F d- MEMYL ’ D - MANNOSDE METABOLISM O POHLUAE AND TRB-‘ALOSE BY BACILLUS Thesis for the Degree of Ph». D. MICHlGAN STATE UNNERSlTY AMARET BHUMIRATANA 1974 -—--— -- .h-_._.,I?:.v~ LIB UR Y ”L: Mich}. an State 1 University This is to certify that the thesis entitled WTABOLI SM 0F a-ME’THYL-D—I‘MNNOSIDE’ AND TREHALOSE BY BACILLUS POPILLIAE presented by Amare t Bhumiratana has been accepted towards fulfillment of the requirements for Ph. D. degree in Microbiology and Public Health l/ Major professor Date ”'12/21/73 0-7639 (fab-1‘5? $3 .‘C I I'll... I III -‘IIIAII‘ r I ABSTRACT METABOLISM OF a-METHYL-D-MANNOSIDE AND TREHALOSE BY BACILLUS PUPILLIAE By Amaret Bhumiratana Previous data have shown that oligosporogenous strains of Bacillus popllliae would sporulate in the presence of high levels of admethyl-D-mannoside (a4MM) while much lower levels of glucose or trehalose strongly repressed sporulation. The objectives of the present study were to characterize the initial steps in the metabolism of a-MM and of trehalose by this organism. a-MM was used by all acetate- oxidizing strains of Bacillus popilliae tested at a growth limiting rate after a prolonged induction period. In contrast, trehalose utilization was constitutive in this organism, and the rates of growth and oxidation of trehalose were higher than with glucose as substrate. The utilization of a-MM was very dependent on concentration; i.e., the length of the induction period was decreased and the rate of growth increased as the a-MM concentration increased. Studies with resting cells and cell extracts demonstrated that an a-glucosidase was induced in B. popilliae by either a-MM or admethyl-D-glucoside (a-MG), and that the latter compound was the more efficient inducer. The data indicate Amaret Bhumiratana that both a-MM and aéMG may be hydrolyzed by the same enzyme, but that the affinity of the enzyme for a-MG is much higher than for a-MM. The slow growth response to the mannoside is apparently due to the slow rate of hydrolysis. Studies of trehalose metabolism indicated that this disaccharide is transported into B. popilliae cells via a phosphoenolpyruvate:sugar phosphotransferase system (PTS). The evidences for this are as follows. (a) Both the oxidation and uptake of trehalose were completely inhibited by sodium fluoride. In contrast, glucose oxidation was only inhibited by about 50 percent. (b) No enzyme which would cleave trehalose per so was detectable in cell extracts, but a phosphotrehalase was present which hydrolyzed T6P into equal moles of glucose and glucose-6-phosphate. This enzyme was partially purified and characterized. (c) A mutant culture lacking phosphotrehalase accumulated T6P. (d) Sugar phosphate formation from trehalose by cell extracts was dependent on phosphoenolpyruvate. No other potential phosphoryl donor tested had any significant effect. Therefore it appears that B. popilliae cells form T6P during transport by the PTS system, and the T6P is then hydrolyzed by a phosphotrehalase. Trehalose was also found to repress the synthesis of B-glucosidase, an inducible enzyme in B. popilliae. Glucose had little or no effect on this induction. Repression of B-glucosidase by trehalose could not be overcome by adding exogenous cyclic adenosine-3',5'-monophosphate (CAMP). It appears that B. popilliae has evolved a unique system for metabolism of trehalose which is the primary source of carbon and energy for the growth of this pathogen in its natural host, the larvae of the Japanese beetle (Pbpilliae japonica). METABOLISM OF a-METHYL-D-MANNOSIDE AND TREHALOSE BY BACILLUS POPILLIAE By Amaret Bhumiratana 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 1974 ACKNOWLEDGMENTS I would like to sincerely thank my major professor, Dr. Costilow, for his guidance throughout my graduate program and his critical review of this thesis. Without his generous advice and financial support, it would have been impossible for me to complete this thesis. I also would like to thank my guidance committee, Drs. R. L. Anderson, R. R. Brubaker, and R. L. Uffen for their suggestions and criticisms during the course of this investigation and during the preparation of this thesis. ii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . Biology of Bacillus popilliae . . . . . . . . . . . . . . . Growth requirements of B. popilliae . . . . . . . . . . Glucose catabolism by B. popilliae . . . . . . . . . . Physiological changes in hemolymph of Japanese beetle larvae during infection . . . . . . . . . . Trehalose metabolism by B. popilliae Trehalose . . . . . . . . . . . . . . . . . . . . . . . . . Synthesis of trehalose . . . . . . . . . . . . . . . . Pathways in trehalose metabolism . . . . . . . . . . . Phosphoenolpyruvate:sugar phosphotransferase (PTS) systems PTS system in Staphylococcus aureus . . . . . . . . . . PTS system in Streptococcus lactis . . . . . . . . . . Catabolite repression and sporulation . . . . . . . . . . . Controls in sporulation . . . . . . . . . . . . . . . . Cyclic adenosine-B',5'-mon0phosphate and catabolite repression . . . . . . . . . . . . . . . Cyclic adenosine-3',5'-monophosphate and controls in sporulation . . . . . . . . . . . . . . iii Page vii 10 ll 13 14 14 15 16 BIBLIOGRAPHY . . . . . . . ARTICLE 1 O O O O O O O O Utilization of a-methyl-D-mannoside by Bacillus popilliae. A. Bhumiratana and R. N. Costilow. Canadian Journal of Microbiology, Vol. 19, Number 2, 1973. ARTICLE 2 . . . . . . . . Trehalose metabolism by Bacillus popilliae. and R. N. Costilow. A. Bhumiratana Manuscript to be submitted to Journal of'Bacteriology. APPENDIX 0 O O O O O O O I Studies of the effect of cyclic adenosine-3',5'-monophosphate on the repression of B-glucosidase synthesis in Bacillus popilliae. iv 35 87 LIST OF TABLES TABLE Page ARTICLE 1 l. Utilization of a a-methyl-D—mannoside by various strains of B. popilliae . . . . . . . . . . . . . . . . . . . . 28 2. Effect of concentration on the extent of oxidation of a-methyl-D-mannoside by B. popilliae 2309MC . . . . . . 31 3. Effect of added acetate on a-methyl-D-mannoside oxidation and of added a-methyl-D-mannoside on acetate oxidation by B. papilliae . . . . . . . . . . . . . . . . . . . . 31 4. Effect of arsenite and iodoacetate on the oxidation of a-methyl-D-mannoside by B. popilliae . . . . . . . . . 31 5. Effect of carbon and energy source in growth medium on the rates of oxygen uptake by B. popilliae cells using various sugars . . . . . . . . . . . . . . . . . 32 ARTICLE 2 1. Rate of oxygen uptake by B. popilliae with various concentrations of glucose and trehalose as substrates . 64 2. Partial purification of phosphotrehalase . . . . . . . . . 65 3. Stoichiometry for cleavage of trehalose-6-phosphate by phosphotrehalase . . . . . . . . . . . . . . . . . . . 66 4. Growth responses of B. popilliae 2309MC and the phosphotrehalase negative mutant (2309MClOO) to various carbon sources . . . . . . . . . . . . . . . . 68 TABLE Identification of trehalose-6-phosphate as an accumulated product in mutant 2309MC100 . . . . . . . . . . . . . Production of trehalose-6-phosphate by a crude extract of cells of the mutant 2309MC100 . . . . . . . . . . Effect of potential phosphoryl donors on the production of sugar phosphates from trehalose by cell extracts of B. popilliae 2309MC . . . . . . . . . . . . . . . Activities of phosphotrehalase and the production of sugar phosphates from trehalose by cell extracts of various strains of B. popilliae . . . . . . . . . APPENDIX Induction of B—glucosidase in B. popilliae by salicin . . vi Page 69 71 73 75 95 LIST OF FIGURES FIGURE ARTICLE 1 1. Effect of various concentrations of a-methyl-D-mannoside on the growth of Bacillus popilliae and the pH changes during growth in a trypticase-yeast extract medium with the phosphate omitted . . . . . . . . . . . 2. Respiration rates of B. popilliae cells on a-methyl-D- mannoside and acetate at various stages of cultural development . . . . . . . . . . . . . . . . . . . . . . 3. Hydrolysis of a-methyl-D-mannoside by a crude non—dialyzed extract of B. popilliae cells . . . . . . . . . . . . . ARTICLE 2 1. Effect of trehalose and glucose on B-glucosidase synthesis. 2. Sodium fluoride inhibition of the oxidation of glucose and trehalose by B. popilliae 2309MC . . . . . . . . . . . 3. Sodium fluoride inhibition of [IACJtrehalose uptake by B. popilliae 2309MC . . . . . . . . . . . . . . . . . . 4. Effect of concentration on [lécJtrehalose uptake by B. popilliae 2309MC . . . . . . . . . . . . . . . . . . S. Trehalose inhibition of the growth of mutant 2309MC100 . . APPENDIX 1. Activity of B-glucosidase versus time . . . . . . . . . . . vii Page 29 32 33 77 79 81 83 85 96 FIGURE Page 2. Activity of B-glucosidase versus protein concentration . 98 3. Lineweaver-Burk plot of B-glucosidase activity . . . . . 100 4. Induction of B-glucosidase in B. popilliae . . . . . . . 102 5A & 5B Effect of CAMP on the synthesis of B-glucosidase in B. popiZZiae o o o o o o o o o o o o o o o o o o o o 104 Uptake of [3H]cAMP by B. popilliae 2309MC . . . . . . . 106 0‘ 0 viii INTRODUCTION Bacillus popilliae is a causative agent of milky disease of Japanese beetle larvae (Pbpilliae japonica). The use of this organism in biological control has not been possible because of the failure to obtain high populations of spores in vitro. The types and quantities of carbohydrates present in the media have been found to affect the ability of many bacilli to sporulate (3,79). In the hemolymph of the Japanese beetle, B. popilliae has been found to sporulate in the presence of large amounts of trehalose (69). However, trehalose at levels above 0.1% inhibits in vitro sporulation of an oligosporogenous strain (81). Some oligosporogenous strains of B. popilliae were recently found to sporulate in the presence of high levels of a-methyl-D-mannoside (a-MM) (16). These findings suggested that there must be major differences in the utilization of trehalose and a-MM by B. popilliae, and instigated the present investigation. Studies of the metabolism of these two compounds constitute the major parts of this thesis. It has been previously proposed by Schaeffer et al. (79) that certain essential spore specific enzymes may be regulated by a mechanism similar to the regulation of B-galactosidase synthesis by catabolite repression in Escherichia coli. Cyclic adenosine-3',5'~monophosphate (CAMP) has been shown to relieve catabolite repression of the lactose enzymes in E. coli (67), and to be of great importance in the differentiation of Dictyostelium (72). Therefore, a few experiments were conducted to determine if it was possible to demonstrate a regulatory role of cAMP in the sporulation or in the repression of an inducible enzyme in B. popilliae. This thesis is organized into four sections. The first is a literature review including discussions of the biology of Bacillus popilliae, known pathways of trehalose metabolism, phosphoenolpyruvate: sugar phosphotransferase systems involved in the active transport of lactose in Staphylococcus aureus, and the control of sporulation by catabolite repression. The second section consists of a published manuscript on the utilization of a-methyl-D-mannoside by B. popilliae, and the third section is comprised of a manuscript prepared for publi— cation on the metabolism of trehalose by B. popilliae. The last part of the thesis is an appendix detailing the studies of the effects of CAMP. LITERATURE REVIEW Biology of Bacillus popilliae Bacillus popilliae was first reported by Dutky (18) in 1940 as a causative agent for milky disease of the Japanese beetle (Pbpilliae japonica). Reviews of the biology of this disease and of the potential of using B. popilliae for biological control have been published (19,89). Large scale production of spores of B. popilliae is the key to extensive use of this organism in biological control. However, attempts to obtain large spore yields of the organism in vitro have been unsuccessful. Some specific strains sporulate at low frequency under specific conditions (48,71,81). Also, growth in a liquid medium containing activated carbon results in limited formation of spores (28). Furthermore, in one medium (15), as much as 50% to 100% of the population formed refractile bodies which appeared to be abortive spores (52). Growth requirements of B.gpopilliae. Various media and condi- tions to grow B. popilliae have been established (15,80,88). Only limited growth results in synthetic media (91). Complex nitrogen sources are required for optimal growth, and there is but little growth in a carbohydrate free medium (4,15,19,70). The addition of glucose, fructose, or trehalose to a complex medium supports good growth (15,19,70). a-Methyl-D-glucoside, a-methyl-D-mannoside, and salicin have also been found to support growth of B. popilliae 3 (4; and Costilow, unpublished results). Pyruvate may be utilized to some extent, since addition of this compound induces growth equal to about half that obtained with an equivalent amount of glucose. Arabinose, ribose, xylose, rhamnose, galactose, lactose, sucrose, cellobiose, melibiose, raffinose, melezitose, starch, inulin, and glycerol are not utilized or attacked. The very limited growth in a trypticase, yeast extract medium in the absence of a fermentable carbohydrate indicates that peptones do not serve as a satisfactory source of energy or as a carbon source for cell proliferation even when present at high concentrations (19). Glucose catabolism in B. popilliae. B. popilliae catabolizes glucose to produce lactic acid, acetic acid, and C02 as major products; and a small but measurable amount of ethanol, glycerol, and traces of acetoin, and acetaldehyde (65). The ratio of acetic acid and lactic acid can be varied over a wide range by changing the oxygen level in the atmosphere. As the percentage of oxygen increases, acetic acid production increases and lactic acid production decreases. Pepper and Costilow (65) demonstrated that the organism catabolizes glucose through Embden-Meyerhof—Parnas and hexosemonOphosphate pathways. There was no evidence of Entner-Doudoroff or phosphoketolase pathways. No glucose oxidase or glucose dehydrogenase activities were detected in the cell extracts. More recently, Bulla et al. (5) investigated the catabolism of glucose by proliferating vegetative cells of B. popilliae and obtained similar results. The presence of tricaboxylic acid (TCA) cycle in B. popilliae was examined by McKay et al. (48). They postulate that B. popilliae has a complete TCA cycle but the activity of the cycle is strongly repressed in wild type strains under the usual conditions used for in vitro cultivation. Somewhat different conclusions on the presence of a complete TCA cycle were reported by Bulla et al. (6). The different strains of B. popilliae used and the differences in methods of cultiva- tion of the organisms may account for the conflicting results. However, B. popilliae cells harvested from larval hemolymph were found to oxidize acetate (personal communication, NRRL., Peoria, Illinois). A functional glyoxylate cycle was not found in B. popilliae (6,65). B. popilliae was originally reported to be a facultative anaerobe and to grow best in highly poised reducing media (18). However, Pepper and Costilow (65) failed to obtain significant growth in the complete absence of oxygen, and found that aeration of broth cultures was very stimulatory to growth (15). Glucose was not dissimilated under anaerobic conditions, although pyruvate was dissimilated anaerobi- cally to approximately equimolar amounts of acetic and lactic acid and CO No hydrogen was evolved (65). 2. There is a cytochrome dependent electron transport in B. popilliae utilizing cytochrome b but not cytochrome c (66). The cytochrome dependent system is particulate, whereas the independent electron transport system is soluble and is stimulated by flavin nucleotides. There is a soluble NADH oxidase which produces H202; and the soluble system is relatively more active than the particulate oxidase in older cells (66). B. popilliae does not have catalase or peroxidase (66), however, it does contain high levels of superoxide dismutase (14,95). Physiological changes in hemolymph of Japanese beetle larvae during infection. A relatively small amount of work has been done on comparing the physiological changes in infected and non-infected hemolymph of Japanese beetles. The pH of the larval hemolymph was unchanged during the course of the disease (87). The dissolved oxygen content of larval hemolymph containing proliferating B. popilliae was one-third that of normal larvae (93). Organic acids (90), and amino acids (82) in healthy and diseased larvae have been examined. The amount of lactate and acetate remained constant throughout the infectious period, and that of pyruvate doubled (90). Trehalose metabolism in B. popilliae. Trehalose is the predom- inant carbohydrate present in the hemolymph of many insects (94). The hemolymph of third instar Japanese beetle larvae does not contain a significant amount of glucose but does contain a relatively large amount of trehalose (69). The amount of trehalose in the hemolymph of diseased larvae was approximately two-thirds the amount in healthy larvae, and may indicate the use of trehalose for proliferation of the pathogen in the host (69). Growth of B. popilliae in liquid medium indicates that trehalose is readily utilized, and the products of its fermentation appear to be similar to those from glucose (70). However, a small amount of glucose in an acetate agar medium severely restricted spore formation, while trehalose was found to be less inhibitory to sporulation of B. popilliae than glucose (71). Costilow and Coulter (16), and Rhodes (69) have suggested that most of trehalose in the hemolymph may not be available to the cells. Trehalose Synthesis of trehalose. Trehalose is a disaccharide (l-a-D-glucopyranosyl-a-D-glucOpyranoside) which is widely distributed in insects (94), fungi (34), and in bacteria (26). Mostly, trehalose exists in these organisms as a storage compound. Synthesis of trehalose has been worked out in locusts (9), Dictyostelium discoideum (38,76), and in Mycobacterium smegmatis (41). The steps in trehalose synthesis involve trehalose-6-monophosphate as an intermediate. It proceeds as follows: Uridine diphosphate glucose (UDPG) or + Glucose-6-phosphate Guanosine diphosphate glucose (GDPG) UDP or + Trehalose-6-phosphate GDP 1 Trehalose + Inorganic phosphate A number of enzymes are known which synthesize trehalose-phosphate but most of these have not been examined in detail. Those reported to utilize UDPG as the glucosyl donor have been isolated in yeast (8,62), silkmoth (55), Dictyostelium discoides (76), locusts (9), and Mycobacterium tuberculosis (26). The synthesis of trehalose in Streptomyces hygroscopicus was found to utilize GDPG as the glucosyl donor (23). Friedman (25) has isolated trehalose-6-phosphate phosphatase from Phormia regina. The enzyme catalyzes the hydrolysis of trehalose-6-phosphate into free trehalose and inorganic phosphate. The enzyme is very specific. It does not hydrolyze any other phosphoryl containing compound except that it attacks glucose-6-phosphate at the rate of about 8% that of its normal substrate (25). Pathways in trehalose metabolism. Metabolism of trehalose, as of present, has been found to proceed in two separate ways. The first and probably the most common route is the hydrolysis of trehalose into two glucose molecules by the enzyme trehalase. Friedman (24) found that the trehalase of Phormia regina is specific for trehalose with no activity toward the following substrates: maltose, sucrose, melibiose, lactose, raffinose, and turanose. Saito (77), and Carnie and Proteous (10) have isolated trehalase from pupae of the silkworm, Bombyx'mori, and rabbit small intestine respectively. Furthermore, trehalase has been examined in several insect species (94), Neurospora (34), and Dictyostelium discoideus (11). Generally, trehalase is found to be quite specific for the u,u linkage of trehalose and not to be a general u-glucosidase (24,47). Clements et al. (13) examined trehalase in fleshfly sarcophage barbata, and reported the enzyme to have an optimal pH of 5.3, and a Km of 1.4 x 10-—3 M. These were the same for both a soluble and a particulate enzyme. Hey and Elbein (33) studied the trehalase from Streptomyces hygroscopicus. This enzyme had a K,m of 1.8 x 10"2 M and an optimal pH of 6.5. Tris (hydroxymethyl) aminomethane buffer was found to inhibit many trehalases (24,33,47). The trehalase from Streptomyces hygroscopicus was purified 80 fold and was found to be very specific for trehalose, with only a slight activity in trehalosamine. The enzyme is present in all of the species of Streptomyces. The trehalase from Drosophila melanogaster is consti- tutive (47). Recently an alternative pathway for metabolism of trehalose has been described in Euglena gracilis (1). The enzyme trehalose phosphorylase catalyzes the following reaction: Trehalose + Inorganic phosphate== B-Glucose-l-phosphate + Glucose This enzyme has been partially purified. The reaction is reversible and either 6-deoxy-glucose or xylose can be substituted for glucose in the reverse reactions forming 6-deoxy-a-D-glucopyranosyl-u-D-glucopyranoside and u-D-xylopyranosyl-a-D-glucopyranoside respectively (2). Trehalose is absorbed and respired by Myrothecium verocaris. It has been proposed that the enzyme trehalase of M2 verocaris may be involved in trehalose transport (45). Further studies with the same organism indicate that trehalose can also be absorbed by another trans- port system which can be induced by exposure to turanose or nigerose (45). This induction needs oxygen and trehalose has no activity as an inducer. Turanose induces the system specific for a-linked glucosyl— glucose or glucosyl-fructose. Trehalose is absorbed against a 10 concentration gradient by the constitutive as well as by induced system. The non-induced system is associated with trehalase, and the rate of absorption of trehalose by the induced system is affected by trehalose concentration. Phosphoenolpyruvate:sugar phosphotransferase systems Only two different pathways for the metabolism of trehalose, involving either hydrolysis or phospholysis have been described. However, two other possible pathways for metabolism of disaccharides have been reported. Both involve phosphorylation of the disaccharide prior to cleavage. The main differences in the two pathways are with respect to the phosphoryl donor, and the nature of the transported disaccharide molecule. Cellobiose and gentibiose have been found to be phosphorylated with adenosine-S'-triphosphate (ATP) by a soluble, inducible B-glucoside kinase after they enter the cell. The disaccharide monophosphates are then subsequently cleaved by a phospho-B-glucosidase (58,59,60,6l). Both enzymes from.Aerobacter aerogenes were partially purified and characterized by Palmer and Anderson (59,60). In the other pathway in which a disaccharide is phosphorylated, the phosphorylation occurs concomitantly with the transport of the molecule inside the cell via the phosphoenolpyruvate (PEP) dependent phosphotransferase (PTS) of Kundig and Roseman (39). The PTS system is widely distributed in bacteria (73), and the extensive work on this system has been reviewed (27,74,75). This portion of the review will include only the PTS systems which involve phosphorylation of lactose in Staphylococcus aureus and Streptococcus lactis. 11 PTS system in StaphylocOccus auerus. Using a lactose negative strain of S. aureus, Hengstenberg et al. (30) showed that this strain of S. aureus accumulates lactose phosphate (a phosphoryl group is on the "6" position of the galactose moiety) when lactose is used as a substrate. They also demonstrated that S. aureus has an inducible enzyme which hydrolyses lactose phosphate into glucose and galactose-6-phosphate. Phosphoenolpyruvate stimulates the hydrolysis of gfnitro-phenyl-B-D-galactoside (ONPG) (31), and 5.0 x 10-2 M sodium fluoride inhibits ONPG hydrolysis by intact cells of S. aureus (36). The data indicate that a continuing supply of PEP formed via the enolase reaction is needed for the hydrolytic process (36). Galactose-6-phosphate is the best inducer and isoprOpyl-B-D- thiogalactoside (IPTG) at 1 x 10"3 M inhibits induction (54). Using specific mutants of PTS systems, Hengstenbert et al. (32) partially purified the four components of the PTS system; Enzyme I, HPr, Factor III, and Enzyme II. Laue and MacDonald (42) found that the product of thiomethyl-B-galactopyranoside (TMG) accumulated in S. aureus is TMG-6-phosphate. The accumulation of TMG as TMG-6-phosphate in whole cells and the phosphorylation of TMG by cell extract were compared in terms of the effect of temperature, inhibitors, and genetic change (43). Egan and Morse (20,21,22) isolated and characterized a car- mutant of Staphylococcus aureus which cannot utilize lactose, maltose, sucrose, galactose, fructose, mannitol, ribose, or trehalose. The ability to ferment glucose was retained but the rate of fermentation was slower in car- mutant. The car- mutant does not accumulate any sugar derivatives 12 (30) and was suggested to be an Enzyme I mutant from a mixing experiment with an Enzyme I mutant of E. coli (31). Roseman and co-workers (29,57,83,84,85,86) have characterized the PTS system in Staphylococcus aureus in much detail. The system catalyzes the transfer of a phosphoryl group from PEP to B-galactosides to yield corresponding galactoside-G-phosphate esters. This transfer requires the participation of four protein components, Enzyme I, two lac lac phospho-carrier proteins (HPr and Factor III ) and Enzyme II These components were separated from one another and subjected to purification procedures (29,86). The four protein components of the PTS system are required for the PEP dependent phosphorylation of lactose and its analogs. The overall reaction proceeds by an ordered sequence of phosphoryl transfer steps, each of which is studied in detail by Simoni et al. (83). The initial phosphoryl transfer from PEP to the phospho-carrier protein HPr is catalyzed by Enzyme I; phospho-Enzyme I appears to be an intermediate in this reaction. Only one phosphoryl group is transferred to each HPr molecule. The subsequent reversible phosphoryl transfer from phospho—HPr to the sugar specific phospho- carrier protein Factor IIIlac is entirely self-catalyzed. The phosphoryl groups in both phospho-carrier proteins are linked to the imidazole moieties of histidine residues; to N-l in HPr and N-3 in Factor III. The final step, formation of galactoside—6-phosphate, is catalyzed by the membrane bound Enzyme II. Kinetic studies indicate that the reaction does not proceed by a 'ping-pong' mechanism, but that a tenary 13 complex is formed by the two substrates, phospho-Factor III, and lactose, and Enzyme Illac. No evidence for a phospho-Enzyme II intermediate is observed. Simoni and Roseman (85) presented evidence for the physiological role of the PEP:lactose phosphotransferase system in the utilization of lactose by Staphylococcus aureus. This organism takes up lactose by group translocation, i.e., the sugar is phosphorylated while it passes through the cell membrane, and that the phosphotransferase system is responsible for this process. Studies of mutants defective in proteins of the PTS systems provide the evidence for this conclusion. Mutants lacking Enzyme I are unable to ferment a variety of sugars, and cannot take up methyl—a-glucopyranoside or thiomethyl-B- galactopyranoside. Mutants defective in Enzyme II or Factor III of the PTS system are also incapable of transporting thiomethyl-B- galactopyranoside, but transport methyl-u-glucopyranoside at normal rates. The mutants do not catalyze a facilitated diffusion process, therefore indicating that the phosphotransferase system is responsible for translocation per se, and that it does not act as a "sugar trap" (85). There are some speculations (75) that all carbohydrates are transported and phosphorylated by PTS system in S. aureus. However, Button et al. (7) have shown that maltose is metabolized principally via diffusion and hydrolysis of the non-phosphorylated molecule. PTS system in Streptococcus lactis. McKay et al. (50) demonstrated that various strains of Streptococcus lactis have an l4 enzyme, B-D-phosphogalactoside galactohydrolase which hydrolyses lactose-phosphate. The latter compound is formed by the PTS system. Lactose utilization by intact cells of S. lactis C 2 2F is inhibited by 3 x 10- M NaF; and phosphoenolpyruvate but no other phosphoryl donors stimulate the hydrolysis of ONPG by extracts of cells of this organism. Also, toluene treated cells need PEP to hydrolyse ONPG. The uptake of [}4G]TMG was studied, and the accumulated product shown to be TMG-phosphate. Complementation studies between cell extracts of S. lactis and of various PTS mutants of Staphylococcus aureus showed that the components in S. lactis are essentially interchangeable with that of S. aureus (49). MOlskness et al. (53) studied B-D-phosphogalactoside galactohydrolase from S. lactis; and Premi et al. (68) partially purified B-D-phosphogalactoside galactohydrolase from Lactobacillus casei. Using ONPG-6-phosphate as substrate the enzyme was found to have a Km of 1.59 x 10"3 M, a pH optimum of 5.0, a temperature optimum of 37 C, and a molecular weight of 1.3 x 10-5. Catabolite Repression and Sporulation Controls in sporulation. The differentiation of vegetative cells to endospores requires changes in morphological events which undoubtedly arise from well controlled changes in biochemical events. The biochemical events in vegetative cells and sporulating cells are believed to be quite different both in the catabolic and biosynthetic pathways. These changes result in the production of enzymes, substances, and structures new or different from vegetative materials (3). The 15 types of controls involved have to account for the ability of an organism to repress the activities of spore specific enzymes during vegetative growth, to shut off certain vegetative enzyme activities and turn on the spore specific enzymes during sporogenesis (46). It has been previously proposed by Schaeffer et al. (79) that certain essential spore specific enzymes may be regulated by a mechanism similar to the regulation of B—galactosidase synthesis in Escherichia coli. The spore specific enzymes may be repressed by a catabolite(s) during vegetative growth. When the amount of the catabolite(s) in the cell decreases, the repression of a key sporulation specific enzyme(s) would be released. The synthesis of a spore specific enzyme may then lead to sequential induction of several other spore specific enzyme(s) needed for the formation of the various spore components (56). Cyclic adenosine-3',5'-monophosphate (cAMP) and catabolite repression. Makman and Sutherland (44) demonstrated the effect of glucose on the level of cAMP in E. coli in 1965. Since then, cAMP and its roles in the control of enzyme synthesis have been demonstrated in several other genera of bacteria (37,64,72). The finding that CAMP relieved catabolite repression and transient repression of the synthesis of B-galactosidase in E. coli (67) led to the discovery of a positive control on the transcription of the lactose operon. Several other enzyme systems beside B-galactosidase have also been found to be affected by cAMP (17) either at the transcriptional level (51,92), translational level (40,63) or at the level of enzyme activity (78). Furthermore, cAMP has been shown to be associated with control of differentiation in Dictyostelium (72). l6 ’ Cyclic adenosine-3',5'-monophosphate and controls in sporulation. The ability of cAMP to relieve catabolite repression and transient repression, and its effect on certain types of differentiation has led to the speculations that cAMP may be involved in the control of sporulation. So far, there have been very few reports on cAMP in Bacillus species. Ide (35) looked for adenyl cyclase and phosphodiester- ase in four Bacillus species and was unable to detect any activities of either enzyme. However, Clark and Bernlohr (12), working with B. lichenifbrmis found that the level of cAMP in the cell increased at the time of sporulation. Yet, they found no significant difference in the activities of adenyl cyclase or phosphodiesterase during sporulation. 7. 9. 10. ll. BIBLIOGRAPHY Belocopitow, E., and L. R. Marechal. 1970. Trehalose phosphorylase from Buglena gracilis. Biochim. Biophys. 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Regulation of lac messenger ribonucleic acid synthesis by cyclic adempsome-B',5'-monophosphate and glucose. J. Biol. Chem. 245: 2259-2267. Wéiner, B. A., W. F. Kwolek, G. St. Julian, H. H. Hall, and R. W. Jackson. 1966. Oxygen concentration in larval hemolymph of the Japanese beetle, Pbpillia japonica, infected with Bacillus popilliae. J. Invert. Pathol. 8; 308-313. Wyatt, G. R. 1967. The biochemistry of sugars and polysaccharides in insects. Adv. Insect. Physiol. 4; 287-360. Yousten, A. A., L. A. Bulla, and J. M. McCord. 1973. Superoxide dismutase in Bacillus popilliae, a catalaseless aerobe. J. Bacteriol. 113: 524-525. ARTICLE 1 UTILIZATION OF a-METHYL-D-MANNOSIDE BY BACILLUS POPILLIAE By Amaret Bhumiratana and Ralph N. Costilow Appears In: canadian Journal of‘Mficrobiology Volume 19, Number 2, 1973 U ' ' tion of a-methyl-D-mannoside by Bacillus popilliae“ 2 AMAREI‘ BHUMIRATANA AND R. N. COSTILOW Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan Accepted October 11, 1972 BHUMIRATANA, A., and R. N. COSTILOW. 1973. Utilization of a-methyl-D-mannoside by Bacillus popil- liae. Can. J. Microbiol. 19: 169—176. a-Methyl-D-mannoside (a-MM) was used by all acetate-oxidizing strains of B. popilliae tested at a growth limiting rate after a prolonged induction period. Those strains which did not oxidize acetate could not use a-MM for growth unless it was present at a high concentration. The period of induction was decreased, the rate of growth increased, and the extent of oxidation decreased as the a-M M con- centration was increased. Theoretical levels of 02 uptake and C02 evolution for the complete oxidation of the mannose moiety of a-MM were observed when the concentration of a-MM was low. Studies with resting cells and cell extracts demonstrated that an a-glucosidase was induced in B. popilliae by either a-MM or a-methyl-D-glucoside (u-MG). The enzyme was induced more rapidly by u-MG than by u-MM. Similarly, the ability of cells to oxidize a-MM was induced more efficiently by a-MG than by the mannoside. The data indicate that the hydrolysis of both u-MG and a-MM may be catalyzed by the same enzyme, but that the affinity for a-MG is much higher than that for the mannoside. The slow hydrolysis of u-MM by this enzyme is believed to account for the type of growth response observed. BHUMIRATANA, A.,et R. N. Cosmow. 1973. Utilization of a-methyl-o-mannoside by Bacillus popilliae. Can. J. Microbiol. 19: 169-176. L’alpha-méthyl-D-mannoside (alpha-MM) est utilise par toutes les lignées de Bacillus popilliae qui oxidant l’acétate et testées a un taux limitant a la croissance apres une période d‘induction prolongée. Les lignées qui n’oxident pas l‘acétate ne peuvent pas utiliscr l‘alpha-MM pour la croissance a mains qu’il soit present a une forte concentration. Lorsque la concentration de l’alplza-MM est augmentee. la période d’induction décroit, le taux de croissance augmente ct l’intensité de l’oxidation diminue. Les valeurs the'oriques de la consommation d’Oz et de l’e'volution du C02 pour l’oxidation complete de la partie mannose de l’alpha-MM ant eté observées lorsque la concentration de l’alpha-MM est diminuee. Des études avec des ccllules au repos et des extraits cellulaires demontrent qu’une alpha-glucosidase est induite chez B. popilliae par l’alpha-MM ou l’alpha-méthyl-D-glucoside (alpha-MG). L’enzyme est induit plus rapidement par l’alpha-MG que par l’alpha-MM. Semblablement, l‘habilité des ccllules a oxider l’alpha-MM est induite plus elficacement par l’alpha-MG que par le mannoside. Les données indiquent que l’hydrolyse de l’alpha-MG et de l’alpha-MM peut étre catalysee par le méme enzyme, mais que l’aflinité pour l’alpha-MG est beaucoup plus élevée que pour le mannoside. Nous crayons 169 que l’hydrolyse lente de l’alpha-MM par cet enzyme explique le type de croissance observe. Introduction An oligosporogenous strain of Bacillus popil- liae (NRRL B-2309M) forms spores during colonial development on agar plates of a special medium devised by Sharpe et al. (12). Spore formation occurs in colonies during a prolonged period of a decelerating growth rate (1). The glucose level in the medium is very low during this time and the slow diffusion of the remaining sugar to the cells probably results in the pro- longed period between exponential and maxi- mum growth. When a-methyl-D-mannoside ((1- MM) was substituted for the 0.05% glucose in this medium, it was found that a much higher level (0.5%) of the mannoside was required to attain a sporulation frequency equivalent to lReoeived June 7, 1972. 2Journal Article No. 5659, Michigan Agricultural Experiment Station. [T raduit par le journal] that in glucose medium. It is known that sporu- lation of Clostridium thermosaccharolyticum occurs in media with carbon sources which greatly limit growth or when glucose is added to the medium at a growth limiting rate (4, 5). It appeared possible that a-MM could be func- tioning in a similar manner during sporulation of the oligosporogenous strain of B. popilliae. Thus, we decided to study the use of this sugar in some detail. The data herein demonstrate that the use of a-MM is inducible in B. popilliae and that the sugar is used at a growth limiting rate. This probably explains the ability of an oligosporo- genous strain to sporulate in the presence of a high level of the mannoside. It is believed that the reason for the slow rate of a-MM utilization by this organism is that the hydrolytic enzyme induced is actually an a-glucosidase with a very low affinity for the mannoside. 170 CAN. J. MICROBIOL. VOL. 19, 1913 Materials and Methods Cultures and Media The sources and characteristics of all the strains of B. popilliae used in this study have been described else- where (9). Unless otherwise specified, all experiments were conducted with B. popilliae NRRL B-2309MC. This is an asporogenic strain derived from the oligo- sporogenous strain NRRL B-2309M described by Sharpe et al. (12). This strain was selected for study since the type of growth response to a-MM was similar to that with 2309M but the cell yields were higher with 2309MC. The basal medium used routinely contained 1.5% trypti- case, 0.5% yeast extract, 0.6% K2HP04 (TY medium). The phosphate was left out of the medium in some ex- periments so that pH changes could be clearly observed. Unless otherwise indicated, carbon sources were added at a level of 0.2%. Cells used in these experiments were grown in TY medium containing 0.2% u-MM (TYM) unless otherwise stated. Cultures were incubated at 30°C on a rotary shaker. Growth Studies Growth was followed by measurements of the optical density (O.D.) at 620nm using a Gilford model 2000 spectrophotometer. The pH was measured with a Beck- man, model G, pH meter. Oxidation Experiments The oxidation of various carbon sources by resting cells was assayed manometrically. The direct procedure (14) was used for determining total CO2 production. The 14C02 produced from [1-14C] acetate was determined by the procedure of McKay et al. (9). The level of a-MM was estimated by measuring the concentration of reducing sugar before and after hydrol- ysis. Hydrolysis was accomplished by adding 0.4 ml of concentrated HCl to a 4—ml sample and incubating 1h at 100°C. The sample was neutralized and made to a standard volume before assaying. Reducing sugar was determined by the method of Neish (10). The amount of free reducing sugar released from u-MM during the oxidation experiments was estimated by determining the amount present initially and at the end of the reaction period. Enzyme Assays Crude extracts were prepared from cells harvested from cultures at the time of maximum growth response to the carbon source added. Cells were disrupted by ultrasonic oscillation for four lS—s intervals with an MSE-IOO probe (Measuring and Scientific Equipment) at 220 V and a peak to peak amplitude of ~ 7 microm- eters (um). large particles were removed by centrif- ugation at 3000 X g for 15 min, and the supernatant was dialyzed overnight against 500 volumes of distilled water. Protein was estimated by the method of Lowry et al. (8). The hydrolysis of u-MM by the extracts was followed by measuring the amount of reducing sugar released (see above for assay). Reaction mixtures of 1.5 ml total volume contained cell extract, 0.05 M potassium phos- phate (pl-1 7.2), and various levels of u-MM. Substrate was added to start the reaction at 30°C. At various inter- vals, 0.25-ml aliquots were removed, diluted to 2 ml, and boiled at 100°C for 10 min. The precipitate was removed by centrifugation and the supernatant solution assayed. Total u-glucosidase was assayed by following the release of p-nitrophenol from p-nitrophenyl-a-D-glucoside (PNPG). The increase in CD. at 420nm was continu- ously measured using a Gilford model 2000 recording spectrophotometer. The assay system was similar to that TABLE 1 Utilization of a-methyl-D-mannoside (a-MM) by various strains of B. popilliae Optical density, 620 nm pH Strain No sugar + a-MM No sugar + a-MM Experiment 1° Uninoculated — — 7.3 7.3 2309 0.216 0.207 7.4 7.3 2309PA 0.241 0.235 7.3 6.8 23098 0.325 0.357 6.8 6.9 2309MC 0.372 1.462 7.5 8.6 2309M” 0.424 0.667 7.2 8.2 Experiment 2° 2309Mb 0.074 0.627 7.1 8.25 MS-9b 0.124 0.697 7.4 8.5 MS-S4b 0.252 0.605 8.1 8.5 MS-69b 0.453 0.937 8.5 8.4 MS-85” 0.459 0.629 8.5 8.7 Experiment 3d 23095 0.173 1.439 7.45 6.6 "The basal medium in Experiment 1 contained 0.5 '2, peptone, 0.5 Pb yeast extract. 0.1 Ci, K2HPO4, and 0.01% MnStu Where indicated. 0.2 ,2, a-MM was added. Readings were taken after 13 days incubation. "()lignspomgcnous strains. 'A 1.5 ;. trypticase, 0.5 5 yeast extract broth was the basal medium in Experiment 2, and 0.2% u-MM was added when: Indicated. Readings were taken after 7 days incubation. “Same as c except that 1“,}, u-MM was added. 'n._— —-—- + a—___— h—_F——s~.—_——~._—‘—F «Ag—“*— -.n"7 BHUMIRATANA AND cosmow: BAClLLUS POPILLIAE 171 of Halvorson and Ellias (3). However, no glutathione or MgClz was added since these reagents failed to stimulate the hydrolysis. Hydrolysis of a-methyl-D-glucoside (ll-MG) was followed by coupling the reaction with hexokinase and glucose-6-phosphate dehydrogenase and measuring the rate of reduction of nicotinamide adenine dinucleotide phosphate (NADP+). Reaction mixtures of 0.5 ml in 5 mM phosphate buffer (pH 7.2) contained 2mM MgClz, 2 mM adenosine triphosphate (ATP), 0.4mM NADP+, excess hexokinase, excess glucose-6- phosphate dehydrogenase, cell extract, and variable u-MG concentrations. The cell extracts contained some ATPase activity and low levels of an NADPH oxidase but the reactions were linear for over 1 min. Chemicals a-Methyl-D-mannoside was purchased from General Biochemicals, a-MG was from Pfanstiehl Chemical Co., and PNPG and p-nitrophenyl-a-D-mannoside were from Mann Research Laboratories. Results Growth Response A number of asporogenous and oligosporo- genous cultures of B. popilliae were tested for their ability to use a-MM for growth. All of the latter cultures and one asporogenic strain (2309MC) derived from 2309M showed a growth response to this sugar (Table 1). Also, the final pH of the medium was higher than that of the control (no added sugar) medium. In contrast, the other three asporogenic strains tested failed to respond to the presence of 0.2% a-MM. When 1% tt-MM was added, the optical density of the culture of strain 23098 did increase greatly, but the final pH of the medium was less than that of the control. The ability to use a-MM at a low concentration is correlated with the ability to oxidize acetate. McKay et al. (9) found that all of the oligosporogenous strains and strain 2309MC would oxidize acetate and that strains 2309 and 2309s would not. While 2309PA was originally found to oxidize acetate, we found that it would no longer do so. With some of the cultures tested, there was an extended lag period of growth (up to 4 days) in an a-MM medium after the optical density had attained the level of a control with no added sugar. This lag was eliminated when the cul- tures were successively transferred in an a-MM 3.0 I T 2.0 |.00 0.50 0.30 0.20 OPTICAL DENSITY 0.05 0.03 0.020 20 40 60 80 100 120 H 0U R 3 FIG. 1. Effect of various concentrations of a-methyl-D-mannoside (a-MM) on the growth of B. popilliae :Illld the. pH changes during growth in a trypticase - yeast extract (TY) medium with the phosphate omitted. e solid lines represent growth and the dashed lines pH. The concentrations of a-MM were: (0), zero; (0). 1%; (A). 0.6%; (A). 0.4%; (v). 0.2%; (V). 0.1%. We} r . 172 CAN. J. MlCROBlOL. VOL. 19, 1973 medium, but was observed again when cells grown in TYM were subcultured in a glucose medium and then subcultured again in TYM. Thus, it did not appear that the prolonged lag was due to the selection of mutants. The concentration of a-MM in the medium had a pronounced effect on the length of the lag period in the growth curve, the rate and extent of growth during the use of a-MM, and on the pH changes observed in the medium (Fig. 1). With 1‘}; a-MM present the lag period was less than 24 h, the generation time during the initial response was from 8 to 10 h, and the final optical density was about 2.0. Correspond- ing values in the presence of 0.1 and 0.2% a-MM were about 52 h, 24 h, and 0.8 optical density, respectively. It is also evident from Fig. 1 that acid accumulated in cultures when a-MM was present in great excess, but there was no ac- cumulation evident when the sugar was added at low levels (0.1 and 0.2%). When 0.2% a-MG was used as the carbon source, there was only a 4- to 5~h lag in the growth response, and the generation time during exponential growth was the same as in a glucose medium (3 to 4 h). Also, the pH of the medium decreased rapidly during the period of rapid growth. Oxidation of a-MM Cells harvested from TYM oxidized a-MM (with a respiratory quotient (RQ) of 1, and, when the sugar was present at low levels, ap- parently oxidized the mannose moiety to com- pletion (Table 2). However, as the concentra- tion of a-MM was increased, the amounts of 02 consumed and C02 produced per umole of a-MM used were progressively lowered. At the highest u-MM level tested, only 2umoles of each gas was consumed or produced per umole of sugar oxidized. However, there was a sig- nificant amount of free reducing sugar released into the medium. The rates of 02 uptake by resting cells were also dependent on the a-MM concentration. The Q02 of one lot of cells from TYM medium was ~ 19 uliters 02 per hour per milligram (dry weight) when measured in the presence of 13 mM a-MM while with either 44 or 130 mM a-MM the Q02 was ~ 38. However, with the highest substrate level the respiration was linear for only ~ 1 h while it was linear for over 2 h with the two lower levels tested. Since the foregoing data indicated that the mannose moiety of a-MM was oxidized com- pletely if the a-MM was at a reasonably low level and that the pH of the growth medium increased throughout the incubation period, it appeared possible that the presence of a-MM might stimulate acetate oxidation. This proved to be true (Table 3). Cells from the stationary phase of a culture in TYM oxidized about 4 times more [1-14C] acetate in the presence of a-MM than in the absence of the sugar. Also, the presence of acetate stimulated the degrada- tion of a-MM by about 4 times. Although the 02 uptake data were corrected for endogenous levels, the levels of 02 uptake observed in the presence of either [1-14C] acetate or a-MM were higher than theoretical. This may have been due to the enhancement of endogenous activity by the presence of added substrate. Respiration of B. popilliae was completely inhibited by iodoacetate and arsenite (Table 4). Since only a small amount of reducing sugar was detectable in the presence of iodoacetate, it is probable that the hydrolytic enzyme was strongly inhibited. a-Glucosidase from yeast is partially inhibited by iodoacetate (3). Arse- nite did not inhibit the degradation of the man- noside, but sharply reduced the amounts of 02 uptake and C02 evolved per equivalent of sugar used. This inhibitor completely inhibits acetate oxidation by this organism (11). Induction of a-M M Oxidation Cells were harvested at various periods during the growth of B. popilliae in TYM with 0.01% glucose added and tested for their ability to oxidize a-MM and acetate. The glucose was added to the growth medium to obtain higher cell yields during early stages of growth. The shape of the growth curve was not changed by the addition of this level of glucose. Cells from very young cultures (10h) failed to show any activity on either substrate (Fig. 2). The maxi- mum Q02 on acetate was observed with cells harvested at the first stationary phase of the diauxie growth curve and decreased thereafter. Some oxidation of the a-MM was observed initially at the same point as acetate, but the Q02 for the sugar continued to increase through- out the incubation period. The increase in the Q02 did not vary significantly from a linear function, but there was some evidence of an .._'__ _fi_ ———-—-—. A.._..‘ _— ,___.. __ —_ BHUMIRATANA AND cosnLow: BACILLUS POPILLIAE 173 increase in the rate corresponding to the time of the secondary growth response in the diauxie growth curve. Cells produced in media containing glucose or trehalose were unable to oxidize either a-MM or a-MG (Table 5). However, a-MG- grown cells oxidized a-MM more rapidly than a-MM-grown cells, and cells grown on a-MM oxidized the glucoside more rapidly than the mannoside. Thus, it appeared that a-MG was the preferred inducer and substrate for the en- zyme(s) involved. The oxidation of maltose was constitutive and cells grown with this sugar did not oxidize a-MG except after a prolonged lag. TABLE 2 Effect of concentration on the extent of oxidation of a-methyl-D-mannoside (a-M M) by B. popilliae 2309MCa Reducing umoles a-MM 02 CO2 umoles 02/ CO2 sugar uptake, produced, per umole a-MM released, Added Degraded umoles umoles utilized umoles Experiment 1 10 2.1 12.5 12.16..0/5 8 —b 50 10.7 65.6 65.01/61 —" Experiment 2 30 9.8 51.4 51.1 5.4/5. 3‘ 0.1 100 22.2 76.2 81.5 3 5/3. 8° 0.5 300 24.0 33.0 33.6 2. 0/2. 0‘ 7.2 °Reaction mixtures in Warburg vessels contained 7 to 8 mg (dry weight) of cells and the indicated level of a-MM 1n 0. 05 M potassium phosphate buffer (pH 7. 2). Those 1n Experiment 1 were incubated for 6 h and those in Experiment 2 were incubated for 5 h I'Not determined. ‘The amount of u-MM degraded was corrected for the amount of free reducing sugar released during the experiment (column 2 minus column 6). TABLE 3 Effect of added acetate on a-methyl-D-mannoside (a-MM) oxidation and of added a-MM on acetate oxidation by B. popilliaea 02 a-MM 14C02 from Acetate uptake, degraded, [1-14C] acetate, used, Substrate(s) umoles" umoles cpm 1,1molesc [1-14C] Acetate (9 umoles) 3 .8 — 9.11 X104 1 .42 a-MM (30 umoles) 9.6 1 .5 — -— Both of the above 26.0 6.1 3.46>< 105 5.39 aThe reaction mixtures were as described in Table 2. Reaction mixtures were incubated for 2.5 h. 5Values reported were corrected for endogenous 02 uptake. ‘Calculated from the amount of 14CO2 using the specific activity of the [l-NC] acetate added (6.43 X 104 cpm lumole). TABLE 4 Efiect of arsenite and iodoacetate on the oxidation of a-methyl-D-mannoside (a-MM) "by B. popilliae“ Reducing a-MM sugar 02 CO2 degraded, produced, uptake, produced, Additive umoles umoles umoles umoles None 10.25 0.03 50.3 49.7 Arsenite (10 mM) 12.51 0 20.1 10.6 Iodoacetate (10 mM) 0 0.55 0 0 “Reaction mixtures in Warburg vessels contained 8.09 mg (dry weight) of cells grown in TYM, 50 mM potas- sium phosphate, pH 7. 2; inhibitors where indicated; and 30 umoles of u-MM. They were incubated for 5 h. All values are corrected for endogenous controls. . ."x ‘3’ , 1;- If it i. l . i I" n . 5, I. 1" . i ~ ‘ ..:,_ ‘ 174 CAN .1. MICROBIOL. VOL. 19. 1913 I O I j l T I F .. 12 >- ’0 0 II )- I '1 '0 / >- D 1: x (I) ~ 8 2 O I, In / Q // ON .1 / q 6 0 < 2 I~ - 4 IL -——A—-——-13 o ,I’e \ \ \\ \ J 2 n a 1 4 O 60 80 I00 I20 HOURS FIG. 2. Respiration rates of B. popilliae cells on u- methyl-D-mannoside (a-MM) and acetate at various stages of cultural development. Cells were harvested from TYM + 0.01% glucose medium at the points indi- cated on the optical density curve (0). and the rates of oxidation of the two substrates determined manometri- cally using the same types of reaction mixtures described in Table 2. (O), u-MM (30 umoles); (A), acetate (9 umoles). In a separate experiment, we observed that glucose-grown cells oxidized mannose as rapidly as glucose. Hydrolysis of a-M M and a-M G Crude extracts of B. popilliae cells failed to hydrolyze p-nitrophenyl-a-D-mannoside (PN PM) to a significant extent under the same conditions used for a-D-glucosidase assays. There was no increase in the CD. at 420 nm with 1 X 10.3 M PNPM and only a very slight increase with 1.4 x 10-2 M. Addition of Zn2+ to the reaction mixture failed to enhance the activity. Similarly, there was no detectable hydrolysis of PNPM when the assay was conducted by the procedure used for mammalian a-mannosidase (7). The rate of release of reducing sugar from a-MM by crude (nondialyzed) extracts of B. popilliae cells was very low except at high substrate levels (Fig. 3). Obviously, the affinity of the enzyme for a-MM was quite low. A Lineweaver— Burk plot of the data for the four highest sub— strate levels tested indicated a Km of ~ 5 X 10"1 M. The reason for the 5- to 7-min lag observed in all reactions is obscure. The addition of aden- osine triphosphate or of phosphoenolpyruvate to reaction mixtures similar to those in Fig. 3 had no effect on the lag or the rates of hydrol- ys1s. Lineweaver—Burk plots for the hydrolysis of PNPG by extracts of cells grown in either a-MM or a-MG were essentially identical, and the hydrolysis in both instances was inhibited by a—MG in a competitive manner. The apparent Km for PNPG with both extracts was ~ 1 X 10“3 M, and the average apparent K1 for a-MG was ~ 2 X 10“1 M. The apparent Km for u-MG as determined with the coupled dehydrogenase assay was ~ 1.9 X 10'2 M. Thus, the apparent affinity of the hydrolytic enzyme for a-MG is ~ 25 X higher than that for a-MM. There was no significant inhibition of PNPG hydrolysis by a-MM in concentrations up to 0.4 M. How- ever, this could be due to the great difference in the affinities for the two substrates. TABLE 5 Effect of carbon and energy source in growth medium on the rates of oxygen uptake by B. popilliae cells using various sugarsa Qozb with: Growth substrates glucose trehalose a-MG a-MM maltose Glucose 17.1 31.4 1.0 0.0 37.0 Trehalose 16.0 33.0 0.0 0.0 —“ u-MGC 34.8 —" 33.5 10.8 18.0 u-MMC 22.4 —'1 23.2 8.8 -“ Maltose 44.5 —" 0.06 0.0 28.3 “RC‘JCIIOH mixtures in Warburg vessels contained cells (7 to 10 mg dry wt.) and the indicated sugars (30 wholes/vessel) in 50111.)! pomssmm phosphate buffer (pH 7.2). i'Qng. “liters oxygen uptake {11 per m1liigram dry weight of cells. ru-MU : u-metliyl-D-glucoside', u-M M = u-mcthyl-D-mannosidc. ”Not determined. cThere was some oxidation after a 30-min lag but this may have been due to induction. BHUMIRATANA AND COSTILOW: BACILLUS POPILLIAE 8_'_1 7— — d 3 \ t 6- .. <1 0 A D 0) 5... _ (D E U 8 4' ‘ lu Q o (I) 3- _. lu 6’ t 2- A _ 1 I- ' d : V 1 o 'v." ”i l I 0 IO 20 30 40 50 60 MINUTES FIG. 3. Hydrolysis of a—methyl-D-mannoside by a crude nondialyzed extract of B. popilliae cells. Reaction mixtures of 1.5 ml total volume contained 1 ml cell ex- tract (26.7 mg protein); 33 mM potassium phosphate, pH 7.2; and the following mM concentrations of a-MM: (O). 102; (O). 76-2; (A). 50.8; (A).25.4; (V). 5.1; (V). none added. Extracts of cells produced in a glucose me- dium hydrolyzed PNPG at a slow rate but there was no detectable hydrolysis of a-MG or a—MM. Therefore, the PNPG hydrolysis in extracts of these cells was probably catalyzed by a constitu- tive maltase enzyme. Both maltase and a—methyl glucosidase from yeast hydrolyze PNPG (6). Discussion It is apparent that a-MM is used by B. popilliae at growth limiting rates, and the rate is dependent on the concentration of the man- noside. However, the minimum generation time achieved with 1% a-MM (~ 8—10 h) was quite slow compared to the time in a glucose medium (3—4 h). This provides a reasonable explanation for the observations that an oligosporogenous strain of this organism sporulates in the pres- ence of 0.5% a-MM (1) while sporulation is strongly inhibited by readily utilizable carbon sources at concentrations over 0.1% ( 12). While there was a prolonged period of a decelerating growth rate in the a-MM broth cultures similar 175 to that observed in colonies on agar plates (1), there were no detectable spores produced in the broth. Thus, there are undoubtedly factors other than the limitation of growth rates by the availability of the carbon source which are im- portant to the sporulation of B. popilliae. The data all indicate that B. popilliae has an inducible a—glucosidase which can be induced at low efficiencies by a-MM. It appears likely that this enzyme will hydrolyze a-MM but has a low affinity for it. These observations are adequate to explain the type of growth response to a-MM observed. Thus, the prolonged lag in growth is correlated with the slow induction of the glucosi- dase, and the growth rate is limited by the rate of hydrolysis of the mannoside. When a-MG is used in the growth medium the lag is greatly reduced and the rate of exponential growth is essentially equivalent to that in a glucose medium. Mannose is used as readily as glucose. It is apparent that a—MM at low concentra- tions does not inhibit the oxidation of acetate by certain strains of B. popilliae. Theoretical levels of 02 uptake and CO2 evolution for the complete oxidation of the mannose moiety of a-MM were observed with 10 mM a-MM. However, with 100 mM a-MM, only sufficient gas exchange was noted to account for the oxidation of a hexose to the level of acetate. The pH changes observed in an a-MM medium were correlated with these observations. Thus, at low substrate levels, the pH continued to increase throughout the growth period, while, at high a-MM levels, the pH of the medium decreased. B. popilliae oxidizes glucose during growth in the presence of unlimited oxygen to acetate and C02 (1 l), but with some strains the cells start oxidizing acetate at relatively low rates as they approach the stationary phase of growth (9). Apparently only those strains which can oxidize acetate obtain sufficient energy to support growth from the oxidation of a-MM when it is present at a low level (0.2%). Those strains which cannot oxidize acetate only grow when a high level of a-MM is present in the medium. The role of a-MM in the stimulation of ace- tate oxidation by B. popilliae may be in sup- plying four carbon intermediates of the tri- carboxylic acid cycle. Previous data indicate that this organism lacks a glyoxalate cycle, and produces a considerable amount of glutamate 176 CAN. J. MICROBIOL. VOL. 19, 1973 from acetate (9). The mechanism for the stimu- lation of a-MM utilization by the presence of acetate is not apparent. Inhibition of acetate oxidation by arsenite had no effect on the extent of a-MM hydrolysis by intact cells. Thus, it is not likely that the stimulation is due to the availability of additional energy. It appears probable that both a—MM and a-MG are hydrolyzed by the same enzyme in B. popilliae. If there was a specific a-methyl man- nosidase present, one would expect it to hydro- lyze p-nitrophenyl-a-D-mannoside, but this was not observed. Also, one would expect that a specific mannosidase would be induced more efficiently by a-MM than by a-MG; but the opposite effect was observed. It would be unique for a single enzyme to be induced which hydrolyzes both a-MG and a-MM. Spiegelman et al. (13) reported the induction by maltose of two enzymes in yeast. One is specific for maltose and the other for a-MG, but both enzymes hydrolyze PNPG (6). There are also two a- glucosidases induced by either maltose or a-MG in Streptococcus pyogenes (2). However, the maltase from this organism does not hydrolyze PNPG while the other enzyme hydrolyzes both PNPG and a-MG. The induction of an a-glucosi- dase by a-MM and the hydrolysis of a-MM by this enzyme would be unique. Studies of a- glucosidases indicate that their specificity is controlled by the entire molecule but there is less tolerance for variation in the sugar moiety than in the aglycone (3). We have found no reports of an a-glucosidase which can hydrolyze a-MM or which is induced by a mannoside. Acknowledgments This research was conducted under a contract with the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois. 1. CosrrLow, R. N., and W. H. Council. 1971. Physio- logical studies of an oligosporogenous strain of Bacil- lus popilliae. Appl. Microbiol. 22: 1076—1084. 2. DoouN, L. E., and C. PANos. 1969. The a-glucosi- dases of Streptococcus pyogenes and derived L-forln. Biochim. Biophys. Acta, 184: 271-280. 3. HALVORSON, H., and L. ELLtAs. 1958. The purifica- tion and properties of a-glucosidase of Saccharomyces italicus Y1225. Biochim. Biophys. Acta, 30: 28-40. 4. H50, E. J., and Z. J. ORDAL. 1969. Sporulation of C lostridium thermosaccharolyticum. Appl. Microbiol. 18: 958—960. 5. Hsu, E. J., and Z. J. ORDAL. 1969. Sporulation of C Iostridium thermosaccharolyticum under conditions of restricted growth. J. Bacteriol. 97: 1511—1512. 6. KHAN, N. A., and N. R. EATON. 1967. Purification and characterization of maltose and a-methyl-D- glucosidase from yeast. Biochim. Biophys. Acta. 146: 173—180. 7. stvv, G. A., and J. CONCHIE. 1966. Mammalian glucosidases and their inhibition by aldolactones. In Methods in enzymology. Vol. VIII. Edited by E. F. Neufeld and V. Ginsburg. Academic Press, Inc., New York. pp. 571—584. 8. LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. 9. MCKAY, L. L., A. BHUMIRATANA, and R. N. Cosrl- Low. 1971. Oxidation of acetate by various strains of Bacillus popilliae. Appl. Microbiol. 22: 1070— 1075. 10. NEISH, A. C. 1952. Analytical methods for bacterial fermentation. 2nd ed. Prairie Regional Laboratory, Saskatoon, Saskatchewan. Report No. 46-8-3. 11. PEPPER, R. E., and R. N. CosrtLow. 1964. Glucose catabolism by Bacillus popilliae and Bacillus lenti- morbus. J. Bacteriol. 87: 303-310. 12. SHARPE, E. S., G. Sr. JULIAN, and C. CROWELL. 1970. Characteristics of a new strain of Bacillus popilliae sporogenic in vitro. Appl. Microbiol. 19: 681—688. 13. SPIEGELMAN, S., M. SUSSMAN, and B. TAYLOR. 1950. Isolation and characterization of two adaptive en- zymes formed by yeast in response to maltose. Fed. Proc. 9: 120. 14. UMBRElT, W. W., R. H. BURRIS, and J. F. STAUFFER. 1957. Manometric techniques. 3rd ed. Burgess Pub- lishing Co., Minneapolis. ARTICLE 2 TREHALOSE METABOLISM BY BACILLUS PUPILLIAE By Amaret Bhumiratana and Ralph N. Costilow Manuscript To Be Submitted To: Jaurnal of’BacterioZogy ABSTRACT TREHALOSE METABOLISM BY BACJLLUS POPILLIAE By Amaret Bhumiratana and Ralph N. Costilow Results from growth and oxidation studies indicated that trehalose was a better carbon source for Bacillus popilliae than glucose. Synthesis of B-glucosidase in B. popilliae was also found to be strongly repressed by trehalose, whereas glucose only exerted a slight effect. Cyclic adenosine 3',5'-monophosphate did not overcome the trehalose repression. The kinetics of [14C] trehalose uptake and its inhibition by sodium fluoride sug- gested that trehalose was transported by an energy dependent system. The crude cell extract of B. popilliae had no trehalase or trehalose phosphorylase activities. However, cell extracts contained a phosphotrehalase which cleaved trehalose 6—phosphate (T6P) into equal moles of glucose and glucose 6-phosphate (G6P). The enzyme was purified 34 fold, and found to have an apparent Km of 1.8 x 10.3 M for T6P, and a pH optimum between 6.5 and 7.0. A B. popilliae mutant lacking phosphotrehalase was isolated, and found to accumulate T6P when trehalose was used as a substrate. Cell extracts of this mutant also produced T6P when it was incubated with trehalose and phosphoenolpyruvate (PEP). PEP was 36 37 found to be the only phosphoryl donor which significantly stimulated the production of [14C] sugar phosphate from [14Cltrehalose by an extract of wild type cells. The data demonstrate that B. popilliae metabolizes trehalose by first phosphorylating it to form T6P, and subsequently hydrolyzing the T6P into glucose and GéP. Although not completely established, it appears likely that trehalose is transported into the cells by a PEP:sugar phosphotransferase system. Cell extracts of various strains of B. popilliae were found to produce [14C]sugar phosphate from [IACJtrehalose and to have phosphotrehalase activities. INTRODUCTION Bacillus popilliae is a pathogen of Japanese beetle (Pbpilliae japonica) larvae and was first described by Dutkey (6). It grows readily in the hemolymph of the larvae and high populations of endospores are accumulated. Numerous attempts to mass produce Spores in the laboratory have failed. Studies of hemolymph compo- sition revealed that there is not a significant amount of glucose present but that trehalose is present in excess (26,28). The trehalose level decreased by only about one-third during the growth of B. popilliae in the larvae (28). Many studies have shown that B. popilliae utilized trehalose readily (2,26,28), and it appears likely that this is the primary source of carbon and energy for growth and sporulation in the natural host. Glucose metabolism by B. popilliae has received considerable attention (3,25) and there is no reason to believe that there should be any difference in trehalose dissimilation after the degradation into two molecules of hexose. Rhodees et al. (27) reported that the products formed during growth of the organism in a trehalose containing medium were similar to those from glucose. How- ever, we observed that the rate of respiration of B. popilliae with trehalose was about twice that with glucose as substrate (2). This observation prompted us to further studies of the utilization of trehalose by this organism. 38 39 To our knowledge, only two types of cleavage reactions have been described for trehalose. A number of organisms have been found to have a trehalase, which hydrolyzes the disaccharide into two molecules of glucose (10,12). A recent report (1) described the phosphorolytic cleavage of trehalose into glucose l-phosphate and glucose by a phosphorylase from Buglena gracilis. Preliminary data indicated that trehalose was not readily hydrolyzed by cell extracts of B. popilliae. Data in this report indicate that trehalose is utilized more readily as a source of energy by B. popilliae than is glucose. Trehalose is transported into the cell by a system forming trehalose 6-phosphate (6—O-phosphory1—a-D-gluc0pyranosyl-a-D—glucopyranoside) which is then cleaved by a phosphotrehalase with the formation of glucose and glucose 6-phosphate. MATERIALS AND METHODS Organisms and culturing procedures. Bacillus popilliae NRRL 2309MC was used in most of the studies. All of the cultures used, including their original source and methods for maintenance, have been described elsewhere (17). The basal media (TY) used throughout the studies contained 1.5% trypticase, 0.5% yeast extract, and 0.6% KZHPO4 (2). The carbohydrates were filter sterilized separately and added to the autoclaved basal medium. In all cases, 50 ml, 100 ml or 200 ml of media were dispensed in 125 ml, 250 ml or 500 ml Erlenmeyer flasks respectively. The cultures were routinely aerated by shaking on a rotary shaker, and the temperature was maintained at 30 C. Growth and oxygen uptake experiments. Each culture contained 50 ml of medium in 125 ml flasks. Samples of 2.0 ml were taken at various intervals and growth was monitored by measurements of the Optical density at 620 nm. One optical density unit equaled approxi- mately 1.6 mg dry weight of cells per ml. For oxygen uptake studies, B. popilliae 2309MC was grown to mid-exponential phase in TY + 0.2% trehalose. The cells were harvested by centrifugation at 10,000 x g and washed twice using 5 x 10-2 M potassium phosphate buffer, pH 7.2. The pellet was suspended in the same buffer. Oxygen uptake was measured in a Warburg apparatus by standard manometric techniques (35). 40 41 Enzyme assays. B-Glucosidase activity was assayed according to the method of Duerksen and Halvorson (5) with slight modifications. Each reaction mixture contained 0.8 ml of cell suspension (125 to 250 ug protein per ml) in 5 x 10-2 M potassium phosphate buffer, pH 7.2, and 0.2 ml of 5 x 10-2 M pfnitrophenyl—B-D-glucopyranoside (PNPBG). The reaction was incubated at 30 C for 20 min and stopped by adding 0.5 m1 of cold 1.0 M NaZCOZ' The cells were centrifuged and the absorbancy was measured at 400 nm. The increase in absoré bancy at 400 nm was linear with time for over 60 min. One unit of B-glucosidase was defined as the amount of enzyme which produced 1 umole of pfnitrophenol per min. The molar absorbance of p-nitrOphenol under these conditions is 1.8 x 104 M -1 cm—l. Phosphotrehalase was assayed spectrophotometrically by coupling the reaction to glucose 6-phosphate dehydrogenase. Each reaction contained 10 umoles of glycylglycine buffer, pH 7.5; l umole MgClz; 0.1 umole nicotinamide adenine dinucleotide phosphate (NADP+); 0.5 umole T6P; a non-rate limiting amount of glucose 6- phosphate dehydrogenase; and a rate limiting amount of the protein to be assayed. The final volume was 0.16 ml. The rate of reaction was determined by monitoring the increase in absorbancy at 340 nm. The rate of increase in absorbancy was constant with time and propor- tional to the enzyme concentration. Unless indicated otherwise, all assays were run at 30 C. One unit of the enzyme was defined as the amount reducing 1 umole NADP+ per min. The molar absorbance 3 1 -1 for reduced NADP+ (NADPH) is 6.2 x 10 M' cm . 42 Partial_purification of phosphotrehalase. All enzyme preparations were in standard 0.1 M tris(hydroxymethyl)aminomethane (tris)-hydrochloride buffer, pH 7.2, containing 1 mM dithiothreitol (DTT). The temperature was maintained at 0-4 C during purification. Step, 1. Prgparation of crude extract. B. popilliae was grown to late exponential phase in TY + 0.2% trehalose medium. Five liters of cells were harvested by centrifugation at 10,000 x g for 15 min, and washed twice with 5 x 10"2 M potassium phosphate buffer pH 7.2. The pellet was suspended in 10 m1 of standard buffer. The cells were disrupted by ultrasonic oscillation as described previously (2). The cell debris was removed by centri- fugation at 10,000 x g for 10 min. The supernatant fraction was referred to as the crude extract. Where indicated, the crude extract was dialyzed against standard buffer at 4 C for 8 h. SteppZ. Streptomycin sulfate precipitation. To 40 ml of crude extract, an equal volume of 5.0% streptomycin sulfate in standard buffer was added slowly with stirring, and the mixture stirred for an additional 60 min. The precipitate was removed by centrifuging at 40,000 x g for 30 min and discarded. The supernatant solution was made up to 100 ml with standard buffer. Step 3. Ammonium sulfate fractionation. Solid ammonium sulfate was added to the supernatant solution from step 2 to obtain 40% saturation. The precipitate was removed by centrifuging at 43 40,000 x g for 30 min and discarded. Additional ammonium sulfate was added to obtain 60% saturation, and the suspension centrifuged as above. The precipitate was dissolved in 14.5 ml of standard buffer and dialyzed overnight against 1 liter of standard buffer with two changes. Step 4. DEAE-cellulose chromatography. A DEAE-cellulose column (2 x 27 cm) was equilibrated with standard buffer to the same pH and the same conductivity as the starting buffer. The dialyzed sample was then loaded on the column, and the column was washed with 160 m1 of standard buffer. The protein was eluted with 300 m1 of a continuous gradient from 0 to 0.2 M KCl in standard buffer. The flow rate of the column was 50 ml per h, and 4.0 ml was collected per fraction. Phosphotrehalase was eluted from the column at approxi- mately 0.14 M KCl. Fractions containing phosphotrehalase activities were pooled and concentrated to 7.5 ml by ultrafiltration through a UM 10 Diaflo membrane (Amicon Corporation, Cambridge, Massachusetts). The concentrated preparation was dialyzed against 500 ml of standard buffer for 15 h with three changes. Step 5. Sephadex G-200 chromatoggaphy. The sample obtained from step 4 was divided into two equal portions and each chromato- graphed on Sephadex G-200. A G—200 Sephadex column (2.3 x 37 cm) was equilibrated with standard buffer. The sample was applied to the column and eluted with standard buffer. The flow rate of the 44 column was 18 ml per h and 3 ml fractions were collected. The fractions containing phosphotrehalase activities were pooled and concentrated by ultrafiltration as described above. This solution was divided into 1.0 ml portions, sealed in ampules, and stored in liquid N2 until used. Isolation of phosphotrehalase neggtive mutants. B. popilliae strain 2309MC was grown to mid-exponential phase in TY + 0.2% trehalose medium and the cells harvested. The cells in 5 x 10"2 M potassium phosphate buffer pH 7.2 were treated with N-methyl-N'— nitro-N-nitrosoguanidine (NTG) at a final concentration of 200 ug/ml for 90 min (approximately 2% survival). The suspension was centri- fuged to remove NTG, and the pellet was resuspended in potassium phosphate buffer. Samples of the suspension were plated on TY + 0.5% trehalose (TYT) and TYG agar plates. The colonies which grew on TYG but not on TYT were picked and purified by restreaking on TYG. Growth of the potential mutants was tested in liquid media containing glucose and trehalose. The trehalose negative mutants were screened for ability to take up [laCltrehalose. The mutants which did not grow on trehalose but did take up [l401trehalose were tentatively identified as phosphotrehalase negative mutants. The crude extracts of these mutants were then assayed for the presence of phosphotrehalase activity. Paper'chromatography. Sugars and sugar phosphates were separated by paper chromatography. Samples were spotted on either 45 Whatman No. l or 3 MM paper, and desCending chromatograms were developed for 12 to 15 h. The two solvent systems used were: system A, butanol-pyridine—water (9:5:4); system B, ethyl acetate-water-glacial acetic acid-formic acid (18:4:3:l). Solvent system A was used to separate glucose (Rf 0.33) from trehalose (Rf 0.22) and sugar phosphates (Rf 0). System B separated trehalose phosphate (Rf 0.36) from glucose 6-phosphate and trehalose (Rf 0.45) and from glucose (Rf 0.54). Uppake of [}4Ctrehalose. Uptake as used in this paper refers to the amount of radioactivity accumulated in cells; and thus, would include both the sugar and metabolic products from it. Cells grown in TY + 0.2% trehalose medium to mid-exponential phase. The cells were harvested, washed twice with 5 x 10"2 M potassium phosphate buffer, pH 7.2, 110 ug/ml dry weight of cells, and 1 x 10-5 M [14C] trehalose (2.6 x 106 cpm/umole). Where indicated, 1 x 10"2 M NaF was added. The reactions were run at 30 C. Samples of 1.0 ml each were taken at intervals, filtered through millipore filters (0.45 um pore size), and washed with 5.0 ml of cold potassium phos- phate buffer. The filter was dried at 60 C for 15 min, and placed in 5.0 ml of toluene based scintillation liquid to determine the radioactivity. Miscellaneous. Protein was routinely determined by the method of Lowry et al. (16) using bovine serum albumin, fraction V, as standard. For determination of protein of the whole cells, the 46 cells were first extracted with hot (90 C) 5% trichloroacetic acid (TCA). The precipitate from the hot TCA extraction was then treated with 1 N sodium hydroxide and held at 40 C for 2 h. The preparation was then neutralized with 2 N hydrochloric acid, and protein was determined using the method of Lowry et al. (16). All optical absorbancy measurements were made with a Gilford, model 2000 spectrophotometer. Radioactivity was determined with a Packard Tri-Carb, model 3320, using a toluene based scintillation fluid (0.01 g 1,4-tris- 2-(5-phenyloxazolyl) -benzene and 6 g 2,5- diphenyloxazole in 1 liter of toluene). RESULTS Repression of §:glucosidase synthesis by trehalose. B-Glucosidase in Bacillus popilliae 2309MC was found to be inducible by salicin. Activity of the enzyme in the induced culture was ten fold higher than that of the non-induced. The plot of the B-glucosidase activity as a function of growth remained linear after the culture was fully induced. Trehalose, but not glucose, was found to strongly repress the synthesis of B-glucosidase (Fig. 1). In the presence of glucose and salicin, B-glucosidase synthesis was delayed but the differential rate of synthesis only decreased slightly as compared to the culture without glucose added. However, when trehalose was added together with salicin, the differential rate of B-glucosidase synthesis decreased by 88% as compared to the culture with salicin alone. In other experiments, it was noted that trehalose also repressed B-glucosidase when trehalose was added after the culture was fully induced. This repression was not affected by the addition of 5 x 10’3 M cyclic adenosine 3',5'-monophosphate to the medium. The results of these experiments and those of previous studies (2) indicated that trehalose was more readily utilized by B. popilliae than was glucose. Growth and oxidation of various concentrations of glucose trehalose by B. popilliae 2309MC. The rate and extent of growth of B. popilliae 2309MC varied greatly when the concentration of 47 48 glucose added to the medium was varied from 2.8 x 10-3 M to 2.8 x 10—2 M. The maximal rate of growth was obtained with glucose at a concentration of 2.8 x 10.2 M with a growth rate of 0.25 generation h-l. However, a growth rate of 0.4 generation h"1 was obtained when only 1.3 x 10-3 M trehalose was used as the carbon source. It has been previously reported that B. popilliae 2309MC grown with either glucose or trehalose as the carbon source oxidized trehalose about twice as fast as glucose when the two sugars were present at the same concentration (0.01 M) (2). When we varied the concentration of the two sugars, it was observed that the rate of 02 uptake by resting cells with glucose as substrate increased as the glucose concentration increased up to a maximum uptake rate at 5 x 10"2 M glucose (Table 1). In contrast, the rate of uptake was maximal with only 5 x 10-24 M trehalose. These data suggest that the difference in growth response to the two sugars might be due to differences in their manner of uptake by the cells. Sodium fluoride is known to inhibit the enzyme enolase in the Embden-Meyerhof-Parnas pathway (13), and previous data (25) showed that it inhibited the rate of glucose oxidation by B. popilliae by 50%. When this inhibitor was tested on trehalose oxidation by resting cells of B. popilliae grown in trehalose, the oxidation was completely inhibited (Fig. 2). Glucose oxidation was only inhibited by 50%, which is the same level of inhibition 49 reported previously with a lower conCentration of glucose (25). Concentrations of trehalose and glucose were adjusted in this experiment so that maximal rates of 0 uptake was obtained in 2 the controls. The results suggested that the uptake of trehalose was energy dependent and dependent on phosphoenolpyruvate (PEP). Inhibition of[l4C]trehaloseuptake by NaF. In order to clarify the complete inhibition of trehalose oxidation by NaF, uptake experiments using [14C]trehalose with and without NaF added were studied. As illustrated in Fig. 3, the uptake of [iaCltrehalose by resting cells of B. popilliae 2309MC was almost completely inhibited by 1 x 10'.2 M NaF. In the absence of the inhibitor, the uptake of [14C1trehalose was found to have saturation kinetics (Fig. 4). Phosphotrehalase in B. popilliae 2309MC. \A number of unsuccessful attempts were made to detect activity of an enzyme(s) in the crude extract of B. popilliae 2309MC which would cleave trehalose. Various methods were used to detect trehalase (7,9) and trehalose phosphorylase (1), but no cleavage of trehalose was detected ((0.0005 umoles glucose formed per min per mg protein). Therefore, it appeared likely that this organism did not contain an enzyme that attacked trehalose per 88. However, these extracts did contain an enzyme which cleaved T6P into equimolar amounts of glucose and glucose 6-phosphate (G6P). This enzyme, phosphotrehalase, 50 was partially purified (34 fold) using the purification steps outlined in Materials and Methods and summarized in Table 2. The kinetic studies of the partially purified phosphotrehalase indicated that the enzyme had an apparent Km for T6P of 1.8 x 10-3 M. The pH optimum of the enzyme was found to be between 6.5 and 7.0. The buffer used in the assay system affected the activity of the enzyme; the activities in tris-HCI and in potassium phosphate buffers were only about 1% and 60% respectively of the activity in glycylglycine buffer at the same pH. As shown in Table 3, the partially purified preparation produced equimolar amounts of glucose and G6P from T6P. Characterization of phosphotrehalase negative mutant of Bp_p0pilliae 2309MC. The above data implied that T6P was probably an intermediate in the metabolism of trehalose by B. popilliae. To confirm that T6P was a true intermediate, and that phosphotrehalase was an essential enzyme in trehalose metabolism, a B. popilliae mutant lacking the phosphotrehalase was isolated. The isolation procedure is described in the Materials and Methods section. The mutant, 2309M0100, did not grow with trehalose as the carbon source but it did take up [14C]trehalose. Also, the crude extract of 2309MC100 did not contain detectable phosphotrehalase activity ((0.0002 umole NADPH formed per min per mg protein by the assay procedure). The mutant behaved similarly to the wild type with respect to carbohydrates other than trehalose (Table 4). 51 In addition, it was similar to the wild type culture in that it was catalase negative, a unique characteristic of this Bacillus. Not only did the mutant fail to grow in TY medium.+ 0.2% trehalose, but also it did not grow in this medium containing both 0.2% trehalose and 0.5% glucose. To show the inhibition of trehalose on growth of the mutant, a culture of 2309MC100 was allowed to grow in TY medium + 0.5% glucose to mid-exponential phase, and 0.2% trehalose was then added to the culture. As shown in Fig. 5, the growth of the mutant stopped shortly after the addition of trehalose. Addition of trehalose to the wild type culture growing in glucose did not affect growth of the organism. Trehalose was not found to interfere with uptake of [@4C1glucose in wild type cells, even when the concentration of unlabelled trehalose was four times greater than that of the [l4élg1ucose. Perhaps, the T6P which was found to accumulate in this mutant was inhibitory. Previous studies have demonstrated the inhibition of microbial growth by the accumulation of sugar phosphates from L-arabinose (9), L-rhamnose (7,8), and D-galactose (20). Identification of T6? accumulation by thg_phosphotrehalose pegative mutant of B. popilliae. Since trehalose was found to be metabolized readily in wild type cells, it appeared likely that the T6P was hydrolyzed as rapidly as it was formed. Therefore, we looked for the accumulation of T6P in the phosphotrehalase negative mutant. A resting cell suspension of the mutant 2309MC100 was incubated with 52 [l4C]trehalose and the cells extracted with 5% cold TCA. The TCA extract was subjected to chromatography with two different solvent systems, and the radioactive spots in both solvent systems were found to migrate to the same location (same Rf's) as authentic T6P. After treating the TCA extract with alkaline phosphatase, the radioactivity appeared at the location corresponding to free trehalose. Furthermore, when it was treated with partially purified phosphotrehalase, the radioactivity appeared equally in both the glucose and G6P spots. These data are summarized in Table 5. The same types of assays indicated that a crude cell extract of mutant 2309MC100 also accumulated [}4C]T6P in reaction mixtures containing [1401trehalose, MgClz, and PEP. Furthermore, the production of T6? from trehalose was indicated by a coupled enzymatic assay (Table 6). In the complete assay mixture with the phosphotrehalase, hexokinase, ATP, G6P dehydrogenase, and NADP+, over 27 nmoles of NADPH were formed, while only 5.5 nmoles and 17.8 nmoles were formed on omission of phosphotrehalase and the hexokinase system respectively. If one corrects for the amount of reduction in the absence of phosphotrehalase, which may have been due to some side reaction(s) in the crude extract, the corrected amount of NADP+ reduced in the complete assay system (Rx. l, 22 nmoles) was approximately double that reduced in the absence of added hexokinase and ATP (Rx. 3, 12.3 nmoles). This would be expected since the phosphotrehalase cleaves T6P to glucose and G6P. 53 ‘ Effect of PEP and othergphOSphorylated compounds on the accumulation of sugar phosphates from trehalose. Since all the previous data indicated that the phosphorylation of trehalose might well occur during transport, we examined the effect of a number of possible phosphoryl donors on the formation of sugar phosphate from [14C1trehalose by a crude cell extract (Table 7). Of the compounds tested, only PEP was found to have a significant effect, although there appeared to be a slight stimulation by ATP and guanosine 5'-triphosphate. Although we have not studied this system in detail, preliminary experiments have demonstrated that the enzyme(s) responsible for the phosphorylation of trehalose is associated primarily with the particulate fraction of cell extracts of B. popilliae. washed cell particles prepared as described by McKay at al. (18) and the soluble fraction of the extract were assayed by the procedure outlined in Table 7. The amount of sugar phosphate accumulated from [é4G]trehalose by the washed particles was about 60% of the amount produced with the combined particulate and soluble fractions, while the activity of the soluble fraction was only 10 to 20% that of the particles alone. Assays of various strains of B. papilliae for the production of spgar phosphate from trehalose and for phosphotrehalase. A number of strains of B. popilliae in our collection including a culture recently initiated from spores harvested from larval hemolymph were 54 tested for the unique activities observed with 2309MC. All of these cultures were catalase negative, which is characteristic of this Bacillus. Cell extracts of all strains were shown to produce sugar phosphate from [1401trehalose and to have phosphotrehalase. The phosphotrehalase activities were about the same in all the extracts, but the amount of sugar phosphate accumulated varied considerably. DISCUSSION B. popilliae is well adapted to the utilization of trehalose which is the principal carbon source available in its natural host, Japanese beetle larvae. The utilization of this disaccharide is constitutive in this organism (2). The comparative rates of growth were about 2x higher in the presence of trehalose than with glucose even when glucose was present in a much higher concentration than the disaccharide. The respiration rate of B. popilliae with 5 x 10'.4 M trehalose as substrate was as high as that attained with much higher levels of glucose. Also, strong repression of the synthesis of an inducible enzyme was evident when trehalose was present along with the inducer while glucose had only a slight effect, if any. Thus, it appears likely that trehalose is the preferred energy source for this organism. The hydrolysis of trehalose by B. popilliae is particularly interesting. No trehalase (10,12) or trehalose phosphorylase activity (1) such as reported in other organisms were detected. Rather, all of the data indicate that trehalose is probably taken up by the cell via a PEP:sugar phosphotransferase system forming T6P; and the T6? is hydrolyzed by a phosphotrehalase. The evidence for a PEP:sugar phosphotransferase system is as follows: (a) PEP- dependent formation of T6P by crude cell extracts and by the particulate fraction of cell extracts, (b) accumulation of T6P in a phosphotrehalase negative mutant, (c) complete inhibition by 55 56 1 x 10-2 M.NaF of [l4C1trehalose uptake by cells, and of respiration with trehalose as substrate, and (d) the failure of ATP to significantly stimulate formation of sugar ph08phates from trehalose by cell extracts. Glucose did not appear to be transported by the same system, since there was only a 50% inhibition of respiration by NaF with glucose as substrate. Neither was there any inhibition of [14C]trehalase uptake by high concentration of glucose. The PEP:glucose phosphotransferase system is widely distributed in bacteria including members of the genus Bacillus (29). The involvement of a similar system in the transport of lactose is also well established in Staphylococcus aureus (11,14,15,30,31,32) and in Streptococcus lactis (18,19). Therefore, it would not be unexpected to find such a transport mechanism for other disaccharides. Although phosphorylated intermediates are involved in the metabolism of the disaccharides cellobiose (21,22,23) and gentibiose (24) in Aerobacter aerogenes, the phosphorylation is catalyzed by a soluble ATP-dependent kinase. No significant kinase activity was observed in these studies. Most of the trehalose phosphorylating activity was associated with the particulate fraction of the extracts of cells of B. popilliae. The cleavage enzyme for which we propose the trivial name phosphotrehalase is a new enzyme which has not been observed previously. The failure of a mutant deficient in this enzyme to metabolize trehalose; the presence of the enzyme in all strains of 57 B. popilliae studied; and the absence of a trehalase or trehalose phosphorylase certainly indicates that the metabolism of trehalose by B. popilliae involves an initial phosphorylation before cleavage. The significance of these findings with respect to the ability of B. popilliae to sporulate in the hemolymph of Japanese beetle larvae is not clear. High concentrations (0.2% of either glucose or trehalose inhibit sporulation of oligosporogenous strains of this organism in vitro (4,27), although trehalose is reported to be less inhibitory than glucose (27). The concentration of trehalose in the hemolymph of the larvae is higher than 0.2% throughout sporulation. It has been proposed (4,26) that the trehalose may be mostly in an unavailable form in the larvae. This should be inves- tigated more thoroughly. ACKNOWLEDGMENT S The authors gratefully acknowledge the council of Dr. Richard L. Anderson, Department of Biochemistry, Michigan State University, during this investigation. Also, we thank Dr. D. L. MacDonald, Department of Biochemistry and Biophysics, Science Research Institute, Oregon State University, for the gift of the trehalose-6-ph03phate. This research was supported by the National Science Foundation, Grant No. GB-32506Xl. LITERATURE CITED Belocopitow, E., and L. R. Marechal. 1970. Trehalose phos- phorylase from Buglena gracilis. Biochim. Biophys. Acta 1285151—154. Bhumiratana, A., and R. N. Costilow. 1973. Utilization of a— methyl—D-mannoside by Bacillus popilliae. Can. J. Microbiol. lgcl69—l76. Bulla, L. A., G. St. Julian, R. A. Rhodes, and C. W. Hesseltine. 1970. Physiology of sporeforming bacteria associated with insects. I. Glucose catabolism in vegetative cells. Can. J. Microbiol. 163243-248. Costilow, R. N., and W. H. Coulter. 1971. 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Rhodes, R. A., M. S. Roth, and G. R. Hrubant. 1965. Sporula— tion of Bacillus popilliae on solid media. Can. J. Microbiol. 113779-783. Rhodes, R. A., E. S. Sharpe, H. H. Hall, and R. W. Jackson. 1966. Characteristics of the vegetative growth of Bacillus popilliae. Appl. Microbiol. 143189-195. Romano, A. H., S. J. Eberhard, S. L. Dingle, and T. D. McDowell. 1970. Distribution of the phosphoenolpyruvate glucose phos- photransferase system in bacteria. J. Bacteriol. 1943808-813. Roseman, S. 1972. Carbohydrate transport in bacterial cells, p. 42-89 in L. E. Hokin (ed.), Metabolic pathways, vol. VI. Academic Press Inc., New York. 31. 32. 33. 34. 35. 36. 63 Simoni, R. D., T. Nakazana, J. B. Hays, and S. Roseman. 1973. Sugar transport. IV. Isolation and characterization of the lactose phosphotransferase system in Staphylococcus aureus. J. Biol. Chem. 1483932—940. Simoni, R. D., and S. Roseman. 1973. Sugar transport. VII. Lactose transport in Staphylococcus aureus. J. Biol. Chem. 2483966-974. Splittstoesser, D. F., and D. F. Farkas. 1966. Effect of cations on activation of Bacillus popilliae spores. J. Bacteriol. 213995-1001. Toennies, G., and J. J. Kolb. 1964. Carbohydrate analysis of bacterial substances by a new anthrone procedure. Anal. Biochem. 8354—69. Trevelyan, W. E., P. P. Procter, and J. S. Harrison. 1950. Detection of sugars on paper chromatograms. Nature .166: 444-445. Umbreit, W. W., R. H. Burris, and J. F. Stauffer. 1957. Manometric techniques. 3rd ed. Burgess Publishing Co., Minneapolis. 64 TABLE 1. Rate of'oxygen uptake by B. popilliae with various concentrations of glucose and trehalose as substratesa Substrate concentration (M) pl 02 per min per mg dry wt. Glucose: 2.0 x 10'3 0.201 5.0 x 10‘3 0.406 1.0 x 10‘2 0.602 2.0 x 10’2 0.738 5.0 x 10‘2 0.820 1.0 x 10"1 0.840 2.0 x 10'1 0.840 Trehalose: 5.0 x 10—4 0.850 1.0 x 10'3 0.850 5.0 x 10'3 0.850 anygen uptake was measured in a Warburg apparatus equilibrated at 30 C. The uptake rate at each substrate concentration was cal- culated from linear plots of oxygen uptake vs. time. All the values ‘were corrected for the endogenous oxygen uptake which was less than 0.05 ul 0 per min per mg dry wt. The cells used were grown in the 2 TY basal medium plus 0.2% trehalose to exponential phase, harvested by centrifugation, and washed twice with 5 x 10.2 M potassium buffer, pH 7.2. Each Warburg flask contained 8.49 mg (dry weight) of cells. 65 TABLE 2. Partial purification of'phosphotrehalasea Step Preparation Volume Protein Total Specific (ml) (mg) activity activity (units) (units/mg) 1. Crude extract 40 1260 100 0.080 2. Streptomycin 100 1300 101 0.075 sulfate 3. Ammonium sulfate 14.5 378 156 0.412 (40% to 60%) 4. DEAE-cellulose 8.0 136 94 0.688 5. Sephadex G-200 7.5 30 85 2.83 aSee Materials and Methods section for detailed procedures. 66 TABLE 3. Stoichiometry for cleavage of trehalose-6—phosphate phosphotrehalasea Products (nmoles) Trehalose-6-phosphate (nmoles)b Glucose Glucose—6-phosphate 3.2 3.2 3.4 6.4 7.1 7.0 12.8 14.1 15.4 19.2 22.9 20.1 aEach reaction mixture (0.25 ml total volume) contained 17 pmoles glycylglycine buffer (pH 7.5), 1.7 pmoles MgClz, 0.85 umole adenosine-5'-triphosphate (ATP) 0.17 pmole NADP+, the indicated amount of trehalose-6-phosphate, partially purified phosphotrehalase (40 pg protein), and excesses of hexokinase and glucose-6-phosphate dehydrogenase. Reactions were incubated at 30 C. The amount of glucose and glucose-6—phosphate produced were determined by calculating the amount of NADPH formed. The reac- tions were first incubated without hexokinase and ATP, and the maximal absorbancy at 340 nm was determined. This value represented the amount of g1ucose-6—phosphate formed. Then, hexokinase and ATP were added and maximal absorbancy at 340 nm was again measured. The latter measurement represented the total amount of glucose and 67 glucose-6-phosphate formed from trehalose-6-phosphate. The changes in volume upon addition of hexokinase and ATP were corrected for. Control reactions without added phosphotrehalase, or without trehalose-6-phosphate showed no NADPH formation. bCalculated on the basis of 91% purity of the trehalose-6- phosphate as determined by the anthrone reaction (34) for total carbohydrate using trehalose as the standard. 68 TABLE 4. Growth response of B. popilliae 2309MC and the phosphotrehalase negative mutant (2309M0100) to various carbon sources a Carbohydrates 2309MC 2309MC100 (0.5%) (Generation/hour) (Generation/hour) None No growth No growth Trehalose 0.400 No growth D-Glucose 0.308 0.250 Maltose 0.133 0.129 Salicin 0.133 0.167 Methyl-a-D-glucoside 0.100 0.154 D-Fructose 0.333 0.200 Sucrose No growth No growth Cellobiose No growth No growth D-Galactose No growth No growth D-Ribose No growth No growth D-Xylose No growth No growth L-Arabinose No growth No growth aAll the carbohydrates were to the basal TY medium. overnight, centrifuged, and the cells resuspended in basal medium before inoculation. tial phase. Growth rates were calculated from the exponen- filter sterilized before addition The inoculum was grown in a glucose medium 69 TABLE 5. Identification of trehalose-6-phosphate as an accumulated product in mutant 2309M0100a Glucose Trehalose Treatments Glucose Trehalose -6- -6- phosphate phosphate (epm) (Cpm) (cpm) (am) No treatment 0 0 116 4492 Boiled phosphotrehalase 0 0 78 4571 Alkaline phosphatase 208 4476 0 8 Phosphotrehalase 1914 0 1586 31 aB. popilliae mutant 2309MC100 was grown to mid-exponential phase in TY medium plus 0.5% glucose. The cells were harvested and washed twice with 5 x 10—2 M potassium phosphate buffer, pH 7.2. The reaction mixtures of 0.4 m1 contained 20 umole potassium phosphate buffer, pH 7.2, 2.38 mg (dry weight) of cells, and 0.2 umole [14C]trehalose with a specific activity of 2.01 x 106 cpm/umole. The reaction mixtures were incubated at 30 C for one hour. The cells were centrifuged, and the pellet extracted twice with 2.0 m1 of 5% trichloroacetic acid (TCA) at 0 C for 30 min. The TCA was removed from the combined supernatant solutions by extracting three times with an equal volume of ether. The resulting solution was evaporated under reduced pressure at 40 C to a small volume. Sugar phosphates were separated from any free sugar by preparative paper chromatography using solvent system A. After elution from the paper with water, the 70 accumulated sugar phosphate was chromatographed in two different solvent systems (A and B) along with the products from alkaline phosphatase treated sub-samples. Glucose, glucose-6-phosphate, tre- halose, and trehalose-6-phosphate were co-chromatographed as standards. The chromatograms were dried and radioactive spots located using a Packard model 7201 radiochromatogram scanner. The standard sugars and sugar phosphates were made visible by alkaline silver nitrate method (35). The areas corresponding to the peak activities were compared to the standards and cut out to quantitate the amount of radioactivity in toluene based scintillation liquid. The data were corrected for background values. 71 TABLE 6. Production of'trehalose-6-phosphate by a crude extract of cells of the mutant 2309M0100a Omissions from assay pmole NADPH reaction mixtures produced None 27.5 Phosphotrehalase 5.5 Hexokinase and ATP 17.8 Phosphotrehalase, hexokinase, ATP, 0.9 and G6P dehydrogenase aFor the production of T6P, reaction mixtures contained 0.5 pmole MgClz, 1.0 umole PEP, 1.0 umole trehalose, and dialyzed crude extract of cells (1.8 mg protein) in a total volume of 0.1 ml. After 30 min at 30 C, reactions were stopped by boiling for 5 min, and the precipitate centrifuged down. A control reaction with boiled enzyme (crude extract) was run concurrently. The super- natant solutions were assayed for T6P as follows. The complete reaction mixture contained 0.03 ml of the test solution, 17 nmoles glycylglycine buffer, pH 7.5, 1.7 pmole MgClz, 0.85 umole ATP, 0.17 pmole NADP+, and excesses of G6P dehydrogenase, hexokinase, and phosphotrehalase. The final volume was 0.25 ml. Omissions were as indicated in the table. The pmoles NADPH formed were calculated from the change in the absorbancy at 340 nm. The values were all 72 corrected for the amount of NADPH formed under the same conditions by supernatant solutions of the control reaction mixture referred to above. 73 TABLE 7. Effect of'potential phosphoryl donors on the production of'sugar phosphates from trehalose by cell extracts of’B. popilliae 2303M0a Phosphoryl donors pmoles/mg protein/min.b (1 x 10"2 M) No phosphoryl donor 40 PEP 470 ATP 57 Guanosine 5'-triphosphate 69 Glucose 6-phosphate 28 Acetyl phosphate 30 Carbamyl phosphate 27 aThe crude cell extract was dialyzed for 8 hours at 4 C in 0.1 M Tris, 1 mM DTT buffer, pH 7.2. Each reaction with a total volume of 0.1 ml contained 6 nmoles of tris-HCl buffer pH 7.2, 0.5 umole MgCl 1 umole of the tested compounds, 2.2 mg cell extract protein, 5 2’ and l umole [iACJtrehalose with a specific activity of 2.2 x 10 cpm/umole. The reactions were started by adding the substrate and incubated at 30 C for 30 min. They were stopped by boiling for 5 min. The precipitate was spun down and 10 uliters of the super- natant solution was spotted on Whatman No. 1 filter paper and chromatographed with solvent system A. The spots corresponding to 74 sugar phosphate were cut out and the radioactivity determined in toluene based scintillation fluid. All values were corrected for background. bCalculated equivalents of [74C1trehalose. 75 TABLE 8. Activities of'phosphotrehalase and the production of sugar phosphates from trehalose by cell extracts of’various strains of’B. popilliae Strainsa PhOSphotrehalaseb Sugar phosphatase (unit/mg protein) (pmole/mg protein/min) 2309MC .080 470 23095 .091 930 2309N .069 490 923B .051 210 L.S. .082 117 2309M .062 283 54 .100 90 aStrains 2309MC, 2309s, 2309N, and 923B were maintained as vegetative cells on slants and cultivated in liquid media prior to use. Strain L.S. was obtained from the Northern Utilization Research and Development Division, Peoria, Illinois, as dried spores. Strains 2309M and 54 were maintained as spores on a sporulation medium (17). Strains L.S., 2309M, and 54 were activated according to the method of Splittstoesser and Farkas (33) before cultivating in liquid medium. All strains were grown in TY + 0.2% trehalose to late exponential phase. Crude cell extracts were prepared as indicated in the Material and Methods section. 76 bPhosphotrehalase was assayed as described in the Material and Methods section. cProduction of sugar phosphates was determined as described in Table 7. Calculated equivalents of [74C1trehalose. 77 FIG. 1. Effect of trehalose and glucose on B-glucosidase synthesis. Three 200 ml cultures in 500 ml Erlenmeyer flasks of B. popilliae were grown on TY medium with the following sugars added: 0 , 0.2% salicin;A , 0.2% salicin plus l.0% glucose; I, 0.2% salicin plus 0.5% trehalose. Ten ml samples were taken from each flask at various intervals, 5.0 ml were used to determine protein, 4.0 ml to measure B-glucosidase activity, and l.0 ml to follow growth of the culture by measuring the 0D620' Methods for the determination of'protein from whole cells and fbr the B-glucosidase assay were outlined in the Materials and Methods section. N 10" UNITS/ml —I 78 FIG. 1 40 so 120 160 200 pg PROTEIN/ml 240 280 79 FIG. 2. Sodium fluoride inhibition of the oxidation of glucose and trehalose by B. popilliae 2309316. The detailed procedures were the same as those outlined in Table 1 except that 1 x l0'2 M NaF was added to the indicated Warburg flasks (closed symbols). Final concentrations of glucose (4,‘) and trehalose ( 0,.) were 5 x l0-2 M and 5 x l0—3 M respectively. All the values were corrected for the endogenous oxygen uptake which was less than 0.15 pl 02 per min. Each Warburg flask contained 5.6 mg dry weight of trehalose grown cells. 80 80 60 400- p 0 O 3 o m 0 2 uzamzoo No _e IOO- 40 MINUTES 20 FIG. 2 81 FIG. 3. Sodium fluoride inhibition of [146:]trehalose uptake by B. popilliae 2309MC. The detailed procedures were described in the Materials and Methods section. Symbols: 0, with NaF; 0, without NaF. 82 mm mw 8 6 4 2 33 Eu 828.085 9.5% MINUTES FIG. 3 83 FIG. 4. Effect of concentration on [14C]trehalose uptake by B. popilliae 2309MC. The general procedure was the same as outlined in Fig. 3 except that no NaF was added, and the trehalose concen- tration was varied as indicated. Each reaction mixture had a final volume of 2.0 ml and was incubated at 30 C for 1 min. 84 32, F6 uEEERoBES 9.49m: 4 6 8 1o 10"M TREHALOSE 2 FIG. 4 85 FIG. 5. Trehalose inhibition of the growth of'mutant 2309MCJOO. Filter sterilized carbon sources were added to TY medium as fellows; O, trehalose (0. 2%); 0, glucose (0.5%); 0, trehalose was added at the time indicated (arrow) to the cells growing in the glucose medium. OPTICAL DENSITY 1.00 86 0.80 E /0/'* T. : .. /. a 0.60 L ./. . .. .. . O a 0.40 —’ Oré'g‘Q—O’L’e/ 4 I— O/. 0.20 - // - o 0.10 t ° 3 0.08 : 0.06 h . ‘ I. . 0.04 I/0 o o o " f o 0 o .- oo 0.02 l l O l 4 l l 0 5 10 15 20 25 HOURS FIG. 5 APPENDIX STUDIES OF THE EFFECT OF CYCLIC ADENOSINE-3',5'-MONOPHOSPHATE ON THE REPRESSION OF B-GLUCOSIDASE IN BACILLUS POPILLIAE By Amaret Bhumiratana and Ralph N. Costilow INTRODUCTION In view of the potential of Bacillus popilliae for biological control of the Japanese beetle, many unsuccessful attempts have been made to obtain good spore yields of the organism in vitro. As discussed in the Literature Review, catabolite repression may be an important mechanism in controlling sporulation. Therefore, it appeared possible that cyclic adenosine-3',5'-monophosphate (cAMP) might be involved in sporulation. Attempts to induce sporulation of B. popilliae by adding cAMP to the medium were unsuccessful (3). we decided to see if cAMP had any effect on catabolite repression in B. popilliae. B-Glucosidase is induced in B. popilliae by salicin, saligenin-B-D-glucopyranoside; and the induction is strongly repressed by trehalose. Therefore, this system was utilized for our studies with cAMP. 88 MATERIALS AND METHODS Cultures and cultural methods. Bacillus popilliae 2309MC was used in all the experiments. The strain used, 2309MC was derived from strain 2309M (7,10). The cultures were maintained on agar slants as outlined by McKay et al. (7). The basal medium (TY) used for all of these experiments contained 1.5% trypticase, 0.5% yeast extract, and 0.6% KZHPO4. This medium was dispensed in 200 ml volume into 500 m1 Erlenmeyer flasks. The carbohydrates were filter sterilized separately and added to the basal medium at the final concentration of 0.5%. All cultures were incubated at 30 C on a rotary shaker. Growth studies. Growth was measured by determining the optical density (O.D.) at 620 nm with a Gilford spectrophotometer model 2000 using the growth medium clarified by centrifugation as a blank. B-Glucosidase assay. B-Glucosidase activity was assayed according to the method of Duerkson and Halvorson (4). The standard assay contained 0.8 ml of cell suspension in 5 x 10.2 M phosphate buffer, pH 7.2, and 0.2 m1 of 5 x 10"2 M p-nitro-phenyl-B-D-glucopyranoside (PNPBG). The protein content of the cell suspension used ranged from 125 ug/ml to 250 ug/ml. The reaction mixtures were incubated at 30 C for 20 min and stopped by adding 0.5 ml of 1.0 M Na CO The reaction 2 3' mixtures were centrifuged and the optical density was measured at 89 90 400 nm using a Gilford spectrophotometer model 2000. One unit of B-glucosidase was defined as the amount of enzyme which produced 1 umole of p-nitro-phenol per min. The absorbance of p-nitro-phenol under these conditions is 1.8 x 104 M.1 cmfl. Protein of the cell suspension was determined by the method of Lowry et al. (6) after prior digestion with 1 N NaOH for 2 h at 40 C (1). Uptake of[3H]cAMPby B.,popilliae 2309MC. B. popilliae was grown in TY + salicin medium to mid-exponential phase, and the culture used for uptake experiments. Each reaction mixture contained 0.2 m1 of [3H]cAMP and 1.8 ml of the culture. The reaction mixtures were incubated at 30 C for 1 min and they were filtered through Millipore filter (0.45 um pore size), washed with 5.0 ml of cold 5 x 10.2 M phos- phate buffer, pH 7.2, and dried at 60 C. The dried Millipore filter was placed in 5.0 m1 of toluene based scintillation liquid (1), and the radioactivity determined using a Packard liquid scintillation spectrom- eter model 3320. RESULTS Validity of B-glucosidase assay system. B-Glucosidase was assayed by measuring the ability of the enzyme to cleave p-nitro-phenyl- B-glucopyranoside (PNPBG). The release of the chromogenic group, p-nitro-phenol, from PNPBG was measured spectrometrically at 400 nm. The reaction was found to be linear over 60 min period (Fig. 1). The B-glucosidase activity was directly proportional to the amount of cell protein in the range of 25 to 250 ug/ml (Fig. 2). A Lineweaver-Burk plot (Fig. 3) indicated the apparent Km of the enzyme was 2.2 x 10.3 M for PNPBG. Induction of B-glucosidase in B. popilliae. The data in Table 1 indicate that B-glucosidase is an inducible enzyme. Very low activity of the enzyme was detected from cells grown in TY + 0.5% glucose medium, whereas high activity was observed from cells grown in TY + 0.5% salicin. When B. popilliae was induced for B—glucosidase with 0.5% salicin and the time course of induction was followed, the culture was fully induced after 2-3 h of incubation. The rate of B-glucosidase synthesis became linear after an initial lag period (Fig. 4). The B-glucosidase activity was not detected when salicin was not added. Repression of B-glucosidase synthesis by trehalose. Data presented earlier in this thesis, demonstrated that the synthesis of B—glucosidase was repressed by trehalose. When trehalose was added to a 91 92 culture which had been fully induced to synthesize B-glucosidase, both transient and catabolite repression could be demonstrated (Fig. 5A). However, cAMP added exogenously to the culture at 5 x 10"3 M did not overcome either the transient or catabolite repression (Fig. 5B). Also, 5 x 10-3 M cAMP did not stimulate the rate of B-glucosidase synthesis. Uppake of [3H1cAMP py B. pgpilliae 2309MC. It was possible that the failure of cAMP to overcome repression of B-glucosidase synthesis by trehalose was due to the inability of cAMP to pass through the cell barrier. Therefore, uptake of [3H]cAMP was studied at various concentrations of [3H1cAMP. The uptake of [3H]cAMP was found to be 3 linear with concentrations of cAMP up to 5 x 10- M (Fig. 6). DISCUSSION There was no measurable effect of adding cAMP to a growth medium on either sporulation (3) or on the repression of B-glucosidase synthesis in B. popilliae. While the cells did take up [schAMP, the data do not rule out the possibility that this compound is involved in the regulation of these processes, since we did not investigate the fate of the cAMP taken up by the cells. It may have been rapidly converted to other forms. There have been few reported studies on the role of cAMP in Bacillus species. Ide (5) failed to find either adenyl cyclase or phosphodiesterase in four species of this genus. However, cAMP has been reported to be present in cells of B. lichenifbrmis, although the levels reported were very low (2). There are no reports of any physiological effect of this interesting compound in spore- forming bacteria. While there is no question of the regulatory role of cAMP in a number of systems (8,9), it is increasingly apparent that the observable effects are different in different organisms. Schechter and Krulwich (Bacteriol. Proc., 1972, 176) reported no effect of cAMP on the repression of one inducible enzyme in an Arthrobacter species. Even more interesting, they observed that very low levels of this compound repressed the synthesis of another inducible enzyme in the same species. Thus, there is much to be learned about the roles of cAMP in various organisms. 93 9. 10. BIBLIOGRAPHY Bhumiratana, A., and R. N. Costilow. 1974. Metabolism of trehalose by Bacillus popilliae. Article 2 of this thesis. Clark, V., and R. W. Bernlohr. 1971. Catabolite repression and the enzymes regulating cyclic adenosine-3',5'-mon0phosphate and cyclic guanosine-3',5'-mon0phosphate levels in Bacillus lichenifbrmis. p. 167-173. In H. O. Halvorson, R. Hanson, and L. L. Campbell (ed.), Spore V. American Society for Microbiology, washington D.C. Costilow, R. N., and W. H. Coulter. 1971. Physiological studies of an oligosporogous strain of Bacillus popilliae. Appl. Microbiol. _2_2_: 1076-1084. Duerksen, J. D., and H. Halvorson. 1958. Purification and pr0perties of an inducible B-glucosidase of yeast. J. Biol. Chem. 233: 1113- 1120. Ide, M. 1971. Adenyl cyclase of bacteria. Archi. Biochem. Biophys. 144: 262-268. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. McKay, L. L., A. Bhumiratana, and R. N. Costilow. 1971. Oxidation of acetate by various strains of Bacillus popilliae. Appl. Microbiol. 11: 1070-1075. Pastan, I., and R. L. Perlman. 1970. Cyclic adenosine mon0phosphate in bacteria. Science 169: 339-344. Roberson, G. A., R. W. Butcher, and E. W. Sutherland. 1971. p. 422- 455. In Cyclic AMP. Academic Press, Inc., New York. Sharpe, E. S., G. St. Julian, and C. Crowell. 1970. Characteristics of a new strain of Bacillus popilliae sporogenic in vitro. Appl. Microbiol. 123 681-688. 94 95 TABLE 1: Induction of B-glucosidase in B. popilliae by salicin.a Sugar in Growth Medium Specific Activity (Units/mg Protein) Glucose .0005 Salicin .0096 aB. popilliae was grown in either TY + glucose or TY + salicin medium to mid-exponential phase. The cells were harvested, washed once with 5 x 10-2 M phosphate buffer, pH 7.2, and resuspended in the phosphate buffer. B-Glucosidase was assayed as described in the Materials and Methods section. 96 FIG. 1. Activity of B-glucosidase versus time. B. popilliae was grown in TY + salicin medium to mid-exponential phase, harvested by centrifuging at 10,000 x g for 15 min, and washed twice with 5 x 10‘.2 M phosphate buffer, pH 7.2. The cells were resuspended in the phosphate buffer. The assay was run as described in Materials and Methods section except that reactions were stopped at various time intervals. Each reaction contained 66 ug protein. 97 _ _ _ _ _ 6. 5. 4. 3. 2. 0 0 0 0 0 AS: 003 >._._me0 4 umsu u H I 0>mmswa A .m .uHm umofipna mm pouonvsoo mums mammmm one .zufi>fiuom mmmwwmoosawlm mo uoan xusm a a we 101 ON 0— all] 1.20328 2:2 9 m V _ 1] a m .on (SIINO)£OI ‘- 102 FIG. 4. Induction of B-glucosidase in B. popilliae. B. popilliae 2309MC was grown in 50 ml of TY + glucose to late-exponential phase. The cells were harvested by centrifugation at room temperature and resuspended in 8 m1 of TY medium. A 1.0 m1 inoculum was added to each experimental flask. One experimental flask contained 50 m1 of TY with 0.5% salicin (O), and the other with only the basal medium, TY (‘0. They were aerated by shaking on a rotary shaker at 30 C. Samples were removed at various intervals for measurements of optical density and B-glucosidase activity as described in Materials and Methods. UNIT PER ML 28 24 20 16 103 FIG. 4 0.2 l | I 0.3 0.4 0.5 0.6 OPTICAL DENSITY 0.7 104 FIG. 5A and SB. Effect of cAMP on the synthesis of B-glucosidase in B. popilliae. Four cultures of B. popilliae were grown in TY + 0.5% salicin medium. After the cultures were fully induced to synthesize B-glucosidase, 0.2% trehalose was added to one culture 6h, FIG. 5A), 3 5 x 10- M cAMP was added to the second culture (.5 FIG. 5B), and 5 x 10.3 M cAMP and 0.2% trehalose were added to the third culture a; FIG. SB). The fourth culture (0, FIG. 5A) served as the control. All additions were made at the time indicated by the arrows, and 1.0 m1 of water was added to appropriate flasks to maintain equal volumes. Sampling, B-glucosidase assay, and determination of growth were as described in FIG. 4. 105 OPTICAL DENSITY FIG. 5 106 FIG. 6. Uptake of [3H]cAMP by B. popilliae 2309MC. The detailed procedure was outlined in Materials and Methods. The amount of cells in each reaction mixture was 0.89 mg dry weight. The specific activity of the [BHJcAMP was 9.45 x 105 cpm/umole. All counts were corrected for background. 107 _ _ PII _ _ _ _ L p O 0 8 6 4 2 ‘4 1| Es :5 82.5802: $3515.13 _ IL 10 ‘ 50 1 '4 M[3HJCAMP 8 FIG. 6