine, it is surrounded by a nutrient-rich medium and has no need to completely degrade tryptophan for carbon skeletons and other building blocks. However, soil organisms such as Bseudomonas sp. or Bacillus sp. might have to utilize all the energy and carbon sources available. 145 Pathway'Controls Ornston (82) has clearly defined many of the terms commonly used in discussions of control systems. According to his definition, an inducer is the meta- bolite which most directly elicits the synthesis of an enzyme. Repression is a relative decrease in the rate of synthesis of a specific apoenzyme resulting from exposure to a chemical substance. A metabolite repressor is the intermediate that most directly re- presses the synthesis of an enzyme. This avoids con- fusion with repressor proteins. Sequential induction is characterized by a shift in the chemical nature of the inducer. Hence, regulatory units that undergo sequential induction are always controlled indepen- dently. Enzymes that are induced by the same meta- bolite are governed by "coincident induction". In coordinate induction, the ratio of the rates of syn- thesis of enzymes remains constant despite extreme fluctuations in their absolute rates of synthesis. Enzymes that are subject to coordinate control are members of a regulatory unit such as the operon. The selective advantage of sequential induction is in the economy of protein synthesis. Enzymes are only induced in the presence of their substrates. The selective advantage of the Operon, a control unit .involving regulator and structural genes, is the 146 regulatory efficiency. The cell has maximal economy of protein synthesis governed by a minimal amount of regulatory information. TryptOphan catabolism in B; megaterium appears to be controlled by coincident and sequential induction. Both tryptophan oxygenase and kynureninase are induced by kynurenine (coincident induction). In nature, tryptophan is a more common growth substrate than L-kynurenine. As the regulation of kynureninase and tryptophan oxygenase evolved, the demand for the in- duced synthesis of the former was almost always accompanied by a demand for the synthesis of the latter. Therefore, the selective pressures favoring unified biosynthetic control overcame those favoring strict sequential induction and these two enzymes were sub- jected to coincident control. Obviously, the gratuitous synthesis of tryptophan oxygenase burdens the cell with a potential hazard. Evidently, this potential hazard is less burdensome than the drain of energy and carbon required for the continuous synthesis of regulatory molecules to govern the enzymes sequentially. There are three ways in which tryptophan could increase tryptophan oxygenase activity as shown in Figure 22: (l) the combination of tryptophan with tryptophan oxygenase could stabilize the enzyme (103) and prevent its degradation (2) the oxidation of tryptophan 147 to formylkynurenine and kynurenine might be effected by "constitutive" tryptophan oxygenase and formamidase, respectively. Kynurenine could then act as a positive feedback signal for the induction of tryptophan oxy- genase (3) tryptophan could be a positive effector for tryptophan oxygenase. Post-Translational Controls The tryptophan pathway was also controlled at the enzyme level. Assays of tryptophan oxygenase indicated that this enzyme reached maximal activity only after binding tryptophan. This was undoubtedly due to its allosteric character. Enough substrate must be present to activate the enzyme. If the allo- steric site has a relatively low affinity for trypto- phan, activation can only occur when the substrate concentration is high. In this manner, low concentra— tions of tryptophan can be conserved for the internal pool and utilized in protein synthesis. Anthranilate hydroxylase is unstable in all systems in which it has been studied. In the absence of anthranilate, the enzyme rapidly disappears from cells. In Nocardia opaca, increasing the temperature from 30 C to 37 C markedly decreases enzyme activity (16). Such instability may have selective advantages for the prevention of the breakdown of internally Synthesized anthranilate. 148 Metabolite Repression Evidence was presented which indicated that kynurenine and anthranilate were inducers in the Bacillus pathway and that sucrose, glucose, and glutamate caused catabolite repression. The latter three carbon sources supported high growth rates of B. megaterium. Carbon sources which did not support high growth rates did not repress the tryptophan enzymes. These results, coupled with the knowledge that cAMP can help relieve the repression of certain bacterial enzymes (53), suggested that the repression of enzymes in the anthranilic acid pathway might in some way be related to energy production. Cox and Hanson (22) studied the catabolite repression of aconitate hydratase in B; subtilis. They found that repression was released when this organism was grown on media deficient in phosphate or nitrogen. Sulfate and tryptophan limitation led to increased repression. The addition of adenosine to cultures growing under nitrogen limitation caused a complete repression of aconitate hydratase synthesis. The data suggested that the metabolite repressor of this system could be ATP or a related compound. There are many other reports which support this hypothesis (29, 88, 89). 149 Pigment Production The break-down of tryptOphan by B; megaterium resulted in the production (or release) of a brown pigment during the stationary phase. This was shown by radioactive labeling studies, tryptophan utiliza- tion Xi' growth and pigmentation studies, and mutant studies. Anthranilic acid could be a pigment precursor. Cain (16) observed that when Pseudomonas and Nocardia 92333 were grown on anthranilic acid, a brown pigment was excreted into the medium. The minimal concentra- tion of anthranilate in the medium required to induce anthranilate hydroxylase was 5-6 micrograms/ml. At 2—5 micrograms/ml no formation of the hydroxylase was observed. When anthranilate was in a concentra- tion above 8 micrograms/ml the enzyme always appeared. The catabolism of g—nitrobenzoate resulted in the excretion of the same brown pigment. Catechol is an intermediate in both the g-nitrobenzoate and anthranilate pathways and it turns brown when left standing. Its formation could completely explain pigment production. Also, dehydrogenation of catechols leads to the pro- duction of quinones which could condense to form the brown pigment. According to these theories, pigment production depended on anthranilate hydroxylase activity. Since hydroxylase activity depends on the amount of anthranilate in the medium, pigment production 150 was also related to kynureninase activity and the concentration of kynurenine. If Bacillus kynureninase was inhibited by its product, alanine (74), the accum- ulation of anthranilate was prevented until alanine was exhausted from the medium. It is proposed that alanine was not exhausted until the late exponential or early stationary phase. At this time there was increased kynureninase activity which led to anthra- nilate accumulation. Hydroxylase was induced which led to the accumulation of catechol; but since catechol oxygenase was product-induced, there was a delay in the oxidation of this intermediate. As the catechol accumulated, it underwent spontaneous changes which led to the formation of the brown pigment. There is evidence presented in this thesis which supports the above proposal: (1) The brown pigment was formed spontaneously upon standing in the cold. Even after cells had been removed, the stationary phase culture supernatant turned brown overnight. (2) Kynurenine was the inducer of kynureninase and accu- mulated throughout the growth cycle of the organism. (3) Anthranilate seemed to induce Bacillus anthranilate hydroxylase. (4) When induced cells were incubated with anthranilate, the reaction mixture became intensely pigmented. (5) The UV spectrum of the purified pig- ment was identical to the UV spectrum of catechol. 151 (6) Mutants which were blocked in anthranilate oxi- dation did not produce the brown pigment but mutants which were blocked in catechol oxidation did. Thus, by understanding the controls and characteristics of a pathway, an observation in nature could be explained. Pigment production was not necessary for sporulation, since non-pigmented mutants sporulated well. The formation of both pigment and spores during the stationary phase was probably a coincidence in which the genes of both systems were turned on by a common but general factor such as the decrease in mRNA or protein synthesis which occurs upon the cessation of the exponential growth phase. Bacillus Tryptophan Permease B; megaterium synthesized an inducible trypto- phan permease. This transport system had affinity mainly for tryptOphan but may have transported pheny- lalanine to a much lesser degree. On the other hand, there may be a separate phenylalanine permease which was repressed and derepressed under the same conditions as tryptophan permease. As shown by the results of competition studies, arginine and leucine were not transported by the per- mease but D-tryptophan was. The transport of D-trypto- phan seemed to be gratuitous since tryptophan oxygenase 152 -0 did not attack the D-isomer and a tryptophan racemase is apparently absent. Arginine caused repression of tryptophan permease, as well as phenylalanine permease and leucine permease. The inducer of Bacillus tryptophan permease was L-kynurenine. Although the possibility remains that kynurenine and tryptophan induced different per- meases, this is unlikely since tryptophan was unable to overcome repression by arginine. The advantage to the cell of having kynurenine as the inducer in- stead of tryptophan is two-fold: (l) Kynurenine will usually be present only when tryptophan is being oxidized. 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