JOURNAL OF BACrERiOLOGY, May 1968, p. 1698-1705 Vol. 95, No. 5Copyright 0) 1968 American Society for Microbiology Printed In U.S.A.

Metabolism of Carbohydrates by Pasteurellapseudotuberculosis'

R. R. BRUBAKER2Department ofMicrobiology, The University ofChicago, Chicago, Illinois 60637

Received for publication 26 February 1968

Cell-free extracts of Pasteurella pseudotuberculosis and P. pestis catalyzed a rapidand reversible exchange of electrons between pyridine nucleotides. Although theextent of this exchange approximated that promoted by the soluble nicotinamideadenine dinucleotide (phosphate) transhydrogenase of Pseudomonas fluorescens,the reaction in the pasteurellae was associated with a particulate fraction and wasnot influenced by adenosine-2'-monophosphate. The ability of P. pseudotubercu-losis to utilize this system for the maintenance of a large pool of nicotinamide ade-nine dinucleotide phosphate could not be correlated with significant participationof the Entner-Doudoroff path or catabolic use of the hexose-monophosphate pathduring metabolism of glucose. As judged by the distribution of radioactivity inmetabolic pyruvate, glucose and gluconate were fermented via the Embden-Meyerhofand Entner-Doudoroff paths, respectively. With the exception of hexosediphospha-tase, all enzymes of the three paths were detected, although little or no glucono-kinase or phosphogluconate dehydrase was present unless the organisms werecultivated with gluconate. The significance of these findings is discussed withrespect to the regulation of carbohydrate metabolism in the pasteurellae, relatedenteric bacteria, and P. fluorescens.

Glucose is catabolized by Pasteurella pestis,the causative agent of bubonic plague, via theEmbden-Meyerhof path (29), whereas gluconateis metabolized by a combination of the Entner-Doudoroff and hexose-monophosphate paths(24). Due to a deficiency of glucose-6-phosphatedehydrogenase (2, 24, 25), P. pestis cannot utilizethe nonglycolytic paths during growth on hexose.In contrast, this key enzyme is present in P.pseudotuberculosis (25), an organism very closelyrelated to P. pestis (27). Few studies have beenreported on the catabolism of carbohydrates byP. pseudotuberculosis; however, the biosynthesisof uncommon sugar compounds by this organismhas received intensive study (23, 31).

Cell-free extracts of P. pestis and P. pseudo-tuberculosis catalyze a rapid oxidation of reducednicotinamide adenine dinucleotide phosphate(NADPH2) at the expense of nicotinamide ade-nine dinucleotide (NAD). The resulting largepool of nicotinamide adenine dinucleotide phos-phate (NADP) cannot, because of the metabolicblock already noted, influence the metabolic

I Presented in part at the 64th Annual Meetingof the American Society for Microbiology, Wash-ington, D.C., 3-7 May 1964.

2 Present address: Department of Microbiologyand Public Health, Michigan State University, EastLansing, Mich. 48823.

fate of glucose-6-phosphate in P. pestis. Thepossibility exists, however, that excess NADPin P. pseudotuberculosis might increase theturnover of glucose-6-phosphate dehydrogenaseand thus permit hexose catabolism to occur viathe nonglycolytic paths (8). The purpose of thisstudy was to define the central paths of carbo-hydrate metabolism in P. pseudotuberculosisand to determine the effect, if any, of the trans-hydrogenase activity on the participation ofthese paths.

MATERIALS AND METHODS

Bacteria. P. pseudotuberculosis strain PBI/+ wasisolated by Burrows and Bacon (6) during a spon-taneous epidemic in laboratory guinea pigs. Theproperties of virulent (V+) and avirulent (V-) cellsof this strain have already been described (5, 6).P. pestis strain EV76, Pseudomonas fluorescens strainB-10, and Escherichia coli strain B were used in com-parative experiments.

Media and cultivation. A modification of the saltcomponent of Higuchi and Carlin (15), consisting of0.025 M K2HPO4, 0.01 M citric acid, 0.0025 M MgC92,and 0.0001 M FeC12, was used in all culture media.Flasks (1-liter) containing 100 ml of this base plus4 g of Casitone (Difco) were autoclaved and neu-tralized with NaOH. Then the medium received 1ml of stock solutions of 0.25 M CaCl2, 0.25 M Na2S2O3,and 1.0 M carbohydrate (sterilized by filtration).After inoculation with 107 cells per rnl, the cultures

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were vigorously aerated at 37 C on a wrist-actionshaker, and the organisms were harvested during theearly stationary phase by centrifugation at 27,000 X gfor 10 min in the cold.

Isotopic experiments. Methods for the isolation anddegradation of metabolic pyruvate were similar tothose used by Fraenkel and Horecker (12) andEisenberg and Dobrogosz (10). Bacteria were culti-vated with glucose or gluconate, washed once with;0.033 M potassium phosphate buffer (pH 7.2), andthen suspended to yield a final concentration of 2 mg(dry weight) per ml in a reaction mixture containing0.05M potassium phosphate buffer (pH 7.0) and'0.002 M sodium arsenite. After aeration for 10 min-at 37 C, the volume was brought to 5 ml by additionof 0.005 M radioactive glucose or gluconate. Utili-zation of substrate and accumulation of pyruvate-were complete after further incubation for 1 hr. Theincubation mixture was then acidified with 1 ml of5 N HC104 and centrifuged at 27,000 X g for 10 min;the clear supernatant fluid was neutralized by drop-wise addition of 10 N KOH. After filtration to removeKC104, pyruvate was assayed in a portion of thesample with lactic dehydrogenase, and other radio--active products were determined by cochromatog-raphy and radioautography.

Essentially all radioactivitity was associated withpyruvate, although insignificant amounts of radio-active mannitol were sometimes noted. Therefore, theradioactivity remaining in the incubation mixture wasestimated directly (as pyruvate), and samples weredegraded with ceric sulfate to decarboxylate a-ketoacids (12, 20). Evolved CO2 was collected in 0.2 mlof 1 N KOH, transferred to a closed vessel, acidifiedwith excess H2SO4, and collected in 1.0 ml of 1 NHyamine hydroxide in methanol. Radioactivity wasmeasured with a Packard Tri-Carb liquid scintilla-tion counter (Packard Instrument Co., Inc., DownersGrove, Ill.) in Bray's scintillation liquid (3). No morethan 0.1 ml of aqueous sample (in 2 ml of methanol)was mixed with scintillation fluid. The same proce-dures were used to determine '4CO2 released duringmetabolism of glucose-1-'4C and glucose-6-14C.

Cell-free extracts. Washed whole cells were sus-pended to a concentration of approximately 101"per ml in cold 0.1 M tris(hydroxymethyl)amino-methane (Tris buffer, pH 7.8). After treatment for1 min with an ultrasonic probe (InstrumentationAssociates, New York, N.Y.), cellular debris wasremoved by centrifugation at 27,000 X g for 10 min,and the resulting clear extract was dialyzed overnightagainst cold 0.01 M potassium phosphate bufferplus 0.001 M mercaptoethanol.Enzyme determinations. Most enzymes were deter-

mined by methods similar to those used by Mortlock(24) and Vandermark and Wood (32). Unless statedotherwise, a volume of 3 ml was used for spectro-photometric assays. Reactions linked to pyridinenucleotides were followed at 340 m/M with a BeckmanDU spectrophotometer (Beckman Instruments, Inc.,Fullerton, Calif.) and were corrected, when necessary,for nonspecific oxidase or reductase activity. In allquantitative determinations, one unit of activity isdefined as that amount of enzyme necessary to con-vert 1 pmole of substrate to product per min at 25 C.

Results are expressed in terms of specific activity orunits of enzyme per mg of protein.The rate of formation of glucose-6-phosphate was

measured in 33.3 mM potassium phosphate buffer(pH 7.2), 3.3 mM MgCl2, 3.3 mM L-cysteine (pH 7.2),0.33 mm NADP, and excess yeast glucose-6-phosphatedehydrogenase. Phosphoglucomutase, glucosephos-phate isomerase, and glucokinase were determined inthis system by the addition of 3.3 mm each of glucose-1-phosphate, fructose-6-phosphate, and glucose,plus adenosine triphosphate (ATP), respectively.The basal incubation mixture, without added glucose-6-phosphate dehydrogenase, was used to determineendogenous levels of this enzyme plus phospho-gluconate dehydrogenase by the method of Glockand McLean (14); gluconokinase was also estimatedin the presence of 3.3 mm potassium gluconate, 3.3mM ATP, and the excess phosphogluconate dehy-drogenase present in 1.5 mg of protein obtained fromglucose-adapted cells of E. coli.

Glyceraldehyde phosphate was estimated in areaction mixture consisting of 73 mm Tris buffer(pH 8.0), 10 mm sodium arsenate, 3.3 mM L-cysteine(pH 7.8), 1 mM MgCl2, and 0.33 mM NADP. Thissystem was used to determine endogenous glyceral-dehydephosphate dehydrogenase after addition of3 mm glyceraldehydephosphate. An excess of thisenzyme in the presence of 3.3 mm fructose diphosphateor dihydroxyacetone phosphate was used to measurealdolase and triosephosphate isomerase, respectively.The production of pyruvate was followed in an

incubation mixture consisting of 33 mm potassiumphosphate buffer (pH 7.2), 3.3 mM reduced gluta-thione, 3.3 mM MgCl2, and 0.17 mm reduced nicotin-amide adenine dinucleotide (NADH2). Pyruvatekinase was determined with this reaction system inthe presence of excess lactic dehydrogenase, plus3.3 mm phosphoenolypyruvate, and 3.3 mm adenosinediphosphate; endogenous lactic dehydrogenase wasassayed directly with 3.3 mm potassium pyruvate.

Phosphofructokinase was determined by estima-tion of triose phosphate (28); pyruvate dehydrogenase,phosphate acetyltransferase, and acetate kinase weremeasured by minor modifications of the proceduresoutlined by Colowick and Kaplan (7). Methods fordetermination of enzymes of the Entner-Doudoroffpath and for qualitative assay of phosphoglyceratekinase, phosphoglyceromutase, and phosphopyruvatehydratase are given in Results. The exchange of elec-trons between pyridine nucleotides was measuredby a procedure similar to that used by Eagon (8, 9).

Chromatography. Reactions involving the destruc-tion of ribose-5-phosphate were stopped by additionof 1 volume of 1 M HC104 to the reaction mixture.After removal of protein by centrifugation, the sam-ples were neutralized with 5 N KOH, filtered, and thenadjusted to pH 5.5 by dropwise addition of 1 Nacetic acid. To each 1 ml of sample was added 1 mgof acid phosphatase and, after incubation for 1 hrat 25 C, the solution was brought to dryness on asteam bath. The free sugars were then extracted in5 ml of pyridine at 100 C for 10 min, the resultingsolutions were filtered, and then pyridine was removedby distillation (22). The residue was dissolved in 10%isopropanol, and samples were chromatographed

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on Whatman no. 1 paper in ascending systems com-posed of n-butyl alcohol-ethyl alcohol-water (13:8:4,v/v), ethyl acetate-acetic acid-water (3:1:3, v/v),ethyl acetate-pyridine-water (2:1:2, v/v), and water-saturated benzyl alcohol-acetic acid-water (3:1:3,v/v). After chromatography, the sugars were locatedby spraying with p-anisidine-HCl or ammoniacalsilver nitrate.

Chemical determinations. Pentose and heptulosewere assayed by the orcinol reaction (16), and thecysteine-H2SO4 method was used for the determina-tion of hexose (1). Triose phosphate was measuredas alkali-labile organic phosphate by addition of 0.5Ml of 1 N NaOH to 0.1 ml of sample; after incubationfor 15 min at 25 C, the samples were neutralized andestimated by the method of Fiske and SubbaRow(11). Protein was assayed by the method of Lowryet al. (21).

Reagents. All purified enzymes, coenzymes, sugarsand phosphorylated intermediates were purchasedfrom the Sigma Chemical Co. (St. Louis, Mo.),with the exception of 2-keto-3-deoxyphosphogluco-nate which was received through the courtesy ofR. P. Mortlock. Radioactive compounds were ob-tained from the Volk Radiochemical Co. (Skokie,Ill.).

RESULTS

Preparations of broken V+ cells utilized aboutsix times as much oxygen in the presence ofexcess NADH2 (0.061 ,umole of oxygen atomsper mg of protein per min) as was used whenNADPH2 served as reductant (0.009 ,umole permg of protein per min). The organisms possessedan active NADH2 oxidase system, whereas

TABLE 1. Evidence for the presence of NADH2oxidase and pyridine nucleotide trans-hydrogenase systems in normal andcentrifuged (144,000 X g, 60 min)extracts ofglucose-adapted cells ofV+ Pasteurella pseudotuberculosisa

Specific activity

Addition to reaction mixtureNormal cedntrietatextract

NADH2......................... 0.074 0.012NADPH2........................ 0.004 0.004NADPH2,NAD.. 0.054NADH2, GSSG.. 0.090 0.027NADPH2, GSSG. . 0.538 1.519NADH2, GSSG, NADP.. 0.235 0.031

a The reaction mixture contained 100 ,umoles ofTris buffer (pH 7.6), 0.4 mg of protein (normalextract) or 0.51 mg of protein (centrifuged ex-tract), excess glutathione reductase, and, whereindicated, 0.5 ,.mole of NADH2 or NADPH2,0.1 jAmole of NAD or NADP, and 10 /Amoles ofGSSG (oxidized glutathione) in a volume of 3.0ml.

NADPH2 was slowly oxidized under comparableconditions (Table 1). Although equine cyto-chrome c was reduced by NADH2 (specific ac-tivity = 0.137), NADPH2 did not promotesignificant reduction of this compound. Sincesimilar results were obtained with analogousartificial electron acceptors, P. pseudotuberculosisprobably possessed a respiratory NADH2 de-hydrogenase, whereas the existence of a NADPH2dehydrogenase appeared unlikely.

Nevertheless, NADPH2 was rapidly oxidizedby NAD, and the reverse reaction could befollowed in the presence of endogenous gluta-thione reductase and added oxidized gluta-thione. This activity was not influenced byadenosine-2'-monophosphate or other nucleo-tides tested, and at least one component of thesystem was removed from solution by centrifuga-tion at 144,000 X g for 60 min or by passagethrough a Millipore filter (Table 1). Similar re-sults were obtained from P. pestis and E. coli,although the difference between specific activitiesin the latter species did not approach that of thePasteurella species (Table 2). In contrast, therate of electron exchange catalyzed by the solublenucleotide-dependent NAD(P) transhydrogenaseof P. fluorescens (18) was similar to that notedfor the particulate activities in P. pseudotubercu-losis and P. pestis. Further study was directedtoward defining the significance, if any, of thesetranshydrogenase systems with regard to theregulation of hexose metabolism.

Radioactive glucose or gluconate was addedto suspensions of resting cells, and the distribu-tion of 14C in metabolic pyruvate was determined.The carboxyl carbon of pyruvate originatingfrom glucose-3,4-14C (in glucose-adapted cells)or gluconate-1-14C (in gluconate-adapted cells)was heavily labeled (Table 3), which indicatedthat these compounds were fermented via theTABLE 2. Determination of NAD(P) transhy-

drogenase in Pseudomonasfluorescensand analogous activities in Pasteurella

pestis and Escherichia colia

Specific activity

Addition to reaction mixture

rescens P. pestis E. coli

NADH2 ................ . 0.126 0.052 0.178NADPH2. .0............. 0.016 0.002 0.010NADPH2, NAD........... 0.087 0.030 0.039NADH2, GSSG ........... 0.140 0.060 0.195NADPH2, GSSG .......... 0.607 0.521 0.538NADH2, GSSG, NADP 0.385 0.215 0.246

a The reaction mixture was identical to thatdescribed for Table 1.

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TABLE 3. Distribution of radioactivity in pyruvate formed during the fermentation of glucose-J-'4C,glucose-3 ,4-14C and gluconate-1-l4C

Percentage of recoveredradioactivity

Carbohydrateingrowth__________a_ PyruvateOrganism Carbohydrate in growt Fermentation substrate" formed

Total Carboxyl (pumoles/ml)pyruvate carbon ofb

Pasterella pseudotubercu- Glucose Glucose-1-14C 31 3 7.3losis Glucose Glucose-3,4-14C 39 34 7.5

Gluconate Gluconate-1-_4C 24 19 8.0Gluconate Glucose-l-14C 38 8 7.4

Escherichia coli Glucose Glucose-1-14C 29 4 6.8Glucose Glucose-3,4-14C 27 22 6.9Gluconate Gluconate-1-_4C 34 26 6.9Gluconate Glucose-1-'4C 32 7 7.3

Pseudomonas fluorescens Glucose Glucose-1-14C 17 13 5.8Gluconate Gluconate-1-'4C 13 10 4.2

a Initial radioactivity for glucose-1-'4C, glucose-3,4-'4C, and gluconate-1-14C was 4.1 Xand 4.1 X 104 count/min per ml, respectively.bMeasured as 14CO2 liberated after ceric sulfate degradation.

Embden-Meyerhof and Entner-Doudoroff paths,respectively. Similar results were obtained incontrol experiments performed with E. coli.P. fluorescens yielded carboxyl-labeled pyruvateafter fermentation of both glucose-J1-4C andgluconate-J-'4C.To obtain an approximate estimation of the

percentage participation of the Embden-Meyer-hof, Entner-Doudoroff, and hexose-monophos-phate paths, "'CO2 released from glucose-1-"4Cand glucose-6-'4C (in glucose-adapted cells)was collected over periods of 30 min. As judgedby differences in rates of evolution (33), a valuenot exceeding 20% participation was obtainedfor the hexose-monophosphate path in E. coliand V+ and V- cells of P. pseudotuberculosis.Glucose- and gluconate-adapted cells released"'CO2 from glucose-1-"4C at similar rates, whereasgluconate was not significantly metabolized byglucose-adapted cells. Gluconate-adapted or-ganisms released 4(CO2 from gluconate-1-14Cat a rate which was essentially identical to thatobtained with glucose-J-'IC.These results indicated that the ability of P.

pseudotuberculosis to maintain NADP in theoxidized state did not, in itself, permit thecatabolism of hexose to occur via the nongly-colytic paths. This restriction, however, may havebeen caused by a metabolic block, perhapsanalogous to that in P. pestis. Thus, the presenceof the individual enzymes of the nonglycolyticpaths was demonstrated by direct or indirectdetermination.

The velocities obtained during a typical de-termination of phosphogluconate dehydrase and2-keto-3-deoxyphosphogluconate aldolase in ex-tracts of gluconate adapted cells are shown inFig. 1. The aldolase appeared constitutive;specific activities of 0.139 and 0.128 were ob-tained for extracts of glucose- and gluconate-adapted cells, respectively. In contrast, thedehydrase was not detected in glucose-adaptedcells, whereas a specific activity of 0.029 wasrecorded for gluconate-adapted cells. Similarly,little or no gluconokinase was found in glucose-adapted cells; this enzyme, however, yielded aspecific activity of 0.304 in extracts of organismscultivated with gluconate.The enzymes of the hexose-monophosphate

pathway did not appear to be under regulatorycontrol. The rate of the formation of glyceralde-hyde phosphate from ribose-5-phosphate (viathe action of ribulosephosphate-3-epimerase,ribulose-phosphate isomerase, and transketolase)was not altered by cultivation with 5 differentsources of energy (Fig. 2). The products formedduring the destruction of ribose-5-phosphate byan extract of glucose-adapted cells are given inTable 4. The generation of hexose, coupled withthe appearance and disappearance of sedo-heptulose as the system approached equilibrium,indicated the presence of transaldolase. Essen-tially identical velocities were obtained when thisexperiment was repeated with extracts of glu-conate- and xylose-adapted cells. Attempts todemonstrate the presence of phosphoketolase in

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0

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0 1 2 3 4 5

MINUTESFIG. 1. Determination of phosphogluconate de-

hydrase and 2-keto-3-deoxy-phosphogluconate aldolasein an extract of gluconate-adapted V+ cells of Pas-teurella pseudotuberculosis. The reaction mixture, ina total volume of 1.0 ml, contained 75 jAmoles of Tris(pH 8.5, 3 ,umoles of reduced glutathione, 0.5 ,umoleof NADH2, 0.14 mg of protein, excess lactic dehy-drogenase, and either 5 jmoles of potassium phospho-gluconate (LI) or 5 j,moles of potassium 2-keto-3-deoxyphosphogluconate (A); 0 represents thecorresponding velocity obtained with potassium py-ruvate.

P. pseudotuberculosis by formation of hydrox-amates were not successful. Significant hexosedi-phosphatase could not be detected in extracts ofV+ or V- cells grown with glucose, gluconate, orglycerol. This determination, based on thegeneration of NADPH2 in the presence of addedfructose diphosphate, glucosephosphate iso-merase, and glucose-6-phosphate dehydrogenase,yielded a specific activity of 0.018 for a similarextract of glycerol-adapted cells of E. coli.Specific activities between 0.163 and 0.106 wereobtained for glucose-6-phosphate dehydrogenasein extracts of V+ and V- cells cultivated with avariety of carbohydrates, and a slightly lowerrange was recorded for phosphogluconate de-hydrogenase.

All glycolytic enzymes were detected andtheir specific activities, with the exceptions ofphosphoglycerate kinase, phosphoglyceromutase,and phosphopyruvate hydratase, are given in

0

a0i

0 1 2 3 4 5

MINUTES

FIG. 2. Formation of glyceraldehyde phosphatefrom ribose-5-phosphate by extracts of V+ cells culti-vated with glucose, galactose, gluconate, xylose, andglycerol. The reaction mixture, in a volume of 3.0 ml,contained 225 ,umoles of Tris (pH 8.5), 10 ,imoles ofL-cysteine (pH 7.0), 2 lumoles of MgCI2, 30 ,imoles OJsodium arsenate (pH 8.5), bacterial extract (I mg),and 10 ,umoles of ribose-5-phosphate. Velocities for allextracts fell within the shaded area.

Table 5. Velocities obtained during qualitativeassays of the latter are shown in Fig. 3. Of specialinterest was the finding that the specific activityof glucokinase in V+ cells was generally aboutfour times greater than that of V- cells.

DISCUSSIONThe factors which control the metabolic fate

of hexose in bacteria are not completely under-stood. Salmonella typhimurium (12) and E. coli(10, 13) utilized the Embden-Meyerhof path forfermentation of glucose, whereas the Entner-Doudoroff path was used during growth ongluconate. Evidence for the existence of thiscatabolic pattern in other organisms has beenreported (13, 30). In contrast, many pseudo-monads and related bacteria ferment both glu-cose and gluconate via the Entner-Doudoroffpath; these species exhibit a glycolytic block,

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TABLE 4. Products formed during degradation ofribose-5-phosphate by an extract of

glucose-adapted cells of V+ Pasteurellapseudotuberculosis'

Time of incubationComponent determinedb

0 min 5 min 10 min 20 min

Pentose............... 10.0 7.1 6.0 4.5Triose phosphate ..... 0 1.2 1.1 1.0Hexosec .............. 0 1.4 1.8 1.0Heptulosed ........... 0 0.5 0.6 0.2

a Reaction mixtures contained 50 ,umoles ofribose-5-phosphate, 12.5 ,umoles of MgC12, 250,umoles of Tris (pH 7.7), and approximately 5.0mg of protein per 5.0 ml. Samples were removedfor assay at the times indicated.

b Pentose equivalents (jumoles of carbon/5).cApproximately 90% fructose and 10% glu-

cose, as judged by chromatographic assay.d Identified as sedoheptulose by chromato-

graphic assay.

TABLE 5. Glycolytic enzymes in extracts ofglucose-adapted cells of Pasteurella

pseudotuberculosis

Specific activityEnzyme

V+ cells V- cells

Glucokinase.0.220 0.052Phosphoglucomutase.... .. .. 0.720 0.684Glucosephosphate isomerase 00.965 1 .065Phosphofructokinase. 0.297 0.324Aldolase.0.193 0.229Triosephosphate isomerase... 0.286 0.317Glyceraldehydephosphate de-hydrogenase. 2.120 2.255

Pyruvate kinase.0.256 0.278Lactic dehydrogenase. 0.226 0.314Pyruvate dehydrogenase. 0.018 0.021Phosphate acetyltransferase. 0.227 0.239Acetate kinase.0.052 0.074

Determined as triose phosphate.

probably at the level of phosphofructokinase(19). However, the percentage of participationof the Entner-Doudoroff or hexose-monophos-phate paths was not increased in mutants of S.typhimurium (12) or E. coli (13) possessinganalogous deficiencies of glucosephosphate isom-erase; the growth of these mutants on glucose,but not gluconate, was significantly slower thanthat of the prototrophs. In both cases, gluconoki-nase and phosphogluconate dehydrogenase weremarkedly induced by gluconate but not byglucose. But Eisenberg and Dobrogosz (10)

.200

.100

.0000 2 4 6 8

MINUTES

FIG. 3. Determination of phosphoglycerate kinase,phosphoglyceromutase, and phosphopyruvate hydratasein an extract ofglucose-adapted V+ cells of Pasteurellapseudotuberculosis. The reaction mixture, in a totalvolume of 3.0 ml, contained 225 ,umoles of Tris (pH7.4), 10 ,moles ofA TP, 2 Mnoles of MgCl2, 10 j,molesof L-cysteine (pH 7.0), 0.5 ,umole of NADH2, 0.21mg of protein, excess glyceraldehydephosphate dehy-drogenase, and 0.1 ml of a 0.1 M (or saturated) solu-tion of either potassium 3-phosphoglycerate (0),potassium 2-phosphoglycerate (A), or potassiumphosphoeniolpyruvate (O ). The latter reaction reflectsdismutation of phosphoenolpyruvate to diphospho-glycerate and pyruvate.

showed that both substrates induced phospho-gluconate dehydrase in P. fluorescens and sug-gested that gluconate, arising from glucose viathe action of glucose oxidase, was the actualinducer.Eagon (8) noted that cell-free extracts of

pseudomonads and related nonglycolytic bac-teria contained NAD(P) transhydrogenase orrespiratory NADPH2 dehydrogenase, whereassimilar preparations of glycolytic bacteria didnot. On this basis, Eagon proposed that therestricted catabolism of hexose via the nongly-colytic paths of the latter species was caused byan inability to maintain a sufficient pool ofNADP to permit saturation of glucose-6-phos-phase dehydrogenase. In accord with this hy-pothesis was the finding that methylene blue andpure oxygen promoted a dramatic and probablyanalogous increase in participation of the hexose-

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monophosphate path in erythrocytes (4) andspecies of Bacillus (26), respectively. Moreover,levels of key enzymes controlling the flow of in-termediates and factors regulating the size of theNADP pool may prove to be equally importantin determining the catabolic fate of hexose.

It is well established that NADH2 is generallyinvolved in catabolic reduction, whereas NADPH2usually supplies reducing power for biosyntheticreactions (17). The presence of a NADH2 de-hydrogenase in P. pseudotuberculosis is con-sistent with this concept and was suggested bythe determinations given in Table 1. However,the unexpected finding that this organism couldrapidly oxidize NADPH2 in the presence ofNADindicated that the former could indirectly initiatethe process of respiration. As a result of thisexchange of electrons, it seemed reasonable toassume that the level of NADP in P. pseudo-tuberculosis might be sufficiently high to favoruse of the nonglycolytic paths during growth onhexose.The low value obtained for the percentage of

participation of the hexose-monophosphate pathin P. pseudotuberculosis probably reflects anessentially anabolic function similar to thatdescribed in E. coli (13); nearly identical resultswere recorded in this study for both organisms.The ability to form glyceraldehyde phosphate(Fig. 2) or hexose, heptulose, or triose phosphates(Table 4) from ribose-5-phosphate did not seemto be influenced by the source of energy usedduring cultivation. One factor that may havelimited the participation of the hexose-mono-phosphate path in P. pseudotuberculosis was theapparent deficiency of hexosediphosphatase, anenzyme necessary for the complete cyclic oxida-tion of hexose. The inability to observe hexosedi-phosphatase probably reflects an instability thatis not uncommon for this enzyme, which, ac-cording to current knowledge, is also essentialfor the biosynthesis of hexose and pentose duringgrowth on triose. During the oxidation of glucoseit is still possible, even in the absence of hexosedi-phosphatase, for accumulated triose phosphateto enter the glycolytic path. Fraenkel andHorecker (12) suggested that the capacity of thehexose-monophosphate path in S. typhimuriumwas constant and limited by the turnover ofphosphogluconate dehydrogenase. This may alsobe true in P. pseudotuberculosis.The ability of P. pseudotuberculosis to main-

tain a large pool of NADP did not permit thefermentation of hexose to occur via the Entner-Doudoroff path, although the organism possessedthe requisite structural genes. The inability ofglucose-adapted cells to utilize the Entner-

Doudoroff path can be attributed to a deficiencyof phosphogluconate dehydrase. However, evenwhen this enzyme was induced, as in gluconate-adapted cells, hexose was almost exclusivelycatabolyzed by the Embden-Meyerhof path(Table 3). Similar results, previously reportedfor S. typhimurium (12) and E. coli (10), mayreflect a respective high and low affinity of phos-phogluconate dehydrogenase and phosphoglu-conate dehydrase for their common substrate(12); the same situation may exist in the case ofP. pseudotuberculosis. It should be noted, how-ever, that the soluble NAD(P) transhydrogenaseof P. fluorescens can directly reoxidize NADPH2generated by the cytoplasmic NADP-linkeddehydrogenases. Less efficient reoxidation mightbe expected in P. pseudotuberculosis, where atleast one component of the transhydrogenaseactivity is particulate in nature and may, there-fore, be associated with the cytoplasmic mem-brane.

ACKNOWLEDGMENTThis investigation was supported by Public Health

Service Graduate Training Grant GM-603 from theNational Institute of General Medical Sciences.

LITERATURE CITED1. ASHWELL, G. 1957. Colorimetric analysis of

sugars, p. 73-105. In S. P. Colowick and N. 0.Kaplan [ed.], Methods in enzymology, vol. 3.Academic Press, Inc., New York.

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