_ _— . ‘- ‘4_ ‘4‘ GLUCOSE. MHABOUSM OF CELLS, SPORES AND GERMENATED SPORES OF CLOSTRSDIUM BOWM Thesis {or the bogus 96 Ph. D. MICHIGAN NATE UNWERSSTY Richard J. Simon: 19.61 ABSTRACT GLUCOSE METABOLISM OF CELLS, SPORES AND GERMINATED SPORES 0F CLOSTRIDIUM BOTULINUM \u‘ By Richard J. Simmons An investigation was made of some of the enzymes of vegetative cells, spores, and germinated spores of Clostridium botulinum, type A, for pur- poses of elucidating a pathway for glucose metabolism and of gaining knowledge on the metabolic potential of spores. Manometric studies with whole cells and spectrophotometric and colorimetric assays of enzymes in cell-free extracts showed that glucose or fructose induce the enzyme(s) which is adaptive in the fermentative system, and that glucose is a better inducer and substrate for fermentation than is fructose. Examin- ation of extracts of cells grown in the presence and absence of glucose indicated the presence of all the Embden-Meyerhof-Parnas (EHP) pathway enzymes in both extracts except glucokinase. Glucokinase activity was detected only in extracts of cells grown in the presence of glucose. All EMP enzyme activities were significantly higher in glucose-adapted than in non-adapted cell extracts. It was concluded that in g. botulinum con- centrations of glycolytic enzymes may be controlled by a sequential induc- tion process mediated by an inducible glucokinase. The hexose monophos- phate pathway may be absent, but this was not conclusively shown. Cell extracts also contained DPN'H oxidase, diaphorase, acetokinase, phospho- transacetylase and coenzyme A transphorase. Richard J. Simmons EMP enzymes, except glucokinase and enzymes of the lower-part of the pathway, and diaphorase, acetokinase, phosphotransacetylase and coenzyme A transphorase were detected in extracts of spores and germinated spores though in much lower activity levels than found in extracts of cells. However, DPN-H oxidase in spore extracts had a higher activity and a considerably higher heat resistance than a similar enzyme in cell extracts. The results indicate that the spore may contain the whole complement of enzymes found in the corresponding cell, and that a specific enzyme such as the heat resistant DPN‘H oxidase may play a significant role in the germination process. GLUCOSE METABOLISM OF CELLS, SPORES AND GERMINATED SPORES 0F CLOSTRIDIUM BOTULINUM by (V Richard JI Simmons 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 1961 L?r"" S' .. (v -.' ;P/ “ . ,x ,u M . I ~", ‘I ’ a 4 A, . ACKNOWLEDGEMENTS The author is indebted to Dr. R. N. Costilow for his interest, generous counsel, and constructive criticisms throughout the investiga- tion and preparation of this manuscript. To my wife, Rose Marie, I owe my thanks for typing and proof-reading this thesis, and for her patience and encouragement throughout my graduate studies. ii TABLE OF CONTENTS INTRODUCTION 0 O O I O O O O O O O O O C O O O O O O O HISTORICAL REVIEW Glucose Fermentation . . Pyruvate Fermentation . . Respiratory Flavoproteins Spore Enzymes . . . . . . MTERIALS AND METHODS O O O O O O O O O O O O O O O 0 RESULTS Activities of Intact Vegetative Cells . . . . . . Utilization of sugars during growth . . . . . . Glucose fermentation by resting cell suspensions Activities of Vegetative Cell-Free Extracts Hexokinase . . . . . . . . . . . . . . Oxidation of hexose phosphates . . . . Aldolase . . . . . . . . . . . . . . . Triose phosphate isomerase . . . . . . . Phosphoglyceromutase, enolase and pyruvate kinase . . . . . . . . . . . . . . . . . Pyruvate fermentation . . . . . . . . . . . DPN-H oxidase . . . . . . . . . . . . . . . Diaphorase . . . . . . . . . . . . . . . . . Enzymes involved in metabolic utilization of acetate . . . . . . . . . . . . . . . . . Role of coenzyme A in fatty acid activation 0 o o o O O O o O 0 O 0 O 0 Activities of Spore and Germinated Spore Extracts DISCUSSION 0 O O O O O O O O O O O O O O O O O O O O O SUMMRY O O O O O I O O O O O O O O O I I O O O O O O wORKS C ITED O O O I O O O O O O O O O O O O O O O O 0 iii 0 O O 0 page \0 \lm-PN l6 l6 17 18 2h 27 27 29 32 39 39 #5 56 61 6h LIST OF TABLES Table Number page l. Growth reSponse of g, botulinum to the presence of various sugars, and the per cent utilization of these sugars during growth . . . . . . . . . . . . . l6 2. Glucose fermentation by cell suspensions of E. botUI‘Inum O O O O O O O O O O O O O O O D O O O O O O O 17 3. Phosphorylation of hexoses by cell-free extracts Of 2. botUIInum O O C O O O O I O O O O O O O O O O O O 23 A. Effect of various agents on aldolase of E. botulinum . . . . . . . . . . . . . . . . . . . . . . . 28 5. Effect of pH on aldolase of E. botulinum . . . . . . . 29 6. Products formed by and the effect of some factors on the phosphoroclastic reaction . . . . . . . . . . . 37 7. Effect of heat on a DPN-H oxidizing system in cell-free extracts of E, botulinum . . . . . . . . . . 38 8. Cofactor requirement for the DPNoH oxidase in cell-free extracts of E. botulinum . . . . . . . . . . Al 9. Diaphorase activity in cell-free extracts of g. botUI‘Inumo O O O O O O O O O 0 O O O O O C O O O O O C A] 10. Phosphorylation of acetate by acetokinase in cell-free extracts of £3 botulinum . . . . . . . . . . #2 ll. Phosphotransacetylase in cell-free extracts of £0 botUIInum . O O O O O O O O O O O O O O O O O O I O 43 l2. Coenzyme A transphorase in cell-free extracts Of 2. botUIInum O O O O O O O O O O O O O O O O O O O 0 AL} 13. Aldolase, phosphohexoseisomerase and phospho- fructokinase in extracts of spores and germ- inated spores of E, botulinum . . . . . . . . . . . . . #6 The Effect of heat on DPNoH oxidase in extracts of spores and germinated spores of E. botulinum . . . . . #7 iv ' Table Number page 14. Effect of heat on DPN'H oxidase in extracts of spores and germinated spores of g. botUIinum O O O O O O C O I O O I O O O C O C O O O O 47 15. Effect of various agents on the DPN-H oxidase in extracts of spores and germinated spores of Co botu‘inm O O O O C O O O O O O O O I O O O O O 0 A9 16. Diaphorase in extracts of spores and germ- inated Spores of g. botulinum . . . . . . . . . . . . 50 17. Acetokinase activity in extracts of spores and germinated spores of £3 botulinum . . . . . . . . 51 18. Phosphotransacetylase in extracts_of spores and germinated spores of E. botulinum . . . . . . . . 52 19. Coenzyme A transphorase in extracts of spores and germinated spores of E, bOtUIinum O O O O O O O O O O O O O O O O O O O O O O 53 20. Comparative activities of some enzymes in extracts of vegetative cells, spores, and germinated spores of E. botulinum . . . . . . . . . . 5h LIST OF FIGURES Figure Number l. 2. 10. ll. l2. Induction of glucose fermentation and its inhibition by chloramphenicol . . . . . . . Fermentation of glucose and fructose by cells grown in the presence of glucose . . Fermentation of glucose and fructose by cells grown in the presence of fructose . . Phosphorylation of hexoses measured by decrease in free reducing_sugar by a cell- free extract of E. botulinum . . . . . . . Glucokinase activity in cell-free extracts of glucose-adapted and non-adapted cells . Reduction of DPN+ by cell-free extracts of E. botulinum with hexose phosphates as substrates . . . . . . . . . . . . . . . . Chromogen formation from fructose-l-6- diphosphate in the presence and absence of hYdraZIne . O O O O O O C I O O C O O O O 0 Conversion of 3-phosphoglycerate to pyrue vate by cell-free extracts of E. botulinwn DPN-H oxidation, and its inhibition by fluoride, by a cell-free extract of C. botulinum with 3-phosphoglycerate as-sub- strate . . . . . . . . . . . . . . . . . . Alcohol dehydrogenase activity by cell-free extracts of 2. botulinum . . . . . . . . . DPN-H oxidation by cell-free extracts of E. botulinum using acetaldehyde as substrate . Reduction of DPN+, formed in the DPN-H oxidase reaction, by alcohol dehydrogenase vi page 19 20 2l 22 25 26 30 31 33 34 36 ho INTRODUCTION Spores of Clostridium botulinum are dormant forms of life that have high heat and radiation resistance. Although a wealth of data has been reported on resistance levels, destruction rates, and some of the changes accompanying the transition from spore to vegetative cell, our understand- ing of the biochemical basis for these changes is limited. Determining the enzyme complement of spores and vegetative cells has been one approach aiding in the resolution of these problems. Studies have been made compar- ing the respiratory and metabolic enzymes of specific cell stages of the aerobic Species, but there is a paucity of data on the anaerobes. Reports have appeared on glucose metabolism of vegetative cells of a few species of clostridia, but relatively little is known regarding the metabolism of E, botulinum, and practically nothing is known of its spore enzyme com- plement. This study was undertaken to elucidate some of the enzymes of vege- tative cells, spores, and germinated spores of E. botulinum. Although emphasis was placed on enzymes of glucose dissimilation, other enzymes were investigated. These included flavoprotein enzymes concerned in terminal respiration, and enzymes involved in metabolic utilization of acetate and energy production. It was hoped that data derived from these studies would not only elucidate a pathway of glucose dissimilation, but would also contribute knowledge to our understanding of the metabolic potential and biology of spores. HISTORICAL REVIEW Glucose Fermentation Knowledge of glucose metabolism in members of the genus Clostridium is incomplete. Bergey's Manual (Breed, Murray and Smith, 1957) states that a majority of clostridia including 2, botulinum ferment glucose. However, since this fact as indicated by Bergey's Manual (Breed, EE.EL°’ 1957) was based on gas evolution and acid production in a medium con- taining glucose, this gives no information on the pathway of glucose metabolism. The early fragmentary evidence on the mechanisms of fermentation suggested a scheme other than the Embden-Meyerhof-Parnas (EMP) pathway. Stone and Werkman (1937), working with Clostridium butylicum, Clostridium sporogenes and Clostridium histolyticum, reported that these organisms did not have an enzyme mechanism capable of producing phosphoglyceric acid in the dissimilation of glucose. Osburn, Brown, and Werkman (1937) showed methylglyoxal formation in the breakdown of glucose by E. butylicum. Methylglyoxal was once regarded as an intermediate in the breakdown of glucose in the so-called Neuberg scheme of alcoholic fermentation. Clifton (1940) reported that glucose fermentation by washed cell suspensions of E, botulinum yielded primarily ethanol and C02, with only traces of H2, acetate and lactate. Lerner and Pickett (l9h5) showed that Clostridium tetani ferments glucose with ethanol and C02 as main products along with small amounts of H2 and lactic acid. Iron was essential for the fermentation, with glucose being fermented in direct proportion to the reduced iron present in the culture medium. They suggested that an iron- containing enzyme or coenzyme is essential for glucose fermentation. Certain clostridia, which normally do not produce lactic acid, are reported to carry out a homolactic fermentation under abnormal conditions. Kempner and Kubowitz (1933) reported that carbon monoxide and cyanide diverted the fermentation by Clostridium butyricum to the homolactic type and that this could be reversed by removal of the inhibitor or by light. Pappenheimer and Shaskan (1944) showed with Clostridium perfrigens that products obtained from glucose dissimilation depended upon iron content of the cells. As iron was decreased, the reaction shifted from an acetic- butyric type with large amounts of C02 and H2 towards a more purely lactic acid fermentation with slight gas formation. Similarly, Hanson and Rodgers (1946) demonstrated that cultures of Clostridium acetobutylicum obtained by serial transfers in low iron medium produced a homolactic fermentation. Such shifts to lactic fermentation would be expected if cytochromes func- tioned in pyruvate breakdown, but no spectnal evidence has been found for these respiratory catalysts in clostridia (Smith, 1954). Lerner and Mueller (1949), working with a mutant strain of E. 533221, showed that cells from an iron-deficient medium and which did not ferment glucose could be activated by glutamine. These data suggest an indirect effect of iron on glucose fermentation. Recent data, though incomplete, suggest that the EMP pathway is present in members of the clostridia. Bard and Gunsalus (1950) reported an iron-requiring aldolase of £3 perfringens, and suggested that since iron is essential for aldolase activity, this affords an explanation of lJTe indiSpensability of iron for clostridia] growth. Furthermore, the _ CAPC 'i' ~J o , Glucose .2 300 Of Fructose a) <1 0: l 50 l l l J 20 4O 60 80 MINUTES Fi ure 1. Induction of glucose fermentation and its inhibi- tion by chloramphenicol. Reaction flasks contained: 1.0 ml of 0.067 M phosphate buffer, pH 7.0; 12 mg dry wt of cells; and 2% Trypticase. Where indicated the flasks contained 10 pmoles of either glucose or fructose, or 30 pg chloramphenicol (CAPC). All flasks had water to 3.0 m1. 20 L Glucose SOOL Glucose +CAPC 400.. FI'UCTOSO 2 .. Fructose +CAPC 5 Control ° 300 b OAPC 'é.‘ ,. 20d- 0) Q Q h 100 - p l 1 I 2J 20 4O 60 M l N U TES Figure 2.. Fermentation of glucose and fructose by cells grown in the presence of glucose. Conditions: see Figure 1. 21 L Glucose 500 P- Fructose - Glucose +CAPC 400 - Fructose +CAPC 2 Control 3 CAPO 9 300 - E“ g? _ In a; 200 r ‘1 . Q) _ / IOO - ,/ 20 4O 60 MINUTES Fi ure 3. Fermentation of glucose and fructose by cells grown in the presence of fructose. Conditions: see Figure 1. 22 '0 0 Controls Fructose \Glucose FREE SUGAR (J1 M) l 1 1 l L 1 J 20 4O 60 MINUTES Fi ure 4. Phosphorylation of hexoses, measured by decrease in free reducing sugar, by a cell-free extract of C. botulinum. Reac- tion mixture contained: 1.0 m1 of 0.067 M phosphate Buffer, pH 7.4; sugars, 10 pmoles; MgTT, 10 pmoles; ATP, 20 pmoles; NaF, 15 pmoles; 25 mg of extract protein, and water to 3.0 m1. Control reaction mixtures contained hexose but no ATP. Results with mannose and galactose in complete reaction mixtures were the same as the control. 23 TABLE 3. Phosphorylation of hexoses by cell-free extracts of E. botulinum. Extracted cells ‘ Substrate Range: Disappearance of substrate grown on: (A pmol es )* Basal Glucose 0 Basal Fructose 0 Glucose Glucose 3.5 - 4.0 Glucose Fructose 0 - 0.9 Glucose Mannose 0 Glucose Galactose 0 Fructose Fructose O Fructose Glucose 2.8 Fructose Mannose 0 Fructose Galactose 0 *Net activity as indicated by the total decrease in free re- ducing sugar over the control without ATP during 60 minutes incubation at 35 C with 25 mg extract protein. medium devoid of sugar did not possess glucokinase, whereas extracts of cells grown in a medium containing glucose possessed a glucokinase that ‘was active on glucose, had a low activity on fructose, but did not phOSphorylate other sugars. Extracts of cells grown in the presence of fructose phosphorylated glucose but not fructose indicating that fruc- tose acted as an inducer for the glucokinase, but was a poor substrate for the enzyme. This type of kinase is similar to the inducible enzyme 24 observed in g. tetani (Martinez and Rittenberg, 1959), and to that found in mutant strains of Pseudomonas putrefaciens (Klein, 1953). 0n the hypothesis that glucokinase, in extracts of cells grown with- out glucose, may be present in amounts too low for detection by the assay used, a more sensitive test was tried. This second assay measures the glucose-6-phosphate (G-6—P) as the end product of the reaction by coup- ling to G-6-P dehydrogenase and then following the reduction of TPN+ at 340 mp. Extracts of glucose-adapted and non-adapted cells were tested in this way. Figure 5 reveals that the kinase was detected only in extracts of cells grown in a medium containing glucose, which substan- tiates the earlier evidence that glucokinase is an inducible enzyme in E, botulinum. Oxidation 2: hexose phosphates. Extracts of cells grown in the presence of glucose catalyzed a rapid reduction of DPN+ when fructose diphosphate (F-l-6-P) was used as the substrate (Figure 6). TPN+ could not be substituted for DPN+ in the reaction. Similar results were obtained when fructose-6-phoSphate (F-6-P) and G-6-P plus ATP and Mg++ were substituted in place of F-1-6-P in the reaction mixture, thus indi- cating the presence of phosphohexoseisomerase, phosphofructokinase, and aldolase in the extract. Iodoacetate, a known inhibitor of glycer- aldehyde-3-phosphate dehydrogenase added at zero time resulted in com- plete inhibition of the reaction. Tests for TPN+ reduction with G-6-P as substrate were negative indicating the absence of G-6-P dehydrogenase in the extract. Therefore, the reaction sequence involved must be that of the EMP system. A reaction system resulting in the reduction of DPN+ with G-6-P, F-6-P, or F-1-6-P as substrate was also demonstrated with extracts of 25 0'20 F Adopted 0.15 ' 0.10 l" 0.05 F OPTICAL DENSITY (340 my) Non -odopted ; “4!""‘f""1L 1 I: 2 3 MINUTES Figure 5. Glucokinase activity in cell-free extracts of glucose-a apted and non-adapted cells. Reaction mixtures con- tained: 1.0 ml of 0.2 M tris buffer, pH 7.4; glucose, 5 pmoles; ATP, 10 pmoles; Mg+*, 10 pmoles; TPNT, 0.9 pmole; glucose-6- phosphate dehydrogenase, 0.01 ml of a 0.01% solution; extract protein, 12 mg; and water to 3.0 ml. The G-6-P dehydrogenase was devoid of hexokinase activity. Reaction temperature was 25 C. 26 0.5- F-I-G-P 3. 0.4- E C) :1; F-6-P+ATP+Mg++ \- 0.3- )~ I; G-6-P+ATP+Mg++ a) E Q 0.2- a‘ 2 I: 0 0.1 P / _ #lAe .. 2 .i 1 l 1 J I 2 3 4 5 6 MINUTES Fi ure 6. Reduction of DPN+ by cell-free extracts of g. botulinum with hexose phosphates as substrates. Reaction mix- tures contained: 1.0 m1 of 0.1 M tris buffer, pH 7.4; hexose phosphates, 5 pmoles; arsenate, 20 pmoles; DPN+, 1.0 pmole; extract protein, 5.4 mg; and water to 3.0 m1. Where indicated, 20 pmoles each of ATP and MgTT, and 3.0 pmoles of iodoacetate (IAc) were added. 27 cells grown in the absence of glucose, but the reaction rate was consid- erably less than that observed with the extracts of glucose-adapted cells. Phosphohexoseisomerase, phOSphofructokinase and aldolase activities were also demonstrated using G-6-P as substrate and measuring DPN-H oxidation in the presence of an excess of cf-glycerophosphate dehydro- genase, and by the colorimetric determination of triose phosphates formed. Aldolase. The clostridial aldolase is inhibited by the metal-bind- ing agent pyrophosphate, and by Zn++ and cysteine at high concentrations (Table 4). Zn++ at a lower concentration had no effect. A lower con- centration of cysteine stimulated aldolase activity which could result from cysteine acting as a reducing agent and thereby protecting the sulfhydryl groups of the aldolase. The inhbition of aldolase at higher cysteine levels was probably due to its ability to act as a metal binder. Ferrous ions caused a three-fold stimulation of activity, and reversed the inhibition caused by cysteine and by pyrophosphate, provided they were added prior to pyrophosphate. Table 5 shows the effect of pH on aldolase activity, the highest activities obtained at pH 7.5 to 8.0. All of these data on aldolase are in complete agreement with those of Bard and Gunsalus (1950) who reported a metallo-aldolase in cell-free extracts of C, perfringens. Extracts of cells grown in the absence of glucose also had signifi- cant levels of aldolase activity. IElSEE phosphate isomerase. This enzyme was determined by using a Inodification of the method of Sibley and Lehninger (1949), as described in the section on methods. Omission of the hydrazine from the assay .should produce about a 1.7 fold increase in chromogen over that of an 28 TABLE 4. Effect of various agents on aldolase of 2. botulinum. Additions 4 . Concn., molar Specific activity* None _ - .83 Fe++ 1 x 10"6 2.40 Fe++ + Cysteine 1 x 10'5 + l x 10’“ 3.12 Fe++ + Cysteine 1 x 10'5 + 4 x 10'3 .87 Cysteine l x lO’h .92 Cysteine 4 x 10"3 .11 Pyrophosphate l x 10"3 .57 Pyrophosphate 4 x 1073. .29 Fe++f# + Pyrophosphate 1 x 10.5 + 1 x 10"3 1.10 Zn++ 1 x 10-3 .25 Zn++ 1 x 10'6 .81 ...... fipmoles F-1-6-P split per hr per mg protein. *AFe++ added before pyrophosphate. Reaction mixtures contained: 1 m1 of.1 titris buffer, pH 7.4; F-l-6-P, 5 pmoles; hydrazine, 56 pmoles; 0.4 mg extract protein using an extract dialyzed against water for 12 hrs; and water to 2.5 ml. After 15 min incubation at 35 C, 1 m1 aliquots were added to 2 m1 of 10% trichloroacetic acid and analyzed for chromogen formed. 29 TABLE 5. Effect of pH on aldolase of E. botulinum. pH of assay . Specific activity* 6.0 .70 6.5 .81 7.0 1.01 7.5 1.19 8.0 1.07 8.5 .95 *Units and conditions same as in Table 4. assay with hydrazine. Data in Figure 7 show that about a 1.6 fold increase in color was obtained when the activity was tested in the absence of hydrazine. Phosphoglyceromutase, enolase Egg pyruvate 513355. In the EMP scheme, 3-phosphoglycerate (3-PG) leads directly to pyruvate, and so it seemed feasible to test for phosphoglyceromutase, enolase, and pyruvate kinase by following the formation of pyruvate from 3-PG. Pyruvate was deter- mined by the double extraction procedure of Friedemann and Haugen (1943), and was tentatively identified as the benzene and carbonate soluble 2-4 dinitrophenylhydrazone. Figure 8 not only shows the conversion of 3-PG to pyruvate, but also its inhibition by sodium fluoride (NaF) which was undoubtedly due to the inhibition of enolase. The extract used was of cells grown in the presence of glucose. 3O 1.2r F-l-G-P 2 Ex _. Q .. ‘t 0.9 K) \ - )~ . F'I‘G'P I: - + "l ‘ H drozine E 0.6- ’ C3 3' - 52 b F~ % 0.3- L J I l I J 5 IO 15 20 MINUTES Figure 7. Chromogen formation from fructose diphosphate in the presence and absence of hydrazine. Reaction mixture contained: 3.0 ml of 0.1 tris buffer, pH 7.4; F-l-6-P, 5 pmoles; 0.75 ml of 0.56 M hydrazine; 2.0 mg extract protein using an extract dialyzed against water for 18 hours; and water to 7.5 m1. Incubation tem- perature was 35 C. One ml aliquots were removed at time intervals indicated, added to 2.0 m1 of 10% trichloroacetic acid, and ana- lyzed for chromogen formation. 31 SD 45 l PYRUVATE FORMED (JIM) .0 no I 3-PG'I-Na F A + Endo eneus ———’L__ —4: '9 I 5 IO 15 MINUTES . Fi ure 8. Conversion of 3-phosphoglycerate (3-PG) to pyruvate b cell-free extracts of C. botulinum. Reaction vessels contained: 0.5 m1 of 0.2 M phosphate_buffer, pH 7.5; ADP, 20 pmoles; Mg++: 10 pmoles; and extract protein, 8 mg. NaF, 5 pmoles, and 3-PG, 25 pmoles were added where indicated. Volumes were adjusted to 2.0 m1. Incubation temperature was 35 C. Reactions were stopped by addition of 1.0 ml of 10% trichloroacetic acid. 32 Figure 9 presents further evidence for enolase by showing fluoride inhibition of DPNoH oxidation by extracts of glucose-adapted cells with 3~PG as substrate in the presence but not in the absence of phosphate (Warburg and Christian, 1942). When 15qpmoles of NaF were preincubated with the reaction mixture containing inorganic phosphate prior to addi- tion of 0PN*H, oxidation of the 0PN°H proceeded at the endogenous rate. With the same system, but minus inorganic phosphate, the oxidation rate was equal to that in the absence of fluoride. Evidence for 3-PG fermentation by extracts of cells grown in the absence of glucose was obtained by measuring the rate of DPN-H oxidation and correcting for endogenous activity. The fermentation rate was much slower than observed with extracts of glucose-adapted cells, but was significant. Pyruvate fermentation. DPN°H oxidation by extracts of glucose- adapted cells was observed when pyruvate was used as the substrate, the ”rate being similar to that seen when 3-PG was used as the substrate. These data do not distinguish the probable routes of pyruvate reduction such as direct reduction to lactate, or decarboxylation to acetaldehyde and then reduction to ethanol, so a more comprehensive study of pyruvate breakdown was undertaken. That pyruvate was reduced to lactate seemed unlikely since all attempts to show lactic dehydrogenase in extracts imere negative. Also, analyses for lactate in completed fermentation scflutions, using whole cell suspensions, were negative. However, a path- imay to ethanol became apparent when it was shown that extracts contained alcohol dehydrogenase. This activity could be shown by following DPN+ reduction at 340 mp with ethanol as substrate (Figure 10), since the 33 0.0 p a. E 0.1 - <3 I?) _ 3-Pc+Po; +NoF \- 93 2 0.2 - Endogenous 3‘» _ ‘l "4 3-PG+NoF o; 8 03- Q . 3-PG l l l J I l I l I 2 3 MINUTES Fi ure 9. DPN H oxidation, and its inhibition by fluoride, by a cell-gree extract of C. botulinum with 3- -phosphog1ycerate (3-PG) as substrate. Reaction .mixture contained: 1. 0 m1 of 0.1 M tris buffer, pH 7. 4; Mg” , 10 pmoles; cysteine, 20 pmoles; ADP, 10 pmoles; DPN H, 0.6 pmole; and water to 3. 0 m1. Where indicated 4 pmoles of 3- PG, 15 pmoles NaF, and 10 pmoles inorganic phosphate were added. 34 0.08 ' 0.06 '- 0.04 '- OPTICAL DENSITY (340mu) .9 (3 BO 1 l I l J J 15 30 45 60 SECONDS Fi ure 10. Alcohol dehydrogenase activity in cell-free extracts of C. botulinum. Reaction mixture contained: 1.0 m1 of 0.2 M pyrophosphate buffer, pH 8.5; ethanol, 200 pmoles; DPN+, 2.0 pmoles; and water to 8.0 ml. No activity was observed in the absence of ethanol. A = 2.0 mg extract protein; B = 4.0 mg extract protein. 35 accumulation of DPN°H without a lag indicated that alcohol dehydrogenase was present in fresh extracts in sufficient amounts to mask the competing endogenous DPN-H oxidation. The activity appeared higher when the reac- tion was run in the reverse direction, following DPNoH oxidation with acetaldehyde as substrate (Figure 11). The pathway of pyruvate breakdown was further studied by testing the ability of cell-free extracts to cleave and oxidize pyruvate by the phosphoroclastic reaction. Table 6 shows the products formed in the cleavage of pyruvate, as well as some of the factors affecting the reaction. It appears that one mole each of C02, H2, and acetyl phosphate is formed from one mole of pyruvate. The slightly higher amounts of C02 evolved is probably due to side reactions since there was good correlation between the amounts of H2 and acetyl phosphate produced. The removal of either DPN+ or phosphate from the reaction mixture resulted in almost complete inactivity, so these factors appeared to be essential. Dialy- sis of the extract resulted in almost complete loss of activity making it impossible to assess the true function of thiamine pyrophosphate and coenzyme A in the reaction. It may be that the crude extract contained excess CoA and ThPP, and so additions of these cofactors would show no effect on the reaction rate. Arsenite inhibits the reaction about 50% when phosphate and DPN+ are present. Exposure of the extract to air resulted in the loss of ability to produce acetyl phOSphate and H2, and only a small amount of C02 was evolved. This finding is similar to that of Wolfe and O'Kane (1955) who worked with cell-free extracts of g, butyricum. They reported that the C02 exchange with pyruvate survived aging, whereas the acetate exchange is only slightly retained indicating more stability of the C02 36 0u0 0.1 Endogenous CL2- DECREASE m o. a. (340 my) ACHO 2 MINUTES Fi ure 11. DPN'H oxidation by cell-free extracts of C. botulinum with acetaldehyde (ACHO) as substrate. Reactionjnix- ture contained: 1.0 m1 of 0.1 M pyrophosphate buffer, pH 7.0; acetaldehyde, 5 pmoles; DPN-H, 1.0 pmole; extract protein, 2.0 mg; and water to 3.0 ml. 37 exchange reaction. Ferrous ion was required, but had no affect on the C02 exchange with pyruvate. They suggested that in the forward reaction Fe++ functions in H2 production after C02 has been liberated. As pointed out in the historical review section, certain clostridia produce chiefly lactic acid in an iron-deficient medium instead of the TABLE 6. Products formed by and the effect of some factors on the phos- phoroclastic reaction. Deletions, additions or pmoles products formed in 30 min special treatments 002 Hz Acetyl phosphate Complete sytem* 12.0 8.2 7.2 - Enzyme 0.0 0.0 0.0 + Arsenite, 5 pmoles 5.9 4.3 4.0 ’ GOA, Tth 110] 709 7.0 - DPN 0.7 0.5 0.5 - Phosphate buffer** l.6 1.3 1.1 Dialyzed extract, 8 hrs at 4 C 1.4 0.6 0.5 Extract exposed to air, 1.5 hrs at 35 C 2.2 0.0 0.0 *Complete reaction mixture contained: 1 ml of 0.2 M phosphate buffer, pH-6.93 pyruvate, 100 pmoles; 25 mg extract protein; DPN, 2 pmoles; coenzyme A (CoA), 0.3 pmole; thiamine pyrophosphate (ThPP), 0.5 pmole; and water to 3.0 m1. Cups for measuring H2 had 0.2 m1 of 20% KOH in the center well; incubation temperature was 35 C. Acetyl phosphate was determined by the hydroxamate method of Lipmann and Tuttle (1945). **Tris buffer used in place of phosphate buffer. 38 usual mixture of H2, C02, solvents, acetic, lactic, and butyric acids. This could be explained by the fact that in iron deficiency, the pyru- vate formed by glycolysis cannot be removed by the phosphoroclastic reaction, a Fe++ requiring system, and hence is reduced to lactate. 22!;fl oxidase. A high endogenous DPN°H oxidation was noted during a number of assay procedures using dialyzed extracts. This suggested the presence of a DPN-H oxidase, and inactivation studies (Table 7) indicated that the activity was enzymatic and not due to non-specific action on the DPN-H by the extract. That the decrease in optical den- TABLE 7. Effect of heat on a DPN-H oxidizing system in cell-free extracts of E. botulinum. Heat treatment ' Relative aCtivity* None 4.80 i 60 C, 1 min 3.30 65 C, l min 1.98 70 C, 30 sec . g y 0-90_ *Change in optical density per min x 102 Reaction mixtures contained: 1 ml of 0.067 M phosphate buffer, pH 7.4; DPN-H, 0.3 pmole; 0.1 ml extract; and water to 3.0 m1. Change in 0.0. at 340 mp was followed; reaction temperature was 25 C. sity at 340 mp was due to DPN‘H oxidation and not degradation of the 0PN°H was shown by testing the DPN+ formed in the reaction as a substrate for 39 alcohol dehydrogenase. Additions of ethanol and alcohol dehydrogenase caused a rapid and complete restoration in absorption at 340 mp (Figure 12), and this was due to DPN+ reduction in the alcohol dehydro— genase system. TPN°H could also serve as electron donor for the oxidase; cyanide, a cytochrome poison, did not inhibit the activity, and cytochrome c was not reduced. Oxygen uptake studies showed that 0.041 pmole 02 per min per mg protein was used in the reaction, while 0.043 pmole DPN°H per min per mg protein was oxidized during spectrophotometric studies at 340 mp. The results indicated a two-electron reduction of oxygen by the DPN'H oxidase which should result in formation of hydrogen peroxide. Proof of flavoprotein catalysis was shown by removal of the flavin component of the oxidase, and then adding back either FAD or FMN. There was no activity without addition of cofactors (Table 8); FAD restored the activity to the original level, and FMN partially reacti- vated the enzyme. Addition of FAD to dialyzed extracts resulted in a 2 to 3 fold increase in activity, while FMN caused only a slight increase. Diaphorase. The ability of cell-free extracts to cause a rapid reduction of 2-6 dichlorophenolindophenol with DPN-H as substrate indi- cated the presence of diaphorase activity (Table 9). Extract alone did not reduce the dye. Addition of FAD or FMN stimulated the activity slightly; heating the extract for one minute at 65 C resulted in loss of one-half the initial activity. Methylene blue and triphenyl tetra- zolium salts could also act as electron acceptors. Enzymes involved igémetabolic utilization g: acetate. Since acetyl phosphate was formed in the phosphoroclastic cleavage of pyruvate, interest 40 0.8 a 0.7 E 0 Extract :5 0.6 4. \. DPN'H : (7, 0.5- E C: ~I 0.4r 11> I: 0 03’" Ethanol+alcohol (— dehydrogenase 0.21- 1 l l 1 l l l i 2 4 6 8 IO 12 l4 l6 MINUTES Figure 12. Reduction of DPNT, formed in the DPN‘H oxidase reaction, by alcohol dehydrogenase. Reaction mixture contained: 1.0 m1 of a 0.2 M phosphate buffer, pH 8.0; DPN-H, 0.5 pmole; extract protein, 6.0 mg; and water to 3.0 ml. After reaction stopped, the test solution was heated to inactivate the oxidase and then 50 pmoles of ethanol and 0.05 ml of a 0.01% solution of alcohol dehydrogenase were added. 41 TABLE 8. Cofactor requirement for the DPN.H oxidase in cell-free extracts of E. botulinum. Cofactor added Concn., pmole Specific activity* None - _ o FAD 0.6 .080 FMN 0.6 .044 #pmoles DPN‘H oxidized per min per mg protein Reaction mixture contained: 1 m1 of 0.067 M phosphate buffer, pH 7.4; DPN-H, 0.3 pmole; 1.2 mg extract protein, plus cofactors as indicated, and water to 3.0 ml. Change in 0.0. at 340 mp was followed; reaction temperature was 25 C. The flavin component was removed from the oxidase by the acid-ammonium sulfate treatment of Warburg and Christian (1938). TABLE 9. Diaphorase activity in cell-free extracts of E, botulinum. Treatment . . . 3. . _ ._Relative acthit¥* None 3 .44 FAD added 1 .56 FMN added .49 Extract heated l min at 65 C E .23 . . ,g, _ . . . ._. I'LL .. _ 2 ,. ._ *Change in optical density per min. Reaction mixture contained: 2.0 m1 of 0.1 M phosphate buffer, pH 7.4; DPN-H, 0.3 pmole; FAD or FMN where indicated 0.06 pmole; 0.05 ml extract; 2-6 dichlorophenolindophenol and water to 4.0 m1. Change in optical density at 600 mp was followed; reaction temperature was 25 C. Extract was dialyzed against distilled water at 5 C for 15 hours. 42 was focused on the enzyme(s) involved in its formation, and the pos- sible role of acetyl phosphate in energy production. Phosphotransacety- lase which catalyzes the transfer of acetyl from acetyl phosphate to coenzyme A, and acetokinase which couples acetyl phosphate to ATP forma- tion have been implicated in these mechanisms, so tests were performed to measure these activities. Data in Table 10 shows that extracts contain high acetokinase activ- ity dependent upon Mg++ and ATP. ADP could not be substituted for ATP, and CoA appeared to lower the activity, but not significantly. TABLE 10. Phosphorylation of acetate by acetokinase in cell-free extracts of E. botulinum. Factor added _ _ ._ _ .. , .,Specific.activity* None 0.0 Mg++ 0.0 ATP 0.0 ATP + Mg++ 2.0 ADP 0.0 ADP + Mg++ 0.0 c.oA+.ATP+,.Mg+“. .. . 3 ‘I_.6_- #pmoles acetyl phosphate formed per min per mg protein. Reaction mixtures contained: 1.0 ml of 0.1 M tris buffer, pH 7.4; potassium acetate, 200 pmoles; hydroxylamine, 600 pmoles; 0.27 mg extract protein; and where indicated, MgClz, 15 pmoles; ATP, 20 pmoles; ADP, 15 pmoles, coenzyme A, 0.03 pmole, and water to 1.0 ml. Extract was dialyzed against water at 5 C for 15 hours. Incubation temperature was 30 C; reaction was stopped by addition of 1.0 ml of 10% trichloroacetic acid. 43 Table 11 demonstrates that cell-free extracts contain an active phosphotransacetylase. Apparently g. botulinum forms acetyl-coenzyme A by the coupled action of acetokinase and transacetylase. TABLE 11. Phosphotransacetylase in cell-free extracts of g, botulinum. . System H . . ,. . ....1 ..l W.Specific activityk Complete 18.9 with 2x protein 20.0 less CoA 0.0 less acetyl phosphate 0.0 *Change in optical density per min per mg protein. Reaction mixtures contained: 0.02 ml of 0.1 M tris buffer, pH 8.0; reduced CoA, 0.06 pmole; dilithium acetyl phosphate, 10 pmoles; 0.32 pg extract protein, or as indicated; and water to 0.2 m1. Reac- tion temperature was 25 C. Change in optical density at 232 mp was followed due to formation of the thiol ester bond of acetyl coenzyme A. Role gf’coenzyme AHLQ fatty acid activation. With the knowledge that cell-free extracts of E. botulinum have enzymes capable of forming acetyl-coenzyme A, it seemed that extracts may also contain enzymes involved in fatty acid synthesis, especially since acetyl-coenzyme A has been implicated in fatty acid activation. 2, kluyveri extracts have been shown by Stadtman and Barker (1950) to contain enzymes that transfer 44 the phosphoryl group from acetyl phosphate to fatty acids containing from 3 to 8 carbon atoms. Furthermore, Stadtman (1953) showed that coenzyme A and phosphotransacetylase are required, along with an enzyme called coenzyme A transphorase that transfers the SCoA group from acetate to several acids such as propionic or butyric, to name a few. Table 12 shows that extracts of E. botulinum contain coenzyme A transphorase. The enzyme catalyzed the transfer of the SCoA group between butyryl-SCoA and acetate. TABLE 12. Coenzyme A transphorase in cell-free extracts of E. botulinum. System Specific activity* Complete 2.7 less enzyme 0 less butyryl-SCoA 0 less acetate 0 *Change in optical density per min per mg protein. Reaction mixtures contained: potassium acetate, pH 8.0, 30 pmoles; potassium arsenate, pH 8.0, 150 pmoles; butyryl-SCoA, 0.01 pmole; phosphotransacetylase, 0.01% of a 300-fold purified enzyme from Beptostreptococcus eldsdenii; 16 pg extract protein; and water to 0.15 ml. Reaction temperature was 25 C. The decrease in optical density at 232 mp due to the arsenolysis of acetyl-SCoA was followed. 45 Activities 2: Spore and Germinated Spore Extracts One objective of this study was to determine whether spores of E. botulinum contain enzymes, and, if so, how their activities might com- pare to those found in cell-free extracts. Secondly, it seemed desir~ able to compare relative enzyme activities of germinated spores to those of the cell and spore, since these data could give information on enzyme synthesis in germinating spores. In general, there is a lack of data on enzymes of spores and germinated spores of anaerobes. Other than the report that a glucose-fermenting system present in spores is activated upon germination (Costilow, 1960), there is no conclusive evidence for a glycolytic pathway in spores of C} botulinum. In this study, glucokinase was not detected in either spore or germ- inated spore extracts even when employing the sensitive assay of coupling to the G-6-P dehydrogenase system. It should be pointed out, however, that the spores studied were produced in a medium devoid of glucose or fructose. Attempts to sporulate the culture in the presence of glucose were not very successful. Attempts to show aldolase activity in spore extracts by coupling to DPN+ reduction at 340 mp in the presence of arsenate were not completely satisfactory. Long lags in activity were often observed, and when DPN+ reduction was noted, the small changes in optical density reflected extremely low activity. It was felt that aldolase activity might be better tested by the more sensitive procedure of coupling to DPN'H oxida— tion in the ¢£1glycerophosphate dehydrogenase system. Any trioses formed should contain more dihydroxyacetone phOSphate (DHAP) than glyceraldehyde- 3-phosphate since the equilibrium of isomerase favors DHAP. While trying this assay, it soon became apparent that Spore-extracts contained an active DPN'H oxidase that greatly interferred with the test. The oxi- dase activity was high enough to mask some of the DPN-H accumulation in the first assay, and appeared as a high endogenous rate in the second assay. The method which overcame the interferences mentioned was the colorimetric aldolase procedure of Sibley and Lehninger (1949). Reac- tion times were selected which allowed accumulation of measurable quan- tities of triose phosphates. Table 13 shows that low, but significant aldolase activity was detected, that Fe++ stimulates activity about four- fold, while controls were essentially negative. When F-6-P or G-6-P plus TABLE 13. Aldolase, phosphohexoseisomerase, and phosphofructokinase in extracts of Spores and germinated spores of E. botulinum. Substrates and cofactors added Range Of specific activities* Spore ‘germinated'3pores** None .01 .02 G-6-P, ATP, Mg++, Fe++ .047 - .07 .05 - .10 F-6-P, ATP, Mg+*, Fe++ .035 - .08 .06 - .11 F-l-6-P .060 - .11 .33 ~ .50 ++ F-1-6-P’ Fe 0250 - el'l's .50 " .60 *pmoles F-1—6-P split per hour per mg protein. **Spores germinated in Trypticase solution for 2.5 hours. "Reaction mixtures contained: 1.0 ml of 0.1 M tris buffer, pH 7.4; substrates, 5 pmoles; hydrazine, pH 7.5, 56 pmoles; 2.0 mg extract protein; and water to 2.5 ml. Cofactors were added as indica- ted: ATP, 5 pmoles; Mg++, 15 pmoles; and Fe++, l pmole. Incubation temperature was 35 C; reactions were stopped by additions of 2.0 ml of 10% trichloroacetic acid, and 1.0 m1 aliquots were removed and analyzed for triose phosphate chromogen. 47 ATP and Mg++ were substituted for F-l-6-P, trioses were formed thus indi— cating the presence of hexoseisomerase and phosphofructokinase in spore extracts. Similarly, these three enzymes were detected in germinated spore extracts. Enzymes of the final steps in glycolysis were not detected using con- ventional methods, but detection of hexoseisomerase, phosphofructokinase and aldolase, plus the demonstration of DPN+ reduction with F-l-6-P as substrate indicates that at least some of the EMP enzymes are present. More sensitive methods might demonstrate the remainder of the enzymes of the glycolytic system. The detection of DPN-H oxidase in spore extracts prompted further study of the enzyme since a similar activity was found in cell-free ex- tracts. Of special interest was the high level of activity and high heat resistance of the spore DPN°H oxidase (Table 14). The extracted TABLE 14. Effect of heat on DPN-H oxidase in extracts of spores and germ- inated spores of E. botulinum. Relativewactivities* Heat treatment "“'SBBFe GeFfiTfiETEE'EBEFES #1 #2 None 5.0 4.1 2.5 70 C, 1 min 5.5 4.0 0.7 80 C, l min 3.5 2.0 O 85 C, 5 min 2.8 0.8 0 90 C,,l min .,H h 2.2 0 0 *Change in optical density per min x 102. '#1 = Spores germinated in acetate solution for 8 hours. #2 = Spores germinated in Trypticase solution for 2.5 hours. -- Reaction mixtures contained: 1 m1 of 0.067 M phosphate buffer, pH 7.4; DPN-H, 0.3 pmole; 0.1 ml extract; and water to 3.0 m1. Change in 0.0. at 340 mp was followed; reaction temperature was 25 C. 48 enzyme from the Spore remained active after heating for 1 minute at 80 C, whereas the enzyme from the cell did not. 0f the spore enzymes demon- strated in this study, DPNrH oxidase was the only one exhibiting a high heat resistance. Evidence presented in Table 14 suggests that the heat stability of the DPN°H oxidase is lost upon germination of the spores. Germination of spores in an acetate solution was slow and incomplete, and hence the heat resistance of this preparation was probably attributable to contam- ination with spore DPN-H oxidase from spores which did not germinate. In the Trypticase germinating solution spores germinated very well in 2.0 to 2.5 hours, and the heat resistance of extracted oxidase was low, similar to that of the oxidase extracted from cells, i.e., these oxidases were not active after heating 1 minute at 80 C. Whether the oxidase of spores, germinated spores, and vegetative cells is the same enzyme with different levels of heat resistance was not determined since this diffi- cult problem was beyond the scope of this study. The DPN-H oxidase of spores and germinated Spores appears to be a flavoprotein since quinacrine (atabrine), a flavin analogue (Hellerman st 31., 1946) inhibits the enzyme and FAD causes a slight stimulation of activity. The stimulation of spore DPN°H oxidase by FAD is not pro- nounced as it is with the cell DPN-H oxidase. KCN, a cytochrome poison, had no affect on activity. Dipicolinic acid (DPA) has been reported to stimulate the spore DPN°H oxidase of E. 255225 (Doi and Halvorson, 1961), but it did not stimulate the clostridial spore oxidase. A summary of these findings is found in Table 15. 49 TABLE 15. Effect of various agents on the DPN’H oxidase in extracts of Spores and germinated spores of E, botulinum. Specific activities* Additions Concentrations, M Spore Germinated spores #1 #2 None - - 5.5 4.1 2-5 FAD 6 x Io-LI 6.0 4.4 3.3 FMN 6 x 10‘“ 5.8 4.1 3.0 KCN 3 x 10‘3 5.6 4.1 2.6 DPA 4 x 10'3 5.5 4.2 2.5 Atabrine 1 x 10'3 3.3 2.0 1.3 Atabrine 1.5 x 10"3 1.8 - - - - Atabrine 2 x 10"3 0.8 - - — - *Change in optical density per min per mg protein x 10 . “#1 #2 spores germinated in acetate solution. Spores germinated in Trypticase solution. All reaction mixtures contained: phate buffer, pH 7.4; DPN°H, 0.3 pmole; extract and water to 3.0 ml. Extracts were dialyzed against distilled water at 5 C for 12 hours. Reaction temperature was 25 C; decrease in optical density at 340 mp was followed. 2 1.0 m1 of 0.067 M phos- 50 Spore and germinated Spore extracts possess diaphorase activity (Table 16) similar to that of vegetative cells in that 2-6 dichloro- phenolindophenol, methylene blue and triphenyl tetrazolium salts can all act as electron acceptors when DPN'H is the substrate. Another TABLE 16. Diaphorase in extracts of spores and germinated spores of .E' botulinum. Relative activities* Treatment Spore Germinated spore #1 #2 None .40 .37 .60 FAD added .45 .40 .64 FMN added .41 .37 .61 Extract heated l min at 65 C .22 .19 .28 *Change in optical density per min. #1 #2 Spores germinated in acetate solution. Spores germinated in Trypticase solution. Reaction mixtures contained: 2.0 ml of 0.1 M phosphate buffer, pH 7.4; DPN°H, 0.3 pmole; FAD or FMN, as indicated, 0.06 pmole; 0.1 ml extract; 2-6 dichlorophenolindophenol and water to 4.0 ml. Change in optical density at 600 mp was followed; reaction temperature was 25 C. Extracts were dialyzed against distilled water at 5 C for 15 hours. similarity is the heat resistance of these systems; the half-life for all diaphorase preparations studied was approximately 1 minute at 65 C. 51 One difference was the apparent lower specific activity of the spore diaphorase as compared with the cell or germinated spore activities which were similar. Acetokinase could readily be detected in both spore and germinated spore extracts. The enzyme is ATP and Mg++ dependent (Table 17), and is significantly lower in activity than that found in vegetative cell extracts. TABLE 17. Acetokinase activity in extracts of spores and germinated spores of E, botulinum. Acetate plus Incubation time, 1 Specific 3¢t1VltY* factor added minutes I Spore Germinated—spore** None 10.0 ' 0 0 ++ Mg 10.0 0 0 ATP 10.0 f 0 0 ++ ATP + Mg 2.5 .26 .35 ++ ATP + Mg 5.0 y .25 .35 ++ ATP + Mg 10.0 .26 .34 ++ ADP + Mg 10.0 0 0 *pmoles acetyl pho5phate formed per min per mg protein. *SSpores germinated in Trypticase solution for 2.5 hrs. Reaction mixtures contained: 1.0 m1 of 0.1 M tris buffer, pH 7.4; 0.9 mg spore extract protein, or 1.0 mg germinated Spore extract protein; potassium acetate, 200 pmoles; hydroxylamine, 600 pmoles; and, where indicated, MgClz, l5 pmoles; ATP, 20‘pmoles; ADP, 15 pmoles; water was added to 1.0 ml. Extracts were dialyzed against distilled water at 5 C for 18 hours. Incubation temperature was 30 C; reactions were stopped by addition of 1 m1 of 10% trichloroacetic acid. 52 Spore and germinated spore extracts contained phosphotransacetylase (Table 18) and coenzyme A transphorase (Table 19) as shown by the very sensitive assays used for their detection. TABLE 18. Phosphotransacetylase in extracts of Spores and germinated spores of g. botulinum. Specific activity* System Spore Germinated spore** Complete 1.09 2.3 with 2x extract protein 1.00 2.2 less CoA I 0.0 _ 0.0 less acetyl phosphate 0.0 0.0 *Change in optical density per min per mg protein. **Spores germinated in Trypticase solution for 2.5 hrs. Reaction mixtures contained: 0.02 ml of 0.1 M tris buffer, pH 8.0; reduced CoA, .06 pmole; dilithium acetyl phosphate, 10 pmoles; 2.2 pg extract protein, or as indicated; and water to 0.2 m1. Reac- tion temperature was 25 C. The change in 0.0. at 232 mp was followed due to formation of the thiol ester bond of acetyl coenzyme A. 53 TABLE 19. Coenzyme A transphorase in extracts of spores and germinated Spores of g. botulinum. System Specific activity* Spore .-,_ »~—.--Germinated.sporekk. Complete 0.9 1.4 less enzyme 0 0 less butyryl-SCoA 0 0 less acetate 0 0 *Change in optical density per min per mg protein. **Spores germinated in Trypticase solution for 2.5 hours. Reaction mixtures contained: Potassium acetate, pH 8.0, 30 pmoles; potassium arsenate, pH 8.0, 150 pmoles; butyryl-SCoA, 0.01 'pmole; phosphotransacetylase, 0.01% of a 300-fold purified enzyme from Peptostreptococcus eldsdenii; 22 pg extract protein; and water to 0.15 ml. Reaction temperature was 25 C. The decrease in optical density at 232 mp due to the arsenolysis of acetyl-SCoA was followed. Table 20 is presented so that the reader can easily compare activi- ties found in the extracts of spores, germinated spores and vegetative cells. Points which should be noted are as follows: a. Glucokinase appears to be an inducible enzyme since it is readily found in cells grown in the presence of glucose, but is absent or of extremely low activity in cells grown in medium devoid of added sugar. The enzyme was not detected in extracts of spores or germinated spores, but it should be recalled that the spores used in this study were not produced in glucose-containing media. 54 TABLE 20. Comparative activities of some enzymes in extracts of vegeta- tive cells, spores, and germinated spores of E. botulinwn. Enzymes Range of specific or relative activities in extracts of: or ’7 Vegetative Cells Spores germinated in: 'Systems Glucose Non- Acetate Trypti- Tzzpti' Spores da t d ada t d c e a p e p e case CAPC GlucokinaseI .25-.44 0 0 0 0 0 Phosphohexoseiso- merase and phos- * phofructokinase .63 .19 .06 .11 .04 .04-.08 Aldolase3 1.6-1.85 .75-.80 .33 .50 .09 .06-.11 Aldolase, triose isomerase and G-3-P dehydro- genase .23 .05 .025 .04 .03 .02 Phosphoglyceromu- tase, enolase, and pyruvate kinase .055 .02 0 0 0 0 6 AcetOkll’laSe 2.0'205 107'200 .2".3 021-033 .20 020-025 Diaphorase7 05-065 05L'63 .32‘037 058-060 .25 020-040 DPN-H oxidasé7 .01-.02 .03-.04 .041 .025 .04 .04-.06 Phosphotransace- not tylase 20 19.3 tested 2.3 1.3 1.1 Coenzyme A trans- not phorase 3.0 2.7 tested 1.4 0.9 0.9 *Assayed with Fe++ in reaction mixture. N . 11 II \IO‘IU't-F‘w II II II ll 11 pMoles glucose phosphorylated per hour per mg protein. Coupled to aldolase reaction since aldolase was in excess. The units of specific activity are same as for aldolase. pMoles F-1-6-P split per hr per mg protein. Coupled to DPN+ reduction at 340 mp; change in 0.0. per min. Coupled to DPNrH oxidation at 340 mp; change in 0.0. per min. pMoles acetyl phosphate formed per min per mg protein. Change in 0.0. per min per mg protein. 55 b. Activity of enzymes of glycolysis from extracts of non-adapted cells were significantlylower than those from glucose-adapted cells. The rate of DPN+ reduction with F-l-6-P as substrate was only 0.05 for extracts of non-adapted cells compared with 0.23 for extracts of glucose- adapted cells. The low activity was probably due to a low level of G-3-P dehydrogenase in extracts of non-adapted cells since aldolase (Table 20) was found to be quite active in these extracts. c. Enzyme activities of spore and germinated spore extracts were much lower than those of cell extracts with the exception of diaphorase and DPN‘H oxidase activities. The specific activity of the DPN'H oxidase was 2x to 4x higher in the extracts from Spores than in cell extracts. d. Activities of enzymes from germinated spores were the same or only slightly higher than those from spore extracts depending upon the conditions of germination. Extracts from spores germinated in acetate solution had activities similar to spore extracts, while extracts of spores germinated in the more complete germinating solution (Trypticase) had activities slightly higher than those from spore extracts. Spores germinated in Trypticase plus chloramphenicol yielded extracts with enzyme activities that were as low as those of spore extracts. These data suggested that very little, if any, synthesis of the enzymes studied is necessary for the initial phases of spore germination. DISCUSSION Demonstration of the individual reactions and enzymes of the EMP scheme is evidence for the presence of this pathway for the dissimila- tion of glucose by E. botulinum. The absence of glucose-6-phosphate dehydrogenase would seem to negate the possibility of the shunt path- way, but isotope studies should be run with intact cells before any final conclusion is reached. Glucose fermentation apparently is mediated by a specific and indu- cible glucokinase, and the presence of glucose in the growth medium results in higher levels of most of the enzymes of glycolysis. Such control of enzyme synthesis by induction is often encountered in catabolic pathways, and is recognized as an important regulatory mech- anism permitting the cell to minimize excessive protein synthesis (Pardee, 1959). As a matter of cell economy, the organism may control the concentrations of glycolytic enzymes by a sequential induction pro- cess mediated by the glucokinase. Sequential induction refers to the increase in activity of a whole series of enzymes on the addition of a new compound such as glucose, in this case. Unpublished work of Costilow (1961) indicated that spores of E. botulinum germinated in an acetate solution fermented glucose slowly, and the rate was not inhibited by chloramphenicol; and data presented in this study indicated that there might be an extremely slow glucose fermentation by intact non-adapted cells. This may be due to the pre— sence of a basal level of a fermentative pathway, probably the EMP path- way, in these spores and in cells. Even assuming that the glucokinase in this pathway is an inducible enzyme, it probably is present at a 56 57 basal level. Pollock (1959) points out that it is unlikely for an induced enzyme to be at a zero level, and that frequently low levels of induced enzymes are detected in a non-adapted system under proper assay conditions. The finding of the above-mentioned fermentative activity on glu- cose by spores germinated in the presence of chloramphenicol adds more credence to the idea that the EMP pathway, although inactive until germination, is present in spores even though all of the enzymes of this pathway were not detected in this study. Failure to detect enzymes of the lower-half of the EMP pathway in Spores may have been due to the lack of adequate sensitivity in the assay procedures used. An active DPN-H oxidase partially masked attempts to assay for enzymes by coupling to DPN-H oxidation, and this was the most sensitive assay used for these enzymes. Enzymes of glucose metabolism have been demonstrated in Spores and germinated spores of the aerobic bacilli. In E, 225223! strain T, a mixture of the hexose monophosphate (HMP) shunt and the Entner- Doudoroff pathways of glucose oxidation have been found in Spores (Church and Halvorson, 19573 Halvorson and Church, 1957); While the isotopic studies of Goldman and Blumenthal (1960), with the same strain, and the studies of Amaha and Nakahara (1959) with B. coagulans have shown that most of the glucose is metabolized via the EMP pathway during germination and outgrowth. However, such enzymes are not required by spores for germination and outgrowth. The deamination of alanine to pyruvate and subsequent oxidation of the pyruvate has been shown to be the primary metabolic activity in spores of B. cereus during germination 58 (Halvorson and Church, 1957). ‘E. botulinum can grow, sporulate and germinate in the absence of glucose, so a glucose fermenting system is not required in Spores and cells of this organism. Such data indicate that spores may contain the whole complement of enzymes found in the corresponding vegetative cells but in much lower activity levels than found in cells. At the same time specific enzymes such as the diaphorase and soluble DPN'H oxidase found in the spores of ‘E. botulinum as well as in the spores of aerobic bacilli (Spencer and Powell, 1952; Doi and Halvorson, 1961) in higher levels than similar enzymes of the corresponding cells, and glucose dehydrogenase (Bach and Sadoff, 1960) found in sporulating E. 255222 but not in vegetative cells may play significant roles in germination and outgrowth of spores. The role of DPN‘H oxidase in spores undoubtedly is that of recycling DPN.H to DPN+. The presence of similar systems and enzymes such as diaphorase and DPN.H oxidase in aerobic and anaerobic spores indicates that spores of widely different species may have similar germination processes. The DPN°H oxidase of £3 botulinum appears to be unlike those reported for C. perfringens, E, kluyveri and B, EEIEEER Dolin (1959) reported that the DPN-H oxidase of C, perfringens catalyzes a four- electron reduction of 02 to water, and this activity is lost upon stor- age at ~20 C. Loss of oxidase activity led to conversion of the enzyme to a cytochrome c reductase. Dolin (1959) was not able to show rever- sible removal of the flavin prosthetic group from the oxidase. g. kluyveri catalyzes both DPN-H and TPNoH oxidation with oxygen as elec- tron acceptor; with DPN-H as reductant, the enzyme catalyzes reduction 59 of cytochrome c. The main pathway of electron transport in spores of 2, 555223 is through a soluble DPN-H oxidase that is stimulated by dipicolinic acid and requires FMN for full activity after the flavin component is stripped from the enzymes, whereas FAD gives 70% of the rate of FMN. By contrast, the oxidase of E, botulinum catalyzes a two- electron reduction of oxygen, is stable to storage, does not act as a cytochrome c reductase, uses DPN-H or TPN’H as reductant, and is not stimulated by dipicolinate. Also, after removal of the flavin portion from the oxidase, FAD is required for full activity and FMN only par- tially restores the activity of the enzyme. The high heat resistance of the DPN-H oxidase in spore extracts provides a system which might be used for studying heat resistance of spores. After germination the Spores as well as the oxidase lose their heat resistance, which indicates that the oxidase of spores, germinated spores, and cells may be the same enzyme with different levels of heat resistance. If the heat resistance of extracted Spore enzymes results from the same physical-chemical phenomena responsible for the much greater heat resistance of spores, then a heat stable spore enzyme and its heat labile form in germinated spores or vegetative cells could be purified and used to obtain basic information on mechanisms of spore resistance. The enzyme has properties of stability upon storage, is in high levels in spores and cells, and is easily assayed. All these are cansiderations favoring the use of the DPN-H oxidase as a system for studying spore resistance. It would also be of interest to follow the appearance of the heat resistant oxidase during the transition of the cell into a heat resistant spore. 60 Apparently very little, if any, enzyme snythesis occurs in the first stage(s) of spore germination. The specific activities of enzymes from spores germinated in the presence of chloramphenicol were not accompanied by increased specific activities over that of enzymes from spore extracts. However, increases in specific activities of enzymes from Spores during germination in the absence of chloramphenicol were noted, and there must have been considerable enzyme synthesis during the swelling and elongation process. The specific activities of enzymes in extracts of vegetative cells ranged from 10 to 20 times higher than those of extracts of spores. SUMMARY A study was made of some of the enzymes of vegetative cells, spores and germinated spores of g. botulinum, type A, emphasizing the elucida- tion of a pathway for glucose dissimilation and the metabolic potential of spores. Growth studies showed that both glucose and fructose increased the amount of growth and were utilized during growth, but that galactose and mannose appeared to inhibit growth slightly and were not utilized. Manometric studies with cell suspensions showed that cells grown in the presence of glucose fermented glucose, whereas cells grown without glucose did not. Glucose-non-adapted cells would adapt to glucose or fructose fermentation in the presence of a rich supply of amino acids, and this induction process was inhibited by chloramphenicol, a known inhi- bitor of protein synthesis. Cells grown in the presence of glucose or fructose fermented these hexoses without the induction lag and these activities were not inhibited significantly by chloramphenicol. The data indicate that glucose or fructose can induce the enzyme(s) which is adaptive in this system, and that glucose is a better inducer and sub- strate for fermentation than is fructose. Results obtained with cell-free extracts demonstrated the presence of an inducible glucokinase. This enzyme was induced by either glucose or fructose, but there was very little activity with fructose as sub- strate, and no activity with mannose or galactose. Further studies with cell extracts demonstrated the presence of practically all enzymes of the EMP pathway with the exception of glucokinase which was found only in extracts of glucose-adapted cells. The enzyme activities of these enzymes 61 62 were much higher in glucose-adapted than in non-adapted cell extracts. The absence of glucose-6-phosphate dehydrogenase seemed to exclude the possibility of the hexose monophosphate shunt being active in E. botulinum, but these data are not sufficient to completely validate this conclusion. Data were presented for the possible routes of pyruvate fermentation. Acetyl phosphate, C02, and Hz were formed from pyruvate in the phosphoro- clastic reaction. Lactic dehydrogenase was not detected, but a pathway to ethanol was apparent as alcohol dehydrogenase was shown. Cell extracts also contained DPN.H oxidase, diaphorase, acetokinase, phosphotransacetylase and coenzyme A transphorase. DPN-H oxidase and diaphorase are believed to function in terminal respiration; coenzyme A transphorase is implicated in fatty acid activation; and, the presence of phosphotransacetylase and acetokinase indicates that E. botulinum can activate acetate as acetyl-CoA or store energy from the oxidation of pyruvate as ATP by these reactions. The spores used in this study were produced in a medium devoid of glucose or fructose, therefore it was not surprising to find them devoid of glucokinase. However, extracts of these spores did have activities attributable to phosphohexoseisomerase, phosphofructokinase, aldolase, triose isomerase and glyceraldehyde-3-phosphate dehydrogenase. 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