Vol. 136, No. 2JOURNAL OF BACTERIOLOGY, Nov. 1978, p. 6038-6460021-919:3/ 78/0 136-0638$02.00/()Copyright (C 1978 American Society for Microbiology Prlnted in U.S.A.

Relationship Between Catabolism of Glycerol and Metabolismof Hexosephosphate Derivatives by Pseudomonas aeruginosa

H. E. HEATH' * AN!) ELIZABETI'H T. G[AtUD)Y2

I)epatment of Microbiology. The Unitier-sity of Alabhama, Utniler.sitv, Alabham a 35486, and Department ofMicrobiology, Oklahonma State Unnit ersitv, Stillovater, Oklahomna 740742

Receive(d for p)ublication 21 September 1977

The relationship between catabolism of glycerol and metabolism of hexose-phosphate derivatives in Pseudomonas aeruginosa was studied by comparing thegrowth on glycerol and enzymatic constitution of strain PAO with these charac-teristics of glucose-catabolic mutants and revertants. Growth of strain PAO onglycerol induced a catabolic oxidized nicotinamide adenine dinucleotide-linkedglyceraldehyde-phosphate dehydrogenase and seven glucose-catabolic enzvmes.The results indicated that these enzymes were induced by a six-carbon metaboliteof glucose. All strains possessed a constitutive anabolic Embden-Meyerhof-Parnaspathway allowing limited conversion of glycerol-derived triosephosphate to hex-osephosphate derivatives, which was consistent with induction of these enzymesby glycerol. Phosphogluconate dehydratase-deficient mutants grew on glycerol.However, mutants lacking both phosphogluconate dehydrogenase and phospho-gluconate dehydratase were unable to grow on glvcerol, although these strainspossessed all of the enzymes needed for degradation of glycerol. These mutantsapparently were inhibited by hexosephosphate derivatives, which originated fromglycerol-derived triosephosphate and could not be dissimilated. This conclusionwas supported by the fact that revertants regaining only a limited capacity todegrade 6-phosphogluconate were glycerol positive but remained glucose negative.

Induction of various glucose-catabolic en-zymes by growth of Pseudomonas aeruginosaon glycerol has been noted repeatedly (9, 10, 20).Similar, though less extensive, observations havebeen reported for P. putida (32) and P. fllores-cen.s (19). This has been interpreted as indicatingthat these glucose-catabolic enzymes are in-duced by triosephosphate or a derivative thereof(9). However, this does not satisfactorily accountfor the fact that most glucose-negative mutantswhich are able to grow on lactate also fail togrow on glycerol (2, 18). Indirect evidence foradditional unselected and as yet undefined de-fects associated with glycerol catabolism in suchmutants has been presented (2).This report specifically addresses the relation-

ship between growth on glycerol and ability todissimilate hexosephosphate derivatives in thewild type strain and glucose-negative mutants ofP. aeruginosa.

MATERIALS AND METHODS

Bacterial strains. P. aeralginosa strain PAO(ATUC 15692, strain 131 of Stanier et al. [251) wasprovided by B. W. Holloway, Monash University.Es(-herichia coli strain B (ATCC 11303) was from thecollection of J. W. l)rake 1.1niversitv of Illinois. All

mutants unable to grow on glucose but capable ofnormal growth on lactate were derived from strainPAO. Strain 707 was isolated from a culture mutagen-ized with ethyl methane sulfonate (15) by replicaplating without enrichment. All other mutants wereobtained after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (1). T'he remaining glucose-nega-tive mutants were isolated by replica plating eitherwithout enrichment (strain 720) or after enrichmentwith i)-cycloserine alone (strains 728 and 732) or incombination with carbenicillin (16). Stock cultureswere maintained by periodic transfer on slants ofnutrient agar (Bacto, Difco Laboratories).Media and cultural conditions. Nutrient broth

(Bacto, Difco) was used for routine growth of cells.Where indicated, nutrient broth was supplementedwith 0.5%/ (wt/vol) glucose (GNB). The compositionof minimal medium has been described previously(29). Compounds used as sole sources of carbon andenergy were added to a final concentration of 0.5%(wt/vol). All cultures were grown at 37°C; liquid cul-tures were aerated by reciprocal shaking. Growth wasmeasured as absorbance at 540 nm.Enzyme assays. Cells were grown to late-exponen-

tial phase in 200 ml of the indicated medium. Mutantcells unable to grow on a particular compound weregrown to late-exponential phase in 200 ml of lactate-minimal medium, washed in minimal medium withoutsubstrate at approximately 22°C, resuspended to vol-ume in mirlinial medium conitaining 0.5% (wt/vol) of

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the desired compound, and incubated an additional 4h for induction. Then, the cells were washed in salineat 0°C, resuspended in 5.0 ml of 20 mM 2-amino-2-(hydroxymethyl)- 1,3-propanediol (Tris)-hydrochlo-ride (pH 7.5), and ruptured by intermittent sonic os-cillation. Residual cells and debris were removed bycentrifugation at 27,000 x g for 10 min at 0°C.

All assays were performed at approximately 22°C.All substrates and coupling enzymes were tested forspurious contributions to reaction velocity by contam-inating substances, and, when necessary, appropriatecorrections were made as indicated. A unit of enzymeactivity is defined as the amount required to convert1.0 jimol of substrate(s) to product(s) per min. Thefollowing millimolar extinction coefficients were usedto calculate enzyme activities: formazan product of 3-(4,5 -dimethylthiazol - 2 - yl) - 2,5 - diphenyl - 2H - tetrazo-lium bromide (MTT) reduction at 550 nm, 8.1 (24);reduced form of nicotinamide adenine dinucleotide(phosphate) [NAD(P)H] at 340 nm, 6.2 (14); and phos-phoenolpyruvate at 230 nm, 3.0 (36). Protein wasdetermined by the method of Sutherland et al. (27).Specific activities are given in milliunits per milligramof protein.The following enzymes were assayed according to

the referenced procedure: glucokinase (28); glucose-6-phosphate dehydrogenase (28); phosphogluconate de-hydrogenase (28); glycerol kinase (11); glycerol-3-phosphate dehydrogenase (11); triosephosphate isom-erase (3); glyceraldehyde-phosphate dehydrogenase(28); glyceraldehyde-phosphate dehydrogenase (oxi-dized nicotinamide adenine dinucleotide phosphate[NADP+]) (phosphorylating) (28); phosphoglyceratekinase (3); enolase (35); pyruvate kinase (30); fructose-bisphosphatase (7, 28); and glucosephosphate isomer-ase (7, 28).

Glucose dehydrogenase was measured as glucose-dependent reduction of MTT to the formazan. T'hereaction mixture contained 67 mM Tris-hydrochloride(pH 7.5), 10 mM KCN, 0.24 mM MTT, 2.5 mM glu-cose, and 0.33 mM phenazine methosulfate. The re-action was dependent on phenazine methosulfatewhich was used to initiate the reaction. Less than 10%of the activity was sedimented during the preparationof crude extracts. For determination of gluconate 2-dehydrogenase activity, 2.5 mM gluconate replacedglucose.

Gluconokinase was assayed under the same condi-tions as glucokinase except that phosphogluconatedehydrogenase (approximately 0.1 [J) and 2.5 mMgluconate replaced glucose-6-phosphate dehydrogen-ase and glucose. The excess phosphogluconate dehy-drogenase was provided by 0.1 ml of an extract of E.coli cells grown in GNB. Such extracts typically haveonly about 0.02 U of gluconokinase per U of phospho-gluconate dehydrogenase for which the observed ie-action velocities were corrected.The combined activity of phosphogluconate de-

hydratase and phospho -2- keto- 3- deoxygluconate(PKDG) aldolase was assayed under the conditionsdescribed by von Tigerstrom and Campbell (33). lo-doacetamide did not affect the reaction and was omit-ted. PKDG aldolase was assayed directly by substitu-tion of PKDG for 6-phosphogluconate in the reactionmixture. In all cases where both activities were pres-

ent, phosphogluconate dehydratase was shown to berate limiting by addition of excess PKDG aldolase(approximately 0.16 U) which was provided by 0.1 mlof an extract of E. coli cells grown in GNB. Suchextracts lack detectable phosphogluconate dehydra-tase activity and were included routinely in the reac-tion mixture to assure an excess of PKDG aldolasewhen assaying for phosphogluconate dehydratase ac-tivity in extracts of mutant cells. A sensitive semi-quantitative test which detects glyceraldehyde 3-phos-phate in addition to pyruvate produced by the actionof PKDG aldolase was devised to screen mutants. Thereaction conditions were modified to include phospho-gluconate dehydratase (approximately 0.05 U), triose-phosphate isomerase (approximately 1 U) and glyc-erol-3-phosphate dehydrogenase (approximately 6 U).Phosphogluconate dehydratase and additional triose-phosphate isomerase were provided as 0.1 ml of anextract of lactate-grown, glucose-induced strain 720cells which lack detectable PKDG aldolase activity.Results obtained by this method were interpretedqualitatively because the resulting activity was lessthan that observed in the direct PKDG aldolase assay.

Fructose-bisphosphate aldolase was measured asfructose-I ,6-bisphosphate-dependent reduction of ox-idized nicotinamide adenine dinucleotide (NAI)+) inthe presence of excess glyceraldehyde-phosphate de-hydrogenase. The reaction mixture contained 67 mMTris-hydrochloride (pH 7.5), 100 mM KCI, 17 mMNa2HAsO,, 10 mM freshly neutralized cysteine, 0.25mM NAI)+, approximately 10 lJ of glyceraldehyde-phosphate dehydrogenase, and 2.5 mM fructose-1,6-bisphosphate. Two moles of NAD+ was assumed to bereduced per mole of fructose- 1,6-bisphosphate cleavedbecause the excess triosephosphate isomerase (ap-proximately 0.5 U) of the extract would convert dihy-droxyacetone phosphate to glyceraldehyde 3-phos-phate. The same consideration applies for determina-tion of 6-phosphofructokinase as fructose-6-phos-phate-dependent reduction of NAD+ in the presenceof excess fructose-bisphosphate aldolase and glyceral-dehyde-phosphate dehydrogenase. This reaction mix-ture contained 67 mM Tris-hydrochloride (pH 8.0), 50mM KCl, 17 mM Na2HAsO4, 5.0 mM MgCl2, 2.5 mMATP, 0.25 mM NAD+, approximately 5 U of fructose-bisphosphate aldolase, approximately 10 U of glycer-aldehyde-phosphate dehydrogenase, and 2.5 mM fruc-tose 6-phosphate.

Chemicals and enzymes. T'he barliuml salt ofPKDI)G, a geinerous gift from WA. A. \Xood of MichiganState Uniiversity, was convert ed to the free acid by! ionexchanige with Dowex-50. mI.-Glvceraldehyde 3-phos-phoric acid was prepared from the barium salt of thediethOlacetal derivative by! ion exchanige with D)owex-50) an(l subsequent hydrolysis for .3 mnil at 1000C.Carbenicillin was donate(d by Beechanm Pharnaceuti-cals. Conmmer cial enzynmes were the highest (qtualityavailable from (Calbiochem or Sigma Chemical Co. Allothei chemicals were reagenit grade or the highestpurity avzailable.

RESULTS

Growth phenotypes of glucose-negativemutants. Twenty independent glucose-negative

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640 HEATH ANI) GAUDY

mutants were isolated. Each was also unable togrow on gluconate or 2-ketogluconate but wasable to grow on lactate. As shown in Table 1,three classes of glucose-negative mutants couldbe distinguished by growth in glycerol-minimalmedium and GNB. Only three strains were ableto grow on glycerol; strain 707 grew well, butstrain 732 grew poorly. The glycerol-negativestrains could be grouped further by growth inGNB. Strain 728 is representative of 13 mutantsthat grew in GNB. Strain 720 is representativeof four mutants that grew normally in NB butbecame severely inhibited and usually lysed inGNB. The other glycerol-positive strain and twoadditional representatives of each glycerol-neg-ative group have been characterized thoroughlybut were omitted here because they gave thesame results as strains 707, 728, and 720, respec-tively.Enzymatic defects of glucose-negative

mutants. The inability of each mutant to growon glucose, gluconate, or 2-ketogluconate sug-gested a defect in catabolism of 6-phosphoglu-conate (Fig. 1). Eight of the ten glucose-catabolicenzymes were assayed. As shown in Table 2,growth of strain PAO on glucose or gluconateinduced all eight enzymes approximately three-fold or more. Growth on glycerol also causedapproximately threefold or more induction of allthese enzymes except gluconokinase.The glycerol-positive strains were specifically

defective for phosphogluconate dehydratase(Table 2); strains 707 and 732 lacked detectableactivity. Both of these mutants possessed phos-phogluconate dehvdrogenase, and the level ofthis activity correlated with their ability to growon glycerol (cf. Table 1). In contrast to the wildtvpe, growth of these mutants on glycerol alsoinduced gluconokinase. Although glucokinaseand gluconate 2-dehydrogenase were not in-duced during the poor growth of strain 732 onglycerol, these activities were increased to 25.2

TABLE 1. Growth phenotypes ofglucose-catabolicmlutants

IGrowth" on:

Strain G(lu- GILi 2-Keto- Glv actate GNBgILucon- att N

cose conate ate erol

PAO + + + + + +707 _ _ - + + +732 - + +728 X _ L+ +

720 +

' Absorbance (A) was determined at 12 h for GNBand 24 h for minimal media: +, A > 0.9; +, A < 0.3;-,A <0.1.

and 54.5 mU/mg of protein, respectively, byexposure of lactate-grown cells to glycerol.

Strain 728 lacked detectable phosphogluco-nate dehydratase and phosphogluconate dehy-drogenase activities (Table 2). Similar resultshave been obtained for cells induced with glu-conate or glycerol. Again, unlike the wild type,glycerol induced gluconokinase to 60 mU/mg ofprotein in this strain.

Strain 720 specifically lacked detectablePKDG aldolase activity (Table 2). Low levels ofglucose-6-phosphate dehydrogenase, gluconoki-nase, and phosphogluconate dehydrogenase alsowere observed upon induction with gluconate orglycerol.Absence of additional defects from

strains with impaired growth on glycerol.Because N-methyl-N'-nitro-N-nitrosoguanidineoften causes multiple mutations (13), strainswith impaired growth on glycerol were examinedfor additional unselected defects specific forglycerol degradation. As shown in Table 3, glyc-erol kinase and glycerol-3-phosphate dehydro-genase were induced by growth of strain PAOon glycerol but not on glucose; triosephosphateisomerase was constitutive. Strain 732, whichgrew poorly on glycerol, and representative glyc-erol-negative strains 728 and 720 did not lackeither inducible enzyme (Table 3). Triosephos-phate isomerase activity was demonstrated forstrain 728 (Table 3) and is inferred for the othermutants from normal growth on glucogenic sub-strates.Enzymes of the triosephosphate pathway

were also examined for additional defects thatcould affect degradation of glyceraldehyde 3-phosphate. Tiwari and Campbell (28) noted thepresence of NAD+- and NADP+-linked glycer-aldehyde-phosphate dehydrogenase activities inP. aeruginosa. As shown in Table 4, strain PAOpossessed inducible glyceraldehyde-phosphatedehydrogenase activity (NAD+-linked) in addi-tion to a constitutive glyceraldehyde-phosphatedehydrogenase (NADP+) (phosphorylating) ac-tivity. Phosphoglycerate kinase, enolase, and py-ruvate kinase were also constitutive (Table 4).NAD'-linked glyceraldehyde-phosphate dehy-drogenase and pyruvate kinase are presumed tobe uniquely catabolic; and, if either were defec-tive, impaired growth on glycerol could result.However, none of the mutants defective forgrowth on glycerol lacked NAD+-linked glycer-aldehvde-phosphate dehydrogenase, pyruvatekinase, or any other enzyme of the triosephos-phate pathway (Table 4). Although not assayedhere, the presence of phosphoglyceromutase ac-tivity was inferred from normal growth on glu-cogenic substrates.

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GLUCOSE-CATABOLIC ENZYMES INDUCED BY GLYCEROL 641

Glucose Gluconate 2-Keto-gluconate

6)

\ T F i ~~~~6-F-2-Keteo-Glucose-6-P )P-Gluconate

g o

0~~~~~~~~~~~~~it /~ ~~~~~~8

Fructose-6-P Pentose-P 6-P -2-Keto-3-deoxy-gluconate

19 < /l < Lactate

Fructose- Gldedera- p Py vate

196-P2 dhyde236

DlhydroxypI

22 3-P-Glyceroyl-P P-Eno/pyruvatesn-Glycerol-3-P

13 15

Glycerol 3-P-Glycerate 2-P-Glycerate

FIG. 1. Metabolism of carbohydrate deriatives by P. aeruginosa. Numbers refer to the following enzymes:1, glucokinase; 2, glucose-6-phosphate dehydrogenase; 3, glucose dehydrogenase; 4, gluconokinase; 5, glucon-ate 2-dehydrogenase; 6, ketogluconokinase; 7, 2-keto-6-phosphogluconate reductase; 8, phosphogluconatedehydrogenase; 9, reversible nonoxidative enzymes of the hexose monophosphate pathway; 10, phosphoglu-conate dehydratase; 11, phospho-2-keto-3-deoxygluconate aldolase; 12a, glyceraldehyde-phosphate dehydro-genase; 12b, glyceraldehyde-phosphate dehydrogenase (NADP+) (phosphorylating); 13, phosphoglyceratekinase; 14, phosphoglyceromutase; 15, enolase; 16, pyruvate kinase; 17, triosephosphate isomerase; 18,fructose-bisphosphate aldolase; 19, fructose-bisphosphatase; 20, glucosephosphate isomerase; 21, glycerolkinase; 22, glycerol-3-phosphate dehydrogenase; and 23, lactate dehydrogenase. The Entner-Doudoroffenzymes are 10 and 11. The triosephosphate pathway (lower portion of the EMPpathway) consists of enzymes12a, 12b, 13, 14, 15, and 16. The anabolic portion of the EMP pathway is composed of enzymes 18, 19, and 20.

Presence of the anabolic EMP pathway. and that mutants with impaired growth on glyc-The facts that growth of strain PAO on glycerol erol apparently had no additional defects sug-induced NAD+-linked glyceraldehyde-phos- gested that growth on glycerol may actuallyphate dehydrogenase and all the glucose-cata- depend on limited metabolism of hexosephos-bolic enzvmes examined except gluconokinase phate derivatives. Therefore, enzymes of the

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642 HEATH AND) GAITI)'

TABl,F 2. Inducible glucose-catabolic enzymes and biochemical (lefects ofglucose-negatiue mltitanttsSp act (rolltJ/mg of protemt

Growth Sob- lck-GIloCOSe-fi- Phospho-fh s)ho-Strall st r.at eG(lokrposphehatevgoloG,Ic l t e glriconat e; )( al-('11ducer") nase dehvdro- kinase dehvdro- deh% dra- dolase

genase drogenasegeniase geniase tase

I'AO Lactate 14.8 5.8 6.0 0)" 4.4 0 1.5 10(.7Glucose 97.5 27f6 16.1 44.0 518 89.5 120 304Gluconate 6f7.7 199 42.6 3'7.4 127 :31.:3 i61.3 '61:.3'Glycerol 82.2 194 126 0 12.7 18.2 80.1 458

707 Glycerol 91.2 '36:3 53.3 74. :32.1 7.6 0 63(0732 Glycerol 18.9 145 178 4.0 2.6G 0.7 0 .42.3'728 (Glucose) :34.4 50.0 23.0 68.5 1.:3 03720)2 (Glucose) 90.9 2.9 28.1 (0.5 :31.() .6 5:3.2

See text for induction procedure.Undetectable activity: <1.5 mU for gluconokinase, phosphogluconate dehydrogenase, and phosphogluconate

dehydratase; <0.5 mU for PKDG aldolase.Minimum specific activity of PKDG aldolase because phosphogluconate dehvdratase was rate limiting in

the assay for the combined activity of these enzymes (see text).d Minimum specific activity obtained by the semiquantitative test for PKDG aldolase (see text).

TABI,F, 3. Formation of the glycerol-catabolicenzvmes by strain PAO and mutants with impaired

abilitv to grolu' on gglycerol| SI act (mlnT/ng of protein)

Gwrowth SuLb- GGlycerol-Strain strate G(;Ivcerol :3-phos- rI'riose-

(inducer") k phate de- phosphatehyvdrogen- isomerase

ase

PAO Lactate (.0:3 Oh 424Glucose 0.4 0 705Gl,cerol 6.:3 3.8 556

7:32 Glvcerol 5.2 2.7 NA'728 (Glycerol) 2.9 12.2 916720 (Glycerol) 4.9 2.1 NA

See text for induction procedure.Undetectable activitv (<1.2 mUJ).Not assaved.

Embden-Meyerhof-Parnas (EMP) pathwaywere examined in the wild type and glucose-negative mutants (Table 5). Lactate- or glucose-gIown cells of strain PAO lacked detectable 6-phosphofructokinase activity. However, this ac-

tivitv was readily detectable in glucose-growncells of E. coli (55.6 mU/mg of protein). Conse-quently, strain PAO lacks a catabolic EMP path-way. In contrast, each of the enzymes involvedin conversion of triosephosphate to hexosephos-phate was constitutive in strain PAO, and noneof the mutants lacked any of these anabolicactivities (Table 5).Reversion of strain 728. Glucose-positive

rev!ertants occurred at a frequency of approxi-mately 4 x 10 in cultures of strain 728. Allglucose-positive revertants were also able togrow on glycerol. These revertants regainedphosphogluconate dehydratase and phosphoglu-

conate dehvdrogenase activities as showni in Ta-ble 6 for one representative, strain A 11. How-ever, glycerol-positive revertants occurred at afrequency of approximately 3 x 10 in culturesof strain 728, and only about 1%' of these wereable to gr-ow on glucose. Like the revertantsselected on glucose, these glucose-positive re-vertants selected on glycerol regained both en-zvme activities as shown in Table 6 for onerepresentative, strain A6. In contrast, strain GI,a typical glycerol-positive revertant that wasunable to grow on glucose, regained low p)hos-phogluconate dehydrogenase activity but hadless than 1I% of the norrmal phosphogluconatedehydratase activity (Table 6). These revertantstherefore riesemble the glycerol-positive, glu-cose-negative mutants described initially. Sinmi-larly, asymimetric reversion to growth on glc-erol or glucose has been observed for two othermutants lacking phosphogluconate dehydrataseand phosphogluconate dehydrogenase.

DISCUSSION

It is clear that in P. aeluginosa strain P'AOthe inducible enzymes for catabolism of glyceroland of glucose constitute three regulatory groupsbased on nutritional inducers. Only growth onglycerol induced glvcerol kinase and glycerol-3-phosphate dehvdrogenase; growth on glycerol orglucose (or gluconate) induced glucose dehydro-genase, gluconate 2-dehydrogenase, glucokinase,glucose-6-phosphate dehydrogenase, phospho-gluconate dehydrogenase, phosphogluconate de-hydratase, PKDG aldolase, and NADW-linkedglyceraldehyde-phosphate dehydrogenase; onlygrowth on glucose (or gluconate) induced glu-conokinase, ketogluconokinase, and 2-keto-6-phosphogluconate reductase (20). 'his study in-

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TABILE 4. Enzymes of the triosephosphate pathway in strain PAO and glucose-negative mutants

Sp act (mtJ/mg of protein)

Glyceralde-

Growth substrate Glyceralde- hyde-phos-Strain ( phate dehv- Phospho-(inducer") hvde-phos- Pvrruvate ki-drogenase glvcerate Enolasephate dehy- nasedrognase (NADP') kinasedrogenase (phosphoryl-

ating)

PAO Lactate 1.8 95.2 363 232 153Glucose 67.7 176 355 240 168Glycerol 74.9 98.4 276 114 101

732 (Glucose) 147h 114 399 147 63.9728 (Glucose) 37.0 74.6 324 82.8 53.2720 (Glucose) 109 98.7 460 172 113

"See text for induction procedure.Glycerol-grown cells.

TABLE 5. Enzymes of the anabolic- EMP pathu'ay in strain PAO an(d glucose-negatite mutantsSp act (mU/mg of protein)

Strain Growth substrate Fructose-bis- t Glucosephos-(inducer") phosphate al Fructose-b- phate isomer- 6-Phosphofruc-phosphatase tokinasedolase ase

PAO Lactate 15.2 14.6 39.6 0")Glucose 16.0 22.9 88.4 0Glycerol 17.2 12.5 81.2 NA'

707 (Glucose) 19.3 19.6 45.6 NA732 (Glucose) 13.1 12.9 72.8 NA728 (Glucose) 9.5 9.3 61.8 NA720 (Glucose) 13.8 18.9 83.2 NA

"See text for induction procedure.Undetectable activity (<0.8 mU). Glucose-grown cells of E. coli had 55.6 mU of 6-phosphofructokinase per

mg of protein.' Not assayed.

TABLE 6. Reversion of strain 728

Revertant phenotype" Sp act (mU/mg of protein)

Strain Growth substrate Phosphoglu- Phosphoglu-Selected Unselected conate dehy- conate dehy-

drogenase dratase

All Glu+ Gly+ Glucose 5.3 34.5A6 Gly+ Glu+ Glucose 11.0 35.2GI Gly+ Glu- Glycerol 1.5 0.7

"Phenotypic symbols: Glu and Gly, utilization of glucose and glycerol, respectively.

dicates that induction of these glucose-catabolicenzymes by growth on glycerol is due to a limiteddependence of glycerol utilization on degrada-tion of 6-phosphogluconate to glyceraldehyde 3-phosphate. Growth of phosphogluconate dehy-dratase-deficient mutants on glycerol is corre-lated with the level of phosphogluconate dehy-drogenase observed (cf. strains 707, 732, and 728in Tables 1 and 2). Moreover, reversion of glyc-erol-negative, glucose-catabolic mutants togrowth only on glycerol is associated with recov-ery of a low phosphogluconate dehydrogenaselevel without an equivalent increase in phospho-

gluconate dehydratase activity.The induction specificity of NAD+-linked

glyceraldehyde-phosphate dehydrogenase pro-vides an explanation for this dependence. Theconstitutive glyceraldehyde-phosphate dehydro-genase (NADP+) (phosphorylating) activity ap-parently is not sufficient for catabolism of glyc-erol or other compounds degraded at least par-tially via glyceraldehyde 3-phosphate becausegrowth on such compounds induces NAD+-linked glyceraldehyde-phosphate dehydrogen-ase. However, this enzyme appears to be inducedby a six-carbon metabolite because it is induced

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644 HEATH ANI) GAUDI)Y

by glucose in mutant strains lacking a fissionmechanisnm (i.e., catabolic EMP pathway, phos-phogluconate dehydratase, and phosphogluco-nate dehydrogenase). Therefore, sufficienttriose-phosphate derived from glycerol presum-ably is converted to hexosephosphate via theanabolic EMP pathway for induction of NAD+-linked glyceraldehyde-phosphate dehydrogen-ase. This also accounts for induction of glucose-catabolic enzymes by growth on glycerol. Glyc-erol degradation proceeds then via the usualtriosephosphate pathway to pyruvate. Any hex-osephosphate produced is dissimilated via theEntner-Doudoroff pathway and to a lesser ex-tent via the hexose monophosphate pathway.

This interpretation is supported by the follow-ing observations. In contrast to the wild type,glycerol induces gluconokinase in phosphoglu-conate dehydratase-deficient mutants, whichcannot dissimilate 6-phosphogluconate well orat all. As previously noted, growth of phospho-gluconate dehydratase-deficient mutants onglycerol correlates with their ability to dissimi-late 6-phosphogluconate. In the extreme case ofmutants completely lacking both phosphoglu-conate dehydratase and phosphogluconate de-hydrogenase, failure to grow on glycerol in spiteof the presence of all the enzymes needed fordegradation to pyruvate is presumably due toinhibition by undegraded hexosephosphate de-rivatives. This is substantiated by the occur-rence of glycerol-positive revertants that re-mained glucose negative and had regained only8%s of the normal phosphogluconate dehydro-genase level but less than 1', of the normalphosphogluconate dehydratase level. Whereas alimited capacity for dissimilation of 6-phospho-gluconate is sufficient to prevent inhibition, thuspermitting growth on glycerol via the usual tri-osephosphate pathway, it is clearly insufficientto support growth of phosphogluconate dehy-dratase-deficient mutants and revertants on glu-cose, gluconate, or 2-ketogluconate. Finally, al-though limited phosphogluconate dehydrogen-ase is necessary for growth of phosphogluconatedehydratase-deficient mutants on glycerol, it isnot a sufficient condition for growth of PKDGaldolase-deficient mutants on glycerol. P.aeruginosa, like E. coli (6) and P. putida (31),apparently is extremely sensitive to accumula-tion of PKDG, as shown by the severe inhibitionof mutants lacking PKDG aldolase in GNB.These mutants would also be expected to accu-mulate PKDG from glycerol and therefore failto grow in spite of the presence of low phospho-gluconate dehydrogenase activity and all thenecessary enzymes for degradation of glycerolvia the triosephosphate pathway.

It is clear that a single mutation caused theloss of phosphogluconate dehydratase and phos-phogluconate dehydrogenase activities in mu-tants such as strain 728 because spontaneousrevertants simultaneously recovering both activ-ities are readilv isolated by selecting for growthon glucose. However, it is equally clear thatphosphogluconate dehydrogenase activity usu-ally is preferentially restored in revertants se-lected for growth on glycerol. These observa-tions are consistent with strain 728 containing apolar or perhaps regulatory mutation, but theorganization and regulation of the genes encod-ing the glucose-catabolic enzymes are presentlyunknown. Consequently, the precise nature ofthe mutation in such strains is not yet clear.Nevertheless, the predominance of this pleio-tropic mutation among the glucose-negative iso-lates probably reflects efficient selection againsteven slightly leaky growth in the enrichmentprocedure (16) used to obtain most of the mu-tants.

Phosphogluconate dehydrogenase plays a crit-ical role in the interpretation of the results re-ported here, and its existence in the aerobicpseudomonads has been questioned recently. Ithas been suggested that this activity in crudeextracts results from coupling of phosphogluco-nate dehydratase, PKDG aldolase, and glycer-aldehyde-phosphate dehydrogenase (2). Thestrongest evidence against the existence of phos-phogluconate dehydrogenase is circumstantial(21; P. V. Phibbs, Jr., and C. McNamee, Abstr.Annu. Meet. Am. Soc. Microbiol. 1976, K184, p.167). As discussed above, simultaneous loss andrecovery of phosphogluconate dehydrogenaseand phosphogluconate dehydratase by mutationneed not preclude the existence of phosphoglu-conate dehydrogenase. Likewise, inhibition ofboth activities by fluoride (21) is inconclusive.Fluoride inhibits a variety of enzymes by diverseand complex mechanisms (8), so phosphogluco-nate dehydrogenase of P. aeruginosa could con-ceivably be inhibited, whereas the enzyme fromother sources is not. Finally, the lack of evidencefor oxidative decarboxylation of 6-phosphoglu-conate may be due to choice of buffer (theenzyme from various sources is inhibited byphosphate [17, 22, 23]) or physiological state ofthe cells employed. In P. aeruginosa, radiores-pirometric analyses indicate that the hexosemonophosphate pathway accounts for up to 30%cof the glucose degraded (26), but this pathwayis not involved in degradation of gluconate (34).As shown in Fig. 1, glucose can be degraded viaglucose-6-phosphate or gluconate. The relativecontribution of these routes is unknown (20) butundoubtedly depends on the physiological state

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GLUCOSE-CATABOLIC ENZYMES INDUCEI) BY GLYCEROL 645

of the cells studied. Thus, under certain condi-tions, glucose may be degraded primarily viagluconate and oxidative decarboxylation of 6-phosphogluconate might not be observed. Suchconditions are in fact known. For example, un-

induced resting cells quantitatively convert glu-cose to gluconate (Heath, unpublished data).Similar results were obtained for P. putida (for-merly P. fluorescens) (5). Furthermore, expo-

sure of fluorescent pseudomonads to high con-

centrations of glucose often results in quantita-tive conversion to gluconate (12). Moreover, Ei-senberg et al. (4) have presented evidence thatalthough P. fluorescens can transport and phos-phorylate glucose and oxidize the resulting glu-cose-6-phosphate to 6-phosphogluconate, thisorganism will not grow on glucose unless it isoxidized to gluconate which is required for in-duction of 6-phosphogluconate dehydratase(19). Vicente and Canovas (31, 32) similarly con-

cluded that glucose catabolism by P. putida isprimarily via 2-ketogluconate in spite of thepresence of glucokinase (4) and glucose-6-phos-phate dehydrogenase in this species also. Theseconsiderations may affect the ability to observeoxidative decarboxylation of 6-phosphogluco-nate.

Several lines of evidence from this study are

incompatible with the coupling hypothesis.Phosphogluconate dehydrogenase activity is de-tectable in mutants completely lacking phospho-gluconate dehydratase or PKDG aldolase activ-ity. Phosphogluconate dehydrogenase and phos-phogluconate dehydratase activities are neitherlost nor restored to the same extent by mutation.NAD+ gives only 1% of the activity observedwith NADP+ in spite of the presence of highNAD+- and NADP+-linked glyceraldehyde-phosphate dehydrogenase activities. Moreover,

these latter activities were totally dependentupon addition of Na2HAsO4 which was not pres-ent in the phosphogluconate dehydrogenase as-

says. Finally, phosphogluconate dehydrogenaseactivity was inhibited 86Wk by substitution ofpotassium phosphate buffer for Tris-hydrochlo-ride of pH 7.5. If NADP+-linked glyceralde-hydephosphate dehydrogenase were involved in

the activity, stimulation would have been ex-

pected because this enzyme requires phosphate(or arsenate), which had been excluded previ-ously. It therefore seems premature to concludethat phosphogluconate dehydrogenase does notexist in the fluorescent pseudomonads. If it werean artifact, then the data presented here clearlyindicate it is not due to the proposed coupling.

Clarification of the role of phosphogluconatedehydrogenase in degradation of glucose and1-elated compounds by P. aeruginosa will require

further study. However, it now seems clear thatgrowth on glycerol depends on limited metabo-lism of hexosephosphate derivatives due to theinduction specificity of the catabolic NAD -linked glyceraldehyde-phosphate dehydrogen-ase. This then accounts for glycerol-induced for-mation of the glucose-catabolic enzymes.

ACKNOWLEDGMENTS

We thank K. K. Brown, C. M. Cowen, and S.-S. Tsay forassaying glycerol kinase, glycerol-3-phosphate dehydrogenase,and triosephosphate isomerase in extracts of certain strains.

T'his work was supported in part hy grants to E.T.G. fronmthe National Science Foundation (xB-4195) and the Okla-homa State tUniversity Research Foundation and to H.E.H.from The tJniversity of Alabama Research Grants Committee(789). H.E.H. was a Public Health Service l'rainee (GM0I 102)during part of this studv.

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