THE ROLE OF BARBITUR‘C ACID IN THE NUTRITION 0F BACILLUS PO'PILLIAE Thesis for the Degree of Ph. D. MICHIGAN STATE umvsasm WILSON HUXLEY comm 1968 TH E518 0-169 LIBRARY Michigan State University This is to certify that the thesis entitled THE ROLE OF BARBITURIC ACID IN THE NUTRITION OF BACILLUS POPILLIAE presented by WILSON HUXLEY COULTER has been accepted towards fulfillment of the requirements for Ph.D. degree in Microbiology /‘7 / "\. q _ / l/ f‘ 1 A, \ sf". (137/ fig; 92/ (K 5 m7} 1..) ‘ / Major professor Date November 18. 1968 ABSTRACT THE ROLE OF BARBITURIC ACID IN THE NUTRITION OF BACILLUS POPILLIAE By Wilson Huxley Coulter Growth studies showed that a high concentration of barbituric acid (0.1%) was required for maximum growth of Bacillus popilliae in a synthetic medium, and that this requirement was not replaced by other pyrimidines. However, only a trace amount of that added (“2%) disappeared from the medium during growth. No detectable amount of barbitu- ric acid was degraded into urea and malonic acid, or oxidized by cells harvested from complex media. No effect of barbituric acid was observed on (a) oxidation of glucose or reduced nicotinamide adenine dinucleotide (NADH), (b) pro- duction of H O by cell extracts during NADH oxidation, or 2 2 (0) loss in viability of cells. A small amount of 1“C from 2-1uC—barbituric acid was consistently associated with both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA); and the amount associated with the RNA increased in a linear manner during incubation. A general distribution of the isotope among cell components was not observed. The isotope found in RNA was uniformly distributed throughout the As, 16s, and 23s RNA fractions, and control experiments indicated Wilson Huxley Coulter that at least 10% of this may occur by nonspecific adsorption. None of the lie from 2-lQC—barbituric acid was found associ- ated With mononucleotides follOWing hydrolysis of the RNA. The presence of barbituric acid resulted in very significant stimulation of both nucleic acid and protein synthesis, and it is believed that the stimulatory effect by barbituric acid may be due to a stabilization of the complexes involved in macromolecular synthesis. THE ROLE OF BARBITURIC ACID IN THE NUTRITION OF BACILLUS POPILLIAE By Wilson Huxley Coulter A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1968 DEDICATION This thesis is dedicated to my parents who wisely instilled in me the values of an education. ii ACKNOWLEDGMENTS I would like to express my deepest appreciation to Dr. R. N. Costilow for his generous and patient guidance during the period of this investigation and for his critical evaluation of this dissertation. I am particularly indebted to Dr. J. A. Boezi of the Department of Biochemistry for his friendly advice and technical guidance with some of the experiments presented in this thesis. The author also wishes to thank Dr. H. L. Sadoff for his helpful suggestions and discussions during the period of this investigation, and Dr. D. E. Schoenhard for his evaluation of this manuscript. In addition, the author is very grateful to the Northern Utilization Research and Development Division, U.S.D.A., Peoria, Illinois, for financial assistance. In conclusion, without the patient understanding and willing sacrifices on the part of my wife, Elaine, this investigation would have been impossible. iii TABLE OF CONTENTS Page DEDICATION. O O O O O O O O O O 0 0 O 0 ii ACKNOWLEDGMENTS . . . . . . . . . . . . . iii LIST OF TABLES . . . . . . . . . . . . . vi LIST OF FIGURES c o o o o o o o o o o o o Viii INTRODUCTION 0 O O O O O O O 1 REVIEW OF LITERATURE . . . . . . . . . 5 Bacillus ngilliae . . . . . . . . . . 5 Barbituric Acid . . . . . . . . . . . 9 EXPERIMENTAL METHODS . . . . . . . . . . . 13 Culture and Cultural Methods. . . . . . . . 13 Growth Studies . . . . . IA Oxidation and Breakdown of Pyrimidines by Resting Cells . . . . 15 Effect of Barbituric Acid on H O Production and on Reduced Nicotinamide AdenIng Dinucleotide (NADH) Oxidation . . . . . . 15. Effect of Barbituric Acid on Permeability and Viability . A' . l7 Incorporation of l C from .2-lQC—Barbituric Acid into Cell Components. . . . 19 Effect of Barbituric Acid on RNA and DNA Synthesis 30 Effect of Barbituric Acid on Protein Synthesis. . 30 RESULTS. 0 O O O O I O O O O O O O O C 31 Growth Studies . . . . . 31 Oxidation and Breakdown of Pyrimidines by Resting Cells . . . . 33 Effect of Barbituric Acid on .H2O2 Production and on NADH Oxidation. . . . . . A0 Effect of Barbituric Acid on Permeability and Viability . . A5 Incorporation of .luC from 2-1AC-Barbituric Acid into Cell Components. . . A8 Effect of Barbituric Acid on RNA and DNA Synthesis 73 Effect of Barbituric Acid on Protein Synthesis. . 78 DISCUSSION. . . . . . . . . . . . . . . 85 iv Page SUMMARY :3 O O O O O I O O O O O O O O O 9 3 BIBLIOGRAPHY . . . . . . . . . . . . . . 95 Table 10. 11. 120 13. 1A. 15. LIST OF TABLES Effect of various pyrimidines on the growth of two strains of B. popilliae in a synthetic medium . . . Effect of glucose level on the growth of B. popilliae NRRL B-2309-P-A in a synthetic medium . . . . . . . . . . . Ability of strain NRRL B-2309—P—A to oxidize barbituric acid and uracil. . . . . Effect of barbituric acid on the ability of resting cells of strain NRRL B-2309-P-A to oxidize glucose . . . . . . . . . Effect of barbituric acid on the oxidative ability of B. popilliae NRRL B-2309M . Production of urea from barbituric acid Effect of barbituric acid on H402 production 2 Effect of barbituric acid on NADH oxidation. Effect of various diluents on RNA excretion and cell viability. . . . . . . . . Distribution of 1”C from 2-luC—barbituric acid among the cell fractions . . . . . . Uptake of 1”C from 2-luC-barbituric acid into the TCA-soluble fraction of cells harvested from TYG cultures. . . . . . . . Alteration of barbituric acid in uninoculated phosphate buffer and S- 5 medium . Effect of ctinomycin D on the uptake of luC from 2-1 C-barbituric acid into RNA. Uptake of 32F and 1”c from 2-luC-barbituric acid into growing cells. . . . Calculated uatoms of 1“C and 32P per mg of RNA and DNA. . . . . . . . . . vi Page 32 3A 36 38 39 Al A2 AA A6 53 57 58 6O 62- 63 Table l6. l7. I8. 19. 20. 21. Microatoms of 32F and 1A0 estimated in various mononucleotides . . . . . . . . . Effect of pancreatic ribonuclease and alkaline hydrolysis on RNA isolated by phenol extraction . . . . . . . . . . . Adsorption of barbituric acid to RNA and DNA Effect of antibiotics on RNA and DNA synthesis. Effect of other pyrimidines and nucleic acid bases on nucleic acid synthesis . . . . Effect of other pyrimidines and nucleic acid bases on protein synthesis. . . . vii Page 65 69 71 76 79 83 Figure 1. 10. LIST OF FIGURES Growth pattern of strain NRRL B-2309M in TYG and 8—5 media. . . . . . . . Effect of barbituric acid on the glutamic acid and proline pools of strain NRRL B-2309M harvested from 20 hr TYG cultures . . Initial experiment on the uptake of label from 2-1AC-barbituric acid into cells of strain NRRL B- 2309M harvested from 2A hr TYG cultures . . . . . . . The effect of chloramphenicol on the uptake of 1“C from 2-1MC-barbituric acid by cells harvested from 2A hr TYG cultures Incorporation of lLAC from 2-luC-barbituric acid into RNA fractions separated by centrifugation through sucrose gradient Electrophoretic separation of 32P- and l”C-labelled RNA mononucleotides Effect of barbituric acid on RNA and DNA synthesis by cells of strain NRRL B-2309M harvested from 11 hr TYG cultures . Effect of age of cells on the stimulation of RNA and DNA synthesis by barbituric acid. Effect of barbituric acid on protein synthesis Effect of culture age at time of harvest on the stimulation of protein synthesis by barbituric acid . . . . viii Page 35 A7 A9 51 67 72 7A 77 80 82 INTRODUCTION Barbituric acid is required by Bacillus popilliae for growth in a synthetic medium and this requirement is not replaced by the common pyrimidines or purines found in nucleic acids (82). However, it is not required for growth in semisynthetic medium containing "vitamin-free" casein hydrolysate plus a variety of vitamins, or in complex media containing yeast extract (18,82). One would not expect barbituric acid to be present in the latter two media. Thus, the actual role of this compound in the nutrition of this organism is obscure. There are a number of possibilities for the nutrition- al response of B. pOpilliae to barbituric acid viz., (a) utilization by the cell as an energy source, (b) an effect on energy generation either by serving as an alternate electron acceptor or by interfering with the transfer of electrons from flavin to oxygen resulting in reduced H202 production, (0) alteration of cell permeability, (d) utili- zation by the cell as a required constituent or precursor of a cell component(s), and (e) regulation of metabolic activity. It does not appear likely that B. popilliae utilizes this compound as a primary energy source since this organism requires sugar for growth and oxidizes glucose rapidly (63,82). However, some microorganisms can grow in a medium 1 2 containing barbituric acid as the main source of carbon and nitrogen (3,86). Also, organisms which were adapted to growth on uracil or thymine could utilize barbituric acid as an oxidizable substrate (3,6,37,50,86). In all of the above cases, barbituric acid was found to be an intermediate in the oxidative catabolism of pyrimidines which these organisms were found to be capable of carrying out. However, it does not appear likely that barbituric acid serves as an energy source in B. popilliae by this pathway since neither barbituric acid nor the other normal pyrimidines were found to serve as energy sources for this organism in media in which barbituric acid was required for growth. Barbituric acid may have an effect on the energy generation by this organism. Previous findings have shown that H202 was produced by the soluble electron transport system in cells from late exponential and stationary phase cultures of B. popilliae (6A), and that these cells were quite sensitive to the lethal effects of 0.01M H202 (18). Since no evidence has been found for the presence of a H202 scavenging system in those cells which produced H202 (6A), the rapid loss in viability observed with this or- ganism may be due to the production of low intracellular concentrations of this compound (18). Hence, one possible role of barbituric acid in synthetic medium was the re— cuction of H202 accumulation. It was thought that bar- biturate might either serve as an electron acceptor from reduced flavins, or interfere with their reaction with molecular oxygen. Since B. popilliae cells loose viability rapidly under some conditions, it appeared possible that barbiturate might stabilize the permeability barrier of the cell; thereby, preventing the leakage of essential metabolites. Amobar- bital has been shown to inhibit the uptake of orotic acid into the pool of B. cereus (55). Also, the stimulation of antibiotic production by barbital with some species of Streptomyces may be due to its prevention of early autolysis of the mycelium; resulting in the extension of antibiotic production (25). A similar role by barbituric acid in the growth of B. popilliae appeared worthy of consideration. Although barbituric acid may be providing the cells of B. popilliae with an essential precursor of a cell con- stituent(s), this does not appear to be very likely in the case of pyrimidine biosynthesis since normal pyrimidines do not replace the barbituric acid requirement in a syn- thetic medium (82). Nevertheless, the possibility existed that derivatives of barbisuric acid may be formed which may serve as essential intermediates in the synthesis of some macromolecule(s) of the cell. In View of the previous findings (82) which suggested that barbituric acid was not utilized as a primary energy source or as a precursor to pyrimidine biosynthesis, the most probable role proposed for this pyrimidine in the nutrition of B. popilliae was in the regulation of metabolic A activity. Barbiturates have been found to stimulate the production of antibiotics by certain microorganisms (25,A6,56), and to result in the production of elevated levels of alanine racemase in Escherichia coli (31). Also, dihydroorotic dehydrogenase has been found to be inhibited by barbituric acid (27,83), and this may be the reason for the increased levels of unaffected pyrimidine biosynthetic enzymes observed on addition of this compound to cultures of Escherichia coli (5). The primary aim of this study is to explore the possibilities for the nutritional response of B. popilliae to barbituric acid. Hopefully, this investigation will result in a clarification of the requirement for barbituric acid for growth in synthetic medium. REVIEW OF LITERATURE Bacillus popilliae.--B. popilliae is a causative agent of "milky disease" of Japanese beetle (Popillia Japonica) (23). The progress of the disease has been described (A,76). Approximately 2 x 109 spores are produced per larvum result— ing in a characteristic milky opacity of the infected larvae. At this time death occurs. Since the bacterial spores are highly infective to beetle larvae, a great deal of interest has evolved in developing media which will support the growth and sporulation of the organism in XEEEQ to obtain high concentrations of spores for the effective biological control of the host. Laboratory media have been devised which support the vegetative growth of the organism in XEEEQ (18,23,39,77, 82). The highest yield of cells (A2 x 109/m1) has been obtained in a liquid medium containing 1.5% trypticase, 0.5% yeast extract, 0.2% glucose, and 0.6% K2HP04(18). Nevertheless, this organism grows poorly when compared to most other members of the same genus, is characterized by a rapid loss of viability after vegetative growth, and sporulates very poorly if at all on laboratory media (18,67, 68,77,78,79). Low concentrations of spores have been ob- tained in XEEEE only by the use of specialized techniques which entailed either the collection of cells from solid media into pastes followed by the subsequent transfer of 6 the pastes to solid sporulation media (78,79), or the addition of activated carbon to a liquid sporulation medium (38). Recently, up to 0.3% sporulation has been attained by spreading dilutions of appropriate strains of vegetative cultures on to acetate agar plates pro- viding there were fewer than 30 colonies per plate (67). Up to 20% sporulation has been observed in the surface colonies of a new varient culture on an agar medium consisting of casein hydrolysate, beef infusion, yeast extract, soluble starch, and trehalose (Sharpe, E.S., and G. St. Julian, Bacterial Proc., p.10, 1967). Due to the lack of success in obtaining adequate yields of spores on laboratory media, a number of studies have been conducted on the nutrition and physiology of this organism. Dutky (23) first described the organism as faculta- tively anaerobic but subsequent investigations have shown that oxygen is required for growth (63,68,77). Larval macerates or extracts supported vegetative growth but not sporulation on laboratory media. -0ptimal growth occurred in the pH range of 7.0 to 7.2 at 32 C. Glucose, mannose, galactose, maltose, and salicin were fermented by the organism (77). The acids produced were adequately neu— tralized providing a buffer consisting of 0.6% K2HP0u was included in the media (68). Consistent growth in synthetic medium was observed only if thiamine, biotin, 11 amino acids, 0.1% barbituric acid, 1.0% glucose, and 0.2% K2HPON were included (82). 7 Barbituric acid had no effect on growth when included in a vitamin rich semisynthetic medium consisting of 1.5% acid hydrolysate of casein, 1.0% glucose, 0.2% K2HP04, 0.01% DL-tryptophan, and the vitamins p-aminobenzoic acid, biotin, pantothenate, folic acid, myoinositol, niacin, pyridoxine, riboflavin, and thiamine; or when included in medium contain— ing 1.5% yeast extract, 0.2% glucose, and 0.6% K2HP0u. Although liquid media which supported vegetative growth, did not support the sporulation of the organism, spore—like bodies were obtained in a medium containing A% trypticase, 0.2% KZHPOQ, 0.1% barbituric acid, 0.02% MnSON-Hgo, 0.02% CaClg, 0.01% DL-tryptophan, and 1 ppm of thiamine-HCL under environmental conditions which resulted in an extension of vegetative cell viability (18). Compari- sons of these bodies with vegetative cells and spores produced in 1319 indicated that they were more similar to spores since they contained catalase; decreased levels of re— duced nicotinamide adenine dinucleotide (NADH) oxidase, pyrophosphatase, and of most of the catabolic and electron transport enzymes; elevated levels of ribosidase; and lower levels of protein, ribonucleic acid (RNA), and deoxyribo- nucleic acid (DNA). However, they were unlike spores since they lacked heat-resistance, were morphologically and cytologically dissimilar, contained higher concentrations of poly-B—hydroxybutyric acid and acid—soluble phosphate, and lacked dipicolinic acid (18,59). It is thought that 8 these spore-like bodies are the end result of abortive sporulation (59). Physiological studies have indicated that both the Embden—Meyerhof pathway and the hexosemonophosphate path- way are operative in this organism depending on the availa- bility of oxygen. The main products of glucose catabolism are lactic and acetic acids, and one strain of B. popilliae was found capable of oxidizing acetate via the tricarboxylic acid cycle (63). Cell extracts contained a nicotinamide adenine dinucleotide phosphate (NADP) dependent glucose —6- phosphate dehydrogenase, lacked reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and NADPH-nicotin- amide adenine dinucleotide (NAD) transfer enzyme, but contained both particulate and soluble NADH oxidase systems. The cytochrome dependent particulate system was prevalent in extracts prepared from younger cells. However, the soluble system was predominant in extracts of older cells and was characterized by H202 production, flavin dependence, and a lack of sensitivity to azide, cyanide, and carbon monoxide. No evidence was obtained for the presence of a H 0 2 2 scavenging system in B. popilliae cells although they produced H 02 (6A); and cells were found to be quite 2 sensitive to the lethal effects of 0.01M H202. The rapid loss in viability observed with this organism may be due to the production of low intracellular concentrations of H202 (18). The cytochrome system was deficient in cytochrome 9 c but was found to contain cytochrome b1 and possibly cyto- chromes a1 and a2 (6A). The physiology of sporogenesis of this organism as well as the role of barbituric acid in the vegetative growth on synthetic medium and in the development of spore- like bodies in sporulation medium remain to be elucidated. Barbituric acid.-—Barbiturates are known for their inhibition of electron transport from NADH to oxygen in the flavoprotein region (12), but their site of inhibition is still controversial (13,2A,A1,61). Studies conducted on the barbiturate inhibition of D-aspartate oxidoreductase indicated that inhibition was competitive with substrate and that a relatively large substituent group at the C-5 position was necessary for inhibition (11). Data obtained from assays of a large number of enzymes suggested that barbiturates may interact with enzyme-bound flavins. How- ever, in some cases barbiturates may interact with the protein moiety of some enzymes since some non-flavoenzymes were found to be inhibited (29). Although unsubstituted barbituric acid had no effect on the enzymes assayed, it was found to have a maximum effect on the light-induced oxida- tion of NADH by flavin or riboflavin, and on the photolytic degradation of flavin mononucleotide (FMN) suggesting a direct interaction of barbiturates with flavin (30). Barbitu— ric acid has been found to be an effective inhibitor of two flavoenzymes; viz., NADP-dependent dihydroorotic dehydrogenase 10 isolated from an aerobic bacterium (83), and the NAD—de- pendent enzyme isolated from the anaerobe, Zymobacterium oroticum (27). Only in the latter case was NADH oxidase activity associated with the enzyme which was inhibited by barbituric acid in the presence and absence of orotic acid. Barbiturates have been found to have other effects on a number of microorganisms. Barbital has been shown to result in a stimulation of antibiotic production by some species of Streptomyces. Rifomycin B was produced in higher concentrations without contamination by other com— plexes in the presence of 0.2% barbital by S. mediterranei (56). Streptomycin production was increased A.5 fold in the presence of 13.5 x 10'3M barbital (25). The role of barbital is not known but it may result in the stimulation of enzyme systems responsible for antibiotic production or may be responsible for the observed delay in autolysis of the mycelium. Barbital did not act as a precursor of the antibiotic (25,A6). Barbital at 0.02M concentration re- sulted in the production of 2.5 times the normal amount of alanine racemase due to increased pool levels of L- and D-alanine by partial inhibition of D-alanine oxidase in E3 391$, without having any effect on the growth of the orga- nism (31). Amobarbital has been shown to inhibit the up- take of orotic acid into nucleic acids of B. cereus by inhibiting its uptake into the pool (55). ll The major known role of unsubstituted barbituric acid in microbial metabolism is as an intermediate in the oxida— tive catabolism of pyrimidines. The organisms in which barbiturates have been found as intermediates in pyrimidine breakdown include Corynebacterium, Mycobacterium (6,36,37, 50), Nocardia (3,50), and a Bacterium species (86). The organisms were isolated by enrichment culture in the presence of the respective pyrimidine. Uracil and thymine were oxidized with the uptake of one atom of oxygen per mole of pyrimidine to barbituric acid and 5—methylbarbitu- ric acid respectively by the same adaptive enzyme (3,36, 37,51). An additional adaptive enzyme was necessary for the deamination of cytosine to uracil (37,87). Barbituric acid and 5-methylbarbituric acid were isolated and identi— fied as the products of purified uracil-thymine oxidase on uracil and thymine respectively (6,37,87). Barbituric acid was degraded anaerobically to urea and malonic acid (3,37, 51), but the corresponding enzyme had no effect on 5-methyl- barbituric acid (6,37). Work carried out by Batt and Woods (3) suggested that the enzymes responsible for the degrada- tion of barbituric acid and 5-methylbarbituric acid were induced only by the respective substrate. Biggs and Doumas (6) have clearly shown that methylmalonic acid and urea were the degradation products of 5—methylbarbituric acid. In many cases considerably less than the theoretical amounts of oxygen uptake and C02 evolution were observed 12 for the complete oxidation of uracil by resting cells (3,37,86). The possibility exists that this may have been due to oxidative assimilation or to the inability of the cells to oxidize malonic acid. However, malonic acid is known to be utilized by some bacteria as a carbon source (37), and bacteria grown on uracil have been shown to be capable of oxidizing malonic acid (86). Nevertheless, the strain of N. carollina-studied by Batt and Woods (3) utilized methylmalonic acid but not malonic acid as a carbon source. In addition, the uracil-grown organism was incapable of oxidizing malonic acid and this acid had no effect on the oxidation of uracil, or barbituric acid. The possibility exists that in some cases an active de- rivative of malonic acid may be formed immediately after the degradation of barbituric acid without the intermediate formation of free malonic acid. or interest, is the re— ported interconversion of the CoA derivatives of malonic and acetic acids with the decarboxylation of malonic acid by extracts of Pseudomonas fluorescens (35). EXPERIMENTAL METHODS Culture and cultural methods.——Cultures used in this study were obtained from the Northern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture, Peoria, Illinois. The experiments were conducted using B. popilliae strains NRRL B-2309-P-A (63) and NRRL B-2309M (Sharpe, E.S., and G. St. Julian, Bacterial. Proc., P.10, 1967). Cultures were maintained on agar slants as outlined by Sylvester and Costilow (82) and in a medium (TYG) con- sisting of 15 g of trypticase (BBL), 5_g of yeast extract, 2 g of glucose, and 6 g of K2HP04 (pH 7.2 to 7.A) per liter as described by Costilow gt al.(18). This medium was dispensed in 250 m1 volumes into 500 ml Erlenmeyer flasks. Cultures were incubated at 30 to 32 C on a rotary shaker and transfers made to fresh medium at intervals of 2A to 72 hr. Some of the experiments were conducted with cultures grown in a synthetic medium designated as 8-5 (82). The composition of the medium was (per liter): glucose, 10 g; K2HP0N, 2 g; barbituric acid, 1 g; biotin, 2 pg; thiamine. HCL, A00 ug; L-arginine, A00 mg; L-asparagine, 800 mg; L-cystine.HCL, 200 mg; glycine, 200 mg; L-histidine.HCL, 100 mg; L-isoleucine, 50 mg; L-leucine, 100 mg; DL-methio- nine, 200 mg; DL-phenylalanine, 200 mg; L—proline, 200 mg; DL-serine, 100 mg; DL-tryptophan, 100 mg; L—tyrosine, l3 1A 100 mg; DL-valine, 200 mg.. The initial pH was 7.2 to 7.A. The synthetic medium was prepared in the absence of glucose, biotin, and thiamine, dispensed in 50 ml volumes into 125 m1 Erlenmeyer flasks unless otherwise specified and autoclaved at 121 c for 15 min. A solution containing 25 g of glucose, 5 ug of biotin and 1 mg of thiamine per 100 ml was sterilized by filtration through an asbestos Seitz filter pad, and the appropriate amount added asep- tically to the cooled medium. A basal synthetic medium (BS) having the same composition of 3-5 but prepared with- out glucose and barbituric acid was used in a number of experiments. Growth studies.--Growth studies were carried out with both strains of B. popilliae to determine the requirement for barbituric acid in 8-5 medium and to see if this re- quirement could be met by other pyrimidines such as uracil, cytosine, thymine, orotic acid, and diethylbarbituric acid (barbital). The media were prepared as described above with the individual pyrimidines substituted for barbituric acid at a concentration of 0.1%. Inocula were prepared in various ways as described in the Results. Growth was measured by determining the optical density (0D) at 620 mu with a Beckman model DU spectrophotometer using the growth medium clarified by centrifugation as a blank. l5 Oxidation and breakdown of pyrimidines by resting ggll§.--The manometric determinations of 02 uptake and C02 evolution were carried out by the direct method as described by Umbreit, Burris, and Stauffer (85). Unless otherwise noted, all components of the reaction mixture were added to the main compartment of the Warburg flask except sub- strate which was tipped in from a side arm after thermal equilibrium was attained. In all cases the experiments were run at 30 C and the total volume per flask was 3 ml. The cells were grown in TYG medium, harvested in the cold, washed twice with cold 0.1M phosphate buffer (pH 7.2) and resuspended in the same medium unless otherwise noted. The production of urea from barbituric acid was es— timated manometrically by measuring the amount of 002 produced after the addition of urease from a side arm of the Warburg flasks. Effect of barbituric acid on H90; production and on reduced nicotinamide adenine dinucleotide (NADH) oxidation.—- Strain NRRL B—2309M was grown in TYG medium, washed twice with cold water, and cell extracts prepared with the Nossal cell disintegrator (McDonald Engineering Co., Cleveland, Ohio) using approximately 2 g wet weight of cells and 10 g of glass beads (2 mm average diameter) in a total volume of about 5 ml. The cells were disintegrated for a total of 2 min. After removal of the glass beads by low speed centrifugation, the soluble and particulate fractions 16 were obtained as described below. Some of the cells were fractionated by the use of a 100 watt Ultrasonic Disinte- grator (Measuring and Scientific Equipment Limited, London, England) for a total of 2 min. Over 90% cell breakage was indicated by observation with a phase-contrast microscope. The soluble and particulate fractions were obtained by centrifugation at 110,000 x g for 3 hr in the Beckman, model L, preparative ultracentrifuge (Spinco Division, Beckman Instruments, Palo Alto, California). The soluble fraction was decanted and the particulate fraction was resuspended in distilled water. The protein content of the fractions was estimated by the method of Lowry gt al.(5A). Standard Warburg techniques (85) were used to measure oxygen uptake. The main compartment of the Warburg flasks contained all the components of the reaction mixture with the exception of NADH which was added to one side arm. The other side arm contained 0.2 ml of AN H230“ and the gas phase was 100% 02. The total volume in the flasks was 3 ml. After temperature equilibration, the NADH was tipped in and the reaction run at 30 C for 1 hr after which the acid was tipped in to stop the reaction. The entire con- tents of the flasks were transferred to test tubes with the aid of 1 ml of water as a wash and the samples titrated for H202 production by the iodometric method as described by Herbert (A0). 17 Experiments were also conducted in cuvettes in which the oxidation of NADH was measured spectrophotometrically by following the decrease in 0D at 3A0 mu with a Beckman model DU spectrophotometer. Effect of barbituric acid on permeability and via- bility.-—Strain NRRL B—2309M was grown in TYG medium for 2A hr and resuspended in various diluents and the amount of RNA leakage determined by the orcinol method (58) as described by Demain, Burg, and Hendlin (20). The cells were harvested and approximately 100 mg wet weight of cells was resuspended in 30 ml of each of the following diluents: distilled water, 0.1M phosphate buffer (pH 7.3), and 0.1M phosphate buffer containing 0.1% barbituric acid (pH 7.3). After 60 min at room tempera- ture, 10 ml aliquots of each suspension were centrifuged at room temperature and the supernatant solutions evapora- ted to dryness. The residue was dissolved in 1 ml of distilled water and the amount of RNA determined as described above. The effect of barbituric acid on permeability was also estimated by measuring the pools of glutamic acid and proline. Cells were grown in TYG medium for 20 hr, har— vested, washed in ES medium containing 1% glucose and with and without 0.1% barbituric acid respectively, and then added in 1 ml amounts to 125 ml Erlenmeyer flasks containing 1A m1 of the same BS medium. U-luC-L-Proline 18 and U—luC—L—glutamic acid were added to separate flasks of each treatment to give a final activity of 2.04 x 105 and A.66 x 106 cpm/umole respectively. The proline flasks contained S—5 medium which contained 5 mg of L—proline per 100 ml instead of the usual 20 mg. Duplicate 0.1 ml ali— quots were removed at various time intervals, one placed into a prechilled tube containing 2 m1 of the respective media and the other into a prechilled tube containing 2 ml of 5% trichloroacetic acid (TCA). The TCA precipi— tate was filtered on to HA type millipore filters (Millipore Filter Corp., Bedford, Mass.) and washed twice with cold 5% TCA. The dried filters were glued to planchets (Planchets, Inc., Chelsea, Mich.) and the cpm determined by using a Nuclear-Chicao thin window gas-flow counter, Model 3037B (Nuclear Instrument and Chemical Corp., Chicago, Ill.) with the counts registered on'a Berkeley Decimal Scaler, Model 100 (Berkeley Scientific Corp., Richmond, Calif.) at a high voltage setting of 1250 volts. The effect of barbituric acid on cell viability was determined by resuspending cells in various diluents followed by the determination of viable counts on solid media by the method of St. Julian gt gl.(69). Strain NRRL B-2309M was grown in TYG medium for 2A hr, and the cells from 5 m1 aliquots harvested aseptically, washed once, and resuspended aseptically in 5 ml of one of the following diluents: TYG medium, S-5 medium, 0.1M phosphate 19 buffer (pH 7.3), 0.1M phosphate buffer containing 0.1% barbituric acid (pH 7.3), and distilled water. After standing at room temperature for 3 hr, the viable count of each 5 m1 aliquot was determined by plating out decimal dilutions of the suspensions in duplicate on TYG agar plates. The colonies on the plates were counted after incubation at 30 C for A days. Incorporation of 14C from 2-14C—barbituric acid into cell components.--An initial experiment was conducted with cells harvested from a 2A hr TYG culture to determine the distribution of the label from 2—1uC-barbituric acid in cell fractions. The cells were harvested, washed twice in cold distilled water and resuspended in the same. A 0.5 ml aliquot of the cell suspension was transferred to a 125 ml Erlenmeyer flask containing 10 ml of 8—5 medium to which had previously been added sufficient 2—luC-barbituric acid to give a total activity of approximately 2.78 x 105 cpm/umole. Aliquots of 2 ml were removed at 0, 1.5, and 3 hr; added to prechilled centrifuge tubes and centrifuged. At the same time intervals, 0.5 ml aliquots were removed and placed on previously weighed millipore filters followed by A washes with 2 ml of cold S—5 medium. The filters were then dried, their dry weights determined and the radioactiv- ity of the intact cells measured. The centrifuged cells were then extracted by the method of Schneider (73) as follows. The pellets were resuspended in 2 m1 of cold 5% 20 TCA, allowed to stand on ice for 15 min, and centrifuged. The supernatant solutions containing the cold TCA-soluble fractions were each decanted into a test tube with the aid of a glass rod. After an additional wash in cold 5% TCA, the pellets were resuspended in 2‘m1 of 5% TCA and placed in a boiling water bath for 30 min. The samples were then chilled on ice, centrifuged, and the hot TCA-soluble fractions containing the extracted nucleic acids were de- canted into test tubes as described above. The pellets were resuspended in 2 m1 of cold distilled water and quantitatively removed with the aid of an additional wash of cold water to previously weighed millipore filters. The filters were then dried, the dry weights of the hot TCA—precipitates determined, and the radioactivities de- termined. The 1“C content of the wall peptidoglycan fraction of the 3 hr sample was determined by the method of Park and Hancock (62). This procedure was as follows. The hot TCA pellet was washed with 0.01N NHuHC03, resuspended in a small volume of the same, and the volume adjusted to A ml after the pH had been adjusted to 8.0. After the addition of crystalline trypsin (A00 ug/ml), the sample was incuba- ted at 37 C overnight, washed with 1N NH3 followed by 3 washes with cold distilled water, and the trypsin-soluble material removed for the determination of radioactivity. 21 The pellet which contained the wall fraction was then quantitatively removed to a preweighed millipore filter, dried, and the radioactivity determined after the deter- mination of the dry weight. Experiments were also conducted using the extraction procedure based on that of Schmidt and Thannhauser (72) to determine the effect of chloramphenicol on the incorpor- ation of the isotope into cell components. Aliquots of 1.5 and 2.0 ml volumes were removed from the reaction mixture at 2, A, and 6 hr, and the samples treated to obtain the cold TCA-precipitates. The precipitates from the 1.5 m1 samples were resuspended in cold distilled water and quantitatively transferred with the aid of two 2 m1 aliquots of cold water as a wash to previously weighed millipore filters for the determination of 14C uptake from 2-luC- barbituric acid into the total nucleic acid fraction. The cold TCA-precipitates from the 2 m1 aliquots were resus- pended in 3 ml of 0.3M KOH and incubated at 37 C for 18 hr. The DNA and protein were then precipitated by the addition of equal volumes of cold TCA (10%). The pellets contain- ing DNA and protein were resuspended in cold 5% TCA and quantitatively transferred with the aid of two washes with 2 m1 of cold 5% TCA to preweighed millipore filters for the determination of 14C uptake from 2-luC-barbituric acid into the DNA plus protein fractions. The TCA was removed from the supernatant solutions containing the cold TCA-soluble fractions by extraction with ether before the determination 22 of the 140 content. An attempt was made to separate the RNA mononucleotides on a Dowex-l-formate column to be described later. Radioactivity determinations were made in the gas—flow counter. An attempt was made to determine the fate of the isotope quantitatively by use of liquid scintillation counting. The cells were harvested from 2A hr TYG cultures, washed twice in cold S-5 medium, and incubated for 6 hr in S-5 medium containing 2-lAC-barbituric acid before the re— moval of samples for fractionation. A determination of the 14C content of the medium was made before and after the in— cubation period in an attempt to measure the depletion of the isotope from the medium. After 6 hr the cells were washed 3 times with cold S—5 medium and resuspended in a total volume of the cold medium equal to that of the reac- tion mixture. To determine the amount of radioactivity in intact cells, a 1 ml aliquot was removed to a tube contain- ing 0.5 m1 of hydroxide of Hyamine 10 X (Packard Instrument Company, Inc., Downers Grove, Illinois), and aliquots re- moved for counting after incubation at 37 C for approximate- ly 3 hr. To determine the distribution of radioactivity in the cold TCA-soluble, ethanol-ether soluble, and TCA- insoluble fractions, a 1 m1 aliquot was removed from the washed suspension and added to a tube containing 2 ml of cold 5% TCA. After standing on ice for 15 min, the precipitate was centrifuged, washed once with 2 m1 of cold 5% TCA, and the supernatants combined for the determination 23 of radioactivity of the cold TCA-soluble fraction. The cold TCA-insoluble precipitate was extracted with ethanol- ether (1:1) for 15 min at A0 C, washed once with an equal volume of the same, and the supernatants combined for the determination of the radioactivity of the ethanol-ether soluble fraction. The precipitate was then dissolved in 0.5 ml of hydroxide of Hyamine as described above and the radioactivity of the acid insoluble precipitate determined. The remainder of the original washed suspension (30 ml) was extracted with cold TCA and the insoluble precipitate fractionated to obtain RNA mononucleotides and the DNA plus protein fraction by the method of Littlefield and Dunn (52) as follows. The acid insoluble pellet was re- suspended in 0.3M KOH, incubated at 37 C for approximately 2A hr, neutralized to pH 7.0 with perchloric acid and the volume determined after the removal of the potassium perchlorate precipitate by centrifugation. Aliquots were removed at this time to determine the radioactivity of the protein plus nucleic acid fraction. The solution was then brought to pH A.0 with acetic acid and absolute ethanol added to a final concentration of 67% (w/v). The precip- itated DNA and protein was removed by centrifugation and the radioactivity of the RNA solution determined. RNA mononucleotides were separated by paper electrophoresis as described later. In this particular experiment, the radio- activities of the fractions were determined by adding aliquots of the liquid fractions to polyethylene vials 2A (Packard Instruments Company, Inc., Downers Grove, Illinois) followed by the determinations of the radioactivities in a Tri-Carb Scintillation Spectrometer, model 3lA—DC (Packard Instruments Company, Inc., LaGrange, Illinois). Then a standard amount of l[AC-glutamic acid was added to each vial, the vials recounted, and the determinations corrected for efficiency, quenching, and background. Experiments were also carried out to determine the amount of label present per mg of RNA and DNA. In these cases the amounts of RNA and DNA present were estimated colorimetrically by the orcinol (58) and diphenylamine (9) reactions respectively. In some cases, the amounts of RNA and DNA synthesized were determined by measuring the amount of radioactive phosphorus (32F) uptake into these fractions. The samples were treated by the method of Schmidt and Thannhauser (72) including an ethanol-ether (1:1) extraction as previously described. Perchloric acid (PCA) was used instead of TCA to acidify the alkaline hydrolysate after alkaline digestion. The procedure used is outlined in Scheme 1. ‘ To determine thetamount of label from 2-18C—barbituric acid in highly purified RNA from cells grown in S-5 medium containing the radioactive pyrimidine, the RNA was extracted from the labeled cells according to the method described by Asano (2). The cells were extracted twice with 1% sodium dodecyl sulphate (SDS) in 0.01N sodium acetate 25 SCIHiMPI l Harvested cells Wash 3 X in A ml of cold medium and centrifuged Supernatant Treated with A ml 5% TCA at 0 C for 15 min and centrifuged. Washed l X with equal volumes of cold 5% TCA and supernatant solu- tions combined. \ Supernatant Residue Extracted TCA A X with ether. Added A ml ethanol ether (1:1) and incubated at Acid—soluble 50 C for 15 min and material. centrifuged. Washed l X with an equal volume of cold ethanol-ether and supernatant solu— tions combined. t \ Supernatant Res1due Lipid-soluble material. Resuspended in 6 ml 0.3M KOH and incubated at 37 C for 18 hr. (2 m1) (A ml) Added equal volume of cold Made 2N with PCA, incubated at 10% TCA, centrifuged, washed 0 C, centrifuged, washed 1 X l X with cold 5% TCA and with 2 ml cold 2N PCA and super- centrifuged. Supernatant ' natant solutions combined. solutions discarded. L v Residue ‘ Supernatant Extracted with A ml of 5% TCA at 90 C for 30 min, centri- fuged, washed 1 X with 2 ml cold 5% TCA and centrifuged. Supernatant solutions combined. Neutralized with KOH, incubated at 0 C, and KClOu centrifuged. Washed KClOu 1 X with A ml cold H20, supernatant solutions com- bined and RNA mononucleotides concentrated to 3 ml. RNA w - Residue Supernatant Extracted TCA A X with ether. DNA 26 (ph 5.0). In each case an equal volume of water-saturated phenol was added and the aqueous layer containing the RNA removed after stirring for 10 min in the cold. The a— queous fractions were then re—extracted twice with phenol, made to 0.3M with sodium acetate (pH 5.0) and the RNA pre- cipitated by addition of an equal volume of cold absolute ethanol. The precipitation was repeated one time. After resuspending the RNA in a small volume of 0.01M sodium acetate and making the solution 0.002M with respect to magnesium chloride, the contaminating DNA was removed by treating with 10 ug/ml of deoxyribonuclease (Worthington Biochemicals, Freehold, New Jersey, electrophoretically purified) at 25 C for 10 min. After making to 0.3M with sodium acetate, the RNA was obtained by two ethanol pre- cipitations as described above. The RNA was then re- dissolved in a small volume of 0.01M sodium acetate containing 0.05M sodium chloride (pH 5.2) and the supernatant solution obtained after centrifugation at 10,000 rev/min for 10 min. After determination of the amount and the spectrum of the RNA present as well as the radioactivity of the RNA, a suitable dilution of the RNA was fractionated on a sucrose gradient (5 to 20% w/v) prepared as described by Britten and Roberts (8). Cen- trifugation was carried out in a SW-39 rotor at 39,000 rev/min for 5.5 hr at 3 C in a Beckman, model L-2 ultra- centrifuge. The RNA fractions were then collected and the OD at 260 mu determined. To determine the radioactivity 27 of the RNA, the RNA was precipitated by the addition of 5 m1 of cold 10% TCA after the addition of 2 drops of salmon sperm DNA as a carrier, and chilled on ice for at least 30 min. The preciptates were collected on B-6 mem- brane filters (Carl Schleicher and Schuell Co., Keene, New Hampshire) and washed 2 X with 5 ml of 001d 5% TCA- The filters were then air—dried, placed in glass scintilla— tion vials and the radioactivity determined in a Packard Tri-Carb Liquid Spectrometer, Model 3003, after the addition of 5 m1 of scintillation fluid consisting of 15.1 g/gallon of BBOT [2,5-bis-2—(5—tert-butylbenzoxozolyl)—thiophene] (Nuclear-Chicago, Des Plaines, I11.) in toluene. For determination of the amount of label present in highly purified DNA from cells grown in S—5 medium con- taining 2—14C-barbituric acid, the DNA was extracted from the labeled cells by the method of Saito and Muira (71). The cells were extracted with a buffer solution containing 0.05M Tris-HCL, 0.5M EDTA, and 0.5% SDS (pH 8.0) in a round bottom flask which was rotated at room temperature for 15 min. An equal volume of water-saturated phenol was added, and the sample centrifuged at 10,000 rev/min after extraction for 15 min. After the removal of the aqueous layer containing the DNA with a large-bore Pasteur pipette, one—half volume of SDS—buffer solution was added to the phenol fraction and the sample extracted for 10 min. Following centrifugation, an equal volume of phenol was added to the combined aqueous fractions and the samples 28 re-extracted. The sample was centrifuged, made to 0.3M with sodium acetate (pH 8.0), the DNA wound on to a glass rod from the interface after the careful addition of a 2 volumes of absolute ethanol, and the DNA redissolved in a small volume of a solution of 0.1M Tris-HCL and mM EDTA (pH 8.0). The contaminating RNA was removed by treatment with 20 ug/ml of pancreatic ribonuclease at 30 C for 30 min. The DNA was then rewound on to a glass rod after the addition of a 2 X volume of absolute ethanol, redissolved in a small volume of a buffer solution con- taining 0.05M Tris—HCL and 0.5mM EDTA (pH 8.0), and made to 0.3M with sodium acetate. The DNA was rewound on to a glass rod after the addition of a 0.5A volume of isopropa- nol, redissolved in a small volume of buffer, and the quantity, spectrum, and radioactivity of the DNA determined as described in the previous paragraph. The remainder of the ethanolic solution from the first ethanol precipitation step was treated as described previously for the extraction of RNA, and the radioactivity of this fraction determined. Where indicated, attempts were made to separate the RNA mononucleotides by anion exchange through a Dowex-l- formate column as described by Cohn (15), and Cohn and Volkin (16), and by paper electrophoresis on Whatman 3MM paper at 32 volts/cm for 1 hr using 0.05M ammonium formate (pH 3.5) as described by Smith (75), and Markham and Smith (57). 29 Incorporation of isotope from 2-luC—barbituric acid into protein was checked by the method of Schneider (73). Samples were removed into test tubes containing 2 ml of 5% TCA, heated at 90 C for 30 min, and the precipitated protein collected on millipore filters with the aid of two washes of 2 m1 of cold 0.01N HCL. The radioactivity on the dried filters was determined in the gas-flow counter. In some experiments, the incorporation of isotope from 2-luC-barbituric acid into nucleic acid was studied by the use of the extraction procedure described by Hanawalt (32). Aliquots were centrifuged, resuspended in 2 m1 of cold 5% TCA on ice for 15 min to remove the cold TCA-soluble fraction, and then resuspended in A ml of ethanol-ether (1:1) for 12 min at 50 C to remove the lipid-soluble material. The sample was then resuspended in 2 ml of cold 5% TCA, collected on B—6 type filters and washed with two 2 ml volumes of cold 0.01N HCL. After the determination of radioactivity in the DNA plus RNA fraction, the filters were placed in polyethylene scintil- lation vials containing 3 ml of 2N KOH and incubated at 37 C for 18 hr. The samples were then acidified by the addition of an equal volume of cold 50% TCA (it was found that the addition of 10% TCA as suggested in the reference was insufficient to acidify the samples). The precipitate was then collected on millipore filters for the determina- tion of radioactivity in the DNA as described above. The 30 The radioactivity of the RNA was then determined by difference. Effect of barbituric acid on RNA and DNA synthesis.-— Cells of strain NRRL B-2309M were harvested from TYG cultures, washed once in cold BS medium with 1% glucose and with and without 0.1% barbituric acid respectively, and resuspended in a small volume of the same medium. Aliquots of these suspensions were then inoculated into flasks containing the same BS medium, and 32F as K2H32P04. At various time intervals, RNA and DNA synthesis was de- termined in filtrates of samples which were removed and extracted by the procedure described by Hanawalt (32) as outlined in the previous paragraph. The radioactivity of the filtrates was determined in a gas—flow counter. Effect of barbituric acid on protein synthesis.-- Cells of strain NRRL B-2309M were harvested from TYG cul- tures as described in the previous paragraph, and inoculated into flasks containing BS medium with 1% glucose and with and without 0.1% barbituric acid respectively, and a luc- 1abe11ed amino acid. Unless otherwise indicated, aliquots were removed into test tubes containing 2 m1 of cold 5% TCA, heated at 90 C for 30 min, chilled on ice, and the pre- cipitates collected quantitatively on millipore filters with the aid of two 2 m1 volumes of 0.01N HCL as a wash. The dried filters were then glued to planchets and the radio- activity of the precipitates determined in a gas-flow counter. RESULTS Growth studies.——The results from experiment 1 (Table 1) indicate that barbituric acid was required by both strains of B. popilliae tested for growth in S-5 medium and that the requirement for barbituric acid was not completely met by any of the other pyrimidines tested. Thymine appeared to partially replace the bar- biturate requirement by strain NRRL B-2309-P-A, but growth in the presence of 0.1% thymine with strain NRRL B—2309M was less than in the control lacking pyri— midine. With both strains, the growth obtained with the other pyrimidines was equal to or less than that ob- served in the control flask. The growth observed with strain NRRL B-2309M in the control medium may have been due to the small amount of barbituric acid added to the test flasks during inoculation, the long incubation time, better adaptation to growth in S-5 medium because of previous growth in this medium, or due to characteristics of the particular strain. Using a shorter incubation time (Experiment 2, Table 1), very little growth of this strain was noted in the absence of barbiturate. Also, it is evident from this experiment that a relatively high level of barbiturate is necessary for maximum growth response . 31 TABLE l.--Effect of various pyrimidines on the growth of two 32 strains of B. popilliae in a synthetic mediuma. Pyrimidine added OD62o NRRL B-2309-P—A NRRL B-2309M Experiment No. l: None 0.021 0.193 Barbituric acid 0.092 0.A67 Uracil 0.012 0.150 Cytosine 0.013 0.183 Thymine 0.0A8 0.088 Barbital 0.022 -- Orotic acid -- 0.172 Experiment No. 2: None -- 0.029 0.1% barbituric acid -- 0.320 0.05% barbituric acid —— 0.161 0.01% barbituric acid -— 0.068 aIn Experiment No. l, the NRRL B-2309-P-A inoculum was prepared by aseptically centrifuging 10 m1 aliquots from 2A hr TYG cultures followed by resuspension in 10 m1 of sterile saline.(0.85%). Two m1 of this suspension was added to flasks of BS medium containing 1% glucose and the pyrimidine to be tested at a concentration of 0.1% and the 0D at 620 mu determined after 72 hr incubation. Flasks containing the same BS media were inoculated with 2% inocula of strain NRRL B-2309M from 2A hr S-5 cultures and the 0D determined after 8A hr incubation. Experiment No. 2 was conducted by observ- ing the growth of a 3A hr culture of strain NRRL B-2309M after 3 consecutive transfers of 10% inocula every 2A hr in the S-5 medium containing the various levels of barbituric acid. 33 The results of experiments to determine the level of glucose required for maximum growth in the presence of 0.1% barbituric acid (Table 2) indicate that a maximum growth response was not observed with glucose levels lower than 1% in the synthetic medium. These results substantiate those presented by Sylvester and Costilow (82). The growth pattern of NRRL B—2309M in S—5 and TYG media (Fig. 1) indicate that the growth rate of this or- ganism is about 3 times greater in TYG medium (generation timecz'2.2 hr) than in S—5 medium (generation time¢56.0 hr). In both media, the cessation of the exponential rate of growth is evident at 12 to 15 hr. Previous work carried out in this laboratory suggested that the exponential rate of growth continued for about 2A hr. However, at that time the cultures were transferred every A8 to 72 hr, whereas for the present experiment the cultures had been trans— ferred every 2A hr for several days in the respective media before inoculation into the test flasks. Microsc6pic examination of the cultures during logarithmic growth indicated that the cells in the S—5 medium consisted of many long and pleomorphic strands whereas the cells in TYG cultures were typical short rods occurring singly and in pairs as shown by Mitruka SE §l°(59)- Oxidation and breakdown of pyrimidines by resting cells.--Strain NRRL B-2309-P-A was incapable of oxidizing barbituric acid as substrate (Table 3) whether the cells 3A TABLE 2.—-Effect of glucose level on the growth of B. popilliae NRRL B-2309-P-A in a synthetic mediuma. Glucose 0D added 620 1.0% 0.092 0.5% 0.070 0.1% 0.03A 0.05% 0.031 aBS medium containing 0.1% barbituric acid and various levels of glucose were inoculated with 2 ml aliquots of strain NRRL B—2309-P—A as described under Table l. The OD of the cultures was determined after 72 hr incubation. ' 35 1‘3 0.8E Inodhun 004 '- 0.2 - - 5-5 ‘ 0.09 : . medium 11£E7r o 0.05 - 0.03 F °°°'o ‘ IO 20 - , so HOURS Figure l.—-Growth pattern of strain NRRL B-2309M in TYG and S-5 media. The media were prepared and dispensed in 125 ml Erlenmeyer flasks at 50 ml per flask. The TYG and S—5 media were inoculated with 5 and 10 ml aliquots from respective 2A hr cultures in the same media. The flasks were incubated on a rotary shaker at 30 C, and l TYG culture and 3 S-5 cultures removed at the designated time intervals for the determination of growth as described in the Experimental Methods. 36 TABLE 3.--Ability of strain NRRL B-2309-P-A to oxidize barbituric acid and uracila. Experiment Experiment No. 2 Substrates cell§e(§:ys) N3. 1 Q Q HQ 02 02 CO2 Endogenous 1 2.88 5.56 8.0A 1.A5 Glucose 13.81 18.09 19.08 1.05 Barbituric acid 2.A7 A.82 6.8A 1.A2 Uracil -- 5.38 7.5A 1.AO Endogenous 3 -— A.51 5.35 1.19 Glucose -- 11.00 11.91 1.08 Barbituric acid -- A.07 A.55 1.12 Uracil -- A.61 5.35 1.16 Endogenous 5 -— 0.73 1.6A 2.25 Glucose -- 2.28 2.68 1.18 Barbituric acid -- 0.36 0.87 2.A2 Uracil -- 0.82 1.32 1.61 aThe reaction mixtures contained 200 umoles of phos- phate buffer (pH 7.2) in the main compartment of the Warburg flasks, 0.2 ml of 20% KOH in the center well, 0.2 ml of AN H280” in one side arm, and the following substrates in the other side arm as indicated above: glucose, 33.5 umoles; barbituric acid, 50.0 umoles; and uracil, A.5 umoles. The experiments were conducted as described in the Experimental Methods and the acid tipped in from the side arm at the end of the incubation period for the determination of evolved C02. Experiment No. l was conducted with cells of strain NRRL B-2309-P-A harvested from TYG cultures whereas Experi- ment No. 2 was done with the same strain grown in sporula- tion medium as described by Costilow et a1. (18). The reaction mixtures contained 21.9 mg (dry weight) of cells in Experiment No. l and l6.A mg of cells in every case in Experiment No. 2. 37 were grown in TYG medium or in a sporulation medium where barbituric acid is required for the development of spore- 1ike bodies (18). In every case the 002 and QCO2 values observed with barbituric acid or uracil as substrate were approximately equal to or less than the endogenoUs values. Barbituric acid did not have a significant effect on the ability of cells of this same strain to oxidize glucose as substrate (Table A). These observations were made both with cells previously grown in TYG medium and in sporulation medium. Barbituric acid did not have any effect on glucose oxidation by cells of strain NRRL B—2309M grown in TYG medium and resuspended in BS medium (Table 5) nor were these cells able to oxidize barbituric acid in the ab- sence of glucose. The latter observation was made whether the cells were resuspended in BS medium or phosphate buffer. It-is also apparent that the cells were not able to oxidize any other component in the BS medium since the 002 observed were less than those observed in the presence of phosphate buffer. Similarly, cells from 2A and A8 hr cultures of strain NRRL B-2309M grown in S-5 medium were not able to oxidize barbituric acid in the absence of glucose and the presence of barbiturate did not have a significant effect on the ability of these cells to oxidize glucose (Ekperiment 2, Table 5). The differences observed may be attributed to errors introduced into the computation of these results since only very small amounts 38 TABLE A.--Effect of barbituric acid on the ability of rest- ing cells of strain NRRL B-2309-P-A to oxidize glucosea. Substrates Culture Q0 Q HQ 2 C02 Endogenous A 2.06 -- -- Glucose + barbituric acid 2A.50 25.00 1.02 Glucose — barbituric acid 20.10 -- —- Endogenous B 1.96 -- -- Glucose + barbituric acid 12.91 13.71 1.06 Glucose - barbituric acid 1A.59 15.90 1.09 Endogenous C 0.A5 1.A8 —- Glucose + barbituric acid A.5A A.79 1.06 Glucose — barbituric acid 5.09 6.12 1.20 aStrain NRRL B-2309-P-A was grown as follows: culture A, harvested after 2A hr growth in TYG medium; culture B, cells were grown for 2A hr in TYG medium, transferred to B-A medium after which a 20% inoculum was transferred to sporu- lation medium after A8 hr and the cells incubated for 2A hr before harvesting; culture C, same as for culture B except that the sporulation medium was inoculated with 10% inoculum and incubated for A8 hr before harvesting. The Warburg cups were prepared as described under Table 3 with the exception that the amount of barbituric acid added where indicated was 7.8 umoles. The dry weights of cells added were: culture A, 22.A mg; culture B, 1A.8 mg; and culture C, 8.1 mg. 39 TABLE 5.--Effect of barbituric acid on the oxidative ability of B. popilliae NRRL B—2309M.a A B Substrates Q02 QCO2 RQ Q02 QC02 RQ Experiment No. 1: Endogenous Control -— —- -— 1.8 2.2 -— Plus barbituric acid -- -- —— 1.5 1.8 -— BS medium (with glucose) Control 12.5 1A.A 1.15 -- -- -- Plus barbituric acid 13.6 15.A 1.13 —- —— -- BS medium (no glucose) Control 1.2 1.9 1.58 0.9 1.7 -- Plus barbituric acid 2.2 3.3 1.50 1.1 2.1 -- Experiment No. 2: Endogenous Control 0 O -— 2.1 0 -- BS medium (with glucose) Control AA.1 29.7 0.67 19.5 18.7 0.96 (A1.9) (AA.3) (0.9A) Plus barbituric acid A9.A 56.7 1.15 2A 8 36.2 1.A6 (A2.8) (A6.3) (1.08) BS medium (no glucose) Control 1.1 0 —- 2.A 1.6 -- ( A.8) ( A.7) ( --) Plus barbituric acid 0 9 0 -- 2.A 1.3 -- (3:8) (8.7) <--> aThe Warburg cups contained 2.3 m1 of BS medium or 0.1M phos— phate buffer (pH 7.3) in the main compartment and 0.5 ml of cell sus- pension was tipped in from a side arm after temperature equilibra- tion. The glucose and barbituric acid were present where indicated at the normal levels for S—5 medium. In Experiment No. 1, strain NRRL B-2309M was grown in TYG medium for 2A hr before harvesting. The Warburg cups contained (A) 26.A mg, and (B) 50.6 mg dry weight of cells respectively. In Experiment No. 2, the Warburg cups con- tained (A) 2.9 mg dry weight of cells harvested from 2A hr S-5 cul- tures, and (B) 8.0 mg dry weight of cells harvested from A8 hr S-5 cultures. Enclosed in brackets are values obtained from a dupli— cate experiment with cells harvested from 2A hr S-5 cultures where 2.8 mg dry weight of cells were present per Warburg cup. A0 of cells (2.9 and 2.8 mg dry weight of cells at 2A hr and 8.0 mg dry weight of cells at A8 hr) were obtainable from S—5 media for these experiments. The 002 and 0002 values observed with cells grown in S-5 medium were approximately 3 times greater than those observed with cells grown in TYG medium. This may be a reflection of the requirement of a higher amount of glucose in the synthetic medium. Urea was not produced from barbituric acid by cells of strain NRRL B-2309-P-A harvested from TYG medium (Table 6). The amount of C02 evolved in the presence of barbituric acid and urease was less than the endogenous 002 evolution. Hence, it seems likely that these cells do not contain an enzyme such as barbiturase capable of de- grading barbituric acid to urea and malonic acid. Effect of barbituric acid on H209_production and on NADH oxidation---Barbituric acid did not have a significant effect on the production of H202 by the soluble extract prepared from cells from 2A hr cultures either in the pres- ence or absence of flavin adenine dinucleotide (FAD) (Ex- periment 1, Table 7). The addition of NaN3 resulted in a significant increase in the percent of observed oxygen up- take accountable for in the H202 produced, thus indicating a slight contamination in the soluble extract of a particu- lar electron transport system. The data indicate that the addition of FAD greatly stimulated oxygen uptake and H202 production by this system. The data obtained with cells A1 TABLE 6.--Production of urea from barbituric acid.a Percent of Substrate ul CO2 evolved Theoretical Endogenous 98.7 -- Barbituric acid, 9.A umoles 8A.7 —— Barbituric acid, 9.A umoles 85.A -- Urea standard, 15 umoles 339.A 101.00 Urea standard, 10 umoles 219.7 98.1 Urea standard, 10 umoles (urease boiled 10 min.) 15.8 7.1 Urea standard, 5 umoles 130.2 116.3 Urea standard, 2 umoles 33.0 73.7 Urea standard, 1 umoles 23.1 103.1 aStrain NRRL B-2309—P-A was grown in TYG medium for 2A hr, washed in 0.1M phosphate buffer (pH 7.3), and added along with substrate and 200 umoles of the buffer to the main compartment of the Warburg flasks. The side arms contained 0.2 m1 of AN H280“ and a solution contain- ing A mg of urease respectively. The flasks were incu- bated at 30 C for 75 min after temperature equilibration, followed by the addition of urease and an additional 10 min incubation period. Acid was tipped in to liberate the C02 produced and the final reading taken after a further 10 min incubation period. The standards were run as described above but in the absence of cells and with the indicated levels of urea. A2 TABLE 7.—-Effect of barbituric acid on H O production.a 2 2 Extract Cofactor umoles 02 Umoles obsgrggd 0 added uptake H2O2 in H O 2 2 2 Experiment No. 1: Soluble +BA A.81 2.01 Al.8 Soluble -BA A.73 2.29 A8.1 Soluble +BA, NaN3 3.57 3.68 103.1 Soluble -BA, NaN3 3.61 3.56 98.6 Soluble +BA, FAD 11.07 9.23 83.A Soluble -BA, FAD 10.80 9.95 92.1 Soluble +BA, NaN3, FAD 10.99 11.AA 10A.1 Soluble —BA, NaN3, FAD 11.16 11.56 103.6 Experiment No. 2: Soluble +BA 2.75 1.A3 51.9 Soluble -BA 2.78 2.A8 89.A Particulate +BA l2.A2 0.9A 7.6 Particulate -BA 12.60 1.00 7.9 aExperiment No. l was conducted with extracts prepared from 2A hr TYG cultures by ultrasonic dis- integration.’ The main compartment of the Warburg flasks contained 5 mg (protein) of soluble extract, 190 umoles of phosphate buffer (pH 7.3) and where indicated 12.5 umoles of barbituric acid (BA), 30 umoles of NaN , and 0.6 umoles of FAD. One side arm contained 2A umoles of NADH as sub- strate and the other side arm contained 0.2 m1 of AN HQSON. Experiment No. 2 was conducted with extracts prepared from 35 hr TYG cultures in the Nossal disintegrator and the particulate fraction of a cell extract (3.A mg protein) and 1A.8 umoles of barbituric acid were included where indi- cated. The amount of H202 produced was determined as described in the Experimental Methods. Endogenous samples were run in the absence of NADH. No oxygen uptake or H2O2 production was observed. A3 from 35 hr cultures (Experiment 2, Table 7) indicate that lower amounts of H202 were produced in the presence of barbiturate, but the differences observed are probably of doubtful significance due to the low values obtained. Similar results were obtained when NADH oxidation was measured spectrophotometrically, with the cell extracts utilized above and with extracts of cells harvested from A8 hr cultures. Again, barbituric acid did not have a significant effect on NADH oxidation in the presence and absence of NaN3 by the soluble extracts tested. (Table 8). The addition of FAD to the soluble fraction of cells ob- tained from 35 hr cultures greatly stimulated the NADH oxidase activity. This extract was prepared by use of the Nossal Disintegrator and this may account fOr the small differences observed in the activities of the various ex- tracts in the absence of FAD. The data obtained with the particulate fraction from cells harvested from 35 hr cul- tures indicate that FAD and barbiturate had slightly in- hibitory effects on the activity of this fraction and that this system was completely sensitive to NaN3. The data presented in this section substantiate. those presented by Pepper and Costilow (6A) and do not give any consistent indication that barbituric acid may be in- volved with the soluble or particulate electron transport systems. The pyrimidine certainly was not oxidized to an extent necessary for it to serve as an energy source, nor did it appear to serve as an alternate electron acceptor. AA TABLE 8.--Effect of barbituric acid on NADH oxidation.a Activities* Extract Cofactor added A B C Soluble +BA 0.091 0.02A 0.052 Soluble -BA 0.092 0.033 0.05A Soluble +BA, NaN3 0.066 0.017 0.029 Soluble -BA, NaN3 0.068 0.02A 0.03A Soluble +BA, FAD 0.AA7 Soluble -BA, FAD 0.520 Soluble +BA, NaN3, FAD 0.507 Soluble -BA, NaN3, FAD 0.A67 Particulate +BA 0.367 Particulate -BA 0.A31 Particulate —BA, FAD 0.3A9 Particulate +BA, NaN3 0 Particulate -BA, NaN3 0 aExperiments were conducted with extracts prepared from (A) 2A hr cultures, (B) 35 hr cultures, and (C) A8 hr cultures grown in TYG medium. With extracts from 2A hr cells, the cuvettes contained 0.21 umoles of NADH as substrate, 1.09 mg (protein) of soluble extract, 300 umoles of phosphate buffer (pH 7.3), and 15.6 umoles of barbituric acid (BA) and 30 umoles of NaN3 where indicated in a total volume of 3 m1. In experiments with extracts from 35 hr cells, 1.0 mg (protein) of soluble extract was included in the absence of FAD whereas 0.05 mg (protein) of soluble extract was included in the presence of FAD which was present at a concentration of 0.6 umoles. With the particulate extract from these cells, the experiments were conducted as described above with the exception that 60 umoles of NaN3 was included where indicated. The amount of particulate extract (protein) included was 0.17 mg. Experiments with the soluble extracts from A8 hr cells were conducted as described for extracts from 2A hr cells with the exception that the cuvettes contained 1.30 mg (protein) of soluble extract. The extracts from 2A and A8 hr cells were prepared by ultrasonic disintegration whereas the extracts from 35 hr cells were prepared in the Nossal disintegrator. *OD at 3A0 mu per min per mg (protein) of extract. A5 Effect of barbituric acid on_permeability and via- bility.--Barbituric acid may have some stabilizing effect on the permeability barrier of B. popilliae since approx- imately 1/3 of the RNA was observed in the supernatant from cells suspended in phosphate buffer alone (Table 9). However, the significance of this difference is questiona- ble; since, (a) the method involved concentrating a relatively large volume of supernatant to 1 ml followed by the determination of RNA, and (b), the viable count of samples resuspended in phosphate buffer alone was the same as in phosphate buffer plus barbituric acid. In contrast, when cells were suspended in water both RNA leakage and loss in viability were dramatically increased. Attempts at measuring the amino acid pool labelled with radioactive glutamic acid or proline were unsuccessful, (Fig. 2), since the counts obtained from samples washed with the S—5 medium were not significantly higher than those samples washed in cold 5% TCA. It should be noted that these cells were previously grown in TYG medium. Hence, the possibility exists that resuspension of these cells in S-5 medium may have been sufficient to release the amino acid pool. Nevertheless, the results indicate that a marked difference occurred in the uptake of labelled amino acid between samples suspended in the presence and absence of barbituric acid irrespective of whether the label was in proline which is required for growth or in glutamic acid which is not required for growth in S-5 A6 TABLE 9.—-Effect of various diluents on RNA excretion and cell viability.a ug RNA/10 m1 Diluent Viable count/m1 supernatant TYG medium -- 1.A x 108 S—5 medium -- 5.A x 107 0.1M phosphate (pH 7.3) 9A 2.6 x 108 0.1M phosphate containing 0.1% barbituric acid 8 (pH 7.3) 32 3.A x 10 Water 238 1.0 x 103 aStrain NRRL B-2309M was grown in TYG medium for 2A hr, harvested, and resuspended in the indicated diluent. After 1 hr standing at room temperature, the amount of extracellular RNA was determined as described in the Experimental Methods. The viable counts were determined as described in the Experimental Methods after standing at room temperature for 3 hr. A7 d 0| .I N . 102 cpm pot mgdry weight of coll: a. 4. .5...’ -—-------g-----------A \ l l J °o9’ I 2 3 HOURS Figure 2.-—Effect of barbituric acid on the glutamic acid and proline pools of strain NRRL E-2309M harvested from 20 hr TYG cultures. The cells were washed in BS media with 1% glucose and with and without 0.1% barbituric acid respectively, and resuspended in 15 ml of the same medium. U-1 C—L—Glutamic acid and l“C-L—proline were present in the media at approx- imate specific activities of A.66 x 106 and 2.05 x 105 cpm/umole respectively. Duplicate 0.1 m1 aliquots were removed at the designated times and treated as described in the Experimental Methods. (0,0) 1“C-glutamic acid with and.without barbituric acid respectively; (A,A) 1"C-proline with and without barbituric acid respec— tively; (———) cells washed with S-5 medium; (-——) cells washed with cold 5% TCA. A8 medium. The large difference in total radioactivity in- corporated between the two amino acids reflects the 20 X difference in specific activity of the acids. These results suggest that barbituric acid may have an effect on the uptake of precursors into cellular macromolecules. Incorporation of 1“C from 2-1uC-barbituric acid into cell components.--Preliminary experiments in which the uptake of barbituric acid from the medium was observed by determining the loss in absorbancy at 257 mu indicated that very little barbiturate was taken up or broken down by the cells since no difference in the absorbancies could be detected after growth in S—5 medium. Hence, barbituric acid uptake was determined by using radioactive barbituric acid. An initial experiment with 2-luC-barbituric acid (Fig. 3) indicated that the label from barbiturate was rapidly taken up into the cold TCA-soluble pool during the first 1.5 hr of incubation in S—5 medium, but the pool level did not change appreciable thereafter. However, a linear uptake of the label from barbiturate into the nucleic acid fraction (hot TCA-soluble fraction) occurred during the entire incubation period of 3 hr. At 3 hr the percentage of the total radioactivity of the cell fractions found in the hot TCA-precipitate was less than 5.2% and this is not believed to be significant. The high, initial counts of the intact cells may have resulted from 6 . Ink“: cells ‘ (,- CO - UCA' E soluble N h o G. 3’ Hot TCA- % soluble '- 2 .. AH.» ‘I'CA- , + “gig“... (I 'L5 3 EKMURS Figure 3.-—Initia1 experiment on the uptake of label from 2-1“c—barbituric acid into cells of strain NRRL B-2309M harvested from 2A hr TYG cultures. The cells were washed with water and added to previously prepared S-5 medium containing approximately 2.78 x 105 cpm/umole of 2—1“C—barbituric acid. Two ml aliquots were removed and fractionated by the Schneider prop cedure (73) as described in the Experimental Methods. The hot TCA-precipitate of the 3 hr sample was treated by the method of Park and Hancock (62) as described in the Experimental Methods to obtain the trypsin— soluble and trypsin—insoluble fractions. Over 9A% of the total radioactivity of the two fractions was pres— ent in the trypsin-soluble fraction. The value for the hot TCA—precipitate at 3 hr is the total of the radioactivities of the two fractions. A 2 m1 aliquot of the cell suspension contained 10.9 mg dry weight of cells. 50 adsorption of the radioactive barbituric acid on the cells during the filtration operation, if the intitial uptake was quite rapid. Further evidence that the incorporation of isotope from 2—1uC-barbituric acid into macromolecules was limited primarily to the nucleic acids was obtained by use of chloramphenicol (Fig. A). The high level (500 ug/ml of chloramphenicol used had no significant effect on the up- take of lLIC from 2—luC—barbituric acid into the cold TCA- soluble fraction of cells, nor into the fraction containing DNA plus protein. However, it did inhibit incorporation into the cold TCA-precipitate, which should have included all the nucleic acids plus protein. The inhibitory effect became greater with time. Such an effect could be accounted for by the inhibition of RNA synthesis after extended in- cubation (A8). Attempts at determining the location of the label in the RNA mononucleotides by separation on a Dowex- l-formate column were unsuccessful although this technique was very successful in the separation of standard solutions of mononucleotides. Results from experiments carried out to give an estimate of the amount of label from 2-luC-barbituric acid incorporated into the RNA and DNA of resting cells indicated that approximately 8.A x 10"2 and 1.2 x 10'2 uatoms of 1”C from barbiturate were taken up per mg of DNA and RNA respectively. These estimates suggest that more of the label was present in the DNA than in the RNA. However, the 51 as.“- T we? 3 a HKNUIS Figure A.--The effect of chloramphenicol on the uptake of 1“c from 2—1“c-barbituric acid by cells harvested from 2A hr TYG cultures, washed in cold S-5 medium and resuspended in S-5 medium containing approximately 5.55 x 105 cpm/umole of 2-'“C-barbituric acid. Two ml aliquots were removed and fractionated by the Schmidt and Thannhauser procedure (72) as described in the EXperimental Methods. A 2 m1 aliquot contained 6.2 mg dry weight of cells at the beginning of the experiment. (0) with chloramphenicol at a final concentration of 500 ug/ml; (0) without chloram— phenicol. 5,. f\.) possibility exists that the DNA fraction was contaminated with low amounts of radioactive RNA or small amounts of 2—luC-barbituric acid carried over in the extraction pro- cedure. Since the amount of DNA present (as assayed color— imetrically) was about one-tenth of the RNA determined, a small contamination of this fraction would result in an over—estimation of the uatoms of 140 present per mg of DNA. A more complete fractionation of cells was then performed in order to obtain a more quantitative estimate of the distribution of the 140 from 2-lAc-barbituric acid in cells. Radioactive measurements were made on these fractions by liquid scintillation counting with appropriate corrections for quenching. About 1% of the label was taken up by the cells over 6 hr (Table 10). The radioactivity present in the intact cells accounted for 81.A% of the observed decrease in the medium and 68.3% of the radioactiv- ity of these cells was recovered in the cell fractions based on the totals.of the acid soluble, ethanol-ether soluble, and the pH 7.0 soluble fractions. The latter fraction represents the constituents in the acid insoluble fraction after alkaline hydrolysis and subsequent adjustment of the pH to 7.0. The radioactivity of the RNA fraction amounted to 89.6% of that of the pH 7.0 soluble fraction which pro- vided further evidence that very little of the isotope was associated with the DNA and protein. 53 TABLE lO.——Distribution of lLAC from 2-luC-barbituric acid among the cell fractions.a dpm/ml of Sample cell. suspens10n S—5 medium, 0 hr 2,8A9,000 S—5 medium, 6 hr 2,820,000 Cell washes 70,110 Intact cells 22,960 Cold TCA-soluble fraction and wash 9,650 Ethanol-ether soluble fraction and wash 950 Cold TCA-insoluble fraction 3,780 pH 7.0 soluble fraction 5,092 RNA ’A,562 CMP + AMP area 1,806 GMP + UMP area 698 "X" area AAO aCells were grown and harvested from TYG cultures as described under Fig. 3. The cells were fractionated by the Schmidt and Thannhauser procedure (72) including an ethanol-ether extraction step, and the RNA obtained by the method of Littlefield and Dunn (52) as described in the Experimental Methods. The 2—1uC—barbituric acid was present in the reaction mixture at a total activity of 3.65 x 105 dpm/umole and the concentration of cells used was 6.26 mg dry weight per ml. The RNA obtained was con- centrated to 3.5 m1, and a total of 0.66 ml electro- phoresed with appropriate standards in 20 ul amounts as described in the Experimental Methods. Areas corres- ponding to those presented in the Table were eluted with water, concentrated to 0.5 m1, and aliquots counted. All counts were corrected to dpm/ml of the original cell suspension. 5A The wet weight of the cells used in this experiment was estimated to be 31.3 mg/ml since the dry weight of cells present in the reaction misture was 6.26 mg/ml. Assuming their specific gravity to be 1.1 g per ml, the volume of this amount of cells would be approximately 2.9 ul. Hence, the dpm per m1 of cell volume would be 5.A x 106 (1.57 x 104/2.9 x 10'3). This estimate was made using the sum of the dpm from the acid soluble, ethanol- ether soluble, and the pH 7.0 soluble fractions; since, it was possible that the method used in solubilizing the intact cells and the acid insoluble fraction for liquid scintillation counting may have resulted in an underestima- tion of the radioactivity in these fractions due to in- complete solubilization. Also, the amount of radioactivity determined in the intact cells may be inaccurate as an estimate of the intracellular concentration of label due to possible adsorption to the cell surface and incomplete washing of the cells on the filter. Since the amount of radioactivity present in the medium at 0 time was 3.65 x 105 dpm per umole of 2-1uc-barbituric acid, the estimated umoles of barbiturate per ml of cell volume was 1A.8 (5.A x 106/3.65 x 105), assuming that the pyrimidine was taken up without degradation of the molecule. The con- centration of barbituric acid initially present in the medium was approximately 8 umoles per ml. This suggests that the cells may be able to concentrate the pyrimidine 55 intracellularly to about 2 X that present in the extracel- lular medium. Although uracil is the most closely related to bar— bituric acid of the pyrimidines in RNA, the results with the RNA mononucleotides (Table 10) indicated that uridylic acid (UMP) was not the major product formed from barbituric acid. In fact, the 140 levels in the area corresponding to cytidylic acid (CMP) plus adenylic acid (AMP) were almost 3 X that in the area corresponding to guanylic acid (GMP) plus UMP. Of particular interest, was the component designated in the Table as "X" which had an electrophoretic mobility in the buffer system of about twice that of UMP. The "X" component did not absorb ultraviolet (u/v) light on the paper. However, the amount of isotope present in the corresponding area from the 20 ul sample which was separated by electrophoresis was only 75 dpm. This was equivalent to the radioactivity in about 2.1 x 10'“ umoles of barbiturate. According to Block, Durrum, and Zweig (7), one would need about A x 10"2 Hmoles of pyrimi- dine per cm2 spot for detection under u/v light. Hence, we would have had to separate the RNA mononucleotides in about A m1 of solution to obtain a sufficient amount of this component in one spot to see u/v absorption. This would correspond to about 15,000 dpm of material. Thus, this component would be very difficult to obtain in suffi- cient quantity and purity for identification by the present methods. 56 An analysis of the acid soluble fraction from cells incubated with radioactive barbituric acid showed that 56% of the activity of this fraction corresponded to barbituric acid (Table 11). However, 22.8% of the 1“C was associated with a u/v absorbing component corresponding to UMP in electrophoretic mobility. Also, 10.6% of the label was associated with the "X" component referred to earlier. The high percentage of recovery indicated that these compo- nents represented essentially all of the isotope present in the pool. Since the associated radioactivity with UMP in the acid soluble fraction was not observed in the UMP derived from RNA after alkaline hydrolysis, an experiment was con- ducted to determine if there was an alteration of the radioactive barbituric acid in the uninoculated medium. These results are shown in Table 12. It is apparent that there was an accumulation of a component in the S-5 medium which had the same electrophoretic mobility as UMP during the duration of the experiment. Since a similar accumula- tion was not noted in the phosphate buffer containing barbituric acid, ‘this component may represent some sort of complex formed between the pyrimidine and some of the amino acids present in the synthetic medium. In any case, these results indicate that the observed radioactivity associated with UMP in the acid soluble fraction may have been due to the formation of a component with similar electrophoretic mobility in the medium, since in some cases 57 TABLE ll.-—Uptake of 1“C from 2-luC-barbituric acid into the TCA-soluble fraction of cells harvested from TYG cultures.a Sample cpm/20 ul Cold TCA—soluble fraction 5AA (After electrophoresis): Barbituric acid area 305 UMP area 12A "X" area 58 Percent recovery 89.5 aCells were harvested from 2A hr TYG cultures and resuspended in S-5 medium as described under Fig. 3. After 6 hr incubation the cold TCA-soluble fraction was extracted and 20 ul electrophoresed as described in the Experimental Methods. 58 TABLE l2.-—Alteration of barbituric acid in uninoculated phosphate buffer and S-5 medium.a Incubation time Percent at Percent at Percent at (a... 02:53“ ESTES? is; Phosphate buffer: 0 (unautoclaved) 1.2 98.2 0.6 0 (autoclaved) 8.3 89.8 1.9 l 7.6 86.3 6.1 2.5 7.5 89.2 3.3 5.5 7.9 85.A 6.7 S-5 medium: 0 (unautoclaved) 1.1 98.1 0.8 0 (autoclaved) 8. 88.1 3.6 l 8. 78.5 13.3 2.5 7.5 7A.1 l8.A 5.5 7.9 57.3 3A.8 aFlasks containing 0.1% barbituric acid in 0.1M phosphate buffer (pH 7.3) and complete S-5 medium were pre- parfid containing approximately 2.52 x 105 cpm/umole of 2-1 C-barbituric acid and sterilized. Aliquots were removed aseptically and 20 ul electrophoresed in 0.05M ammonium formate (pH 3.5) along with standard solutions of barbituric acid and UMP. Strips were then cut from these samples and the radioactivity corresponding to the areas occupied by barbituric acid and UMP as well as that present at the origin determined. The percentages in the Table are the percentages of the total count found in each of the three areas. 59 the synthetic medium was not used on the same day that it was made up. The association of a small amount of radio- activity at the origin may help to explain the presence of radioactivity at the origin of electrophoresis conducted with RNA mononucleotides if this component was capable of associating with the RNA in some manner. The formation of these components in the medium is not believed to have any relation to the ultimate effect of barbituric acid on B. popilliae since no variations were observed in the growth of this organism in fresh media as compared to growth in media after several days storage. Results from parallel experiments on the effect of actinomycin D on RNA synthesis and on the incorporation of label from 2-1uc—barbituric acid into the RNA fraction are shown in Table 13. The antibiotic inhibited RNA synthesis as measured by 32F incorporation by 90.2% whereas the up- take of the1“c label into this fraction was inhibited by only 52.3%. The reason for this difference may be that a relatively lower amount of the 18C was taken up into the RNA. The previous experiments had been conducted with cells which had been freshly transferred from a complex medium to a synthetic medium.' This could interrupt the synthesis of a number of macromolecules. Therefore, a series of experiments were conducted with cells grown in both a synthetic and complex medium. Parallel studies of nucleic acid synthesis were conducted using 32P as 60 TABLE 13.—-Effect o actinomycin D on the uptake of 1“c from 2—1 C-barbituric acid into RNA.a Sample Time (hr) cpm, 32P cpm, 1“G Control 0 106 52 Actinomycin D 75 A2 Control 3 10,291 532 Actinomycin D 1,006 253 aCells were grown for 6 hr in TYG medium and resus- pended in 15 ml of S-5 medium with the phosphate decreased to 20 mg per liter, and the buffer increased by adding Tris.HCL, 0.01M (pH 7.3). Duplicate flasks were prepared which contained 2-1Ac-barbituric acid and 32P at final concentrations of 2.33 x 105 and 1.01 x 107 cpm/umole respectively. Flasks were also included which contained 25 ug of freshly prepared actinomycin D per flask. Ali- quots of 0.3 and 0.5 ml were remove at 0 and 3 hr from the flasks containing the 32P and l C isotopes respect- ively and processed by Hanawalts method (32) as described in the Experimental Methods to obtain the RNA + DNA frac- tion for the determination of radioactivity. 61 K2H32POA and 2-luC-barbituric acid. -In all experiments in which cells were grown in the presence of radioactive barbituric acid, the medium was used within 2A hr of prep- aration. Very little of the isotope from barbituric acid was taken up by cells grown in either S-5 or TYG medium as evidenced by the depletion of approximately 2 and 0% re- spectively of the radioactivity during the growth of the organism. The cells grown in TYG medium took up only about one-third of the 1"’C taken up by cells grown in S-5 medium (Table 1A). This might be expected since barbiturate is not required for growth in TYG medium. There were also differences in the distribution of the label among the fractions. Thus, with TYG grown cells, the percentage of the 140 in the cold TCA-soluble and lipid fractions was higher and the percentage in the nucleic acids lower than with cells grown in S-5 medium. Approximately the same relative amount of 32F was taken up by the cells whether grown in S-5 or TYG medium with or without barbituric acid. This is also reflected in the similarities of the percentage of the total cpm found in the various fractions of cells from the two media. Since the amount of RNA and DNA present was assayed by the orcinol and diphenylamine reactions, the uatoms of 1“C from 2-1uC-barbituric acid and the uatoms of 32P in- corporated into RNA and DNA per mg of the respective nucleic acid could be determined as shown in Table 15. The uatoms 62 TABLE lA.--Uptake of 32P and 114C from 2-1uC-barbituric acid into growing cells.a Percent of total cpm in Total cpm of Media cell fractions/ Cold DNA + mg cell RNA TCA- Lipids RNA . protein soluble With 2-1uC- barbituric acid: S-5 ‘ 6,52A 16.6 2.2 52.3 28.9 TYG + barbituric acid 2,1A9 32.3 12.9 39.8 15.0 With 32P: S-5 263,7A6 9.2 5.0 73.3 10.A TYG + barbituric acid 232,215 9.3 5.0 76.7 9.1 TYG - barbituric acid 2AA,A7A 10.2 5.8 75.9 8.1 aStrain NRRL B-2309M was grown in TYG medium (the phosphate level was reduced to 0.2% and the medium contained 0.1% barbituric acid where indicated) and S-5 medium with the respective isotope for 2A hr and the cells fractionated as shown in Scheme 1 in the Experimental Methods. The radioactivities were determined after drying the samples on planchets in the gas-flow counter and the amounts of RNA and DNA present assayed colorimetrically as 1A described in the Experimental Methods. 3The cpm/umole of 2- C- bagbituric acid was 1.1 x 105, and for. P was 0.7 x 105 — 0.9 x 10 . 63 TABLE 15.--Calculated Uatoms of 1MC and 32P per mg of RNA and DNA.a RNA DNA (A) (B) (c) (A) '(B) (0) With 2-1uC- barbituric acid: s-5 J 3.2 x lo‘2 - 1.A 5.2 x lo‘2 - 1.0 TYG + barbituric _3 3 _2 acid 8.1 x 10 - 0.3 2.7 x 10 - 1.0 With 32P S-5 - 2.3 - - 5.1 - TYG + barbituric acid - 2.5 - - 2.6 - TYG - barbituric acid — 2.6 - - 2.3 — aThe calculations were made from data obtained from the experiment described under Table 1A. (A) uatoms of 1”C per mg. (B) uatoms of 32P per mg. (C) (fl)/(B) x 100: This calculation represents the uatoms of C from 2- 1A6- barbituric acid taken up into the nucleic acid per 100 uatoms of 32F incorporated. 6A of label from barbituric acid taken up per 100 umoles of RNA and DNA mononucleotide formed was then calculated by 32P data were a true measure of the assuming that the amounts of RNA and DNA formed. This assumption was par- ticularly accurate in the case of RNA. By taking the average molecular weight of a mononucleotide to be A00, one would expect 2.5 umoles of mononucleotide per mg of RNA, a value which corresponds very closely to that ob— served from the 32F data. The reason for the 2 X higher value obtained for DNA from cells grown in S-S medium is not known. The cells grown in the synthetic medium con- tained approximately one-fourth as much DNA as RNA. Since the diphenylamine reagent has a fairly high specificity for deoxypentose (21), a small amount of contamination of the DNA fraction with RNA would result in an overestimation of the amount of32P taken up per mg DNA. It is apparent that cells grown in TYG medium took up only one-fourth as much 1A0 from-2-14C-barbituric acid per 100 umoles of RNA mononucleotide as cells grown in S-5 medium. Table 16 shows the results of an attempt to separate the RNA mononucleotides electrophoretically. From the 32F data it is apparent that an amount of "X" similar to that of the other mononucleotides was synthesized. However, if this component corresponded to a barbiturate mononucleotide or if barbiturate was a required precursor for its formation in synthetic medium, one would expect the corresponding value obtained from the 1”C data to be much higher since 65 Li TABLE 16.--Microatoms of 32P and 1‘C estimated in various mononucleotides.a cpm/20ul sample found in Media areas corresponding to: CMP AMP GMP UMP "x" Percent recovery With 2—luc- barbituric acid: S-5 21 7 9 23 27 91.2 TYG + barbituric acid 27 17 1A 26 13 71.2 With 32P: S—5b 750 633 966 717 606 8A.5 TYG + barbituric acidb 3A95 395A 5778 3660 3381 80.9 TYG - barbituric acid 10Al 938 l6A6 1008 1291 82.5 aThe mononucleotides were isolated frdm the RNA of cells treated as described under Table 1A by the methods described in the Experimental Methods. bSince the 32P-labelled mononucleotides were con- centrated to 3.0 ml and the 1 C-labelled mononucleotides were concentrated to 1.0 ml, the actual values obtained with the former were multiplied by 3 so that the values would be comparable. 66 the unknown component would contain one uatom of 1”C from barbituric acid per Amole of mononucleotide. Since this was not observed, the fate of the label from barbituric acid in RNA remains unknown. To further confirm that the isot0pe from 2-14C- barbituric acid was truly incorporated into the nucleic acid rather than just associated with the cell fraction in which it was found and to determine the component(s) of RNA with which it was associated, highly purified RNA was prepared from cells grown in fresh S-5 medium with radioactive barbituric acid and fractionated on a sucrose gradient. The spectrum of the purified RNA was very similar to that reported by Swift (81), and the 260/280 ratio was found to be 1.82. The results (Fig. 5) revealed that the 1”C from 2-1uC-barbituric acid was taken up uni— formly into the As, 16s, and 23s RNA. This is obvious from the fact that the radioactivity peaks corresponded so closely with the absorbency peaks. This would be possible only if the specific activity of each fraction was essen- tially the same. In addition, these results give the most conclusive evidence so far that a small but significant amount of the label from 2-1uC-barbituric acid is either incorporated or very tightly bound to the RNA. The amount of barbituric acid equivalent to the 1”C incorporated per mg of RNA was estimated from the average cpm (A27) per OD260° Since one OD260 unit corresponds to about 50 ug of RNA, the activity of the RNA was approximately 67 $3 25 27 29 3133 35 37 39 4|" FRACTION Figure 5.—-Incorporation of 1‘'C from 2—‘“C-barbituric acid into RNA fractions separated by centrifugation through a sucrose gradient. Cells of strain NRRL B—2309M were grown in 200 m1 of S-5 medium containing 2-1“C-barbituric acid at a final approximate activity of A.16 x 105 cpm/Umole. The cells were harvested after 2A hr growth and the RNA extracted and fractionated on a sucrose gradient as described in the Experimental Methods. The radioactivities of the fractions were determined by liquid scintillation counting. The OD at 620 mu of the cell suspension after 2A hr was 0.153. t 68 8A20 cpm per mg of RNA. The specific activity of the radioactive barbituric acid was approximately 3.0 x 105 cpm/umole, hence, the uatoms of 14C from barbiturate taken up per mg of RNA was about 2.8 x 10‘2. .This value is very close to that obtained in the previous experiment. Highly purified DNA was also prepared from cells grown in fresh S-5 medium containing radioactive barbituric acid and analyzed for radioactivity. The 260/280 ratios of the RNA and DNA isolated from these cells were 1.87 and 1.68 respectively. In this eXperiment the Uatoms of 1("C from 2-luC-barbituric acid taken up per mg of RNA and DNA 2 and 1.5 x 10"2 into these macromolecules were 2.6 x-lO- respectively. These results suggest that less radio- activity was associated with the DNA than with the RNA fraction. However, the small amount of DNA analyzed could result in considerable error. Control experiments were conducted with the RNA isolated by phenol extraction and these results are shown in Table 17. It is apparent that the label in the isolated RNA was completely released from polymer by pancreatic ribonuclease and alkaline hydrolysis. Similar experiments could not be conducted with the DNA since only 13.2 ug was obtained and the complete sample was precipitated with TCA for the determination of 1“C content. Since the amount of 1“C from the labelled barbiturate found in the RNA and DNA of cells grown in synthetic medium in which barbiturate was required for growth was extremely 69 TABLE l7.--Effect of pancreatic ribonuclease and alkaline hydrolysis on RNA isolated by phenol extraction.a RNA cpm Percent of control Experiment No. 1: Control 396 __ Enzyme—treated 29 7.3 KOH-treated 31 7.8 Experiment No. 2: Control 198 -— KOH—treated 2 1.0 aRNA isolated by phenol extraction was incubated in 25 ul aliquots with 10 ug/ml of pancreatic ribonuclease at 30 C for 30 min and with 0.3M KOH at 37 C for 13 hr. The samples were then precipitated and collected on membrane filters as described in the Experimental Methods, and the radioactivities determined. Experiment No. l was conducted with the RNA isolated for sucrose gradient centrifugation, and Experiment No. 2 was conducted with the RNA isolated from cells from which DNA was isolated. 70 small and did not appear to be associated with a specific RNA or mononucleotide, the function of the barbiturate could n0t be that of a required precursor. In fact, the possibility still remained that none of the label was in- corporated. Such small amounts could be tightly bound to the nucleic acids. Accordingly, experiments were conducted with RNA and DNA obtained from commercial sources and these results are shown in Table 18. It is apparent that a sig- nificant amount of the barbituric acid was adsorbed to the RNA and DNA, and that it remained bound through a number of treatments. ‘While the levels of 2-luC—barbituric acid bound in this experiment were only about 0.1 of those found in the nucleic acids from B. popilliae, the conditions were different. This does not exclude the possibility of some incorporation of the label, since previous experiments in- dicated a linear uptake into the RNA. However, if incorpo- ration does occur, it is so low that it is of doubtful significance. Further evidence that barbituric acid was not specifically incorporated into RNA monoucleotides is pre- sented in Fig. 6. It is apparent that the lAC peaks are not aligned with the 32P peaks as would be expected if the 2-1uC-barbituric acid had been incorporated into the RNA mononucleotides. Moreover, since l4C peaks are present which appear closely related to those present in uninocula- ted S-5 media after several days storage, and since this experiment was conducted with cells harvested from TYG 71 TABLE 18.--Adsorption of barbituric acid to RNA and DNA. Sample umoles of barbituric acid per mg RNAa 1.2 x 10'3 DNAa 3.9 x 10‘3 RNAb 3.0 x 10'3 DNAb 6.0 x 10‘3 aTwo mg of yeast RNA was incubated with and without 2 mg of DNA in 2 ml of 0.2% phospgate buffer (pH 7.3) containing approximately 5.6 x 10 cpm/umole of 2—1 - barbituric acid. The samples were precipitated by the addition of an equal volume of cold 10% TCA and washed twice with 2 m1 of cold 5% TCA. The samples containing both RNA and DNA were treated with 0.3M KOH to hydrolyze the RNA followed by the addition of an equal volume of cold 50% TCA to precipitate the DNA. The DNA was washed as described above and the precipitate collected on mem- brane filters with the aid of 3 washes of 2 m1 of cold 0.01M HCl. The filters were glued to planchets and counted in the gas-flow counter. bThe experiment was carried out identically as des- cribed above with the exception that the samples were suspended in complete S-5 medium containing the radio- active barbituric acid. Both the phosphate buffer and the S-5 medium were sterilized before use. Io. " *5 . i . . .‘ d I E H. i 9 i' ‘. c: F6~ 13 2 i ‘, /\ 2 ‘ l | \ ‘ A? I \ (3' A l A . '. ’ 0 l l 0 I \ ' l \ \ I’ \ V 2- V \ I I \ I \\ ’ s I “u i 3 f2 18 I (""7” CENTIMETERS Figure 6.—-Electrophoretic separation of 32P— and ‘“C— labelled RNA mononucleotides. The remaining suspen— sions of the control flasks of the experiment described under Table 13 were fractionated as outlined in Scheme 1 in the Experimental Methods to obtain the RNA mono- nucleotides. 20 ul aliquots of the labelled mononucle— otides were electrophoresed at the same time on the same paper with mononucleotide standards as described in the Experimental Methods. The u/v absorbing areas were marked as shown above, and 1 cm strips were cut and placed in glass scintillation vials for the determination of radio— activity by liquid scintillation counting after the addition of 5 m1 of toluene—base scintillation fluid (PPO, A g; POPOP, 50 mg; toluene to 1 liter). 73 cultures in 8-5 media of unknown storage age, it appears that not only barbituric acid but the components represented by the other two peaks may be capable of binding to RNA even though they separate from the mononucleotides follow— ing alkaline hydrolysis and electrophoresis. The one 140 peak at 24 cm does not correspond to any of those observed after storage of uninoculated media containing barbituric acid. This may or may not correspond to the "X" component noted earlier.' In any event, it is obvious that this does not represent a mononucleotide, since no 32F peak corre- sponded to it. 'These results indicate that although bar- bituric acid may adsorb to RNA, it does not adsorb to RNA mononucleotides. Effect of barbituric acid on RNA and DNA synthesis.—— Since only a very small amount of isotope from labelled barbituric acid was associated with the nucleic acids and this appeared to be uniformly distributed, it was apparent that cells could synthesize the required nucleotides in synthetic medium without barbiturate. Thus, this did not appear to be the major role of barbituric acid in the nu- trition of Q. popilliae. However, this did not exclude the possibility that barbiturate may be controlling the rate of synthesis of these macromolecules. This was the next possibility investigated. The results shown (Fig. 7) indicated that barbituric acid did result in a strong stimulation of both RNA and DNA synthesis (29.1% and 78.8% respectively after 2 hr 7H .moozpmz Hapcmsfinmoxm on» CH omnfipommo mm cosmopo paw om>oEmL who: mposvfiam .o cHomEOpHE mo He\wn H spa: pan .mam>auomanop ofiom oHLSpfinpmn psozpfis 6cm Spa: Au.Iv mo :Hooncfipom mo HE\w1 m Lows pan .zam>fipomammp ofiom capsufinhmn psozpfiz cam npfiz A<.HpoQOmp ofiom oapzpfinmmn psonpfiz ocm spas Aoaov .wE mm.o mm: mAprHE coapommm mo as Log psommhq maaoo Mo pnwfimz zap one HE m.o .HE ma mo oESHo> Hmqu m Ca mfioan\smo 50H x mn.a mo >9H>Hpom oahaomam Hmcfim m pm mum mcacfimusoo EdHomE meow oz» 0ozfi ompmHSoocfi cam .%Hm>fiuooammp ofiom oaLSpfinhmn xa.o psonpfiz cam npflz UCm mmoosrw sa Sufi: Ezfioos mm CH omnmmz ohms mHHmo one .mmpSpHSO owe Ln flfl Eon“ ooumm>hm£ Emommlm ammz campum mo mHHoo an mammnpcmm «za cam