JOURNAL OF BACTERIOLOGY, June, 1966Copyright @ 1966 American Society for Microbiology

Vol. 91, No. 6Printed in U.S.A.

Carbohydrate Catabolism of Mima polymorphaII. Abortive Catabolism of GlucoseADRIENNE MARUS AND EMILY J. BELL

Department of Biological 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 ofglucose. J. Bacteriol. 91:2229-2236. 1966.-Mima polymorpha, unable to grow inthe 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. Variousenzymes of hexose catabolism have been assayed in this organism and in M. poly-morpha M, a mutant obtained by ultraviolet irradiation. The parent strain containsa functional glucose dehydrogenase, glucose-6-phosphate dehydrogenase, diphos-phofructoaldolase, and a 2-keto-3-deoxy-6-phosphogluconate aldolase, but islacking in glucokinase, gluconokinase, 2-ketogluconokinase, and 6-phospho-gluconate dehydrogenase. The enzymes present indicate partially functioning hexosediphosphate and Entner-Doudoroff pathways. The absence of kinases explains theinability of the strain to grow on glucose and an absence of 6-phosphogluconatedehydrogenase would indicate the absence of the complete pentose pathway. Themutant strain, M. polymorpha M, possesses, in addition to those enzymes producedby the wild type, both gluconokinase and 6-phosphogluconate dehydrogenase. Thepresence of the former explains the mutant's ability to grow on glucose, and thepresence of the latter indicates a more complete pentose shunt. The supplementalenergy obtained from partial glucose catabolism (to gluconic acid) may be obtainedfrom a cytochrome-linked reaction of the glucose dehydrogenase.

Although Mima polymorpha is unable toutilize glucose as a sole carbon and energy source,it has been shown (1) that an energy-linkedpartial catabolism of this substrate must occur,as the addition of supplemental glucose to agrowth medium results in a greater rate ofgrowth and a larger cell crop, an induction oftransport mechanisms for slightly permeablephosphorylated intermediates, and a more rapidinduction to a higher level of specific activity ofan inducible enzyme, isocitrate lyase E.C. 4.1.3.1.(isocitritase). The purpose of the present studywas to survey various hexose catabolic enzymesin an attempt to locate the metabolic lesion(s)occurring in the strain. A glucose-utilizing mutantwas studied also so that comparative assays mighthelp to pinpoint the catabolic pathways fol-lowed.

MATERIALS AND METHODS

M. polymorpha ATCC 9957 was maintained andcultured as described previously (1). The mutantstrain, designated M. polymorpha M was obtained in

the following manner. A suspension of acetate-grown,wild-type cells was spread over agar plates preparedfrom the mineral salts medium with 0.03 M glucoseincorporated as sole carbon and energy source. Theplates were exposed to ultraviolet irradiation for 10or 15 sec from a germicidal lamp (Precision ScientificCo., Chicago, IlJ.) which emits radiation from 2,500A and peaks at 3,660 A. A colony was obtained froma plate which had been exposed for 10 sec and inocu-lated into minimal salts medium containing eitherglucose or fructose as sole carbon and energy source.It was found that good growth occurs on either of thehexoses, as well as on acetate. This mutant strain hasbeen maintained on a synthetic mineral salts, glucosesemisolid medium.

Since some of the enzymes of glucose catabolismare inducible, M. polymorpha was grown routinelyfor all assays in the basal salts medium supplementedwith 0.03 M acetate and 0.03 M glucose. It should bepointed out, however, that, with the exception of theisocitrate lyase studies (1), possible metabolic controleffects of double substrate growth have not beenelucidated for this organism. When used, M. poly-morpha M was grown on the same medium, to whichglucose alone was added. Growth conditions of the

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MARUS AND BELL

control organisms will be designated where appro-priate.Growth was followed by periodic optical-density

determinations in a Klett-Summerson photoelectriccolorimeter. The organisms were grown in Beliconepheloflasks so that frequent turbidimetric readingscould be made, and were incubated with shaking in a

New Brunswick incubator shaker, model G 26, at28 C and 250 rev/min. Cell-free extracts for all en-

zyme analyses were prepared by disruption of twice-washed cells in a Raytheon 9-kc, 50-w sonic oscillatorat 150-v output for 15 min. Cell debris was removedby centrifugation for 10 min at 10,000 X g in thecold. Protein content was measured by the procedureof Lowry et al. (15). Most of the major reagents wereobtained from the sources listed previously (1). Inaddition, nicotinamide adenine dinucleotide (NAD)and adenosine triphosphate (ATP) were obtainedfrom Mann Research Laboratories, New York, N.Y.;lactic dehydrogenase, glucose-6-phosphate dehy-drogenase, and reduced nicotinamide adenine dinu-cleotide (NADH2), from Nutritional BiochemicalsCorp., Cleveland, Ohio; and nicotinamide adeninedinucleotide phosphate (NADP) and glyceraldehyde-3-phosphate dehydrogenase, from Sigma ChemicalCo., St. Louis, Mo. Enzyme assays were carried outby use of either a Gilford model 2000 recording spec-trophotometer or a Bronwill Warburg apparatus. Theassays performed will be described as used. Intra-cellular gluconic acid was detected, after deproteiniza-tion of a sonic extract and concentration, by thechromatographic method of Norris and Campbell(18).

REsULTS

Since M. polymorpha is unable to grow on

nonphosphorylated sugars, the first enzymeassayed was glucokinase (hexokinase). The firstmethod employed was the manometric pro-cedure of Colowick and Kalckar (3) in whichcarbon dioxide evolution from bicarbonate in thepresence of ATP is measured. The controlorganism, Escherichia coli, was grown on theusual basal medium to which 0.03 M glucose was

added. After 20 hr of incubation, both M. poly-morpha and E. coli were harvested, washed, andsonically treated. The results in Table 1 show thatthe amount of CO2 released from bicarbonate inthe presence of glucose and ATP by the cell-freeextract of M. polyinorpha is not significantlyhigher than the amount released in the presenceof ATP alone. High adenosine triphosphataseactivity in cell-free extracts of certain micro-organisms has been a difficulty noted by otherinvestigators using this method (9, 21). Theamount of CO2 released, however, in the presenceof glucose plus ATP by the sonic extract of E. coliis much greater than that produced in any of theother flasks, indicating the presence of an activehexokinase in that organism. These data wereconsidered to be an indication of the absence of

TABLE 1. Manometric hexokinase assay*

Total,umoles of C02 released

after Mima polymorpha Escherichia colitipping extract extract(min)

+ ATP + ATP + ATP + ATP+ glucose + glucose

5 1.1 1.5 3.0 2.010 6.0 7.0 5.2 6.320 7.0 7.0 5.3 8.4

* The vessels contained in a total of 2.2 ml: 40,moles of NaHCO3 (pH 7.5), 10,umoles of MgCl2,and 1.5 ml of sonic extract (M. polymorpha, 3.9mg of protein/ml; E. coli, 3.8 mg of protein/ml).ATP, 20,uM, and 16.6 Mm glucose were added fromthe side arm. Incubation was at 30 C in an atmo-sphere of N2. The total amount of CO2 released inan endogenous control vessel for M. polymorphawas 1.6, and for E. coli was 1.1. The total amountof CO2 released in a glucose vessel without ATPwas 1.3 ,umoles for M. polymorpha and 1.9 ,molesfor E. coli.

hexokinase in M. polymorpha. To confirm this, aspectrophotometric assay (16) was performed.M. polymorpha M, grown in the presence ofglucose, was tested also. This method determinesthe ability of the cell to form glucose-6-phosphate(G6P) by measuring NADP reduction in thepresence of added G6P dehydrogenase. Opticaldensity changes, recorded at 340 mI,, are pre-sented in Fig. 1. The data confirm the absence ofhexokinase activity in cell-free extracts of M.polymorpha as well its absence in extracts of themutant organism, at least under these experi-mental conditions.

Since the organism is unable to phosphorylateglucose but yet can obtain energy from it, it wasassumed that a direct oxidation might occur withthe formation of gluconic acid or 2-ketogluconicacid. Accordingly cell-free extracts were assayedfor glucose dehydrogenase activity by comparingglucose disappearance (by the Glucostat method)in the presence of an enzyme preparation addedwith and without ATP (16). The control organismHerellea vaginicola, was grown in the presence ofacetate and glucose, and M. polymorpha and theM mutant were grown in the usual media. Allorganisms were incubated for 18 hr and harvested.To insure that no residual glucose from thegrowth media remained adsorbed to the cells, thesupernatant fluids from the washed cells wereassayed for glucose, until none could be detected.Also, in the assay itself, one tube was includedas an endogenous intracellular glucose control foreach organism. The results (Table 2) indicate that

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CARBOHYDRATE CATABOLISM OF MIMA POLYMORPHA. II

1.5 -

1.0-

0.5 -

0

E. coli

M. polymorpha Mla la

M. polymorpha

blankIn _ - _-A

0 6 12 18 i24TIME (min.)

FIG. 1. Hexokinase assay in sonic extracts ofMima polymorpha and M. polymorpha M, and thecontrol, Escherichia coli. The reaction mixture con-tained: 1.0 ml of cell-free extract (1.5, 1.7, and 0.9mg of protein per ml, respectively), 0.8 ,umoles ofNADP, 0.06 mg ofglucose-6-phosphate dehydrogenase,5.0 ,umoles ofATP, 0.5 ,umoles of MgCl2, 10.0 ,umolesof glucose, and 0.2% NaHCO3 buffer (pH 7.5) to3.0 ml. The reduction ofNADP was followed spectro-photometrically at 340 m,u at a temperature of28 C.

both M. polymorpha and the mutant, M. poly-morpha M, contain glucose dehydrogenaseactivity. Further, since the disappearance ofglucose is not enhanced by the addition of ATP(as it is in the case of H. vaginicola), these datarepresent further confirmation of the absence ofglucokinase activity in these organisms. Mano-metric experiments support the evidence for aglucose oxidase system. In the presence of glu-cose, both whole cell suspensions and sonicextracts consistently consume more oxygen(about 40%) than do endogenously respiringcontrols. Chromatrographic evidence also sup-ports the formation of gluconic acid. A spotmigrating with known gluconic acid was ob-served. No 2-ketogluconate could be detected.

Since M. polymorpha may oxidize glucosedirectly, its inability to grow in the presence ofthis substrate must depend, then, not only upon

TABLE 2. Glucose dehydrogenase activity in cell-free extracts of Mima polymorpha, M.

polymorpha M, and Herelleavaginicola*

Unitst of enzyme activity/mg ofprotein

Extract from With ATP Without Differ(hexoki- ATP Difrnaae + (guoe enceglucose dgehcase (hexoki-dehydro- dehydro- naae)genaae) gea)

M. polymorpha ...... 0.002 0.002 0.0M. polymorpha M... 0.003 0.003 0.0H. vaginicola ........ 0.006 0.005 0.001

* The reaction mixture contained in a total of2.0 ml: 100 ,uM phosphate buffer (pH 6.5), 1.1,umoles of glucose, 1.0 ,M MgSO4, and 0.2 ml ofsonic extract. ATP was added (1.0 umole) whereindicated. The amount of glucose at time zero andafter 20 min of incubation at 28 C with shaking wasdetermined by the Glucostat method.

t One unit causes the disappearance of 1 ,umoleof glucose per min.

the absence of glucokinase, but also upon theabsence of enzymes functioning in the dissimila-tion of gluconate. Gluconokinase was assayed bythe manometric method of Colowick and Kalckar(3). Since M. polymorpha M is able to grow in thepresence of glucose as sole carbon and energysource, in the absence of glucokinase, the organ-ism must be able to phosphorylate an aldonicacid and, therefore, was tested also. The controlorganism selected, Pseudomonas fluorescens, wasgrown in the basal salts medium containing 0.03M gluconic acid. The data obtained for M. poly-morpha and P. fluorescens are presented in table3. Because of apparent high ATP-splitting activityin the sonic extract of M. polymorpha, the dataare hard to interpret. However, the amount ofCO2 released in the presence of both gluconateand ATP by enzymes in the Pseudomonas extractis higher than in the control vessels withoutgluconate. Therefore, the absence of stimulatedCO2 release by M. polymorpha in the presence ofboth ATP and gluconate is considered to beadditional evidence for the absence of thisenzyme. The experiment was repeated with theuse of varying amounts of the enzyme prepara-tion; however, no kinase activity was detected.M. polymorpha M, which is able to grow well

with gluconic acid as the sole carbon and energysource, was assayed for enzyme activity by thesame method after 24 hr of growth in the presenceof this substrate. The results (Fig. 2) indicate anactive gluconokinase in this organism, and mayexplain the ability of the mutant, in contradistinc-

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2MARUS AND BELL

TABLE 3. Manometric gluconokinase,

Total pmoles of C02 release

after Mima polymorpha Pseudomonatipping extract ext(min)

+ ATP +gluconate + ATP

10 4.0 3.1 3.315 4.5 3.2 3.620 5.0 3.3 3.825 3.6 4.030 3.6 4.235 3.7 4.3

* The vessels contained in a total ofAmoles of NaHCO3 (pH 7.5), 10 /AM MgCml of sonic extract (M. polymorpha,protein/ml; P. fluorescens, 3.9 mg of pr4ATP, 20 ,umoles, and 20 sAmoles of gluceadded from the side arm. Incubation wEin an atmosphere of nitrogen.

2.7

2.1

G luconate

0 10 20 30 40TIME (min.)

FIG. 2. Manometric determination ofgluoactivity in a sonic extract of Mima polynThe Warburg vessels contained in a total of,umoles NaHCO3 (pH 7.5), 10 jAmoles of A1.5 ml of sonic extract containing 2.7 mgper ml. Where indicated 20 ,umoles of A],umoles of potassium gluconate were addedioxide production was measured at 30 C inphere ofnitrogen.

assay* tion to the parent organism, to grow in thepresence of gluconate as carbon and energysource. At the present time, no reason can begiven for the negligible adenosine triphosphatase

'ract activity in the mutant in contrast to the very highactivity in the wild type. Assays for 2-ketogluco-

+ ATP nokinase were performed with preparations from+ gluconate both the wild-type and mutant organisms, but no

3.9 activity could ever be demonstrated.415 The data presented can explain, then, the4.7 inability of M. polymorpha to grow in the presence5.2 of glucose as sole carbon and energy source. The5.6 organism, however, can grow in the presence of5.9 various phosphorylated intermediates (1) and,

accordingly, must possess enzymes of one or more2.2 ml: 4. of the major catabolic pathways. To obtain pre-12,and 1.5 liminary information as to which pathway(s) is3.4 mg of followed by the wild-type organism and by thenate'were mutant, several of the key enzymes were assayed.as at 30 C Diphosphofructoaldolase activity was estimated

by the method of Warburg as described byChristian (4), in which the aldolase reaction iscoupled to the subsequent NAD-linked oxidationof glyceraldehyde-3-phosphate. The reduction ofNAD was followed spectrophotometrically at 340

*- m,ur in the presence of fructose-i ,6-diphosphateand sonic extracts of the two organisms. Bothstrains showed definite activity; the M. poly-morpha extract (2.5 mg of protein per ml) causedan optical-density change of 0.04/min whereasthat of the mutant (2.0 mg of protein per ml)caused a change of 0.09/min. The presence ofthis enzyme indicates a functional portion, atleast, of the glycolytic pathway.

Glucose-6-phosphate dehydrogenase activitywas determined by following NADP reduction at340 m,u (10). For this assay, a prepared reactionmixture was obtained from Calbiochem, and thecontrol was a commercial preparation of glucose-6-phosphate dehydrogenase. The data presentedin Fig. 3 show that both strains possess enzymeactivity and can oxidize G6P to 6-phospho-gluconate (6PG). Accordingly, 6PG dehydro-genase activity was determined according to themethod of Horecker and Smyrniotis (8). In this

-ATP procedure, NADP reduction is followed in the-..---- presence of substrate and enzyme prepared ingo 60 tris(hydroxymethyl)aminomethane (Tris) buffer.

The substrate was prepared according to theiconokinase method of Seegmiller and Horecker (20). Thenorpha M. control organism, E. coli, was grown for 20 hr in2.2 ml: 40 a stationary culture in 0.03 M glucose. The optical-VIgC12, and density changes (Fig. 4) show that M. polymor-of protein pha, under these conditions of growth, does notd. Carbon possess an active 6PG dehydrogenase, althoughan atmos- the mutant organism does possess an active

enzyme. No activity of the wild type could be

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1.5

C I

0D

0.5-M. polymorpho

blank0

0 6 12 18Time (min.)

FIG. 3. Glucose-6-phosphate dehydrogenase assayin sonic extracts of Mima polymorpha and M. poly-morpha M. The control was a preparation of purifiedglucose-6-phosphate dehydrogenase (Calbiochem). A1-ml amount of sonic extract (3.3 and 2.5 mg ofpro-tein per ml, respectively) was added to 2.0 ml ofa pre-pared reaction mixture containing glucose-6-phosphate,NADP, and MgCl2 (Calbiochem). The reduction ofNADP was followed spectrophotometrically at 340m,uat 28 C.

demonstrated by varying the amount of sonicextract protein, by using preparations withdemonstrable activity for G6P dehydrogenase,or in the presence of NAD. It would appear thatthe hexose monophosphate pathway is incompletein M. polymorpha. However, it has been shown(1) that slight growth does occur in the presenceof 6PG as the sole carbon and energy source.Since the enzyme for the conversion of thiscompound to pentose and CO2 is absent, acombined assay was used to measure the activityof 2-keto-3-deoxy-6-phosphogluconate aldolase,the presence of which would indicate, possibly, apartial Entner-Doudoroff pathway. The proce-dure was as follows. The action of 6PG dehydrasein the presence of6PG was coupled to the aldolaseassay devised by Kovachevich and Wood (11).The reduction of the pyruvic acid formed from2-keto-3-deoxy-6PG by this latter step in the

0

0

0.cl

0

0.5

M

0 3 6 9 12Time (min.)

F FIG. 4. 6-Phosphogluconate dehydrogenase assay insonic extracts ofMima polymorpha and M. polymorphaM. The control organism was Escherichia coli. Thereaction mixture contained 0.4 ml of sonic extract(2.1, 2.5, and 5.1 mg ofprotein per ml, respectively),0.0027 X NADP, 0.02 M MgCl2, and Tris buffer (pH7.6) to a volume of 2.9 ml. The reaction was initiatedby the addition of0.1 ml (5 pa) of6-phosphogluconate.The reduction of NADP was followed spectrophoto-metrically at 340 m,u at 28 C.

presence of lactic dehydrogenase may be meas-ured by following the oxidation of NADH2 at 340m,I. M. polymorpha was grown in the presence of0.03 M acetate and 0.03 M glucose with supple-mental 0.001 M 6PG added as inducer. M. poly-morpha M and Pseudomonas fluorescens, thecontrol organism, were grown in the presence of0.03 M glucose. Specific inducer was not added tothese flasks because, if the Entner-Doudoroffpathway is functioning in glucose dissimilation,6PG and 2-keto-3-deoxy-6PG will be formed andact as internal inducers of the enzyme. The reac-tion mixture was prepared to contain: sonicextract, 300 Amoles of Tris buffer (pH 7.65), 3.0,umoles of reduced glutathione, and 6.0j,moles ofFeSO4. Distilled water was added for a calculated3.0-ml final volume. After 2 min, 10.0 ,umoles of

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6-phosphogluconate was added and, after anadditional 2 min, 0.33 ml of lactic dehydrogenase(1.5 mg of purified enzyme per ml; NutritionalBiochemicals Corp.) and 0.14 pAmoles of NADH2were added.One of the difficulties inherent in the assay is

the presence of endogenous pyruvate in the cell-free extracts. This causes an increase in opticaldensity in the minus-substrate control which,however, ceases after approximately 30 min.After this period, continuing optical-densitychanges occurred in the experimental cuvettes.Although small, the changes were approximatelyequal in the three preparations which containedcomparable amounts of protein. The M. poly-morpha extract (1.3 mg of protein per ml) causedan optical density change of 0.001/min; that ofM. polymorpha M (1.3 mg of protein per ml), achange of 0.0016/min; and that of Pseudomonas(1.4 mg of protein per ml), a change of 0.0013/min. Pending fractionation and purification, it isimpossible to assert that the enzyme is functionalin M. polymorpha. However, the facts that therewas definitely a change in optical density afterthe endogenous activity had ceased and that theknown positive control showed an equivalentshift in optical density indicate that pyruvate wasbeing formed by the reactions occurring in theexperimental cuvettes.Table 4 lists the enzymes assayed and the pres-

ence or absence of each in M. polymorpha and inM. polymorpha M.

DiscussIoNThe data show that the inability of M. poly-

morpha to grow in the presence of nonphos-phorylated sugars and polyols may be due to theabsence of functional kinases for these substrates.This, indeed, appears to be the case with glucose.

TABLE 4. Enzyme activities detected in Mimapolymorpha and in M. polymorpha M

M.y poly-Enzyme ply morphamorpha M

Hexokinase...................... - -

Glucose dehydrogenase .......... + +Gluconokinase................... - +2-Ketogluconokinase .............Glucose-6-phosphate dehydro-genase ........................ + +

6-Phosphogluconate dehydro-genase................-........_ +

2 - keto - 3 - deoxy - 6 - phospho-gluconate aldolase............. + +

Diphosphofructoaldolase......... + +

Hexokinase activity was not detected in sonic ex-tracts of either M. polymorpha or M. polymorphaM. P. fluorescens (22) and P. fragi (14), physio-logically similar in many aspects to M. poly-morpha, also lack this enzyme, as do the gram-negative coccus studied by Taylor and Juni (21)and Veillonella sp. (19). The entry of glucose intothe glycolytic pathway is blocked, then, althoughgrowth of the organism on phosphorylated inter-mediates suggests that several other enzymes ofthis pathway may be present. Diphosphofructo-aldolase activity was shown to be present in boththe wild type and the mutant. The presence ofaldolase in the absence of a full complement ofglycolytic enzymes has been demonstrated invarious bacteria (19, 21). An active enzyme couldaccount for the good growth of the organisms onfructose- , 6-diphosphate. Both the aldolase anda F1,6P phosphatase presumably would be es-sential for the synthesis of polysaccharides,pentoses, and aromatic amino acids duringgrowth on various carboxylic acids. Assays forthe F1 ,6P phosphatase and for the acid hexosephosphatase have not been performed. However,in light of the control studies by Englesberg et al.(5) and of Fraenkel and Horecker (6), an investi-gation of the levels of these two enzymes formedin the presence of the various utilizable sub-strates by these two strains of M. polymorpha wiUlbe of interest.Both the wild-type organism and the mutant

possess glucose dehydrogenase activity, and in-tracellular gluconic acid accumulation was de-tected chromatographically in concentrated sonicextracts of M. polymorpha, indicating that lac-tonase is present for the hydrolysis of glucono-6-lactone to gluconic acid. It is possible, however,that the lactone may have been converted to glu-conic acid during the procedures required for thepreparation of chromatography samples. Theabsence of gluconokinase activity in enzymepreparations of M. polymorpha and the presenceof this activity in the mutant extract would explainthe inability of the former and the ability of thelatter to grow on glucose and gluconic acid. Theexistence of the dehydrogenase in the parentstrain can explain the effect of ancillary glucosein a growth medium (1). Most of these dehydro-genases, similar in many organisms, have beenshown to be cytochrome-linked, although thenature of the linkage is, in some instances, notelucidated. Several investigators (2, 7, 17) haveshown them to be particle-bound and linked tothe cytochrome system by an, as yet, uncharac-terized cofactor(s). Pediococcus pentosaceus (12),however, possesses a NADP-linked glucose de-hydrogenase. Although the M. polymorpha en-

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VOL. 91, 1966 CARBOHYDRATE CATABOLISM OF MIMA POLYMORPHA. II

zyme has not yet been subjected to such studies,a linkage to the cytochrome system must be pres-ent, and the energy resultant from glucose oxida-tion must be available for the increased rate ofgrowth and the more rapid induction of isocitri-tase and permeation systems seen with this or-ganism (1). A preliminary observation that theglucose dehydrogenase of M. polymorpha willnot reduce triphenyltetrazolium chloride in thepresence of glucose suggests that there is no flavinlinkage in this organism.G6P dehydrogenase activity was detected in

sonic extracts of both organisms. The presence ofthis enzyme in the wild type represents somewhatof an anomaly, especially as 6PG dehydrogenaseactivity is lacking. As these enzymes are the firstand second catabolic enzymes in the hexosemonophosphate pathway, it is difficult to under-stand why one enzyme is present, apparentlyconstitutively. It may be that, as in the case of thegram-negative coccus of Taylor and Juni (21), anincomplete pentose pathway exists in M. poly-morpha. Since growth does occur on ribose-5-phosphate, certain of the enzymes of this pathwaymust be present. No inhibitor could be demon-strated in the wild-type extract for the 6PG de-hydrogenase present in the mutant. This agreeswith the failure of Taylor and Juni (21) todemonstrate an inhibitor of 6PG dehydrogenaseactivity in extracts from their organism. 2-Keto-3-deoxy-6-phosphogluconate aldolase activity wasdemonstrated in both M. polymorpha and in M.polymorpha M. This indicates that the Entner-Doudoroff pathway plays some role in the me-tabolism of these organisms, and possibly explainsgrowth on 6PG in the absence of the dehydro-genase by the wild-type. The relative roles of thevarious partial and complete pathways in themetabolism of both organisms are being studied.The results have shown that M. polymorpha M

has, at least, two enzymes functioning in itsmetabolism which are not operant in the wildtype. These enzymes are gluconokinase and 6-phosphogluconate dehydrogenase. The currentgenetic concept (13) suggests that, in each case ofa gain in function, the initial mutation has in-volved a derepression of an enzyme which al-ready possessed the requisite catalytic propertyrather than an alteration of inducer specificityor a modification of an enzyme present in thecells. Without additional studies, it can only behypothesized that the mutation involved a dere-pression of Eluconokinase synthesis and that the6-phosphogluconate formed would then inducethe formation of the appropriate dehydrogenase.However, no detectible 6PG dehydrogenase ac-tivity could be found in the wild type which grew

slightly upon 6PG. This latter substrate, however,was never added to induce this enzyme specifi-cally. It is entirely possible, of course, that theenzyme is present and has escaped detection bythe methods employed. Should future experimentsconfirm its absence, then enzymes of the phospho-fructoketolase type will be sought as a possibleroute to pentose formation. Further studies onthe intermediary metabolism and metaboliccontrol mechanisms of these two strains are inprogress.

ACKNOWLEDGMENT

This investigation was supported by National Sci-ence Foundation grant GB-2300.

LITERATURE CITED

1. BELL, E. J., AND A. MARUS. 1966. Carbohydratecatabolism of Mima polymorpha. I. Supple-mental energy from glucose added to a growthmedium. J. Bacteriol. 91: 2223-2228.

2. BENTLEY, R., AND L. SLECHTA. 1959. Oxidationof mono- and disaccharides to aldonic acidsby Pseudomonias species. J. Bacteriol. 79:346-355.

3. COLOWICK, S. P., AND H. M. KALCKAR. 1943.The role of myokinase in transphosphoryla-tions. I. The enzymatic phosphorylation ofhexoses by adenyl pyrophosphate. J. Biol.Chem. 148:117-126.

4. COLOWICK, S. P., AND N. 0. KAPLAN [ED. ]. 1955.Methods in enzymology, vol. 1, p. 315. Aca-demic Press, Inc., New York.

5. ENGLESBERG, E., J. A. WATSON, AND P. A. HOF-FEE. 1961. The glucose effect and the relation-ship between glucose permease, acid phospha-tase and glucose resistance. Cold Spring Har-bor Symp. Quant. Biol. 26:261-276.

6. FRAENKEL, D. G., AND B. L. HORECKER. 1965.Fructose-1, 6-diphosphatase and acid hexosephosphatase of Escherichia coli. J. Bacteriol.90:837-842.

7. HAUGE, J. G. 1961. Glucose dehydrogenation inbacteria: a comparative study. J. Bacteriol.82:609-614.

8. HORECKER, B. L., AND P. Z. SMYRNIOTLS. 1951.Phosphogluconic acid dehydrogenase fromyeast. J. Biol. Chem. 193:371-381.

9. KATZNELSON, H. 1958. Hexose phosphate metab-olism by Acetobacter melanogenum. Can. J.Microbiol. 4:25-34.

10. KORNBERG, A., AND B. L. HORECKER. 1953. Tri-phosphopyridine nucleotides. Biochem. Prepn.3:24-28.

11. 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.

12. LEE, C. K., AND W. J. DOBROGOSZ. 1965. Oxi-dative metabolism in Pediococcus pentosaceus.

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