JOURNAL OF BACTERIOLOGY, JUly 1973, p. 268-276Copyright ( 1973 American Society for Microbiology

Vol. 115, No. 1Printed in U.S.A.

Phenotypic Suppression of a

Fructose-1.,6-Diphosphate Aldolase Mutation inEscherichia coli

RENATE SCHREYER AND AUGUST BOCK

Fachbereich fur Biologie der Universitat Regensburg, 84 Regensburg, Germany

Received for publication 13 April 1973

Strain NP 315 of Escherichia coli possesses a thermolabile fructose-1, 6-diphos-phate (FDP) aldolase; its growth on carbohydrate substrates is inhibitedprobably as a consequence of the accumulation of high intracellular levels ofFDP. Studies of one class of phenotypic revertants of strain NP 315 which haveregained their ability to grow on C. substrates at 40 C showed that in thesestrains the buildup of the inhibitory FDP pool is prevented by additionalmutations in enzymes catalyzing the conversion of the substrate offered in themedium to FDP. For example, mutations affecting 6-phosphogluconate dehy-drogenase activity (gnd-) may be selected in great number without anymutagenesis and enrichment simply by isolating revertants of strain NP 315 ableto grow on gluconate at 40 C. Similarly, an additional mutation in phospho-glucose isomerase (pgi-) restores the ability of these fda- gnd- strains to grow on

glucose at 40 C. Glucose metabolism of these fda- gnd- pgi- strains was

investigated. The enzymes of the Entner-Doudoroff pathway are induced to an

appreciable extent upon growth of these mutants on glucose medium; furtherevidence for glucose degradation via this route (which normally is induced onlyin the presence of gluconate) was provided by following the fate of the Cl labelof radioactive glucose in L-alanine. Predominant labeling of the carboxyl-carbonof L-alanine was observed, inciating a major contribution of the Entner-Doudoroffpath to pyruvate formation from glucose. Chromatographic analysis of theintermediates of glucose metabolism showed further that glucose apparently is atleast partly metabolized via a bypass consisting of the accumulation ofextracellular gluconic acid which arises by dephosphorylation of 6-phospho-gluconolactone and possibly of 6-phosphogluconate. This extracellular gluconateis then taken up and metabolized in the normal manner via the Entner-Doudor-off enzymes.

The inhibitory effects of high sugar phosphateconcentrations under conditions of any iinbal-ance in carbohydrate catabolism is well known(1) and is usually ascribed to a general toxicityof these components. Thus, growth of a mutantof Escherichia coli with a defective fruc-tose-1, 6-diphosphate (FDP) aldolase (EC4.1.2.13) is severely inhibited under conditionswhere high intracellular concentrations of FDPaccumulate (1, 2).

In an attempt to more closely characterizethis growth inhibition, we have isolated revert-ants of the original temperature-sensitive FDPaldolase strain. One class of revertants could beidentified as strains possessing mutations inenzymes leading from the C6 or C5 substrate

26E

of the medium to FDP, thereby preventing ac-cumulation of FDP. For example, growth inhibi-tion on gluconate as carbon source is reversedby mutations (gnd-) affecting activity of gluco-nate-6-phosphate dehydrogenase (EC 1.1.1.44);similarly, glucose-supported growth was some-what restored by mutations in gnd and phos-phoglucose isomerase (EC 5.3.1.9) (pgi-). Oneof these apparent fda- gnd- pgi- mutants wasused in the investigation of the path of glucosedegradation because carbon flow through gly-colysis and the hexosemonophosphate shuntshould be prevented by the mutational blocksand the Entner-Doudoroff-path (5) (Fig. 1)should be repressed upon growth on glucose(10). The results show that in these mutants the

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GLUCOSE METABOLISM IN E. COLI MUTANTS

Entner-Doudoroff path contributes to glucosedegradation.

MATERIALS AND METHODSThe following strains were used: K-10, a proto-

trophic Hfr strain (rel-); strain NP 315 (fda-), aderivative of strain K-10 with a temperature-sensitivefructose-1,6-diphosphate aldolase (1); strain NP3151 (fda- gnd-) is a spontaneous revertant of strainNP 315 scored for growth at 40 C on gluconate mini-mal plates. Strain NP 31515 (fda- gnd- pgi-) is a de-rivative of strain NP 3151; it was selected for growthat 40 C on glucose minimal medium plates.The minimal medium used was a modified salt

solution P of Fraenkel and Neidhardt (9). Onlyone-half of the original phosphate concentration wasemployed, and it was supplemented with 0.2%(NH4)2SO4 and 0.4% of the indicated carbon source.Plates contained 1.5% agar (Serva). Cultures weregrown in gyratory water bath shakers; the bacterialgrowth was measured by following the optical density(OD) increase at 420 or 405 nm in a Zei,B PMQ II or anEppendorf photometer.

Preparation of cell-free extracts and enzymeassays. For preparation of cell-free extracts, thecultures were grown to an OD420 of about 1.5 andharvested by centrifugation. The sedimented cellswere washed once with the respective extractionbuffer and subsequently broken by sonic treatmentwith a Branson sonic oscillator for three 1-min periodsat setting no. 3. The extracts were cleared by centrifu-gation for 20 min at 20,000 x g, and their proteinconcentration was determined quantitatively by themethod of Lowry et al. (18) with bovine serumalbumin as standard protein. The activities of phos-phoglucose isomerase, glucose-6-phosphate dehy-drogenase, gluconate-6-phosphate dehydrogenase,and gluconokinase were assayed in the reaction mix-tures by the method of Fraenkel and Levisohn (10);the activities of 6-phosphogluconate dehydrase and2-keto-3-deoxy-6-phosphogluconate aldolase were de-termined by employing the procedure of Kovachevichand Wood (16) as modified by Fradkin and Fraenkel(6).

Analysis of sugar phosphates in the soluble pooland in the medium. The amount of sugar phosphatesaccumulated in the cellular pool and excreted into themedium was determined as described by Fraenkel (8).The cells from 2 ml of the cultures were separatedfrom the medium by filtration through Sartoriusmembrane filters (pore size 0.45 1sm), extracted byboiling in 2 ml of water for 4 min, and cleared bycentrifugation. The sugar phosphate contents of thisextract and of the medium were measured by theprocedures of Bergmeyer. Thus, fructose-1,6-diphos-phate was assayed quantitatively by the aldolase-,triosephosphate isomerase-, and a-glycerophosphatedehydrogenase-mediated oxidation of reduced nico-tinamide adenine dinucleotide (NADH) (4); glucose-6-phosphate was measured by glucose-6-phosphatedehydrogenase-coupled NADP-reduction (13); simi-larly, gluconate-6-phosphate was measured with theaid of 6-phosphogluconate dehydrogenase (14). Glu-conic acid was determined quantitatively via glucono-

Glucose

IGlucose-6-P b 6-phosphog uconolactone

I pgl g6 - Gluconatepgi 6-phosphogluconate =

/ edd ~~ i4 DPG

8 >/ R~~~~~~ndeds

pfkJ

Fructose-1,6-diP Tricese-P + Pyruvate

j l fds

2 Triose-P~IrGlycolysis Pentose-phosphats cycle Entner-Doudoroff-

Pathway

FIG. 1. Pathways of glucose and gluconate dissimi-lation by Escherichia coli (17). Gene designations arefda, fructose-1,6-diphosphate aldolase; pgi, phospho-glucose isomerase; gnd, 6-phosphogluconate dehy-drogenase; pgl, 6-phosphogluconolactonase; edd, 6-phosphogluconate dehydrase; eda, 2-keto-3-deoxy-6-phosphogluconate aldolase; pfk, phosphofructoki-nase; glk, gluconokinase. Dashed arrows indicateinducible enzymes.

kinase and gluconate-6-phosphate dehydrogenase byusing the assay system of Moellering and Bergmeyer(19). It was found that high levels of fructose-1,6-diphosphate greatly interfered with the quantitativedetermination of gluconate and gluconate-6-phos-phate by inhibiting gluconate-6-phosphate dehy-drogenase activity (3). Prior to these determinations,fructose-1,6-diphosphate was, therefore, removed byincubation in the presence of FDP-aldolase, triose-phosphate isomerase, a-glycerophosphate dehydro-genase, and NADH. All the enzymes employed inthese determinations were obtained from Boehringerand Sons, Mannheim.Labeling pattern of L,alanine in cultures grown

on [1-14C]glucose or [1-'4C]gluconate. The contribu-tion of the Entner-Doudoroff path to the degradationof glucose or gluconate was assessed by following thelabeling pattern of L-alanine in the protein of cellsgrown on [1-'4C]glucose or [1-'4C]gluconate as sub-strate (10). The isolation and degradation of alaninewas carried out by the method of Fraenkel andLevisohn (10) except that alanine was separated fromthe other amino acids of the protein hydrolysate bythree successive descending chromatographies onWhatman no. 1 paper. The solvent systems used inthe given sequence (20 h each, 20 C) were isopropanol-water-glacial acetic acid (75: 15: 10), a-picoline-water-ammonia (78:20:2), and phenol-water (88: 12).

Chromatography. Separation of sugar phosphateswas achieved by chromatography on Whatman no.3MM paper, using a solvent system of 1 M ammo-nium acetate (pH 5.0), 95% ethanol, and 0.1 Methylenediaminetetraacetate disodium salt (30:70: 1,vol/vol/vol), descending at 30 C (20, 17). Separation ofgluconolactone from other intermediates was accom-plished by chromatography on Merck cellulose thin-layer plates under the conditions given by Grassetti et

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SCHREYER AND BOCK

al. (12). The location of radioactivity on chromato-grams was detected by means of a Berthold chromato-gram scanner, model LB 280, for the paper chromato-grams and with a Berthold scanner II, model LB 2723,for thin-layer plates. Standards were run on the samechromatogram and detected with the periodate benzi-dine spray (11). Quantitative evaluation of the radio-activity was done by cutting out or scraping off therespective area from the paper or plates and bycounting in a toluene-0.5% diphenyloxazole scintilla-tion mixture using a BF 5001 liquid scintillationspectrometer.

RESULTS AND DISCUSSIONGrowth studies. It has been shown previ-

ously that blocking carbon flow through glycol-ysis by a mutational alteration of FDP aldolase(Fig. 1) results in the complete and immediatecessation of growth on C6 carbon sources includ-ing gluconate (1, 2). On the other hand, Korn-berg and Smith (15) reported that mutants of E.coli lacking phosphofructokinase activity are

able to utilize the hexosemonophosphate path-way as a degradative route during glucose-supported growth if a sufficient supply of phos-phoenolpyruvate (PEP) is provided for the up-

take of glucose via the PEP phosphotransferasesystem. In contrast to these pfk- strains, theFDP aldolase mutant grows neither at therestrictive condition with glucose-6-phosphateas substrate nor under conditions where cellswere pregrown on pyruvate which results in theinduction of PEP synthase (Fig. 2). Cessation ofgrowth of strain NP 315 at 40 C, therefore,seems not to be due to shortage of PEP. Sincethe main difference between pfk- and fda-strains should reside in the lack of the intracel-

0,75-

0,50 C400C

300 C 0- 0

6L 0,25- /oO (30) Pyr

L- Gluc (400 C)

0,1

-1 -2 0 1 2 3TIME (hrs)

FIG. 2. Growth of mutant NP 315 in glucose-6-phosphate minimal medium at 30 C and after a

shift to 40 C, 0; the effect ofpregrowth of mutant NP315 in pyruvate minimal medium at 30 C on subse-quent growth with glucose as carbon source at 40 C,.

lular accumulation of FDP in the pfk- strainswhereas high levels of this intermediate can bedemonstrated in strain NP 315, it is likely thatFDP accumulation, and not that of any othersugar phosphate, is responsible for the growthinhibition observed.

Isolation of revertants. To gain more infor-mation about the site of growth inhibition byFDP, revertants of strain NP 315 (fda-) wereisolated. At first, strains were selected whichregained the ability to grow on gluconate mini-mal medium plates at 40 C. Spontaneousrevertants to temperature-resistant growth ongluconate arose quite frequently. About 50 colo-nies per 108 cells plated appeared at 40 C.Fifty-six of these 40 C revertants were analyzedfor the enzymes of gluconate metabolism andfor inhibition of gluconate-6-phosphate dehy-drogenase by FDP (3). Fifty-four of them turnedout to possess a drastically reduced activity ofgluconate-6-phosphate dehydrogenase (Table1). No change of the pattern of inhibition ofgluconate-6-phosphate dehydrogenase by FDPcould be found in the two residual strains.These apparent gnd- strains were used to se-lect spontaneous revertants able to grow at 40C on glucose minimal medium plates. A hightemperature dependency of appearance ofphenotypic glucose-positive revertants couldbe observed. At 35, 38, and 40 C about 8,000,600, and 340 temperature-resistant coloniesappeared per 3 x 108 cells plated, respectively.Most of the low-temperature revertants

showed improved FDP aldolase activities,whereas enzymatic analysis of a total of nine40 C revertants revealed that, in addition to thefda and gnd mutations, seven of them showeda drastically reduced activity of phosphoglu-cose isomerase (Table 1). Growth on fructose of

TABLE 1. Specific activities of 6-phosphogluconatedehydrogenase and phosphoglucose isomerase in

cell-free extracts of strains NP 315, NP 3151, and NP31515 grown on gluconate minimal medium at 30 C

6-Phosphogluco- PhosphoglucoseStrain nate dehydrog- isomerase

enase sp acta sp acta

NP 315 (fda- 116 660NP 3151 (fda- <4 646gnd-)

NP 31515 (fda < 1 < 1gnd- pgi-)

a Specific activities are expressed in nanomoles ofsubstrate converted per minute per milligram proteinat 20 to 22 C. All extracts were devoid of fructose-1, 6-diphosphate aldolase activity and showed wild-type-like glucose-6-phosphate dehydrogenase activity.

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GLUCOSE METABOLISM IN E. COLI MUTANTS

these apparent fda- gnd- pgi- strains was stillstrictly temperature-sensitive. Table 2 lists thegrowth rates of some of these revertants withglucose and gluconate as substrate. It showsthat gluconate-supported growth is near nor-

mal; this confirms the result of Fraenkel (7) thatthe gnd function is dispensable during gluconate-supported growth. Doubling times of strain NP31515 (fda- gnd- pgi-) on glucose were found tobe variable within the indicated range in differ-ent experiments. Colony formation on glucoseminimal medium was first visible within 3 daysat 40 C.

These results show that strain NP 315 fda-,therefore, provides a convenient means for rap-idly selecting mutants in the gnd and pgi lociwithout any mutagenesis and enrichment proce-dure. This technique should work for selectionof mutants in any other enzyme of the Cf- or

C5-carbohydrate metabolism because release ofgrowth inhibition seems to be a result of theprevention of FDP accumulation, so any muta-tion blocking carbon flow from the substrateofferred in the medium to FDP should releasegrowth inhibition. As expected, the gnd muta-tion of strain NP 3151 (fda- gnd-) prevents thebuild-up of a detectable FDP pool on gluconatemedium and the gnd and pgi lesions in NP31515 (fda- gnd- pgi-) the accumulation ofFDP both on glucose and gluconate medium(Table 3). Fructose, which at 40 C cannot bemetabolized via the hexosemonophosphateroute nor by glycolysis, does not give rise to highFDP values in strain NP 31515 (fda- gnd- pgi-)probably because of lack of energy for uptakeand phosphorylation.Route of glucose degradation in fda- gnd-

pgi- strains. Under the assumption that the invitro-determined enzyme activities reflect thein vivo conditions, the lesions in the fda- gnd-pgi- strains separate the glycolytic and thepentose phosphate sequence from a hypotheti-cally possible route for glucose degradation viaglucose-6-phosphate 6-phosphogluconolac-tone gluconate-6-phosphate and the Entner-

Doudoroff enzymes. The assumption of such a

pathway, however, does not agree with thebehavior of gnd- pgi- double mutants isolatedby Kupor and Fraenkel (17; and D. G. Fraenkel,personal communication) which are completelyunable to grow on glucose. The elucidation ofthe pathway for glucose dissimilation in our

strains, therefore, seemed desirable. In a firststep the specific activities of gluconokinase(glk) and of 2-keto-3-deoxy-6-phosphogluconatealdolase (eda) were determined in order toassess any contribution of these enzymes toglucose degradation. The cells were grown for

TABLE 2. Mean doubling times of strains NP315, NP3151, and NP 31515 on glucose and gluconate minimal

medium

Growth Doubling times (min)Strntemp (C) Glucose Gluconate

NP 315 30 165 135NP 315 40 NGa NGNP 3151 30 180 150NP 3151 40 NG 100NP 31515 30 210-270 150NP 31515 40 240-330 105

aNo growth.

this purpose at 30 C to preclude any interfer-ence of aldolase inactivation with any possibleinduction of the enzymes of gluconate metabo-lism. Gluconokinase levels of strains NP 3151and NP 31515, upon growth on glucose at 30 C,nearly reach those which are observed in crudeextracts of gluconate cells (Table 4). Strain NP315 possesses an appreciably induced level, too,which is in accordance with the evidence forgluconate excretion during glucose-supportedgrowth of this strain (1). 6-Phosphogluconatedehydrase and 2-keto-3-deoxy-6-phosphoglu-conate aldolase activities, which were deter-mined in a coupled assay, are significantly in-duced in glucose cells of strains NP 3151 andNP 31515, whereas in the parent strain NP 315their level is almost completely repressed.

Direct evidence for the use of the Entner-Doudoroff pathway for glucose degradationcomes from the labeling pattern of L-alaninefrom cells grown at the expense of [1-"4C]glucose or [1- 4C]gluconate, respectively(10). The wild strain K-10 almost exclusivelydegrades glucose via glycolysis and the hexose-monophosphate route (Table 5). The slightlyincreased labeling of the carboxyl group ofL-alanine derived from protein of glucose-growncells of strain NP 315 indicates a minor contri-bution of the Entner-Doudoroff path to pyru-vate formation. In contrast, pyruvate is synthe-sized in strains NP 3151 and NP 31515 to a con-siderable extent via the Entner-Doudoroffpathway.The induced level of gluconokinase observed

in strains NP 3151 (fda- gnd-) and NP 31515(fda- gnd- pgi-) suggests gluconic acid-theinducer of this enzyme-to be an intermediateof glucose degradation by these cells. Such arole of gluconate in glucose metabolism hasbeen described by Kupor and Fraenkel (17) forphosphogluconolactonase mutants of E. coliwhich excrete gluconolactone and rephosphory-late its extracellular hydrolysis-product glu-

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TABLE 3. Intracellular FDP concentration in strains K-10, NP315, NP3151, and NP31515 at different growthconditionsa

nmol of FDP/mg of cell proteinStrain

Glucose, 30 C Glucose, 40 C Gluconate, 30 C Gluconate, 40 C

K-10 8 (<2)" <2 (<2) 5 (5) 2 (3)NP 315 49 (16) 260 (55) 31 (5) 236 (62)NP 3151 39 (14) 244 (43) 4 (<2) 2 (8)NP 31515 7 (4) <2 (8) 14 (<2) <2 (<2)

a Cells were grown in minimal medium with 0.4% carbon source; in the case of of the 40 C values the cultureswere exposed to the high temperature for 60 min.

b Numbers in parentheses represent nanomoles of FDP leaked into the medium from an amount of cellsequivalent to 1 mg of cellular protein.

TABLE 4. Specific activities of gluconokinase and of 6-phosphogluconate dehydrase and KDPG aldolase incell-free extracts of strains K 10, NP 315, NP 3151, and NP 31515 grown at 30 C.

Sp act of glu- Sp act of Entner-

conokinasea ~~~Doudoroff enzymes"Strain conokinasea (edd and eda)

Glucose Gluconate Glucose Gluconatecells cells cells cells

K-10 10 72 9 132NP 315 (fda- 45 118 14 88NP 3151 (fda- gnd) 68 133 39 123NP 31515 (fda- gnd- pgi-) 112 118 51 110

a Expressed as nanomoles of gluconate phosphorylated per minute x milligrams of protein.b Expressed as nanomoles pyruvate formed per minute x milligram of protein.

TABLE 5. Distribution of label from [1-_4C]glucose inCl and C3 of L-alanine in the protein of strains K 10,NP 315, NP 3151, and NP 31515 grown at 30 C

dpm in the carboxylStrain Substrate group of L-alanine

(%)

K-10 Glucose 8.9K-10 Gluconate 95.6NP 315 Glucose 16.8NP 315 Gluconate 96.3NP 3151 Glucose 35.9NP 3151 Gluconate 96.8NP 31515 Glucose 70.3NP 31515 Gluconate 96.1

conic acid. The existence of a similar bypass instrains NP 3151 (fda- gnd-) and NP 31515(fda- gnd- pgi-) was tested by growth on

[1- '4C]glucose and chromatographic analysis ofany detectable intermediates (17). Figure 3shows the results for strain NP 3151. At 30 C(Fig. 3A) levels of glucose-6-phosphate, 6-phos-phogluconate, gluconolactone, and gluconateare small, but detectable. At 40 C (Fig. 3B) a

preferential accumulation of gluconate takesplace. Figure 4A shows the results of the same

experiment done with strain NP 31515 at 40 C.

Again, an accumulation of gluconate and also ofglucose-6-phosphate and 6-phosphogluconatecan be observed. No gluconolactone could bedetected on the thin-layer plates in this experi-ment. 30 C cells of strain NP 31515 (not shown)essentially yield the same result.

Figure 4B more directly demonstrates theintermediate role of gluconic acid in glucosemetabolism by strain NP 31515. In this experi-ment, glycerol-grown cells were transferred to[1- 4C]glucose in the presence of chloram-phenicol. This treatment prevents the inductionof gluconokinase (which is absent in cell-freeextracts of glycerol cells) and also causes glu-conate to accumulate to a much higher levelthan that observed in the absence of chloram-phenicol. The experiments presented thereforeshow that a bypass sequence

Piglucose -. glucose-6-phosphate -. I -;

adenosine 5'-triphosphategluconate-*u 6-phosphogluconate

is followed in cells from strain NP 31515 (fda-gnd- pgi-). It is not yet clear, however, fromthese data if the intermediate I which is dephos-phorylated is 6-phosphogluconolactone or 6-phosphogluconate.

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30 40 t

TIME (min)FIG. 3. Chromatographic separation of intermediates from glucose metabolism. A culture of strain NP 3151

was grown in glycerol minimal medium to an OD40, of 1.18, washed with basal salts solution, and incubatedwithout carbon source for 30 min at 30 C. A 0.4-ml amount of this culture was incubated with 0.01 ml of[12Cjglucose (2.6 mg/ml) and 0.01 ml [1-14CJglucose (57 ,gCi4smol) at 30(A) or 40 C (B). Samples of 0.02 ml weretaken at the indicated times into 0.02 ml of 0 C acetone and chromatographed in the systems described. Valuesfor glucose (0), gluconate (0), glucose-6-phosphate (A), and 6-phosphogluconate (0) are from the paperchromatograms; those for gluconolactone (A) are from the cellulose thin-layer plates. Values in A for gluconate,gluconolactone, 6-phosphogluconate, and glucose-6-phosphate are multiplied with a factor of 2.

Dephosphorylation of 6-phosphogluconolac-tone would lead to increased levels of 6-gluconolactone which has been shown to beresponsible for conferrring to lactonase-deficientcells the "maltose-blue" phenotype (17). StrainNP 31515 was checked for this property andshowed the maltose-blue characteristic on mal-tose-glycerol plates (17) both at 30 and 40 C.The preferential dephosphorylation of 6-phos-

phogluconolactone which is indicated by themaltose-blue phenotype should also be cor-related with a decreased concentration of 6-phosphogluconate in the cellular pool. Indeed,strain NP 31515 accumulates less 6-phosphoglu-conate than glucose-6-phosphate in the solublepool, which could (amongst other possible ex-planations) be interpreted as a preferentialdephosphorylation of 6-phosphogluconolactone

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SCHREYER AND BOCK J. BACTERIOL.

u ~~0Q 3.10

2.10'~~~~~~~~~

10'

a~

6.104' El

5.10'-

2.10'2 -°/

E

0.~ ~ ~

10*'0 o

10 20 310 L 50 60 70 60 m 10

TIME (min)FIG. 4. A, Intermediates of glucose metabolism of strain NP 31515 at 40 C. A 0.8-ml amount of a

glycerol-grown culture (OD 1.22) of strain NP 31515 (washed and preincubated as described in the legend toFig. 3) was mixed with 0.02 ml of [1-14Clglucose (57,uCi/,umol) and 0.02 ml ofglucose (2.6 mg/ml) and incubatedat 40 C. Samples (0.02 ml) were taken, mixed with 0.02 ml of acetone, and chromatographed (17). Nogluconolactone could be detected on thin-layer plates. Symbols: 0, glucose; *, gluconate; A, glucose-6-phos-phate; 0, 6-phosphogluconate. B, The experiment was done as described underA except that chloramphenicolwas given together with the labeled glucose to cells of strain NP 31515 in a final concentration of 100 ,g/ml.Samples (0.04 ml) were taken, the cells were removed from it by centrifugation, and 0.02 ml of the supernatant

fluid were spotted directly onto the chromatograms. Symbols: 0, glucose; 0, gluconate; 0, 6-phosphoglaco-nate. No gluconolactone could be detected on cellulose thin-layer plates.

(Table 6). The high level of 6-phosphogluconatein the medium which could also be detected as

metabolite in the experiments illustrated byFig. 3 and 4, however, provides additionalevidence for 6-phosphogluconolactone excretionwithout any preceding dephosphorylation.

Several possible explanations could account

for the observation that the fda- gnd- pgi-strain metabolizes glucose via an apparentlysimilar route as 6-phosphogluconolactonase-deficient strains (17). (i) Either some metabo-lite which accumulates in these mutants couldinhibit the activity of the lactonase (ii) StrainNP 31515 (fda- gnd- pgi-) also could carry a

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TABLE 6. Intracellular pools of glucose-6-phosphate and 6-phosphogluconate in strains NP 3151 (fda- gnd-)and NP 31515 (fda- gnd- pgi-)

Glucose-6- 6-phospho-Strain Substrate Temp (C) phosphate gluconate(nmol/mg of (nmol/mg of

protein) protein)

NP 3151 Glucose 30 11 (24)b 37 (31)NP 3151 Glucose 30/40a 140 (36) 100 (36)NP 3151 Gluconate 30 13 ( <2) 26 (15)NP 3151 Gluconate 30/40 < 2 (20) 18 ( <2)NP 31515 Glucose 30 89 (255) 6 (200)NP 31515 Glucose 30/40 100(258) 10 (199)

a Pools measured 60 min after a shift from 30 to 40 C.b Numbers in parentheses represent nanomoles of sugar phosphates in the medium from an amount of cells

equivalent to 1 mg of cellular protein.

lesion in the pgl structural gene; this possibility,however, seems improbable because the gluco-nate and glucose positive revertants arose spon-taneously with a frequency incompatible withthe assumption of a double mutation; or (iii) thegnd mutation causes all metabolites prior to thelesion to accumulate, and the phosphataseinvolved in dephosphorylation preferentiallyacts on 6-phosphogluconolactone.

It still needs to be discussed why the pgi-gnd- strains of E. coli isolated by Kupor andFraenkel (17) by selection for inability to formcolonies on glucose plates (17) do not exhibit thesame pattern of glucose metabolism describedfor our fda- gnd- pgi- mutants (which wereselected for ability for glucose supportedgrowth). At the moment, no definite answer ispossible. In vivo leakiness of the mutationalblocks of our strains which could facilitateinduction of the Entner-Doudoroff enzymes bythe extracellular gluconate might be responsiblefor the different behaviour. The assumption ofphysiological leakiness is supported by the ex-periment of Fig. 4B. Conversion of glucose togluconate in the absence of a functioning Ent-ner-Doudoroff path may only be explained ifsome phosphoenolpyruvate necessary for glu-cose uptake may be formed either via glycolysisor the hexosemonophosphate route.

ACKNOWLEDGMENTSWe are very much indebted to Dan G. Fraenkel for

valuable suggestions and for reading the manuscript.

LITERATURE CITED

1. Bock, A., and F. C. Neidhardt. 1966. Properties of amutant of Escherichia coli with a temperature-sensi-tive fructose-1,6-diphosphate aldolase. J. Bacteriol.92:470-476.

2. Bock, A., and F. C. Neidhardt. 1966. Isolation of amutant of Escherichia coli with a temperature-sensi-tive fructose-1,6-diphosphate aldolase activity. J. Bac-teriol. 92: 464-469.

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