JOURNAL OF BACrERIOLOGY, Mar., 1967, p. 941-949 Vol. 93, No. 3Copyright © 1967 American Society for Microbiolog Printed in U.S.A.

Gluconate Metabolism in Escherichia colilROBERT C. EISENBERG2 AND WALTER J. DOBROGOSZ

Department ofMicrobiology, North Carolina State University, Raleigh, North Carolina

Received for publication 15 November 1966

On the basis of information available in the literature, gluconate dissimilationin Escherichia coli is thought to occur via the hexose monophosphate pathway.Evidence is presented in this study that gluconate is catabolized in this organismvia an inducible Entner-Doudoroff pathway. This evidence is based on chromato-graphic examination of end products produced from 14Clabeled gluconate or glu-cose, distribution of 14C in the carbon atoms of pyruvate formed from specificallylabeled 14C-glucose and "4C-gluconate, and the ability of cell-free extracts to pro-duce pyruvate from 6-phosphogluconate. Degradation of gluconate by an Entner-Doudoroff pathway occurred simultaneously with a glycolytic cleavage of glucose.A relationship between gluconate-induced, Entner-Doudoroff pathway activityand catabolism of glucose in Escherichia coli and other bacterial species is dis-cussed.

In some bacteria, gluconate but not glucose ismetabolized via an inducible Entner-Doudoroff(ED) pathway. Thus, Salmonella typhimuium (9),Pseudomonas natriegens (8),. and Streptococcusfaecalis (25) are known to be induced to dis-similate gluconate by the ED pathway. Thissystem involves a gluconbkinase that phos-phorylates gluconate to 6-phosphogluconate, adehydrase that converts 6-phosphogluconate to2-keto-3-deoxy--phosphogluconate (KDPG),and an aldolase that cleaves KDPG to pyru-vate and glyceraldehyde-3-phosphate.

In Escherichia colt, gluconate is believed to bemetabolized by way ofthe hexose monophosphatepathway (4, 5, 24). Furthermore, DeLey (6)indicated on the basis of available data tbat,although 6-phosphogluconate dehydrase andKDPG aldolase were detected in E. coli (16),these enzymes play no role in the overall metabo-lism of carbohydrates by this organism.Recent studies in our laboratory, however,

provided evidence conflicting with these views(23). Our findings indicated that the ED systemconstituted the primary route of gluconate metab-olism in E. coli ML30. On the basis of theseinvestigations, it was apparent that the ED

'Contribution from the North Carolina Agricul-tural Experiment Station, Raleigh. Published withthe approval ofthe Director ofResearch as Paper No.2297 of the Journal Series. This work is part of adissertation submitted by the senior author in partialfulfiment of the requirements for the Ph.D. degreefrom the North Carolina State University.

2Present address: Department of Dairy Science,University of Illinois, Urbana.

system and gluconate metabolism in E. coli werein need of a re-evaluation. The present com-munication describes the results of our studies ongluconate metabolism in E. coli and other bac-terial species. A preliminary report of theseinvestigations was presented (R. C. Eisenbergand W. J. Dobrogosz, Bacteriol. Proc., p. 77,1966).

MATmL4S AND MEMODS

u-Glucose-lC (uniformly labeled) and D-glucose-J.14C were products of New England Nuclear Corp.,Boston, Mass. D-Gluconic acid-14C (uniformlylabeled) and D-gluconic acid-1-14C were purchasedErom Nuclear-Chicago Corp., Des Plaines, Ill., andpyruvic acid-1 4C, -2-4C, and -3-14C were products ofthe Volk Radiochemical Co., Chicago, Ill. The 2-keto-3-deoxy-6phosphogluconate was a gift through thecourtesy of J. Ingram, Department of Biochemistry,Michigan State University.

,E. coli ML30 was obtained through the courtesy ofJ. L. Ingaham (University of California, Davis). E.coli B srains and Salmonella typhimurium LT2 werefrom the laboratory collection of the Department ofMicrobiology, North Carolina State University,Raeigh. Pseudomonas fluorescens FS and Serratiamarcescens FS were from the laboratory collectionsin the Department of Food Science of the same uni-versity. E. coli ATCC 11303 and 8739, Enterobacteraerogenes ATCC 12658 and 8724, S. marcescensATCC 13880, Erwina carotovora ATCC 138, andP.fluorescens ATCC 13525 were purchased from theAmerican Type Culture Collection (ATCC), Rockville,Md.

All cultures were maintained on Trypticase SoyAgar (BBL) slants and were transferred approximatelyevery 2 months to the same medium. All cultures were

941

on January 17, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

EISENBERG AND DOBROGOSZ

grown under aerobic conditions at 37 C exceptPseudomonas cultures which were incubated at 25 C.The basal medium contained (grams per liter):

K2HPQ-3H20, 36.8; KH2PO4, 8.0; MgS04-7H20,0.1; and (NH4)2SO4, 1.0. Substrates were sterilizedseparately and added at a level of 0.02 M. In somecases, 0.25% casein hydrolysate (acid-hydrolyzed,vitamin-free, Nutritional Biochemicals Corp., Cleve-land, Ohio) was added to the medium just prior toinoculation. In some experiments, the cultures weregrown in radiorespirometer vessels (27) so that 14CO2formed during growth on labeled substrates could bedetermined.

Preparation of cells and cell-free extracts. Cultureswere collected by centrifugation at 10,000 X g for 10min and washed twice with either 0.05 M sodium phos-phate buffer, (pH 7.0) or 0.40 M tris-(hydroxymethyl)-aminomethane (Tris) buffer (pH 7.7). Cell-free ex-tracts were prepared by sonic disruption (BransonUltrasonic Corp., Stamford, Conn.), followed byretention of the supernatant fraction after centrifuga-tion at 20,000 X g for 30 min. All manipulations werecarried out in the cold. In some experiments, extractswere prepared with use of the French pressure cell(Aminco, Silver Spring, Md.) rather than by sonicdisruption.

Enzymatic assays. Glucose-6-phosphate and 6-phosphogluconate dehydrogenase activities were de-termined spectrophotometrically by following thereduction of nicotinamide adenine dinucleotide phos-phate (NADP) at 340 mu as a function of time. Thereaction mixture included: Tris chloride (pH 7.65),400 pumoles; glucose-6-phosphate or 6-phosphoglu-conate, 10,moles; NADP, 0.3 ,umole; extract andwater to 3 ml. The reaction was conducted at roomtemperature. Gluconokinase activity was measuredunder the same conditions as 6-phosphogluconate de-hydrogenase except that 0.1 ,Amole of MgSO4, 10pmoles of adenosine triphosphate (ATP), and 10pmoles of gluconate were substituted for 6-phospho-gluconate. Extracts of glucose-grown E. coli ML30were employed as a source of 6-phosphogluconatedehydrogenase. Hexokinase activity was measured bytaking advantage of glucose-6-phosphate dehydro-genase activity and measuring NADP reduction.Hexokinase was rate-limiting in this assay. The reac-tion mixture included: glycylglycine buffer (pH 9.0),200 umoles; NADP, 1.5 jumoles; MgSO4, 10 ;&moles;ATP, 10 ,moles; glucose, 10 ,Amoles; and cell-freeextract prepared with a French pressure cell. Totalvolume was 3 ml, and the reaction was carried out atroom temperature. Glucose dehydrogenase activitywas measured by the amount of NADP reductionwhich occurred in the hexokinase assay when ATP wasomitted. KDPG aldolase and 6-phosphogluconatedehydrase were assayed essentially as described byKovachevich and Wood (15, 16) for P. fluorescens.Conditions of the assay are described in Results.

Determination ofend products and ceric sulfate deg-radation of 14C-pyruvate. End products producedduring aerobic and anaerobic growth of E. coli on14C-glucose and '4C-gluconate were isolated by silicicacid chromatography and determined as described

elsewhere (7). Pyruvate formed either as an endproduct in growing cultures, or accumulating duringsubstrate dissimulation with arsenite-treated, non-proliferating cells, was prepared and chromato-graphically isolated in an identical manner. Fordegradation studies, chromatographic fractions con-taining labeled pyruvate were pooled and extractedwith 0.1 N NaOH. The extracted pyruvate was acidi-fied, and the isotope level was determined. A sample ofthis preparation was added to a 50-ml Erlenmyer flaskequipped with a side arm and a center well containing1 ml of monoethanolamine. The flasks containingpyruvate-'4C (0.5 ml), water (0.5 ml), and 18 NH2SO4 (0.2 ml) were sealed with serum stoppers, and4 ml of 10 N H2SO4 saturated with Ce(S04)2 was addedto the main compartment with a syringe. The reactionmixture was allowed to incubate for 2.5 hr at 25 C,and 1.5 ml of 0.4 M FeSO4 in 1.0 N H2SO4 was thenadded with a syringe to neutralize excess Ce+4. Thecontents of the flask were quantitatively removed, anddiluted to known volume with absolute ethyl alcohol;samples were then taken for scintillation counting.The counts found in the ethanolamine fraction werecalculated as 14CO2. The above is a modification of theprocedure described by Krebs and Johnson (17) fordecarboxylation of a-keto acids with ceric sulfate.

Counting procedures. All samples were countedwith a Packard Tri-Carb Liquid Scintillation Counter(Packard Instrument Co., La Grange, Ill.), by use ofa standard scintillation fluid consisting of toluenecontaining 0.4% PPO (2, 5-diphenyloxazole) and0.01% POPOP [1 ,4-bis-2-(5-phenyl-oxazolyl)ben-zenel. Aqueous samples (no more than 0.1 ml) wereadded to 2 ml of ethyl alcohol in the counting vialsbefore addition of the scintillation fluid. Samples(1-ml) of eluates from silicic acid columns were addedto 1 ml of t-butanol in counting vials before addition ofscintillation fluid. Corrections were made for activityquenching by chloroform present in the fractionsobtained by chromatography.

Miscellaneous. Protein concentrations were esti-mated by a modification of the procedure describedby Lowry et al. (20). Since Tris gives a positive testwith this method, appropriate blanks were used whenTris buffer was employed.

Pyruvate was determined by the direct method ofFriedemann and Haugen (11) and identified by theabsorption spectrum of its 2,4-dinitrophenylhydra-zone chromogen as well as by silicic acid chroma-tography.

Gluconate was estimated colorimetrically by incu-bation of 0.02 to 0.2 umole of gluconate with 5 jumolesof sodium periodate and 40 jrnoles of acetic acid for10 min, followed by another 10-min incubation with40,umoles of sodium arsenite (total volume, 1 ml).Color was developed by addition of 6.0 ml of chro-matropic acid reagent (200 mg of chromatropic acidper 100 ml of 19.2 N H2S04) and incubation at 100 Cfor 30 min. Absorbance was measured at 570 m,u, andconcentration was determined from a standard curverelating concentration to absorbancy. Glucose wasdetermined by the procedure of Nelson (22).

942 J. BACrERIOL.

on January 17, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

GLUCONATE METABOLISM IN E. COLI

RESULTS

Oxidation ofglucose and gluconate. To establishthe nature of gluconate dissimilation in E. coliML30, preliminary manometric experiments wereconducted on the oxidation of glucose andgluconate by this organism. These data showedthat glucose-grown cells were not capable ofoxidizing gluconate to any appreciable extent inthe presence of chloramphenicol. Gluconate-grown cells, on the other hand, readily oxidizedboth glucose and gluconate. When glucose cellswere used in the absence of chloramphenicol,gluconate oxidation occurred after an initial lagperiod. These data suggest that gluconate dis-similation is regulated by one or more inducibleenzymes.As a means of further establishing the inducible

nature of gluconate metabolism in E. coli ML30,extracts of both gluconate- and glucose-growncells were prepared, and the activities of thoseenzymes responsible for initial oxidation of thesesubstrates were examined. The results of theseexperiments are summarized in Table 1. Thesedata clearly show that there were no significantdifferences in the levels of glucose dehydrogenase,hexokinase, glucose-6-phosphate dehydrogenase,and 6-phosphogluconate dehydrogenase betweencell-free extracts of glucose-grown and gluconate-grown cells. The amount of gluconokinase activityin gluconate cell extracts exceeded that in glucosecell extracts by approximately 40-fold and demon-strated the inducible nature of this enzyme. Thus,both whole-cell and cell-extract data were con-

TABLE 1. Enzymes involved in dissimilation ofgluconate and glucose

Specific activitya

Enzyme Glucose Gluco-cellSb natecl celtls

Glucose dehydrogenase .......... 0.8 0.4Hexokinase...................... 15.8 13.1Gluconokinase................... 5.3 234.3Glucose-6-phosphate dehydro-genase......................... 129.5 113.5

6-Phosphogluconate dehydro-genase......................... 38.6 38.2

a Enzyme specific activities expressed as milli-micromoles of NADP reduced per minute permilligram of protein. Assays were performed asdescribed in Materials and Methods using cell-free extracts of Escherichia coil ML30.

b Cultures were grown aerobically for 10 hr inbasal media containing either 0.02 M glucose or0.02 M gluconate.

sistent with the findings of Cohen and Scott (5),who concluded that the enzymatic compositionof glucose-grown and gluconate-grown E. coli isessentially the same except for gluconokinase.

Radioisotope studies. Analysis of end productsformed during growth on specifically labeledsubstrates can yield evidence concerning thenature of the pathways employed for carbo-hydrate catabolism. Nongaseous end productssuch as formate, acetate, ethyl alcohol, and lactateare known to occur when E. coli is grown onglucose (2, 26). Acetate and ethyl alcohol arederived from carbons 2 and 3 of pyruvate.Formate is derived from the carboxyl group ofpyruvate. Glucose-1-14C catabolized via theglycolytic pathway would yield 14C-acetate and'IC-ethyl alcohol. Catabolism of glucose-1-14C viathe ED pathway would yield 14C-formate, andthe two carbon fragments derived from pyruvatedegradation would not contain isotope.With these parameters in mind, experiments

were designed to determine the end productsproduced when E. coli ML30 was grown in thepresence of uniformly labeled glucose, glucose-1-14C, uniformly labeled gluconate, and gluconate-1-14C. The results of these experiments are pre-sented in Fig. 1. Evaluation of these data providedinformation as to the nature of gluconate dis-similation in E. coli ML30. Comparison of thoseproducts containing '4C derived from glucose-U-14C and glucose-1-14C indicated a pattern oflabel distribution consistent with the operationof the glycolytic pathway in these cells. Bothethyl alcohol and acetate derived from glucose-1-14C contained isotope, whereas very littlelabeled formate was present compared with cul-tures grown on glucose-U 14C. The end-productprofiles from gluconate-U-14C and gluconate-I14C clearly showed that, although it was a majorend product of gluconate dissimilation, acetatedid not contain isotope when gluconate-1-14Cserved as the substrate.To provide a more complete picture of the dis-

similation of gluconate by this organism, carbon-recovery data were obtained for the utilizationof gluconate- U- 4C and gluconate-1-14C underboth aerobic and anaerobic conditions. The re-sults of these experiments are shown in Table 2.The products of gluconate metabolism underaerobic conditions were primarily CO2, ace-tate, and pyruvate, whereas, under anaerobicconditions, ethyl alcohol, formate, lactate, andsuccinate, but not pyruvate, accumulated. Thedifferences in product labeling between gluconate-U-14C and gluconate-1-14C under both aerobicand anaerobic conditions were consistent with

VOL. 93, 1967 943

on January 17, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

EISENBERG AND DOBROGOSZ

20

10

Ethanol Formate Lactate S

.A IAicetate I

20 -

to [

10

-0------- 0".- _N

0 10 20 30 40 50 60Tube No. (12 MI/Tube)

FIG. 1. Anaerobic end products of Escherichia coliML30. Glucose-grown and gluconate-grown cells wereharvested during exponential growth, washed in basalsynthetic medium, and then resuspended in the mediumat a concentration of 100 jug of cells (dry weight) perml. The cultures were then made anaerobic by bubblingwith 95% Nr-5% CC2, and growth was initiated byaddition of 0.02 m substrate. Gluconate-U-'4C andgluconate-1-14C were added at a concentration of 1.50,Ac per ml of culture; glucose-U-_4C and glucose-1_14Cwere added at a level of 1.0 j.c per ml ofculture. Cellswere incubated anaerobically for 3 hr at 37 C. Growthwas stopped by acidification with H2S04 and a mixtureof carrier acids at 4 C, and cells were removed bycentrifugation. The remaining supernatant fractionswere chromatographed on silicic acid columns as de-scribed in Materials and Methods. In each case, the 14Cin the supernatant fractions could be accounted for asend products elutedfrom the silicic acid columns or 14Cpresent as unused substrate. (A) Glucose-U-14C; (B)glucose-i-14C; (C) gluconate-U-14C; (D) gluconate-1_14C.

cleavage of gluconate via an ED pathway as seenby (i) absence of 14C-acetate in both aerobic andanaerobic gluconate-1-14C end products and (ii)high levels of 14C-formate as an end product ofanaerobic gluconate-1_14C catabolism.At this point in the investigation, the evidence

for an ED system in E. coli ML30 centered aroundthe assumption that pyruvic acid was producedfrom the C-1, C-2, and C-3 positions of gluconateand that the carboxyl group of gluconate wasretained as the carboxyl group of this pyruvate.In this regard, it was desirable to determine thedistribution of label in pyruvate formed fromgluconate-1-14C, glucose-J-14C, and glucose-3,4-14C. The results of these experiments are sum-marized in Table 3. In these experiments, E. coliwas grown under the various conditions indicated

in the table. Cells were harvested, washed in 0.05Iuccnate M sodium phosphate buffer(pH 7.0), and suppliedwith appropriately labeled substrate in the pres-ence of 2 mm sodium arsenite and the same buffer.After 1 hr of incubation, the reaction mixtureswere acidified and the supernatant solutions werechromatographed on silicic acid columns. Pyruvicacid was extracted from these chromatographiceluates and degraded with ceric sulfate to acetate

< and CO2. The 14CO2 and the supernatant fractionscontaining acetate-14C were collected and counted.These counts were calculated as the percentageof total counts added to the ceric sulfate reactionmixtures. Commercial preparations of pyruvate--J14C, -2-14C, and -3-14C were chromatographed,extracted, and degraded under conditions iden-tical to those employed with the metabolicallyderived pyruvate-14C as described in Materials

70 80 and Methods.

TABLE 2. Distributionz and recovery of 14C aftergrowth of Escherichia coli ML30 on gluconate

Aerobic Anaerobicgrowtha growtha

DeterminationGluco- Gluco- Gluco- Gluco-nate- nate- nate- nate-U_C14 I_J4C U-14C 1JJ4

Cells ................ 9.8 2.1 5.6 1.1CO2 ................ 10.1 23.3 2.5 10.0Supernatant fluid ..... 74.6 70.4 86.9 84.8Recovery (%)b...... 94.5 95.8 95.0 95.9

Supernatant analysiscGluconate.......... 82.1 92.2 34.1 29.6Ethyl alcohol -.6.3Acetate... 15.1 - 36.6Pyruvate ........... 2.4 1.5Formate .. 11.9 41.7Lactate ....... 4.8 5.3Succinate ...... 4.8 1.2Recovery (%) 99.6 93.7 98.5 77.8d

a Cells were grown in medium containing 0.02 Mgluconate and 0.5 uc of 14C-gluconate per mlIncubations were in radiorespirometer flasks at37 C with continuous flow of compressed air foraerobic conditions or 5% C02-95% N2 for anaer-obic conditions.

b Values presented are percentage of countsadded as gluconate-14C.

c Values listed are percentage of counts foundin the culture supernatant fraction. Gluconatecounts were determined as unused substrate.

d Only those counts in the formate, lactate, andsuccinate region could be identified when anaer-obic gluconate-1_14C supernatant fluids werechromatographed (see Fig. 2). Low levels ofradioactivity appearing in the ethyl alcohol-acetate-pyruvate elution region were poorlyresolved and were therefore not calculated. Thisaccounted for the low recovery in this experiment.

0=20FBIUE.a oBM.L .[Uo.

a-C.)I

0

944 J. BACTERioL.

on January 17, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

GLUCONATE METABOLISM IN E. COLI

TABLE 3. Characterization ofpyruvate-14Cproduced from specifically labeled

substrates"

14C distribution14C-labeled in pyruvate"Expt substrates

CO2 Acetate Total

1 Gluconate-1-14C 89.9 5.8 95.7Glucose-1-14C 7.3 86.3 93.6Glucose-3,4-14C 86.1 10.6 96.7

2 Glucose-1-'4C 5.9 91.2 97.1Glucose-3,4-14C 86.4 10.5 96.9

3 Gluconate-1-14C 87.5 5.6 93.1+ C12-glucose

Glucose-1-14C + 7.5 84.5 92.0CQ2-gluconate

4 Gluconate-1-14C 90.9 3.6 94.5

Stand- Pyruvate-1-14C 85.9 9.4 95.3ards Pyruvate-2-14C 9.0 90.5 99.5

Pyruvate-3-14C 4.0 96.6 100.6

a In all experiments, E. coli ML30 was grownand incubated under aerobic conditions. Thegrowth substrates were (0.02 M): gluconate (ex-periment 1), glucose (experiment 2), and glucoseplus gluconate (experiment 3). In experiments 1and 2, cells were grown in basal medium, washed,and resuspended to 2 mg (dry weight) per ml in0.05 M phosphate buffer (pH 7.0) containing 2 mmNaAsO2. Cells were incubated in the presence ofarsenite for 15 min before adding substrate to0.833 uc/ml (specific activity was 0.167 ,uc/,Amole).Total volume was 6.0 ml at 37 C. After 1 hr ofincubation, suspensions were acidified and ac-cumulated pyruvate, about 5 ;&moles/ml, was ex-tracted. Experiment 3 was performed as aboveexcept that 8 ,umoles of each substrate per ml wasadded (5 ,uc of glucose and 10 pc of gluconate),and incubation was for 75 min before acidificationand extraction of pyruvate (ca. 7 umoles/ml). Inexperiment 4, the culture was grown aerobically inmedium containing gluconate-1-14C and caseinhydrolysate. Under these conditions, pyruvate ac-cumulated in the medium and was isolated di-rectly from the medium. Conditions for isolationand degradation of accumulated pyruvate-14C aredescribed in Materials and Methods.

bDistribution (%) of 14C in pyruvate deter-mined by ceric sulfate oxidation of pyruvate toCO2 and acetate. The "4C-pyruvate in each casewas formed from the indicated "4C-substrates.

It was shown that carboxyl-labeled pyruvate isproduced from (i) gluconate-grown cells providedwith gluconate-1-'4C, (ii) glucose-grown cellsgiven glucose-3, 4- C, and (iii) cells grown on acombination of glucose and gluconate suppliedwith gluconate-1-14C. Pyruvate isolated from anaerobic culture growing in medium containingcasein hydrolysate and gluconate-1-'4C was also

labeled in the carboxyl position. These labelingstudies combined with the data on end productsdemonstrated that gluconate metabolism in E.coli does in fact involve an inducible ED pathway.The following enzyme studies provided additionalevidence along this line.

Occurrence of ED pathway enzymes in E. coli.Cell-free extracts of E. coli ML30 prepared fromcultures grown on glucose and gluconate wereprepared by sonic disruption. These preparationswere tested for their ability to convert 6-phospho-gluconate and KDPG to pyruvate. As seen inFig. 2, the accumulation of pyruvate from 6-phosphogluconate occurred much more readilywith extracts of gluconate-grown cells than withglucose-grown cells. Omission of reduced gluta-thione and FeSO4 from the reaction mixtureresulted in complete loss of activity, althoughslight activity was found when only FeSO4 waspresent. Comparison of the specific activities(micromoles of pyruvate produced per minuteper milligram of protein) obtained in the 6-phos-phogluconate assay and the KDPG assay showedthat the dehydrase enzyme was rate-limiting. Thisrelationship was also seen from the data pre-sented in Table 5. When gluconate, Mg, andexcess ATP were substituted for 6-phospho-gluconate and the reaction was allowed to go tocompletion, 1 ,umole of pyruvate was producedfor every micromole of gluconate consumed.When gluconate-1-14C was added and the pyr-uvate-14C formed was degraded and character-ized as described previously (Table 3), 83% ofpyruvate-'4C counts were found in the CO2 frac-tion. These data are in agreement with thosedescribed by Kovachevich and Wood (15, 16) forglucose-grown cells of P. fluorescens.Although extracts of E. coli ML30 accumulated

pyruvate from 6-phosphogluconate at a ratedependent on protein concentration, this rate wasnot a direct function of the amount of extractadded. This lack of complete proportionality wasfound in several different preparations, and allexperiments reported were thus conducted underconditions of comparative protein concentrations.Extracts of E. coli ML30 cells grown on varioussubstrates were prepared and tested for theirability to convert 6-phosphogluconate to pyr-uvate. The data in Table 4 showed that cellsgrown on glucose, lactate, glycerol, and succinatedid not contain appreciable amounts ofED path-way enzymes and again indicated the induciblenature of the ED mechanism when gluconateserved as growth substrate.At this stage of the investigation, information

was sought concerning the existence of ED path-way enzyme activities in other strains of E. coli

VOL. 93, 1967 945

on January 17, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

EISENBERG AND DOBROGOSZ

and in other representative strains of gram-nega-tive bacteria. Such information would establishwhether induction of ED pathway enzymes bygluconate in E. coli is a general phenomenon.

6r .

'

c04-0

a

w

cc

E

E

0LL

4O.-

CL

0~

0)

E

5

4

3

2

2

0

A

1 2Minutes

FIG. 2. 6-Phosphogluconate dehydrase and KDPGaldolase in extracts of Escherichia coli ML30. (A)6-Phosphogluconate dehydrase. Curve 1, extract fromgluconate-grown cells; curve 2, extract from gluconate-grown cells with FeSO4 and glutathione omitted fromthe reaction mixture; curve 3, extract from glucose-grown cells. The reaction mixture contained extract (3mg of protein per ml); Tris buffer (pH 7.65), 200pmoles; reduced glutathione, 3 pmoles; FeSO4, 6pmoles. After 10 min ofincubation at 37 C, 7 pmoles of6-phosphogluconate was added to start the reaction,and pyruvate formation was measured. The total volumeofthe reaction mixture was 1.0 ml. (B) KDPG aldolase.Curve 1, extract from gluconate-grown cells; curve 2,extract from glucose-grown cells. The reaction mixture(1 ml total volume) contained KDPG, 5 pmoles; Trisbuffer (pH 7.65), 200 pmoles; extract, 0.5 mg ofproteinper ml. Incubation was at 37 C, and pyruvate forma-tion was measured after adition of substrate.

TABLE 4. Effect ofgrowth substrate on conversionof 6-phosphogluconate to pyruvate in cell-free

extracts of Escherichia coli ML30Growth substrate Pyruvate formed per mg

(0.02 mi) of protein per miinAmo16S

Gluconate ................ 0.13Glucose.................... 0.02Lactate.................... 0.01Glycerol.................... 0.01Succinate................... 0.01

a The assay employed was the same as describedin Fig. 3 with 6-phosphogluconate except thatreaction mixtures containing 2 mg of protein perml were used. Cells were grown aerobically, andextract was prepared with a French pressure cell.

Comparison of E. coli with other bacterial species,such as Pseudomonas which contains both dehy-drase and aldolase activities when grown onglucose, could provide a basis for better under-standing of gluconate catabolism in E. coli. Theresults of experiments designed to explore thesepossibilities are presented in Table 5. The abilityof gluconate to induce 6-phosphogluconatedehydrase and KDPG aldolase in all E. colistrains tested was apparent. In other experiments(not shown), various E. coli B derivatives grownon gluconate were also found to contain theseenzymes. In addition, these enzymes were shownto be present in either aerobically or anaerobicallygrown cultures. In contrast, P. fluorescens con-tained approximately equivalent levels of theseenzymes when grown in the presence of eitherglucose or gluconate. The level of these enzymesin extracts of P. fluorescens grown only on caseinhydrolysate is comparable to that found in E. coligrown in glucose medium, i.e., noninduced levels.Separate experiments (not shown) with P.aeruginosa were consistent with those obtainedfor P. fluorescens; i.e., the dehydrase enyme waspresent in glucose- and gluconate-grown cells butnot in cells grown on casein hydrolysate alone.The data in Table 5 also showed that growth ongluconate, but not glucose, resulted in inductionof ED pathway enzymes in Enterobacter (Aero-bacter) aerogenes and Salmonella typhimurium.This pathway does not appear to be induced bygrowth on gluconate in Erwinia carotovora orSerratia marcescens. The presence of high levelsof KDPG aldolase in Serratia suggested that anonphosphorylated route leading to KDG (1)may exist for catabolism of gluconate in thisspecies. When extracts of gluconate-grown Ser-ratia species were tested for ability to accumulatepyruvate from gluconate, in the presence of ATP,Mg++, and components of the 6-phospho-

946 J. BAcrERIO)L.

I

I

on January 17, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

GLUCONATE METABOLISM IN E. COLI

TABLE 5. Comparison of 6-phosphogluconatedehydrase and KDPG aldolase activities inEscherichia coli and selected gram-negativeorganisms grown on glucose and gluconate

Pyruvate formedper mmper%mgof proti

Organism Growthsubstrate5 6-Pho-

sphoglu- KDPGconate aldo-dehy- lasedrase

pmoles pAmoles

Pseudomonas fluo- Glucose 0.34 0.99rescens ATCC Gluconate 0.38 0.7613525 No additions 0.02 0.15

P.fluorescens, FS Glucose 0.18 0.25Gluconate 0.23 0.21No additions 0.00 0.04

E. coli ML30 Glucose 0.00 0.53Gluconate 0.78 1.64

E. coli ATCC 11303 Glucose 0.02 0.39Gluconate 0.40 0.76

E. coli ATCC 8739 Glucose 0.02 0.54Gluconate 0.47 1.17

Enterobacter aero- Glucose 0.11 0.92genes ATCC Gluconate 0.85 1.8812658

E. aerogenes ATCC Glucose 0.02 0.388724 Gluconate 0.73 1.31

Salmonella typhi- Glucose 0.01 0.79murium LT-2 Gluconate 0.87 1.60

Erwinia carotovora Glucose 0.00 0.53ATCC 138 Gluconate 0.00 0.26

Serratia marcescens Glucose 0.00 0.12ATCC 13880 Gluconate 0.04 0.53

S. marcescens, FS Glucose 0.00 0.23Gluconate 0.03 0.94

The medium in each case contained 0.25%casein hydrolysate, to which the indicated sub-strates were added at a concentration of 0.02 M.

b One-ml assays performed as described inFig. 4 except that approximately 2.5 pmoles ofKDPG was added per ml of reaction mixture inthe KDPG aldolase assay. Amounts of 3 mg ofprotein per ml and 0.5 mg of protein per ml wasemployed for 6-phosphogluconate and KDPGassays, respectively. Activity was determinedfrom the amount of pyruvate accumulated be-tween 1 and 2 min of incubation.

gluconate reaction mixture, no activity was found.Extracts of gluconate-grown E. coli readily con-verted gluconate to pyruvate under these condi-tions.

DIscussIoNThrough use of radioisotope experiments and

enzyme studies, we have demonstrated that afunctional ED pathway is specifically induced

in E. coli ML30 during growth on gluconate.These findings, summarized in scheme 1, are con-sistent with those of Horecker (12), who describedin S. typhimurium an inducible ED pathway thatwas the major route of carbon flow from gluco-nate with the pentose phosphate cycle servingonly as a source of precursors for biosyntheticreactions. In the present study, high levels ofgluconate-induced, ED pathway enzymes werefound in E. coli, E. aerogenes, and S. typhimurium.These results indicate a wide-spread role for theED system in gluconate catabolism by theseclosely related members of the Enterobacteriaceae.Evidence has recently been provided, however,that the ED system in these bacteria need notalways be inducible. Loomis and Magasanik (19)recently found constitutive ED activity in theirstrains of E. coli. It should be noted, however,that these authors did not employ Fe++ andglutathione in their assay system. These compo-nents were found necessary for optimal activityin our E. coli extracts. On this basis it is clearthat further studies are needed before decidingwhether this system is generally an inducible ora constitutive pathway in E. coli.The ED pathway is generally considered to be

a constitutive system of carbohydrate metabolismin many pseudomonads such as P. fluorescens.This was observed to be the case in the presentstudy when P. fluorescens was grown in mediumcontaining glucose or gluconate (Table 5). Whenthese substrates were omitted from the medium,however, and the cultures were grown only in thepresence of casein hydrolysate, the 6-phospho-gluconate dehydrase concentration in P. fluores-cens dropped to a basal level. This would indicatethat the ED system is not constitutive in these or-ganisms, but rather that a supply of inducer suchas gluconate is readily formed during catabolismof various carbobydrate substrates. It is likelythat gluconate or a closely related analogue is thespecific inducer of the ED system. Kovachevichand Wood (15) have postulated that gluconate isan intermediate in glucose metabolism via the EDsystem in P. fluorescens. Gluconate and 2-keto-gluconate are known to accumulate during glu-cose dissimilation in a variety of pseudomonadsand related bacteria (3, 13, 14, 18). In addition,growth on glucose leads to a sequential inductionof enzymes involved in dissimilation of gluconateand 2-ketogluconate (10, 13, 21). These findingsprovide some foundation for explaining inductionof the ED system in the Escherichia-Enterobacter-Salmonella group. It can be viewed that theselatter organisms are capable of only limited endog-enous production of an inducer such asgluconate from other carbohydrate substrates.

947VOL. 93, 1967

on January 17, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

EISENBERG AND DOBROGOSZ

Major carbon flow

Gluconate

I6-Phosphogluconate

Minor carbon flow

Entner-Doudoroffpathway

2 Keto-3-deoxy-6-phosphogluconate

19Triose-phosphate +

Embde,

pyruvate

r-Meyerhof

Tricarboxylic acid.L-- __-- cycle

Pentose-phosphatecycle

Ribulose-5-phosphate + C02

1Synthesis of required

intermediates

PathwayScHEtm 1. Summary ofpathways ofgluconate metabolism in Escherichia colt.

Thus, the ED system is repressed until the cellsare exposed directly to gluconate.

It has been known for some time (16) thatcultures of E. coli grown on glucose contain a lowlevel ofED activity despite the fact that glucose ismetabolized primarily by glycolytic reactions withsome dissimilation occurring via the oxidativebexose monophosphate shunt. This point was em-phasized in the present study by the finding of alow but usually detectable level of 6-phospho-gluconate dehydrase in glucose-grown cells (Table4), and by the finding that low levels of 14C-for-mate accumulated during anaerobic growth onglucose-4-'IC (Fig. 1). This basal level of EDpathway activity in glucose-grown cells could beexplained by the production or maintenance oflow levels of gluconate during glucose metabolismvia glucose dehydrogenase activity (Table 1).With gluconate-induced cultures, glucose and

gluconate are degraded simultaneously and inde-pendently by both the ED and the glycolytic path-ways (Table 3). A similar situation was describedfor gluconate-grown cultures of S. typhimurium(9) and was explained on the basis ofa low affinityof 6-phosphogluconate dehydrase for 6-phospho-gluconate and a much greater activity of glucono-

kinase than of glucose-6-phosphate dehydrogen-ase, gluconate therefore being more readilyconverted to 6-phosphogluconate than glucose.Another possibility in this regard would be thatthese two systems are compartmentalized intospatially discrete complexes.

LrrATURE Cr1. ASHWELL, G., A. J. WAHBA, ANm J. HicKMAN.

1960. Uronic acid metabolism in bacteria. I.Purification and properties of uronic acid isom-erase in Escherichia coli. J. Biol. Chem. 235:1559-1565.

2. BLACKWOOD, A. C., A. C. NEISH, AND G. A.LEDINGHAM. 1956. Dissimilation of glucose atcontrolled pH values by pigmented and non-pigmented strains of Escherichia coli. J. Bac-teriol. 72:497-499.

3. CAMPBELL, J. J. R., F. C. NORRIS, ANm M. E.NORRIS. 1949. The intermediate metabolism ofPseudomonas aeruginosa. H. Limitations ofsimultaneous adaptation as applied to theidentification of acetic acid, an intermediate inglucose oxidation. Can. J. Res. Sect. C 27:165-171.

4. COHEN, S. S. 1951. Utilization of gluconate andglucose in growing and virus-infected Escher-ichia coli. Nature 168:746-747.

948 J. BACTERIOL.

Im-

on January 17, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

GLUCONATE METABOLISM IN E. COLI

5. COHEN, S. S., AND D. B. M. Scorr. 1950. Glu-conokinase and the oxidative path for glucose-6-phosphate utilization. Nature 166:781-782.

6. DE LEY, J. 1962. Comparative biochemistry andenzymology in bacterial classification. Symp.Soc. Gen. Microbiol. 12:164-195.

7. DOBROGOSZ, W. J. 1966. Altered end-productpatterns and catabolite repression in Escher-ichia coli. J. Bacteriol. 91:2263-2269.

8. EAGON, R. G., AND C. H. WANG. 1962. Dissimila-tion of glucose and gluconic acid by Pseudo-monas natriegens. J. Bacteriol. 83:879-886.

9. FRAENKEL, D. G., ANm B. L. HORECKER. 1964.Pathways of D-glucose metabolism in Sal-monella typhimurium. A study of a mutantlacking phosphoglucose isomerase. J. Biol.Chem. 239:2765-2771.

10. FRAMprON, E. W., AND W. A. WOOD. 1961. Carbo-hydrate oxidation by Pseudomonas fluorescens.VI. Conversion of 2-keto-6-phosphogluconateto pyruvate. J. Biol. Chem. 236:2571-2577.

11. FRiEDEMANN, T. E., ANm G. H. HAUGEN. 1943.Pyruvic acid. II. The determination of ketoacids in blood and urine. J. Biol. Chem. 147:415-442.

12. HORECKER, B. L. 1965. Pathways of carbohydratemetabolism and their physiological significance.J. Chem. Educ. 42:244-253.

13. Krros, P. A., C. H. WANG, B. A. MoHLER, T. E.KING, AND V. H. CHELDELIN. 1958. Glucose andgluconate dissimilation in Acetobacter sub-oxydans. J. Biol. Chem. 233:1295-1298.

14. KOESPELL, H. J., F. H. STODOLA, AND E. S.SHARPE. 1952. Production of alpha-ketoglu-tarate in glucose oxidation by Pseudomonasfluorescens. J. Am. Chem. Soc. 74:5142-5144.

15. KOVACHEVICH, R., Am W. A. WOOD. 1955.Carbohydrate metabolism by Pseudomonasfluorescens. IHI. Purification and properties of a6-phosphogluconate dehydrase. J. Biol. Chem.213:745-756.

16. KOVACHEVICH, R., AND W. A. WOOD. 1955.Carbohydrate metabolism by Pseudomonasfluorescens. IV. Purification and properties of2-keto-3-deoxy-6-phosphogluconate aldolase. J.Biol. Chem. 213:757-767.

17. KREBS, H. A., AND W. A. JOHNSON. 1937. Metabo-lism of ketonic acids in animal tissues. Biochem.J. 31:645-660.

18. LOCKWOOD, L. B., B. TABENKIN, Am G. F. WARD.1941. The production of gluconic acid and 2-ketogluconic acid from glucose by species ofPseudomonas and Phytomonas. J. Bacteriol.42:51-61.

19. LOOMIS, W. F., AND B. MAGAsANIK. 1966. Natureof the effector of catabolite repression of P-galactosidase in Escherichia coli. J. Bacteriol.92:170-177.

20. LOWERY, 0. H., N. J. ROSEBROUGH, A. L. FARR,AND R. J. RANDALL. 1951. Protein measure-ment with the Folin phenol reagent. J. Biol.Chem. 193:265-275.

21. NARROD, S. A., AND W. A. WOOD. 1956. Carbo-hydrate oxidation by Pseudomonasfluorescens.V. Evidence for gluconokinase and 2-keto-gluconokinase. J. Biol. Chem. 220:45-55.

22. NELSON, N. 1944. A photometric adaptation ofthe Somogyi Method for the determination ofglucose. J. Biol. Chem. 153:375-380.

23. OKINAKA, R. T., AND W. J. DOBROGOSZ. 1966.Enhanced catabolite repression in Escherichiacoli by growth on combined substrates. J.Bacteriol. 92:526-527.

24. Scorr, D. B. M. 1956. The oxidative pathway ofcarbohydrate metabolism in Escherichia coli.3. Glucose-6-phosphate dehydrogenase and6-phosphogluconate dehydrogenase in cellsgrown under different conditions. Biochem. J.63:587-593.

25. SOKATCH, J. T., AND I. C. GuNsALus. 1957. Al-donic acid metabolism. I. Pathway of carbonin an inducible gluconate fermentation byStreptococcusfaecalis. J. Bacteriol. 73:452-460.

26. STOKEs, J. L. 1949. Fermentation of glucose bysuspensions of Escherichia coli. J. Bacteriol.57:147-158.

27. WANG, C. H., I. STERN, C. M. GnmouR, S.KLUNGSOYR, D. J. REED, J. J. BIALY, B. E.CHRISrENsEN, Am V. H. CHELDELIN. 1958.Comparative study of glucose catabolism by theradiorespirometric method. J. Bacteriol. 76:207-216.

949VOL. 93, 1967

on January 17, 2020 by guesthttp://jb.asm

.org/D

ownloaded from