JOURNAL OF BACTERIOLOGY, July, 1966Copyright © 1966 American Society for Microbiology

Vol. 92, No. 1Printed in U.S.A.

Nature of the Effector of Catabolite Repression of,B-Galactosidase in Escherichia coli

WILLIAM F. LOOMIS, JR.,1 AND BORIS MAGASANIKDepartment of Biology, Massachusetts Institute of Technology,

Cambridge, Massachusetts

Received for publication 1 April 1966

ABSTRACTLOOMIS, WILLIAM F., JR. (Massachusetts Institute of Technology, Cambridge,

Mass.), AND BORIS MAGASANIK. Nature of the effector of catabolite repression off-galactosidase in Escherichia coli. J. Bacteriol. 92:170-177. 1966.-Many carbonsources were found to give rise to catabolite repression of,-galactosidase in a mu-

tant strain of Escherichia coli lacking hexose phosphate isomerase activity. Com-pounds containing glucose or galactose cannot be formed from several of thesecarbon sources in this mutant strain, and, therefore, appear not to be required forcatabolite repression of ,B-galactosidase. Glucose was observed to elicit catabolite re-

pressionoff-galactosidase in another mutant strain under conditions in which the for-mation of compounds of the citric acid cycle is inhibited. If catabolite repressionof the lac operon is mediated by a single compound, it appears that the compoundis related to the pentoses and trioses of intermediary metabolism. The repression of3-galactosidase by galactose in galactokinase negative strains was shown to be inde-

pendent of the gene, CR, which determines catabolite sensitivity of the lac operon,and to be dependent on a functional i gene.

The theory of catabolite repression (18) postu-lates that the degree of repression of an operondepends on the concentration of one or morespecific catabolites which can be formed frommany different carbon sources, including the sub-strate of the repressible enzyme. Many biochem-ical steps in catabolism are reversible. It is, there-fore, very difficult to determine which specificcompound is involved in the control of an enzymeby analyzing the repression elicited by differentcarbon sources. However, the relative facilitywith which a carbon source gives rise to thecatabolite can be determined. This approach hasbeen used for study of the control of j-galacto-sidase (8, 17). Glucose was found to be a bettersource of the compound related to the lac operonthan is glycerol. Pyruvate was found to be a poorsource of the catabolite related to control of f-galactoside. However, it was shown that pyruvateis a good source of the catabolite related to con-trol of tryptophanase; although the specific com-pound involved in the control of either of thesegenes is unknown, it appears that the two genesare controlled by different catabolites.McFall and Mandelstam (17) suggested that a

1 Present address: Department of Biology, BrandeisUniversity, Waltham, Mass.

high concentration of galactose, galactose-1-phosphate, or uridine diphosphate (UDP) galac-tose per se gives rise to catabolite repression off-galactosidase. They found that the presence ofgalactose in the medium would repress ,B-galacto-sidase in galactose-negative mutants blocked ingalactokinase, galactose transferase, or UDPgalactose epimerase. Since the conversion ofgalactose to glucose is impaired in these strains,galactose compounds per se appeared to exertrepression. McFall and Mandelstam (17) showedthat glucose gives rise to the same degree ofcatabolite repression of f-galactosidase in galacto-kinase- and galactose transferase-negative mutantsas in wild-type strains. They concluded that UDPgalactose alone could give rise to catabolite re-pression. When glucose was present in the mediumof a UDP galactose epimerase-negative mutant,catabolite repression of f-galactosidase could beobserved, but appeared weaker than in the wild-type strain. They concluded that only the galac-tose compounds are involved in cataboliterepression of ,B-galactosidase and that the glucose-elicited repression observed in the epimerase-negative mutant was the result of residual epimer-ase activity.Our studies with a mutant lacking hexose phos-

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CATABOLITE REPRESSION

phate isomerase suggest that compounds otherthan those related to galactose can rise to catab-olite repression of ,B-galactosidase.-The characterization of a gene, CR, which

determines catabolite sensitivity specific for thelac operon, and the isolation of a CR- mutantstrain (15), allowed us to determine whether therepression of ,B-galactosidase by galactose ingalactose-negative mutants is dependent on a

functional CR gene. The results suggest thatgalactose can inhibit induction of the lac operon

independently of catabolite repression.

MATERIALS AND METHODS

Chemicals. Isopropyl-thio-,3-D-galactoside (IPTG)and o-nitrophenyl-,B-D-galactoside (ONPG) were ob-tained from Mann Research Laboratories, New York,N.Y.

Media. Minimal medium was made by adding to 1

liter of water 10 g of K2HPO4, 0.2 g of MgCl2, 0.2 gof Na2SO4, 5.0 g of NaCl, 0.2 g of sodium citrate, tracesof FeSO4 and CaCl2, and 0.5 mg of thiamine. The pHwas adjusted to 7.0 with concentrated HCl. Carbonsources were added to 0.4%. (NH4)2SO4 was presentat 0.4% as nitrogen source. Individual amino acidswere added to 20 ,ug/ml when required.

EMB-galactose medium was prepared by adding to1 liter of water 8 g of tryptone, 5 g of NaCl, 1 g ofyeast extract, 15 g of agar, 400 mg of eosin, 64 mg ofmethylene blue, 2 g of K2HPO4, and 10 g of galactose.Tryptone medium was made by adding to 1 liter ofwater 10 g of tryptone, 5 g of NaCl, and 5 g of yeastextract. ThepH was adjusted to 7.0 with 5 N NaOH.

Phage and bacterial strains. Transducing phagePlkc was obtained from the collection of S. E. Luria.The bacterial strains used in this study are listed inTable 1.

f3-Galactosidase assay. ,B-Galactosidase activity wasmeasured by the method reported by Loomis andMagasanik (14).

Hexose phosphate isomerase. The specific activityof hexose phosphate isomerase was determined incell extracts by observing the rate of reduction ofnicotinamide adenine dinucleotide phosphate (NADP)at 37 C on a Beckman DU spectrophotometer at-tached to a Gilford absorbance recorder at 340 m,u.Washed, concentrated bacterial cultures in 0.04 M

glycylglycine-HCl buffer (pH 7.4) were disrupted inan ultrasonic oscillator (Measuring & Scientific Equip-ment, Ltd., London, England). The extracts werefreed from debris by centrifugation. To 2.6 ml of0.04 M glycylglycine buffer (pH 7.4) was added 0.1 mlof a solution 6 X 10-l M MgCl2, 0.1 ml of a solution4.5 X 103 M NADP, 0.1 ml of extract, and 0.1 ml ofa solution 2 X 10-2 M fructose-6-phosphate. Theamount of NADP reduced was calculated from thechange in optical density with 6.2 X 103 as the molarextinction coefficient of reduced NADP (NADPH2).The assay depends on endogenous glucose-6-phos-phate dehydrogenase activity and so gives a minimalvalue for the enzyme.

6-Phosphogluconate dehydrase. The activity of 6-

TABLE 1. Bacterial strains

SourceK-12 strain Pertinent genotypea or

refer-enceb

C600. lac- (i+z+y-) CR+ gal+ TL- aJR-11 lac- (i+z+y-) CR+ gal- TL- glut- b

HI-JR-1 I-R. lac- (i+z+y-) CR+ gal+ TL- H1-JR-11-I. .. lac- (i+z+y-) CR+ gal+ TL- HI-3.000 .. lac+ (i+z+y+) CR+ gal+ aLA-12 lac+ (i+z+y+) CR- gal+ cA-263 lac+ (i+z+y+) CR+ gal- aA-263-1... lac+ (i+z+y+) CR- gal- dA-413 .. lac+ (i-z+y+/i-z+y+) CR+ gal- aA-460.. lac+ (i+z+y+) CR+ acet- a

a The lac symbols are identical to those ofLoomis and Magasanik (14); CR-, catabolite in-sensitivity of the lac operon; TL-, threonine andleucine requirements; glut-, glutamate require-ment; acet-, acetate requirement; H1-, hexoseisomerase-negative; gal-, galactokinase-negative(except strain JR-I1 which is galactokinase-posi-tive but unable to ferment galactose).

b (a) S. E. Luria; (b) J. Rothman; (c) Loomisand Magasanik (15); Loomis and Magasanik (inpress). All other strains were isolated during thisstudy.

phosphogluconate dehydrase was determined accord-ing to the method of Fraenkel and Horecker (4) byobserving the NADP-independent pyruvate forma-tion from 6-phosphogluconate.

Tryptophanase. The activity of tryptophanase wasdetermined according to Pardee and Prestidge (22)by observing the formation of indole from L-trypto-phan. One unit is defined as that amount which willform 1 m,umole of indole in 1 min at 37 C at pH 7.0.

Amylomaltase. Amylomaltase activity was esti-mated according to Lund and Magasanik (unpublisheddata). Cell extracts were prepared by disruption of awashed, concentrated culture of the various strains inphosphate buffer (pH 7.0) with an ultrasonic oscilla-tor (Measuring & Scientific Equipment, Ltd.). Theextracts were freed from bacterial debris by centrifu-gation. To 1 ml of extract in 0.1 M potassium phos-phate buffer (pH 7.0) was added 0.1 ml of 10% mal-tose. After exactly 30 min at 37 C, 0.5 ml of 0.114 NKOH and 0.5 ml of a 2% solution of ZnSO4-7H20were added. The precipitate was removed by cen-trifugation, and the supernatant fluid was assayedfor glucose by use of the Glucostat reagents (Worth-ington Biochemical Corp., Freehold, N.J.).

Protein. The concentration of protein was esti-mated by the method of Lowry et al. (16). Proteinsynthesis was measured by determining the rate ofC14-leucine uptake according to Nakada and Maga-sanik (20).

RESULTSStudies on hexose phosphate isomerase-negative

mutants. Strain JR-11 was isolated from an

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ethylmethane sulfonate (EMS)-mutagenized cul-ture of Escherichia coli strain C600 as a mutantwhich ferments galactose poorly on EMB-galac-tose plates. It was shown that this phenotype is notrelated to a mutation in the gal operon since, afterthe introduction of a functional gal operon bytransduction or F-duction into strain JR-11, thecells are still unable to ferment galactose well

(J. Rothman, personal communication). Theactivities of UDP glucose pyrophosphorylase andphosphoglucomutase were found to be normalin strain JR-11. However, the strain was found tocontain less than 1% of the wild-type hexosephosphate isomerase activity (5).

It has been shown that T-even phage lysates ofbacterial strains lacking UDP glucose are host-restricted (5). Such lysates are termed T*. NormalT4 phage lysates can be made from strain JR-11if glucose is present in the medium. However, inan amino acid medium lacking glucose, T* phagelysates are produced. It appears that the onlybiochemical pathway from amino acids to UDPglucose has been lost in this strain.

Although the strain grew well in an amino acidmedium, it grew poorly and only after a lag ofmany days in minimal medium supplementedwith the amino acids, threonine and leucine,which the parent strain is known to require. Wefound that the addition of glutamate to the me-dium shortened the lag to about 1 day and thenallowed reasonable growth of the strain on manycarbon sources (Table 2). L-Glutamate (20,g/ml)was added to the media in all subsequent experi-ments. However, strain JR-1 1 was found to growmore slowly than the parent strain on severalcarbon sources even with added glutamate and

TABLE 2. Growth rates of various strains ofEscherichia colia

StrainCarbon source

C600 JR-11 JR-11-R JR-11-I

Glycerol ......... 90b 105 95 95Glucose .......... 70 115 110 105Gluconate ........ 78 80 75 75Fructose 120 >300 120 120

Xylose ........... 75 85Succinate ......... 85 85

a The growth rates were determined at 37 C inminimal medium containing 20,Ag/ml of L-threo-nine, 20 Ag/ml of L-leucine, 20 ,g/ml of L-gluta-mate, and the various carbon sources at 0.4%.The optical density at 530 mlA was measured on aKlett-Summerson photoelectric colorimeter for atleast two generations in the exponential phase ofgrowth.bMass doubling time (minutes).

was found to grow extremely slowly on fructose,as shown in Table 2. To elucidate the nature ofthis growth response, we selected derivatives ofstrain JR-11 for ability to ferment galactose on

EMB-galactose agar from an EMS-mutagenizedculture (13). We also isolated similar derivativesafter infection of strain JR-11 with phage P1 froma lysate of the gal- strain, LA-12. Single colonyisolates of the revertant, JR-11-R, and the trans-ductant, JR-11-I, were gal- on the EMB plates.Both strains grow well on glycerol and fructoseand require threonine and leucine but not gluta-mate for growth (Table 2).The activity of hexose phosphate isomerase in

these strains was determined (Table 3). To our

surprise, neither strain JR-1 1-R nor strain JR-1 1-Ihad more than 0.1 % of the activity of the enzymefound in strain C600. To test for in vivo isomeraseactivity, T6 phage lysates of strain JR-11-R wereprepared. As can be seen in Table 4, T* phagecharacteristics were found in the lysates of cellsgrown on amino acids and on amino acids plusfructose. Normal phage lysates were formed whenglucose was present in the medium. It appearsthat strain JR-1 1-R lacks hexose phosphateisomerase activity in vivo as well as in vitro. The

TABLE 3. Hexose phosphate isomerase activitya

Activity (mpmolesStrain per min per mg of

protein)

C600 .................. 154JR-lI ................. 0.24JR-11-R ............... 0.0JR-1 -I ................ 0.09

a The activities were determined in extracts ofovernight cultures of the strains grown on glycerolminimal medium.

TABLE 4. Production of T* in strain JR-11-Ra

Efficiency of plating on

MediumShigella Escherichia

dysenteriae coli B

Tryptone. 1.0 104Tryptone + 0.4% glucose.... 1.0 0.8Tryptone + 0.4% fructose. 1.0 104

a Phage T6 lysates of strain JR-ll-R in thevarious media were prepared by incubating 2 X107 T6 phage with 3 X 108 bacteria at 37 C for 10min, collecting the bacteria by centrifugation, andresuspending them at 3 X 104 bacteria per milli-liter in the various media. After 80 min at 37 C, adrop of chloroform was added. Dilutions weremade for titering phage on S. dysenteriae 16 andE. coli B.

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CATABOLITE REPRESSION

biochemical basis for poor galactose fermentationand lack of growth on fructose in strain JR-11 isnot understood, but does not appear to be relatedto hexose phosphate isomerase activity. In anycase, strains JR-11-R and JR-11-I appear com-pletely normal except for the loss of hexose phos-phate isomerase activity and the concomitantinability to form glucose derivatives from carbonsources not containing glucose or galactose.A similar hexose phosphate isomerase-negative

mutant of Salmonella typhimurium has been iso-lated (4). In contrast to the isomerase-negativestrains of E. coli, the Salmonella mutant growsvery slowly on glucose. In an effort to determinethe basis for this difference, we investigated theEntner-Doudoroff pathway. E. coli is known tocontain 2-keto-3-deoxy 6-phosphogluconate al-dolase, the second enzyme of the pathway (12).We found that E. coli forms 6-phosphogluconatedehydrase, the first enzyme of the pathway, whengrown on glucose, gluconate, or glycerol (Table5). Thus, it appears that E. coli forms the Entner-Doudoroff enzymes constitutively in contrast toSalmonella, which forms 6-phosphogluconatedehydrase only when grown on gluconate. Theutilization of this pathway for glucose metabolismin the isomerase-negative mutants of E. coli mayaccount for the relatively rapid growth on glucose.

Catabolite repression in strains JR-lI, JR-II-R,and JR-HI-I. We determined the differential rateof $-galactosidase synthesis in strains C600,JR-11, JR-11-R, and JR-11-I after induction inminimal medium minus a source of nitrogen, inthe presence of various carbon sources. The cul-tures were all preadapted to growth on the testcarbon source. Thus, complications due to tran-sient repression of ,-galactosidase were not pres-ent in these experiments (Loomis and Magasanik,in press). Likewise, IPTG was present in all experi-ments at I0-3 M concentration, a concentration

TABLE 5. 6-Phosphogluconate dehydrase activity'

Activity (mumolesStrain Carbon source per min per mg of

protein)

3.000 Glucose 78

3.000 .......... Gluconate 180C600 ...... Glycerol 50LA-12 ...... Glucose 62LA-12 ...... Gluconate 156

a The strains were grown overnight in minimalmedium with the various carbon sources. Thebacteria were collected by centrifugation, washed,and suspended in glycylglycine-HCl buffer (pH7.4). The enzyme was measured in sonic extractsas described in Materials and Methods.

known to give optimal induction of cells growingin glucose-containing medium (14). The repres-sion observed under these conditions is completelyrelieved by a mutation in a gene, CR, which hasbeen shown to determine catabolite repressionspecific to the lac operon (15; Loomis and Maga-sanik, in press).Under these conditions, extremely strong

catabolite repression of ,B-galactosidase is exertedby gluconate, xylose, lactate, succinate, andfructose in all strains tested (Table 6). Thesecarbon sources cannot give rise to either glucoseor galactose nor to their derivatives in the isom-erase-negative strains. Thus, it appears thatcatabolite repression of,-galactosidase can re-sult from a high concentration of compoundsother than those related to glucose or galactose.

If one assumes that lactate gives rise to a catab-olite which represses ,B-galactosidase independ-ently of hexose phosphate isomerase activity,one would expect glycerol to give rise to the samecatabolite independently of hexose phosphateisomerase activity. However, it can be seen inTable 6 that, although glycerol in the absence ofa nitrogen source elicits strong repression of,-galactosidase in the wild-type strain, it does notrepress f3-galactosidase in the isomerase-negativemutants. The weight of evidence, however, sug-gests that the lack of repression by glycerol inhexose phosphate isomerase-negative mutants isnot due to the fact that the formation of glucose-6-phosphate is blocked in these mutants. Atpresent, the problem remains a mystery.

Nevertheless, we decided to determine whetherglycerol would give rise to catabolite repressionof tryptophanase and amylomaltase in strainJR-1 1 in the absence of a nitrogen source. It canbe seen (Table 7) that, although gluconate re-presses amylomaltase strongly in both strain

TABLE 6. Catabolite repression of l-galactosidasea,a S 8-

0

0 a Cd

C600 ...... 4 1 1 7 4 14Jr-l ...... 7 - 2 90 4 13JR-11-R... 1 5 - 112 1JR-11-I - - - 80

a Values are the differential rate of ti-galactosi-dase synthesis in minimal medium lacking a nitro-gen source, containing various carbon sources at0.4% concentration. The rate in the absence ofboth a carbon and a nitrogen source is taken as100. The rate of synthesis was determined over aperiod of 80 min after the addition of 10-3 MIPTG.

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TABLE 7. Catabolite repression of tryptophanaseand amylomaltase

TryptophanaseaStrain Carbon source Amylomaltaseb

+NH4+ -NH4+

C600 Glycerol 100 7Glucose 26Gluconate 37 0

JR-1I Glycerol 100 20 81Glucose 32Gluconate 43 0

a The values in nitrogen-containing medium arethe relative rates of enzyme synthesis after induc-tion with 1 mg/ml of L-tryptophan. The rate withglycerol as carbon source is taken as 100. The ratewas determined over a period of 60 min. All val-ues are the average of at least two determinations.The values in the absence of a nitrogen source arethe relative differential rates of synthesis. Therate when no carbon source is present is taken as100. The rate was determined over a period of 80min.

b Values are the relative specific activities ofamylomaltase after incubation overnight in thepresence of 0.4% maltose. The specific activity ofa culture incubated overnight without added car-bon source is taken as 100

JR-il and the parent strain C600, glycerol onlyrepresses this enzyme in the parent strain.Glycerol exerts strong repression of trypto-phanase in strain JR-il under this condition.It appears that the catabolite related to tryp-tophanase can be formed in the mutant from bothglycerol and gluconate, and the catabolite relatedto amylomaltase and ,B-galactosidase can beformed in the mutant from gluconate but notfrom glycerol.

Repression by glucose in strain JR-Il. Since wefound that strain JR-l1 grows more slowly onglucose than on glycerol (Table 2), we wonderedwhether glucose would give rise to cataboliterepression of ,B-galactosidase in a growing culture.The fully induced differential rate of f-galacto-sidase synthesis in a culture of strain JR-i1 wasfound to be reduced by the presence of glucose inthe medium to 55% of the differential rate inglycerol-containing medium. It appears that glu-cose can give rise to catabolite repression of f-galactosidase, even when the growth rate onglucose is reduced by loss of hexose phosphateisomerase activity.When the oxidation of glucose to gluconate in

Aerobacter aerogenes is impaired by mutation,glucose no longer represses f3-galactosidase (21).Since an impairment in utilization of the pentoseshunt, but not a block in the Embden-Meyerhof

pathway, reduces catabolite repression of ,B-galactosidase, it seems that the specific catabolitemay be a derivative of the pentose shunt.

Studies in a mutant unable to form acetyl-coen-zyme A (CoA). To define further the compoundsinvolved in catabolite repression of j3-galacto-sidase, we investigated the repression elicited byglucose in a mutant unable to form compoundsof the citric acid cycle in the absence of exogenousacetate.

E. coli strain A-460 requires acetate for growthand has been shown to have only 0.5% of thewild-type lipoic reductase-transacetylase activity(6). A culture of this strain growing exponentiallyin glycerol minimal medium with 0.4% acetatewas induced with 10-3 M IPTG, and glucose wasadded to one half of the culture. The rate of f3-galactosidase synthesis was reduced in the glucosemedium to 50% of the rate in the glycerol me-dium. To exclude the Krebs cycle intermediatesfrom a role in catabolite repression of (-galacto-sidase, a glucose-grown culture of strain A-460was collected on membranes, washed, and re-suspended in minimal medium, but acetate wasomitted. Glucose was added to one half of theculture, and the other half remained free from acarbon source. The cells could not grow underthese conditions. It was found that the differentialrate of ,B-galactosidase synthesis was reduced to5% of the rate in the carbon-free culture by thepresence of glucose.

It has been shown that, when the concentrationof acetyl-CoA is low, very little of the 4-carboncompounds of the Krebs cycle can be formed (1).Thus, it appears that neither compounds of theKrebs cycle nor the derivatives of these com-pounds are required for complete catabolite re-pression of ,B-galactosidase.

Apparent repression by galactose. We haveshown that several carbon sources cause cataboliterepression of 3-2galactosidase in a mutant inwhich none of the galactose compounds can beformed from these carbon sources. Therefore, weinvestigated the repression elicited by galactosein strains able and unable to metabolize galactose.

Unlike McFall and Mandelstam (17), we didnot observe repression by galactose of,-galacto-sidase in growing cultures of a galactose-positivestrain, 3.000. McFall and Mandelstam utilized astrain, 58-161, which had been selected for strongrepression of (-galactosidase by glucose, and it isconceivable that galactose is also a better sourceof catabolites in strain 58-161 than in strain 3.000.We did observe a severe repression by galactoseof f-galactosidase when anabolism was reducedby the lack of a nitrogen source in the wild-typestrain, 3.000, but not in the strain carrying a muta-

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tion in the CR gene, LA-12G (Table 8). A muta-tion in the CR gene has been shown to render astrain insensitive to catabolite repression of thelac operon by several carbon sources even underconditions of nitrogen starvation (15). Therefore,we conclude that the repression elicited by galac-tose in the wild-type strain is dependent on afunctional CR gene and appears to be an expres-sion of catabolite repression.

In agreement with the observations of McFalland Mandelstam (17), galactose was found togive rise to repression of ,3-galactosidase in agalactokinase-negative mutant, A-263 (Table 8;Fig. 1). However, repression of fl-galactosidaseby galactose was also observed in a CR7 deriva-tive of the galactokinase-negative mutant, A-263-1(Table 8; Fig. 1). Thus, the repression of f-galactosidase elicited in these mutants, unable tometabolize galactose, appears to be independentof the gene which determines catabolite sensitivityspecific to the lac operon.

Galactose appears to repress p-galactosidasein growing cultures of a galactose-negative strainbut not in growing cultures of a wild-type strain.It has been shown that the internal concentrationof galactose is much higher in galactokinase-neg-ative cells than in wild-type cells (7). The re-

TABLE 8. Repression by galactose

Rate of,B-galactosidase

Pertinent genotype synthesis ingalactose-containing

Strain medium

lac gal+

3.000..... i+z+y+CR+ + 100 1LA-12G.. i+z+y+CR- + 100 106A-263... i+z+y+CR+ - 58A-263-1... i+z+y+CR - 56 -A-413.. F'i-z+y+1/iz+y+ - 100 -

CR+

a The values are the differential rate of enzymesynthesis in minimal medium containing 0.1%Casamino Acids, 0.4% glycerol, 2 X 10-2 M galac-tose, and 5 X 104 M IPTG. The differential rate ofsynthesis in the absence of galactose is taken as100, and was similar in all strains. The rate wasdetermined over a period of 90 min. The galactose-negative strains lack galactokinase activity.bThe values are the differential rate of enzyme

synthesis in minimal medium minus NH4+, con-taining 2 X 10-2 M galactose and 10-3 M IPTG.The differential rate in the absence of a carbonsource is taken as 100 and was similar in bothstrains. The rate was determined over a 90-minperiod.

7.Uf)

z02400

w

0

0

< 2000

25 50 25 50OPTICAL DENSITY (KLETT UNITS)

FIG. 1. Repression by galactose in CR+ and CR-galactose-negative strains. A culture of cells in the ex-ponential phase of growth in glycerol minimal mediumwas induced with 5 X JO4-M IPTG. The culture wasimmediately divided and IO-2M galactose was added toone half. Samples were taken for determination of /3-galactosidase activity. The optical density at 530 mAwas observed on a Klett-Summerson photoelectriccolorimeter. (a) Strain A-263 lac+ (i+z+y+ CR+)galactokinase-; (b) strain A-263-1 lac+ (i+z+y+ CR-)galactokinase-.

pression of f3-galactosidase in galactokinase nega-tive strains growing in the presence of galactosemay result from this increased internal galactose,per se.When glucose is added to fully induced lac+

cells growing on carbon sources other than glu-cose, 3-galactosidase synthesis is strongly re-pressed for about 40 min. After this period ofgrowth in the presence of glucose, #3-galactosidaseis synthesized at the rate determined by cataboliterepression in the particular strain. The repressionby galactose in galactokinase-negative mutantsdoes not appear to be a case of this transient re-pression, since the repression was relatively weakand appeared constant during 80 min of growthin the presence of galactose (Fig. 1).Glucose is also able to repress severely ,B-

galactosidase synthesis in glucose-grown cellswhen induced by low levels of inducers. Thissevere repression can be overcome in ,B-galacto-side permease-positive cells by a short period ofpreinduction of the lac operon (Loomis andMagasanik, in press). It appears that the presenceof glucose in the medium reduces the internalconcentration of inducer so that only partialinduction of the lac operon can occur. The ability

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to concentrate inducer in the cells, i.e., permeaseactivity, overcomes this effect of glucose (9;Loomis and Magasanik, in press). The repressionby galactose of ,B-galactosidase in galactokinase-negative mutants is not relieved even after 1 hr ofinduction of the lac operon. Since ,B-galacto-sidase activity and, presumably, permease activityincreased markedly during this period, it appearsthat increased permease activity does not relievethe repression by galactose in these galactose-negative mutants. It has been shown that galac-tose inhibits the entry of galactosides intoinduced permease-positive cells (11). Thus, galac-tose may give rise to repression of f3-galactosidasein induced cells of a galactokinase-negativestrain by competing with inducer for entry intothe cells. Therefore, f-galactosidase synthesis ina galactokinase-negative strain constitutive forf3-galactosidase might not be repressed by galac-tose, since the rate of ,B-galactosidase synthesisis independent of internal concentration of in-ducer in such a strain. When the differential rateof,-galactosidase synthesis was determined instrain A-413 (F i-zy+/i-z4y+, gal-), it wasfound that the presence of galactose in the me-dium did not repress the enzyme in this lac con-stitutive strain (Table 8). Thus, it appears thatgalactose gives rise to repression of 3-galac-tosidase in galactokinase-negative mutants by aninhibition of induction and not by cataboliterepression of the operon.

DISCUSSIONWhen anabolism of wild-type E. coli is reduced

by nitrogen starvation, the presence of any carbonsource in the medium reduces the differential rateof f3-galactosidase synthesis by a factor of morethan 10 (19). It is thought that the concentrationof one or more catabolites in the cells determinesthe degree of repression of a specific operon (18).Our results with the hexose phosphate isom-

erase-negative mutant indicate that cataboliterepression of the lac operon does not requireglucose, galactose, gluconate, or their directderivatives, since these compounds cannot beformed by any known pathway from fructose,lactate, xylose, or succinate in an isomerase-negative strain, and yet a strong repression of,B-galactosidase by these compounds can be ob-served when anabolism is reduced. Likewise, thecompounds related to the Krebs cycle do notappear to be required for catabolite repression ofthe lac operon, since glucose gives rise to strongrepression in a mutant under conditions wherethe formation of Krebs cycle compounds isseverely restricted.McFall and Mandelstam (17) reported re-

pression of f-galactosidase by galactose in mu-tant strains unable to grow on galactose. Theysuggested that the degree of catabolite repressionof the lac operon is determined by the concentra-tion of galactose compounds. We have presentedevidence that the repression by galactose observedin galactokinase-negative strains is the result ofan inhibition of induction which is independentof the gene, CR, which determines cataboliterepression specific to the lac operon. Thus, galac-tose does not appear to be involved in cataboliterepression of f-galactosidase.

If catabolite repression of the lac operon iseffected by a single compound, we suggest thatthe compound is related to the pentoses andtrioses of intermediary metabolism.

ACKNOWLEDGMENTS

One of us (W.F.L.) has been a trainee under PublicHealth Service training grant STi GM 602 from theNational Institutes of Health to the Department ofBiology, Massachusetts Institute of Technology. Thisinvestigation was supported by National ScienceFoundation grant GB-265 and Public Health Servicegrant GM-07446.

LITERATURE CITED1. CANOVAS, J., AND H. KORNBERG. 1965. Fine con-

trol of phosphopyruvate carboxylase activity inEscherichia coli. Biochim. Biophys. Acta 96:169-172.

2. COHN, M., AND K. HORIBATA. 1959. Inhibition byglucose of the induced synthesis of the $l-galac-toside-enzyme system of Escherichia coli. Analy-sis of maintenance. J. Bacteriol. 78:601-612.

3. COHN, M., AND K. HORIBATA. 1959. Physiologyof the inhibition by glucose of the induced syn-thesis of the ,B-galactoside-enzyme system ofEscherichia coli. J. Bacteriol. 78:624-635.

4. FRAENKEL, D., AND B. HORECKER. 1964. Path-ways of D-glucose metabolism in Salmonellatyphimurium. J. Biol. Chem. 239:2765-2771.

5. HArTMAN, S. 1964. The functioning of T-evenphages with unglucosylated DNA in restrict-ing Escherichia coli host cells. Virology 24:333-348.

6. HENNING, V., AND C. HERZ. 1964. Ein Struktur-gen-Komplex fur den Pyruvat-Dehydrogenase-Komplex von Escherichia coli K12. Z. Verer-bungslehre 95:260-275.

7. HORECKER, B., J. THOMAS, AND J. MONOD. 1960.Galactose transport in Escherichia coli. I. Gen-eral properties as studied in galactokinaselessmutant. J. Biol. Chem. 235:1580-1585.

8. KENNEL, D., AND B. MAGASANIK. 1964. The con-trol of the rate of enzyme synthesis in Aero-bacter aerogenes. Biochim. Biophys. Acta 81:418-434.

9. KEPES, A. 1960. Etudes cinetiques sur la galacto-side-permease d'Escherichia coli. Biochim. Bio-phys. Acta 40:70-84.

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10. KESSLER, D., AND H. RICKENBERG. 1964. A new

method for the selection of mutants of Escheri-chia coli forming /-galactosidase constitutively.Biochim. Biophys. Acta 90:609-610.

11. KOCH, A. 1964. The role of permease in transport.Biochim. Biophys. Acta 77:177-200.

12. KOVACHEVICH, R., AND W. WOOD. 1955. Carbo-hydrate metabolism by Pseudomanas fluores-cens. IV. Purification and properties of 2-keto-3-deoxy-6-phosphogluconate aldolase. J.Biol. Chem. 213:751-767.

13. LIN, E. C. C., S. A. LERNER, AND S. E. JORGENSEN.1962. A method for isolating constitutive mu-tants for carbohydrate-catabolizing enzymes.Biochim. Biophys. Acta 60:422-424.

14. LOOMIS, W. F., JR., AND B. MAGASANIK. 1964. Therelation of catabolite repression to the induc-tion system for ,B-galactosidase in Escherichiacoli. J. Mol. Biol. 8:417-426.

15. LOOMIS, W. F., JR., AND B. MAGASANIK. 1965.Genetic control of catabolite repression of thelac operon in Escherichia coli. Biochem. Bio-phys. Res. Commun. 20:230-234.

REPRESSION 177

16. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR,AND R. J. RANDALL. 1951. Protein measure-ment with the Folin phenol reagent. J. Biol.Chem. 193:265-275.

17. MCFALL, E., AND J. MANDELSTAM. 1963. Specificmetabolic repression of three induced enzymesin Escherichia coli. Biochem. J. 89:391-398.

18. MAGASANIK, B. 1961. Catabolite repression. ColdSpring Harbor Symp. Quant. Biol. 26:249-256.

19. MANDELSTAM. J. 1962. The repression of constitu-tive ,B-galactosidase in Escherichia coli by glu-cose and other carbon sources. Biochem. J.82:489-493.

20. NAKADA, D., AND B. MAGASANIK. 1964. The rolesof inducer and catabolite repressor in the syn-thesis of ,B-galactosidase by Escherichia coli.J. Mol. Biol. 8:105-127.

21. NEIDHARDT, F. 1960. Mutant of Aerobacter aerog-enes lacking glucose repression. J. Bacteriol.80:536-543.

22. PARDEE, A., AND L. PRESTIDGE. 1961. The initialkinetics of enzyme induction. Biochim. Bio-phys. Acta 49:77-88.

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