JOURNAL OF BACTERIOLOGY, Oct 1971, p. 137-144Copyright 0 1971 American Society for Microbiology

Vol. 108, No. IPrinted in U.S.A.

Lethal Synthesis of Methylglyoxal by Escherichiacoli During Unregulated Glycerol Metabolism1

W. B. FREEDBERG, W. S. KISTLER,2 AND E. C. C. LIN

Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115

Received for publication 28 May 1971

In Escherichia coli K-12, the conversion of glycerol to triose phosphate is regu-

lated by two types of control mechanism: the rate of synthesis of glycerol kinaseand the feedback inhibition of its activity by fructose-1,6-diphosphate. A strainwhich has lost both control mechanisms by successive mutations, resulting in theconstitutive synthesis of a glycerol kinase no longer sensitive to feedback inhibi-tion, can produce a bactericidal factor from glycerol. This toxic factor has beenidentified by chemical and enzymological tests as methylglyoxal. Methylglyoxalcan be derived from dihydroxyacetone phosphate through the action of an enzymewhich is present at high constitutive levels in the extracts of the mutant as well as

that of the wild-type strain. Nine spontaneous mutants resistant to I mm exoge-nous methylglyoxal have been isolated. In all cases the resistance is associated withincreased levels of a glutathione-dependent enzymatic activity for the removal ofmethylglyoxal. Methylglyoxal-resistant mutants derived from the glycerol-sensitiveparental strain also became immune to glycerol.

The initial steps in the dissimilation of glyceroland L-a-glycerophosphate (L-a-GP) in Esche-richia coli are outlined in Fig. 1. Glycerol entersthe cell by facilitated diffusion (34) and is re-tained as L-a-GP after its phosphorylation byglycerol kinase, whose activity is subject to re-mote feedback inhibition by fructose- , 6-di-phosphate (21, 44). L-a-GP enters the cell via anactive transport system (20). Intracellular L-a-GP may be acted upon by two specific pyridinenucleotide-independent L-a-GP dehydrogenases:an aerobic enzyme associated with the particu-late fraction, and an anaerobic enzyme found inthe soluble fraction whose activity is stimulatedby flavine adenine dinucleotide and flavine mon-onucleotide (25, 30). The expression of the genesfor all the above proteins is under the control ofthe glp regulator gene (10, 28).

In previous communications (6, 44), it hasbeen shown that cells of a mutant E. coli strainconstitutive in the gip system and producing analtered glycerol kinse which is insensitive to inhi-bition by fructose- I, 6-diphosphate are killedwhen exposed to glycerol during growth on suc-cinate or casein amino acids. Zwaig and Dieguezhave further shown that succinate-grown cells of

' Part of this work was presented before the American So-ciety of Biological Chemists, San Francisco, Calif., June 1971.

2 Present address: Ben May Laboratory for Cancer Re-search and the Department of Biochemistry, The University ofChicago, Chicago, III. 60637.

this strain, suspended in an unbuffered solutionof glycerol, excreted a dialyzable product whichis toxic to a variety of bacterial cells, includingboth gram-positive and gram-negative species(43). These workers found the bactericidalproduct to be stable to heating at 100 C for 10min and to have a molecular weight of less than500 by gel-exclusion chromatography (43).

In this paper we present chemical, enzymologi-cal, and genetic evidence to show that this lethalfactor is methylglyoxal (2-oxopropanal). Cooperand Anderson (7) have discovered in E. coli anovel enzymatic activity catalyzing the formationof methylglyoxal from dihydroxyacetone phos-phate (DHAP). Data will be presented whichindicate that this enzyme activity plays a role inthe lethal synthesis of methylglyoxal in strainswith uncontrolled dissimilation of glycerol.

MATERIALS AND METHODSBacterial strains. All bacterial strains described are

ultimately derived from strain 1, an E. coli K-12 strainwith a deletion in the alkaline phosphatase structuralgene (28). Strain 7, derived directly from strain 1, syn-thesizes the gene products of the gip regulon constitu-tively, rather than inducibly (28). Strain 43 in turn wasderived from strain 7 as a mutant which produces analtered glycerol kinase insensitive to feedback inhibi-tion by FDP (45). Methylglyoxal-resistant derivativesof these three strains were obtained by direct plating onglucose minimal media containing 1.0 mm methyl-

137

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

b on

25

Nov

embe

r 20

21 b

y 19

0.14

1.20

0.15

7.

FREEDBERG, KISTLER, AND LIN

glyoxal, or an appropriate concentration of the lethalfactor from culture filtrates of strain 43 cells that hadbeen challenged with glycerol.

Growth of cells and preparation of extracts. The min-imal medium buffered by phosphate at pH 7.0 was thatpreviously described (38). Solid media with the samemineral pomposition were prepared by the addition ofagar (Difco) to a final concentration of 1.5%. Concen-trations of carbon sources in liquid or solid growthmedium were: glucose, 10 mM; glycerol, 20 mM; sodiumsuccinate, 15 mM. Succinate minimal medium was sup-plemented with 0.04% casein hydrolysate. Bacterialgrowth at 37 C with vigorous aeration was monitoredturbidometrically with a Klett colorimeter (no. 42 fil-ter, I unit = 3 x 10" bacteria per ml during exponen-tial growth).

For preparation of extracts, cells growing in 250-mlcultures in 2-liter Erlenmeyer flasks were harvested atthe late exponential phase, washed once by centrifuga-tion at 3 to 4 C with 0.9% NaCl, and suspended in 5ml of 25 mm imidazole-hydrochloride buffer (pH 6.6).The cells were disrupted by treatment for 5 min with a60-w sonic disintegrator (Measuring & ScientificEquipment, Ltd., London). A dry-ice-ethanol bath at-10 C served to prevent overheating of the extractsduring sonic treatment. The sonically treated crude ex-tracts were clarified by centrifugation at 12,000 x g for20 min at 3 to 4 C. Extracts were stored at -20 C.

Enzyme assays. Glycerol kinase was measured by aspectrophotometric assay at pH 7.5 (45). The aerobicL-a-GP dehydrogenase was assayed by coupling theoxidation of L-a-GP to the reduction of a tetrazoliumdye to its formazan (30) under a condition where thereis little or no contribution from the flavine-stimulated,anaerobic enzyme (25). Enzymatic production of meth-ylglyoxal from DHAP was assayed by monitoring theincrease in absorbancy at 240 nm due to the formationof S-lactylglutathione in the presence of excess reducedglutathione and glyoxalase I. The complete reactionmixture contained 25 Amoles of imidazole-hydrochlo-ride buffer (pH 6.6), 1.6 gmoles of glutathione, 0.8jumole of DHAP, 50 jg of glyoxalase I, and E. colicrude extract in a final volume of 1.0 ml. The reactionwas initiated by the addition of DHAP or crude ex-tract. Extracts were diluted in 25 mM imidazole buffer(pH 6.6).

MEDIUM

CELL

GlycrolDi ffusig

L-w-GPI

Transro

~ed61I i Frucfose l.6 JiP

> hAnerobic-% L-o-GP Aerobic DHAP GAP

DelyJro3eneses

FIG. 1. Pathways for glycerol and L-a-glycero-phosphate (L-a-GP) dissimilation in Escherichia coli.DHAP, dihydroxyacetone phosphate; GAP, D-glycer-aldehyde-3-P.

Enzymatic determination of intermediates. L-a-GPwas measured spectrophotometrically with the aid ofrabbit muscle nicotinamide adenine dinucleotide(NAD)-linked L-a-GP dehydrogenase. Reduced NAD(NADH) production from L-a-GP-containing sampleswas monitored at 340 nm in 0.3 M sodium carbonate,30 mm hydrazine, and 20 mM NAD, with 20 Mg of en-zyme, in a final volume of 1.0 ml at pH 9.5. For enzy-matic determination of glycerol, the same reactionsystem was used but with the addition of 10 mMMgCI2, 10 mm adenosine triphosphate, and 20 jug ofcrystalline Candida mycoderma glycerol kinase per mlto convert glycerol to L-a-GP. L-Lactate was estimatedin the same manner as L-a-GP, with 50 Mg of crystal-line rabbit muscle NAD-linked L-lactate hydrogenasein place of L-a-GP dehydrogenase. DHAP concentra-tions were determined by measuring the extent of oxi-dation of NADH in a reaction system containing 0.1Mmole of NADH, 50 umoles of NaHCO, (pH 8.3),and 30 Mg of rabbit muscle L-a-GP dehydrogenase in afinal volume of 1.0 ml. Crystalline triose phosphateisomerase (50 Mg) was added to this reaction systemfor the determination of D-glyceraldehyde-3-phosphate.Methylglyoxal was determined spectrophotometricallyby monitoring the formation of S-lactylglutathionecatalyzed by glyoxalase I as described by Klotzsch andBergmeyer (26).

Preparation, concentration, and assay of the lethalfactor. Late-logarithmic-phase cells of strain 43growing in succinate minimal medium (160 to 170Klett units) were harvested by centrifugation, sus-pended in an equal volume of sterile 10 mm glycerolsolution made up in deionized distilled water, and incu-bated with shaking for 3 hr at 37 C. The incubationsolution was recovered and freed from the cells by apreliminary low-speed centrifugation, followed by fil-tration through a membrane filter (0.45 Mim pore size,HAWP type, Millipore Corp., Bedford, Mass.). Thefiltrate was usually concentrated 50-fold by rotary flashevaporation with reduced pressure at a bath tempera-ture of 40 to 50 C. For factor bioassay, 0.1-ml portionsof a 1:100 dilution of an overnight broth culture ofstrain 1 were plated on glucose minimal medium.Wells, 10 mm in diameter, were made in the seededplates with a sterile cork borer. When 0.1 ml of 50-foldconcentrated lethal filtrate was added to a well, a zoneof inhibition 35 to 40 mm in diameter was observedafter overnight growth at 37 C. This assay provided aconvenient semiquantitative test for assessing the effectof various physical and chemical treatments on theunknown lethal factor. To prepare growth media con-taining the lethal factor at its original concentration,appropriate amounts of the membrane-filtered concen-trated fluid were diluted into liquid or solid culturemedium made up as described above.

Chromatography. In system A, Whatman no. Ipaper was developed in the ascending direction with n-butanol equilibrated with an equal volume of 1.5 MNH4OH. In system B, Whatman no. I paper was de-veloped in the descending direction with n-butanolethanol-water (3: 1: 1). In systems C and D, Eastmanterphthalate-backed silica gel chromatogram sheets(no. 6061) were developed in the ascending directionwith chloroform and benzene, respectively. To demon-

138 J. BACTERIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

b on

25

Nov

embe

r 20

21 b

y 19

0.14

1.20

0.15

7.

LETHAL SYNTHESIS OF METHYLGLYOXAL

strate glycerol and methylglyoxal, chromatograms weresprayed with or dipped in AgNO, reagent (saturatedAgNO,-acetone, 1: 200), dried, and sprayed with ordipped in ethanolic NaOH. Carbonyl compounds werelocated by spraying chromatograms with 25 mm 2,4-dinitrophenylhydrazine reagent and 90 mm HCI inmethanol. Carbonyl compounds chromatographed astheir 2,4-dinitrophenylhydrazine derivatives were ren-dered visible by ethanolic NaOH.

Cbemical methods. The 2,4-dinitrophenylhydrazonesof carbonyl compounds present in culture filtrates wereprepared by the addition of an equal volume of 10 mM2,4-dinitrophenylhydrazine and 2 N HCI to a sample ofappropriately diluted or concentrated filtrate. Proteinwas determined by the biuret method (17).

Cbemicals. Rabbit muscle L-a-GP dehydrogenase, L-lactate dehydrogenase, and C. mycoderma glycerolkinase were obtained from C. F. Boehringer & Soehne,Mannheim, Germany. Glyoxalase I (from yeast), tri-ose phosphate isomerase (rabbit muscle), and methyl-glyoxal were obtained from the Sigma Chemical Co.,St. Louis, Mo. Methylglyoxal was purified by directsteam distillation. Uniformly labeled "4C-glycerol wasa product of ICN Chemical and Radioisotope Division,Irvine, Calif. Casein amino acids (salt-free, vitamin-free) were obtained from the Nutritional BiochemicalsCorp., Cleveland, Ohio. Glutathione was obtained fromSchwarz BioResearch, Inc., Mt. Vernon, N.Y.

RESULTSCharacterization of the bactericidal product as

a carbonyl compound. Although it was stable totreatment with strong acid (I N HCI at 50 C for15 min), the bactericidal product formed bystrain 43 was readily inactivated by treatmentwith weak alkali, as measured by the bioassaydescribed above. All bactericidal activity was lostwhen 50-fold concentrated preparations of thelethal factor were adjusted to pH 10 with 0.1 MNa2CO8. The extreme alkaline lability suggestedan essential sulfhydryl or carbonyl group. Theformer possibility, however, was eliminated bythe resistance of the biological activity to treat-ment with N-ethylmaleimide. The involvement ofa carbonyl group was confirmed by the followingfindings. Treatment of concentrated lethal factorpreparations with NaHSO, (final molarity: 0.1M) completely inactivated the factor. A concen-trated solution of the bactericidal product pre-pared from strain 43 gave copious orange precip-itates when treated with the 2, 4-dinitrophen-ylhydrazine reagent, whereas a control solutionprepared from strain I contained no lethal factorand gave no precipitate. A similarly preparedconcentrated filtrate from strain 43 pregrown onsuccinate and challenged with 0.2% glucose in-stead of glycerol was shown to have no bacteri-cidal activity and gave no precipitate.

Characterization of the 2,4-dinitrophenyl-hydrazine derivative. When bactericidal factorpreparations were treated with excess 2,4-dini-trophenylhydrazine reagent, only a single spotwas detected on chromatograms developed insolvent systems A and D. This single spot gave abright red color on exposure to ethanolic NaOH.RF values of the derivative were 0.85 and 0.28,respectively, in systems A and D. When bacteri-cidal factor preparations were treated with lim-iting concentrations of 2,4-dinitrophenylhydra-zine, the formation of two additional derivativeswas observed. Thus it seems that the unknownfactor is a dicarbonyl compound; under condi-tions of reagent excess, a bis-2, 4-dinitro-phenylhydrazone was formed, and under condi-tions of limited reagent mono-derivatives wereformed. Bis-2, 4-dinitrophenylhydrazones of re-distilled authentic methylglyoxal and of the un-known, prepared in parallel in the presence ofexcess reagent, were found to have indistin-guishable chromatographic properties. Moreover,melting points of both preparations (recrystal-lized from water) were identical (292.5 to 293.5C, corrected), and no depression of melting pointwas observed when the preparations were mixed.Both derivatives were readily dissolved in ethyla-cetate but could not be reextracted from ethyla-cetate into 1 M Na2CO3. The absorption spectraof the derivatives of the bactericidal product andauthentic methylglyoxal were identical, both be-fore and after treatment with alkali.Enzymatic confirmation of the identity of the

lethal factor. The release of methylglyoxal intothe incubation fluid of strain 43 cultures chal-lenged with glycerol was confirmed by specific a-ketoaldehyde assay using the glyoxalase I assayof Klotzsch and Bergmeyer (26). Enzymaticallydetermined methylglyoxal concentrations in thefiltrates (before concentration) ranged between0.8 and 1.4 mm. The average concentrationfound in four separate experiments was 1.2 mM.Control filtrates from glycerol-challenged cells ofstrain 1 contained less than 0.01 mm methyl-glyoxal. Further evidence that the toxic com-pound was methylglyoxal was obtained from thefollowing experiment.An aqueous methylglyoxal solution and a

preparation of the lethal factor were each dividedinto two portions, one maintained at neutral pHand the other adjusted to pH 11.0 with NaOH.The four solutions were kept at 3 to 4 C over-night and then assayed for a-ketoaldehyde and L-lactate content. Treatment of the methylglyoxalsolution or the lethal factor preparations withalkali resulted in the loss of all a-ketoaldehydeaccompanied by the formation of L-lactate, oneof the predicted products of the alkaline rear-

139VOL. 108, 1971

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

b on

25

Nov

embe

r 20

21 b

y 19

0.14

1.20

0.15

7.

FREEDBERG, KISTLER, AND LIN

rangement of methylglyoxal, in 20% yield (Table1).Furthermore, system B chromatograms of le-

thal factor-containing filtrates from suspensionsof strain 43 cells challenged with "4C-glycerolrevealed the presence of only two radioactivespots: untransformed glycerol and methylglyoxal.Enzymatic determinations of intermediates werealso performed on such a filtrate, the results ofwhich are summarized in Table 2. In view ofthese findings, and of the demonstration belowthat strain 43 contains high levels of all threeenzymes required for the rapid conversion ofglycerol to methylglyoxal, it seems unlikely thatthe toxic carbonyl compound is any other thanthe 3-carbon a-ketoaldehyde, methylglyoxal.

Enzymatic pathway leading to methylglyoxalformation. It was shown previously that the pro-duction of the lethal factor required not only afeedback-insensitive glycerol kinase but also thesynthesis of this altered enzyme at a high level(44). It has been shown also that cells possessingthe above characters but lacking a functional L-a-GP dehydrogenase could no longer produce thebactericidal product (44), thus indicating thatmetabolism of glycerol to a product distal to L-a-GP was required. Anderson and Cooper recentlydemonstrated that E. coli mutants lacking triosephosphate isomerase could still convert glycerolto glycogen and that the extracts catalyzed theconversion of DHAP to methylglyoxal and inor-ganic phosphate (1, 7). We found that strains 1,7, and 43 grown on succinate contained a similaractivity at a level of 0.1 to 0.3 Amole per min permg of soluble protein. Figure 2 presents data onsome kinetic properties of the enzymatic activityfor synthesis of methylglyoxal from DHAP. Therelatively high Km for DHAP observed (0.7 mM)and the complex kinetics of inhibition by inor-ganic phosphate are strikingly similar to thosepresented by Hopper and Cooper (7, 23), leavinglittle doubt that the same enzyme was involved inboth cases (Table 3).

Although succinate-grown cells of strains 1, 7,

TABLE 1. Effect of alkali on methylglyoxal and on thelethal factor

Prepn a-Keto- L-Lactatcealdehyde~

Lethal factorpH 7 51 0.5pH 11 0.4 10

MethylglyoxalpH 7 34 0.6pH 11 0.1 6.3

a Expressed in micromoles per milliliter.

TABLE 2. Concentration of3-carbon intermediates infiltrates containing the lethalfactor

Compound Concna

Glycerol ........................ 9.6 1.2L-a-Glycerophosphate ...... ...... < 0.02Dihydroxyacetone phosphate ....... <0.02D-Glyceraldehyde-3-phOsphate .... <0.02Methylglyoxal ................... 1.4 + 0.1

aExpressed as micromoles perminus average deviation.

V

milliliter plus or

1000 2000 3000 000 5000 6000(DHAP)-'

FIG. 2. Kinetic properties of the enzymatic activityconverting dihydroxyacetone phosphate to methyl-glyoxal found in crude extracts of strain 1. DHAP,dihydroxyacetone phosphate.

and 43 all contained high levels of methylglyoxalsynthase activity, only the constitutive strains 7and 43 had the necessary high levels of glycerolkinase and aerobic L-a-GP dehydrogenase re-quired to produce a flood of DHAP, the sub-strate for methylglyoxal synthase (Fig. 3). Butunder physiological conditions, inhibition of thekinase by fructose- 1, 6-diphosphate apparentlyprevented cells of strain 7 from the lethal syn-thesis.

Strain 7 cells grown on succinate medium andchallenged with aqueous glycerol can indeed pro-duce methylglyoxal (Table 4). Zwaig (personalcommunication) previously observed productionof the toxic factor by this strain. Presumably, theosmotic shock removed the fructose-I,6-diphos-

140 J. BACTERIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

b on

25

Nov

embe

r 20

21 b

y 19

0.14

1.20

0.15

7.

LETHAL SYNTHESIS OF METHYLGLYOXAL

TABLE 3. Kinetic parameters ofEscherichia colimethylglyoxal synthase

Determination Hopper and PresentCooper (23) studies

Km for DHAP 0.5 mm 0.7 mM(PJ)05a 0.6 mM

Response to DHAP in 2.9c 3.0dpresence of Pi, slope ofHill plotb

Response to Pi, slope of 2.7'Hill plote

a Concentration of modifier giving one-half maximalinhibition. See reference 5.

Log [Vobserved/(Vmaximum - Vobserved)] plottedagainst log (DHAP).

c (P,), 0.3 mM.d (Pi), 1.0 mM.e Log of the ratio (V,)/(V - V,) plotted against log

(PI), where V, is the rate of reaction measured in thepresence of orthophosphate, and V is the rate observedin the absence of orthophosphate. (PI), concentrationof inorganic phosphate.

f Dihydroxyacetone phosphate (DHAP), 0.8 mm.

0.

-0-

I

a.-

ELa

-t

._;0

IL7ycerol L-w-OP MethylglyoaalKinase Dehydroenase Synthase

FIG. 3. Activities of glycerol kinase, aerobic L-a-glycerophosphate (L-a-GP) dehydrogenase, and meth-ylglyoxal synthase in Escherichia colt strains 1, 7, and43 grown on succinate.

phate necessary for curbing the activity of thekinase. Strain I cells uninduced in the glp systemproduced essentially no methylglyoxal, even in ahypotonic solution, as one would expect.

The production of methylglyoxal by strain 43is not restricted to cells which had been pregrownin a succinate medium. Table 5 shows a varietyof media which permitted the excretion of meth-ylglyoxal upon challenge with glycerol. Succi-nate, lactate, and casein amino acids were amongthe most effective carbon sources for this pur-pose. The determining factor in the production ofthe toxic product thus appears to be a high levelof the kinase rather than the nature of thecarbon source per se during growth.Mechanism of methylglyoxal resistance. When

any strain of bacteria was plated on glucose min-imal media containing 1 mm methylglyoxal or anequivalent concentration of the toxic product asmeasured by the glyoxalase I assay, spontaneousresistant mutants were found at a frequency ofapproximately 10- 7. Mutants selected fromstrains 1, 7, and 43 for growth in the presence ofauthentic methylglyoxal were found to be re-sistant also to the bactericidal product derivedfrom glycerol, and vice versa. Unlike their par-ents, the resistant mutants actually were able toform small colonies on minimal agar containing1 mm methylglyoxal as sole carbon source.

TABLE 4. Methylglyoxal formation by strains 1, 7, and43 during hypotonic exposure to glycerol

Straina Methylglyoxalformed"

I <<0.027 0.69

43 1.45

a Cells grown on succinate minimal medium.b Millimolar concentration found in the incubation

fluid after 3 hr of hypotonic exposure to glycerol (10mM) at 37 C.

TABLE 5. Formation of methylglyoxal by strain 43grown on various carbon sources

Carbon source Methylglyoxalfor growtha formed'

Casein amino acids ...... ......... 1.85Succinate ........................ 1.45D-Lactic acid .................... 0.88Threonine ....................... 0.45

Mannitol ........................ 0.34L-a-Glycerophosphate ..... ....... 0.26Glycine ......................... 0.22

Lactose ......................... 0.20

Glucose ......................... 0.17Glycerol ........................ 0.13

a Carbon sources present at 0.06 M carbon, with theexception of casein amino acids, present at 1.0%.

b Millimolar concentration found in incubation fluidafter 3 hr of hypotonic exposure to glycerol (10 mM)at 37 C.

2 Strain 1

.1

3

I Strai 7

3 7 I,f^stloOv.fS 3<-vfosbacl IrwklWiftnI Strain 3

r D sSsLl

VOL. 108, 1971 141

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

b on

25

Nov

embe

r 20

21 b

y 19

0.14

1.20

0.15

7.

FREEDBERG. KISTLER, AND LIN

To see if the methylglyoxal resistance is asso-ciated with a new or increased capacity to utilizethis compound, crude extracts of resistant andsensitive strains were examined for the presenceof an enzymatic activity capable of removingmethylglyoxal. Table 6 shows that such an ac-tivity could be demonstrated in vitro for all thestrains, but the mutant strains were found tohave from four to eight times more activity thanthe wild type. The activity in every case de-pended on glutathione, which points to glyox-alase as the responsible enzyme (27, 31). Thus itseems that resistance against methylglyoxal isacquired through an increased capacity for de-toxification rather than simple tolerance for thecompound.

Effect of methylglyoxal resistance on the re-sponse of strain 43 to glycerol. The questionarises as to whether resistance to methylglyoxalat an external concentration of I mm is sufficientto protect cells of strain 43 while they generatethe compound internally during exposure to glyc-erol. To answer this, strains 1, 7, 43 and threeresistant derivatives of strain 43 were grown onsuccinate. At mid-exponential phase, glycerolwas added to each culture. Only cells of strain 43were killed, whereas the rest continued to grow(Fig. 4). Examination of the culture filtrates re-

TABLE 6. Glutathione-dependent transformation ofmethylglyoxal by extracts of methylglyoxal-sensitiveand methylglyoxal-resistant strains of Escherichia coli

Strain Parent Sensitive/ Relativeresistant activitya

I K-12 Sensitive 1.0291 1 Resistant 4.4292 1 Resistant 6.9293 7 Resistant 4.8294 7 Resistant 3.8295 7 Resistant 7.7296 7 Resistant 5.3297 43 Resistant 5.9298 43 Resistant 5.7299 43 Resistant 4.0

a The activity of strain 1 (0.011 umole transformedper min per mg of soluble protein) is set at 1.0. Theactivity observed in the absence of glutathione was lessthan 0.002 uimole per min per mg of protein. The reac-tion mixtures contained 0.25 gmole of methylglyoxal,3.3 jsmoles of reduced glutathione, 50 jumoles of potas-sium phosphate buffer (pH 6.6), and E. coli crude ex-tract containing 50 to 300 ug of protein in a finalvolume of 0.5 ml. After 15 min of incubation at 30 C,the reaction mixtures were treated with 2,4-dinitro-phenylhydrazine reagent for 15 min as described. Fi-nally, 2.0 ml of 2.5 N NaOH was added, and theamount of methylglyoxal remaining was quantitated onthe basis of the absorption of the bis-2,4-dinitrophen-ylhydrazone in alkali at 550 nm.

3000 j

1000 [

E

U.

-0

300 [

100 F

30 [

10 F

30 2 3

Hours

FIG. 4. Immunity of methylglyoxal-resistant deriva-tives of strain 43 to glycerol. Succinate cultures ofstrains 1, 7, 43, 297, 298, and 299 were grown at 37 C.At mid-exponential phase, glycerol was added to aconcentration of20 mM. The number of viable bacteriaper milliliter in each culture was determined by platingon rich nutrient agar immediately before and at 1, 2,and 3 hr after the addition of glycerol to each culture.Symbols: strain I (0), strain 7 (A), strain 43 (0),strain 297 (x). The behavior of strains 298 and 299,independently isolated methylglyoxal-resistant deriva-tives ofstrain 43, was identical to that ofstrain 297.

vealed significant quantities of methylglyoxal(0.20 mM) only in the case of strain 43; the otherfiltrates contained less than one-tenth thisamount of methylglyoxal.

DISCUSSIONFor many years, methylglyoxal was considered

to be a possible intermediate in normal glycolysis(8, 19, 33, 35). However, the experiments ofLohmann (31), in which NAD rather than gluta-thione restored the ability of dialyzed muscleextracts to convert glycogen to lactic acid, andthe observed broad specificity of the glyoxalasesystem for a large number of a-ketoaldehydes(11) led to the denial of methylglyoxal as an in-termediate of the major glycolytic pathway (9,27). Cooper and his co-workers (7, 23) recentlydemonstrated that methylglyoxal synthase ispresent in E. coil and that this enzyme can pro-vide a dephosphorylating pathway for DHAP

142 J. BACTERIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

b on

25

Nov

embe

r 20

21 b

y 19

0.14

1.20

0.15

7.

LETHAL SYNTHESIS OF METHYLGLYOXAL

and thus is responsible for gluconeogenesis fromglycerol in a mutant lacking triose phosphateisomerase. Hence, both their studies and thosepresented here show that under certain circum-stances methylglyoxal is an important interme-diate in a side pathway of glycolysis (Fig. 5). Asstated by Hopper and Cooper (23), the strikinginhibition of methylglyoxal synthase by inorganicphosphate suggests that the physiological task ofthis enzyme is to replenish intracellular inorganicphosphate when its concentration is low at theexpense of phosphorylated glycolytic intermedi-ates. The presence of such an enzymatic activityin higher organisms might explain the conversionof FDP to methylglyoxal and inorganic phos-phate observed by a number of workers some 40years ago (4, 24, 39, 40).

In bacteria, methylglyoxal can also be formedfrom glycine and threonine via aminoacetone(16, 18, 22, 41, 42). Subsequent catabolism ofmethylglyoxal in these cases has been shown totake place by means of the glyoxalase system, orin some cases by way of an NAD-linked dehy-drogenase (22, 42).

It is beyond reasonable doubt that the toxicproduct excreted by E. coli strains hyperactive inglycerol dissimilation is methylglyoxal. The tox-icity of millimolar levels of this compound to E.coli was noted and studied several years ago byEgyud and Szent-Gy6rgyi (12, 14), although noresistant mutants were found (13). Because ofthe growth inhibition properties, methylglyoxal(or another a-ketoaldehyde) was thought to bethe universal inhibitor "retine," and the ubiqui-tous glyoxalase system was thought to be thepromoting element "promine" in their theory onnaturally occurring regulators of cell growthand division (36, 37). Some preliminary chemo-therapy studies showed that methylglyoxal couldindeed inhibit the growth of ascites tumors (2, 3,15).Whatever the advantages of producing methyl-

glyoxal from DHAP or from other metabolites,the toxicity of the a-ketoaldehyde requires thatthe enzymatic pathways leading to its formationbe well controlled, or that the cell be wellequipped to dispose of the compound. The main-tenance of a high constitutive level of methyl-glyoxal synthase, as found in E. coli cells under avariety of growth conditions by Cooper and hisco-workers (7, 23) and by us, is probably madepossible by the combination of a high Km of theenzyme for DHAP, the dramatic third-powerinhibition of its activity by inorganic phosphate,and the presence of the glyoxalase system.The target of the lethal effect of methylglyoxal

is likely to be a component of protein synthesis,as shown by the in vitro work of Szent-Gy6rgyi

DHAP%

D-Glycraldehyde-3-P_ _ - DPN

DPNH

yoxal ADP

ATP3 PGA

2 PGALactate PEP

ADP

-2H Pyruvate, ATP

FIG. 5. Methylglyoxal by-path in glycolysis in Esch-erichia coli. DHA P, dihydroxyacetone phosphate;GSH, glutathione; DPN, nicotinamide adenine dinucle-otide; DPNH, reduced nicotinamide adenine dinucleo-tide; PGA, phosphoglyceric acid; ADP, adenosinediphosphate; A TP, adenosine triphosphate; PEP, phos-phoenolpyruvate.

and collaborators (14, 32) and in the in vivoexperiments of Zwaig and Dieguez (43). How-ever, there is also evidence for methylglyoxal in-hibition of succinate oxidase and a number ofother enzymes (13, 29). SulThydryl enzymes maybe particularly sensitive to methylglyoxal (29,36). It has also been proposed that the charge-transfer properties of methylglyoxal might causethis compound to have drastic effects on the sec-ondary structure of cell membrane and cyto-plasmic proteins at high concentrations (36).

Methylglyoxal may in fact kill cells by morethan one mechanism. What is certain is that thewild-type cells must possess an array of controlmechanisms to prevent the suicidal synthesis ofthis compound. In the case of glycerol dissimila-tion, it took the removal of two safeguards, feed-back inhibition and specific repression (44), toreveal the latent danger of improperly channeledmetabolic flow.

ACKNOWLEDGMENTSWe are grateful to Roger Jeanloz for his counsel during the

initial phases of this investigation.Work in this laboratory is supported by National Science

Foundation grant GB 5854, by Public Health Service grant 5RO1 GM1 1983 from the National Institute of General MedicalSciences, and by American Heart Association grant AHA69-991. E. C. C. Lin was supported by a Research Career Devel-opment Award from the Public Health Service (3-K3-GM-17,925), and W. B. Freedberg was supported by a postdoctoral

143VOL. 108, 1971

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

b on

25

Nov

embe

r 20

21 b

y 19

0.14

1.20

0.15

7.

FREEDBERG, KISTLER, AND LIN

feliowship (1 F02 AM 37625-01 BNB) from the National In-stitute of Arthritis and Metabolic Diseases.

LITERATURE CITED

1. Anderson, A., and R. A. Cooper. 1969. Gluconeogenesis inEscherichia col: the role of triose phosphate isomerase.FEBS Letters 4:19-20.

2. Apple, M. A., and D. M. Greenberg. 1967. Arrest ofcancer in mice by therapy with normal metabolites. I. 2-Oxopropanal (NSC-79019). Cancer Chemother. Rep.51:455-464.

3. Apple, M. A., and D. M. Greenberg. 1968. Arrest ofcancer in mice by therapy with normal metabolites. II.

Indefinite survivors among mice treated with mixtures of2-oxopropanal (NSC-79019) and 2,3-dihydroxypropanal(NSC-67934). Cancer Chemother. Rep. 52:687-696.

4. Ariyama, N. 1928. The formation of methylglyoxal fromhexose phosphate in the presence of tissues. J. Biol.Chem. 77:395-404.

5. Atkinson, D. E. 1966. Regulation of enzyme activity.Annu. Rev. Biochem. 35:85-124.

6. Berman, M., and E. C. C. Lin. 1971. Glycerol-specific re-

vertants of a phosphoenolpyruvate phosphotransferasemutant: suppression by the desensitization of glycerolkinase to feedback inhibition. J. Bacteriol. 105:113-120.

7. Cooper, R. A., and A. Anderson. 1970. The formation andcatabolism of methylglyoxal during glycolysis in Esche-richia coli. FEBS Letters 11:273-276.

8. Cori, C. F., and G. T. Cori. 1933. Carbohydrate metabo-lism. Ann. Rev. Biochem. 2:129-146.

9. Cori, C. F., and G. T. Cori. 1934. Carbohydrate metabo-lism. Annu. Rev. Biochem. 3:151-174.

10. Cozzarelli, N. R., W. B. Freedberg, and E. C. C. Lin.1968. Genetic control of the L-a-glycerophosphatesystem in Escherichia coli. J. Mol. Biol. 31:371-387.

11. Dakin, H. D., and H. W. Dudley. 1913. On glyoxalase. J.Biol. Chem. 14:423-431.

12. Egyiid, L. G. 1967. Studies on cell division: the effect ofaldehydes, ketones, and a-ketoaldehydes on the prolifer-ation of Escherichia coli. Curr. Mod. Biol. 1:14-20.

13. Egyud, L. G., and A. Szent-Gy6rgyi. 1966. Cell division,SH, ketoaldehydes and cancer. Proc. Nat. Acad. Sci.U.S.A. 55:388-393.

14. Egytid, L. G., and A. Szent-Gy6rgyi. 1966. On the regula-tion of cell division. Proc. Nat. Acad. Sci. U.S.A. 56:203-207.

15. Egyiid, L. G., and A. Szent-Gy6rgyi. 1968. Cancerostaticaction of methylglyoxal. Science 160:1140.

16. Elliot, W. H. 1960. Aminoacetone formation by Staphylo-coccus aureus. Biochem. J. 74:478-485.

17. Gornall, A. G., C. J. Bardawill, and M. M. David. 1949.Determinations of serum proteins by means of the biuretreaction. J. Biol. Chem. 177:751-766.

18. Green, M. L., and W. H. Elliot. 1964. The enzymatic for-mation of aminoacetone from threonine and its furthermetabolism. Biochem. J. 92:537-549.

19. Harden, A. 1932. Alcoholic fermentation, 4th ed., p. 109-146. Longmans, Green and Co., London.

20. Hayashi, S., J. P. Koch, and E. C. C. Lin. 1964. Activetransport of L-a-glycerophosphate in Escherichia coli. J.Biol. Chem. 239:3098-3105.

21. Hayashi, S., and E. C. C. Lin. 1965. Capture of glycerolby cells of Escherichia coli. Biochim. Biophys. Acta 94:479-487.

22. Higgins, I. J., and J. M. Turner. 1969. Enzymes of methyl-glyoxal metabolism in a Pseudomonad which rapidlymetabolizes aminoacetone. Biochim. Biophys. Acta 184:464-467.

23. Hopper, D. J., and R. A. Cooper. 1971. The regulation of

Escherichia coli methylglyoxal synthase; a new controlsite in glycolysis? FEBS Letters 13:213-216.

24. Inoue, H. 1931. The formation of methylglyoxal from hex-osephosphate in the presence of animal tissue. J.Biochem. (Tokyo) 13:369-387.

25. Kistler, W. S., C. A. Hirsch, N. R. Cozzarelli, and E. C.C. Lin. 1969. A second pyridine nucleotide-independentL-a-glycerophosphate dehydrogenase in Escherichia coliK-12. J. Bacteriol. 100:1133-1135.

26. Klotzsch, H., and H. U. Bergmeyer. 1965. Methylglyoxal,p. 283-284. In H. U. Bergmeyer (ed.), Methods of enzy-matic analysis. Academic Press Inc., New York.

27. Knox, W. E. 1960. Glutathione, p. 253-294. In P. D.Boyer, H. Lardy, and K. Myrback (ed.), The enzymes,2nd ed., vol. 2. Academic Press Inc., New York.

28. Koch, J. P., S. Hayashi, and E. C. C. Lin. 1964. The con-trol of dissimilation of glycerol and L-a-glycerophos-phate in Escherichia coli. J. Biol. Chem. 239:3106-3108.

29. Kun, E. 1950. Inhibition of succinic dehydrogenase bymethylglyoxal. J. Biol. Chem. 192:187-289.

30. Lin, E. C. C., J. P. Koch, T. M. Chused, and S. E. Jor-gensen. 1962. Utilization of L-a-glycerophosphate byEscherichia coli without hydrolysis. Proc. Nat. Acad.Sci. U.S.A. 48:2145-2150.

31. Lohmann, K. 1932. Beitrag zur enzymatischen Umwand-lung von synthetischen Methylglyoxal in Milchsaure.Biochem. Z. 254:332-354.

32. Otsuka, H., and L. G. Egyud. 1968. Locus of the inhibi-tion of protein synthesis by aldoketones. Curr. Mod.Biol. 2:106-1 10.

33. Parnas, J. K. 1932. The chemistry of muscle. Annu. Rev.Biochem. 1:431-456.

34. Sanno, Y., T. H. Wilson, and E. C. C. Lin. 1968. Controlof permeation to glycerol in cells of Escherichia coli.Biochem. Biophys. Res. Commun. 32:344-349.

35. Shaffer, P. A., and E. Ronzoni. 1932. Carbohydrate me-

tabolism. Annu. Rev. Biochem. 1:247-266.36. Szent-Gy6rgyi, A. 1968. Bioelectronics, a study in cellular

regulations, defense, and cancer. Academic Press Inc.,New York.

37. Szent-Gyorgyi, A., L. G. Egyiid, and J. A. McLaughlin.1967. Ketoaldehydes and cell division. Science 155:539-541.

38. Tanaka, S., S. A. Lerner, and E. C. C. Lin. 1967. Replace-ment of a phosphoenolpyruvate-dependent phospho-transferase by a nicotinamide adenine dinucleotide-linked dehydrogenase for the utilization of mannitol. J.Bacteriol. 93:642-648.

39. Toenniessen, E., and W. Fischer. 1926. Methylglyoxal alsAbbauprodukt der glykose. Z. Physiol. Chem. 161:254-264.

40. Vogt, M. 1929. Bildung und Isolierung von Methylglyoxalbei der Glykolyse durch tierisches Enzym. Biochem. Z.

211:17-39.41. Willets, A. J., and J. M. Turner. 1970. Threonine metabo-

lism in a strain of Bacillus subtilis: enzymic oxidation of

the intermediate DL-lactaldehyde. Biochim. Biophys.Acta 222:234-236.

42. Willets, A. J., and J. M. Turner. 1970. Threonine metabo-lism in a strain of Bacillus subtilis: enzymes acting on

methylglyoxal. Biochim. Biophys. Acta 222:668-670.43. Zwaig, N., and E. Dieguez. 1970. Bactericidal product

obtained from a mutant of Escherichia coli. Biochem.Biophys. Res. Commun. 40:1415-1422.

44. Zwaig, N., W. S. Kistler, and E. C. C. Lin. 1970. Glycerolkinase, the pacemaker for the dissimilation of glycerol inEscherichia coli. J. Bacteriol. 102:753-759.

45. Zwaig, N., and E. C. C. Lin. 1966. Feedback inhibition ofglycerol kinase, a catabolic enzyme in Escherichia coli.Science 153:755-757.

144 J. BACTERIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

b on

25

Nov

embe

r 20

21 b

y 19

0.14

1.20

0.15

7.