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-<Arginine-Histidine Kediuin with

Different Concentrations of Amino Acids 113

2ii» A Study of Special Ajnino Acid I^edia 12k

26. The Effect of L-Serine on Riboflavin Synthesis in

Glutamic Acid-Arginine and Glutamic Acid-Aiginine-

Histidine Media • I30

27. Bie J-ffect of Different Concentrations of Asparagine

on Growth and Riboflavin Formation in a Peptone

tlediua 132

IV. SUMHART ANC CaiCLUSIOHS 13^

BIBLIOQRAPBy H^l

BIOGRAPHICAL ITaiS 11^7

CCflKITTEE REPORT 11^8

vl

IIST OF TABLES

table Page

X Ihe Fluorescence of Standard Riboflavin Solutions tt

i The Rf Values of Amino Acids and Peptides in Butanol-

Acetic AcidJ^ater and Pyridine-Tertiary Aniyl Alcohol- Water Solvents on Circular Chromatograms 33

3 "Qxe Per Cent Transmittance, pH and Riboflavin Content of

Cultures of A, gossypil. and Dry Weights of Their Extracted Ce5j.s, at "different Ages Grown in a Peptone Medium 37

lb- The Rf Values of Chromatocraphically Separated Fractions

of Peptone hX

$ Ihe Grovth and Pigmentation of Ashbya R03s:^T)ii on Media

Containing ?eptone Fractions as Kitarogen Sources ||2

4 ^e Rf Values of the Rings and Possible Amino Acids in

the Hydrolyzates of Peptone Fractions

t The Amino Acids ^ranpoaition of Synthetic Kedia ||7

8 2ie Effect of Amino Acid Combinations on Growth and

Pigmentation by Ashbya goss.,T'ii III

f The Effect of Added Amino Acids on the Growth and

Riboflavin Formation by Aghbya gossypii in a Histidine

Base Medium JH

11 The I'ffect of Added Amino Acids on Growth and Riboflavin Synthesis in a L-Glutamic Acid-Histidine HCl Base Medium 55

11 Ihe Effect of Added Amino Acids on Growth and Riboflavin

Synthesis in a Glutamic Acid-iiistidine-Arrrinine Base

I'ledium 5U

12 Ihe Ccanposition of Media for Studying the Effect of

Serial Transfer of Cells on Growth and Riboflavin

Foimation tift

vii

Table Page

II The Effect of Serial Transfer of Cells of Ashbya gosaypll on Growth and Riboflavin Formation on Peptone and Synthetic Kedia ff

lib '^^e Effect of Serial Transfer on the Growth and Pigmentation of £. gossypii on the Olutaadc Acid-Arginine Base Kedium xdLth Added Amino Acids 63

liS Groorth and ^ibofl?.vin Formation of A, goagypil on a

Peptone Kedim Inoculated vri.th Serial Transfer Cells * 6S

i$ She Growth and PiCTientation on the Glutainic Acid-Arginine- Glycine (GAC-ly) Kediuin Inociilated with Cells Washed a Different Nimber of Times %

If The Growth and Riboflavin Foinatlon on Media Containing

Untreated and Pemiutit Treated Peptone 0

in The Rf Values and Possible Amino Acids Present in a

Chromatosraphed, Hydroly^ed Pemutit Extracted Fraction

of Peptone 69

15> 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 <x-ketoglutaric acid and acetate via (x-aminoadipic acid,

The biosynthesis of histidine is not too clearly understood at the present time. Purine bases have been reported to be related to histidine biosynthesis in lactic acid bacteria (9), Fries (29) suggested that asparagine may function as a nitrogen donor in soiae step of histidine bio- synthesis since either histidine or asparagine vers reoolred by a mutant of the mold, Ophiostoma, In accumulation studies vith Neurospora. Haas, et al,, (38) have proposed a path of synthesis via imidazole glycerol phosphate, imidazole acetol phosphate, histidinol phosphate, h3.9tidinol and histidine. In yeasts grown with labeled acetate, the carboxyl group of histidine was reported to be derived from the methyl group of acetate (25), Ames and Mitchell (k) have suggested that the five carbon chain of histidine might ccane from pentose-5-phosphate. Using isotope distribution analysis. Wolf (ll5) showed that histidine could be degraded to glutaaic acid through some unknown path of reactions.

Tryptophan, phenylalanine and tyrosine are biosynthetically related throogh the common precursor, shikimic acid (16), Davis (l8) reported tiiat phenylalanine was synthesized from shikimic acid via prephenic acid and

If

phenylpyruvic acid, Prephenic acid has also been shown to be « prectirsor for tyrosine biosynthesis in a tyrosine requiring mutant of Neurospora (20), Srinivasan, et al,, (96), in experiments with labeled glucose, hare suggested that the side chains of phenylalanine and tyrosine may arise from a glycolysis product. In tryptophan biosynthesis, shikimic «cld was converted to anthranilic acid (102), Rafelson, et al., (71*) showed that acetate was added to anthranilic acid to give indole, Tatian and Shemin (lOl) reported that serine combined with indole to form tryp- tophan in the presence of the enzyme, tryptophan desmolase.

Serine and glycine are known to be related in their metabolisms but there is some question whether serine or glycine is formed first. Shrensvard, et al,, (2^) reported that the pathway, pyruvate, serine to glycine, probably existed in Tprulopsis utilis, since evidence indicated that the same isotopic distribution in alanine, serine and glycine occur- red when labeled acetate was used as the carbon source. Abelson (l) also reported the synthesis of glycine frcan serine in E, coli using labeled serine and glycine in the presence of glucose, Wang, et al., (ill) ob- tained evidence that serine was synthesized from glycine in baker's yeast vhen pyruvate-2-Cll+ was the sole carbon source. Low (I43) has presented evidence that the carboxyl group of glycine is incorporated into the pentoses of ribonucleic acids. Glycine has also been shown to be incorpo- rated into purine bases (98) •

""^^ cysteine are two sulfur containing amino acids which are important in metabolism. Abelson (l) reported that inorganic siOfur and serine combined to form cysteine which was further converted to cystine In E, coli. Pierpoint and Hughes (6l!) have pointed out that in

u

Lactobacillus arabinosus Coenzyme A formation from pantothenic acid would proceed only if cystine was present in the medivim.

Ehrensvard, et al., (25) proposed that threonine synthesis origin- ated with aspartic acid in Torulopsis utllls and E, coll since the isotope distribution with labeled acetate was the same in aspartic acid and threo- xilne. Woods, et al,, (ll6) have reported that threonine can spare aspartic acid reoiiirenents in certain lactic acid bacteria. The work of Abelson, et al,j (2) has shown that homoserine can serve as a precursor for threonine. Black and Wright (8) have suggested that ^-aspartyl phosphate and aspartic acid /^'-semialdehyde may be intermediates between aspartic acid and homo- serine in the pathway of threonine synthesis. The formation of homoserine froan ^-aspartyl phosphate has been shown to be inhibited by glutamic acid in Leuconostoc dextranicum (80).

The biosynthesis of valine has been shown to proceed from pyruvic •old via <»<-ketoisovaleric acid (l, 3)« Abelson (l) has suggested that leucine may be derived from pyruvic acid via c<-ketoisovaleric acid and <<-ketoisocaproic acid. He proposed that o(-4s:etoisovalerate may contribute carbons- 3 and 6 to combine with acetate in the formation of o(-ketoiso- caproic acid. Ehrenarard, et al., (25) and Cutinelli, et al,, (l5) have established that the o< -carbon atom and carboxyl group of leucine are derived directly from acetate in yeasts and bacteria. Abelson and Vogel (3) reported that isoleucine is formed from threonine via the intermediates" oC-ketobutyric acid and o<-keto-^-mcthylvaleric acid.

Methionine is a stilfur containing amino acid which is Important in transmethylation reactions. Homoserine and cysteine combine to form cysta- thionine which is converted to methionine (ItO, I|l). Cystine can spare but

not replace methionine as a growth factor (8U),

The relationships of aaiino acids are sunraarized in i'igure 1,

C, Chromatography Chromatography is an ii^ortant technique which is used in the •eparation and analysis of biological materials. The first application of chromatography is usually attributed to Tswett (103) who separated green leaf extract into different fractions by means of a column of pow- dered calcium carbonate. The introduction of paper-partition chromato- graphy by Consden, Gordon and Martin (Ub) in 19hh was one of the most important recent advances in chrcjmatography. Paper chromatography is probably one of the most widely used methods in the biological research of today,

33iere hare been oaoy variations of the original technique which resolred mixtures on paper strips or sheets by means of various solvents, Rutter (87, 88) introduced a method which he called circular chromato- graphy, which allowed a separation of a mixture by the movement of the solvent system through a small wick cut from the center of a filter paper disk, thus giving a series of rings making up the conponents of the mix- tare. The method gives the advantage of rapid separation of mixtures but the resolution is not very clear cut.

The solvent system is important for the proper separation of com- ponents In paper chroraatography. One of the first solvents was composed of water saturated phenol solution (Hi). Slotta and Primosigh (93) intro- duced the solvent system, n-butanol-acetic acid-water, vhlch is widely used for the separation of many groups of coo^wunds. The conponents of the solvent vere mixed in the ratio of Usli^, respectively, by volume and

•m BIOSIHTHETIC REUTIOMSHIPS OF MilKO ACIDS

prolixw

lysine

pyruvate

Citric Acid

exaloftcetate

(I) - — glutanate ■* o<-ketoglut«rat€

gtvtaRilae

Cycle

/^-alanine

A

xaethlonlne

■ aspartate — flicanoserine

(II)

lysine aqMuraiglnt

(I) ondthine >-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 <v> 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.

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Since the growths and riboflavin formations were about the same for the second and third transfers, it seems that the substances were re- duced in concentration by transfer.

On the agar slants, all of the amino acids, except valine, showed some stiimilation of riboflavin formation when they were added to the GA medium^ however, there were variations in degree of effect on riboflavin fonaation. Of the amino acids used, glycine, DL-alanine, h^tyrosine, SL-tryptophane, L-cystine, L-lycine HCl, L-proline and L>leueine 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<E

KediuE		Dry Weight Cells {wJ2Q ml)			Biboflavin (WmI)
	1	2	3	Ave.	1	2 3	Ave.
Untreated Peptone	78.0	79.8	92.0	83.2		67.5 110.8	750
Penmitit Treated Peptone	120,0	110 .li	118,2	116.2	li.8	5.1 5.3	5.1

now sgjproximately 25 per cent greater than that in the imtreated peptone nedixjic which indicated that Pennutit did not remove anything f roK the peptone vhich whs CEsential for the growth of the organism. In fact, the treatment of the peptone rasy have removed sometliing that was inhibitory to the growth of the organism.

lim affect on jciboflavin formation waa eren more pronoxmced than the effect on growth. The average yield of riboflavin was 5.1 ug. per ml, for the treated peptone medium and 75.3 ug, per ml, for the tmtreated peptone medium or fifteen times as much riboflavin formed ±n the imtreated over the treated media. When Permutit w?s treated with a 1 M solution of KOH, the absorbed substances from the extraction were freed into the so- lution. The eluate, firom the Permutit, used in the peptone trcatroent, showed the presence of ninhydrin positive substances when spotted on filter paper and sprayed with the ninhydrin developing reagent. 2Ms verified that something was removed frcan the peptone when treated with

Pernutit and the factors removed may be concerned in the synthesis of riboflavin. A circular chromatogram of the eluate from the Penmitit showed that only CHie wide ring was present and this heavy ring had an Ef value of 0,30 - 0.31 ifdiich corresponds with one of the Rf values of the components in the riboflavin stimulating combined fraction found in the colvann separation of peptone. .

A portion of the Pemutlt eliiate was hydrolyzed with 6 M HCl in a sealed glass tube by autoclavlng the tube at 15 pounds steaa pressure for 1 hour. A sample of the eluate and hydrolyzed eluate were chro- ittatogrsqphed by iiie two dimensional techniques on Whatman number 1 sheet filter paper with the n-butanol-acetic acid-water and pyridine-tertiary amyl alcohol-water solvents. The chromatogram of the unhydrolyzed eluate showed one large ninhydrin positive «pot which had Rf values of 8?)proximately 0.13 and 0,21, respectively, with the two solvents. The chromatogram of the bydrolywd eluate showed rdne distinct spots. Their Rf TBlues irere calculate and the possible amino acids identified by conpaidson of the values with those for pujre aid.no acids. The Rf values and corre^nding possible amino acids are given in Table 18,

To study this absorbed fraction further, a medium was prepared from the i-egular GA medium by adding 70 mg. of L-lysine HCl, 20 mg. of L-histidine HCl, IS mg, of glycine, UO mg. of DL-threonine, 30 mg. of L-proline, 10 mg, of L-leucine and 5 mg, of phenylalanine to 100 ml, of the medium. These amino acids were selected froa those in the above table because in previous experiments all of them gave some indication of being related to riboflavin synthesis. The medium was adjusted to pH 6.5 with a solution of KOH, sterilized and inocxilated with a U8 ho\ir washed

69

■y ■ TABLE 18

THE Rf VALUES AKD POSSIBLE AMIWO ACIDS PHESERT IN A CHECMATOGHAPHED, HYDHOLYZED PLMUTn EXTRACTED FRACTION OF PEPTONE

ouxanol oolvent		Composition
0,16	oa?	Lysine Arglnine
OA?	0,29	Hj^stidine .
0.20	0.39	Serine .
0.26	0.19	Aspartlc acid - , Glutamic acid
0,2U	0.27	glycine
0.30	0.38	threonine
0,35	0.12	proline
0,61	0.59	▼aline
0,72	0.68	leucine isoleucine phenylalanine

cell suspenaion. After incubation on the shaker for h days at 28® C,, the flasks were examined visually for groirUi and riboflavin forsmation. Ihia aedium gave excellent growth of the organism but did not show any appre- ciable riboflavin formation. These results indicate the strong effect due to interrelationships of amino acids since under other conditions these amino acids showed an effect on riboflavin synthesis^ however, when they were together in these concentrations there was no appreciable effect on the formation of the vitamin. It appears that further study is necessary

70

In order to detenaine an amino acid medium vhich will give good yields of ilboflavin.

11, The Growth and Riboflavin Synthesis on the Glutamic Acid- Arginine Kedium with Different Concentrations of Glycine, Since glycine was one of the «nino acids which stimulated the formation of riboflavin in the GA medim, a series of GA media were prepared to deterndne the effect of different concentrations of glycine on growtti and riboflavin formation when the glycine was added to the GA medium. The GA mdla were prepared in the same way as that in Baperiment number 7 except different amounts of glycine were also included in the media. The GA medium with- out added glycine served as a control for the experiment. The media were •t^ilized and duplicate flasks inoculated with a US hour washed ceU attspension of A, gossypii. After Incubation for h days on the shaker at 28® C,, the growth was determined turbidime trie ally and the riboflavin was determined fluorometrically as in previous eixperlments. The con- centrations of glycine in the media and the results of the experiment are reported in Table 19,

The addition of different concentrations of glycine had very little appai'ent effect on growth, as shown by the turbidity readings for the different fXadc8| even though there «u a considerable Increase in the total nitrogen In the media, as congjared with the GA control medium.

The addition of glyrine to the GA medium resulted in an increased foimation of riboflavin. The Increase In riboflavin was rather gradual and reached a maximum value of 15.2 ug. per ml, when 30,0 mg, of glycine was added to 100 ml, of the GA medium, "When UO.O mg, or 50.0 ii^, of glycine was added to the GA medium, there was no further increase in riboflavin

TABLE 19

THE OEOWTH im RIBOFUVIN SINTHESIS OF A. 00S5YPII IN GLOTAIHC ACID - ARGININE (OA) MEDIA WITH ABTSSD AMOUNTS OF GLTCIHE

Glycine Added to GA Kediian (ing/lOO ml)	Turbidity Transmit tance)		Riboflavin (ug/ml)
1	2	Ave.	1	2	Ave,
0	•	9.2	9.5	0.61	0.59	0,60
l.O	9.9	10.2	10.0	1.1	1,1	1.1
2.5	10.2	8.5	9.3
5,0	ii.2	1U.2	12,6	3.8	3.8	3.8
10.0	' 10.1	11.2	10.6	2.8	2.8	2.8
15.0		12.1	11.U	7#S	7.5	7.5
20.0	10.0	9.2	9.6	11.2	10.3	10,8
30.0	10.0	10.8	10.U	15.3	15.0	15.2
Uo,o	10,0	33.8	11.9	10.6	10.0	10,3
50.0	11.0	12.7	11.8	12.5	12.5	12.5

fomatlon i»hich wight indicate that a limiting condition had been reached and, as a resxilt, further increases of other substances might be necessary for further conversion of glycine to the intermediate in riboflavin syn- thesis.

These results indicate a relationship of glycine to riboflavin formation in the OA medium^ however, it appears that other substances wttst be necessary for ftirther riboflavin synthesis in this mediiim. Ihis re- lationship of glycine to riboflavin fonaation in A, goggypii vas in agrement with the results of Plant (6?) lAo showed that tagged glycine

72

vas incorporated into the rir^ portion of the riboflavin molecule.

12. She Effect of Different Concentrations of Peptone on Grovth and Riboflavin Formation in a Qlutamic Acid » Arginlne - Glycine Medium. Uie proceeding experiment shcfued that glycine stimilated riboflavin for- mation in the OA medium but the yield was considerably less -Uian that vhich could be obtained fSrom peptone at approximately the same nitrogen concentration. If peptone contained sme factor which vas responsible for the formation of riboflavin, the addition of peptone to the glutamic acid- airgininc-glycine (QAGly) medium should show an increased formation of riboflavin* To tMt this, the QAQly medium vas jan^9r9d by adding 20 mg. of glycine to 100 ml, of the regular GA medium nhich has been described previously. The peptone was added to the GAGly medium in amounts ranging from 0*001 mg. to 100 mg. per 100 ml, of the medium, duplicate flasks being set up for each amount of peptone added, as well as, a duplicate •et of flasks containing the QAGly mediun as a control, I!he madlA were sterilised in the usual nanmr and inoetilated vith a 1(8 hoar vuhed cell suspension of A, gossypii. The inoculated flasks were incubated on the shaker for k days at 28** After incubation, the flasks were rranoved and the resulting growth and riboflavin were r^asured using the dry weight of cells and fluorometric analysis, respectively. The concentrations of the peptone added and the results obtained are presented in Table 20.

The addition of increasing amounts of peptone to the QAGly medium resulted in a gradual increase in the growth of the orgajiian^ except for the two lovrer anasunts of adcted peptone where the growth ma erratic. The addition of 100 mg, of peptone to the GAGly medium resulted in the largest IncWNMMi la growth, aanotinting to approximately two times that of the GAGly

TABIE 20

■EHE GROWTH AITD iOBOFLAVIS SYNTHESIS IN A GLUTAMIC ACID - AKGININE - GLYCINE (QAGly) MEDIUM CONTAININO

VARIOUS cgnce:thations gf peptoke

Concentration (ngAOO ml)	Dry Weight Cells (mg/20 ml)	Riboflavin tug/ini;	\ ug/ing dry cexxs/
1	2	Are.	1	2	Ave.
	53 •©	53,8	53.7	lO.O		lo.O
0.001	81.2		81i.2	7.5		7.5	1.8
0.01	36.0	32.8	3hJi	6.0		8.9	5.2
0,10		M W X	53.2	10.8	***	10,8	U.1
1.00	63.0	7!i.6	68.8	16.0	'8.6	12.3	3.5
5.00 .	85.2	73.8	79,5	5.7	5.7	5.7	1.U
. 25.00	97.0		97.0	5.7		M	1.2
5b. 00	77.U	85.1*	8l.i»	5.7	2.8		1.0
100,00	97.8	lOij.l; 101,1	2.2	2.1*	2.3	0.5

♦ GAGly control.

*♦ Lost because of breakage.

♦*» Contaminated Flask.

control medixjm.

The addition of different concentrations of peptone to the QAGly control nedium did demonstrate a marked effect on riboflavin formation. The GAGly control showed the formation of 16,0 ug. of riboflavin per ml. iriiich Has the maxireum produced ia this eatperlment. The addition of peptone to the GAGly medium restilted, in general, in a decreased fomatitm for riboflavin, vith the lowest value of 2,3 ug. per ml. being obtained

7h , ; , •

in the medium to vhich 100 mg. of peptone had been added per 100 ml, of medium. The addition of the 100 mg, of peptone reduced the riboflavin fomed to about one-eighth the value of the control. There appears to be an inverse relationship between growth and riboflavin f onnation, since the increased growth was accong)anied by the decreased formation of ribo» flavin. This is especially evident as shown by the amount of riboflavin produced per mg. dry weight of cells which decreased as the amount of

■

peptone was increased. The addition of the peptone to the OAGly medium might have increased the growth metabolism of the organian, therefore, decreasing the availability of those intermediates necessary for ribo- flavin formation. The results of this erperl'nent serve to reiterate the importance of the interactions of nitrogen sources in the formation of riboflavin by A, gossypii. .

13. The Effect of the Components of the Glutamic Acid - Arginine - Glycine Medium on the Synthesis of Riboflavin in a Peptone Kediua. The unexpected inhibition of riboflavin formation by the addition of peptone to the GAGly medium raised the question as to which part of the ^thetic medium might be causing the effect, if this weire the reason for the effect, Thl» •jqaerlment was performed in an effort to obtain some infor- mation concemiiu^ the effects of the different cor.ponent8 of the QAGly medium on the riboflavin formation in a peptone medium,

A peptone medium was prepared by adding 100 mg, of peptone to 100 ml. of the basal, which was used for the preparation of other media. There were three media used for this eaqperiment and their composition was as follovrsj Medium number 1, the control, was the peptone medium prepared as just described, I^edlum number 2 wa« th« peptone medium to irtilch 0.21 g.

of glutamic acid and 0,01 g, of arginine HCl had been added per 100 ml, of B»ditoQ and Kedlttm number 3 vas the peptone medium supplemented with 0,02 g, of glycine per 100 ml, of medium, Duplicate f lades of the media vere sterilized and inoculated with a 36 hour washed cell suspension of £, gossypli. The inoculated media were incubated for $ days on the shaker at 28® C, !Ihe growth was exajnined \risually and the riboflavin detenained fluorometrically in the usual maimer, Bie results of the ex- periment are given in Table 21.

TABLE 21

m SYNTHESIS OF RIBOEL/IVIN IN A PEPTONE IffiDIUK CMiTAINING COKPCMTS OF THE OLUTAJIIC ACID • AHQIKIIiE - GUCINE (OAGly) MEDIUK

Amino Acids Added Riboflavin

to Peptorw KediuB (up/ml)

(g/100 ml) 1 2 Ave,

Cf 10,3 9.U 9.9

L-glutamic acid (0,2l) 0,8 0,8 0,8

Wrginine HCl (0,0l)

Glycine (0,02) 13.1 10.6 11,9

Since growth in all the media was very good, the possibility of ai^r inhibition effects due to the addition of components of the GAGly medium was eliminated. The riboflavin formation averaged 9,9 ug, per ml. in the peptone medium which was a reasonable value for the amount of nitrogen present in the medium. The addition of the glutamic acid- arginine components of the GAGly medium to the peptone medium caused the riboflavin formation to be reduced to an average of 0,8 ug, per ml,.

76

whereas, the addition of gljncine alone produced a slight increase in ribo- flavin formation ae compared to that foxsied in the p^tone medium. These results indicated that the gltitamle acid-ax^nlne portion of the OAGly aediura must have caused the inhibition of riboflavin formation by A, gossypii in the peptone medium and that this was probably the effect ob- served in Table 20 of the preceeding experiment. Van Lanen, et al,. (105) reported that dicarboxylic acids and their derivatives interferred with the synthesis of riboflavin by A, frpssypii. Under the condition of growth iriien peptone and glutamic acid arc both present in the same medim, glu- tamic acid loay be converted to 0<«ketoglutaric acid through deamination, and thereby giving a sufficient concentration of the dicarboxylic acid to •xert an inhibitory effect on the formation of riboflavin^

The Effect of a Cell Extract of A, gossypii on Growth and Riboflavin Formation. Since the experiments on serial transfer (Experi- ments 13, Hi and 1^) had indicated the possibility of a factor being carried over with the yeast extract-peptone agar slant cells, an etjt- traction of this type cell was made. The extract was prepared from pig- menting cells of A, gossypii which were ramoved with an inoculating loop trm several U8 to 72 hour yeast extract-jpeptone agar slants. The cells were placed in a mortar and ground in a small amount of distilled water with a mixture of ground glass and sand, in order to break up the cells. The ground mixture was centrifuged and the centrifugate, about 10 ml,, ifattoved as the cells extract which was stored in the fraeaing ccaspartaent of a refrigerator.

Two media were prepared with the following compositions i l-iedium number 1, daaignatod GA, contained 0,210 g, of glutamic acid and 0,010 g.

77

of arginine HCl per 100 ml, of the basal and Medium number 2, designated GAGly, contained 0,020 g. of glycine per 100 ml. of the GA Bedim* Before sterilization of the GA and GAGly media, 0.1 nl. of the A, gogaypil cell extract was added to each 20 ml, of the different media in Erleoneyer flasks. After sterilization, duplicate flasks of the media vere inocu- lated with a 36 hour washed cell suspension of A, p^ogsypii and incubated on the shaker for h days at 28® C,

At the coirpletion of the incubation period growth was observed and riboflavin determined fluorometrically. Growth was very good in all but one flask which showed no gxtnrth at all, this flask being one con- taining the OA medium plus the cell extract. The results of the fluoro- metric deteminations of riboflavin are shown in Table 22..

TABLE 22

THE SYNTHESIS OF RIBOFLAVIN BY A. GQSSIPn IN GLUTAMIC ACID - ABGBONE (GA) AND GLUTAMIC ACID""- ARGE-IINE - GLYCm (QAGly) MEDIA CONTAH'IING 0.1 MILLILITER OF CELL EXTRACT

Medium		Riboflavin (u^ml)
	1	2	Ave.
	2.7	No gixnrth	2.7
QAGly	21.8	26.9	2U.3

Ihe addition of the cell extreust to the GA medium resulted in a riboflavin yield which was only slightly greater than that lAich has been reported for the OA medium in oth^ ejiperiments. This increased yield may have been due to the small amount of riboflavin added with the 0,1 ml.

78

of the cell extract which <Jld contain a small aino\mt of the vltaain, The effect of the cell extract was rery evident in the QAGly medium vhere a riboflavin yield of 2h»3 ug, per ml, of medium vas reported. This was approximately double that which was normally produced in this medium vlthout the latMience of added extract and therefore indicated the presence of some factor In tte extract which stimulated the fomatiem of riboflavin. In observations of the progress of growth in this experiment, it was noted that « perceptible amount of pigmentation had formed in Medium number 2 after only 12 hours of iiwubation which was several hours sooner than previously observed. Since the cell extract was added to the medium before sterilization, the stimxilation factor must be some heat stable compomd or compounds, contained in the cell extract or resulting from the autocl«vl33g of the cell extract^ which accounts for the stlmulatioa of riboflavin formation in the presence of glycine, Autoclaving would also eliminate the probability "Uiat an enayme or enzyme system in the cell extract was stimulating the synthesis of the vitamin. The relationship of glycine to riboflavin synthesis was more firmly established as a result of these findings since the increased riboflavin formation was noted only in the GAGly medium,

15, The Effect of Chromatographic Fractions of an A. gossypii Cell Extract on Riboflavin Formation, The previous experiment had showx that the extract of A, gossypii cells stimulated the fonaation of ribo- flavin in the GA medium with added glycine. In an effort to detprmin if the whole extract or only part of the extract was responsible for the in- crMied riboflavin foination, the extract was chroma tographed on filter paper strips and fractions were tested.

n

The extract was chromatofrraphed on strips of Whatman number 1 filter paper vith the n-butanol-acetic acid-water solvent mixture as the resolving system. After 0.05 ml. of the cell extract had been applied to each of three filter paper strips and dried, the strips were placed in the solvent containers and chromatographed for approxijcately 6 hours. At this time, the strips were removed from the solvent and allowed to air dry. Two of the strips were sprayed with indicator reagents and the third was scanned with ultraviolet light. Strip number 1 was sprayed with a ninhydrin solution to test for the presence of ninhydrin positive comp- onents, such as, amino acids, peptides and some proteins. Strip number 2 was developed with a phenol red indicator solution which would locate •ome of the basic components since the indicator turns red at pH values ever 8,5. Strip nvimber 3 was examined under ultraviolet light to locate •say fluojrescent spots, especially riboflavin which gives a strong yellow fluorescence. Strip number 3 was also used in the preparation of special media for determining the effects of the substances (on riboflavin for- »ation) located at the different spots as shown by the developing agaits on strips 1, 2 and 3. llie results of the chromatographic examination of the strips, together with the fractions to be incorporated into the media, are shown in Table 23 ,

The OA and GAGly media were prepared fran the basal as described In the preceeding experiment. No cell extract was added to the GA medium or one flask of the GAGly medium; however, 0,05 ml. of the whole extract was added to one flask of the GAGly medium, thus giving three controls for the eaqperiment. Since only one chratiatographic strip was used in this experiment, only one fla«k was used for each fraction to be tested. The

80

THE CHROMATOGRAPHIC COMPOSITION OF Aii A. GOSSn'II CELL EXTRACT FROM A BUTANOL - ACETIC ACID - WATER SOLVEMT SEt^ARATION

Fraction Number Developing Agent Rf Values Color Fomed

		If	none
2	Ninhydrin	0.23	Light orange
3	Ninl^rdria	0,31	Purple
?	Ninhydrin	0.58	Pink
■ 6 ' ■ [' '	Ninhydrin	0.7$	Idght pink
3	Phenol Red	0.28
3	W light	0.30	Blue fluor.
	m light		lellofir fluor.
7^ (top) '

« Riboflavin.

•« Portion of strip from last ?pot to solvent front.

chromatographic fractions, numbered according to Table 23, were cut from Btrip number 3 and placed into s^arate flaaks of the GAGly medium. These media, together with the GA and GAGly controls, were sterilized and the sterile media inoculated with a hh hour old washed cell suspension of A. gossypii. After an incubation period of 108 hours on the shaker at 28° C., the fluorometric determination of the riboflavin was made. The results are shown in Table 2U.

Since filter paper had been lntro<^ced into the flasks with the chromatographic fractions, the dry weight of cells was not detennined.

81

TABLE 2h

THE EFFTCT OF CHRtMATOGlAPHIC CELL EXTRACT FRACTIOTS ON RIBOFLAVIN SYNTHESIS BY A. QOSSYPII IN A GLUTAllIC ACID • ARGININE"- GLYCINE (GAGly) I-iEDIUM

fu • WM Jib	P^n't T\nt i^m A ZUJlCJipcLL ricCLiuin	Added	Hiboflavin
1 ' 1	OA* QAGly*	V	0*6
1	GAGly plus 0.05 ral» extract		19.4
1	GAGly	t-- ,	16.9
f	QAGly
	GAGly	1
f	GAGly		M - ■
t	GAGly .
f	GAGly	6	2,8
	•OAOly	7

* Control media.

however, observations of growth indicated that it was excellent in all of the media. The routine daily examination of the progress of the experi- Kent shovred that pigmentation had appeared in tledia 3 and k after 36 hours of incubation. As would be expected, the riboflavin formed in the OA medium was rather snail, irtiile that fonned in the QAQly medium, although less than had previously been reported, was noticeably greater than the GA medium control. The stimulation of riboflavin foiiaation was again apparent irfien only 0.05 ml. of the whole cell «cti«ct was added to the

82

GAOly laedlum before sterilization. An examination of the results when the chromatographic fractions were added to the GAGly medium showed that Fraction number 1 stimulated riboflavin formation to approximately the earn eoctent as the whole cell extract. This was xuiexpected since it was felt that the stimulation would probably come from nlnhydrin positive portions of the cell extract. Since only 0,05 ml. of the cell extract was aqpplied to the origin of the paper strip, the amount of substances re- maining at the point of application after the finish of the chromatogram vast have b«en very small because the density of the ninhydrin color developed on the spots would indicate a rather heavy concentration of substances, especially at the Rf values of 0.31 and 0,58, Fractions numbered k and 6 gave some slight indication of being inhibitory to ribo- flavin formation in the OAGly medium. The remainder of the fractions seemed to show little effect on riboflavin synthesis, Fraction number 1 »ay have contained some of the larger molecular cell cooiponents, such as, proteins, nucleotides, etc., irtiich would show little, if any, migration in the n-butanol-acetic acid-*ater solvent system,

16, A Further Study of the i^ff ect of Cell Kxtracta of A, gossypii on Growth and Riboflavin Formation. The data presented in the two pro- ceeding ^cperiments, showed that the extract of A, gossypii cells from yeast extract-peptone agar slants stimulated the formation of riboflavin in the presence of glycine, W"hether this same stirnilatory effect could be shown with extracts from shaker cells grown on the yeast extract- peptone medixan, remained to be demonstrated. A large flask was set up as a feimenter in order to grow a large number of A, gossypii under aerated conditions. Three liters of medium were prepared by adding 1.5 g, of

83

DifcoHpeptone, 1.5 g* of Dif co-yeast extract and 6,0 g, of glucose to 3 liters of distilled tmter. The sterilized inedium was inoculated with a U8 ho\ir old cell suspension of A, gossypii and incubated at room temp- erature with continuous aeration by a stream of sterile diffused air.

After Qh hours of growth, a portion of the inedium was removed and the cells filtered off to give 18, it g, of wet cells. These cells were ground in a small cmtount of distilled water with sand and powdered glass to disrupt the cells and the resulting extract of the cells was obtained by centrifugation. This extract will be referred to as the 8U hour aqueous cell extract.

The remainder of toe nwdlum was removed after 132 hours and the cells ronoved by filtration to yield $8,8 g, of wet cells. A l5 g, portion of the cells wes dried with cold acetone to yield 2,8 g, of acetone dried cells and an acetone extract of the cells. The rranaining li3«8 g, of cells was extracted in the same manner as the Qk hour cells to give a 132 hour aqueous cell extract. The aqueous extracts of the different age cells were »ade in order to deteznine at which phase of growth the stimulatory factor or factors were most predominate. All ex- tracts were stored in the refrigerator when not in use, A 20 ml, portion of toe 132 hour aqueous cell extract was lyophilized to give 0,1? g, of dried solids or 8,$ mg, per ol, of extract. The OA and GAGly media for testing the extracts wei-e prepared from the basal according to previoue procedures. The otoer media for the experiment were prepared by the ad- dition of toe cell extracts and dried cells, to the GAGly i»dium. After the addition of the cell extracts and dried cells, the media were steril- ized, inociaated with a 2ii hour old washed cell suspension of A. gossyppi

and incubated on the shaker for U days at 28** C, The results of the dry might of cells and the fluorometric riboflfnrin d«termination8 are shown In table 2$, In addition, the riboflavin content of each of the added

extracts was determined and the average yields corrected for the ribo- flavin added initially, although much of the riboflavin would have been destroyed in au toe laving the media at a near neutral pH,

TABLE 25

THE aaCWTH AMD RIBOFLAVIN H33iMATIG9T IN A GLUTAMIC ACID - ABGDilHE . GLYCINE KEDHJl^ WITH ADDED CELL EXTRACTS AND ACETONE DRIED CKLLS

Medium Used Component Dry Weight Cells Riboflavin

Added/20 ml, (ag/io ml) (ug/ml)

		1	2	Ave.	1	2	Ave.
QA«*			91.6	69.0	1.1	3.7	2.U
OAGly**		50.U	72.8	61.6	10.7	16.2	13.5
OAGly	0.1 ml. of 8U hr. extract	66.1*	63.6	65.0	16.5	10.0	13.1 12.5*
GAGly	0.1 ml. of 132 hr extract	50.8		U7.8	38.1	101.3	ia.2 39.2*
OAGly	0.1 ml. acetone extract	hh,2	35.it	39.8	5.5	10.5	8.0 6.k*
QAGly	1.0 Dig. acetone dried cells	76.5	62.1	69.0	5.5	11,7	8.5

* Corrected averages for riboflavin present in the ext-ects, «♦ Control media

The results of this experiment verified the previous findings con- cerning the stimulation of riboflavin synthesis by aqueous extracts of A, gossypii cells and showed that cells grown on a liquid yeast extract-p^tone

85

medim couLd produce the riboflavin stimulating factor. The data shoved that the stlHulation factor vas present only in the 132 hour aqueous cell extract which may indicate that it was formed in the older cells after appreciable riboflavin had been produced, %is does not eliininate the possibility that it could be in the 8U hour aqueous cell extract but if there, the concentration imust be less than that which could be demon- strated under the conditionB ezqployed. The acetone cell extract and th« «c«tone dried cells i^parently did not contain the stimulation factor but rather appeared to contain factors which were inhibitory to the synthesis of riboflavin in the GAGly medium. Although the 132 hour aqueous cell extract increased the formation of riboflavin, it produced a decreased growth of the organism, again showing the inverse relstiooship between growth and riboflavin formation,

17. The Colorimetric Analysis of Riboflavin. Since the fluoro- metric jaethod required a considerable amount of time, it seemed desirable to make an effort to develop a colorimetric assay method to reduce the time necessary to make an analysis of the riboflavin produced in a set of experimental rcedia. On this basis some of the acciiraoy of the fluo- rometric determinations could be sacrificed in order to gain speed through the colorimetric detennination, A colorimetric method would be especially suited to these studies since the riboflavin produced by cells in liquid media v.-as ccanparatively free from interf erring substances. The use of a colorimetric method for the assay of riboflavin had been reported by Yaw but no details were given for the procedure. To test the colorimetric method a standard curve was prepared, different size aliquots of a ground cell extract were assayed and the recoveries of riboflavin added to

m

allqiiots vere checked, -

An Evelyn colorimeter, which vas fitted with a U20 millimicron filter, was standardized at 100 per cent tranaaidttance with a distilled water - dithionite solution which contained approximately 30 mg, soditnn dithionlte per 10 ml, of distilled water. An air blank was then re- corded and the instrument adjusted to this blank throughout the r«Eain» der of any test determinations. The riboflavin samples or standard so- lutions of 11 to 12 ml, volmne were added to calibrated colorimeter tubes end the tubes inserted into the colorimeter. The per cent transmission of the sample was read to the nearest 0,25 per cent transmlttance unit and recorded as the sample reading. After the senile was i^d, approxi- mately 30 mg, of graniilar sodium dithionite was added to the tube, the solution mixed thoroughly and the mixed solution allowed to stand for 30 to 60 geconds, While one tube was standing, another tube in the assay was read and treated in a similar manner, thus no time was lost in waiting for reduction of the riboflavin. After reduction, the dithionite treated sample was also read on the galvanometer scale to the nearest 0,25 per cent transmittance unit and recorded as the correction reading, Tlie sample and correction readings were then converted to absorbance values and the correction subtracted from the sample to give the cor- rected absorbance value produced by the riboflavin in the sample. The riboflavin in the sample was determined from a standard curve idilch re- lated the absorbance value to known concentrations of riboflavin. An riboflavin determinations that were acccmipllshed colorimf trically in •ucceeding experiments followed the above procedure. The data in Table 26 was obtained from a colorlmetric analysis of a series of standard

87

riboflavin solutions by the above procedure. jEhe data are plotted as a standard curve in Figure h»

TABLE 26

ABSORBANCE VALUES K)R KNOWN RIBOFLAVIN SOLUTICSIS

Riboflavin (ug/ml)	Per Cent Transjcaittance		Absorbance
San^le	Correction	S8it9}le	Correction	Corrected Value
0		200			0
1	58.75	87.00	0.2310	0.0605	0.171
It	3h.75	78.75	0,li59	O.lOh	0.355
	21.00	70.00	0.678	0.155	0.523
m	13.75	62.75	0.862	0.202	0.660
•$	9.25	55.75	1.03U	0.25b	0.780

An examination of Figure k shows a linear relationship between absorbance and riboflavin concentrations v$ to 15 ug. per ml., beyond uhlch the curve falls off slightly. However, the relationship's are suf- ficiently linear throughout the curve to allow for a reasonable estimate of the riboflavin concentration.

In order to test this method further, the riboflavin content of a cell extract, obtained by grinding cells of A, gossyrij^ in a small amount of distilled water with ground glass and sand, wag detemdned using different aliquots of the cell extract. The aliquots of the cell extract were tested to check the reproducibility of the method in the presence of increasing concentrations of interferring substances which

FIGUHS U

A STA1JHARD cum fm tTHF. COLOaiKETKIC mtrSIS OF illBOFUVIN

Riboflavin (ug/ml)

vere introduced by the addition of the alicpiots of the cell extract. Four unknown ssonples were prepared, by adding 0,1, 0,3> 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

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unit dzy weight of cells. The effect vas also shoim at higher concm* trations of histidlne tn the GAGly aediim, 5he data seen to Indicate that there nay be two conditions under which ilboflavin was synthesized, namely, synthesis after cells had reached roaximuju growth and synthesis by histidine inhibited cells which show only negligible growth, With the lower histidine concentrations, there appeared to be an increased growth accoinpanied by riboflavin synthesis. It has been shown previously that riboflavin synthesis under conditicms of rapid growth occured after cells had reached aaxiimaB grovth^ that is to eay^ in this experiment riboflavin was syrithesiaed by a relatively largB number of cells for a relatively short period of time with the lower histidine concentrations. As the concentration of histidine was increased, inhibition of growth occu3?ed and less riboflavin was fomed by the decreased nmber of cells that hs.d reached the maxisiuBi growth phase. At the higher concentration of histidine, growth was inhibited fujrtherj however, appreciable ribo- flavin was synthesized by a relatively small number of inhibited cells duxlng the entire incubation period. It has been noted that pigmentation appeared earlier in the media containing the higher concentrations of histidine. At the liistidine concentration of 25 ag, per 100 ml,, the growth was inhibited in the QAGly medium to the extent of one-twentieth of the control but the synthesis of the riboflavin was not retarded, as might be expected, Ihe high synthetic rate of the inhibited cells is shown by the ratio of riboflavin synthesized to the dry weight of cells.

The other test concerning the effect of histidine on growth and riboflavin formation was performed in the saiae way as the first test, using the sane GAGly medixm as ^ust described^ however, higher concentrations

of histidine were used to confirm the Inhibition Toy histidine and to observe any other effects of the higher concentrations. Control laedia of GA and GAGly without histidine were also included. In this teat, a 28 hour washed cell suspeiision was inoculated into duplicate flarics of the i!iedia and the inoculated flasks incubated for k days at 28° C, on the shcker* Ilie amounts of added histidine^ the dry weight of cells and the colorlmetric determinations of riboflavin are shown in Table 30 »

The results indicate an even stronger inhibition on growth by higher concentrations of L-histldine HCl, At concentrations of histidine greater tlian 30 rag. per 100 ml, of lacdim, there seems to be less total riboflavin synthesis but this may not be significant, A concentration of ho mg, per ml, seems to be about the ittaxiraun concentration necessary, since ^ mg, per 100 ml, showed only a small difference in effect on growth or riboflavin formation over the kO mg. concentration. However, there was a steady increase in the amount of riboflavin produced per 1^. dry weight of cells, indicating the same high rate, if not higher, of riboflavin biosynthesis in the small number of inhibited cells. Since inhibition of growth can be shown without stopping riboflavin synthesis, this effect may provide a means of separating the growth and riboflavin synthesizing metabolisms of A, gossypii,

20. She Effect of Initial yH of the Itedium on Growth and Riboflavin Formation in the Glutamic Acid«Ar^nlne Medium and the (XLutamic Acid» Arginlne.Glyclne«Hlstldine Medium. Ihe inhibition of the growth of A. gossypii by histidine in the glutamic acid-arginine (GA) and glutamic acid-arginine-glycine (GAGly) media without accorapanying inhibition of riboflavin synthesis, presented a very interesting problem since no

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report of this inhibitixm effect had been noted in the llterfttxu^. Since mny iiMbitlons Are a fonetion of the pH at idilch they occur, the effect

of different Initial pH values, on the histidiiie inhibition effect, to- gether with growth and riboflavin forraation, were studied. The effect of pH on growth and riboflavin was studied in the OA mediiaa; however, the histidine inhibition, as affected by pH, was studied only in the GAGly medium. The GA and GAGly media were prepared in the eatperiment as pre- viously described. The glutamic acid-arginine-glyclne-histldine (GAGlyH) ffledixsB, used in this experiment, was prepared by adding 25 ng, of L- histldine JK^ to 100 ml, of the GAGly medium. The media were adjusted to the different initial pH values with a solution of NaOH and duplicate flaske of each laedium were steiiliaed in the usual maxmer. After sterili- zation the media were inoculated with a 2h hour washed cell suspension and incubated for h days on the shaker at 28® C, After incubation, growth and colorimetric riboflavin determinations were made in the usual manner* These results and the initial pli values are presented in Table 31.

the growth of A, goggypii was moderately affected by different initial pH values in the GA medium^ however, the changes were jiot large. In the GA medium, the data show a slight increased growth from pH 6,0 to a maximum growth at pH 6»k wliich was followed by decreased growth to pH 6,8, The initial pH of the mediun exhibited a more pronounced effect on growth in the GAGlyH medium. At the initial pH of 6,0 in the GAGlyH medium, the inhibitory effect of histidine was completely eliminated and grotrth of A, p:os8ypii was considerably higher than even the maximum growth in the OA medium. This might indicate that at this -inHt-iaT pH the histidine was in a form vtdch decreased its active concentration and

101

TABLE 31

THE EFFECT OF BIITIAL pH OF MEDIA ON GiJOWTH AND RIBOFLAYITT FOKMATIOH IN THE GUJTAiaC ACID « ARGU^INE (OA) AND OLUTAI-IIC ACID - AFiJINITIE • GLYCINE - HISTIDIKE (GAGlyH) MEDIA

Medium	Initial pH	Dyy Weight Cells (inK/20 ml)	Riboflavin (Wal) (Wag cells)
		1	2	Ave.	1	2	Ave.	» Ave.
Qdk	6,0	^3,6	U6.U	50.0	1.0	0.5	0.8	0.3
m	6,2	52.0	U2.a	hi, 2	1#3	0.5	0,9	OA
	6.U	53.6	65.2	59.lt	1.3	0.8	1.0	O.li
m	6,6	50.2	li7.2	U8.8	2.0	1.3	1.7	0.7
OA	6»8	UO.0	l42.Ii	ia.2	3.5	3.5	3.5	1.7
GAGl^yH	6.0	78.0	80.0	79.0	6.3	7.5	6.9	1.7
GAGljfH	6.2	21.2	10.6	15,9	7.8	8.5	8.2	11.7
OAGl^	6.h	5.6	U.O	li.8	6.3	8.0	7.2	31.3
GAG2jfl	6.6	10.2	10.lt	10.3	16.7	15.0	15.9	30.9
GAGlyfi	6.8	8.ii	l4.8	6,6	11.8 12.0	U.9	39.1

therefore It acted to stimulate growth as was shown in B^eriment nmber 19. It could also indicate that glycine was more readily used in the jgPWth metabollan of the organism at this pH, When the initial pH was raised to 6.2, the inhibition effect once again was apparent and the growth was decreased to approximately one-fifth of that obtained at the initial pH 6.0. The inhibition increased further at pli 6.U but was slightly less at the initial pH values of 6.6 and 6,8. Ihe data would indicate a pH dependency for the marbrnm inhibitoty effect by histidine on the growth of A. gossypii.

102

Hm effect of the initial pH of the media on riboflavin fomation Wts erident in both series of media. In the GA aedixan, the nonaal values for riboflavin fonnation, in the range of 0,8 ug. to 2,0 ug. per jnl., were noted at initial pH values of 6,0 to 6,6, idaich included the normal initial pH range around pH 6,5 used in most of the previous eagseriments. At the initial pH of 6,8, the riboflavin fomed in the OA medium rose to 3.5» ug, per ml, which -wae a rather high r&biB for this medium without additional amino acids. It is possible that pH 6,8 was approaching the optimum pH for the synthesis of riboflavin anri even on the GA medium the metabolism may shi.ft somewhat to the sjTithesis of the vitamin. It should be pointed out, that the growth in the GA medium was least at the initial pH where the highest value of riboflavin was produced in GUL medium. Once again, notice is made of the formation of riboflavin at the eiqpense of the growth of the organism. The riboflavin which was f omed in the GAGlyH medium with the initial values of 6,0 through 6.1t, wat slightly below the normal values usually found in this medium; however, at the initial pH of 6,6, the riboflavin fomed was approximately twice the value of yields in the GAGlyH medium at lower initial pH values. The initial pH of 6,6 seems to be the optimum for riboflavin synthesi* in the GAGlyH medium which is in contrast to the pH 6,8 optimum for the OA medium, ,

21, Ttie Effect of Imidazole on Growth and Riboflavin Synthesis. Imidazole is a chemical compound which is structurally related to his- tidine in that it makes up the ring poition of the amino acid molecule. Because of this relationship, imidazole wes used to determine irfiether this portion of the histidine molecule contributed to any of the inhibition

103

effect produced histidlne. The GA «nd QAGly media were prepared as previously described and served as controls for the experiment. The effect of imidazole was tested in media, prepared by adding 20.0 mg« and 30,0 lag, of Imidaaole, respectively, to separate 100 ml. volumes of the GAOly medium. For a conparison with histidine, two other media were prepared in the same manner as the imidazole media, except L-histidine HCl was used in the place of imidazole. All media were adjusted to pH 6.5 with NaDH and duplicate flasks of each medium were sterilized in the usual maxiner. 2he sterile nedia w«re inoculated with a 28 hour washed cell suspension and Incubated for U days on the shaker at 28® C. The dry weight of cells and the coloriraetric riboflavin values were determined* !Qiese values are shown in Table 32,

.The controls were run in addition to the imidazole media in order to give a better con5)arison of some of the factors involved. Ihe effect of ijnidazole was different than expected, for the addition of either con- centration used, to the QAGly medium produced an increased growth irtiich was approximately 20 per cait greater than the growth on the GA or the GAOly control media. It Is evident that imidazole does not demonstrate any of the inhibition effect shown by histidine on A, gossypii.

microscopic examination of the cells in the imidazole media showed that a large percentage of the cells were the "btab forms" re- ported by Pridham and Raper (72) who showed the presence of riboflavin crystals within these cells. The number of this type cell in the luddazole media would ^proximate the number of the same tvpe cells found in a 0.5 per cent peptone medium. In the other synthetic media, where good growth occurred, there was only a small percentage of these swollen cells

m

. TABLE 32

A CGMPARISOK OF THE EFFECT OF BilDAZOLE WTTIi mT OF HISTBmn?: Off QROWTH AHD RIBOFLAVITJ SBITHESIS BJ Tffi GLUTAMIC ACID - AEGEIINE - GLICDffi (OAGly) HEDIUK . : .

Medium	Compound Added	Diy Weight Cell* (mg/20 ml)		Riboflavin (WbI)
	(ae/lOO ml) \tm^M JmV\J tiU*/	1	2	Ave.
	•	77.2	8U.8	81,0		2.5	2.6
GAOly*	' ■'»-■ ^	93.6	77.8	85.7	12.1}	16.7	li4.3
GAGljr	L-histidlne HCl (20.0)	^9.2	79.8	69.5	13.8	15.5	lii.6
GAGly	L-histidine KCl (30.0)	8.8	7.6	8.2		11.8	13.2
GAGly	Imidazole (20.0)	110.0	96.0	103.0	13.8	18.0	15.9
GAGly	Imidazole (30,0)	m.o	102.0	108,0	20,8	21.0	20,9

* Control media.

present, with the lower riboflavin yielding media having the fewer nimber of the cells. Iliere w«re a very few of these riboflavin producing cells in the histidine inhibited mediaj however, those present wer« 5 to 10 times larger than the nonnal "bulb form" cells, which may accotmt, to some extent, for the formaticai of reasonably large amounts of riboflavin wder the inhibition conditions.

There was increased riboflavin formation in the imidazole media as compared to the other media but this was probably due to the increased number of cells present. As a result of these data, it is apparent that wore than the imidazole portion of the histidine molecule Is necessaiy

105

in the inhibition effect of histidine on the growth of A, goesypii,

22, The Effect of Different Concentratione of Glutainic Acid or Aspartic Acid on Growth, Riboflavin Fprroation and Hjstidine Inhibition in Synthetic Media. Some of the earlier experiments using the pqptone medium indicated that riboflavin was formed principally after the growth of the organism had reached the naximim growth phase. Among other thiiogs, this could indicate that riboflavin was not formed until the nitrogenous nutrients iiad been nearly metabolized, thereby producing a nitrt^en deficient condition in the Biedium, Host of the synthetic media twed in these studies contained an appreciable amount of the main nitro- gen source, namely glutamic acid or aspartic acid, vtolch might prevent a physioli^cally suitable condition for riboflavin syn'Uiesis after maximum growth. This experiment was performed to deteitaine if lower concentrations of the main nitrogen sotirce would have any effecto on the metabolism of A, goeff>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<m of cystine was increased the amount of riboflavin formed was shown to decrease, acccwipanied by an increase in growth of the organism. At the higher concentrations of cystine, the conditions in the medium, with respect to riboflavin synthesis, became s.-'jnilar to those in the GAH control which indicates again the possible dual function of histidine as discussed in the case of methionine.

Large effects on riboflavin fomation were dosaonstrated by the addition of the amino acids glycine, L-leucine, L-serine or L-threonine to the GAii medium. Glycine, leucine or threonine showed a gradual in- creased synthesis of riboflavin with increasing amounts of the amino aclda to the GAH medium. Serine showed increased riboflavin formation at concentrations up to and including 10.0 mg, per 100 ml,; however, at higher concentrations of the amino acid no further stiiaulation or ribo- flavin synthesis was shown. Of all the amino acids leucine showed the largest amount of riboflavin formed when it vas added at a concentration

of ltO«0 Bg, per 100 ml, of the QAil medlun. '

I

The interrelationships of these araino acids vith respect to ribo- flavin formation anchor growth offer a considerable field for speculation. The effect of cystine and methionine on the histidine inhibition has al- ready been discussed} however, it is worth noting in Figure 1, page 18, that these two amino acids can be related to each other in the amino acid metabolic scheme. The fact that cystine was less effective than raethio^ nine in reliexdng the histidine inhibition may indicate only a partial converaioa of cystine to whatever is necessary to completely relieve "Wie Mstidine inhibition. The Initial addition of $^0 mg, of cystine per 100 ml, may give rise to increased riboflavin because of the possible formation of serine which has been shown to stimulate riboflavin formation^ however, the rate of the fonnation of serine may be a limiting step which would allow only a certain conversion of cystine to serine. Therefore, ■when increased amounts of cystine are added the large excess present forces the f omation of the inhibition relieving substance which in turn inhibits the formation of any riboflavin. This seems to be verified by ttie fact that Incremed concentrations of cystine produced an increased inhibition of riboflavin synthesis even though appreciable growth had occured. On the other hand, methionine probably went directly into the system which relieved the histidine inhibition and as a result ribo- flavin formation v&a inhibited as a result of the processes involving the growth of the organism.

The other amino acids, vhlch had a slicht relieving effect on the histidine inhibition, have no direct interrelationahips with one another or with methionine (see F±g[xre l). Threonine may hanre been able to show

122

its slight relief of iiMbition through methionine due to the relation- ship of the fonner to homo serine i^ch in turn may fom a eRiall amount of methionine.

As a rerult of the effects of the ajnino acids on the synthesis of riboflavin, some interesting facts were brought to light, TjTosine and tryptopliane, trhich are related in Figure 1, did shov a stimulating effect on riboflsvin formation, with tryptophane beii^ more readily metabolized than the tyrosine. The limited solubility of tyrosine may have been another factor vhLch limited its effectiveness toward ribo- flavin formation. ...

• Ihe stimulation of riboflavin si^-nthesis by 5.0 mg, of L-proline and the reversal, on the addition of 10.0 mg. of the amino acid was rather difficult to e3q)lain. However, in the case of cystine, if proline, at low concentrations, did metabolize to some precursor by a slow re- action, the presence of higher concentraticai of proline may force the reaction back to glutamate with the resultant fonnation of inhibitory metabolites of the dicarboxylic acid type reported by Van Lanen, which would effectively reduce the formation of riboflavin.

Lysine and Beta-alanine also showed a moderate stimulation of riboflavin formation which is of interest since both have been shown to be related to aspartic acid which, as has been pointed out previously, may be closely related to riboflavin synthesis. Another possible ex- planation might be based on the fact that lysine and Bcta-alanlne have a terminal amino group iriiich might be a factor in riboflavin synthesis. This appears to be the first time that Beta-alanine has been reported in connection with riboflavin syntheols.

353

liireonine shoved an appreciable effect on riboflavin foiwation and this may result tram its relationship to aspartic acid, via horao- serine. This was furi^her verified by the slight effect it had on re- versing the histidine inhibition since aom hoisoserine may be metabolized to inethicmine. The fact that isolettcine showed no stlunilation of ribo- flavin synthesis tends to eliminate the formation of isoleucine from threonine as a pathway of effect on riboflavin formation.

In the histidine inhibition medixun, the amino acids, serine and glycine, showed a stiriulatinc effect on riboflavin fomation which was rather surprising, since in previous experiments serine had not shown any effect on riboflavin synthesis in the GA medium. These data appear to substantiate the fact that serine and glycine are related In their metabo- liams. The effect of glycine or serine may indicate a closer relation- ship to riboflavin formation since the other amino acids which can be formed from gljclne and serine do not show any significant stimulation of riboflavin synthesis.

Leucine showed the greatest stimulation of riboflavin fonoation, with an effect vhLch seems to be separate from any of the other amino acids, since there were no relationships with riboflavin stimulating aaino acids noted. The effect of leucine on riboflavin formation may be ttore closely related to riboflavin synthesis than smy effects shown by the other riboflavin stimulating amino acids since it exerted the greatest effect.

An examination of the data seems to shovr that the amino acids, studied with respect to riboflavin synthesis, can be classified into three groups on the basis of their metabolic interrelationships and effect on

12li

riboflavin fonnatdon, namely, those related to aspartic acid, the glycine and serine group and the individual amino acid, leucine. It might be aesvaned that more than one amino acid may be involved in the formation of the larger aoounts of riboflavin,

2S« A Study of Special Amino Acid Media. The study of the effect of amino acids on riboflavin formation in the OAH medium offered infor- mation vhich might be used as a guide to develop a synthetic medium con- taining several amino acids. In previous esqjeriiaents, the atterapts to cranbine a nmber of amino acids usually led to a moderate formation of riboflavin or no appreciable yield at all, even though the growth was satisfactory. It was decided to test media containing aljnost all of the amino acids used in the proceeding ^eriment using concentrations genei^ly based on those that appeared to be most efficient for riboflavin formation. Since the actual stimulation of riboflavin formation caused by an amino acid is usually based on an increase over a control, the amounts of riboflavin produced above that in the GAH control medium were used in the calcTilations, From the yields of riboflavin above that in the GAH medium obtained by the addition of each concentration of amino acid to the GAfi medium, a yield of riboflavin per mg, of amino acid added was calculated, to determine at which concentration the added amino acid was BKJBt efficiently converted to riboflavin. The results of these calcu- lations are shown in Table 37,

^ asdno acid base medium was prepared from the basal by adding certain concentrations of the amino acids listed in Table 37 as well as concentrations of L-arginine HCl, L-histidine HCl and L-aetliionine, The concentrations of the amino acids to be added were chosen, in most cases.

12^

TABLE 37

THE RESULTS OF CALCUIATIONS TO DETEH-HNE THE HIBOHAVIH SYNTHESIZED PER KILLIQRAM OF AKEIO ACID ADDED TO THE GLUTAMIC ACID - AEQINIlffi - HISTIDINE }W)I\M

Concentration

Amino Acid Added Riboflavin*

(mg/lOO ml) Ug/lOO ml) tug/mg of saaino acid)

Glycine 5.0 ttl ' 28.0

10.0 W 19.0

20.0 lH 17.5

1|0.0 S50 13.1

L-prollne ■ , • . :':IM> , 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<?.ta appear to show that the amino acids closely related to the citric acid cycle caused a decreased syn- thesis of riboflavin, at the higher concentrations. At the lower concen- trations of these amino acids, the riboflavin could be synthesised more readily because there might be a lesser concentration of inhibitory products resulting from excesses of the principal nitrogen sources, since there was little differences In growth. Even though tlie principal

129

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130

nitrogen source vas reduced ty one-foTirth in three of the media, there was no corresponding growth decrease which had been noted in an earlier experiment when the nitrogen supply was decreased in a medium i^ch lacked amino acids. This was very stood evidence that many of the amino acids were being effectively used for the synthesis of cellular material.

The highest yield of riboflavin occured in Medium nvimber 6 where 0»05 g. of aaparagltte per 100 ml, of medium was the principal nitrogen source. This yield was more than three times that of the peptone control, even though they both contained approximately the same amount of total nitrof^en« In these studies, this was the first instance that riboflavin was produced in a synthetic medium to give a higher yield than the cor- responding peptone medium of approximately the same nitrogen content,

26, The Effect of L-serine on Riboqavin Synthesis in Glutamic Acid-Ar^inine and Glutamic Acid«Arginine-Histidine Media. Previous ex- periments had shown that serine had no effect on riboflavin formation in the GA medim whereas in the GAH medium it stimulated riboflavin syn- thesis. Because these results were unexpected, this experiment was per- fomed to verify the previous results. The regular GA and GAH media were prepared with 0,21 g, of glutamic acid per 100 ml, in the GA medium and kO mg, of histidine HCl per 100 ml, of the GA medium to give the GAH medixim. In addition, a second set of media were prepared in which

the glutamic acid concentration was reduced to 0,05 g, per ;j,pO ml, idiich

Mi- was the concentration in the histidine medium when serine stimulated

riboflavin formation. A concentration of 0,01 g. of arginine HCl per

100 ml, was used for all the media. Other media were prepared by adding

28,0 mg, of serine per 100 ml, of the OA and GAH media of both glutamic

acid concentrations. Glycine controls were also prepared by adding 20,0 ing, of glycine per 100 ml, of each OA and GAli media. Duplicate flasks of sterile media vere Inoculated vith a 28 hour vashed cell suspension and incubated for h days at 28*^ C, on the shaker, The dry weight of cells and the colorimetric detenninations of riboflavin that were made and the conucentrations of glutaaic acid are shown in Table UO,

table: lo

THE EJFECT OF SERINE ON RIBOFLAVIN FOSJIATION IN GLUTiOCC ACID - • AEGININE (GA) AIID GLUTAl^C ACID - ABGIIilNE - HISTIDINE (GAH) MiHIA WITH DIFFERENT CONCENrriATIONS 0? GLUTAMIC ACID

Glutamic Acid Bzy Weight Cells Riboflavin Medium Added (m^20 ml) (ug/ml)

	(g/100 ml)	1	2	Ave.	1	2	Ave.
GA	0,21	72.8	62.U	67.6	1.0	1.2	1.1
QAH	:.. 0.21	1,6	2.U	2.0	U.o	h.5	lt.3
GAGly	0.21	67.0	55.6	61.3	n.o	9,3	10,2
GAHGly	. • . : 0.21	1.6	2.1	2.0	11.8	10.0	10,9
GASerine	\. 0.21	50,2	h6.2	U8.2	1.2	1.2	1.2
GAHSerine	. 0.21	2.6	1.8	2.2	6,8	6.5	6,7
OA	0.05	12.9	12.2	12.6	0.5	0.5	0.5
QAH	0,05	l.U	1.0	1.2	li.3	U.O	U.2
GAGly	0.05	12,1	9.9	11.0	5,0	8.5	6.8
GAHGly	0.05	1,0	1.0	1.0	7.5	8.3	7.9
GASerine	0,05		5.0	8.2	0,5	0.8	0,7
OAHSerine	' 0.05	1.0	1.0	1,0	5.3	5.3	5.3

- 132

Uxe results of this expcrfjaent confirm the previous findings and did shov that serine stiimilated riboflavin formation in the GAH medium, whereas, it did not shofw this effect under gxwth conditions in the GA medium. The data showed that glycine was more effective than serine in stimulating jriboflavin formation in either media which may indicate that in the GAH medium with serine, the serine was converted to glycine rather than the reverse. In ihe rapidly grotdi^ aediura the concentration of glycine that may be formed from serine may never reach the value necessary for riboflavin formation but in the inhibition medium, a sufficient concentration of glycine may be reached since the glycine can not be used for growth and thus is available for riboflavin synthesis. These results farther establish the usefulness of the histidlne inhibited mediion for studies of riboflavin synthesis, and that the addition of serine to media xasy also be helpful* » >

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. The possibility of enzjTsies or eneyiae systems being introduced in the aqueous cell extract was eliminated by autoclaving of the media to iriiich the extracts had been added. The chromatographic separation on filter paper strips and the subsequent testing of the fractions found indicated that the factor or factors in the aqueous cell extract, which stimxdated riboflavin formation, did not migrate in the butanol-acetic acid.H<ater solvent system*

The addition of relatively large concentrations of glutamic acid or aaperagine to a peptone medium resulted, in the decreased fonaation of riboflavin and a corresponding increased growth. The results of manj' of the experiments sho»; this inverse relationship between growth and

138

riboflavin formation which probably indicates that conditions for good growth of fhe organism might not be conditions for good riboflarin syn- thesis.

Other nitrogen sources vere tested for growth and riboflavin formation with A, gossypii and it was found that NHj^N03, KNO3, (NH^)2S0^, aranonium acetate, ammonium citrate and urea would not serve as singl« nitrogen sources for either growth or riboflavin formation. The presence of relatively high concentrations of nitrate ions in a peptone medium was found to be inhibitory to the formation of riboflavin by A, goss.^mi* but apparently had no effect on the growth of the organism.

In synthetic media with glutamic acid, aspartic acid or aspara- gine as the principal nitrogen sources, the ac'dition of glycine resulted in an increaswl formation of riboflavin, especially in the presence of aspartic acid or aspara^ine. The addition of L-serine or DL-threonine, instead of glyc'.ne, to the same media resiilted in no increased riboflavin formation. Under the conditions used, it appeared that the synthesis of riboflavin was more closely associated with aspartic acid than glutamic acid and that glycine was associated in sosiie way with the formation of riboflavin.

The addition of L-liistidine HCl to a synthetic medium ccaitaining glycine had a varied effect on growth and riboflavin formation by A, ^ofTsypii, In low concentrations, 2,5 mg. to 10,0 mg, per 100 ml,, it had a stimulatory effect on growth which was accoirpanied by a slight decrease in formation of riboflavin; however, at higher concentrations, 2^,0 mg, to U0,0 rag, per 100 ml,, it inhibited the grow^h of the organism but did not inhibit the formation of riboflavin. By this inhibition effect.

339

the grovth metabolism of the organism aod the riboflavin ayiithesizing metabolism had been apparently separated* When imidazole, a compound making up part of the histidine molecule, was added, in the same approximate con- centrations as histidine, to the synthetic media without histidine, no in- hibition of the organism was noted,

A system of histidine inhibited cells of A^ gossypii was used to stuc^ the effects of 15 amino acids on riboflavin synthesis and growth, The amino acids, glycine, proline, tyrosine, alpha-alanine, beta-alanine, phenylalanine, isoleucine or valine had no effect on the growth of the organism in the presence of histidine; however, the amino acids leucine, tryptophane, lysine, serine and threonine did relieve the histidine in- hibition slightly, usually at the highest concentrations used. The amino acids cystine and methionine relieved the inhibition of histidine wiiich seemed to relate the inhibition by histidjjie to the metabolism of the sulfur containing amino acids. Ihe amino acids alpha-alanine, phenyl- alanine, Isoleucine, valine and methionine showed no effect on riboflavin synthesis in the histidine inhibited cell system, whereas proline and tyrosine showed a slight effect and cystine, lysine, tryptophane and beta-alanine showed a moderate effect, Ihe greatest effect on riboflavin formation was shown by the amino acids glycine, serine, threonine and leucine, with leucine showing the most effect of the four amino acids listed,

A synthetic medium containing 17 different amino acids was found to allow the production of considerable riboflavin and peimitted good growtli of A. gossr/pii. The riboflavin foiroed was over three times that foimed in a peptone medium of approximately the same percentage nitrogen.

Ho

This stu(fy of the metabolism of riboflavin in Ashbya gossypli has pointed out that the biosynthesis of riboflavin is related to many factors. It spears that the synthesis of riboflavin can be placed into three dif- ferent group functions as followst one, aspaartic acid and those compounds related to it, two, glycine and serine, and three, leucine.

Various conditions have been developed vhich perndt the study of the metabolism of K ^03s:rpii under different circumstances, namely, growth with little or no riboflavin formation, growth with different degrees of riboflavin formation and no growth with riboflavin formation. It is evident that further work, such as, studies of radio active tracers, short time chranatographic analyses, amino acid antagonists, the mechanisms of histidine inhibition, etc., must be done in order to more conqpletely understand the pathways of riboflavin synthesis in microorganisms.

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BICX>RAmCAL ITFKS

Ihe writer vas bom on July 12, 1921 in Cincinnati, Ohio. 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<q)ared imder the direction of the chair-

num of the candidate's supervisory camrittee and has been approved by all members of the coiranittee. It vas subanitted to the Dean of the College of Arts and Sciences and to the Graduate Council and vas approved as partial fulfillment of the requirements for the degree of Doctor of Philosqphy,

June ht 1956

Bean, Graduate School

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