JOURNAL OF BACTERIOLOGY, June 1970, p. 753-759Copyright a 1970 American Society for Microbiology

Vol. 102, No. 3Printed in U.S.A.

Glycerol Kinase, the Pacemaker for theDissimilation of Glycerol in Escherichia coli'

N. ZWAIG,2 W. S. KISTLER, AND E. C. C. LIN

Department of Bacteriology and Immunology and the Department of Biological Chiemistry,Harvard Medical School, Bostoni, Massachusetts 02115

Received for publication 12 March 1970

The activity of glycerol kinase is rate-limiting in the metabolism of glycerolby cells of Escherichia coli. A mutant strain producing a glycerol kinaseresistant to inhibition by fructose-I , 6-diphosphate grows faster than its wild-typeparent on glycerol as the sole source of carbon and energy. The amount of intra-cellular fructose-1 ,6-diphosphate was determined for wild-type cells growing ex-

ponentially on glycerol. The water content of such cells was also determined,allowing calculation of the intracellular concentration of fructose-1 ,6-diphosphate.This value, 1.7 mm, is adequate to exert substantial inhibition on the wild-type glyc-erol kinase. The desensitization of glycerol kinase to feedback inhibition alsoenhances the power of glycerol to exert catabolite repression, both on the enzymes

of the glycerol system itself and on those of the lactose system. However, desensi-tization of glycerol kinase alone does not eliminate the phenomenon of diauxicgrowth in a glucose-glycerol medium. Biphasic growth in such a medium is abol-ished if the altered enzyme is produced constitutively. The constitutive productionof the mutant kinase at high levels, however, renders the cells vulnerable to glyc-erol. Thus, when the cells have been grown on a carbon source with a low power

for catabolite repression, e.g., succinate, sudden exposure to glycerol leads to over-

consumption of the nutrient and cell death.

Cells of Escherichia coli do not have a mecha-nism for the transport of glycerol against a chemi-cal gradient (9). The passage of the compoundacross the cell membrane is governed by facili-tated diffusion, an energy-independent process(23). Once inside the cell, glycerol may be re-tained as L-a-glycerophosphate (L-a-GP) by theaction of a kinase dependent on adenosine tri-phosphate (ATP). This kinase is subject to non-competitive inhibition by fructose-i, 6-diphos-phate (FDP; references 1, 29). This unusualcombination of a freely reversible entry processwith an irreversible metabolic step under kineticcontrol (Fig. 1) suggests that the kinase mayserve as a pacemaker for glycerol utilization. Theexperiments presented herein were devised toexplore conditions under which this may be true.

MATERIALS AND METHODSBacterial and phage strains. All the bacterial strains

were derivatives of E. coli K-12 Hfr C (3). The originsof strains 1 and 7 are described by Koch et al. (10)and strain 43 by Zwaig and Lin (29). Strain 44 was

I Part of this work was presented before the American Societyof Biological Chemists, Atalntic City, N.J., April 1968.

2 Present address: Escuela de Biologia, Facultad de Ciencias,Universidad Central de Venezuela, Caracas, Venezuela.

constructed by transducing the met B+ and glp K'alleles of strain 43 into a recipient carryingmet B- andglp K+ (see reference 6) and selecting for the metB+ marker.The genotypes of the bacterial strains are given in

Table 1.The transducing phage Plkc was donated by S. E.

Luria. Transduction was carried out by the method ofLuria, Adams, and Ting (14).

Growth of cells and assays of enzyme activities.Mineral medium buffered by phosphate at pH 7.0(25) was used. Unless otherwise indicated, caseinhydrolysate was used at 1% concentration and singlecarbon sources at 0.2%. Growth rates at 37 C wereturbidimetrically determined in a Klett colorimeterwith a no. 42 filter. During exponential growth, oneKlett unit corresponds to about 4 X 106 cells per ml.

For the assays of glycerol kinase, L-a-GP dehydro-genase (11) and ,6-galactosidase (27), cells were grownfor at least three generations in a given medium andwere extracted as described by Koch et al. (10). Allstrains with glycerol kinase activity were examinedfor sensitivity of the enzyme to FDP at pH 7.5 (29).Protein was determined by the biuret reagent (8).Specific activities are expressed as micromoles ofsubstrate converted per minute per milligram of pro-tein at 25 C.

Determination of intracellular FDP levels. Cells ofstrain 7 were grown in 35 ml of glycerol mineral me-

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CELLFocilitoted

Glycerol GlycerolDiffusion Feedback- Kin-se

Inhibition--- FDP F-s-PPhosytotase

Kinasoe Adolose

Active

L-a-GP _-A L-a-GP -a (DNA-P) GA-3-P _Transport Dehydrogenose Isomeraoe

IIFIG. 1. Metabolic scheme for the dissimilation of

glycerol and L-a-GP by E. coli.

dium contained in a 300-ml Erlenmeyer flask. Incuba-tion was carried out at 37 C on a rotatory shaker. At adensity of 100 Klett units, 5-ml samples were rapidlypipetted into individual tubes containing 0.45 ml ofice-cold 60% HCI03 and 0.055 ml of 0.1 M ethylene-diaminetetraacetic acid. Samples were also withdrawnand immediately filtered through Millipore filters toprovide controls of cell-free medium. A third set ofsamples for determination of cellular protein waspipetted into chilled tubes and immediately frozen.The first set of samples in the perchlorate-containingtubes was chilled on ice for 2 hr, after which cellulardebris was removed by centrifugation at 40,000 X gfor 10 min. The supernatant fluid was decanted andneutralized with a calculated volume of 3.5 M K2CO3.The precipitated KC103 was allowed to settle over-night at 0 C. Samples of the neutralized perchlorateextract and of the Millipore filtrate were assayed forFDP (see below). As internal standard, 2.5 nmoles ofFDP was added to one of the perchlorate extractiontubes just after the addition of cells. Recovery was 90to 100%. Protein content of the frozen cell sampleswas determined by the method of Lowry et al. (13),by using the culture filtrate as blank.FDP was assayed by enzymatic coupling to the

oxidation of reduced nicotinamide adenine dinucleo-tide (NADH), which was followed in a Turnerfluorometer (12). To a mixture containing a sample ofthe unknown, 4 nmoles NADH and 40 nmolesimidazole (buffered at pH 7.0) was added sequentiallyto 10 ,ug of each of the following enzymes: glycerolphosphate dehydrogenase, triosephosphate isomerase,and aldolase. The final volume of the assay mixturewas 2.5 ml. The reactions were allowed to proceed at25 C. After the addition of each enzyme, the fluores-cence was monitored periodically until a plateau wasestablished. The decrement in fluorescence after theaddition of aldolase was used to calculate the amountof FDP present. A solution of standard FDP wasused for calibration.

Determination of cell water. Cells of strain 7 weregrown in 500 ml of glycerol mineral medium con-tained in a 2-liter Erlenmeyer flask which was incu-bated at 37 C on a rotatory shaker. At 100 Klettunits, a 200-ml sample was centrifuged at 6,000 X gfor 15 min at 4 C. The supernatant fluid was carefullydecanted, and the tube was drained. The pelleted cellswere resuspended in a small volume of mineral me-

dium and quantitatively transferred by several rinsesto a tared glass tube. One microcurie of "4C-inulinwas added and thoroughly mixed with the cell sus-pension (final volume about 5 ml). The suspensionwas immediately centrifuged at 1,000 X g for 20 min,and the supernatant fluid was carefully poured offand saved for determination of '4C-inulin concentra-tion. The tube was weighed to determine the wetweight of packed cells. The cells were then quanti-tatively transferred to a tared planchet by three rinsesof distilled water (total volume, 2 ml). Samples of theresulting suspension were withdrawn for determina-tion of '4C-inulin. The contribution of extracellularwater to the wet weight, that is, the "4C-inulin-avail-able volume, was calculated by counting samples ofthe supernatant fraction and the resuspended cells in agas flow counter (Nuclear-Chicago Corp., Des Plaines,Ill.). For both the supernatant fraction and resus-pended cells, proportionality between counts andsample size was established. Radioactivity in thepacked cells was the same whether cells were centri-fuged immediately after addition of 14C-inulin orwhether they were incubated for 30 min at room tem-perature before centrifugation; thus, the labeledinulin was not taken up by the cells. To determine dryweight, the remaining material on the planchets wasevaporated and then dried at 110 C for 20 hr beforeweighing. The value obtained was then corrected bothfor the removal of samples for determination of 14C-inulin (4%) and for the contribution of inorganic saltspresent in the mineral medium comprising the inulin-available volume (6%). The cellular dry weight thuscalculated was consistently about 16% of the wetweight. Cellular water [total wet weight of the pelletminus (dry weight plus the weight of the water in theinulin-available space)] was found to be 2.18 i 0.03uliters per mg (dry weight) or, equivalently, 0.374 i0.004 ,uliters in 1 ml of culture at 100 Klett units(mean of four determinations ±t standard error).This value is slightly lower than that reported byWinkler and Wilson [2.7 uliters of cell water per mg(dry weight) for cells grown on casein hydrolysate;reference 281 and that calculated from the dataof Schultz, and Solomon [2.55 ,lditers of cell

TABLE 1. Genzotypes of the strainis

glp Markers'Strain

Kb T D Rc

+ + ± +44 + + + +7 + + + -

43 1 + + -

a The L-a-GP system is abbreviated as gip.Letters K, T, D, and R refer to glycerol kinase, theL-a-GP transport system, L-a-GP dehydrogenase,and the regulator, respectively.

b +, Wild-type allele, and i, the allele for akinase which is insensitive to feedback inhibition.

c +, Inducibility, and -, constitutivity.

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GLYCEROL DISSIMILATION

water per mg (dry weight) for cells grown on glucose;reference 241. Our lower value may reflect a more com-pact structure of the smaller glycerol-grown cells.

Chemicals. Disodium DL-a-GP, ATP, isopropyl-,8-D-thiogalactoside (IPTG), and tetrasodium D-FDPwere from Sigma Chemical Co., St. Louis, Mo. Acidhydrolysate of casein was from Difco. CrystallineL-a-GP dehydrogenase, triosephosphate isomerase,and aldolase were from Boehringer Mannheim Corp.,New York, N.Y. '4C-inulin was from New EnglandNuclear Corp., Boston, Mass.

RESULTSRole of kinase on growth rate. To discover

whether inhibition of glycerol kinase could limitthe metabolism of glycerol, we compared thegrowth properties of wild-type cells (strain 1)with cells producing a glycerol kinase refractoryto feedback inhibition (strain 44). With glycerolas carbon source, mutant cells grew reproduciblyfaster than wild-type cells. In contrast, whenL-a-GP was employed as the nutrient, the growthrates were indistinguishable. The disparity ofthe growth rates on glycerol between the twostrains is not attributable to a difference in theinducibility of the glp system (L-a-GP system),because similar results were obtained in a pairof constitutive mutants, strains 7 and 43, whichproduce, respectively, normal and desensitizedglycerol kinase. Growth rates for the four strainsare given in Table 2. In another experiment to besubsequently described, with glycerol as carbonsource, extracts of cells of strain 7 actually con-tained a higher specific activity of glycerol kinasethan those of strain 43, despite the fact that theformer grew more slowly. Hence, it was probablythe partial inhibition of the enzyme by FDP whichprevented the wild-type cells from growing morerapidly on glycerol, not the number of kinasemolecules per cell.To determine whether FDP is actually present

during growth on glycerol at levels sufficient toaccount for such inhibition, we have measuredthe necessary parameters to calculate its approxi-mate internal concentration. For this purpose,cells from an exponential culture were initiallycollected and washed on Millipore filters beforethey were extracted. This process required atleast 10 sec. Further examination showed that, asthe interval between harvest and extraction ofthe cells was deliberately lengthened, FDP levelsrose sharply and were almost doubled by 2 min.To avoid any such changes in the FDP concentra-tion during manipulation, samples were extracteddirectly with perchloric acid, as detailed in Mate-rials and Methods. Cultures of strain 7 growingexponentially in glycerol minimal medium andextracted at a density of 100 Klett units werefound to contain 0.62 i 0.06 nmoles of intracellu-

TABLE 2. Doublinig times of cells

Carbon sourceStrain

Glycerol L-a-GP

min min

1 90 9044 70 907 90 90

43 70 90

lar FDP per ml of culture (equivalently, 6.9 0.7nmoles per mg of cellular protein). This value isthe mean of five determinations i standard error.Since 1 ml of culture at 100 Klett units also con-tains 0.37 ,uliter of cell water, the intracellularconcentration of FDP may be estimated to be 1.7mm. Determinations made at 50 and 150 Klettunits gave similar values. This concentration maybe a slight underestimate, since the cell watercalculation probably includes water presentbetween the cell wall and the plasma membraneand bound water inside the cell which is unavail-able as solvent. Nevertheless, the magnitude ofthis concentration is sufficient to cause significantinhibition of the kinase, which has a Ki value of 2mM (29).

Effect of desensitization on the catabolite repres-sive power of glycerol. If feedback inhibition ofthe kinase prevents achievement of the potentialmaximal growth rate on the substrate, it mightalso limit the catabolite repressive power ofglycerol, inasmuch as growth rate on a compoundand its catabolite repressive power can often becorrelated (22). Accordingly, the effect of glycerolon the induction of the lactose system by IPTGwas examined in strains producing normal orinsensitive glycerol kinase. It was found that,whereas in wild-type cells the ability of glycerol toantagonize the induction of,-galactosidase wasfeeble in comparison to that of glucose, in cellswith the mutant kinase the repressive power ofglycerol approached that of glucose. This is trueirrespective of whether the gip system wasconstitutive or inducible (Table 3). Furthermore,the pattern of ,B-galactosidase induction was notaltered by increasing the external concentration ofIPTG from 5 X 10-4 to 5 X 10-3 M (Table 4).Thus, the critical difference in the behavior of thetwo strains should not be attributed to differencesin the intracellular concentration of the inducer(4, 7).The abolition of the allosteric control of the

kinase also permitted glycerol to exert greaterself-repression of the glp system which is illus-trated by a pair of constitutive strains (Table 5).Glycerol kinase and L-a-GP dehydrogenase were

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both more strongly repressed by glycerol in cellscontaining the feedback-resistant kinase than inthose containing the normal enzyme. The moresevere repression of the two enzymes in strain 43by glycerol was specific since, with L-a-GP orglucose as the carbon source, the levels of thekinase and the dehydrogenase did not vary withthe nature of the glycerol kinase protein produced.

Glucose-glycerol diauxie. It has long beenknown that the phenomenon of diauxie can beobserved with E. coli in a medium containingglucose and glycerol (15, 16). Because glucosedissimulation gives rise to FDP and FDP canprevent glycerol utilization at the level of enzymecatalysis, and, therefore, also at the level of en-zyme synthesis (through interference with theformation of the inducer L-a-GP), an experimentwas performed to test whether both types ofaction are necessary for this diauxie. Strain 44(in which the kinase is desensitized), strain 7(in which the specific repressor system is non-functional), and strain 43 (in which both kineticcontrol of the enzyme and the specific repressorcontrol are absent) were compared with wild-typecells (Fig. 2). The results show that removal ofeither feedback inhibition or specific repressionwas insufficient to abolish diauxie. Biphasic

TABLE 3. Induction of f3-galactosidase in the pres-ence oJ various carbon souircesa

Carbon source

GlycerolL-a-GP

Glucose

GlycerolL-a-GP

Glucose

GlycerolL-a-GP

Glucose

GlycerolL-a-GPGlucose

Specific activity of,6-galactosidase(units/mg ofprotein)

8.09.73.2

3.79.53.2

8.79.53.4

3.69.43.3

a The inducer, isopropyl-g-D-thiogalactoside(IPTG), was added at a concentration of 5 X 10-4M when the culture density was at 15 Klett units.Cells were harvested when the density attained200 Klett units. Glycerol was initially present at0.04 M, DL-a-glycerophosphate (DL-a-GP) at 0.08M, and glucose at 0.04 M. All cells were pregrownfor several generations on the respective carbonsource before IPTG was added to minimize con-tributions from transient repression (2, 17).

TABLE 4. Inductionl of ,3-galactosidase by two con-centrations of isopropyl-j-D-thiogalactoside

(IPTG)a

Specific activity ofStrain IPTG ,-galactosidase(units/mg of

protein)

7 5 X10-4 8.85 X 10-3 8.9

43 5 X 10-4 3.65 X I0-3 3.3

a Cells were grown in minimal medium con-taining glycerol at an initial concentration of 0.04M. The inducer was added when the culture densitywas at 15 Klett units; cells were harvested at adensity reading of 200 Klett units.

TABLE 5. Repressioni of glycerol kinase and L-ae-glycerophosphate (L-a-GP) dehydrogenasea

Specific activity Specific activityof glycerol of L-a-GP

Strain Carbon source kinase dehydrogenase(units/mg of (units/mg of

protein) protein)

7 Glycerol 1.1 0.21L-a-GP 1.1 0.23Glucose 0.25 0.15

43 Glycerol 0.27 0.16L-a-GP 1.2 0.21Glucose 0.21 0.14

a All cells were harvested when the cultures wereat a density of 200 Klett units.

growth was eliminated only when both types ofcontrol were lifted.

Lethal effect of unregulated consumption ofglycerol. High intracellular concentrations ofL-a-GP are known to cause growth stasis (5).Hence, the question arises as to how cells withdesensitized glycerol kinase can avoid over-phosphorylation. In the case of strain 44, whichmakes the desensitized kinase inducibly, the cellsencounter no difficulty when transferred from asuccinate ora caseinhydrolysate medium to a glyc-erol medium. After a lag period, growth graduallyresumes as the glp system becomes progressivelyinduced. Attainment of a harmful level of thekinase was prevented by autocatabolic repression.On the other hand, cells which are constitutivein the glp system but which produce normalglycerol kinase can also protect themselves duringtransitions from succinate or casein hydrolysate toglycerol, presumably because the kinase at itsinitial high concentration is prevented from cata-lyzing excessive phosphorylation of glycerol by

Strain

1

44

7

43

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GLYCEROL DISSIMILATION 757

HOURS MVUK5

FIG. 2. Growth patterns of wild-type and mutant strains in a medium containing glucose (2.5 mM) andglycerol (5 mM). Cells pregrown onz glucose alone were used to inoculate the cultures.

feedback inhibition. As growth continues, thelevel of the kinase should gradually be reduced byautocatabolite repression, and the degree of inhi-bition of the enzyme should be concomitantly re-

laxed. However, when both the kinetic control ofthe kinase and the control by the regulatory geneare absent, acute transition from a succinate or a

casein-hydrolysate medium to a glycerol mediumis no longer compatible with life. A dramaticillustration of such a metabolic embarrassmentis provided by an experiment in which glycerolwas suddenly introduced to a culture of strain 43cells growing on succinate. Growth was promptlyarrested (Fig. 3) and cell death rapidly ensued(Table 6). To verify that the nature of the glycerolkinase was responsible for the lethal effect, a sim-ilar culture grown on succinate was exposed toL-a-GP instead of to glycerol; in this case a slightstimulation occurred (results not shown).The bacteriocidal effect of glycerol on strain 43

was evidently not due to an inordinate buildup ofL-a-GP. Separate experiments revealed that adouble mutant with the insensitive kinase butwithout the L-a-GP dehydrogenase was subjectto growth inhibition by glycerol but not to killing.The metabolite (or metabolites) causing death ofcells of strain 43 must, therefore, be distal toL-a-GP in the catabolic pathway.

Cells lacking L-a-GP dehydrogenase but pro-ducing normal glycerol kinase could be rescuedby glucose when their growth was inhibited byglycerol (5). Cells of strain 43 poisoned by glyc-erol could not be thus rescued, a result to be ex-pected since FDP is ineffective in suppressingglycerol phosphorylation.The only known way for cells of strain 43 to

survive and grow on glycerol is for them to havebeen initially grown on a carbon source such as

glucose, which reduces the production of the

z 60 -

z~~ Glucose

F / ~~~Glycerolz

w

3

lo 2 4 6 24HOURS

FIG. 3. Effect of glycerol on cells of strain 43growing on succinate. At the time indicated by thefirst arrow, two cultures received 0.2% glycerol (A\and X), and a third culture an equal volume of water(O). Al the time indicated by the second arrow, glucosewas added at 0.2% to one of the cultures which receivedglycerol (X).

glycerol kinase to a safe level (Table 7), beforecontact with glycerol.

DISCUSSIONIt might at first seem surprising that a catabolic

enzyme should be subject to remote product

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TABLE 6. Killing of cells of strain 43 by exposure toglycerola

Exposure time Viable cells Culture density

hr no./ml Kleti units

0 1.4 X IO" 402.5 6X 103 495 0 46

20 0 43

a Cells were grown on succinate as carbonsource. Glycerol was added to a concentration of0.02 M at zero time.

TABLE 7. Glycerol kinase and L-a-glyceroplhosphate(L-a-GP) dehydrogenase activities in cells of

strain 43 grown on succinate and on glucosea

Specific activity of Specific activity ofCarbon source glycerol kinase L-a-GP dehydro-(units/mg of genase (units/mg

protein) of protein)

Succinate 0.50 0.34Glucose 0.08 0.21

a Cultures were harvested at the density of 40Klett units after more than three doublings in eachmedium.

inhibition. The establishment of such a regulatorymechanism in the glycerol system is probablyrelated to the fact that the kinase not only initiatesthe metabolism of glycerol, but also serves toextract the compound from the medium by phos-phorylation (9). Under conditions in which theexogenous supply of glycerol is very low (i.e.,below 10-6 M, the Km of the enzyme), the efficacyfor glycerol capture can be improved by produc-ing additional enzyme. On the other hand, whena cell possesses a high level of glycerol kinase, itbecomes vulnerable to poisoning if the supplyof the substrate suddenly increases. Repressionalone would not be effective in this situation, sincereduction of the enzyme level requires both thecessation of enzyme synthesis and the dilution ofthe preexistent enzyme molecules by growth.However, precisely because of the toxic condition,growth could not take place. The introduction of akinetic feedback mechanism circumvents thisdifficulty.That the kinetic control in the kinase should be

poised at a level which prevents achievement ofmaximal growth rate on the substrate should alsobe no surprise. It may be recalled that bacterialcells rarely encounter only one utilizable organiccompound in their natural environments.Whatever might have been the basis for the

selection ofFDP as the negative modifier of glyc-erol kinase (29), the establishment of this relation-ship assures the hegemony of glucose as a pre-

ferred carbon source. By giving rise to FDP,glucose metabolism can suppress the phosphoryl-ation of glycerol. In so doing, it also weakens theinduction of the glp system, because L-a-GP,the product of the reaction, is the inducer. Finally,glucose exerts strong catabolite repression onglycerol kinase. The power of glucose, exercisedthrough FDP, was strikingly demonstrated byBock and Neidhardt (1) in their study of a mu-tant with a temperature-sensitive aldolase. At40 C, the aldolase was unstable, and the additionof 6 X 10-6 M glucose halted growth on glycerol.

In biosynthetic systems, the destruction offeedback inhibition could lead to a superfluoussynthesis of end products, which are excreted intothe growth medium. Such a phenomenon has beenobserved in studies of histidine (19-21), trypto-phan (18), and leucine biosynthetheses (26). Incontrast, the loss of feedback inhibition in acatabolic pathway, as exemplified by the glycerolsystem, could have variable consequences fromtrivial to lethal, depending on the particularcondition. A biological trait need not have ex-clusively negative or positive values. A loss muta-tion leading to the overproduction of an endproduct in the case of amino acid biosynthesiscould protect a population against certain sub-strate analogues noxious to the organism (18-21,26), until an improved mechanism for protectioncould be evolved. The loss of a feedback controlin a catabolic system, on the other hand, couldpermit the organism to increase its growth rate ona given nutrient or to utilize it preferentially toother compounds, a choice which might be bene-ficial if the preferred compound suddenly becameabundant.

ACKNOWLEDGMENTS

E. C. C. L. was supported by a Public Health Service ResearchCareer Development Award from the National Institute ofGeneralMedical Sciences, and W. S. K. by a National Science Foundationpredoctoral fellowship.

This investigation was supported by grant GB-5854 from theNational Science Foundation and Public Health Service grantGM-11983 from the National Institute of General MedicalSciences.

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