Metabolism of Carbohydrates by Pasteurella Pseudotuberculosis' R

JOURNAL OF BACrERiOLOGY, May 1968, p. 1698-1705 Vol. 95, No. 5 Copyright 0) 1968 American Society for Microbiology Printed In U.S.A. Metabolism of Carbohydrates by Pasteurella pseudotuberculosis' R. R. BRUBAKER2 Department ofMicrobiology, The University ofChicago, Chicago, Illinois 60637 Received for publication 26 February 1968 Cell-free extracts of Pasteurella pseudotuberculosis and P. pestis catalyzed a rapid and reversible exchange of electrons between pyridine nucleotides. Although the extent of this exchange approximated that promoted by the soluble nicotinamide adenine dinucleotide (phosphate) transhydrogenase of Pseudomonas fluorescens, the reaction in the pasteurellae was associated with a particulate fraction and was not influenced by adenosine-2'-monophosphate. The ability of P. pseudotuberculosis to utilize this system for the maintenance of a large pool of nicotinamide adenine dinucleotide phosphate could not be correlated with significant participation of the Entner-Doudoroff path or catabolic use of the hexose-monophosphate path during metabolism of glucose. As judged by the distribution of radioactivity in metabolic pyruvate, glucose and gluconate were fermented via the Embden-Meyerhof and Entner-Doudoroff paths, respectively. With the exception of hexosediphospha- tase, all enzymes of the three paths were detected, although little or no gluconokinase or phosphogluconate dehydrase was present unless the organisms were cultivated with gluconate. The significance of these findings is discussed with respect to the regulation of carbohydrate metabolism in the pasteurellae, related enteric bacteria, and P. fluorescens. Glucose is catabolized by Pasteurella pestis, fate of glucose-6-phosphate in P. pestis. The the causative agent of bubonic plague, via the possibility exists, however, that excess NADP Embden-Meyerhof path (29), whereas gluconate in P. pseudotuberculosis might increase the is metabolized by a combination of the Entner- turnover of glucose-6-phosphate dehydrogenase Doudoroff and hexose-monophosphate paths and thus permit hexose catabolism to occur via (24). Due to a deficiency of glucose-6-phosphate the nonglycolytic paths (8). The purpose of this dehydrogenase (2, 24, 25), P. pestis cannot utilize study was to define the central paths of carbo- the nonglycolytic paths during growth on hexose. hydrate metabolism in P. pseudotuberculosis In contrast, this key enzyme is present in P. and to determine the effect, if any, of the trans- pseudotuberculosis (25), an organism very closely hydrogenase activity on the participation of related to P. pestis (27). Few studies have been these paths. reported on the catabolism of carbohydrates by P. pseudotuberculosis; however, the biosynthesis MATERIALS AND METHODS of uncommon sugar compounds by this organism Bacteria. P. pseudotuberculosis strain PBI/+ was has received intensive study (23, 31). isolated by Burrows and Bacon (6) during a spon- Cell-free extracts of P. pestis and P. pseudo- taneous epidemic in laboratory guinea pigs. The tuberculosis catalyze a rapid oxidation of reduced properties of virulent (V+) and avirulent (V-) cells of this strain have already been described (5, 6). nicotinamide adenine dinucleotide phosphate P. pestis strain EV76, Pseudomonas fluorescens strain (NADPH2) at the expense of nicotinamide ade- B-10, and Escherichia coli strain B were used in com- nine dinucleotide (NAD). The resulting large parative experiments. pool of nicotinamide adenine dinucleotide phos- Media and cultivation. A modification of the salt phate (NADP) cannot, because of the metabolic component of Higuchi and Carlin (15), consisting of block already noted, influence the metabolic 0.025 M K2HPO4, 0.01 M citric acid, 0.0025 M MgC92, and 0.0001 M FeC12, was used in all culture media. I Presented in part at the 64th Annual Meeting Flasks (1-liter) containing 100 ml of this base plus of the American Society for Microbiology, Wash- 4 g of Casitone (Difco) were autoclaved and neu- ington, D.C., 3-7 May 1964. tralized with NaOH. Then the medium received 1 2 Present address: Department of Microbiology ml of stock solutions of 0.25 M CaCl2, 0.25 M Na2S2O3, and Public Health, Michigan State University, East and 1.0 M carbohydrate (sterilized by filtration). Lansing, Mich. 48823. After inoculation with 107 cells per rnl, the cultures 1698 VOL. 95,1968 CARBOHYDRATE METABOLISM IN P. PSEUDOTUBERCULOSIS 1699 were vigorously aerated at 37 C on a wrist-action Results are expressed in terms of specific activity or shaker, and the organisms were harvested during the units of enzyme per mg of protein. early stationary phase by centrifugation at 27,000 X g The rate of formation of glucose-6-phosphate was for 10 min in the cold. measured in 33.3 mM potassium phosphate buffer Isotopic experiments. Methods for the isolation and (pH 7.2), 3.3 mM MgCl2, 3.3 mM L-cysteine (pH 7.2), degradation of metabolic pyruvate were similar to 0.33 mm NADP, and excess yeast glucose-6-phosphate those used by Fraenkel and Horecker (12) and dehydrogenase. Phosphoglucomutase, glucosephos- Eisenberg and Dobrogosz (10). Bacteria were culti- phate isomerase, and glucokinase were determined in vated with glucose or gluconate, washed once with this system by the addition of 3.3 mm each of glucose- ;0.033 M potassium phosphate buffer (pH 7.2), and 1-phosphate, fructose-6-phosphate, and glucose, then suspended to yield a final concentration of 2 mg plus adenosine triphosphate (ATP), respectively. (dry weight) per ml in a reaction mixture containing The basal incubation mixture, without added glucose- 0.05 M potassium phosphate buffer (pH 7.0) and 6-phosphate dehydrogenase, was used to determine '0.002 M sodium arsenite. After aeration for 10 min endogenous levels of this enzyme plus phospho- -at 37 C, the volume was brought to 5 ml by addition gluconate dehydrogenase by the method of Glock of 0.005 M radioactive glucose or gluconate. Utili- and McLean (14); gluconokinase was also estimated zation of substrate and accumulation of pyruvate in the presence of 3.3 mm potassium gluconate, 3.3 -were complete after further incubation for 1 hr. The mM ATP, and the excess phosphogluconate dehy- incubation mixture was then acidified with 1 ml of drogenase present in 1.5 mg of protein obtained from 5 N HC104 and centrifuged at 27,000 X g for 10 min; glucose-adapted cells of E. coli. the clear supernatant fluid was neutralized by drop- Glyceraldehyde phosphate was estimated in a wise addition of 10 N KOH. After filtration to remove reaction mixture consisting of 73 mm Tris buffer KC104, pyruvate was assayed in a portion of the (pH 8.0), 10 mm sodium arsenate, 3.3 mM L-cysteine sample with lactic dehydrogenase, and other radio- (pH 7.8), 1 mM MgCl2, and 0.33 mM NADP. This -active products were determined by cochromatog- system was used to determine endogenous glyceral- raphy and radioautography. dehydephosphate dehydrogenase after addition of Essentially all radioactivitity was associated with 3 mm glyceraldehydephosphate. An excess of this pyruvate, although insignificant amounts of radio- enzyme in the presence of 3.3 mm fructose diphosphate active mannitol were sometimes noted. Therefore, the or dihydroxyacetone phosphate was used to measure radioactivity remaining in the incubation mixture was aldolase and triosephosphate isomerase, respectively. estimated directly (as pyruvate), and samples were The production of pyruvate was followed in an degraded with ceric sulfate to decarboxylate a-keto incubation mixture consisting of 33 mm potassium acids (12, 20). Evolved CO2 was collected in 0.2 ml phosphate buffer (pH 7.2), 3.3 mM reduced gluta- of 1 N KOH, transferred to a closed vessel, acidified thione, 3.3 mM MgCl2, and 0.17 mm reduced nicotin- with excess H2SO4, and collected in 1.0 ml of 1 N amide adenine dinucleotide (NADH2). Pyruvate Hyamine hydroxide in methanol. Radioactivity was kinase was determined with this reaction system in measured with a Packard Tri-Carb liquid scintilla- the presence of excess lactic dehydrogenase, plus tion counter (Packard Instrument Co., Inc., Downers 3.3 mm phosphoenolypyruvate, and 3.3 mm adenosine Grove, Ill.) in Bray's scintillation liquid (3). No more diphosphate; endogenous lactic dehydrogenase was than 0.1 ml of aqueous sample (in 2 ml of methanol) assayed directly with 3.3 mm potassium pyruvate. was mixed with scintillation fluid. The same proce- Phosphofructokinase was determined by estima- dures were used to determine '4CO2 released during tion of triose phosphate (28); pyruvate dehydrogenase, metabolism of glucose-1-'4C and glucose-6-14C. phosphate acetyltransferase, and acetate kinase were Cell-free extracts. Washed whole cells were sus- measured by minor modifications of the procedures pended to a concentration of approximately 101" outlined by Colowick and Kaplan (7). Methods for per ml in cold 0.1 M tris(hydroxymethyl)amino- determination of enzymes of the Entner-Doudoroff methane (Tris buffer, pH 7.8). After treatment for path and for qualitative assay of phosphoglycerate 1 min with an ultrasonic probe (Instrumentation kinase, phosphoglyceromutase, and phosphopyruvate Associates, New York, N.Y.), cellular debris was hydratase are given in Results. The exchange of elec- removed by centrifugation at 27,000 X g for 10 min, trons between pyridine nucleotides was measured and the resulting clear extract was dialyzed overnight by a procedure similar to that used by Eagon (8, 9). against cold 0.01 M potassium phosphate buffer Chromatography. Reactions involving the destruc- plus 0.001 M mercaptoethanol. tion of ribose-5-phosphate were stopped by addition Enzyme determinations. Most enzymes were deter- of 1 volume of 1 M HC104 to the reaction mixture. mined by methods similar to those used by Mortlock After removal of protein by centrifugation, the sam- (24) and Vandermark and Wood (32). Unless stated ples were neutralized with 5 N KOH, filtered, and then otherwise, a volume of 3 ml was used for spectro- adjusted to pH 5.5 by dropwise addition of 1 N photometric assays.

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