ABSTRACT GLUCOSE METABOLISM BY BACILLUS POPILLIAE by Rollin E. Pepper Investigations were conducted to determine the metabolic products resulting from the catabolism of glucose. the metabolic pathways used for glucose catabolism, and the types of electron transport systems used by Bacillus popilliae. Efforts were made to elucidate the reasons for the relatively low cell yields. limited viability. and the lack of sporulation l£.!iE£2 by this organism. The major glucose products were shown to be lactic acid. acetic acid. and C0 the minor products were found 2; to be glycerol. ethanol, acetoin. and acetaldehyde. The ratios of lactate to acetate may be controlled by adjusting the oxygen levels available to the cell. The enzymes in the upper half of the Embden-Meyerhof Pathway were demonstrated in cell free extracts and the hexose monophosphate pathway was implicated by the dissimilation of ribose-S—phosphate with the reduction of TPN. Essential enzymes for the Entner Doudoroff and Rollin E. Pepper phOSPhOketolase routes were found absent. Inhibitor and radioactive isotope data were consistent with enzyme assays. The percent participation of the hexose monophosphate pathway is dependent upon oxygen availability. The strain of g. popilliae used in most of these studies is apparently a mutant which oxidizes acetate through the TCA cycle. The selection for such a mutant was possible because of the continuous cultivation in liquid media in which acetate was present as the major product of glucose metabolism. Another variety of the same strain maintained on solid medium did not oxidizeacetate. Both strains proved capable of producing the typical fmilky disease? of the Japanese beetle larvae. The lack of the acetate oxidizing characteristic may be one of the reasons for the organism's not sporulating in yitrg. since this characteristic has been shown to be necessary for sporulation in other bacilli. An electron transport system through cytochrome oxidase to oxygen was demonstrated in cell-free extracts. Sensitivity to cyanide, azide, and carbon monoxide was demonstrated as well as characteristic cytochrome peaks in a spectrophotometric analysis. Hydrogen peroxide is pro- duced but no catalase or peroxidase was found. Production Rollin E. Pepper 0i hydrogen peroxide combined with the accumulation of organic acids may be basic causes of low cell yields and limited viability . GLUCOSE METABOLISM BY BACILLUS POPILLIAE BY Rollin Ey Pepper 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 1963 v :3 "J ‘ ACKNOWLEDGEMENTS The author is particularly indebted to Dr. R. N. Costilow for his generous and wise counsel during the course of this investigation and during the preparation of this manuscript. He is also grateful to the Northern Utilization Research Division. U.S.D.A., Peoria. Illinois. for their financial assistance and to Dr. H. H. Hall for his interest. encouragement. and his sense of humor during his very welcome visits. I am grateful to my wife, Lucille. for her encourage- ment throughout my graduate studies and for the patience of our children. Roger. Barbara. and Susan whose special interest in this thesis was the final punctuation mark. ii ACKNOWLEDGEMENTS The author is particularly indebted to Dr. R. N. Costilow for his generous and wise counsel during the course of this investigation and during the preparation of this manuscript. He is also grateful to the NOrthern Utilization Research Division. U.S.D.A., Peoria. Illinois. for their financial assistance and to Dr. H. H. Hall for his interest. encouragement. and his sense of humor during his very welcome visits. I am grateful to my wife. Lucille. for her encourage- ment throughout my graduate studies and for the patience of our children. Roger. Barbara. and Susan whose special interest in this thesis was the final punctuation mark. ii TABLE OF CONTENTS INTRODUCTION I I I I I I I I I I I I I I I I I I I I I REVIEW OF LII'EMTUM I I I I I I I I I I I I I I I I I EXPERIMENTAL METHODS . . . . . . . . . . . . . . . . . Cultures and Cultural Methods Protein Determination OAR Determination Manometric Studies Isotope Studies Chemical Assays Enzyme Assays mSULTS . O O I I I O O O I I O O O O O O I O I I I 0 Products of Glucose Metabolism Pathways of Glucose Catabolism Terminal Oxidation of Acetate Electron Transport System DISCUSSION I O I O I 0 I O O I O O I O C O O I I O I I SUMMARY . I I D O O O O O C O Q Q . O C U . I O O O O BIBIJIOGRAPHY I I I I I I I I I I I I I I I I I I I I I Page 13 14 17 17 18 18 20 22 28 71 73 Table 10. 11. LIST OF TABLES Composition of media used for growing cells of g. popilliae . . . . . . . . . Scintillator solution for counting carbon-l4 disintegrations . . . . . . . Effect of oxygen availability and pH control upon the growth of g. popilliae . . . . Acids produced by a growing culture of E. EoEillj—ae Q 0 I O I 0 O I O O O O I 0 Separation of glucose oxidation products into volatile and non-volatile acids . . Carbon balance of glucose oxidation products Acid production by concentrated and diluted cells of the same harvest . . . . . . . Oxidation of acetate by a growing culture by g. EOEilliae 0 O O O I O O O O O O O I I Acetokinase activity in extracts of g. Eoeilliae. C I I I O I O O O O O O O O C Phosphotransacetylase and condensing enzyme activities in a g. popilliae extract 0 C C O I I C O I O O O O O O O Assay for catalase in a g. popilliae extract iv Page 15 19 30 32 33 35 35 51 54 55 57 Figure 1. 10. 11. 12. LIST OF FIGURES Glucose utilization. acid accumulation. and pH changes in a broth culture of g. popilliae . . . . . . . . . . . . . Effect of cell concentration upon the molar ratio of lactate to acetate . . . . . . . Effect of various oxygen levels upon the type of acid produced . . . . . . . . . . Effect of fluoride upon glucose oxidation . . Glucose-6-phosphate dehydrogenase activity in cell free extracts . . . . . . . . . . Ability of log phase and stationary phase harvested cells to oxidize acetate . . . . Glucose oxidation characteristics of acetate oxidizing (A) and acetate non-oxidizing (D) varieties of g. popilliae . . . . . . Comparison of glucose oxidation with oxygen consumption and titratable acidity . . . . Effect of pH upon the oxidation of acetate. succinate. fumarate. and malate by resting cells of g. popilliae . . . . . . DPNH oxidase activity in whole cell free extracts and in the soluble and particulate fractions . . . . . . . . . . . . . . . . Oxidation of DPNH in the Warburg respirometer by a whole cell free extract and by the soluble and particulate fractions . . . . Peroxidase activity in extracts of Streptococcus faecalis and g. popilliae . Page 29 37 38 41 44 47 48 50 53 56 58 60 Figure Page 13- Effect of cyanide and azide on DPNH oxidation by soluble and particulate fractions of a cell free extract as measured by oxygen consumption . . . . . . 61 14. Effect of carbon monoxide upon DPNH oxidation by the particulate fraction of a cell extract . . . . . . . . . . . . . . . 63 15. Difference spectrum of an oxidized vs. reduced particulate fraction of a cell extract . . 64 vi INTRODUCTION Bacillus popilliae and Bacillus lentimorbus are unique in that they are selectively pathogenic for the Japanese beetle and some related insect pests. The infection. causing what is known as the ”milky disease.’ usually involves the larval stage of the insects (Angus and Heimpel, 1960). Since these bacteria are not pathogenic for mammals or many other insects. particularly beneficial types, great interest has been generated in the propagation of these two bacilli for insect control. Unfortunately. great difficulty has been encountered in growing and maintaining viability of these organisms in vitro even though growth is easily effected in artificially inoculated beetle larvae; and efforts to sporu— late these bacilli in vitro have failed. Reports concerning these organisms have been mostly concerned with the milky disease itself (Beard, 1945; and Angus and Heimpel. 1960) or growth characteristics and conditions in yitrg (Dutky. 1947; and Steinkraus. 1957). Both Dutky and Steinkraus list carbohydrates which these organisms will utilize and both indicate that acids accumulate by their references to the drop in pH and the need for highly buffered media to obtain optimal growth. Other than this. little has been rePOrted on substrate metabolism. This study involved the characterization of metabolic products appearing from glucose. the tracing of metabolic pathways. and determining the type of electron transport system used by B. popilliae. Certain environmental factors were studied as controlling influences on product ratios and metabolic pathways. An effort was made to elucidate factors which might be limiting growth and cell viability. It is hoped that the findings detailed herein will supply basic information which will aid in future investigations with g. popilliae. REVIEW OF LITERATURE The literature was reviewed in the light of surveying the various pathways of glucose catabolism to pyruvate; the types of metabolic products produced. and the nature of various electron transport systems in microorganisms. Attention was focused on methods of demonstrating pathways as well as on factors which influence their usage. A brief review of the environmental and nutritional factors which cause a qualitative or quantitative alteration of glucose metabolic products was made as well as the cultural variations which influence the make—up of the electron transport system. The review is not intended to be complete since it is so voluminous. but an attempt was made to give representative examples for the areas mentioned. Metabolic Pathways The longest known and most frequently used route for glucose dissimilation is the Embden-Meyerhof pathway (EMP). Some tissues and organisms use the EMP exclusively. In mammalian tissues. for example, Bloom. Stetten and Stetten (1953) reported that there is no evidence for a non-glycolytic pathway in rat diaphram slices. In support of this, Bloom 4 and Stetten (1953) measured ratios of C140 from glucose 2 labeled in the 1 and 6 positions and found these to be in equal announts. However. they reported that C1402 from glucose l—Cl4 is preferentially higher when kidney and liver slices are used indicating a non-glycolytic pathWay is used to at least some extent with these tissues. Departures from the EMP pathway came to light when some data could not possibly fit known patterns. Evidence for the phosphoketolase cleavage was reported by DeMoss et a1. (1951) who found that Leuconostoc mesenteroides produced 1 mole each of lactate. ethanol. and CO2 from 1 mole of glucose; and that aldolase. triosephosphate isomerase, and carboxylase were absent. These workers (1953) later found a glucose-6- phosphate (G—6—P) dehydrogenase in L. mesenteroides and they postulated that G-6—P was oxidized to 6-phosphogluconate (6-PG) which. after decarboxylation to a 5 carbon compound. was converted to lactic acid and an ethanol precursor upon a 3:2 split. DeMoss (1954) found the 6-PG dehydrogenase to be DPN dependent in this organism and identified ribulose-S- phosphate as one of the compounds resulting from the 6-PG decarboxylation. Krichevsky et a1. (1955) demonstrated EMP enzymes in Microbacterium lacticum but also reported an alternative pathway leading to a direct cleavage of a pentose which 5 involved riboas-S-phosphate (R—S—P) or ribulose—S-phosphate (Ru—S—qp). Evidence for the phosphoketolase cleavage appeared when RFS‘P l-Cl4 yielded unlabeled pyruvate, and Ru—t-P—2.3—Cl4 yielded carboxy labeled pyruvate. Data indicating a phosphoketolase in Acetobacter xylinum were reported by Schramm et al. (1958). Washed suspensions of this organism synthesized cellulose from glucose in the presence of con— centrations of iodoacetate which completely inhibit glyceraldehyde-3—phosphate (G-3-P) dehydrogenase. With the phosphoketolase mechanism. energy for synthesis was obtained through the formation of acetyl phosphate. Schramm also reported that an induced phosphoketolase appears in Lactobacillus plantarum grown on pentose which will cleave xyulose-S-phosphate (Xu-S—P) to G-3-P and acetyle phosphate but will not cleave fructose-6-phosphate (F-6-P). Purified F—6-P phosphoketolase will cleave Xu-S-P. Inhibitors such as fluroide. iodoacetate. and arsenite will not affect organisms dependent upon the phosphoketolase mechanism. Entner and Doudoroff (1952) described another path— way which departs from the traditional EMP route through a G—6-P dehydrogenase, as does the phosphoketolase route. How- ever. in the Entner Doudoroff (ED) pathway. there is no de— carboxylation prior to the formation of pyruvate. Instead. the 6-PG is transformed by a 6—PG dehydrase to a 2-keto-3 deOXY‘S—phosphogluconate (KDPG) which is split by a KDPG aldolase to pyruvate and G—3—P. Pyruvate is formed from the first three carbons and G-3—P from the last three. The pyruvate resulting from the KDPG action has carboxyl carbons originating from both carbons l and 4 of glucose. This route was reported by Claridge and Werkman (1954) in Pseudomonas aeruginosa. These workers found this to be a strictly aerobic mechanism. According to Holzer (1959) pseudomonads metabolize 70 to 100% of glucose by this route. Evidence for the hexose monophosphate pathway (HMP) was provided by de la Haba and Racker (1952) when they demon- strated triose phosphate formation from a phosphate ester of ribose and ribulose with yeast extracts. They also reported hexose phosphate generation. This deviation from the EMP pathway which also involves G-6-P dehydrogenase and 6-PG dehydrogenase with glucose as substrate was shown operative in Erwinia (Sutton and Star. 1960) based on the reduction of TPN through the oxidation of RrS-P. This suggested the formation of hexose phosphate through transketolase and transaldolase. Enzymes for the EMP pathway were also found to be present. Cochrane et a1. (1953) found that Streptomyces coelicolor and Streptomyces scabies lack early steps in the EMP system. but the HMP sequence is present. They also mention that an alternate route exists in which pentose phosphate is 7 5911*: iJTto triose phosphate and an unidentified two carbon fragment, Their data include the information that 0.01 M iodoacetate and 0.02 M fluoride did not inhibit G-6-P oxidation. This. particularly the inhibitor data. indicates that an active phosphoketolase mechanism is operating by— passing inhibited enzymes. Santer et a1. (1955) found EMP and HMP enzymes active in Pasteurella pestis. Their evidence shows that the HMP is particularly active during growth. In a study of several phytopathogenic bacteria. Katznelson (1955) found that only Erwinia used the EMP pathway; the others used strict oxidative routes. Agrobacterium, Xanthomonas. and Pseudomonas produced pyruvate from 6-PG by the ED route or the HMP route or both. Corynebacterium apparently uses the HMP. Later, Katznelson (1958) found that only Erwinia possess an intact glycolytic system. The others lack one single enzyme. phosphohexokinase. to compkate this sytem. It is possible, however, that this enzyme was present but not found since it is notably unstable. Allen and Powelson (1958) reported that Escherichia 921i oxidized glucose by both the HMP and EMP during the growth phase and shifts to the EMP exclusively during the stationary phase. Daws and Holms (1958) found that Sarcina leutea oxidized glucose by both the EMP and HMP pathways and employed the tricarboxylic acid (TCA) cycle in terminal o$ldat ion , Glucose was not utilized anaerobically and the ED Patinnay was not used. Doi and Halvorson (1959) demon- strated the HMP in vegetative cells and spores of Bacillus cereus var terminalis. They reported hexokinase and gluco- kinase were absent in spores. A direct oxidation of glucose to 2—ketogluconate yields 2-keto-6—phosphogluconate upon phosphorylation. The latter is either converted to pyruvate via an unknown pathway or reduced to 6-PG and oxidized via the HMP. The vegetative cells possess a complete glycolytic system as well as a TCA cycle. Goldman (1961), however. reported the key EMP enzymes hexokinase. phosphohexoisomerase. phosphofructokinase. and aldolase to be present in spores of Bacillus subtilis at various stages. Wang et al. (1958) examined several organisms for pathway participation. He found that E. 991i. Saccharomyces cerevisiae. B. subtilis. Aspergillus ni er. Penicillium digitatum. Penicillium chr so enum. and Streptomyces griseus utilize the EMP and HMP routes to various degrees; Pseudomonas reptilivora uses the HMP and the ED routes; and nggggmgggg saccharophilia the ED route only. Metabolic Products Products from glucose range all the way from CO2 and H20 to a variety of metabolic intermediates. Incomplete 9 degradation of glucose is due to oxygen availability, enzyme b10CKage, pH. nutrition of cells. and substrate concentration. Puziss et a1. (1957) found that Bacillus anthracis and B, cereus anaerobically dissimilated glucose to lactate. succinate. acetate, formate. acetoin. 2,3 butanediol. and glycerol. While examining aerobic and anaerobic glucose products of B. subtilis. Neish et a1. (1945) observed that air increased the production of acetoin. acetate. and CO2 but decreased 2.3 butanediol. glycerol. ethanol. lactate. and formate. Ehrlich and Segel (1959) examined glucose products of Bacillus megaterium under conditions of continuous and intermittent shaking. Increased aeration depressed lactate and increased C02. They found no striking changes in acetoin. 2.3 butanediol. glycerol. pyruvate. and acetate. Gray and Bard (1952) grew B. subtilis cells on complete and simple media and allowed resting cells to dissimilate glucose aerobically and anaerobically. They found a homolactic fermentation with both groups under anaerobic conditions. Aerobically, the cells from the simple medium oxidized glucose completely to CO and H 0; cells from the complete 2 2 medium also accumulated acetate and acetoin but no lactate. Pierce (1957) reported that pH affected glucose fermentation products of Streptococcus pyogenes. At pH 5.0, most of the glucose was converted to lactate; at pH 9.0. 10 laCtatEB dinfinished and more acetate. formate. and ethanol were produced. Similarly. Paege (1961) found that B. E fermented glucose principally to lactate at pH 5.0 and 8.0. However. with the increased pH, lactate diminished from the pH 5.0 value and increased amounts of ethanol, formate. succinate. and CO2 appeared. Christensen (1958) noted that sugar concentration had an effect on acid ratios produced by lactic acid bacteria. He found that an increased sugar concentration decreased the acetate/lactate ratio. Electron Transport Dehydrogenation of a substrate results in the reduction of an organic acceptor. a non-organic acceptor such as nitrate, or one or more pigments (cytochromes and/or flavin) leading to oxygen. Normally. anaerobes will reduce other organic compounds with the hydrogen transported by the pyridine nucleotides. However, they have the potential to reduce oxygen to H202 through flavins (M'Leod and Gordon. 1923). Lactic acid bacteria will transport hydrogen to oxygen in this manner and will dispose of the H202 via a peroxidase. Most aerobes possess a cytochrome electron transport system to oxygen. Although the H 02 potential is present. most of 2 these organisms produce catalase which converts this harmful 11 PIOduCt into H20 and 02. Several workers have studied cytochromes by reducing them with various substances and observing their representa- tive peaks with split beam spectrophotometry. Chance (1952) recognized cytochrome a after cells had become reduced by 3 their own respiration. Smith (1954) using the same method stated that bacterial cytochromes may differ from those found in yeasts and mammalian cells in that (a) a broad band of cytochrome b1 may replace those for b and c and (b) cytochrome a is often replaced by a1 or a2. Wood (1955) reduced cytochromes in Pseudomonas fluorescens with glucose, gluconate. or sodium hydrosulfite and found a peaks for cytochrome c and b (558 and 565 mu) and fl absorption for b and c (530 mu). The lack of peaks in the 600 to 625 mu range and no cyanide inhibition seemed to indicate the lack of a cytochrome oxidase. Chance (1957) reduced cytochromes with carbon and b in monoxide and demonstrated cytochromes a a 1' 3' Aerobacter aerogenes. In Aerobacter pasteurianum, he found an a peak at 427 mu. He also reported cytochromes b and c 1 in Rhodos irillum EEBEQE but no distinguishing bands for the type a. Work on Haemophilus parainfluenzae (White. 1962; White and Smith. 1962; and Smithand White. 1962) showed cytochromes b. c. o. a. and a2 after reduction with sodium hydrosulfite. No peroxidase was found and very little 12 cYtochromes. Dobrogosz (1962), using sodium hydrosulfite as true reductant. demonstrated no absorption peaks in the 400 to 700 mu range for Streptococcus faecalis and Pediococcus but definite bands for fpositive? controls; i.e., B. subtilis and B. fluorescens. The presence of cytochromes not only is a character— istic of a particular organism but is also a function of age and cultural conditions. Smith (1954) found that the level of cytochromes present increases with age. Gary (1954) found that B. subtilis grown on complex media lacked cyto— chrome oxidase whereas cells produced on a simple medium contained a cytochrome oxidase. EXPERIMENTAL METHODS The following abbreviations will be used: OAR for oxygen absorption rate expressed as mmoles of oxygen absorbed per liter per min; ATP for adenosine triphosphate; DPNH for reduced and DPN for oxidized diphosphopyridine nucleotide; TPNH for reduced and TPN for oxidized triphosphopyridine nucleotide; CoA for coenzyme A; TPP for thiamine pyrophosphate; G-6-P for glucose-6-phosphate. KDPG for 2-keto—3-deoxy-6— phosphogluconate; RPS-P for ribose-S-phosphate; Ru-S-P for ribulose-S—phosphate; Xu-S—P for xyulose-S—phosphate; F-6—P for fructose-6-phosphate; F-1.6—P for fructose—1.6-diphospate; G-3—P for glyceraldehyde-3-phosphate; DHAP for dihydroxy- acetone phosphate; EMP for Embden—Meyerhof pathway; HMP for hexosemonophosphate pathway; ED for Entner-Doudoroff pathway; TCA for tricarboxylic acid pathway; and OD for optical density. Optical densities at wavelengths in the visible range were measured using a Bausch and Lomb Spectronic 20 colorimeter (Bausch and Lomb Optical Co., Rochester. N.Y.) and those in the ultraviolet range were made using the Beckman Model DU spectrophotometer. 13 l4 Cultures and Cultural Methods Cultures used: All cultures used in this study were obtained from the Northern Utilization Research Division. U.S.D.A.. Peoria, Illinois. Culture designations are those used by the U.S.D.A. Initial studies involving substrate utilization and acid accumulation in growth media were made with strain NRRL B-2043. Later. strain NRRL B-2309—P was used since the U.S.D.A. reported that this strain attained higher populations lg yipgp. All metabolic studies were made with the latter strain. Cultural media and maintenance: Two media were used throughout this study. The first. designated as B—l. was suggested by Hall (1961) as a medium in which good cell yields of B. popilliae had been obtained. This medium was used in the studies conducted with B. popilliae B—2043. A later modification of this medium was made. again through a suggestion from Hall (1961). to obtain higher cell yields and to retain viability over a longer period of time. This medium. designated as B-4. was used principally for the purpose of producing cells for metabolic determinations. Both of these media were used at various times for maintenance of strains by adding 1.5% agar and preparing slants. The composition of these media is given in Table 1. 15 ¢able 1—- Composition of media used for growing cells of _B. popilliae. Ingredients Medium Medium B-l B—4 Yeast extract (DIFCO) 15.0 g. 15.0 g. Glucose 3.0 g. 2.0 g.* KZI-IPO4 3.0 g. 6.0 g. Tryptone 3.0 g. --- Distilled water 1000 ml 1000 m1 pH 7.0 _ 7.2 7-3 — 7-4 *Actually 2.5 g of glucose were added. but it was found by experimentation that 2.5 grams of glucose per liter of medium would assay as 2.0 grams per liter when 250 ml amounts were autoclaved for 15 min at 15 pounds pressure. B. popilliae was maintained principally in B—4 broth. Cultures were transferred at 48 to 74 hr intervals by inoculating flasks containing 250 m1 of medium with a 10 ml inoculum. They were incubated at 30 C on a rotary shaker. Cultures on agar slants were incubated in parallel to assure that the strain would not be lost. This method of maintenance was necessary since viability is easily lost if cultures are stored fin stoc V as is the custom with most other species of bacteria. Production of cells and cell free extracts: Cells for metabolic studies were obtained either by inoculating several flasks of media or a 10 liter carboy. Flasks were shaken at 200 RPM on a New Brunswick shaker1 and cultures 1New Brunswick Scientific Co.. New Brunswick. N.J. 16 in carboys were agitated by forced filtered air. All flask cultures were inoculated with 10 ml of a 24—hr broth culture and carboys were inoculated with four 250 ml 24-hr cultures (1 liter inoculum). After the desired growth had occurred. cells were removed from the medium by centrifugation. washed twice. and resuspended in distilled water. Extracts were prepared by breaking cells with No. 100 glass beads2 in a high speed Servall3 omnimixer. Approxi- mately 12 g (wet weight) of cells were suspended with 40 to 45 g of glass beads in 50 ml of distilled water. The mixture was chilled for 30 min in an ice bath and the blender cup maintained in the ice bath throughout the breaking period which was completed in 10 to 15 min. The extract was centrifuged at 1500 x g for 15 min to remove beads and whole cells. These preparations will be referred to as whole extracts. Separation of the whole cell-free extracts into soluble and particulate fractions was accomplished by centri- fugation at 110.000 x g for 3 hr in the Beckman Model L Preparative Ultracentrifuge.4 Particles thus obtained were 2Minnesota Mining & Manufacturing Co.. St. Paul. Minn. 3Ivan Sorvall. Inc.. NOrwalk. Conn. 4Spinco Division. Beckman Instruments. Palo Alto. Calif. 17 feSuSPended in 0.2 M phosphate buffer. pH 7.4. Protein Determination The Folin-Ciocalteu test for protein determination (Lowry et al.. 1951) was performed on cell extracts in order to quantitate activity in terms of protein content. This reagent reacts with phenol groups (tyrosine and phenylalanine) to give a color intensity which is correlated directly with the protein content of the extract. Bovine serum albumin was the standard. OAR Determination OAR's were determined according to the method of Coreman et a1. (1957). These were run in dimpled 500 m1 Erlenmeyer flasks with urethane stoppers containing 50 m1 of 0.41 N sodium sulfite and 0.001 M copper sulfate. The flasks were shaken at various rates on a New Brunswick shaker. Five ml samples, taken initially and at various time intervals. were pipetted directly into tubes containing dry ice chips which served to stop the oxygenation as well as to agitate the solution during titration. The solutions were titrated with standardized iodine using starch as an indicator. Titration differences were used in the following formula to calculate the OAR: 18 OAR = nfl. titration difference x iodine normality x 1000 x 1 4 5 min Manometric Studies Oxygen consumption and CO2 evolution were determined manometrically with the Warburg respirometer by the procedures outlined by Umbreit et a1. (1957). Flask contents were examined chemically for initial and residual substrate as well as for metabolic products. Isotope Studies Glucose-l—C14 and -6-Cl4 and acetic acid-l-Cl4 were mixed with cold substrate and oxidized by resting cells in the Warburg respirometer. The activity of radioactive sub— strate did not exceed 0.1 uc per vessel except where other- wise noted. Durham tubes cut in half and containing 0.2 m1 of 20% KOH were placed in the center well of the Warburg flasks to trap CO . After the reaction was stopped with 2 4 N H2SO4. the tube was removed with forceps and the contents transferred to a 20 ml sample vial with a bulb pipette. The tube and pipette were successively washed out with measured quantities of distilled water and added to the radioactive sample. Distilled water was added to 1.0 m1. Likewise. samples were taken from the residue and similarly made up to 1.0 m1 volume in the sample vial. 19 Using the method of Goldman (1961). CO was collected 2 fr°H\Ei growing culture of B. popilliae during the oxidation of acetic acid-l—Cl4 in B—4 medium. Five hundred m1 flasks containing 25 m1 of medium and 0.5 uc of label were stoppered with rubber plugs. Each plug was outfitted with a bent glass rod to which a 1.2 x 3.5 cm tube was secured with a rubber band. The tube. suspended above the medium. contained 0.5 ml of 40% KOH to trap all CO from oxidized acetate. Flasks 2 were shaken at 200 RPM at 30 C on a New Brunswick shaker. Individual flasks were removed periodically to examine the KOH for the presence of the label. The composition of the 9scinti11ator solution? (Baldwin. 1961) used for counting carbon-l4 disintegrations is given in Table 2. Table 2. Scintillator solution for counting carbon-l4 disintegrations. Ingredient Amount* xylene 192 ml p-dioxane 192 ml ethanol (absolute) 115 m1 PPO** 2.5 g aNPO** 25 mg naphthalene 40 g Cabosil** 20 g *The ingredients are combined in a Waring Blender and mixed at high speed for three min. **Packard Instrument Company. LaGrange. Illinois. 20 Fifteen ml of the scintillator solution were trans— ferred turneans of a syringe into the 20 m1 vial containing 1 ml of diluted sample. The vial was closed and vigorously shaken to mix the contents. The disintegrations were counted with Tri-carb Liquid Scintillation Spectrometer.5 Benzoic 1 . acid—C 4 was used as a counting standard. Chemical Assays Glucose: The colorimetric determination of reducing sugars as described by Neish (1952) was used for quantitatively determining glucose. As reducing sugars are heated in an alkaline curpic copper solution. the copper is reduced and precipitated as curpous oxide. The cuprous oxide. proportional to the reducing sugar. is determined colorimetrically by the formation of molybednum blue upon the addition of arseno— molybdate. Agigp: Total acids were determined by titration with standard NaOH using measured samples taken from growing cultures or Warburg flasks and centrifuged to remove cells. A celite column described by Wiseman and Irvin (1957) containing celite and sucrose with alpha amine red as the internal indicator for acid detection were used for separation and identification of acids. Solvents consisting of mixtures 5 . . Packard Instrument Co.. LaGrange, IllinOis. 21 of Skally Solve B and acetone are used to elute acids after :introduction of the sample. Eluted acids were titrated with 0.0045 N Ba(OH)2 standardized against potassium acid phthalate. volatile acids were separated from samples of media and warburg flask contents by steam distillation in the presence of H2804 and magnesium sulfate and the distillates titrated with 0.1 N NaOH. Lactic acid was determined by the method of Neish (1952). In the presence of concentrated sulfuric acid. lactate is oxidized quantitatively to acetaldehyde which is determined colorimetrically in the presence of copper sulfate and p-hydroxydiphenyl. Ethanol: Ethanol was determined by the microdiffusion method described by Neish (1952). Using sealed Conway plates, ethanol diffused from the outer section into the center well thereby reducing the dichromate. Ethanol is calculated from the amount of dichromate reduced. Glycerol: Upon oxidation by periodate. 1 mole of glycerol forms 2 moles of formaldehyde and one of formic acid. The method for periodate oxidation outlined by Neish (1952) was followed. The formaldehyde formed in this oxidation was quantitated by a method described by Nash (1953). Glycerol was calculated from the formaldehyde. 22 Acetaldehyde: Acetaldehyde was estimated by the bisulfite binding method detailed by Neish (1952) . Bisulfite. quantitatively bound by acetaldehyde is released upon the addition of sodium bicarbonate. The released bisulfite is titrated with iodine. Acetoin: The method of oxidizing acetoin by alkaline iodine as described by Neish (1952) was employed. After acidification. the excess iodine is measured by thiosulfate titration. The iodine reduced by reaction with acetoin is calculated. Pyruvate: The colorimetric method described by Umbreit et a1. (1957) was used for pyruvate determinations. Pyruvate is reacted with 2,4—dinitropheny1hydrazine and extracted successively with ethyl acetate and socium carbonate. The color. which is directly proportional to the pyruvate content is measured at 520 mu. Enzyme Assays Glucose-6zphosphate dehydrogenase: G—6—P dehydrogenase was assayed according to the method of Kornberg and Horecker (1955). With G—6—P as the substrate. TPN reduction was followed by an increase in OD at 340 mu. Magnesium chloride as well as TPN were supplied as cofactors in a pH 7.5 glycylglycine buffer. 23 Hexose mono hos hate athwa HMP) enz es: The Presence of the complete HMP enzyme system was assayed by supplying R—5-P as the substrate and TPP. MgClz. and TPN as cofactors. Where the complete HMP system is present. TPN is reduced as evidenced by an increase in OD at 340 mu. This enzyme system generates F-6-P and in the absence of ATP. G-6-P will be formed through the action of phosphohexoisomerase. As the G—6-P is generated it is oxidized by the G-6-P dehydrogenase. and TPN is reduced. This method was reported by Sutton and Star (1960). DPNH oxidase. TPNH oxidase. and TPNHEDPN transfer enzyme: DPNH oxidase was assayed by measuring the decrease in OD at 340 mu in the presence of the extract. Likewise. TPNH oxidase was assayed by replacing DPNH with TPNH. The TPNHHDPN transfer enzyme was assayed by placing these two cofactors in the presence of the extract and observing OD readings at 340 mu.. Since no TPNH oxidase was present. a decrease in OD would occur if this enzyme were present. KDPG aldolase: KDPG aldolase (Heath et a1. 1958) was assayed by supplying KDPG. 0.5 umole; DPNH. 1 umole; imidazole buffer. pH 8.0. 10 umole; 0.0002 ml of extract and an excess of lactic dehydrogenase in a total volume of 0.15 ml. If this enzyme is present. pyruvate is generated and DPNH oxidized at a rate in excess of the DPNH oxidase alone. 24 Phosphoketolase: Xu-S-P was used as the substrate ior tfliis enzyme assay. Cuvettes contained Xu-5—P. 0.2 umole; phosphate buffer pH 6.5. 50 umole; DPNH. l umole; MgClz. 0.5 umole; TPP. 0.1 umole; glutathione. 10 umoles,extract. 0.0002 ml; and a-glycerophosphate dehydrogenase and triose- phosphate isomerase in excess. The total volume was 0.15 ml. This enzyme generated G-3—P which. in the presence of supplied enzymes. forms a—glycerophosphate with the oxidation of DPNH. As with the KDPG aldolase assay. the oxidation of DPNH would be in excess of the DPNH oxidase activity. This method was described by Kovachevich and Wood (1955). Aldolase. triosephosphate isomerase. and ppppphofructo- kinase: Aldolase was assayed by the method of Sibley and Lehninger (1949). The action of aldolase converts one mole of F—l.6-P into one mole each of G-3-P and DHAP. The trioses are trapped by hydrazine in a 1:1 proportion as they are formed. The hydrazones are treated with alkali and colori- metrically assayed at 540 mu after the addition of NaOH and 2.4-dinitropheny1hydrazine. Triosephosphate isomerase was assayed by a modifi— cation of the aldolase assay. Hydrazine added initially traps G-3-P and DHAP as they are formed in approximately a 1:1 proportion. By adding the hydrazine after incubation. the triosephosphate isomerase is allowed to act which V:— 2'3“. ‘u‘: u- "1__ 25 results in a G—3-P:DHAP ratio of 4:96. Since DHAP reacts more: strongly with dinitrophenyl hydrazine than does G-3—P. the enzyme is demonstrated by the color differential. Phosphofructokinase was determined by the aldolase assay method with F-6-P and ATP substituted for F-1,6—P. Acetokinase: Acetokinase was determined by a color- metric procedure described by Rose (1955) in which acetyl phosphate. formed from acetate and ATP. is converted to the hydroxamic acid derivative. The reaction of the latter compound with iron (FeCl3) gives a colored compound which is measured at a wavelength of 540 mu. Phosphotransacetylase and the condensing enzyme: The method of Ochoa (1955) was employed for assaying phosphotrans- acetylase and the condensing enzyme at the same time. Acetyl phosphate disappearance proportional to extract concentration in the presence of CoA and oxalacetate was used as evidence for the presence of these two enzymes. Controls in which CoA and oxalacetate were omitted separately were used to demonstrate specificity. Acetyl phosphate was determined by the method of Lipmann and Tuttle (1945). Glucose oxidase and glucose dehydrogenase: The manometric determination of glucose oxidase described by Bentley (1955) was used. The direct oxidation of glucose by cell extracts is measured by respiratory oxygen consumption. 26 An alteration of the method of Strecker (1955) for glucose dehydrogenase was made. Instead of observing an increase in OD at 340 mu caused by the reduction of DPN in the presence of glucose and the enzyme. these elements were supplied and oxygen consumption was measured. If the enzyme were present. DPN would be reduced to DPNH and DPNH would be oxidized by the DPNH oxidase known to be present resulting in oxygen uptake. Catalase: Catalase activity was assayed by three methods: ‘yig.. (a) dropping H202 on agar colonies of B. popilliae. (b) tipping H202 into buffered cell free extracts and manometrically observing oxygen release. and (c) by the iodometric titration method of Esrbert (1955) in which residual HIZO2 is determined after various periods of time in the presence of the extract. Peroxidase: A method described by Dolin (1957) for assaying a flavin linked peroxidase was used to establish the presence or absence of this enzyme. Dolin used this method for establishing peroxidase activity in Bpgppppgppgppigpgpglip and Walker (1963) used it with lactobacilli. Briefly. DPNH oxidase activity was assayed with a decrease in OD at 340 mu. The same test was then run with peroxide added. A faster rate of OD decrease indicates the presence of this enzyme. The assay was run at pH 5.4. 27 Hydrogen_peroxidepproduction: Whole cell free extracts as well as soluble and particulate fractions separated by ultracentrifugation were used to oxidize DPNH in the‘Warburg respirometer. When oxygen consumption ceased. catalase was tipped and oxygen return was measured. Net return of oxygen (less endogenous) indicates the presence of hydrogen peroxide. Cytochromes: Suspensions of extract particles (110.000 x g) were reduced by a few crystals of sodium hydrosulfite as described by Dobrogosz et a1. (1962). A difference spectrum between the oxidized and reduced particles was obtained by the split beam method employing the use of the Cary Model 15 Spectrophotometer.6 Absorbancy differences at wavelengths varying from 400 to 700 mu were determined. 6Applied Physics Corp.. Monrovia. Calif. RESULTS Initial studies with B. popilliae involved establish- ing growth curves from liquid shake cultures. Shaking 50 m1 of medium in 500 m1 flasks at 200 RPM resulted in growth approximating 2 to 3 x 108 cells per ml in 24 to 36 hr. The viability dropped approximately 99.9% between 48 and 72 hr. Growing cultures were examined for pH changes. The steady drop in pH indicated a continuous accumulation of acid. When acid production was compared with glucose utilization. it was found that the glucose oxidation products were quanti- tatively accumulating as acid intermediates rather than proceeding through to terminal oxidation (Fig. 1). Since one of the overall objectives of this project was the achievement of high populations of cells with pro- longed viability. the effects of oxygen and pH on growth and cell viability were studied. Efforts were made to determine whether a specific level of oxygen was necessary for high populations. Furthermore. the rapid drop in pH was studied as a possible inhibitor of increased growth as well as a factor which affects overall viability. The results (Table 3) 28 ,_.. ._ -_ .w i' ‘0 .- - a, 29 25-1} 1' 7.5 4L “‘L q \ of; 20.. I ‘L 7.0 Q: 1’ .1 p" v 1’ g x . a. '5 1 ’I "' 6. 5 .3 ,z k. ACID J 3 0X ACCUMULATIO i g GLUCOSE .’ IO .. UTILIZATION X 2 ,’ .. 6 .0 O) '1‘. ‘\ O \‘s 2 E 5- «5.5 1? l l 0 l2 24 36 48 HOURS Figure 1. Glucose utilization. acid accumulation. and pH changes in a broth culture of B. popilliae. Fifty m1 of B—l medium contained in a polystyrene plugged 500 m1 dimpled f1a$. CH3COOH C02 2H CH3COCOOH + 2H+ _I- CH3CHOHCOOH 2 CH3COCOOH + H20 —u- CH3COOH + CH3CH0HCOOH + CO2 Uhder aerobic conditions, molecular oxygen can serve as the hydrogen acceptor, and there is much less lactate produced per mole of substrate dissimilated. Pathways of Glucose Catabolism With the products from glucose established, efforts were made to determine the metabolic routes for glucose catabolism. The first approach involved the use of metabolic inhibitors. Although not definitive. inhibitors give strong presumptive evidence of metabolic pathways involved. Next, estimation of respirative C1402 resulting from equal amounts of glucose—l-C14 and —6-Cl4 gives strong indications of the various pathways. Little or no radioactive CO2 signifies purely EMP whereas the appearance of the label from glucose-l- C14 but not from -6—Cl4 in the CO indicates HMP or a hetero- 2 lactic pathway. Large amounts of isotope from glucose-6-Cl4 in the CO2 would indicate terminal oxidation. The third method involved the direct assays for individual enzymes. With essential agreement of evidence gathered from all three approaches, the establishment of metabolic routes for 40 the organism can be made with confidence. Inhibition of glucose oxidation: Two inhibitors, iodoacetate and fluoride, were used to gain preliminary information concerning pathway participation. Iodoacetate inhibits G-3—P dehydrogenase and fluoride inhibits enolase. Inhibition of glucose oxidation was measured by decrease in oxygen consumption as compared with the non-inhibited control. Iodoacetate (0.01 M) inhibited glucose oxidation 100%. 0.01 M fluoride inhibited approximately 50%. and 0.1 M fluoride inhibited 100%. Fig. 4 shows the effects of various levels of fluoride on glucose oxidation. These data indicate that the ED and the phosphoketolase pathways are unlikely routes for glucose oxidation in B. popilliae since they should both be insensitive to iodoacetate and low level of fluoride. Both the EMP and the HMP systems would be greatly affected by both of these inhibitors. Distribution of i§ptope frqmrgluchg-l-Cl4 and -6—Cl4: Equal amounts of glucose-l-Cl4 and -6-Cl4 were catabolized by B. popilliae resting cells and CO was collected for radio- 2 activity counting. Relatively little label from the 6th carbon was obtained. However. when the incubation period was extended, increasing amounts of CO2 appearing from carbon 6 were observed. Lowering the pH to about 6.0 essentially stopped the formation of all C1402 from glucose I000- 900" 800" 83‘ 99 y! 02 cowsuueo O 3 3 Figure 4. 41 O GLUCOSE CONTROL 0 o 0.00!!! J NaF ° )0 I O // o/ ,’ 0.0! M /._.s— a F / 0 1' O [’0 /” ,0 0.l M I NoF ,0- 0” C 0"- LEndoq. L l 1 IO 20 30 4O 50 60 TO 80 SO IOO MINUTES Effect of fluoride upon glucose oxidation. Reaction vessels contained 32.25 mg dry wt. of cells; 0.5 ml of 0.3 M phosphate buffer, pH 7.0; 0.5 ml of 0.0667M glucose; and fluoride as indi- cated. One side arm held 0.2 ml of 2 M H2804 and the center well contained 0.2 ml of 20% KOH. Water was added to 3.0 ml. The gas phase was air. ‘ 42 "6 l4 . . _ , —C» . A Significant amount of label appeared in the CO2 from glucose-l-C14. and the relative quantity of CO2 from the first carbon remained the same during extended incubation. The method of Wang et a1. (1958) was used to estimate glucose participation in phosphogluconate decarboxylation. Calculations for this participation were made according to the following formula: 61—66 G =—G— p t where G = the fraction of glucose participating in the phosphogluconate decarboxylation G and G = total activity of respiratory CO 1 6 . . . utiliZing equal amounts of Cl and C6 labeled glucose 2 Gt = total activity of administered substrate The participation of phosphogluconate decarboxylation was quite small (2.1%) when the vessel contained 60 mg (dry wt) of cells and an atmosphere of air. However. this could be altered by varying the cell concentration and/or the atmosphere. The effect of the atmosphere upon the participation of the HMP enzymes in oxidizing glucose was determined by allowing cells (48 mg dry wt per vessel) to oxidize glucose in the presence of air and pure oxygen. The percent participation was 9.05% in air and 39.05% in oxygen. 43 Thus. the availability of oxygen obviously controls the pathway used to a great degree. Enzymes of catabolic pathways: A key enzyme in HMP system is the G—6—P dehydrogenase. The presence of this enzyme was established as evidenced by the reduction of TPN with G-6—P as substrate (Fig. 5). The soluble nature of the enzyme was demonstrated when an extract was separated into its soluble and particulate fractions by ultracentri- fugation (110.000 x g for 3 hr). The activity was concen— trated in the supernatant. The possibility of direct oxidation of glucose to gluconate was investigated by manometric tests for both glucose oxidase and glucose dehydrogenase as described in the section on methods. Negative results were obtained for each. Evidently. glucose has to be phosphorylated by this organism before it can be further oxidized. Since G—6-P oxidation can lead to one of three pathways. the possibility of each was examined. The presence of the ED pathway was tested by assaying for one of its key enzymes. KDPG aldolase. No increase in DPNH oxidation occurred beyond that of DPNH oxidase alone. Likewise. the extract was examined for the presence of phosphoketolase and this was also found missing. However. when RPS-P 44 O 0.25-- / ’/;3 0-6-P o TPN L, 0.20" Mg+§ O / . E 0.:5 -- o 6 o/ 3, o e-e-P ‘2 0:0- ‘3 o' I 0.05- TPN SIMQ++ o o O o o o o' 2 3 MINUTES Figure 5. G1ucose—6—phosphate dehydrogenase activity in cell free extracts. All vessels contained dialized extract, 1.0 m1 of 0.04 M glycyl glycine buffer, pH 7.4. Glucose—6—phosphate. 2 umoles; TPN, 0.9 Hmole; and MgCl , 20 umoles were added as indicated. Water was added to 3.0 ml. 45 ‘Nas used as the substrate in the presence of the proper co- factors. TPN was reduced to TPNH indicating the presence of the HMP enzymes (phosphoriboisomerase. phosphoketopentoepime- rase. transketolase. and transaldolase) and of phospho- hexoisomerase. Efforts to demonstrate the presence of the upper half of the EMP pathway were made by assaying for each enzyme. The presence of aldolase was demonstrated by the colorimetric procedure of Sibley and Lehninger (1949). By omitting the hydrazine until after the incubation period. a more intense reaction was obtained. thus demonstrating the presence of triosephosphate isomerase. With the substitution of F—6—P and ATP for F-l.6—P. phosphofructokinase was shown to be present in the extract. The presence of phospho- hexoisomerase has already been referred to in the establish— ment of the HMP system. No attempts were made to assay for the enzymes of the lower half of the EMP system since they are required for both this system and the HMP. and all of the data indicate the participation of both pathways. Terminal Oxidation of Acetate Initial attempts to oxidize acetate with resting cells of B. popilliae failed. Later. it was found that an alkaline pH was necessary rather than an acid one. Further— more. cells harvested during the logarithmic phase of growth 46 CDKidized acetate very poorly whereas cells harvested in the Stationary phase oxidized acetate more rapidly. Incorpor- ation of acetate in the growth medium failed to alter this characteristic of log phase cells. Fig. 6 shows the difference in acetate oxidation by similar concentrations of cells harvested after 9 hr and 24 hr incubation. During the course of investigating acetate oxidation by B. popilliae (designated as the rA? strain). another culture of this strain (designated as FD?) was obtained from the Nbrthern Utilization Research Division. U.S.D.A.. Peoria. Illinois. The PD? strain was found not to have the ability to oxidize acetate. at least under the same conditions as the TA? strain; i.e.. at an alkaline pH using stationary phase harvested cells. Both of these strains oxidized glucose in the Warburg respirometer (Fig. 7) with two obvious differences; viz.. strain PAP had a 002 of 13.2 pl 02 per hr per mg cells and continued to utilize oxygen after the glucose had disappeared. while strain TD? had a 002 of 21.5 ul 0 per hr per mg cells and abruptly stopped con- 2 suming oxygen with the exhaustion of the glucose. Apparently. a strain with the acetate oxidizing characteristic was selected by the method of maintaining the cultures. The ?A? strain was maintained on B~4 broth. and transferred every 48 - 72 hr with continuous incubation. I Figure 6. 47 o 220T 200-- 24-Hn I80-- CELLS 1. 0 I60-- 0 0 I40"- h: s g '20" C) 9 loo:- 0 6“ N 80-- a 60-- S-Hn 40-- CELLS i «- O / 20-- C/ .,o/' . J 1 L 1 l l 30 60 90 I20 I50 l80 2l0 MINUTES Ability of log phase and stationary phase harvested cells to oxidize acetate. Reaction vessels con— tained 9 hr harvested cells (35.2 mg dry wt.) or 24 hr harvested cells (32 mg dry wt.); 0.5 ml of 0.3 M phosphate buffer, pH 7.4; 120 Umoles of acetate. 0.2 ml of 2 M H2804 was contained in a.side annand0.2 m1 of 20% KOH in the center well. Water was added to 3.0 ml. The gas phase was 100% 02. I 8001 ISOO' I400- IZOO‘ IOOO 600' Jul 02 cowsuuso 400' 200' L Figure 7. .// o/° "A" STRAIN \ o/ .— V". 'g ./ o " 0" STRAIN / O - / 1 I I 1 1 l 1 40 80 l20 I60 200 280 280 MINUTES Glucose oxidation characteristics of acetate oxidizing (A) and acetate non-oxidizing (D) varieties of B. popilliae NRRL B—2309P- Reaction vessels contained 38 mg ("A" variety) and 35.3 mg ("D" variety) of cells; 1.1 ml of a 0.3 M phosphate buffer, pH 7.4; and 0.5 m1 of 0.0667 M glucose. 0.2 m1 of 2 N H2804 was contained in a side arm and 0.2 m1 of 20% KOH in the center well. Water was added to 3.0 ml. The gas phase was 100% 02. 49 During most of this time. acetate was present in substantial announts with the result that any mutant with the ability to oxidize acetate would have a selective advantage. The PD? strain. on the other hand. had been maintained on agar slants. Metabolic products in this situation would more easily diffuse away from the immediate vicinity of the cells. The rA? strain is apparently a mutant since it retained most of the growth characteristic of the original culture and it was checked by the U.S.D.A. for its patho- genicity and was found able to cause the Fmilky disease? in the Japanese beetle larvae. The PD? strain was not used for any other experiments during these investigations. To determine the rate of oxidation during growth. acetate-l-C14 was incorporated in the B—4 growth medium. The CO2 was trapped in alkali and periodically examined for radioactivity. No significant amount of label was detected in the CO through the first 12 hr of incubation. Only 2 after the stationary phase had begun was there any accumu— lation of C1402 (Table 8). Resting cells of g. popilliae were induced to oxidize glucose primarily to acetate by supplying a pure oxygen atmosphere. Fig. 8 shows the relationship between oxygen consumption. glucose oxidation. and acetate accumulation. The acetate was oxidized after the glucose had disappeared as evidenced by the drop in Jl HOLES and y EOUIVALEN TS Figure 8. 50 llO" ,,—"’o (DA/b OXYGEN IOO-- - -—o LGLUCOSE x 2 9dr "‘~‘ ‘\ 80-1 ~51. ACID I \A II 70» I I 60.. I o” 50-- 1 ,0 4o-- 30.. 20.:- I 0 IO-- J l l l l l 50 IOO I50 200 250 280 MIN UTES Comparison of glucose oxidation with oxygen consumption and titratable acidity. Reaction vessels contained 46.2 mg dry wt. of cells, 1.0 ml of 0.3 M phosphate buffer, pH 7.1; and 0.5 ml of 0.0667 M glucose. 0.2 ml of 2.0 N H2SO4 was contained in side arms except for those vessels which were used to determine titratable acidity. The well contained 0.2 ml of 20% KOH and water was added to 3.0 ml. The atmosphere was 100% 02. 51 tidaratable acidity with a continued oxygen consumption. Table 8. Oxidation of acetate-l—Cl4 by a growing culture of Q. popilliae. Incubation Time %’of Label in C02 6 hr 0.053 12 9 0.103 24 V 2.25 36 9 8.70 48 F 16.00 120 9 31.00 0.05 umole of acetic acid l-Cl4 (0.5 uc) was incorporated into B—4 broth. The flasks were stoppered with rubber plugs and the C02 was collected in 40% KOH. Individual flasks were removed periodically to examine the KOH for radioactive label. The percent label contained in the 002 was based on the total amount initially present in the medium. When glucose was oxidized at pH 6.0, oxygen consumption ceased at the time when glucose was depleted. Likewise, no 14 significant amount of C140 appeared from glucose-6-C . 2 This is further evidence of the lack of terminal oxidation of the accumulated acetate at this pH. A number of observations were made which strongly indicate the presence of an operative TCA cycle in g. popilliae. First, during the oxidation of acetate, oxygen and CO2 were liberated in a 1:1 ratio which is the theoretical relationship for the TCA cycle. Arsenite (0.01 M) inhibited oxidation of acetate 100%” Since this inhibitor blocks the 52 deuzarboxylation of d-keto acids, the point of inhibition is most likely the decarboxylation of d-ketoglutarate. The complete inhibition is evidence against a glyoxylate shunt. Finally, three TCA intermediates were oxidized by whole cells. The three, succinate. malate. and fumarate were oxidized at both pH 6.1 and 7.6. However, most rapid oxidation took place at pH 6.1, probably because the acids are less dissociated at this pH; and, thus, the cell is more permeable to them. Fig. 9 shows the oxidation of these three acids compared with the oxidation of acetate at both pH values. It will be seen that only acetate responded more favorably in the alkaline range. More direct evidence for the TCA cycle was obtained when enzymes linking acetate to the production of citrate were demonstrated. Using the method of Rose (1955), acetokinase was demonstrated in a cell free extract of g. popilliae. In the presence of this enzyme, acetyl phosphate is formed from acetate and ATP, and this is converted into its hydroxamic acid by reacting with neutral hydroxylamine and the color is developed by complexing with FeCl The 3. development of color, read at 540 mu, was directly propor- tional to enzyme concentration (Table 9). Evidence for both phosphotransacetylase and the condensing enzyme was obtained by measuring the disappearance r“! 53 MW (0) o pHOJ / IZO” o Suoclnay 80‘! 4o. / Malena al..-«3:120 g o.‘é8:0/ —Fumoran} a) \. Acetate» § \1. -1_—. Q -40 . 3 30 4O 60 80 I00 I20 g, ' MINUTES 0 2 I60 “” " puts IzoJL - 80 Acetonu 0/ F ./ 40., /Succlmto,‘ /’ _guualatc /:_..-=='""—-—-P___3r""" __r_u-;---9Jummto 20 4O 60 80 DO I20 MINUTES . Figure 9. Effect of pH upon the oxidation of acetate, succinate, fumarate, and malate by resting cells of g. popilliae.° Reaction vessels con- ‘tained 41.4 mg dry wt. of cells (48 hr harvest), 0.5 ml of 0.2 M phosphate buffer (final pH shown above); and 120 umoles of"substrate. 0.2 ml of 2 M H2804 was contained in the side arm and 0.2 ml of 20% KOH in the center well. Water was added to 3.0 ml. The atmosphere was 100% 02. \l-lx, 54 Off acetyl phosphate in the presence of the extract, CoA. and oxalacetate (Table 10). Where either CoA or oxalacetate were omitted, the level of acetyl phosphate remained essenti ly the same. The reaction was proportional to the concen- tration of the extract. Assaying for acetyl phosphate was performed by the method of Lipman and Tuttle (1945). Table 9. Acetokinase activity in extracts of g. popilliae. Extract Concentration CD at 540 mu ane (control) 0 3 mg protein 0.34 6 mg protein 0.70 The reaction mixture contained 0.3 ml of stock sub- strate (3.2 M potassium acetate; 1.0 M tris buffer, pH 7.4; and 1.0 M Mgclz in a volume ratio of 25:5:1), 0.35 ml of neutral hydroxylamine, 0.1 ml 0.1 M ATP, and water to 1.0 ml. The mixture was incubated at 29 C for 2 min in a water bath and stopped by the addition of trichloroacetic acid. Color was developed by the addition of 4.0 m1 of FeCl3 (5% in 0.1 N HCl). Electron Transport System Methods used to characterize the electron transport system involved enzyme assays. inhibitors. and direct spectrophotometric absorption patterns. Initially, assays were made for oxidases of reduced pyridine nucleotides. 55 ‘Taflale 10. Phosphotransacetylase and condensing enzyme activities in a g. popilliae extract. Extract Concentration OD at 540 mu 28 mg protein. no oxalacetate 0.54 28 mg protein. no CoA 0.58 14 mg protein 0.43 28 mg protein 0.35 The reaction mixture contained 25 umole phosphate buffer, pH 7.4; 10 umole of cysteine; 20 umole oxalacetate; 10 umole acetyl phosphate. and extract. Water was added to 10 ml. Omissions were made as indicated. After incubation at 25 C for 10 min. trichloroacetic acid was added and acetyl phosphate was determined by the method of Lipmann and Tuttle (1945). Examinations for a DPNH oxidase. TPNH oxidase. and a TPNH-DPN transfer enzyme were made. An active DPNH oxidase was readily demonstrated but neither of the latter two was found. DPNH was oxidized by both the particulate and soluble fractions of the cell extracts. The activity was highest in the particulate fraction (Fig. 10). Assays for the presence of catalase were made by dropping 3% H202 on agar colonies, tipping hydrogen peroxide into warburg vessels containing cell extract, and by iodometric titration. No bubbles appeared when H202 was dropped on g. popilliae colonies nor was there any oxygen release measured when 0.5 ml of 1.5% H20 was tipped 2 into a Warburg vessel containing cell extract. Neither was there any significant evidence of catalase as 56 0'50? ‘\\\b a \o 0 “\~CL\\~ \ O 0.40" X. \o\ x SOLUBLE °\o E‘ X o FRACTION/ ‘0 \ o ‘- 3) oso-- \ k \0 WHOLE EXTRACT ‘ \. k/ 8 0.20,, PARTICULATE \ FRACTION J ‘1 ‘\~.'.K‘~ "<1 0J0 l l l 1 L n n 4 0.25 0.50 0.75 l.0 I25 L5 I75 2.0 MINUTES Figure 10. DPNH oxidase activity in whole cell free extracts and in the soluble and particulate fractions. The following extract concen- trations were used: whole extract, 0.5 mg protein; soluble fraction, 1.6 mg protein; and particulate (110,000 x g) fraction, 0.35 mg protein. Cuvettes also contained 0.5 umole DPNH and 0.5 ml of 0.2 M phosphate buffer, pH 7.0- Water was added to 3.0 ml. / .— / . I ‘ 57 determined by iodometric titration (Table 11). Table 11. Assay for catalase in a g. popilliae extract. Minutes contact between ml 0.0172 N thiosulfate H202 and cell extract to titrate 0.00 0.76 0.25 0.74 0.50 0.74 0.75 0.74 1.00 0.72 The reaction mixture contained 5.0 m1 of H O . in 0.01 M phosphate buffer. pH 6.8. and 1.0 ml of extract. The reaction was run at 25 C and stopped with 2.0 m1 of 1.0 N H SO . The H O was determined by adding 0.5 m1 of 10% K1 and one drop of 1% ammonium molybdate and titrating with sodium thiosulfate. Since catalase was apparently missing, it was particularly important to determine if hydrogen peroxide was produced. DPNH was oxidized by the whole extract as well as by the particulate and soluble fractions in the Warburg respirometer; and the production of H202 determined by oxygen release upon the addition of catalase. It will be seen (Fig. 11) that H was produced in the presence of 202 the soluble but not the particulate fraction. On tipping in catalase, there was only 2% oxygen return with the whole extract, 36.5% with the soluble fraction. and none with the particulate fraction. If all 02 consumed was converted into H202, a 50% return would be theoretical. However. 58 CATALASE TIPPED \| ISO- O’O—O— \ I40 O’O’O’O’ ..- "' o/ WHOLE EXTRACT :' l 3 '20“ PARTICULATEI : FRACTION”: | a g IOOJ- ’9 e 9 I " .800 SOLUBLE /8/ I w FRACTIOIQIa I O I V 600 .. 8/ I. . x I I 40. f/ I I . I I I ' ' 20 I . l l I I I I I I I IO 20 30 4O 50 so MINUTES Figure 11. Oxidation of DPNH in the Warburg respirometer by a whole cell free extract and by the . soluble and particulate fractions. The following concentrations were used: whole extract, 15 mg protein; soluble fraction, 1.2 mg protein; and particulate (110,000 x g) fraction, 21 mg protein. Reaction vessels also contained 20 umoles of DPNH and 0.5 m1 of 0.2 M phosphate buffer, pH 7.6. A side arm contained 0.5 m1 of catalase and the center well 0.2 m1 of 20% KOH. Water was added to 3.0 ml. The atmosphere was 100% 02. No substrate was tipped into particulate fraction until 10 min before catalase was tipped. 59 'there is always a problem with substrate inactivation of «catalase; and. thus, the percentage of oxygen going to peroxide cannot be estimated from these data. 'With the absence of catalase and the obvious pro- ciuction of hydrogen peroxide, a peroxidase would be of much importance to the cell. The assay described by Dolin (1957) has been used extensively for assaying flavin linked 'bacterial peroxidases. The activity of the DPNH oxidase is established and a net difference observed when peroxidase is present. Fig. 12 shows that peroxidase activity was present in a Streptococcus faecalis extract but no activity was found in the g. popilliae extract. Cyanide (0.01 M) and azide (0.01 M) inhibited completely the oxygen consumption by the particulate extract fraction (Fig. 13a). Hewever, where the same inhibitors were used with the soluble fraction, no inhibition of oxygen uptake occurred (Fig. 13b). Upon tipping catalase. about 30% oxygen return was observed with the control and the cyanide containing flask. No return occurred in the azide flaSk. waever, azide may have inactivated the supplied catalase. Extract particles were treated with carbon monoxide and were compared with non—treated particles for their ability to oxidize DPNH. The carbon monoxide inhibited this 60 .33 o S. FAECALIS O .340 . °\o o. y \o OXIDASE ~.‘~‘~ \OLO O .“~~ 9 5“ \o ' ~~ .2 3- PEROXIOASE \0 ‘.~“‘, .200 ~~. 0 ~- .“s‘. N \°\O xx. OXIDASE .220 ‘N. 4+ PEROXIOASE \~‘2 :‘.IBCI I l I l 1 I |~‘\- E 0.25 0.50 0.75 I.OO l.25 L50 Ln 2.00 0 MINUTES " In L. w: .340 Q B.POPILLIAE ° .30 05.. 3\8\ 026° ‘9‘ a\ .220 ‘~g\ \3 \a Figure 12. ‘— faecalis and g. popilliae. I I l 1 I I I n 0.25 0.50 0.75 l.00 L25 L50 I.75 2.00 MINUTES Peroxidase activity in extracts of Streptococcus To assay for DPNH oxidase, cuvettes contained 1.0 ml of 0.1 M acetate buffer, pH 5.4, 0.5 uM DPNH, and 0.5 mg protein. To assay for peroxidase, 4 umoles of hydrogen peroxide were present in addition. Water was added to 3 m1. 61 I a I CATALASE TIPPED RARTICULATE FRAC o— —o I40" 7 I Izo-I /° CONTROL I 0 I00" 0 ‘i‘ l a .. a, BOI I k 0 O u so" I ON 40 V A 0 I :‘ J 2° ’ CYANIDEN I 0 o—O—o—‘o—‘O‘ o 0 <:— AZIOE cl , . 9 9 .g?--9—— 9 ‘5 IO I5 20 25 30 35 5 MINUTES 0 “” CATALASE III ‘ TIPPED AZIDE : SOLUBLE FRACTION / “’ 60- ""' g ’3 (CYANIDE Q) ggfl‘ I N 40" O 3/' SeCONTROL ‘N’ 2(Jv .d’f’ ‘ I 5 IO I5 20 25 30 35 5 MINUTES . Figure 13. Effect of cyanide and azide on DPNH oxidation by soluble and particulate fractions of a cell free extract as measured by oxygen consumption. Re- action vessels contained extract protein (soluble, 1.2 mg; particulate 4.2 mg), 20 umoles of DPNH and 0.5 ml of 0.2 M phosphate buffer, pH 7.6. ' A side arm contained catalase and the center well 0.2 ml of 20%.KOH. Water was added to 3.0 ml. The atmosphere was 100%.02. Cyanide (30.umoleS) and azide (30 ILmOleS) were added as shown above. 62 reaction nearly 100% (Fig. 14). A difference spectrum was obtained with the Cary Model 15 Spectrophotometer by the split beam method. One cuvette contained particulate extract reduced with sodium hydrosulfite and the other cuvette contained oxidized extract. The results (Fig. 15) give positive evidence for the presence of cytochromes. The peaks at 562 mg and 530 mu correspond to the a and B peaks of cytochrome b; the peaks at 602 mu and at 426 mu are probably due to cytochrome al. The trough at 452 to 462 mu is likely indicative of flavo- protein. The 555 mu a peak for cytochrome c is missing. Hewever, cytochrome components differ between organisms and even within an organism depending upon cultural conditions and age. It is important. however. to note positive evidence for this mode of electron transport. «h‘m’ at- ‘¢ w..— _ _ H ‘.---_L 63 .SOOTo-‘n‘r‘. . r ' ~0‘0 260" CO TREATED K - O E 220" Q . V' ' . "’ .IaO-- 0 I. _ \ 4‘ o Q .l40I- O \ CONTROL IOO“ °/\ ° 0 . \o .060" .0207 I I I I L I 0.25 0.50 0.75 I.OO I25 I.50 MINUTES Figure 14. Effect of carbon monoxide upon DPNH oxidation by the particulate fraction of a cell extract. Cuvettes contained 0.5 umole DPNH, 0.7 mg particulate extract protein, 0.5 m1 of 0.2 M phosphate buffer, pH 7.0 and water to 3.0 ml. CO was bubbled through the treated reaction mixture for 1 min before adding substrate. I .kumfiouonm ouuowmm ma H0002 ammo wsu nuHS uomuuxm pmuwcflxo may umsflmmm UOHSmOOE Esnuummm mus can VONmmmz Suez OOUSUOH OHOB mmfionaooumo .uomuuxm HHOU m mo coauumum mamasofluumm OOUSUOH .m> anacaxo cm mo Esnuowmm mosmummman .ma mnsmsm 3.. z. :Szunuis a. a. a. a. a: an as .3 .8 .7 .7 .7 .7 a. .6 nu a. a. .7 .6 .7 O m o O O o m. o m m m. m m. m d u q q 1 q n m u $ u n :86 4 6 :«od :30 nu a cod :30 nxo . 1......— h'wfifi' .7"- . ' \' DISCUSSION g. popilliae catabolizes glucose to pyruvate via the EMP and HMP routes. The evidence for this, provided by enzyme assays, isotopic studies, and inhibitor data, was in complete agreement. Thus, the pattern is the same as wi most aerobic species of bacteria studied to date. Wang et a1. (1958), using isotopic techniques studied pathway participation by several aerobic and facultative microbial species. Of the nine species studied, seven catabolized glucose via both the EMP and HMP routes, one by the HMP and ED routes, and one by the ED route only. The HMP route was found to be used primarily during growth, and is un— doubtedly useful in the production of TPNH for synthesis of lipids, etc. The extent of the participation of the HMP route is influenced in g. popilliae by the atmosphere provided. Increased oxygenation increases the percent participation. It is undoubtedly very active in shake cultures since it was shown that these conditions resulted in a purely respiration type of metabolism. Although g. popilliae contains the essential enzyme for dissimilating glucose gy glycolysis. glucose is not fermented in the absence of air by resting cell suspensions 65 a“..- - y: 5' 7 66 IX small amount of oxygen is an absolute essential require— ment for glucose to be dissimilated. Apparently some need is being supplied by the presence of oxygen that otherwise is not being met. It is possible that sufficient ATP is not generated by glycolysis and the presence of oxygen may provide enough through oxidative phosphorylation for the continued movement of glucose through glycolytic system. The organism may contain an active ATPase which is respon- sible for such a limitation. Oxygen affects the type of acid produced by this organism. With increased oxygen, acetate is produced almost exclusively whereas with minimal oxygen present, lactate is preferentially produced. Although the lactate producing potential is present in an oxygen atmosphere, apparently oxygen is the preferential hydrogen acceptor over possible organic acceptors such as pyruvate. This is not an unusual phenomenon. Neish et a1. (1945) found that the presence of air increased acetate and depressed lactate when com- paring aerobic and anaerobic dissimilation of glucose by g. subtilis. Similarly, Ehrlich and Segel (1959) found that increased aeration depressed lactate production. Gary and Bard (1952) also found corresponding relationships between the type of acid produced and the presence of oxygen in the atmosphere when working with g. subtilis. .45 "T" I 67 The E. popilliae strain used in this study was found to have the ability to oxidize acetate whereas a more recently acquired culture of the same strain was found not to have this ability. Both, however, were able to cause the Pmilky disease? of the Japanese beetle larvae and had similar cultural characteristics. The acetate oxidizing strain was maintained in liquid culture where acetate accumu— lates and thus an acetate oxidizing strain would have a selective advantage. It would be of considerable interest to study the mutation rate of this organism since it produces H202 and apparently lacks an enzyme to break it down. This is a known mutagen and the organism may be more susceptable to mutations than most. Nakata and Halvorson (1960) reported that g. cereus accumulates acetate and pyruvate during the log phase of growth, dropping the pH of the growth medium to about 4.9. It apparently does not have the constitutive enzyme comple- ment in vegetative cells at this stage to oxidize the acetate. However. when the growth phase:h completed. acetate is oxidized and the pH rises. These workers found that the oxidation of acetate during this phase not only takes place simultaneously with sporulation. but that the presence of acetate is apparent- ly necessary for this cellular change. The route for acetate oxidation was described by Hanson. Srinvasan. and 68 Halvorson (1962) as through acetate kinase and transacetylase. g. popilliae sporulates readily in the Japanese beetle larva but very little success has been achieved in 23339. The oxidation of acetate appears to proceed by the same route as that used by g. cereus. i.e.. via acetokinase and phosphotransacetylase. waever. g. cereus oxidized acetate at pH 4.9 and g. popilliae will perform this operation only near neutrality or above. This seems to indicate that an active transport for acetate is involved since acetate is largely in the undissociated state at neutral pH levels, and thus, could not be likely to enter the cell by diffusion. This is in contrast with the oxidation of some TCA inter— mediates which were oxidized by whole cells best in the acid pH range, probably because these acids enter the cell by diffusion. Recent work by Costilow (1963) indicates that acetate oxidation in g. popilliae is related to sporulation. Of course. it is possible that the cell is so permeable to acetate at low pH levels that the acid accumu- lates in the cell to a high extent. This could cause a pH change in the cytoplasm and result in an inhibition of the oxidizing enzymes. There may be several reasons as to why growth and viability are limited in g. popilliae. The accumulation of 69 hydrogen peroxide and acids may be in part or wholly responsible for this. Although the cell has the potential to produce hydrogen peroxide, it produces no catalase or peroxidase. Consequently, this harmful product can accumu- late in this organism whereas most other aerobes and facul- tative aerobes have mechanisms for its breakdown. Dutky (1963) has reported that he has achieved high populations of cells of this organism which retain their viability over a long period by culturing in deep broth cultures with added riboflavin. Here, where there is a low oxygen level, another final electron acceptor may be utilized circumventing the requirement for oxygen and eliminating the production of hydrogen peroxide. This may possibly simulate conditions present in the larvae. Certainly, studies should be conducted utilizing other potential electron acceptors. The accumulation of acids may be detrimental to the cell. It is well known that acetate is more harmful to most bacteria than is lactate. As the pH is lowered, most cells are more permeable to undissociated acids and more harm is effected. Acetate obviously gets into the cells at alkaline pH values since it is oxidized by at least the one strain; and, as mentioned above, may be more readily taken up in acid pH ranges. Therefore, mere neutralization may not result in much retention of viability. Data were 70 presented which indicated that this indeed was the case. Little acetate is produced when oxygen is greatly limited. This might explain the prolonged viability observed by Dutky (1963) in deep broth cultures. The apparent need of acetate for sporulation and its possible detrimental effect on the cell is an apparent paradox. It is possible that the larvae may supply acetate oxidation products to the bacterium which may be as active as acetate in stimulating and supplying nutrients necessary for sporulation; or the larvae may control the levels of acetate and the pH at just the proper level for sporulation to occur. Such an approach may well be a fruitful one in vitro. SUMMARY Initial studies with g. popilliae showed that glucose is catabolized primarily to acid intermediates. Because of the rapid pH drop in growing cultures, tests were performed to determine whether neutralization of acids with sterile NaOH would increase cell yields and improve viability. Shaking cultures at various speeds also provided a series of oxygen levels. Results showed that neutralizing acids and maintaining an OAR of 1.0 increased cell yields about 100%Ibut no significant increase in viability was achieved. Carbon balances demonstrated lactic acid, acetic acid, and CO to be the major metabolic products from 2 glucose while glycerol, ethanol, acetoin, and acetaldehyde comprised the minor products. Lactate to acetate ratios were controlled by adjusting the oxygen levels; lactate predominated while oxygen was limited and acetate when oxygen was in excess. The total acid production was un- affected by oxygen level changes, however. Glucose was not catabolized in the absence of oxygen while pyruvate was dissimilated both aerobically and anaerobically. 71 72 The action of inhibitors, radioactive isotope studies, and enzyme assays gave ample evidence for the catabolism of glucose via the EMP and HMP routes. The per— cent participation of the HMP system is very dependent upon oxygen availability. Approximately 40% of the glucose was catabolized by this route in an atmosphere of pure 0 N while only 2%»participation of the HMP was observed in air with high cell concentrations. Terminal oxidation of glucose and acetate oxidation takes place at neutral pH levels or above. Enzymes connecting acetate with the TCA cycle were shown to be operative in cell extracts. This evidence combined with the oxidation of succinate, malate, and fumarate by resting cells indicated participation in the TCA.cycle. Inhibition of the particulate extract fraction by azide, cyanide, and carbon monoxide as well as spectro- photometric data demonstrated electron transport through the cytochrome system. Hydrogen peroxide production was demon- strated but no catalase or peroxidase was found. It is believed that hydrogen peroxide production may be an important factor in limiting the in vitro growth of g. popilliae; and, also,in destroying the viability of vegetative cells. BIBLIOGRAPHY Allen. S. H. G. Jr., and D. Powelson. 1958. Pathways of glucose oxidation in dividing and nondividing cells of Escherichia coli. J. Bacteriol. 15:184-189. Angus, T. A., and A. M. Heimpel. 1960. 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Hematin enzymes of Hemo- philus parainfluenzae. J. Biol. Chem. 237:1332-1336. Wiseman, H. G. and H..M. Irvin. 1957. Determination of organic acids in silage. Agricul. Food Chem. 5:213-215. Wbod, W. A. 1955. Pathways of carbohydrate degradation in Pseudomonas fluorescens. Bacteriol. Rev. 12:222-233. answ— \ \ \ \ I. X \ . . \ ~ \ r — \ K ’ \ 1 " \ \ ‘ \ \ \ I \ \ \ ~ \ .-'- lexmmr ; V‘IFLJ.‘ -- . I _m'-F~ 7 v" I? I ._ . ' { 3 I ' 1‘ . ‘ .' t - .. .1. I Rl M“ 8‘! Ull" u H" “Ml! R“ u " III III IIIII'I 03142 8752 3 1293