STUDIES ON THE METABOLISM OF RIBOFLAVIN IN ASHBYA GOSSYPII By ROBERT LAWRENCE STEPHENS A DISSERTATION PRESENTED TO THE GRADUATE COUNQL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA June, 1956 ACKNOWLEDGMENT The writer vishes to express his appreciation to Dr. T, W, Steams, under whose direction this work was carried out j to the Tennessee Eastman Company for the Kodak Fellowship funds which permitted a more extensive study of the problemj to the Chemistry Department of the University of Florida ^rtiich provided supplies and laboratory space) and to Dr. L, J, Wickerham of the Agriciiltural Hesearch Service, United States Department of Agriculture, irtxo supplied the culture of Ashbya gossypii* 11 TABLE OF CONTENTS . ; •• Page ACKNC»JLEDC5MiI?TS *^ ii LIST OF TABLES . vil LIST OF FIGURES xi I. ROTI2W OF THE LITERATORE • , 1, A, The Microbiological Synthesis of Riboflavin % B, The Bioaynthe^c Relationships of Amino Acida 11 C, Chromatograpty 17 D, Ribofla-rin Assay ■ v ' 19 II. STATEMENT OF THE PROBLEM 21 m, EJCPEiOMENTAL 22 A, Apparatus 22 1. The Colorimeter 22 2. The Shaker 22 3. Tiie pH Meter 22 k» 35ie Fluorometer 23 B, General Procedures 23 1. I^ntenance of the Culture 23 2. Jbcperimental Media 23 3. Inoculation and Incubation of Media 2li it* Wbarorement of Growth 2^ C, Riboflavin Determinations 26 ill 1. Preparation of the Sample for Riboflavin Beteminationa 26 2. Fluorometric Detennination of Riboflavin 26 Chromatography /" 31 1. Paper Sheets and Strips 31 2. Circular Chromatography . 31 3. Goluntn Chromatography 32 E« iaqperiaenta 3^ 1. Ihe Relationship of Riboflavin Synthesis to Growth In a Peptone liedium 35 2, llie FractioDation of Peptone on a Cellulose Coluam UO 3# ^e Growth and Riboflavin Production of A, goasypii with Peptone Fractions as Nitrogen Sources Ijl The Proximate Qualitative Chromatographic Analysis ■ of the Amino Acids in the Peptone Fractions h3 $9 !Ihe Effect of Various Amino Acids C(»nbinations on the Growth and Riboflavin Fomation of L» Eossypii , U6 6# Further Studies on the Effects of Amino Acids on the Growth and Biosynthesis of Riboflavin by A. gossypii k9 7. The Effect of Serial Transfers of Cells of g^gypll on Synthetic liedla, on Growth and ?a.boflavin Formation 8, The Growth and Riboflavin Formation of A. gossypii as Affected by Inocula Prepared fro!m'"Cells Subjected to Different Numbers of Washings 63 9» The Effect of Miscellaneous Nitrogen Sources on Growth and Riboflavin Formation 6l< 10, The Treatment of Peptone with Peimitit 66 It Page 11. The GTowth and Riboflavin Syniihesla on the Glutamic Acld-Arginine Medium vitb. Different Concentrations of Glycine 12. The Fffect of -different Concentrations of Peptone on Growth and Riboflavin Formation ia a Glutaaic Acid-Arginine-Glycine Keditun 72 13. Tlie Effect of the Components of the Glutamic AcidU Arginine-Glycine Hedl;jm on the Synthesis of JBiboflavin in a Peptone Modluai Hi Hi. The Effect of a Cell Extract of A, gosgypii on Growth and Riboflavin Formation . 1^, The Effect of Chrcanatographic Fractions of an A, gosa:;/pii Cell Ext.ract on Ribollavin ^bimation 78 16* A Further Stud^ of the Effect of Cell Fjctracts of ^» gfOS3,vpii on Growth and uiboflavin Formation 82 17. The Colorimetric Analysis of JCiboHavin ' U 18. The Effect of Glycine, L-Serlne and DL-lhreonine on Growth and .■-iboflavin Synthesis xdien Added to Different Synthetic Media ft 19. The Effect of L-flistidine HCl on Growth and Riboflavin Formation in Glutamic Acid-Arginine and Glutamic Acid-Arginine-Glycine Media f$ 20. The Effect of Initial pH of the Medium on Growth and Idboflavin Formation in the Glutamic Acid- Arginine Medium and the Glutamic Acid-Arginine- COycine-Histidine Medium 98 21. The Effect of Imidazole on Growth and Riboflavin Synthesis 102 22. The Effect of Different Concentrations of Glutamic Acid or Aspartic Acid on Growth, Riboflavin Formation and Histidine inhibition in Synthetic Media 105 23. Studies on Growth and Riboflavin Synthesis in the Glutamic Acid-A3?ginine-Histidine Medium with Different Concentrations of Amino Acids 108 2U» Studies on Growth and Riboflairln SyntJiesis in th* Glutainic Acid- The Growth and Riboflavin Synthesis of A, gossypii in a Glutamic Acid-Arginine (GA) Media with Added. Amounts of Glycine ft M "Hie Orowth and Riboflavin Synthesis in a Olutajnlc Acid- Aj-ginine-Glj-eine (C-AGly) liedium Containing Various Concentrations of Peptone d Bie Synthesis of Riboflavin in a Peptone Kediua Containing Cooponents of the Glutamic Acid-Arginine-Glycine (GAGly) Medivan |3f tt jEhe Synthesis of Riboflavin by A. gossypii in Glutajnic Acid- Arginine (GA) and Glutamic A~id-^rginine-Glycine (GAGly) Media Containing 0.1 Milliliters of Cell Extract 77 $$ The Chromatographic Composition of an A, goss:)'pii Cell Ebctrp.ct from a Butanol-Acetic Acid-«ater Solvent System 80 fil The Effect of ^ronatogrjqpliic Cell ioctract Fractions on Riboflavin Synthesis by A, gossypii in a Glutamic Acid- Arginiae-Glycine (GAGly) "tledium 81 0 The Growth and Riboflavin Fomation in a Glutamic Acid- Arginine-Glycine Medium with Added Cell inxtracts and • Acetone Dried Cells 8ll viii Table Pag* Abcorbsmce Values for Kno;«i Riboflavin Sol-u.tiona tf ff Ihe Cetenrdnation of Hlboflavin in Aliquots of an Jbctract of Cells and a Check of Recovery of Added Riboflavin 91 Ihe Effect of Glycine, L-Serine or DL-Threonine on (Irowth and riboflavin Synthesis in QLutaitdc Acid-Arginine, (GA) Asparbic Acid-Arginine, (AA) and Asparagine-Ai^liiine (Aspul) Media 9h 2^ Ihe Effect of Histldine HCl on Gro^rbh and Riboflavin Formation in Glutaraic Acid-Arginine (GA) and Glutamic Acid-Arginine-JC-lycine (GAOly) Media 96 |9 The Effect of Histidine HCl on Growth and Riboflavin Formation in the Glutamic Acid-Arglnine-iJlycine (GAGly) Kedium ■ 99 31 The Effect of Initial pH of Media on Grovrth and Riboflavin Fomation in the Glutanic Acid-Arginine (GA) and Glutamic Acid-Arginine-Glycine-Histidine (GAGlyH) Kedia liSl j9 A Comparison of the Effect of Imidazole vith that of Histidine on Growth and Riboflavin Synthesis in the Glutamic Acid-Ai^inineX-lycine (GAGly) Kedium . UBk 3© the Effect of Different Concentrations of L-Glutamlc Acid and L-Aspartic Acid on Growtli and Riboflavin Synthesis in Synthetic Media 106 % The • f f ect of Glycine on C-rowth and Riboflavin Formation During Eovir Days Incubation in Sjmthetic l^edia xdth Glycine Added at Different Times During Incubation 109 If The Effect of Delajied Addition of Glycine on Growth and Riboflavin Formation in Synthetic ^edia 112 36 The Growth and Riboflavin Formation in the Glutamic Acid- Arginine-Histidine (GAH) Mediijin with Different Concen- trations of Different Amino Acids 115 37 Results of Calculations to Determine the Riboflavin Synthesized per Milligram of Amino Acid Added to the Glutamic Acid-Argixiine-ilistidine iiediun 125» Jtf The Amino Acids Added to the Basal in the Amino Acid Base Medium Hfy ix ^^le Growth and Riboflavii! Synthesis on a %)ecial Amino Acid Medium with lUfferent Concentrations of Principal Nitiyjpen Soiirces The Effect of Serine on Riboflavin Formation in Olutandc Acid-Arginine (GA) and Glntainic Acld-Arginine-Histidine (G^.H) Media with Different Concentrations of Glutamic Acid . , . . The Effect of Different Concentrations of Asparagine on Growth anc^ Riboflav5.n Formation in a Peptone Kedium LIST OF FirrUnES Figure Pag« 1 The Biosynthetic Relationships of Amino ^c±da 2i 2 A Standard Riboflavin Cnrre . ; 30 3 The Relationship of Growth, pH and Riboflavin Content of Cultures of A, ?;ossv'pii Orown in a Peptone Keditjai and the T)ry Weights of Their Extracted Cells 39 Ix A Standard Curve for the Colorimctric Analysis of Riboflavin 8f 4" ad 1. RE7IEW OF THE LITERATURE A, The Microbiological Synthesis of Riboflavin Riboflavin, one of the B complex vitamins, vas first discovered by Goldberger and Lillie (3U) while studying the nature of pellagra. They reported the eristance of a heat stable substance vhich prevented certain deficiency syrqjtoms in rats which were on special diets. Kuhn, et al,, (U9) isolated a yellow-green fluorescent substance froB egg vhlte and demonstaTated that it was similar to the factor reported by Goldberger and Lillie. Ellinger and Koschara (26) reported the isolation of a similar substance from whey, liver, kidney, muscle, yeast and cer- tain plant materials. These isolated materials were named ovoflavin, lactoflavin, heptoflavin, etc., depending on the source from which they had been isolated. l*hen it became known that they contained the same active substance, the various names were discarded and the name, ribo- flavin, was adopted. Karrer, Schopp and Benz ikh) described the first chemical method for the synthesis of riboflavin which has the following structure I CH2-CHOH-CHOH-CHOH-CH2OH II 0 X i Ton Baler, et al., (108) establiahed that synthetic riboflavin and the natural substance were identical. Althotigh the chemical synthesis of riboflavin has been veil estab- lished, the methods by which organisms synthesize riboflavin have not been elucidated iq) to the present time. Riboflavin has been reported to be synthesized in appreciable amounts by several nlcroorganiemSf naael/^ Clostridium acetobutylicinn (117), Candida gnlHermondia (90), Cairiida flareri (99), Kycobacterium smepjiatis {$3)$ Aspergillus flavus (70), rusariian species (62), Saceharomyces cerevisiae (33), Eremothecium ashbyii find Ashbya ^ossypii (37). The closely related microorganisms, Eremothecixm ashbyii (E, ashbyii) and Ashbya gossypii (A, gossypii), are used in the commercial microbiological synthesis of riboflavin due to their capacity to produce large amoiints of the vitamin. For this reason, the conditions for microbiological synthesis of riboflavin have been studied more iiidely in these organisms than in other known producers of the compound. The microorganisms, E, ashbyii and A, gossypii, are plant patho- gens, having been isolated from the infected boll of the cotton plant, Ouillemond, Fontaine and Raffy (37), in 1935, reported that the organism, E* ag-l^t)yii« produced a yellow-orange fluorescent pigment when cultured on Sabouraud's Medium at 25° C. They found that A, gossypii produced only anall amounts of the pigment when cultiired under the same conditions, Raffy and Fontaine (76) produced large amotmts of the flavin pigment by growing E, ashbyii on Sabouraud's Agar Medium and Raffy (75) showed by r»t experiments that the pigment had vitamin properties similar to riboflavin. Miriraanoff and Raffy (59) demonstrated that, microchemically, physiologi- cally, spectrographically and fluororaetrically, the flavin pigment was Identical vith riboflavin. In 1930, Farries and Bell (27) stated that A. i^ossypii voxild not grow on artificial media containing KNO^ and «Bmonium salts unless sup- plemental growth factors were present. However, hydrolyaed natural pro- telnaceous material would serve as a good nitrogen source for growth, Buston and Pramanik (12) established that the factor necessary for growth was a complex. One component was inositol and the other was a snabstance vhich was similar to a then little known factor called biotin, Kogl and Fries (li7) and Fries (28) showed that three factors, biotin, thiamine and Inositol, were necessary for the growth of A, gossypii, Buston, Kasinathan and Wylie (ll) reported that inorganic and organic aHimoni\im salts would not give satisfactojry growth when added to a medium of natural lentils extract, ndneral salts and inositol. However, when /^'-alanine or L-asparagine was used, moderate growth was obtained. The first systematic study of riboflavin production by E, ashbyii vas made by Schopfer (8?) ■^o used a basal liquid medivan containing, 1 per cent glucose, 0,1 per cent glycine or asparagine, 0,05 per cent i'5gSO|j,7H20 and 0,15 per cent KH2P0^, to test the effect on riboflavin synthesis when different natural extracts were added to the basal meditun. Liver eoctraet, at a concentration of 3 per cent, produced a marked stimulation of ribo- flavin synthesis in the basal medixxm. There was a considerable variation In the amount of riboflavin produced when peptones from various sources were tested, Norite treatment of peptone rendered it inactive for the synthesis of the vitamin, Biotin was reported to be essential for growth and riboflavin synthesis with thiamine aiK3 inositol acting as complemen- tary growth factors. It Renaud and Lachaxuc (81, 82), in experimenta with E, ashbyli grown on a liquid medium of peptone, glucose and NaCl, reported that the depth of the medium was a factor influencing yields of riboflavin, highest yield* being obtained vhen the depth was less than 1 centimeter, Hiey obtained highest yields on the peptone-glucose medium when 1,5 - 3.0 per cent glu« cose and 1,2 per cent peptone were used. The addition of 0,1 per cent NH}jfi2PCJ^was benificial, Schopfer and Chiilloud (9l) reported that leucine or arginine, alone or in combination, supported the growth of E, ashbyli in a medium to which biotin, thiamine and inositol had been added, Moore, de Becze and Schraffenberger (6o) studied the synthesis of riboflavin by E, ashbyii using the shake flask method of growth. The optimum temperature was found to be betv/een 26 - 30® C, with an optimum of 5,5 - 6,5. The pH was reported to increase to the alkaline side in the later stages of growth. Refrigeration of the culture at U° C, for more than 7 days reduced the ability to produce riboflavin, Lyophil- iaation destroyed the vitamin synthesizing ability completely, Hadert (86), using E. ashbyii, reported that riboflavin could be synthesized on a carbohydrate free nutident medium containing a metabo- lizable lipid, such as com oil, egg albumin an^ mineral salts, as long as sufficient air was supplied to the culttire, Deseive (21) observed that 0,001 per cent cystine reduced the growth of E, ashbyli and inhibited the riboflavin formation. Chin (13) studied the growth and riboflavin formation of E, ashbyii in a medium of 1 per cent glucose, 0,2 per cent KH2P0^, 0,1 per cent J%SOi^, 0,1 per cent llaCl, 0,1 per cent agar, 5.0 per cent rice germ extract and 5,0 per cent of a suitable nitrogen source. Peptone was the best nitrogen sotaree, folloved by glvitaalne, asparagine, alanine and (NK2j)2S02j in decreasing order of suitability as a nitrogen source. Tryptophan, tyro- sine, cystine, cysteine, histidine and urea were not satisfactory. Glu- cose, sucrose and glycerol were good carbohydrate sources but glycol, aylose and mannitol could not be used, Phelps (63) grew E, ashbyii on a medium with solubilized casein as the nitrogen source. He reported that 0,2^ - 2,5 per cent fatty acid glycerides increased the synthesis of riboflavin with the greatest stimu- lation being shown by butter fat, , In 19lj6, VJickerham, Fleckinger and Johnston (II3) reported that a yellow-orange varient strain of A, gossypii resulted from continual transfers of the original light yellow strain. This new strain was able to produce appreciable quantities of riboflavin when grown under proper conditions, When grown in a medium of 0.3 per cent yeast extract, 0.5 per cent peptone and 2,0 per cent glucose at pH 6,8 - 7.0, under aerated culture conditions and at 28° C,, abundant a»ounts of riboflavin were produced. They reported that sucrose and maltose but not lactose could be used as a substitute for glucose. Tanner, Vojnovich and Van Lanen (lOO) made an extensive study of the conditions for growth and riboflavin synthesis by the organism. A, gossypil. The organism grew well over a wide temperature range but highest yields of riboflavin were obtained when the culture was incubated at 26 - 28° C, Tewperatures higher than 28° C, decreased the riboflavin synthesis sharply, whereas tonperatures below 26° C, caused a lesser decrease. The best initial pH range was found to be between 6,0 - 7,0, Glucose at a concentration of 0,25 - 3.0 per cent served as an adequate carbohj^ate source. Sucrose and maltose in relatively pure form could replace glucose but xylose and arabinose vere not metabolized. They fouztd tlaat 0«5 per cent com steep liquor 0»5 per cent peptone, as nitrogen sources, gave very high yields of riboflavin. The fermentation was reported to be conqjrised of two distinct phases. In the first phase, which occurred during the first 2lj - 36 hours, glucose was assimilated, the medium became more acid and very little riboflavin was formed. The second phase occur- red after glucose consumption was substantially complete and was charact- erized by an increase in the pH of the medium and a rapid production of riboflavin, Pridham (7l) found that riboflavin synthesis by A. gossypti was increased by the addition of 1 - 2 per cait of a fermentable sugar to the medixan after the fermentation had progressed for 2li - 72 hours, usually during the period of pH increase between pH 5.3 and 6,It, Most of the early woric on riboflavin synthesis by E, ashbyli and £• gossypll was mairJ.y designed to bring about a greater commercial pro- duction of the vitamin under more economical conditions. It wag not until about 1950 that any interest was directed toward how these microorganisms produced riboflavin and what chemical components were involved In this synthesis, Bulaney and Grutter (2k) studied the nutritional requirements of E, ashbyii grown on a medium of glucose, hydrolyzed casein, inositol and mineral salts. They found that inositol was necessary for growth and riboflavin production and that low levels of glutamine, pantothenic acid, vitamin Bx2, nicotinic acid, pyridoxine, choline, p-aminobenzoic acid and folic acid had no effect when added singly or together, L-proline, DL- glutamic acid and L-arginine with inositol gave growth and riboflavin T formation, Minoura (56) reported that ashed peptone had no growth pro«Boting not riboflavin produclnfr effect on E. aahbyll. Hydrochloric add hydro- Ijzed peptone was more effective in promoting growth and riboflavin syn- thesis that vmtreated peptone, Asparagine and arginine prOTioted the growth of the organism but no riboflavin was produced, Pridham and Raper (72) gave an extensive morphological and cyto- logical description of A, goasypil. They described the growth cycle of the organism from the vegetative stage, through the sporulation stage and to the spore germination stage. Fhotcsnicrographs illustrating the different growth phases included the "bulb forms" which acco^^3any ribo- flavin synthesis. In another report, Pridham and Ra^er (73) also sub- jected A, gossypii cultures to ultra violet light, x-ray radiations and chenilcal mutagenic agents In an att«Bq>t to get higher riboflavin producing strains. In general, the trend of mutation effect was the fomatlon of de- generate strains which produced less riboflavin. The addition of 0,1 per cent sodium dithionite to tiie stock medium enhanced pigment formation and tended to maintain the organisms at a high flavinogenic state, law (118) reported that methionine was important in the gmrth of ashbyii, Histidine was thought to be related to riboflavin synthesis either by direct incorporation into the vitamin molecule or by an in- direct effect on the synthesizing system. The medium for these studies was completely defined being composed of glucose, asparagine, histidine. Inositol, biotin, thiamine and mineral salts, KacLaren (^O) studied the effect of various purines and pyrlmldines on the growth and riboflavin synthesis of E, ashbyli in a variation of Tair^s mediTia. The purines, adenine, xanthine and guanine increased ribo- flavin synthesis without increasing the grovth of the organism. The pyriffiidines, uracil and thymine, did not effect the growth of the organism but uracil inhibited the synthesis of riboflavin, KacLaren postulated that a "purine pathway" aay exist in the process of riboflavin biosyn- thesis, : ' . . Dikanshaya (22, 23) observed that more active growth of E, ashbi^di produced leas riboflavin. Folic acid and p-aminobenzoic acid were needed for growth and biotin and inositol wer^ needed for riboflavin synthesis. A low vitamin content of the medium along with sparse mycelial growth contributed to favorable condition* for riboflavin formation. Strains of E. ashbyii most effective in riboflavin synthesis had weaker cytochrome systems but more active dehjrdrogenase and proteolytic enzyme systeins than low riboflavin producing strains, Kinoura (57, 58) reported that ai^inine was most effective in prwnoting riboflavin synthesis in E, ashbyii . Other amino acids, in de- creasing order of effectiveness were, aspartic acid, glutamic acid, aspaira- gine, proline, lysine, methionine, phenylalanine, leucine and tyrosine. Serine and norleucine were inferior to (NH|^)2S0^, Asparagine was most effective in increasing growth. The addition of 0,01 - 0,1 milligram* of V -benzene hexachloride per 100 milliliters of medium increased the synthesis of riboflavin; however, concentrations greater than 0,1 milli- gram were inhibitory to the vitamin synthesis. Van Lanen, Smiley and Stone (105) devised an amino acid medium for the growth and riboflavin synthesis of A. gossypii. Glycine, alanine, threonine and. proline added to a sucrose, vitamin, mineral salts medium gave a noticeable jdeld of riboflavin, Bicarboxylic acids and related com- pounds were reported to interfere vith riboflavin synthesis, . Qoodwin and Pendlington (35), tising a 0,(X)5 per cent peptone basal mediian, reported that L-serine, L-threonine or L-tyrosine stimulated ribo- flavin synthesis in E, ashbyii . L-glutaudc acid, L-aspartic acid and L-asparagine stimulated both growth and vitamin synthesis, vhereas L-cystine inhibited both. The other axaino acids and (NH^)2S0^ vere without effect. Xanthine, adenine and adenosine increased riboflavin synthesis but pyrijni- dines had no effect. Alloxan inhibited growth and riboflavin synthesis, Klungsoyr (U5, U6) obtained riboflavin synthesis by cells of E, ashbyii suspended in a buffer composed of NaCl, HCl, KgSOij,7H20, Na2HP0ij adjusted to pH 6,9. He proposed that the riboflavin was synthesized in prefonned cells and that growth and riboflavin s;^Tithesis may be regarded as independent processes, VJhen acetic acid-l-C^ was added to the syn- thesizing cells, less than 0,01 per cent of the added activity was recovered in the riboflavin and this was found to be in the caition-2 atoai of the ring portion of the molecule. The activity from added cUt-formate was also found to be in the carbon-2 atom of the ring. McNutt (51, 52) continued the work of KacLaren on E, ashbyii^ using uniformly labeled adenine. He reported that the adenine was prin- cipally incorporated into the 6,7-diinethylisoalloxazine portion of the riboflavin molecule, further substantiating the postulated "purine path- was" of riboflavin synthesis, Natvirally occurring purine nucleosides and nucleotides were no more effective than the parent purines in in- creasing riboflavin synthesis. Brown, Goodwin and Pendlington (lO) reported that pyruvate was the only non-nitrogen cojnpoimd related to serine and threonine which gave an increase in riboflavin foiaation with E, ashbyii. Aminopterin, at a con- centration of 2 micrograiBS per 100 milliliters inhibited the growth of the organism but stiimilated flavogenisis, . . Plaut (66, 67, 68) reported on the location of tagged at(Mns in the riboflavin molecule when cl^-formate, C^~bicarbonate, acetic acids- 1-C^ and glycine s-l-d^ and 2JC'^, and totally labeled glucose were added to a growing culture of A, goss^'pli in a medium of com steep liquor, peptone and glucose, ile found that the C^-fonoate activity was concentrated in the carbon-2 atom of the riboflavin, whereas the carbonate activity was in the carbon-h atom of the molecule. Acetic acid- 1-C-^ produced activity in the carbon-1; atom but to a lesser degree than C^-bicarbonate, The acetic acid-2-Clit activity was in the »ethyl groups, cart>ons-S»,6,7,8,8a and 10a and in the rtbityl side chain of the molecule, A similar distribution of activity was shown >7ith unifonaly labeled glucose. The activity of the labeled glycines was located predominantly in carbons- Ua and 9a of the ring portion of the riboflavin molecule. The addition of glucose-l-C^^ and gluco8e-6-Cl^ insulted in the activity being primarily located in the methyl groups and carbons- 5 and 8. Pl«it expressed the possibility of a two carbon fragment being involved in the synthesis of the cyclic carbon ring of the riboflavin molecule. Plant and Broberg (69) have reported further studies on the incorporation of C^ii- acetate and glucose into the ribityl side chain of idboflavin. The abundajoce of research on the problem of riboflavin synthesis by microorganisms is shown by the number of reports in the literature, however, the question of the nature of the pathway of synthesis remains 11 tuianswered. Further research on the growth and metabolism of these ribo- flavin synthesiiBing organisms will no doubt eventually provide the answer to this question, , ' B, The Biosynthetlc Relationships of Amino Acida Aaiinc acids are often referred to as the "building blocks" of pro- tein, but they are also essential in the biosynthesis of other Ijnportant nitrogen containing biological compounds. The structure of riboflavin in- dicates that there are foxir nitrogen atoms per molecTole and, therefore, amino acids might be expected to play a key role in the biosynthesis of the molecvile. The relationship of amino acids to riboflavin biosynthesis is emphasized by the fact that foiroation of the vitamin by microorganisms is dependent on a suitable organic nitrogen source as shown by many refer- ences in the preceeding section. The review of the biosynthetic relation- ships of amino adds in this section will be of general nature and, for the most part, restricted to processes in microorganisms. A more compre- hensive survey of the subject may be obtained from recent reviews (l9, • One of the iB5>ortant amino acids involved in transamination re- actions is -alanine which is present in a wide variety of natural pro- teinaceous materials. It is related to pyruvic acid through the trans- amination reaction sequence, glutamate ■♦• pyruvate o<-ketoglutarat© + o<-alanine, which is reversible (36), Ujia reaction serves as the mechanism for the microbiological synthesis and degradation of c<-alanine. The compound, ^-alanine, is one of the less common amino acids but it is ii^ortant as a part of the vitamin, pantothenic acid which makes up part of coenayne A. Gale (3I) reported that -alanine could be fomed b7 the decarboxylation of aspartic acid. It has been shown by Roberts, et al,, (83) that ^-alanine vas associated with transamination in Aspergillus fumigatusj however, the reaction mechanism has not been estab- lished. In rat feeding experiments with isotopic compo\inds, Phil and Fritsaon (6S) found that /^"-alanine was rapidly oxidized and part of th« carbon chain conrerted to acetic acid. They were of the opinion that oxidation took place after the amino acid had been deaminated and de- carboxylated, Aspartic acid, like o< -alanine, is Important in nitrogen transfer reactions involving transamination. It is synthesized in many micro- organisms by the amination of fumaric acid which is a reversible reaction catalyzed by the enzyme aspartase (30), It has also been reported to be formed by a transamination reaction involving oxaloacetic acid (lj8), Abelson and Vogel (3) stated that aspartic acid wss the precursor for the amino acids, homoserine, methionine, threonine and isoleucine, in Yorulopsis u tills and Neurospora crassa. Aspartic acid has been re- ported to be associated with the biosynthesis of CoenzjTne A (31), purines (no) and pyrimidines (77). Asparagine is formed by the amidation of aspartic acid; however, the reaction mechanism is not known. Black and Gray (?) reported that ^-aspartyl phosphate may be a precursor of aspara- gine. In a rat liver preparation, >i«iit©r and Fr«aer (^5) found that asparagine could be formed by the transamination of o<-keto8uccinamlc acid with glutamine which was the most active amino group donor. Aspara- gine is important as a nitrogen source for many microorganisms. Another amino acid, glutamic acid, occupies an Important position in the nitrogen metabolian of microorganisms and animals. It is primarily formed through the transamination of o<-ketoglutaric acid (32) and the reaction of o<-ketoglutarate with anaaonia catalysed by the enzyme glu- tamic dehydrogenase, Cilutainic acid can also result from the degradation of proline, arginine or histidine. Sheain and Russel (92) reported the biosynthesis of glutasiate by the reaction of glycine and succinate via (T -aminolevulenic acid, Abclson (l), using isotqpe inhibition analysis, claimed that glutaiaic acid vas the precursor of proline, ornithine, citrulline and arginine, Glutamine, the amide of glutamic acid, is syn- thesized by the COTibination of glutaaaic acid and ammonia in the presence of ATP (U2, 112). Arginine is synthesiaed from glutamic acid, via ornithine and citrulline, in E, coli and raanj- other microorganisms. Anderson-Kotto, et al,, (5) established that in E, coli N~acetylglutaid.c acid was one of the intermediates in the synthesis of ornithine from glutamate, Srb and Horowitz (95) reported the formation of citrulline from ornithine in Neurospora, the mechanism not being known, Citrulline and aspartic acid form ai^inine via the intermediate, arginlno-succinate (79, 78). Vogel, et al., (106) reported that the activity of labeled N-acetylglutaraic acid was heavily concentrated in the arginine synthesized by E, coli, verifying the glutamate to arginine sequence, Vogel and Davis (l07) have reported that proline, an imino acid. Wis foraed fsrom glutamic acid via glutamic y -semialdehyde and ^i-pyrroline- 5-carboxylic acid in two mutant strains of E, coli. The biosynthesis of lysine is believed to follow different pathways in bacteria and fungi. Davis (17) reported that in E, coli x,oc'-diamino- pimelic acid is an intermediate in the synthesis of lysine from aspartic acid. In Neurospora, I'^indsor (llli) shoved that oC-aminoadiplc acid covlA be converted to lysine, Strasaman and Weinhouse (97) established that the alpha carboxj'^1 carbon of lysine was derived from acetate as shovn by the use of labeled acetate as the sole carbon source for a yeast and Neurospora. They proposed that carbons- 3 and 6 resembled the succinyl moiety of o<-ketoglutarate and that acetate probably condensed irtth suc- cinyl-Coenzyme A in the initial step of Ij^ine biosynthesis, Rothstein and Millar (85), using lysine-6-Clii proposed that lysine degraded to -eltrttlUne ►arginine (II) threonine >- i soleucine -valine (III) o<-ketoi8ovalarate <;;^ o< -ketoi socaproate > leucine ? — > histidinol phosphate — hiatldinol — »> histidina arc*;atic akiso acids -shikimic acid: rprephenic acid<^ anthranilic acid phenylpyruvlc acid tyrosine >- indole j^iMiyl* alanine ■tryptophan Figure 1 the top layer used as the solvent. Another iioeful solvent is composed of pyridine, tertiaiy aniyl alcohol and water in the ratio of 3.5»3«5«3 by Toltmie (6l), A comprehensive survey of the voluminous literature on chromatography is beyond the scope of this review, The references pre- sented include only those considered to be pertinent to the problem, D, Riboflavin Assay After riboflavin was established as a growth factor, it was neces- sary to provide a means of analyzing for the vitamin. Von Euler and Kalmberg (l09) introduced the Mological assa.. method which was designed to meesurc riboflavin by ccmparing changes in growth rates of test animals, usually rats, treated with the xmknown sample and animals given standard amounts of riboflavin. 3nell and Strong {9k) developed the microbiological method of ribo- flavin assay which is extensively used today. It is based on the growth response to riboflavin by the microorganism, Lactobacillus casei, which produces lactic acid in proportion to the amount of the vitamin present under certain conditions. The lactic acid produced with the unknown is titrated and conqjared with that produced with standard amounts of the pure jriboflavin, The assay is very reliable but it must be carried out under carefully controlled conditions and requires the preparation of special cultxxre media. The flTiorometrlc analysis of riboflavin was Introduced 1^ Van Eckelen and Emmerie (lOl^) . Under the influence of ultraviolet light, ribo- flwrin eahibita a characteristic fluorescence which is proportional in intensity to the a»ount of riboflavin in dilute solutions, Hodson and m Korris (39) modified the method by introducing the use of sodium dithionite to reduce the riboflavin to the colorless foiw without effecting the other fluorescing substances present, thus increasing the accuracy of the pro- cedure. The Association of Vitamin Chemists (6) has described a standard fluororaetric procedure, including extraction techniques and methods of performing the assay. Tanner, et al,, (lOO) extracted the riboflavin from A, ^ossypii cells by autoclaving them for 30 minutes at 1$ pounds steam pressure in a 0,123 M sodium acetate buffer at pH U,7, law (ll8) used a colorimetric assay method to determine riboflavin extracted from E, ashbyii cells. The method, using a Klett-Summerson colorimeter, was reported to agree within plus or minus $ per cent of the microbiological method; however, there were no details given for the procedure. II. STATEMENT OF THE PROBLEM Although the conditiona for the commercial synthesis of ribo- flavin are well established, the mechanisms by which microorganisms synthesize it have not been fully elucidated* Thin study of the netabo- lism of riboflavin by Ashbya gossypil was undertaken in an effort to gain more facts pertaining to the mechanism of riboflavin synthesis in microorganisms and, thereby, bring about a more complete understanding of the pathway and intermediates participating in the synthesis. 21 m. EXPERIKEKm A. Apparatus 1« Ihe colorimeter* An Evelyn Photoelectric Colorimeter, manu- factured by the liabicon Company of Fhiladelplila, Pennsylvania, was the instrument used in turbidimetric measurements of grovth and colorimetric determinations of riboflavin. The instrument is a single-photocell, direct reading, photoelectric photometer equipped with light filters and a light beam galvanometer. 2, The shaker. A platform reciprocating shaker, constructed in the Chemistry Department shop at the University of Jlorida, was used to maintain an efficient aeration of the cialtures during incubation in liquid media. The shaking platform accommodated fifty 2^0 ml. narrow aoTith Erlenmeyer flasks which were held in place by a removable plywood top containing ^ two and one-half inch holes to separate the flasks. A second layer of fifty flasks could be added by attaching a special tray to the main shaking platform. The shaking rate could be ad;Justed by using different size pulleys; however, the size pulley used provided a shaking rate of 90 three inch strokes per minute. The shaker was housed in a constant temperature room at 28° C, 3. The pH meter. A Beckman Model H-2, line operated, glass electrode pH meter with attached constant voltage transformer was used for all pH measurements. The pH meter was standardized before use against a pH 7.0 buffer prepared from a Beckman concentrated liquid buffer. Ihe 22 23 Instrumftfit was allcwed to iranB tip for at least a half an hour before standardization. %4 the fluorometer. A Colonan Universal Spectrophotometer, Model Ih, was used for the fluorometric determinations of riboflavin. The instrument was adapted to fluorometric analysis with a Universal Ultraviolet Illuminator and a set of filters, UV-2 and PC-2, specific for riboflavin analysis. Round, fluorometer microcuvettes which held 10 ml. of sample were used with a special miczxifluorcunetric cuvettt 1. I^aintenance of the culture. The culture of A, gossypil, strain KRRL I-10?6, obtained from the Northern Regional Research Labora- tory of the Department of Agriculture in Peoria, Illinois, was main- tained on stock agar slants idiich were prepared from a medium containing 2.0 per cent glucose, 1.0 per cent Dif co-pep tone, 0,5 per cent Dlf co- yeast extract and 1,8 per cent Difco-agar, The medium was sterilized by autoclaving at 15 pounds steam pressure for 30 minutes and slanted. Transfers were made to new slants every I48 hours in order to maintain an actively pigmenting culture. Every two or three weeks, a U- day- old, pigmenting ctilture was placed in the refrigerator at 8 - 10® C, so that a pigmenting strain would be available in case of contamination or loss of the ability to produce constant pigmentation. The c\altures were in- cubated in a constant temperature room at 28° C, 2. Experimental media. Liquid media were prepared by adding different nitrogen sources at different concentrations to a general basal B. (^neral Procedures 21» •ediun containing 2,0 g, glucose, 0,1 g. KH2P0^, 0,1 g, K2HP0^, 0,1 g, NaCl, 0.05 g. MgS0^.7H20, 3.0 itig. inositol, 0.1 mg. thiainine-HCl and 2.0 ug biotin in 100 ml. of distilled water. In the early experiments, 0,05 g. NaCl was used in place of the 0,1 g. NaCl as shown in the gen- eral basal medium. All media were adjusted to pH 6,5 - 6,6 with 0,1 - 1,0 molar solutions of either NaOH or KOH unless otherwise specified. In each shaker flask for inoculation, 20 ml, of liquid mediian was placed. When solid media were used in an experiment, 1.8 g, of Difco-agar was added to each 100 ml, of liquid media. Such solid media were dispensed in 10 to 12 ml. volumes per tube and slanted after autoclaving. All »edia were sterilized by autoclaving at 15 pounds steam pressure for 17 minutes. 3, Inoculation and incubation of media. The inoculum for the shaker flasks was prepared from cells of A, gossypii grown on stock agar slants for 2it to 72 hours depending upon the experimental conditions being studied. In the early experiments, the inoculum was prepared by washing the cells from each slant with approxinately 10 ml. of sterile distilled water and breaking up clumps with the inoculation loop. This cell suspension was transferred to a sterile, 15 ml, centrifuge tube, centrifuged, washed twice with 10 ml. of fresh sterile distilled water and resuspended in 10 - 12 ml, of sterile distilled water. A volume of 0,2 - 0,3 ml. of the washed cell suspension was used to inoctilate each shaker flask. In later experiments, the suspension, before being washed, was shaken with sterile glass beads to provide a more uniform inoculum. The flasks of inoculated experimental media were incubated on the 25 shaker in a constant temperature room at 28° C, for a given period of time, in most cases, for 96 hours. Solid media slants were incubated at the same temperatiire as the flasks for the desired length of time, 1}. Measurement of growth, Giwth was measured by turbidimetric Beaaurements or by obtaining the weight of the dry cells, Turbidimetric measurements were carried out in the Evelyn photoelectric colorimeter using the ^0 millimicron green filter. The instrument wis set at 100 per cent transmittance with a distilled water blarik and the value of an air blank was determined with this setting. Further adjustments of the instrvanent were made by setting the instrument on the air blank reading throughout each set of determinations. After incubation each culture was adjusted to the original volume of 20 ml. with distilled water and then added to a colorimeter tube which was placed in the colorimeter, Th« growth was measured as per cent transmittance which varied in- versely as the amount of growth present. The readings were made within a few seconds after the suspensions were prepared because the cells tended to pack to the bottom of the tube. The method was rapid and reasonably accurate at lower concentrations of cells, however, at higher concentrations of cells it was difficult to measure differences in growth, ^ Ihe determination of the dry weight of cells was accomplished as follows I the medium containing the cells was diluted to the original volume of 20 ml. After th*»ugh mixing, a 10 ml, aliquot of a suspension was removed for riboflavin deterroination and the remaining portion of the suspension was suction filtered onto a previously dried and weighed filter paper. After the cells and filter paper were washed with distilled water, they were placed in an oven at 105® 0, to dry for 12 hours, ^e dried cells and paper vere reweighed and the dry weight of cells per 10 ml, of aliquot was determined by difference. The dry weight method provided a more accurate means of measuring growth than the turbidi- metric method, - C, Riboflavin Determination • !• Preparation of the sample for riboflavin determination. Th« riboflavin was extracted from the cells of A, soss?,'pii by autoclaving than for 30 mdnutes at 15 pounds steam pressure. Before extraction, 2,5 ml, of 0,123 M sodium acetate b\iffer, at pH 1^,7, was added to each 10 ml, of the giwth medium containing cells. The buffer was added to stabilize the riboflavin during extraction, since it would be rapidly destroyed in near neutral or alkaline solutions. The autoclaved mixture was filtered to remove the cells and the filtrate was diluted to an ^propriate volume, usually 25 ml,, with distilled water. The necessary dilutions were made from this solution before the riboflavin deteimn- ations were made, 2, Fluoroanetric detemination of riboflavin. The fluorometric detemination of riboflavin described by the Association of Vitamin Chemists (6) was modified to adapt it for use with the Universal spectro- photometer. Fluorescence was measured by readings on the per cent trans- mittance scale of the instrUiTent, The standard solutions were made from a standard stock solution which was prepared by dissolving 25 mg, of riboflavin in 1000 ml, of a solution containing 2 ml, glacial acetic acid. Ten milliliters of each diluted unknown riboflavin solution and of the 27 standard solution were added to separate test tubes each of vhich contained 1 ml, of glacial acetic acid. After thoroughly mixing the solutions, each solution was transferred to a special inicrocuvette and fluorescence mcaa- uremen+s were made, THiplicate determinations were made for each unknown or standard riboflavin solution. The cuvettes were rinsed thoroughly with distilled water and dried after each determination. The fluoroinetric determination of riboflavin was made on a specially adapted Universal Spectrophotometer. The instrument was adjusted to eero per cent tranamittance xxsing a water blank, then using a solution containing 50 ug, of sodivan fluorescein per liter of distilled water as a substitute standazxi, the Bal dial vnm Mt at a per cent tranemittance for reference, usually between UO to 60 per cent, and then the index of the galvanometer scale was adjusted to read eero with the fluorometer Icnob of the instrument, During the assay, frequent checks were made with the sodium fluoroscein solution by readjusting the instrument to the reference transmission selected in order to maintain standardized conditions, A water-dithionite blank value was determined on a solution of 10 ml. of distilled water and 1 ml, of glacial acetic acid containing approximately 20 mg, of sodium dithionite. After the fluorescence of a riboflavin sample was measured, appixocimately 20 mg, of sodium dithionite was added to the sample in the cuvette and thoroughly mixed. The dithionite reduced the riboflavin to the colorless form and the sample was again measured to determine the fluorescence hy substances other than riboflavin in the reduced sample. "Phe water-ditdiionite blank value was subtracted from the fluorescence of the reduced riboflavin sample to give the correction for the fluorescence 28 of non-rlboflavin substances present in the untreated sample, "Che fluo« rescence due only to riboflavin in the san^le was determined by sub- tracting the correction for the non-riboflavin fluorescence from the fluorescence of the untreated sample, A series of standard riboflavin •olutions were measured in order to calibrate the instrument guad to prepare a standard curve. The standard stock solution was diluted to give duplicate solutions ranging in concentration frwa 0 ug. to 1 ug. of riboflavin per ml. Hie results of a standard calibration determination are shown in Table 1 and the data plotted as a standard curve in Figure 2. ■ ■ ' " TABLE 1 j ^ THE FI^ORESCENCE OF SXILN'DABD BIBOFLAVIH SOLUTIONS " ^n«ani,Liiiii yiiii,""i ssaggaeagg niu Riboflavin Fluorescence Solutions (Dial Readings) (ug/inl) Untreated Reduced Correctioij^ iiiboflavin Average 0.05 0.05 76.2 76.6 73.9 73.8 0.1 I 0.0 i l~ t : 1 76.1 76.6 76.3 ' 0,10 0.10 ' 79.2 79.1 73.9 71.0 0.1 0,2 79.1 78.9 79.0 0.30 0.30 89.1 89.0 73.9 7lt.0 0.1 : h 0.2 I 89.0 88,8 88.9 0.50 0.50 96.0 98.2 73.9 7li.0 0.1 0.2 97.9 98.0 98.0 0.75 0.75 108.7 109.2 73.9 7li.O 0.1 1 0.2 ^ 108.6 109.0 108,6 1.0 1.0 119.6 119.8 73.9 7ii.O 0.1 i 0.2 , ! 119.5 119.6 119.6 * "» Average water-dithionite blank value 73,8, •» - Correction for non-riboflavin fluorescence^: A STANDAHD RIBomviK CURTB 30 130 0 0.25 0.50 0.75 1.0 Riboflavin (ug/ml) n Since the curve In Figure 2 showed that the most desirable con- centrations of riboflavin for the determination vere between 0,3 ug, and 1,0 ug, per ml,, dilutions of unknown samples were made to give concen- trations which would fall within this desired range. The unknown samples were treated in the same manner as the standard samples above, fluores- ceiice readings were made and «&ount0 of riboflavin were read from the standard curve prepared for each series of unknown deterHiinations, Although the fluorometric method gave good results, it required U to 6 hours to make one complete assay of a series of samples, therefore, it placed a limit on the number of experiments which could be performed at one time, D, Chromatography 1, Paper sheets and strips. The conventional methods of ascending paper chromatography were employed with sheets or strips of V^hatman No, 1 filter paper, The twtanol-acetic acid-water and the pyridine-tert, amyl alcohol-water solvents were used as the resolving systems. Small sheets, 18 cm, by 18 cm., were formed into cylinders and run in large mouth jars. The cylinder was held in form by a nichrome wire shaped in the form of a small crescent. The spots were developed by ultraviolet light, nin- hydrin solution or other standard developing solution. The small two- dimensional chroraatograms could be completed in 7 to 8 hours as compared to at least U8 hoiirs for the standard large sheets, 2, Circular chromatography. The technique of circtilar chromato- graphy was employed as a rapid means of chromatographic separations and for the identification of component fractions present in a solution. 32 Circles of V^tman No, 1 filter paper, 20 cm, in diameter, were cut into quarters and a vriLck, approximately 2 mm, wide, was cut from the arc side of the quarter to the center of the quarter, Ihe solutiorv to be chromato- graphed, was applied at the bend where the wick and the paper connected. As in all paper chromatograms, the smaller the spot of application, the better the resolution of the components. The chromato^rraphic chamber was made of two 9 cm. fluted watch glasses, one to serve as the solvent reser- voir and the other as the top. After ^ to 6 ml, of solvent had been added to the reservoir, the wick was bent down and placed in the solvent with the remainder of the paper resting on the watch glass. The watch glass top was put in place and the chromatogram allowed to flow as a circular migration from the wick in the center of the paper* 33ae chromato- gram was removed from the solvent when the solvent front had progressed 25 to 30 mm, frcaa the point of application. The solvent front was marked and the paper dried, sprayed and developed according to standard pro- cedures, Ihe Rf values of the ring spots were measured in the conven- tional manner by dividing the ring distance by the solvent front distance. Solutions of amino acir's and peptides, prepared by adding 50 ng, of the individual amino acid or peptide to 100 ml. of distilled water were chromatographed. In Table 2, the Rf values of amino acids and pep- tides are recorded for circular chromatograms using the solvent systems, n-butanol-acetlc acid-water and pyridine-tertiary amyl alcohol-water, TlM ring spots were developed with a spray solution of ninhydrin, 3. Column chromatograohy. Column chromatography was used in the separation of peptone into different fractions. 'The column was made of a •tandajxi 50 ml, burette, however, a pyrex glass condenser tube, 250 mm. 3) TABLE 2 THE Rf VALUES OF A1»!IN0 ACIDS AND PEPTIDES IS BUTANOL-ACETIC ACID^TER IND PIRIDINI-TERTIART AKYL ALCOHOLJ^AT^Ti SOLVENTS ON CIHCUUR CHHOMATOORAIffl Amino Acids and Peptides BuOH-JlAc-W'ater Pyridinc-t-amyl alcohol-Water Ninhydrin Color alpha-Alanine beta-Alanine Arginine-HCl Asparagine Aspartic Acid Cystine-ilCl Glutamic Acid Glycine liistidine-aCl Isoleucine Leucine Lysine-HCX Methionine Phenylalanine Proline Serine Threonine Tryptophane Tyrosine Valine Alanylphenylalanine Glycylglycine Glycylalanine 0.U7 0.li3 0.3ii 0.39 0.38 0.33 0.50 O.hl 0,36 0,7h 0.77 0,38 O.6I4 0.72 0.^ o,hh 0.50 0.68 0.60 0.6lt 0.77 0.10; 0.70 0.50 o.hh o.3h 0.38 0.37 0.31 0.U1 0.1i3 O.hh 0.70 0.70 0.31 0.66 0.71 0.5h 0.U6 0.51 0,69 0.66 0.61 0.70 0.36 0.61 Hed-purple Pvirple-blue Purple Brown Purple Purple lied-purple Purple Blue-purple Purple Purple Red-purple Purple Purple lellow Purple Red-purple Purple Red-purple Red-purple Brown, tume pink Brown, turns pink Brown, turns pink - Average of duplicate measurements. by 10 mm,, can be used when fitted with a removable stopcock at the narrow end, Approxiinately one-half inch of glass wool was packed into the bottom of the column to retain the material foming the staUonary phase of the column. In the eoqjeriments with peptone, Whatman powdered cellulose was I 3k used ft8 the statlonarj^ phase. Approximately 8 gmms of powdered cellulose Iras suspended in the butanol-acetic acic'-irater solvent and carefully added to the colurcn. The column was allowed to pack by the flow of the solvent l^iroagh the cellulose, takinft care to tap the air bubbles out as the column formed. It is Important to keep the solvent level above the top of the cellulose since once the cellulose becon^es free of the solrent^ a pocket of air forms thus destroying the unifonaity of the stationary phase. After •11 of the cellulose was packed into the column it was washed with 50 to 60 ml, of fresh solvent. The solvent was allowed to run through the column until it was about 1 cin. frcwi the top of the cellulose and at this point the stopcock was shut off, '. Bie peptone was prepared by dissolving about 1,0 g, of Dif co-pep tone in « winlaua araoimt of water. The peptone solution was mixed with powdered cellulose to form a paste which was dried at 7?° C, for about 30 minutes, Ihe peptone-cellulose mixture was added to the top of the cellulose column and carefully packed. Fresh solvent was run into the column and the stop- cock opened to allow a flow of the solvent to pass through the cellulose. Samples of the effluent from the column were checked periodically by wetting a filter paper strop, drying it and spraying it with ninhydrin solution to show the appearance of the ninhydrin positive portions of peptone. After the first indication of ninhydrin positive material in the effluent, fractions were collected from the column every 3 to 1« ml. Circular chromato- grajia were made of each fraction as it was collected irtiich provided an in- dication of the progress of the separation on the column. This speeded up the entire procedure, so that similar fractions could be combined as the separation was taking place. This appUcation of circular chromatography served to stress the utility of the technique in cases vhere a rapid chromatographic interpretation of mixtures is desired, S* lixperinents 1, The Relationship of Riboflavin Synthesis to Qrovth in a Peptone MedijOT,, The vork of Tanner, et al., (lOO) vdth Ashbya Kossypii has shown that riboflavin synthesis d3.d not begin until after the organism had grown for approximately U8 hours. They suggested that riboflavin may be syn- thesized only after the organism had reached a certain stage of growth but gave no data to indicate at what stage of growth the synthesis started. This esqperiment was uMertaken to show the relationship of riboflavin syn- thesis to the growth of A, gossypii. A liquid medium was prepared which contained 2,0 per cent glucose, 0,5 per cent peptone, 0,1 per cent KH2P0^, 0,1 per cent K2HP0^, o,o5 per cent FigS0^.7H20 and 0.0^ per cent NaCl, in 100 ml. distilled water. The medim was adjusted to pH 6,5 and autoclaved in 2^0 ml, Erlenmeyer flasks. Twenty flasks containing the sterilized medium were inoculated with a U8 hour washed cell suspension of A, goss;:^T?ii in the usual manner. Two flasks ■mate used for the dsqplicate wro tim« determinations, %e rest of the flarfcs were Incubated on the shaker, %to flasks were removed from the shaker after each of the following time intervals? 8, 12, 18.5, 2U, 29, 36, 1*8, 72 and 96 hours. The contents of each flask were diluted to 20 ml,, the original vol\)m€^ and transferred to a colorimeter tube and the turbidity detemLned by measuring the per cent transmittance. Hie pH of the contents of each tube was determined next, followed by the addition of 5 ml, of acetate buffer and extraction of the riboflavin, 'Ihe extracted cells were filtered onto prefviously weighed filter papers and dried for dry veight determinationa of the extracted cells. Each filtrate vas removed and diltfed to the appropriate concentration range and the riboflavin vas detennined fluorometrically, Ihe results of the eatperiment are given in Table 3 and are shown graphically in Figxire 3» ^he growth of A, gosgypii follows a pattern typical of most ndcro- organisms, as shown by the turbldometric growth curve in Figure 3. After a 6 hoxir lag period, the growth increases rapidly for a 10 to 12 hoxir period which corresponds to the logarithmic phase of growth. At this phase, growth slowly begins to level off and reaches a maxiimua between 38 and US hotcrs. The growth, as determined by the dry weights of extracted cells, follows a pattern similar to that detemlned turbidimetrically, reaching a maximum dry weight at 36 hours. The dry weights of extracted cells decrease rapidly from 36 hours to the final determination at 96 hours, which is approximately 5U per cent of the maximu» dry weight. This probably indicates a greater disruption of the cells during the extraction procedure with the resultant loss of intracellular materials, l^iis is to be expected since older cells are known to be more easily affected by physical environment. In the work of Mac Laren on E. ashbyii^ the dry weight of extracted mycelia was used as an indication of growth for com- paring the effects of various purines and pyrimidines in different media. On the basis of the experiment Just discussed, there may be some question whether the dry weights of extracted cells could be validly used as a true measure of growth. A better indication of the true growth of the 03rganism might be obtained by taking the dry weights of an aliquot of cells before the extraction of xdboflavin. TABLE 3 THE PER CENT TPANSMTTANCE, pH AND RIBOFLAVIN CONTraTT OF CULTi'RES OF A. GOSSYPII^ Aim DRY WEIGHTS OF THEIR EXTRACTED CELLS, AT DIFFERENT AGES GROWN IN A PEPTONE MEDBM Hiboflavin Age Transnittioice (hrs) i%) pn (»g) AXIQUO b (ug/ml) Factor ■lotaJ. (ug/ml) 0 ■ 97.8 6.5d 6^ 0.6 1.1 0,02 • 0.03 2.5 2.5 0.05 0.08 Ave, 97*9 6.50 0,9 0.07 • 93,0 6.30 6.^5 1.7 44- 0,0U O.QU 2.5 2.5 0.10 0.10 Ave. 93.3 6.33 1.^ 0.10 72.0 70.2 6.00 5.90 7.5 6.2 0,06 0,08 2,5 2.5 0.15 0,20 Ave, 71,1 % 0.17 18.5 23.0 22,1 6.00 37.6 l2.0 0,13 0.15 5,0 5.0 0,65 0.75 Ave, 22.6 39.8 0.70 Ave, lli.O l3.9 5,65 5.63 66.5 68,0 ^7.3 0.16 0,17 5.0 5.0 0.80 0.85 0,83 Ave, 10,2 10.6 5.50 89.1 0.25 0.37 5.0 5.0 1.3 1.9 lo,l 91.1 TT Ave, 9.1 8.0 5.U0 5.20 5.30 87,3 102.0 9ii.7 0.3li 0.56 5.0 5.0 * 1.7 2.8 2.3 7.3 J4- - 5.30 5.20 93, h 91.1 0.53 0.56 5,0 5.0 2.7 2.8 Ave. ITT 5.25 92.8 2.8 n Ave. 6,9 6,9 6,9 6.a5 6.1iO 6.h3 78.1 85.7 &2,0 0.09 0.16 50,0 50.0 U.5 8,0 6.3 H 9.2 ■i^ ■ 7.50 7.U0 7.ii^ h9.6 5h.2 0,81» 0,23 250.0 250,0 2Ut.O Air«« 51.9 13^.8 THE RELATKMSIUP OF OHOm, pH AKB BIBOFLAVBT CO^IOT OT CDLTORES OF A. QOSSYPII OaOIW IN A PEPTOSS KEDIOM AH) THE DRT WEIOHTS OF ISiEIR EXIBACISD CSXiLS 39 There is no indication of riboflavin synthesis until after the first 2li hoiirs. The synthesis proceeds slowly xrntil at 72 hours there la a very rapid increase in the production of the vitaain. As Figure 3 shows, riboflavin is not synthesized in appreciable amounts until several hours after the laaxiramn groirth has been reached. The data leares little doubt that riboflavin synthesis occurs after maximum growth in a glucose- peptone fflediun. The changes in pH during growth were the same as those described by Tanner, et al., (lOO), Duidng the first 36 to 1^0 hovirs of growth, there is a gradual decrease in pH to a value of approxdmately 5.3. The pH shows a rapid increase after hours reaching a value of pH "J, US at 96 hours. It is to be noted that the pH increase closely parallels the increase in riboflavin synthesis. It has not been determined whether the pH increase is a result of riboflavin synthesis or riboflavin syn- thesis is a result of the changes ca.xising the pH increase. It is pos- sible in the peptone medium that the concentration of some intermediate related to riboflavin synthesis must reach a critical value before the vitamin production can begin and that the cells must be of a certain age or stage of growth before this limiting intermediate can be made available to the riboflavin synthesizing mechanisms of the cells, ^he Fractionation of Peptcaie on a Cellulose CpluBm. A sample of Bifco-peptone was fractionated on a Whatman powdered cellulose column, in order to determine which amino acid or peptide components were asso- ciated with the formation of riboflavin by A, gossypii. The column was prepared according to the procedure previously given. The cluate was collected in tubes, manually, eveiy 3 to li ml, until a total of I4O separate volimes had been collected. The liquid in each tube was chromatographed "With the circular chromatography technique as it was collected from the coluKn, Rf values of components were calculated and tubes with compo- nents having the same Rf value were combined, Ihe Rf values and the tubes that were combined are shown in Table U, TABLE k TOE Rf VALUES OF CHRCKATOGRAPHICALLI SEPARATED mCTIONS OF PEPTONE Fraction Number Tubes Combined Average Rf Values of the Rings I 1-7 0,78 ': ' n 8-10 0.75, 0.57 JOES 11-13 0.72, 0,57, 0,Ul m ■ 15,16,18 0.58, O.la, 0.30 f 19-30 0.36, 0.27 ii 31-3U ApproK. 0.27 fix 35-UO Very faint ring The fractions were concentrated to dryness over CaCl2 in a vacuum desiccator at room temperature. The Rf values of the fractions indicated that peptone contained at least four distinct components, 3. The Growth and Riboflavin Production of A, gossypii with Peptone Fractions as Nitrogen Sources, The peptone fractions obtained in the previous esqieidment were used as nitrogen sources for studying the growth and the foiroation of riboflavin by A. gossypii. The standard basal It medliim vith 0,0^ per cent instead of 0.1 per cent NaCl vas prepared as previously described and the different peptone fractions were added to the basal medlxun to give individual media vi:ilch were atorllized by auto- claying. The sterile media were inoculated with a washed cell suspen- sion of A, gossypii and. placed on the shaker in the constant temperature room. A control with peptone as the nitrogen source was run in the same way. After 96 hours, the flasks were removed frcm the shaker and examined visually for growth and riboflavin production (the formation of yellov pigment) . The growth and pigmentation were estimated on the basis of an arbitrary maximum value of *h for the peptone control, Ihe lower values were in proportion to the control value, with the designation ♦ indic- ating a value slightly less than the corresponding + value. The results obtained are shown in Table 5, > TABLE 5 BIE GROWTH AfJD PIGMENTATION OF ASHBYA QOSSIPII ON MEDIA COKTAINIKG PEPT(aiE SRACHONS AS NITROGEN SOURCES Fraction Number Growth Pigmentation I + U t 1 ♦ 3 HI • t sv + h i 1 ? ♦ h VI * h + 3 VII * 2 • Peptone Control + h li The growth compared favorably with the control in all fractions except III and VII, Fraction VI vas the only fraction lAlch showed any appreciable pigmentation, however, there was evidence of slight pigmen- tation in fractions I and IV, It appeare that fraction VI contains the component or components which account for the stimulation of riboflavin synthesis when peptone is used as a nitrogen source. It is to be noted that this fraction contained the low Rf value ninhydrin positive c«n- ponents which would indicate the presence of basic amino acids and/or peptides, under the chromatographic conditions rar^jloyed, ll. ^tte Proximate Qualitative Chromatographic Analysis of the Amino Acids in the Peptone 1 ractions. In order to obtain some indi- cation of the amino acids present in the peptone fractions, portions of the fractions were hydrolyaed with 6 N HCl in sealed glass tubes by autoclaving at 15 povinds steam pressure for 30 minutes. Portions of the hydrolyzates were chromatogrs^jhed by the circular technique and the Rf values calculated for the resulting rings. These Rf values were CQiqp>ared with those for the knoim amino acids in Table 2 to find the wdno acids that were possibly present. No effort was made to identify the individual amino acids. The results are presented in Table 6, The Rf values of these rings may vary slightly from the values of the pure amino acids since interactions among the amino acids and their hydro- chloride salts will affect the migrationi however, relative locations of the rings can be compared with the known locations of the pure aadno acids , The chromatogram of fraction VI, tiie ft-action which allowed ap- preciable pigmentation, showed that the concentration in the three rings ♦ 1 1 t 1 ♦ t 1 1 1 t 1 1 1 i 1 Phansrlalanin* ■ w 1 f • • Kethlonlne ▼ 1 w 1 1 1 1 1 4> 1 1 t 1 1 1 1 f t Hi8ti(!iB« 1 1 «■ 4 • 1 i {OMUmc Acid t ♦ 1 t Cystln* 1 f 1 ♦ Aipttrtlc Acid 1 ♦ 1 t • t • 1 Alanine 1 1 t t ill! I t I I I t •♦III I4>llll lllll I I « i III I t t «t ft I • ♦ lit llltll ♦lilt Itll 4>lllll •♦III ltl« Itll^l lll^l ♦ lit liiiii ♦iiti lift ♦ittii •♦III ft iii^i I f ♦ I t I I ♦ f I I I ♦ t t ittt iiiiii ti^ii III! ittit^ tiii^ till I » 1 t t I t I ♦ I I ••♦I llltll lllll Itll I I ♦ t I t I t I I I t^Nor^cj so xi^ «n r4 h'-Of^ctri • ••• •••••• ••••« O O O O O O O O O O O ,o P P O h * f 1 1 » • t ♦ * 1 fyremim P f * * J# If . Sarins J It » • » • # j» • 1 Btttlilonlne t # * # t^ti « t J Ly»in» 4 * f *J • # ,» . t Lvttciiw # > t • . » ,1 . ft laolMieia* f P 1 M 1 HiaUdin* t P # M ♦ Olydnn 1 i i If * .f It* » Cyetlae .( * * « J # . A4Q>artic Acid i 1 » t * t . i 1 1 # ♦ >• M • ♦ ^ » t » »J • ,1.1 1 oc?oc>c?o ooooo eoo<9 m of lower Rf value was much greater than the coiaparable rings of any of the other fractions. These rings indicate the possible presence of the di- carboxylic and basic amino acids, which would indicate a relationship between these amino acids and idboflavin formation in A, ^ossypii. however, congjounds other than amino acids could be present in this fraction and exert some effect on the synthesis of the vitamin* The Effect of Various Amino Acid Ccanbinations on the Qrowth and Riboflavin Formation of A. gossypii. The previous experiment pre- sented some evidence that certain amino acids were concentrated in the fraction of peptone which gave appreciable pigmentation with A, ^ossypii. These amino acids, in various conbinations, were used as nitrogen sources In a sflxdes of media in order to determine if combinations of pure amino acids could stimulate the growth and pigmentation obtained on peptone. The basal medium was prepared with 0.05 per cent NaCl and the amino acids were added in various concentrations to give a constant UO rag, of nitrogen per 100 ml, of each medium. The nitrogen was detenidned on the basis of L-amino acids present in each medium. After the addition of the amino acids, the pit was adjusted to 6,5 • 6,8 and the media sterilized by auto- claving. The amino acid ccsiqposition of the media are given in Table ?• Hiwie arginlne seemed to be present In each peptone fraction it wat arbitrarily chosen as the initial amino acid of the series which was tested, * The sterile media were inoculated with a 36 hour washed cell suspension of A, gossypii and incubated on the shaker in the constant \ teugjerature room. The media were examined in the same iranner as in th« previous experiment at various time intervals to estimate the growth and pigmentation* The results are presented in Table 8« TABLE. 8 THE EFFECT OF AMINO ACID CCMBINATIONS ON GROWTH AND PIOJffiNTATION OF ASiiBIA GOSSIPII Time Medium 2U hoxxrs U8 hours 72 hours 96 hours No. Growth Pigment Growth Pigment Growth Pigment Growth Pigment t A 0 t 1 0 ♦ 2 0 ♦ 3 0 i *l 0 ♦ 2 ■1 ♦ 2 tH ♦ k + 1 1 '0-- ' m 0 ♦ 1 0 * 2 0 1 A 0 * 1 0 ♦ 1 0 ♦ 3 si 5 ♦ 1 0 ♦ a 0 ♦ 3 0 • 6 + 2 0 ♦ 3 0 ♦ I 0 si T ♦ t 0 + 3 0 * h 0 ♦ h si 8 ♦ t 0 ♦3 0 * h si si * 3 si ♦ U ♦ 1 ♦ h ♦ 2 ♦ It + 3 10 ♦ 3 0 * h 0 * h 0 ♦ ? • At the end of the first 2U hours, medium nonber 6 showed an in- creased growth over the previous media, indicating that L-glutaraic acid is probably associated with the growth of the organism, A similar in- creased growth w"8 noted with medium number 9 when L-histidine HCl was added. Medium number 9 also gave evidence of slight pigmentation which may relate histidine to riboflavin formation. After IjS hours, growth had increased in all the media except nmber 3 which may have been inhibited by DL-phenylalanine since the presence of D-amino acids does sometimes w •xert 0uch effects. At hours, pigmentation continued in medium number 9 and a slight coloration appeared in medium number 2 vhich contained only arginine and leucine as nitros-en sources. The final examination of the media at 96 hours indicated good growth in all cases except medium number 3. There was slight pigmentation in media numbers 6, 7 and 8| moderate pigmentation in medium number 2 and appreciable pigmentation in medium number 9. The final results seem to indicate interaction effects of the amino acids which are present in the media. Ihese effects could be the results of interferring concentrations or merely the presence of the amino acids themselves. When L-leucine was added to L-arginine HCl, pigmen- tation resulted which would seem to relate leucine to riboflavin for- mation but the addition of BL-phenylalanine to the leucine-airginine medium prevented the pigment formation which should have been expected to con- tinue. An even greater effect on riboflavin synthesis vms shown by the addition of methionine which completely reversed the effect of histidine and inhibited the riboflavin fonaation which had been demonstrated in medium number 9. It is evident that interactions must be taken into con- sideration whenever combinations of amino acids are used for studying riboflavin synthesis in A, goss^joii, 6, Further Studies of the Effects of Amino Acids on the Qrowth and Biosynthesis of Riboflavin by A, gossypii. In the preceeding experi- ment it was noted that the addition of histidine to the other amino acids resulted in an increased formation of riboflavin, Iliis experiment was designed to study the effect of histidine further and to determine which amino acid relationships were directly concerned with the fozMtion of riboflavin by A, fypssypli. The standard baaal mediisin with 0,05 per cent NaCl was used throughout the experiment and was designated as "basal". Since histidine seemed to be a stimulator for riboflavin synthesis, it vas chosen as the base amino acid in the first part of the CKperiment and iras added to the bai^al as the hydrochloride in a concentration of 0.012 g* or an equivalent of 2#2U mg. nitrogen, per 100 ml, of medium to make the base medium for studying the effects of other added amino acids. This base medium was distributed into flasks, into each of which except one was added singly the other amino 4cids tised. Thus one flask contained histidine as the nitrogen source, wiiereas the other flasks con- tained histidine plus another amino acid. Control flasks were also set up for peptone as the nitrogen soxirce and the basal alone without a nitrogen source. The media were adjusted to pH 6.5 with NaDH and steri- lized. The sterile media were inoculated with a 36 ho\ir washed cell suspension and incubated on the shaker for I4 days at 28° C, After incu- bation the growth was estimated visually in the same manner as the pre- vious e^qjerlment. The riboflavin was estimated visually by examining the flasks in a beam of ultraviolet light and estimating the amount of fluorescence as compared with that from peptone as + U, This allowed the detection of a vuch smaller concentration of pigment than could be de- termined by color. The composition of the media and the effects on growth and riboflavin formation are shown in Table 9, ■ Glutamic acid was the only amino acid which showed a substantial stimulation of the growth of A, gossypii, however, the addition of leucine, alanine, threonine or asparagine did increase the growth slightly over the histidine alone. Only leucine and threonine exhibited any stimulating 51 • TABLE 9 • THE EFFECT OF ADDED AMINO ACIDS ON THE GROWTH AUD RIBOFLAVIN FQRJIATION BY ASHBYA G05SYPII IN A HISTIDINE BASE MEDIUM Medium Composition Pluor- Kedivon Base Mediian Nitrogen Source Concentration Growth escence No. Added (g/lOO ml) (ribo- ' flavin) l2 Basal L-histidine HCl 0.012 0 Med. #1 L-glutamic acid 0.21 ♦ U Hed. #1 L-leucine 0.19 . + 1 si Med. #1 L-arginine HCl 0«08 si 0 Ked. #1 Glycine 0.11 si 0 Med. #1 DL-aspartic acid 0.39 tH Med. #1 DL«^anine 0.26 ♦ 1 • Hed. #1 DL-threonine 0.3k + 1 si Med. #1 DL-^erine 0.30 0l 0 tied. #1 Asparagine 0.09 + 1 0 Basal Peptone O.IU ♦ u + h Basal None 0 0 effect on pigment formation^ however, the effect was small. At the con- centration used, it is evident that histidine cannot serve as the sole nitrogen source for growth and riboflavin formation by A, g;ossypii. Since these results indicated that good growth was obtained with L-glutamic acid and L-histidine HCl as nitrogen sources, the next part of this experiment was performed using these amino acids as the principal nitrogen sources. L-glutamic acid and L-histidine HCl were added to the ' «r ■ basal to give 20.0 lag. and 2,1} mg,, respectively, of L-amiao acid nitrogen per 100 ml, of meditm to make the bas« medium for studying the effects of other added amino acids in this part of the experiment, This base medium vas distributed into flasks, into each of which except one was added singly the other amino acids used. Thus one flask contained glutamic acid and histidine as the nitrogen sources, whereas the other flasks contained glutamic acid, histidine and another amino acid. The media were adjusted to pH 6,5 with NaCa, sterilized, inoculated with a 36 hour washed cell suspension and incubated on the shaker for k days at 28° C, The compo- ■ition of the media, and the results deteraiined in the same way as in the first part of the experiment are presented in Table 10. The addition of L-histidine HCl to the glutamic acid medium re- sulted in decreased growth as compared to the glutamic acid mediiim. There was also decreased growth when DL-serine or glycine v&s added to the glu- tamic acid-histidine medium, whereas the addition of L-arginine HCl in- creased the growth noticeably, Pdboflavin formation occured to a slight extent when L-leucine or DL-threonine was added to the glutamic acid- histidine medium, however, the addition of DL-alanine or glycine resulted in a moderate foimation of the vitamin. The effects of alanine or glycine •ire of interest, since neither of these amino acids has previoxisly shown «ny effect on riboflavin formation irtien added to the histidine medium in the first part of the experiment. In the last part of this experiment, a base medium was prepared by adding L-glutamlc acid, L-histidine HCl and L-arsinlne HCl to the basal for the nitrogen sources, since this combination of amino acids had given the best growth without riboflavin formation above. This base medium was «5 TABLE 10 SHE EFFECT OF ADDED AKHIO ACIDS ON OROl'JTH AND RIBOFUVIN SYNTHESIS IN A L-GLUTAMIC ACID - HISnDINE HCl BASE MEDIUM Medium No. HedluBi Composition Growth Fluor- escence (ribo- flavin) Base Medium Niti^jgen Source Concentration Added (g/100 ml) 1 Basal L-glutamic acid 0.210 + 3 0 t Ked. #1 L-hisUdine IICl 0,012 + 2 ' Med. #2 L-leucine 0.025 ♦ 1 ii i Med. #2 DL-threonine O.QbO + 3 8l 1 Ked. #2 DL-serine 0,032 1 2 1 Med. #2 L-arginine HCl 0,010 ♦ li • r IfwS. #2 DL-isoleucine 0.0^ + 1 % ■ • Ked. 02 I3i<»alanine 0.035 ♦ 3 ♦ 1 f Med. #2 Glycine 0,015 t 2 ♦ I 10 Med. #2 L-lysine HCl 0,016 + 3 0 11 Basal Peptone 0.155 ♦ii 12 Basal None # 0 used to study the effects of other added amino acids and was distributed Into flasks, into each of which except one was added singly the other andno acids used. This gave one flask which contained glutamic acid, histidine and arginine as the nitrogen sources, whereas the other flasks contained glutamic acid, histidine and arginine plus another amino acid. A peptone control flask was set up also. The media were adjusted to pH 6,5 with NaOH, sterilized, inoculated with a 36 hour washed cell Buspension and incubated on the sliaker for k days at 28^ C, Ihe cos^> sltion of the media, and the restilts determined in the seme way as in the first part of the experiment are presented in Table 11, mBLE 11 THE EFFECT OF ADDi3) AlCENO ACIDS ON GECWTH AND RIBOFUVIK SB^THESIS IN A GLUTA>1IC ACID - HISTIDINE - AKGEilKE BASE MEDUM Medium Composition Fluor- Medium No. fease Medium Nitrogen Source Concentration Added (g/lOO ml) Grovth escence (ribo- flavin) 1 Basal L-glutamic acid L-histidine HCl In-arginine HCl 0.210 0.012 0.010 ♦ 3 • . ? Ked. #1 L-leucine 0.025 + 2 + 3 1 Med. #1 DL-threonine o.oUo ♦ 3 1 Ked. #1 DL-alanine 0.035 + 3 8l s Ked. 1^1 Q]grcine 0.015 + 3 Basal Peptone 0.172 ♦ h ♦ 3 The growth of A, gossypii was good in all media except medium flSidMr 2 ^ere the addition of L-leucine decreased the growth slightly. However, the addition of L-leucine increased the riboflaTin formation sharply, approximately equaling that of the peptone control. The ad- dition of DL-threonine, PL«alanine or glycine to medium number 1 gave only a slight formation of riboflavin. This wcperlment. has again served to point out the Interaction effects of amino acids on the growth and riboflavin synthesis by Ashbya gossypil. The resiilts seem to indicate that riboflavin synthesis Is not just the result of one or two amino acids in the medium but rathM* the result of the combined action of several amino acids and other factors. Iw^lutaaic acid, alone or in combination with L-arginine HCl and small concentrations of I»-histidine HCl, has been shown to adequately serve as a nitrogen source for the growth of A. goss./pii. however, it appears to have little direct effect on the sjmthesis of riboflavin. L-leucine showed some relationship to riboflavin synthesis as indicated by the in- creased riboflavin fomation when it was added to the glutajaic acid- histidine-arginine mediuffi{ however, a decreased growth was noted in this medium when appreciable riboflavin was formed. The results obtained with DL-threonine, DL-alanine and glycine indicate some relationship to ribo- flavin formation but the effect of these amino acids may be masked by the presence of the other amino acids in the medium. If one can assume that threonine, alanine and glycine are related to riboflavin formation, their lack of greater effect may be due to limited production of an essential link in the riboflavin synthesizing system, this link not being limiting when leucine was the amino acid added. 7. The Effect of Serial Transfers of Cells of A. goss3T)ii on •ynthetic Media, on Growth and Hjboflavin Fpimation. Since the inocula for the different experiments were prepared from cells grown on yeast extract-peptone agar stock slants, tiiere was a possibility that soms Bub- •tance affecUng riboflavin fomation was being carried over in the in- oculum even though the cells were washed two times with sterile distilled water. In order to test this possibility, it was decided to see if such substances would be diluted by serial transfer on two types of synthetic Biedia, media that support growth but not riboflavin foznation and media that svqoport both. Several solid media were prepared frran the basal with 0.0^ per c«xt NaCl, by adding different nitrogen sources including peptone as a control to give the compositions shown in Table 12, TABLE 12 THE CaiPOSITION OF MEDU FOR SIUDUNG THE EFFECT OF SESIAL TEANSFER OF CELLS ON GRCWTH AMD RIBOFUVIK FCEMTION Medium No. BaM Meditmi Nitrogen Source Added Concentration Nitrogen Source (g/100 ml) X . . - • Basal Peptone 0.178 Basal L-glutamic acid 0.210 L-arginine HCl 0.010 1 Med. #2 L-leucine 0.025 % Med. #3 L-histidin« HCl 0,012 The media were made solid by adding 1,8 per cent I'ifco-agar and were slanted after sterilization. Two slants of each media were inocu- lated with cells of A, gossypii taken directly from a U8 hour yeast extract-peptone agar slant without washing and were incubated at 28** G, in the incubator roam. After four days, cells on these slants were transferred to fresh slants of the same medium composition and were incu- bated. This procedure was repeated after another k days, making trans- twB to fWsh slants giving a total of three transfers including the original transfer from the yeast extract^peptone agar slant. The growth n vas «caB)lned visually and the riboflavin formation vas estimated by ob- serving the yellow fluorescence of the vitamin under ultraviolet light on all slants after being incubated 6 or 7 days. The results of the study are shown in Table 13. • TABLE 13 ■ ■ » , THE EFFECT OF SE"1AL TRANSFER OF CELLS OF ASHBIA GOSSYPII ON GROWTH AND RIBOFUVm FORMATTCK ON FEfTOtE AHD SlfNTHEHC WSDU Medium First Transfer* Second Transfei^ Third Transfer* No. Growth Fluorescence Growth Fluorescence Growth Fluorescence 1 * k ♦ h ♦ k ♦ 3 ♦ h ♦ 3 2 * k 0 0 ♦ It 0 3 + 2 * k ♦ 2 + 2 1^ ♦ k ♦ 2 ♦1 + 2 ♦ u • ♦ 2 * Growth and fluorescence after 6 d^s incubation. ** Growth and fluorescence after 7 days incubation, Ihe results suggest that there may be some carry over in the original transfer to the peptone medium since there appeared to be a de- creased pigmentation in the first and second transfers. In the other media there was little indication of a carry over effect. In media numbers 3 and h the pigmentation was noticeably scattered throughout the growth on the slant rather than being generally distributed as the pig- mentation on ihe peptone slants. This may indicate a localiaation of riboflavin fonnatlon on these synthetic media as a result of local depo- sition of some flavogcnic substance carried over from the preceeding medium when the cells were streaked on the slantsi however, this spotty pigmentation was approximately equal in distribution for all transfers. There vas no pipientation xAth ejny transfers on medium nianber 2} however, the growth vas equal to that of the other media. To investigate further the possible carry over of substances that alght have effects on growth or riboflavin formation, a similar teat was made using liquid media instead of the solid media. The procedure was the same as that for the solid media except the agar was oioitted and incu- bations were carried out on the shaker. In this case there was little difference noted among the first, second and third transfers in peptone flasks, both with respect to growth and pigmentation. The other media showed good growth; however, in contrast to the slants, there was no evi- dei2ce of riboflavin formation, . Since the first test indicated that there was a possibility of a carry over, another test was carried out on a series of solid synthetic oAdia containing different amino acids. The amino acids, L-glutamic acid and L-arginine HCl, In concentrations of 0,210 g, and 0,010 g,, respec- tively, per 100 ml, of medium, were added to the basal with 0,0^ per cent NaCl to give a ccai5)letely synthetic medixim which would BtQ)port good growth of A, nossypii; however, it would not allow. pigmentation. For purposes of dearity and brevity this glutaadc acid-ai^sinine medium will be desig- nated "OA", The OA medium served as the l»se medium and was distributed into flasks into each of which except one was added o"Uier amino acids singly and at a concentration of 0,015 g, per 100 ml, of base medium. Thus one flask, as a control, contained the GA mediiim and the other flasks contained the OA medium plus single amino acids. The media were made into solid form by adding 1,8 per cent agar and the sterilised agar was slanted. 0 The original inoculation of the media wa« made from a hS hour old yeast extract-peptone stock agar slant and subsequent transfers of the growth on the special media vcre every U8 hours onto fresh special media of the same composition, A total of three transfers, including the original, wre made. The growth and pigmentation vere examined as previously des- cribed except that a ♦ 5 value was arbitrarily chosen as the maximum growth value. The ccsnposition of the media and the results are recorded in Table Ih. , In general, the resvilts indicated that there was decreased growth in the second and third transfers as compared with the original transfer. The riboflavin formation followed a sinilar pattern, occurring approx- imately 2k hours later than the appearance in the original transfer on similar media. Since the media in this experiment could not be con- sidered optimum for the growth of the organism, it would be expected that the second transfer after the original would be slower in starting growth since the necessary conditions for a rapid initial cell growth may not be available. The appearance of the growth on the synthetic media slants was also very much diffCTent than the growth on the peptone raediim slants. On the synthetic media the growth was very often tough and wrinkled with the mycelium growing tenaceously to the agar, making it very difficult to transfer the cells to fresh slants, In contrast, the growth on a peptone slant was very soft and spongy, making it easy to remove cells from a slant with an inxjculation loop for transfer. These results show that there ■was apparently a carry over of some substance or substances that had a slight effect on growth and riboflavin formation, as was shown by the dif- ference between the first and second transfers. £0 Os ir CM O t oofcj c o I C3 r CM Jo I CO cu a ieo 5 •d rj O O O H, O O O O O H O O + + + + + + + + ♦ + ♦ + ♦ + + ♦ oooooooooooooooo CNJHHMHCMCJCVICSICMCSieMWCjCMCM ♦ + + + + + + + + + + + + ♦ CJ CJ O + + ■lA lr\ ^ ^ V\ \A try V\ XTv \A "lA V\ vn lA "lA + ♦♦♦ + + ♦ + ♦♦ + ♦■♦• + * + r40000H»-400r-tOHOHOO +• +1 +1 +« +1 +1 + + + + + + ■♦■•♦• + + + + ♦ + + ■♦■ OOOOOOOOOOOOOOOO f^CMHC\JHC\IO400C\JCVJOJr^CVje\ICleueine exhibited the greatest stiimilation of riboflavin synthesis* In addition to the test just described, a study was made of the effect on riboflavin synthesis of serial transfers of A, go8a?/pii cells in liquid GA med3.uin. The glutardc acid-ai^inine ir.ediuia was prepared from the basal medium with 0,05 per cent KaCl as previously described for the preparation of solid media except the 1.8 per cent Difco-agar was omitted. The washed cells from a U8 hour culture of A, gossypii. grown on a y»Mt extract-peptone stock agar slant, were inoculated into the GA aedlTan and shaken for U8 hours at 28° C. After this U8 hour period, the cells from the GA medium were removed, centriftiged, washed and re suspended as the inoculum for the second transfer to fresh GA medium which was again shaken for h8 hours. At the end of the second 1^8 hour period of incubation the growth from the second transfer of cells was reraoved, centrifuged, washed and resuspended as the new inoculum. These cells were inoculated into a peptone medium iai which the basal had been supplemented with 0,5 per cent Difco^eptone as the nitrogen source. At the same time that these serially transferred cells were inoculated into the peptone medium, a peptone con- trol was inoculated with washed cells frm a yeast extract-peptone slant. The flasks vere incubated for ? on the shaker at 28° C, tdth a get of five flasks used for each type of inoctiltaji* 3ie tia-bidimetric growth and fluorometric riboflavin determinationa ii«r» wade on the flaakt and the results obtained are shown in Table 15« • , ' ■ • ' ' ' TABLE 1^ ' THE GRWm AND RIBOFIAVIN FORMATION OF A. QOSSYPII ON A PEPTONE MEDIOTl INOCULATED WITH SERIAL TRAJISPEH CELLS Control Cells Serial Transfer Cells Number Turbidity Riboflavin Turbidity Riboflavin (% trans,) (us/ml) (% trans.) (xig/inl) 1 8.U 22.1 8.3 30.0 • 8.2 1»8.3 8.5 16.9 31.8 9.5 22.5 k 18.7 . 8.9 16.9 $ Contaminated 8.1i .: 20,8 Average 8.U 30.2 8,6 21.lt The data in Table 15 shov that the average growth vas i^roxiinately the same In ^ther eaae. The average yield of riboflavin vas approximately 29 per cent greater in the peptone medium inoculated vith the yeast extract- peptone slant cells than in the peptone mediiam inoculated vith the serial transfer cells. The highest individual yield was also obtained with the yeast extract-peptone grown cells. It is to be noted, however, that al- tliough there was a large variation in the amounts of riboflavin found in the samples from either source of inoculum, the variation vas less in the medium inoculated with the serial transfer cells. These data seen to indicate that scwie factor or factors may be carried over the yeast ejctrac t-p ep tone agar grown cells} hoirever, the effect woiild be expected to be constant throughout a particvuLar eaqseriment, i^i yhe Grovth and Riboflavin Formation of A, gossypii as Affected by Inocula Prepared fron Cells Subjected to Different Hxambers of V'ashinf;s » The studies on serial transfers of inocula indicated the pos- sibility^ of a carrj' over effect on riboflavin synthesis from cells grown on the yeast extract-peptone agar slants. If this factor vere loosely bound to the surface of the cells, it might be expected that differences in cells, obtained after a different number of vashings, might be demon- strated. This experiinent vas performed to see if such an effect could be deraonstrated, ' The medium for this experiment i»as prcfpared fjrom the GA medium to . which was added 0.015 g. of glycine and 1,8 g. of Difco-agar per 100 ml, of the medium. The test cells were taken from a h8 hour stock slant of A, frossypii grown on y«ast extract-peptone agar. Same of the cells from this stock slant vere transferred to duplicate slants of the test jnedium. The rest of the cells were washed with 10 ml, of distilled water, centri- fuged and resuspended in distilled water. The washings were repeated to obtain a total of four washings. Transfers of cells after each washing wre made to duplicate slants of the test medittm. AH of the slants w«rc incubated at 28° C, for 72 or 102 hours. After incubations, cultures wers examined for growth and riboflavin foraation in the same manner as in the prcfvious eaperiment. The results are given in Table 16, The number of washings had no effect on growiii or riboflavin for- mation. This would seem to indicate that any factor being carried over 6k TABLE 16 TIffi GHOWTH AND PIGMENTATION ON THE OLUTAKIC ACID - ABOmNE - GLYCINE (GAGly) KEDIOM INOCULATED WITH CELLS WASHED A DIFFERENT NUKBER OF HUES Period of Incubation Number of h5 hrs. 72 hrs. 102 hrs. Washings Growth Pigment (Srowth Pigment Growth Pigment e + 3 0 ♦ 3 ♦ 1 ♦ 3 0 I + 3 t # s ' # 3 t ♦ 3 ♦ 3 H + 3 0 ♦ ♦a t 4> t 1 ♦ 5 ♦ t f • ■ ♦ t ♦ t t 1 + 5 ♦t + 5 ♦ t + 1 + 5 ♦ 2 0 + 5 ♦ 2 0 + 5 ♦ t . + 5 ♦ a i ♦ 5 ♦ t onrer by the cells from the yeast extract-peptone agar slants must be either bound relatively tightly to the cells or contained within the cells. 9» Ihe Effect of Miscellaiwoue Nitrogen Sources on Grovrbh and gtbofla^in Formation. Several different nitrogen sources were tested to ascertain their effects on the growth and riboflavin formation by A. goesypii. A medim was prepared for each nitrogen source by adding 0,5 g, of the single nitrogen source to 100 ml. of toe basal, the nitrogen sources being NHj^N03, KNO3, (Niij^)2S0j|^, araaonium acetate, ammonium citrate and urea. The results in all the media showed no growth or riLboflavin formation, in- dicating that A, gossypii probably cannot grow or produce riboflavin on Bin^le nitrogen sources but must require more complex organic nitrogen 6$ sources, Althofugh the inorganic nitrogen sourcea did not siqpport giowth «r riboflavin forwation vhen used as the only nitrogen source in a »edium, it was possible that they might have some effect when added to a medium containir^ organic nitrogen sources. In order to test this possibility, a medium was prepared by addii^ 0,5 g. of peptone and 0,5 g» of NH{|N03 to loo ml, of the basal, The medium was sterilized and in- oculated in tha usual manner and Incubated on the shaker at 2S° C, AftMr U days of incubation the flasks showed a good growth of A, gossypii but ▼ery little riboflavin formation, as apparent finm the lack of pigmen- tation. It was evident that the addition of III^NO^ to the medium in th© presence of peptone resulted in a decreased synthesis of riboflavin, since the peptone alone had been shown previously to support garowth and riboflavin formation, . This effect was studied further with KNO^ and aiamonixun acetate, A peptone medium was prepared by adding 0,5 g. of peptone to 100 ml, of the basal and the resulting medium was xiaed to prepare two other media, one containing 0,5 g. of KHO3 per 100 ml. of the medium and the other containing 0,5 g» of ammonium acetate per 100 ml, of the medium, Ihe media tfere inoculated, sterilized aad incubated in the usual manner and examined visually for growth and pigmentation. The growth in both media WKS very goodj however, in the medium containing the KTIO^ there was no appreciable pigmentation, whejreas in the msdiua ccaataining the ammonium acetate there was a normal yellow pigmentation present. From these re- sults it appeared that the nitrate ion, at the concentration used, might act in some way to inhibit the formation of riboflavin when the ion was - 66 in the presence of peptone. This fact raay be of use in farther studies of the riboflavin metabolism of A, fijossypii. 10« ^he Treatment of Peptone vith Pennutit. It has been shown previously in Experiment number 3 that the more basic components of pep- tone seemed to be concerned vith riboflavin formation in A, goss^iJiii rather than the other coarponenta. If these basic ccsaponents could be removed from the peptone in scxne sijnple manner, then their relationship to idboflavin synthesis could be further demonstrated. Since Pemutit has been used to resnove ammonia in urine analysis, it might also remove "Uie Bore basic components of pcpton«» ■ '■ , Ihe experiment vas carried out on a solution containing 0.^ g« of peptone dissolved in 50 ml, of distilled ifater. Five grams of washed Pennutit vas added to the solution vhlch vas stirred for several minutes. The Penmitit vas removed by filtration and the filtrate containing the unabsorbed parts of peptone vas made into a medium by adding the other coeqoonent parts of the basal to the filtrate and diluting it to 100 ml. • An untreated peptone medium was prepared by adding 0,5 pei* cent of pep- tone to the standard basal. TIob Penautit treated and untreated media were inoculated with a washed U8 hour cell suspension of A, gossypll and incubated for i; days at 28® C. on the shaker. The growth was then measured by obtaining the dry weight of an aliqaot of cells before ex- traction of riboflavin and the riboflavin was measured fluorometrically in the usual manner. The results of the experiment are shown in Table 17. data show a great contrast between the treated and the un- treated peptone media. The growth in the treated peptone medium «u 67 TABLE 17 THE GfiOWTH AND RIBOFLAVIN TOWJiTlCM (M lEDU CONTAINING UNTREATED AND PERMUTIT TfrEAIED PEPTQI 0,5 and 1,0 lal, of the cell extract to approximately 15 ml, of distilled water, and autoclaved in the presence of sodium acetate buffer, as preriousl}'- des- cribed, in order to solubilize any bound riboflavin in the cell extract. After autoclaving, the samples vere diluted to 25 nil, with distilled vater before being teSfted, A second set of four samples was prepared as just described, except 1 ml, of a standard riboflavin solution, containing 25 ug, of ribo- flavin per ml,, was added to each aliquot sample of the cell extract be- fore autoclaving. This second set of san^jles was used to test the method for recoveries of known amounts of riboflavin which were added to the unknown samples, Ihe results for the determinations are given in Table The data show good agreenient between different aliqaots of the unknown cell extract and adequate recoveries of added riboflavin, When the Tal\xes for the riboflavin content of the original cell eoctract were «rer«^ed, a value of 3h.h ug, of riboflavin per ml. was obtained. Twenty days prior to the colorimetidc determinations on the cell extract, this same extract had been assayed fluorometrically and found to contain iiO,5 ug, of riboflavin per ml. A c<»T,parison of these two values indicates the colorimetric method gave restilts about lb per cent below the fluorometric method. Considering the storage period of 20 days in the refrigerator and the fact that some riboflavin was destroyed during storage, tlie colorimetric assay of the cell extract was in satisfactory ^reement with the fluorometric determination, at least for making ft appimlmate assays. The results of the assay above indicate that the col- ©ilTOtric determination of riboflavin is feasible by this method, especially vhere approxirate measureaents are of intez*est rather than aanall accurate quantitative determinations. It is believed tiiat the method could be re- fined to a point where it could be used as a more quantitative rcethod of assay for riboflavin in culture media, ' ' . v 18, The Effect of Glycine, L-serine and TTL-threordne on Grovth ^ ^^mt^mm I III .III II i>ii I. . II. I 1,11, I a and Riboflavin Synthesis vhen Added to Different Synthetic Media. Glycine, iriien added to the synthetic glutamic acid-arginine mediuia, has been shosn to be effective in stimulatdnp the foimation of riboflavin by A, go3s:T)ii (Experiment number 9). Ihe iaterrelationahips of glycine and serine hav» been reported in the metabolisms of Microorganisms, therefore, the sub- stitution of L-serine for glycine might be expected to stimulate ribo- flavin formation also. DL-threonine vas used because of its structural similarity to serine and the sli ht effect on riboflavin synthesis it had previously shown. The media were prepared from the basal as follows t the glutamic acid-arginine (GA) medium, 0,210 g. of L-glutamic acid and 0.010 g, of L-arginine HCl per 100 ml, of basal, the aspartic acid-arginine (AA) medium, 0.210 g, of L-aspartic acid and 0.010 g, of L-arglnine HCl per 100 ml. of basal, and the asparagine-arginine (AspA) medium, 0,210 g, of asparagine «nd 0,010 g, of L-arginine KCl per 100 ml. of basal. The other media were prepared by the addition of 20.0 mg. of glycine, 28,0 mg. of L-serine or 63.? mg, of DL-threonine to each of the GA, AA and AspA media, the amino acids being added on the basis of the sane L-alpha amino acid nitrogwi content. The media were adjusted to pH 6,$ with NaOH and sterilized in the \i8tial manner. After steriliaatlon duplicate flasks of each media ■were inoc\alated vith a 36 hour washed cell suspension and incubated for li days at 28® C, on the shaker. The dry weiphts of cells and colori- metric riboflavin dctenninations on the different media were madej these Talues are shown In Table 28. The growth vas approxiraately the same in the case of the GA and AA media without added amino acids but the yields of riboflavin were very small. The fact that the vitamin synthesis in the AA medixim was twice that in the GA medium, may or may not be significant. However, in the AspA medium without added amino acids appreciable riboflavin was synthesized although there was decreased growth of the organism. It has been observed that there wm usually less growth in a medium cor^ared with the control, when riboflavin was produced. When glycine was added at a concentration of 20 mg. per 100 ml. to each of the three media, a noticeable increase in the riboflavin fonned was observed, especially in the AA and AepA media. This would further substantiate the relationahip of glycine to riboflavin synthesis with aspartic acid balng probably more closely related to the synthesis than glutamic acid. The results with asparagine would seem to agree with this, since asparagine is known to be metabolically related to aspartic acid. The increase in riboflavin ■ynthesis shown by asparagine without glycine may be due, in part at least, to the amide nitrogen which is present. Again in the GA and M media, there was decreased growth when appreciable riboflavin was fonned. The substitution of L-serine or CL-threonine for glycine failed to stim- ulate vitamin synthesis to the same extent as the glycine. This aay in- dicate that serine and threonine are not as directly involved in riboflavin ■ ■ ■ TABLE 28 THE EJFiXT OF GLYCDffi, L-SEHEJE OR DL;-3HRE(mE C3N GBCWTH AND PJBOFIAVIN SINTHESIS IH QLaTA>ac ACID - ARGINliai;, (OA) ASPARTIC ACID - ARGINIRE (AA) AHD ASPAiJAGINE - ARGIl'^BiE (A»pA) KEDIA Hediiaa Aadno Acid Dry Weight Cells (rJK/20 ml) Riboflavin (ufi/ml) Added 1 2 1 2 Ave, Qk None 106-6 t«t Glycine 96 8 13.3 n L-^erine 102.2 91.6 96.9 2.5 0.5 1.5 DL-threonine 126.li 123.6 125.0 2.8 2.0 2.1t Hone 109.6 IOU.8 107.2 2.8 2.0 2.1i m Olycine 89.8 80.2 85*0 20,2 28,8 2li.5 M L-serine 103.8 99.2 101,5 !».8 h.3 h.6 m DL-.threonine 136.8 83.il 110.1 li.3 2.8 3.6 AspA None 57.8 67.0 62.1 7.8 12.2 10.0 A^pA Glycine 73.U — * 73.1» 30.5 — — * 30.5 AspA L-serine 91.2 65.6 17.5 10,5 lli.Q DL-threonine 66,6 6b.8 65.7 9.5 9.5 9.5 ♦ Contaminated flask. synthesis or it may be due to the fact that \mder the conditions of the experiment they were not utilized as readJ.ly as glycine. If glycine is jrequired for ariboflavin formation, the rate of its fonaation may be a factor} therefore, serine Kight not stimulate the formation of riboflavin, even though it is thought that glycine is derived from serine in mnj •icroorganisms, because tlie glycine might not be formed fast enough. 9i Serine tcad threonine hare been reported by Goodwin and Pendlington to be related to riboflavin synthesis in £, aahbyii, whereas, gl^^xine had no effect. The data from this experiment woiiLd indicate that the reverse may be the case with A, eoss:/pii. !?• Ihe Iffect of L-histidine HCl on Growth and Hiboflavin ^or- Mtttion in Glutaadc Acid«Arginine and Qlutandc Acid«Arginine-01ycine Media. Taw had reported that histidine was effective in stiriulating riboflavin synthesis in E, aahbyii but the addition of histidine to experiinental media in previous experiments of this study failed to show any appreciable effect on riboflavin f orruation with A, frogaypij. Since onlj' one con.cen- tration of histidine had been used in the previous experiment, the effect of histidine might not have been apparent j therefore, tests were performed using different concentrations of histidine in the GA medium and the Qk Eiediuja with glycine (OAGly) of the preceeding experiment, The GAGly medium contained 20 rac, of glycine per 100 ml. of the GA medi\im. In the first of two tegts, t«ie inoculum wfs prepared from a 36 hour washed cell suspension of A, gossypii which was added to the sterile riedia and incubated on the shaker for h days at 28° C, After incubation the dry weight of cells and colorSmetric riboflavin deteminations were made. These values and the amounts of histidine added are reported in Table 29, ^e lower concentrations of histidine showed a stimulation of growth in both media and an inhibition on riboflavin synthesis in the OAOly aeditaa. At higher concentrations of histidine there was a decreased growth of the organism, Histidine may have some effect on riboflavin synthesis in the OA medium especially at high concentrations where a slight stimulation of synthesis was shown on the basis of riboflavin per 96 o o o -it -a CM m \o CO • • • o o o -3 • o CM * CM CO CM p- O O Q -^r CM 3 • « • vO 0\ vo 3« o 6-i O is H 3 as _ CO w I r IK CO CM H CM CO CM • • H H H o 00 CM -a- CM CO CM H CM CM CO CM d CO o CO CM CM « O H CM T)ii in non-riboflavln producing media and in riboflavin producing inhibition media. The media were prepared from the basal, containing 0,01 g, of L-arginine HCl per 100 ml,, and by the addition of L-glutamic acid or L-espartic acid in different concentrations to form the GA or AA media. Three GA media were prepared by the addition of 0,21 g,, 0,10 g, or 0,05 g. of L-glutamic acid, rei^ectively, to three different 100 ml, volumes of the basal plus arginine. Two M media were prepared in a slfflilar manner using 0.1? g. or 0,10 g, of I«aspartic acid. The glutamic acid-arginine-glycine-histidine (GAGlyil) and the aspartic acid-arginine- glycine-histidine (AAGlyH) media were prepared frtan each individual OA or AA mediiim by the addition of 20 mg, of glycine and IjO mg, of 106 L-histidine HCl to 100 ml, of each individual mediton. The pH of each medium vas ad;5uEted to 6,5 with NaOH and duplicated flasks sterilized in the usual manner. The sterile media vere inoculated with a 28 hour washed cell suspension and incubated for h days on the shaker at 28® C, After incubation, the dry ireight of cells and colorlraetric ldbofla^^in values were determined. These values and the concentrations of glutamic and aspartic aoida are given in Table 33* TABLE 33 THE WYECf (Sr DIFFERENT COHCENTRATIONS OF t-GLUTAKIC ACID MD t-ASPARTIC ACID ON OSCWTH AND RIBOFLAVIN SYNTHESIS US SYIJTffiTIC MEDIA Concentration Dry V/eight Cells Riboflavin MediuB Kain N So\irce (ng/20 ml) [ (ug/mg (g/lOO ml) 1 2 Ave. 1 Ave, , — n cells) Ave, OA Glutamic Acid GA 0,21 75.0 78.0 76.5 2,0 1.8 1,9 GA 0,10 35.6 Uo.o 38.8 1.5 0.5 1.0 0.1 OA 0.05 25.li 2li.O 2U.7 0.3 0.3 0.3 0.3 QAQ]yH 0,21 ^ 2.2 3.h 2.8 7.5 8,5 8.0 2.6 OAQlyH 0,10 Lit 2.6 2.0 ii.5' 5.3 U.9 3.1i GAGlyH 0.05 1.2 1.3 9.5 7.5 8.5 7.6 AA Aapartic Acid AA 0.19 73.1 83.1i 78.i» 2.3 1.8 2.1 O.li AA 0.10 ho,h 37.8 39.1 1.5 1,8 1.7 0.2 AAOlyH 0.19 2.2 2.8 2.5 9.0 8.5 8.8 7.2 AAGlyH 0.10 1,0 1.2 1.1 li.O U.5 U.3 5.3 107 Ihe reduction of the amount of the aain nitrogen source in the media pro*iced a decreased growth £K)proxiniately equal in degree to the reduction of the nitrogen source. In the C5A and AA media, a reduction of the concentrations of glutamic and aspartic acids by one-half pro- duced jqjproximately one-half of the growth obtained at the higher con- centrations of 0,21 and 0,19 g*f reipectively, for the amino acids. A further decrease in glutamic acid concwttration to 0.05 g. per 100 al, of media did not produce as great a reduction in growth; however, a moderate reduction was errldent, A reduction in the riboflavin a^^nthesized occured in the OA and GAGlj^ media, as well as the AA and AAGlyH media, when the concentrations of the main nitrogen sources were reduced to 0,10 g, per 100 ml, of media. At a glutamic acid concentration of 0,05 g. per 100 ml. of medium in the GAGlyH medium, the amount of riboflavin produced was approximately the same as that produced at the highest concentration of glutamic acid in the experiment. It is possible that at this low level of glutamic acid, the producto of the glutamate ijietAbolissn may not have been at a high enough concentration to interfere with the formation of riboflavin, assuming the inhibitory effect of dicarboxylic acid derivatives on ribo- flavin formation is a causative factor as was previously reported. The etimulatory effect of aspartic acid on riboflavin synthesis in the presence of glycine, ^rtiich was noted in a previous experiment, did not manifest itself under the inhibited condition of growth in the AAOlyii medium. This may indicate that the aspartic acid was not metabolized as readily under these condiUons to the necessary inteimediates for ribo- flavin synthesis. However, on the basis of the amount of riboflavin per m mg. dry velght of cells, the AA media did ahow a slightly greater effect on ribofla\rin formation than the corresponding CJA nedia of the same rel- ative composition, 23. Studies on Growth and Riboflavin Formation vith the Addition of glycine to Synthetic Media at different Tines During Incubation. In studies an metabolic pathways of biosymthesis for biological compounds, it often becomes necessary to resort to tracer technicpies to follow the progress of a certain coii5)ound which may be thought to be related to the pathway, Ihe development of the synthetic GA nedium has increased the possibility of using such techni<3ues to stuify riboflavin formation under defined conditions, especially since a compound, such as glycine, stimu- lateo the formation of riboflavin when added to the medium. If a tagged compoxmd were to be used, it would be helpful if it could be added to the medim after the growth of the organism had progressed for a period of tine, and thus decrease the scattering of the tagged a tan into other pathways which would be probably functioning more extensively during the early phases of growth than would the riboflavin synthesizing metabolism, With this idea in mind, it was decided to study the effect of adding glycine to several synthetic media at different times during incubation. The base mediurri for the first test was prepared from the basal by the addition of 0,21 g, of L-glutamic acid and 0,01 g. of L-arginine HCl per 100 ml. to give the GA medium. The other media were prepared as follows t the QAGly medium, by adding 20 mg. of glycine per 100 ml, of GA medium} the GAOlyH medium, by adding hO mg. of L-histidins HLl per 100 JBl, of the GAGly medium, and the glutamic acid-srginine-histidine (GAE) medium, by adding I4O mg. of L^stidine HCl per 100 ml. of the GA medium. 109 All media vere then sterilized, inoculated with a 28 hour washed cell suspension and incubated for li days on the sliaker at 28° C, For the delayed action flasks of media, a sterile glycine solution, containing h Btg. of glycine per ml. of distilled water, was added to the cultures in the GA and GAH media to give a concentration of 20 Bg« of glycine per 100 ml. of media, tiie same concentration as that in the GAGly and GAGlyH media. After incubation, the dry weight of cells and the colorimetric riboflavin values were determined in the usual marmer. These values and the times of the additions of glycine are given in Table 3U» TABLE 3k THE EFFICT OF QLYCINE ON GROWTH AND RIBOFLAVBi FORMATION DURING POUR DAYS mCUBATION IN SYNTHETIC KEDIA WITH GLYCINE ADDED AT DIFITPiHT TE'iS DURING INCUBATION Medium Time Glycine Added Dry Weight Cells ■ (mg/20 ml) Riboflavin (ug/ml) (hours) 1 2 Ave. 1 2 Ave, « t 66.8 62.1 M 2.0 2J GAGly « 57.8 6Ii.2 61.0 15.5 16.5 16.0 9k 51.U 60.0 55.7 6.0 5.7 , OA 11 66.8 87.6 77.2 5.5 7.2 GAH • 6.8 h.h 5.1 5.5 5.5 GAGlyH • llt.l 12.8 13.5 16,3 IU.3 15.3 GAH ill 8.1, 12,1 10.3 8.3 7.1 QAH 7.2 6,2 6.7 M 6,8 6.k data show that riboflavin was simthesized in the GA flasks no to irtiich glycine was added after 2h hours, or hB hours* incubation, to ap- proximately the same extent for either tine of addition of the ctanpound. The yield of riboflavin resrilting from the late addition of glycine was considerably lefuer than that obtained for the GAGly medixm in which the glycine was present fl^jin the beginning of the incubation. This could be explained if the gljncine was necessary in the early stages of growth in order to make same intermediate in riboflavin synthesis which could not be fonned in an effective concentration in the GA medium alone. However, on this basis the medium with glycine added at 2h hours would be expected to yield more riboflavin than the meditm vhere the glycine was added at 1*8 hours, but this was not the case according to the results. The U8-hour addititm of glycine to GA Biedium produced an increased growth -idiich may IjKlicate a more direct incorporation of glycine into growth vhen added to this medium after appreciable grovrth was already present, - • . • This test of additions of glycine to the GA and GAH media after growth had been underway for a time was repeated, using more controls in order to obatin a clearer picture of the effect of added glycine at dif- ferent times during incubation. In addition, longer incubation tljnes after the addition of glycine were used. The sterile GA, GAGly, GAGlyH and GAH media and the sterile solution of glycine used for additions Of glycine to the OA and GAH media during grovrth were prepared as des- • cribed above. The sterile media were inoculated with a 28-hour-washedT cell suspension and incubated on the shaker at 28® C. After the addition of glycine to the incubated GA and GAH media at 2h and hB hours, the growth was allowed to continue until a full 96 hour incubation period had been attained after each addition, which meant that there were 120 hours Ill ? ■ • and Ihk hours total incubation periods, respectively, for the flasks with gljrcine added during growth. Some of the flasks of OA and GAH aiedia to which glycine had been added were ronoved after 96 hours total incubation in order to give short period incubations to cor^are with tliose media where glycine was added initially. After incubation, the dry cell weight and the coloriraetric riboflavin values were determined in the usual manner. These values and the times of the additions of glycine are reported in Table 35. ^ The delayed addition of glycine to the GA medium showed approxi- mately the same yield of riboflavin for either the 2h hour or the U8 hour addition of the glycine. The riboflavin produced in these media was less than that produced in the GAGly medium incubated for 96 hours even though the media with delayed addition of glycine did have the same incubation time with glycine in the media. This nay indicate that glycine, which was added after the growth had pro ressed, entered into the growth metab- olism of the organism rather than into the synthesis of riboflavin since the growth in the 96 hour GA medium after delayed addition of glycine was greater than that of the ftOl llili hour incubation of the G/>Gly medium. SoBie of the inhibition of the synthesis of riboflavin in the GA medium with delayed addition of glycine may have been due to the formation of some riboflavin inhibitory metabolic products during the early growth of the orgeni-sm. In the histidine inhibited GAH medium, there was a alight increase in riboflavin over the GA control which may indicate a relationship of histidine to riboflavin synthesis; however, due to the inhibition of growth, the metabolism of glutamic acid and arginine may be forced into 'Hi TABLE 35 THE ^FFTCT OF DELAYED ADDITION 0? GLYCINE ON QROfeTil AND RIBOFLAVK FCSlMnON IN SYNTHETIC MEDIA an Hour Glycine Added Incubation Time I hours ; Dry Weight Cells (inR./20 ml) Riboflavin (ug/inl) 1 2 Ave, 1 2 Ave. GA 96 5U.3 36.0 lii.7 2.5 3.2 2.9 uA ■mm ti i 31 .a 33.8 32.6 2,5 3.2 2,9 ■Mk 9 Jjo 29,6 27.il 2o.5 2,0 3.0 2.9 w ft 3u*2 3a»2 '31 O 3a. Z 0.3 9.0 0.7 M- 3a«0 39. o 3o.9 7.5 10,3 0.7 n 1 lili 37. o u2,o 1 n 1 aO.l 0,0 12,5 10.3 GA mt . 75 ft 35. y 33.2 3a .3 5.0 5.5 5.6 OA 19 XcU 49.0 2U.2 36,6 6.0 u.o 5.0 ft* Ikft ho 35.14 37.2 36,3 ii.8 5.3 5.1 Ua ii m 1 1.I Ilia 1j3.6 51t.8 li9.2 5.3 5.8 5.6 UAH 96 3.U 3.6 3.5 5.3 3.5 U.U GtAH Mil liiU 2.8 U.8 3.8 5.3 5.8 5.6 GAGlyH 0 1»8 6.1j 6.6 3.U 3.5 3.1* GAGlyH 0 72 S.O liio m 12.5 ll.b n.U GAGlyH 0 96 h.h h.2 U.3 7.5 9.3 8.U QAQlyH 0 UOi 3.8 3.8 3.8 10.0 11,3 10.7 OAfi 2h 72 k.B lt.2 9.3 9.5 9,U OAH 2h 120 5.6 2.6 li.l 8.3 8.5 8. It GAH hs h» 3.2 6.0 ii.6 6.5 8.0 7.3 GAH i|8 8.2 7.U 7,8 7.8 11.0 9.U 113 the direction of riboflavin formation. In the GAH media vhere glycine was added at 2k and hR hours, the riboflavin formed was apprasdmately eqml to that produced in the C-AGlyH medium where glycine was present at t^ tiraie of inoculation. This would indicate that the late addition of glycine to the histidine inhibited media had little effect on the extent of riboflavin synthesis. The small yields of riboflavin obtained after the delayed ad- «!ltion of glycine to the GA medium would make difficult the use of tagged compounds in studies on the bioayntheeis of riboflavin under these conditions unless yields could be Increasedi however, their use in the histidine inhibited media might have possibilities. 2h» Studies on Growth and Riboflavin Synthesis in the Glutamic Acid-Arginine-Hlstidlne Medium with lUfferent Concentrations of Amino Acids. The discovery of the inhibition effect of histidine offered an opportunity to study the effects of amino acids on riboflavin synthesis by eliminating, to a large extent, the growth metabolism of A, gossypii while riboflavin synthesis continued. Under these conditions, the ad- dition of precursors or other compounds may be studied with the hope that a clearer pattern of the ccanpounds which are related to the syn- thesi* of riboflavin wovdd appear. The following series of tests was ■uch an attempt, in which a number of amino acids were studied for their effect on riboflavin formation in a histidine inhibited cell system, GA medium was prepared fSpom the basal and contained 0,05 g. of L-glutamlc acid and 0,01 g, of L-arginine HCl per 100 ml, of the basal. Ihe lower concentration of glutamic acid as compared with 0,21 g, per 100 ml. in previous media was used to eliminate possible inhibitory effects on riboflavin synthesis that might result from the higher conctntration usually used. The lower concentration of glutamic acid was shown in a previous experiment to be satisfactory for the foimation of riboflavin in the histidine inhibition medium. The QAH medium was prepared from the OA medium by adding UO mg, of L-histidine HCl to 100 ml, of the OA medium just described. The GA and GAH media and the QAH medium plus 20 mg, of glycine per 100 ml. were used as controls throughout the experiment. Other special media except tiiat with methionine were prepared from the GAH medium by the addition of 5.0, 10,0, 20.0 and ijO.O mg. quantities of the individual amino acids to be studied and were sterilized. The methionine, which was used in one part of the esj)eriment , was prepared as a solution and sterilized by Seitz filtration before the addition to the sterile media. A 28 hour washed cell suspension of A, gpssypii was used for each inocxilation throughout the experiment. Because of space limitations on the shaker, the experiment was carried out with three amino acids and controls for each test. The inoculated media were shaken for h days at 28*^ C,, after which they were removed from the shaker and the dry weight of cells and coloarimetric determination of riboflavin made in the us\ial manner. Ihe results along with the amino acids used, with their concentrations, are presented in Tttbls 36» In all of the ejqjeriments, the riboflavin produced in the GA medium controls was of a low level as had been expected. The growth in the OA Bidium controls was within reasonable agreanent among the different experiments, ranging from 22,9 mg. to 36.6 mg, dry weight of cells per 20 ml. of medium. The QAH control gave the expected inhibition of growth in all of the experiments with the c3ry weight of cells ranging from less 115 TABLE 36 THE GROWTH AND RIBOFLAVIN: F(3lKAnc»I IN THE GLUTAMIC ACID - ARGItilNE - mSTiriNE (GAH) MEDIUM WITH DIFFEREMT CONCENTHATIONS OF DIFFERENT AMINO ACIDS rieOluRl ^Ajnino Acid AaQeu Dry Weight Ceils (mR/20 ml) 'Riboflavin (ug/ml) (ng/lOO ml) 1 2 Ave, 1 2 Ave, GA* 31.8 30.1 30,9 14 1.5 1.5 GAH^ 1.0 <1.0 <1,0 $^ 1*.7 5,0 GAH CUjrcine 5.0 10.0 20.0 Uo.o <1.0 1.1* 3.0 <1.0 1.2 1.8 1.1* <;l.o <1.1 1.6 2.2 <1»0 6.8 6.8 7.8 10.8 6.0 7.0 9.2 10.3 6.1* 6.9 8.5 10.5 GAH L-proline 5.0 10.0 20,0 1*0.0 l.l <1,0 <1.0 1.0 1.8 1.2 1.0 i*.o 1.6 <1.1 <1.0 2.5 6.5 1*.5 1*.5 ii.l* 6.0 5.0 5.0 5.2 6.2 1*.8 1*.8 1*.8 GAIi L-leucine 5.0 10.0 20.0 liO.O 1.6 1.8 5.1 1.1* 1.0 2 6 5.8 1.5 1.1* It. 2 5.6 l*.o 6.2 11 8 12.5 U.5 5.8 7 fi lli.5 U.3 6,0 9.8 13.5 OA* 22.1} 23. It 22.9 0.5 0.8 0.7 GAPf* <1,0 <1.0 <1,0 3.3 U.8 a.i GAH* Glycine 20.0 1.2 1.0 1.1 8.3 8.U GAH L»tyrosine 5.0 10.0 20.0 1*0.0 <1.0 <1.0 1.0 <1.0 1.0 <1.0 <1.0 1.0 <1.0 <1.0 <1.0 <1.0 5.3 6.5 5.8 6.0 6.5 6.0 5.8 7.3 5.9 6.3 5.8 6.7 GAH DL-trypto- phane 5.0 10.0 20.0 liO.O <1.0 1.2 1*.0 3.8 <1.0 1.0 7.6 3.0 <1.0 1.1 5.8 3.1* 5.3 6.0 8.5 8.5 7.3 5.8 10,0 8.0 6.3 5.9 9.3 8.3 GAH L-lysine 5.0 10.0 20.0 1*0,0 U.8 1.2 1.0 1.2 2,8 2.6 1.0 1.1* 3.8 1.9 1.0 1.3 7.0 h 5.8 7.8 6.1* 7.1* (Table continued on following page) 116 TABLE 36 (continued) Amino Acid Dry Weight Cells fiiboflavin Mediiun Added (ing/20 ml) 1 11 £7 /ml J (ma/lOO ml) 1 2 Ave. 1 2 # M Aw 32.2 26.1i 29.U 0,5 0.8 0.6 GAH^ 3.8 1.8 2.8 3.8 5.0 U.U Glycine 20.0 1.2 3.1 2.3 9,0 8,7 GAH L-^stine 5.0 7.8 19.0 i3.lt 9.3 8.5 8,9 10.0 19.8 23. V 21.6 Ii.8 8.3 6.6 20,0 21.1 21,6 21,5 3.0 li.O 3.5 IjO.O 22,U 21,2 21.8 3.0 3.0 3.0 GAfi L»e«rine 5.0 2,8 5.2 ii,0 8.8 11.0 9.9 10.0 1.8 2.2 2.0 11.0 11.0 U.O ... - 20.0 3.6 3, It 3.5 9.3 13,0 11.2 liO.O 1 1 U.U 5,U U.9 11,8 10,2 ■b«»tni*eQnine 5.0 3.0 5.2 U,l 7,0 7,3 7.2 XU.U 3.0 a.2 3.6 0,0 0,0 ii 0 0,0 . . 5.8 U.8 5,3 10.0 9.5 9.0 liO.O 5.2 U.U t Q U.O 11.0 9.3 10.2 Qir ~— — 37.8 35.1i 36.6 1.0 0.8 0.9 III ■■ . -. 2.6 3.0 2.8 I1.3 U.l GAH* Glycine 20.0 2.8 li.2 3.5 S.8 6,5 6.2 u&ti T "1 J L«alanine 5.0 a. 3 2.6 3.0 M 2,8 3.3 10,0 2.8 2.I4 2.6 : 3,0 3.8 cU.U 2.2 1.8 2,0 1.0 3.9 Uo.o l.li 2,0 1.7 1*,8 U.6 QAH IHL-phenyl- 5.0 2.0 U.8 3.U U.O U.5 U.3 alanine 10.0 1.8 l.U 1.6 1,5 U.5 20.0 1.6 2,ii 2.0 U.5 U.5 llO.O U.o 3.0 3.5 5.3 U.8 DL-iso- 5.0 2.6 3.0 2.6 5,0 U.5 leucine 10.0 3.0 1.8 2.U U.3 U.U 20.0 3.0 2.U 2.7 5.3 5.3 Uo.o 3.2 1.8 2,5 5.3 5.3 (Table continued on folloving page) 'it Added Amino Acid. (mp/lOO ml) Dry Weight Cells (ing/20 ml) Riboflavin (ufi/ml) X o Av6» X o c Ave. — — 30.0 32.0 31.0 1»0 1.0 1.0 GAH* 2.6 2.0 2.3 5»0 5.0 GAH* Glycine 20.0 1.6 1.8 1.7 10.8 9.7 10,3 18.0 15.8 16,9 lt«8 Ii.5 U. 1 nine 10.0 22.2 IS.O 18.6 li.7 20.0 30.1 32.8 3*5 5.0 U.3 1*0.0 5o,U ^ . U5,2 U7.8 h.2 h.k QAH L-Taline 5;o 3.0 3.6 3.3 5.3 5.2 10.0 3.0 3.2 3.1 6,0 5.3 5.7 20.0 2.0 2.0 2,0 ?.8 5.5 5,7 Uo.o 3.2 3.6 3.U 5.8 6.3 6.1 GAH Beta- ■ 5.0 1.8 2.6 2.2 6,5 5.8 6.2 alanine 10.0 2.2 2.U 2.3 7.5 8.3 7.9 20.0 \.2 2.2 3.2 7.5 8.0 7.8 liO.O 2.6 2.6 2.6 7.0 9.0 8.0 * Control media. than 1,0 fflg, to 2.8 mg, per 20 ml, of medium. The riboflavin fomed in ■ the GAH control medium was in the range of U,l to 5.0 ug, per ml, which | was an averape of 3.5 ug. per ml, higher than the riboflavin ja-oduced in ' the GA control medium. This would indicate that histidine, at the con- centration used in the GAH medium, may have some effect on riboflavin synthesis. If the growth metabolism of A. t^oasypil is considerably disturbed \mder these inhibited conditions, the metabolism of glutamic acid and arginine may be forced into other path-, ays than those for growth, thus some riboflavin may be formed as a resiilt of their changed metabolic \ products. If such were the case, it would indicate an indirect fianction j of histidine on riboflavin synthesis; however, under these same i j ■j j I 118 eonditions histidine may alao be aasuned to be more directly involved in riboflavin fonaation* A total of 15 amino acids vrere studied, in this series of tests, with respect to their effects on formation of riboflavin in the histidine inhibited cell system. In keeping Tri.th the other experiments, growth was also deteiroined in conjunction with riboflavin synthesis. The growth in- hibition by histidine was not significrntly affected by the amino acids, glycine, L-proline, L-tyrosine, L-alanine, TjL-phenylalanine, '^L-isolevicine, L-valine or Beta-alanine when added to the GAH media. Assvaning the con- centration of these amino acids is high enot^h to overcome any histidine inhibition of their metabolisms, the data would seem to indicate that histidine was not inhibitory to the growth of A. goasypii by blocking the metabolisms of anj'- of these amino acids « L-leucine, t'L-tryptophane, L-oerine or L-threonine showed a alight effect in the histidine inhibition when the 20,0 lag, or U0,0 ng, concen- trations were added to the GAH medium as shown by the slightly increased growth enrer the GAH control medium. When only 5.0 mg. of L-lysine HCl wae added to the OAH medium, there was a slight increaae in growth over the GAH control, but at the higher concentrations of lysine there waa no longer any such effect noted. The sulfur containing amino acids had a large effect on the his- tidine inhibition of A, f^ossypii cell growth. Hie addition of 5,0 mg. or 10,0 mg. of L-cystine to 100 ml. of the OAH medium resulted in 13,U mg, or 21,6 11^, dry weight of cells, respectively, which was approximately one-half or two-thirds, of the uninhibited cell growth in the GA control medium. L-methionine was even more effective in relieving the histidine 119 inhibition of growth. The growth in the GAH medium when 20 rag, of L-metMonine per 100 ml, was added, equalled that formed in the OA control raedixra. At the concentration of hO mg, of methionine per 100 b1» of the GAH medium, the growth in the GAH medium exceeded that of the uninhibited GA control medliijn by approximately 50 per cent. This strongly suggests that the histidine inhibition may be due to a blocking of the soilfur metabolism of A, gossypii as related to either the for- mation of methionine or the function of meth-ionine in the growth raet^b- olism. An examination of the effects on riboflavin synthesis showed that the amino acids L-alanine, UL-phenylalanine, DL-isoleucine, L-valine and L-methionine apparently had no stimulating effect on the synthesis of riboflavin iji the GAH medim. It is of interest to note that methionine did not stimulate riboflavin synthesis even though it relieved the histidine inhibition of growt,h. This would create a con- dition similar in some respects to growth in the GA medium without histidine^ however, the yield of riboflavin Is considerably higher than that in the GA control which seems to indicate an effect by histidine on fonnation of riboflavin. Thus, it would e^pear that histidine might have a dual effect, one as a grofwth Inhibitor and the other as a stim- ulator for riboflavin synthesis. L-proline and L-tyrosine appeared to show a slightly increased riboflavin synthesis over ihe QM control when 5.0 mg. of the amino acids was added to 100 ml, of the medim. At higher concentrations of these amino acids, tyrosine showed no further effect but in tiie case of proline there was a reduction to the value of the control. These amino 120 acids showed no significant effect on histidine inhibition. There was a moderate but significant increase in riboflavin for- mation when L-lysine HCl, 3eta-alanine or DL-tryptophane was added to the GAH mediiim. In the cases of lysine and tryptophane, the synthesis in- creased as the amount of the amino acid added was increased; however, in i;he case of Beta-alanine, the maximum amount of riboflavin vas formed when 10*0 mg« of the amino acid v.-as added to 100 ml* of the GAH medium and further additions produced no increase. One of the most striking amino acid effects was shown by L-cystine which stimulated both growth and formation of riboflavin when 5.0 mg, of the amino acid was added to 100 ml, of the OAU medim. As the concen- trati , Hi - 2ii,0 L-leucine ,10,0 Wf^ 10,0 20.0 Ml 19,0 lt0,0 li9 21.2 L-tyro8ii» 5.0 Hi 36.0 10.0 m 22.0 20.0 m 8.5 ho.o m 1^. DL-tryptophane 5.0 $0 Ult.O 10.0 m 18.0 20.0 26.0 Uo.o jgl 10.5 . L-lysine HCl 5,0 . U6.0 10.0 Mi 33.0 20.0 m U.5 Uo.o tm ■ 10.0 L-cystine 5.0 |^ 90.0 10.0 Hi . . 22.0 L-serine 5.0 ^ 110.0 10.0 m 66.0 20.0 49i 3U.0 «o,o lii il,,5 (Table continued on follovdj:^ page) 126 TABLE 37 (continued) ii2!u.nO ACXu V Ullt' 61 ill X^cl 1/ X AUUcCl Riboflavin* Ii*threonine 5.0 280 56.0 10.0 2U0 2U.0 20,0 5Uo 27,0 ■ - • ' . .- * ■ \ • ■ ■ •■■ ■ • ■ 580 IU.5 L-alanlne Uo.o 50 1.3 nr.— •nhftnvl a1 nni WA 20 U.O 10,0 Uo U.O 20,0 UO 2.0 UO.O 70 1.8 DIwi soTeuc ine 5.0 Uo 8,0 10,0 30 3.0 20,0 120 6,0 hO.O 120 3.0 L-valine 5.0 20 U.O 10.0 70 7.0 20,0 70 3.5 iiO.O no 2,8 Beta-alanine 5.0 120 ,. 2U.0 10,0 290 29.0 20.0 280 lU.O UO.O 300 .7*5 ♦ Riboflavin in excess of GAH controls in Table 36. from the data in Table 37 vhlch. showed the concentrations giving the highest yield of riboflavin per mg. of andno acid, as recorded in the last colimin of the table except that none uere used above 20 ng. per 100 ml. Certain derviations were made with some amino acids because of prior knowledge of their effect on riboflavin formation and other in- formation related to th«n. Glycine was added at a concentration of 10 Mg. per 100 ml., although the calculations in the above table indicate 127 that 5»0 Kg, per 100 ml. is more efficient, because it vo felt that this amino acid was truly involved in riboflavin formation. The concentration changes f rcan the apparently most efficient values of any of the other amino acids were for sonewhat similar reasons. The concentration of arginine HCl was increased to 20 m--, per 100 ml, because of the apparent iBxportaxice of the basic amino acida in the riboflavin synthesizing netab» olism. The concentration of hlstldine HCl and methionine for the base medium was 5.0 mg. per 100 ml, since, in eaperiments number 19 and 2h, respectively, both of these amino acids showed some effect on growth at these concentrations and it was believed that concentrations higher than these might have prox'ed detrimental to riboflavin formation. The coo- position of the amino acid base medium is shown in Table 38, * TABLE 38 urn AMINO ACIDS ADDED TO THE BASAL IM THE A^IINO ACID BASE MEDIUIi Amino Acid Concentration (ffig/lOO ml) Amino Acid Concentration (ng/100 ml) L-arginine HCl 20.0 I*-leucine 20.0 L-alanine 10.0 L-lysine HCl 10.0 Beta-alanine 10.0 I>-wethionlne ^.0 L-cystine 5.0 L-proline 5.0 Glycine 10.0 DL-phenylalanine 5.0 L-histidine HCl 5.0 L-serine 5.0 i-threonine 5,0 Dl.-tryptophane 2,5 L-tyrosine 5.0 128 Eight media were prepared hy adding different concentrations of the principal nitroren sources, I«-glutainic acid, L-aspartic acid or Mparagine to the amino acid base medium -without methionine, A peptone control medium was prepared by adding 200 mg, of peptone to 100 ml, of the basal. A solution of methionine, for addition to the media after sterilisation, was prepared and sterilized by 3eitz filtration. Dupli- cate flasks of media were sterilized, the required amount of methionine added to all flasks but the peptone controls and all flasks were inocu- lated witii a 28 hour vaslwd cell suspension. The inoculated flasks were Incubated on the shaker for 1^ days at 28° C, After incubation, the dry weight of cells and colorimetric riboflavin values were obtained. These results and the concentrations of the principal nitrocen sources are shown in Table 39. At the higher concentrations of the principal nitrogen sources, the riboflavin formed was lower in amount than the peptone meditaa in all except Medium number 3 which contained asparagine only as the principal nitrogen sovirce. When lower concentrations were used, the yield of ribo- flavin was approximately the same as that in the peptone medium, except again the asparagine medium, l edium number 6, which showed considerably more rilooflavin than the control, The d 27. The Effect of Idfferent Concentrations of Aspara^ine on Growth and iboflavin Formation in a Peptone Medium. Asparagine, the amide of «|Q>artic acid, may be associated with the biosynthesis of riboflavin since previous experiments have shown that the greatest stimulation of riboflavin formation occured in media containing this compound. In order to test this possibility further, different concentrations of as- paragine were added to a peptone medium composed of 0,2 g, of peptone per 100 ml, of the basal. Duplicate flasks of the sterilised media were inoculated with a 28 hour washed cell suspension and incubated on the shaker for h days at 28° C, The dry weight of cells and the colorLmetric riboflavin values w«rc determined. These values and the concentrations of asparj^ine added are shown in Table hi. 133 '•■ ^ TABLE I4I THE EFFECT OF BIFFIPJLTIT CONCENTBATIOKS OF ASPARAOniE ON GHOWTH AHD RIBOFLAVIN FOEMAnON IN A PEPTONE mmiM Asparagine Dry Weight Cells ^boflavin Added (mg/20 ml) (Wml) (ng/lOO ml) 1 2 Are 1 2 Ave. 0* 784 . 83.3 18.5 13,0 15,8 ' . . . * . 82.8 79.6 81,2 36.3 33.5 31.9 83,0 811.8 83.9 3i».6 30.0 32.3 10.0 80,2 72,6 ,. 27,6 22,0 2U.8 20,0 : 9I1.2 66,6 |).o 17.0 2?, 6 21.3 ItO.O ' 86.2 78.2 82.2 11,0 8.8 9.9 * Peptone control. The addition of only 2.5 mg, of asparagine per 100 ml, of th« peptone medium resulted in riboflavin yield -which was more than double that found in the peptone medium alone. These data further substantiate the relationship of asparagine to the fonaation of riboflavin in A, gossvEii. Asparagine may itself be related to the synthesis or it may be involved in the foMMition of some other compound which is involved in the synthesis process. The formation of riboflavin was inhibited when ItO mg, of aspara^^ine was added to 100 ml. of the peptone medium, which was probably the same sort of an effect which was noted when the glutamic acid medium was ad-ed to peptone. The addition of even high concentrations of asparagine to the peptone medium failed to cause any significant change in the growth of the organirai which was further evidence that asparagine must function very closely to the riboflavin synthesizing metabolisra of gossypii. In studying biosynthesis path- ways for riboflavin in media supporting growth and riboflavin formation, further research on addition of asparagine might yield useful infonnation. " IV, SUMI4fcRI AND COMCLUSItaJS Studies on the growth and riboflavin metabolism of Ashbya poasypii •were undertaken in an effort to gain more facts pertaining to the mech- anism of riboflavin synthesis in microorganisps anc* thereb; bring about a more complete understainding of tJhe pathway and intermediates partici- pating in the synthesis. During these studies the circular chromatographic technique vas introducee and its utility demonstrated in con;}unction vith the rapid, ^alitative chromatogi-aphic separation of unknown mixtures. The observed Rf values of amino acids and peptides were reported for the n-butanol- acetic acid-fwater and pyrldine-tertiary amyl alcohol-water solvents em- ploying the circular chromatographic technique. The use of this tech- nique in relation to further studies on the metabolism of A, goss:vT3ii is indicated. A colorimetric method for the analysis of riboflavin was developed in order to provide a more rapid assay of the results obtained in the different experiments. 'I^s method, although not as accurate as the fluo- rometric method, did give Indications that it could be refined further, for use as a quantitative method for riboflavin determinations, especially in culture media for the study of riboflavin synthesis, where there is only a small amount of interf erring substances present. A check of the method showed reasonable recoveries of added riboflavin in unknown samples could be obtained, further demonstrating that the method could be used 13^ 136 for approximate esti ations of riboflavin foination. The ribo lavin formation by A, gossypii in a yeast extract-peptone rnediim was shoxm to occur almost enti3?ely after the organism had reached maxiiffum growth. The formation of riboflavin' in the medium was also ac- companied by an increase in pH of the medium, the pH having reached a minimum value at approximately the age of maximum growth. During the initial phases of growth, the organism must apparently have to produce some special conditions and/or intermediates for the fonnation of ribo- flavin, since the morphology of the organism showed narked changes during this period. The con^jlex nitrogen source, peptone, was fractionated on a powdered cellulose column, using a n-butanol-acetic acid-water solvent, end the resulting fractions tested for growth and riboflavin fonnation with A, £oss^£ii. All, except one, of these fractions gave good growth of the organism; however, only one fraction gave good growth and allowed the formation of appreciable riboflavin. The chromatographic analysis of the hydroljzate of this riboflavin stimulating fraction indicated that there vas a predominance of basic amino acids present, as well as di- carboxylic amino acids. A study of the possible amino acids present in this fraction resulted in the development of a synthetic medium, con- taining glutamic acid and arginine as the nitrogen sources, which sup- ported good grovrth of A. gossypii but allowed no significant riboflavin formation. Since the inocula for these studies were prepared from cells grown on yeast extract-peptone stock agar slants, the possibility of substances being carried over in the inoculations was investigated by a seides of 137 studies involving the serial transfer of cells, on solid and in liquid, natural and synthetic media. It was apparent that sc»ne factor or factors vere being carried over from the yeast extract-peptone agar slant in the first transfer} hox^ever, in subsequent transfers the factor or factors seemed to have been diluted out to a point where there was little effect, Although this carry over effect cannot be overlooked, the effect of it should be rather constant under sny one particular set of experimental conditions and the results of a particular experiment can be discussed with respect to added known factors. Aqueous cell extracts of A, gossypii. obtained by grinding the cells in a small amoT:int of distilled water with ground glass and sand, were shown to stimulate riboflavin synthesis in a synthetic medium containing glycine. This stimulation effect was shown to be present principally in older cells, since the effect W8.s demonstrated with 132 hour cells but could not be demonstrated with 8U hour cells from the MM growth medium. 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His early education was obtained throut?;h the Public School System of Cin- cinnati, Ohio, He served as a commissioned officer with the United States Marine Corps during VJorld VJar II, He pursued his undergraduate studies at the Urlversity of Cincinnati, The University of Tampa and the University of Florida, receivia^ the Bachelor of Science Degree, with honors, frora the Unitersity of i^orida in August, 19U9» ) He continued graduate studies in Biochemistry at the University of Florida and received the Begree of I'laster of Science in February, 1951. He attended Purdue University from February, 19^1 to August, 19^2, doing graduate work in bacteriology, ile returned to the University of Florida in September, 1952, as an Assistant in Research at the iingineering and Industrial Fxperiirent Station, He became a teaching assistant in the Chemistry T)epartinent of the University of Florida in February, 195U, serving in that capacity until he wes awarded the Tennessee Eastman Kodak Research Fellowship in September, 1955. After his return to the University of Florida in 1952, he pursued graduate studies in biocheaistry leading to the Degree of Doctor of Philosophy in Chemistry, He is a member of Phi Kappa Phi, Phi Sigma Biological Society, Sigma ii, the Ajnerican Chemical Society and the Aaiericcji A«8ociation for the Advancement of Science. li»7 This dissertation vaa pr