Carbohydrate Catabolism of Mima Polymorpha II
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JOURNAL OF BACTERIOLOGY, June, 1966 Vol. 91, No. 6 Copyright @ 1966 American Society for Microbiology Printed in U.S.A. Carbohydrate Catabolism of Mima polymorpha II. Abortive Catabolism of Glucose ADRIENNE MARUS AND EMILY J. BELL Department ofBiological Sciences and Graduate Division of Microbiology, University of Cincinnati, Cincinnati, Ohio Received for publication 6 January 1966 ABSTRACT MARUS, ADRIENNE (University of Cincinnati, Cincinnati, Ohio), AND EMILY J. BELL. Carbohydrate catabolism of Mima polymorpha. II. Abortive catabolism of glucose. J. Bacteriol. 91:2229-2236. 1966.-Mima polymorpha, unable to grow in the presence of glucose as a sole carbon and energy source, is able to obtain sup- plemental, utilizable energy from the partial catabolism of this substrate. Various enzymes of hexose catabolism have been assayed in this organism and in M. poly- morpha M, a mutant obtained by ultraviolet irradiation. The parent strain contains a functional glucose dehydrogenase, glucose-6-phosphate dehydrogenase, diphos- phofructoaldolase, and a 2-keto-3-deoxy-6-phosphogluconate aldolase, but is lacking in glucokinase, gluconokinase, 2-ketogluconokinase, and 6-phospho- gluconate dehydrogenase. The enzymes present indicate partially functioning hexose diphosphate and Entner-Doudoroff pathways. The absence of kinases explains the inability of the strain to grow on glucose and an absence of 6-phosphogluconate dehydrogenase would indicate the absence of the complete pentose pathway. The mutant strain, M. polymorpha M, possesses, in addition to those enzymes produced by the wild type, both gluconokinase and 6-phosphogluconate dehydrogenase. The presence of the former explains the mutant's ability to grow on glucose, and the presence of the latter indicates a more complete pentose shunt. The supplemental energy obtained from partial glucose catabolism (to gluconic acid) may be obtained from a cytochrome-linked reaction of the glucose dehydrogenase. Although Mima polymorpha is unable to the following manner. A suspension of acetate-grown, utilize glucose as a sole carbon and energy source, wild-type cells was spread over agar plates prepared it has been shown (1) that an energy-linked from the mineral salts medium with 0.03 M glucose partial catabolism of this substrate must occur, incorporated as sole carbon and energy source. The plates were exposed to ultraviolet irradiation for 10 as the addition of supplemental glucose to a or 15 sec from a germicidal lamp (Precision Scientific growth medium results in a greater rate of Co., Chicago, IlJ.) which emits radiation from 2,500 growth and a larger cell crop, an induction of A and peaks at 3,660 A. A colony was obtained from transport mechanisms for slightly permeable a plate which had been exposed for 10 sec and inocu- phosphorylated intermediates, and a more rapid lated into minimal salts medium containing either induction to a higher level of specific activity of glucose or fructose as sole carbon and energy source. an inducible enzyme, isocitrate lyase E.C. 4.1.3.1. It was found that good growth occurs on either of the (isocitritase). The purpose of the present study hexoses, as well as on acetate. This mutant strain has was to survey various hexose catabolic enzymes been maintained on a synthetic mineral salts, glucose to locate the metabolic semisolid medium. in an attempt lesion(s) Since some of the enzymes of glucose catabolism occurring in the strain. A glucose-utilizing mutant are inducible, M. polymorpha was grown routinely was studied also so that comparative assays might for all assays in the basal salts medium supplemented help to pinpoint the catabolic pathways fol- with 0.03 M acetate and 0.03 M glucose. It should be lowed. pointed out, however, that, with the exception of the isocitrate lyase studies (1), possible metabolic control MATERIALS AND METHODS effects of double substrate growth have not been M. polymorpha ATCC 9957 was maintained and elucidated for this organism. When used, M. poly- cultured as described previously (1). The mutant morpha M was grown on the same medium, to which strain, designated M. polymorpha M was obtained in glucose alone was added. Growth conditions of the 2229 .2230 MARUS AND BELL J. BACTERIOL. control organisms will be designated where appro- TABLE 1. Manometric hexokinase assay* priate. Growth was followed by periodic optical-density Total,umoles of C02 released determinations in a Klett-Summerson photoelectric colorimeter. The organisms were grown in Belico after Mima polymorpha Escherichia coli nepheloflasks so that frequent turbidimetric readings tipping extract extract (min) could be made, and were incubated with shaking in a New Brunswick incubator shaker, model G 26, at + ATP ++glucoseATP + ATP ++glucoseATP 28 C and 250 rev/min. Cell-free extracts for all en- zyme analyses were prepared by disruption of twice- 5 1.1 1.5 3.0 2.0 washed cells in a Raytheon 9-kc, 50-w sonic oscillator 10 6.0 7.0 5.2 6.3 at 150-v output for 15 min. Cell debris was removed 20 7.0 7.0 5.3 8.4 by centrifugation for 10 min at 10,000 X g in the cold. Protein content was measured by the procedure * The vessels contained in a total of 2.2 ml: 40 of Lowry et al. (15). Most of the major reagents were ,moles of NaHCO3 (pH 7.5), 10,umoles of MgCl2, obtained from the sources listed previously (1). In and 1.5 ml of sonic extract (M. polymorpha, 3.9 addition, nicotinamide adenine dinucleotide (NAD) mg of protein/ml; E. coli, 3.8 mg of protein/ml). and adenosine triphosphate (ATP) were obtained Mm from Mann Research Laboratories, New York, N.Y.; ATP, 20,uM, and 16.6 glucose were added from lactic dehydrogenase, glucose-6-phosphate dehy- the side arm. Incubation was at 30 C in an atmo- drogenase, and reduced nicotinamide adenine dinu- sphere of N2. The total amount of CO2 released in Biochemicals an endogenous control vessel for M. polymorpha cleotide (NADH2), from Nutritional was 1.6, and for E. coli was 1.1. The total amount Corp., Cleveland, Ohio; and nicotinamide adenine of CO2 released in a glucose vessel without ATP dinucleotide phosphate (NADP) and glyceraldehyde- was 1.3 ,umoles for M. polymorpha and 1.9 ,moles 3-phosphate dehydrogenase, from Sigma Chemical for E. coli. Co., St. Louis, Mo. Enzyme assays were carried out by use of either a Gilford model 2000 recording spec- trophotometer or a Bronwill Warburg apparatus. The hexokinase in M. polymorpha. To confirm this, a assays performed will be described as used. Intra- spectrophotometric assay (16) was performed. cellular gluconic acid was detected, after deproteiniza- M. polymorpha M, grown in the presence of tion of a sonic extract and concentration, by the glucose, was tested also. This method determines chromatographic method of Norris and Campbell the ability of the cell to form glucose-6-phosphate (18). (G6P) by measuring NADP reduction in the REsULTS presence of added G6P dehydrogenase. Optical density changes, recorded at 340 mI,, are pre- Since M. polymorpha is unable to grow on sented in Fig. 1. The data confirm the absence of nonphosphorylated sugars, the first enzyme hexokinase activity in cell-free extracts of M. assayed was glucokinase (hexokinase). The first polymorpha as well its absence in extracts of the method employed was the manometric pro- mutant organism, at least under these experi- cedure of Colowick and Kalckar (3) in which mental conditions. carbon dioxide evolution from bicarbonate in the Since the organism is unable to phosphorylate presence of ATP is measured. The control glucose but yet can obtain energy from it, it was organism, Escherichia coli, was grown on the assumed that a direct oxidation might occur with usual basal medium to which 0.03 M glucose was the formation of gluconic acid or 2-ketogluconic added. After 20 hr of incubation, both M. poly- acid. Accordingly cell-free extracts were assayed morpha and E. coli were harvested, washed, and for glucose dehydrogenase activity by comparing sonically treated. The results in Table 1 show that glucose disappearance (by the Glucostat method) the amount of CO2 released from bicarbonate in in the presence of an enzyme preparation added the presence of glucose and ATP by the cell-free with and without ATP (16). The control organism extract of M. polyinorpha is not significantly Herellea vaginicola, was grown in the presence of higher than the amount released in the presence acetate and glucose, and M. polymorpha and the of ATP alone. High adenosine triphosphatase M mutant were grown in the usual media. All activity in cell-free extracts of certain micro- organisms were incubated for 18 hr and harvested. organisms has been a difficulty noted by other To insure that no residual glucose from the investigators using this method (9, 21). The growth media remained adsorbed to the cells, the amount of CO2 released, however, in the presence the washed cells were of glucose plus ATP by the sonic extract of E. coli supernatant fluids from is much greater than that produced in any of the assayed for glucose, until none could be detected. other flasks, indicating the presence of an active Also, in the assay itself, one tube was included hexokinase in that organism. These data were as an endogenous intracellular glucose control for considered to be an indication of the absence of each organism. The results (Table 2) indicate that VOL. 91, 1966 CARBOHYDRATE CATABOLISM OF MIMA POLYMORPHA. II 2231 TABLE 2. Glucose dehydrogenase activity in cell- 0 free extracts of Mima polymorpha, M. polymorpha M, and Herellea vaginicola* E. coli Unitst of enzyme activity/mg of 1.5 - protein Extract from With ATP Without Differ (hexoki- ATP Difr naae + ence glucose (guoe (hexoki- dehydro- dehydro-dgehcase naae) 0 genaae) gea) c 0 1.0- M.