JOURNAL OF BACTERIOLOGY, Feb. 1969, p. 535-543 Vol. 97, No. 2Copyright © 1969 American Society for Microbiology Printed in U.S.A.

Control of Mixed-Substrate Utilization inContinuous Cultures of Escherichia coli

RICHARD S. SILVER1 AND RICHARD I. MATELESDepartment of Nutrition and Food Science, Massachusetts Institute of Technology,

Cambridge, Massachusetts 02139

Received for publication 14 October 1968

The chemostat culture technique was used to study the control mechanisms whichoperate during utilization of mixtures of glucose and lactose and glucose and L-aspar-tic acid by populations of Escherichia coli B6. Constitutive mutants were rapidlyselected during continuous culture on a mixture of glucose and lactose, and the,B-galactosidase level of the culture increased greatly. After mutant selection, thespecific ,B-galactosidase level of the culture was a decreasing function of growth rate.In cultures of both the inducible wild type and the constitutive mutant, glucose andlactose were simultaneously utilized at moderate growth rates, whereas only glucosewas used in the inducible cultures at high growth rates. Catabolite repression wasshown to be the primary mechanism of control of,3-galactosidase level and lactoseutilization in continuous culture on mixed substrates. In batch culture, as in thechemostat, catabolite repression acting by itself on the lac enzymes was insufficientto prevent lactose utilization or cause diauxie. Interference with induction of the lacoperon, as well as catabolite repression, was necessary to produce diauxic growth.Continuous cultures fed mixtures of glucose and L-aspartic acid utilized both sub-strates at moderate growth rates, even though the catabolic enzyme aspartase waslinearly repressed with increasing growth rate. Although the repression of aspartaseparalleled the catabolite repression of,-galactosidase, L-aspartic acid could beutilized even at very low levels of the catabolic enzyme because of direct anabolic in-corporation into protein.

Epps and Gale (9) demonstrated that the pres-ence of glucose in the nutrient media prevents thecatabolic dissimilation of many other substrates.Monod (30) showed that when microorganismswere cultivated on a variety of substrate mixturesdiauxic growth occurred: the most rapidly ca-tabolized substrate was assimilated first; a lagperiod followed during which adaptive enzymeswere produced; and finally the secondary sub-strates were utilized. Recently, many workershave studied the control mechanisms which causemicrobial cells to utilize one substrate in prefer-ence to another. Magasanik and his co-workers(26, 33) developed the theory of catabolite repres-sion, which postulated that adaptive enzymesnecessary to catabolize the secondary substratewere repressed through the interaction of anoverfed catabolite pool and a genetic system.Other workers have claimed that, although thecatabolite repression model suffices to explainmany aspects of diauxie in the lac system of

IPresent address: Gulf Research and Development Co., P.O.Drawer 2038, Pittsburgh, Pa. 15230.

Escherichia coli, controls involving the transportof inducer or substrate predominate in otherenzyme systems and may play a strong supportingrole in the glucose-lactose diauxie (1, 2, 15).Transport-level controls such as these have beendescribed for the gal (1, 2, 15) and the lac (4, 7)systems of E. coli.Another mechanism of control is the inhibition

of preformed catabolic enzymes by an enlargedcatabolite pool, as demonstrated for the glucoseand glycerol system in E. coli by Lin and co-workers (11, 12, 18, 39, 40).Most studies of controls operating during

growth on mixed substrates have been carried outin batch cultures and are limited both by thechanges in medium and physiological state of thecell which occur during the course of batch growthand by the inability to vary growth rate inde-pendently of medium composition. In this study,continuous chemostat cultures of E. coli B6 werefed mixtures of glucose and lactose or glucoseand L-aspartic acid. The effect of growth rate onthe control of enzyme level and substrate utiliza-

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tion was examined during prolonged steady-state operation at various growth rates.

MATERIALS AND METHODSOrganisms and media. E. coli B6 was used in all

experimental work. This strain is inducible for thelac operon and produces both relatively high basaland fully induced levels of j-galactosidase.

The basal medium M63 (4) was autoclaved in 8-liter batches. L-Aspartic acid was autoclaved with thebasal medium. Glucose and lactose were sterilized byfiltration through membrane filters (Millipore Corp.,Bedford, Mass.) and were added aseptically to thebasal medium.

Continuous-culture apparatus. The equipment forcontinuous culture was similar to that previouslydescribed by S. K. Chian (Ph.D. Thesis, MassachusettsInstitute of Technology, Cambridge, 1967). Themagnetically agitated fermentor, with a culture volumeof 200 ml, was kept in a thermostatically controlledbath at 37 C and fed sterile medium by a tubing pump.Air was sterilized (by passing it through a fiberglassfilter) and introduced into the fermentor headspaceat a rate of about 1 liter/min. All flexible tubing wassilicone rubber (Esco Rubber Ltd., London, England),and all fermentor surfaces in contact with mediumwere silicone-coated with Siliclad (Clay-Adams, NewYork, N. Y.) to reduce cell adhesion.

Continuous culture techniques. A batch culture wasinitiated in the culture vessel, and continuous mediumfeed and culture withdrawal were begun when theculture became visibly turbid. The medium feed rate,f (milliliters per hour), was monitored by a pipetteflowmeter. The growth rate becomes equal to the dilu-tion rate, D (hours-'), defined as f/v (v = culturevolume in milliliters), when the culture reaches steadystate. Existence of the steady state was confirmed bystabilization of the cell density measured at 640 nm(red filter) with a Klett-Summerson colorimeter(Klett Manufacturing Co., New York, N. Y.). Tochange the grov%th rate, the medium feed rate waschanged, and the culture was maintained until a newsteady state had been achieved. At least six residencetimes (v/f) at each dilution rate were allowed to elapsebefore samples were collected.

Sample preparation. Samples were withdrawn fromthe culture effluent stream through an ice-packedstainless-steel heat-exchanger coil to chill to below 5 C.The cells were separated from the medium by batchcentrifugation at 0 C and 48,200 X g; the packed cellswere washed and resuspended in the appropriatebuffers described under Analytical techniques.

Batch culture techniques. Cells were inoculatedinto 50 ml of sterile M63 medium in 500-ml baffled,Klett side-arm Erlenmeyer flasks. Cultures were incu-bated at 37 C with shaking. Samples were withdrawnwith sterile pipettes, chilled rapidly, and processed inthe same manner as samples from continuous culture.

Analytical techniques. Glucose was determined byuse of the enzymatic Glucostat micro procedure(Worthington Biochemical Corp., Freehold, N.J.).Lactose was assayed by an enzymatic procedure.Crude yeast ,j-galactosidase (30.6 o-nitrophenol galac-

toside units/mg; Mann Research Laboratories, NewYork, N.Y.) was suspended in cold sodium phosphatebuffer (0.1 M, pH 7.0) by vigorous agitation to give aconcentration of 2.0 mg of crude enzyme per ml. Un-dissolved matter was removed by filtration, and 0.1ml of the enzyme solution was added to 1.0 ml of cell-free culture sample, diluted to contain less than 200,ug of lactose per ml. Incubation with agitation for 1 hrat 37 C resulted in complete hydrolysis of the lactose,and the glucose released was determined by the Gluco-stat micro procedure, with correction for enzymeabsorbance made with a reagent blank. For samplescontaining both glucose and lactose, Glucostat assayswere applied to duplicate samples, one of which hadundergone FB-galactosidase hydrolysis; lactose wascomputed by difference.

L-Aspartic acid was determined by a modificationof the method of Matsushita et al. (29). To 2.0 ml ofsample containing less than 200 J,g of L-aspartic acidper ml was added 0.5 ml of 0.5% CoSO4*7H20 in 1%sodium tartrate. Then 0.2 ml of Folin phenol reagent(1.0 N, Fisher Scientific Co., Medford, Mass.) wasadded, and the mixture was agitated. After the mixturewas allowed to stand for 30 min at room temperature,2.0 ml of a solution consisting of 2% Na2CO3 in 0.1N NaOH and 0.01% Gum Ghatti (Fisher ScientificCo.) was added, and the mixture was agitated well.The mixture was again allowed to stand for 30 min atroom temperature, and then the absorbance was readat 750 nm, with correction made for the reagent blank.

Protein was measured by the method of Lowry etal. (24). The culture sample was centrifuged, and thepacked cells were washed and resuspended in an equalvolume of sodium phosphate buffer (0.1 N, pH 7.0).A 1.0-ml sample of cell suspension was mixed with 1.0ml of a solution containing 1% sodium deoxycholate(Difco) and 0.5% NaOH; the mixture was then keptat 100 C for 5.0 min, which resulted in complete diges-tion of the cells. This digest was subject to the Lowryprocedure, and absorbance was read at 740 nm, withcorrection made for the reagent blank. Protein wasdetermined from a standard curve prepared withbovine serum albumin (Pentex, Inc., Kankakee, Ill.).A modification of the method of Lederberg (20)

was employed for assay of ,3-galactosidase. Packedcells were washed and resuspended in a reducingbuffer (37) and were diluted with this buffer if theinitial enzyme titer was high. To a 2.0-ml sample ofcell suspension, 0.05 ml of toluene and 0.05 ml of 1%sodium deoxycholate were added, and the mixture wasincubated with shaking at 37 C for 1 hr. The cell sus-pension was brought to 30 C; 1.0 ml of 3 X 10' Mo-nitrophenol galactoside in reducing buffer wasadded; and the mixture was incubated with agitation at30 C for 10.0 min. A 2-ml amount of 1 M Na2C03 wasthen added, and the absorbance was read at 420 and550 nm. ,3-Galactosidase activity was computed withthe use of the correction for cell absorbance (37) andis reported as international enzyme units per milligramof protein (abbreviated as SEU). It was found thatcellular protein expressed as micrograms per millilitercould be approximated by 2 X Klett units. Therefore,the latter was occasionally used to compute SEU.

Aspartase was determined by a modification of the

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method of Farley and Lichstein (10). Packed cells werewashed and resuspended in sodium phosphate buffer(0.1 M, pH 7.0). To 2.0 ml of cell suspension, 1.0 ml of0.015 M L-aspartic acid was added, and the mixture wasincubated at 37 C for 1.1 hr. The reaction was stoppedby adding 0.3 ml of 25% trichloroacetic acid, and thecells were removed by centrifugation. Releasedammonia was determined by nesslerization, and thespecific aspartase activity was reported as internationalenzyme units per milligram of protein (SEU).

RESULTS

Continuous culture on mixed glucose and lactose.When a chemostat culture was initiated with E.coli B6 and fed a medium containing 0.5 g of glu-cose per liter and 0.5 g of lactose per liter, theresults at first appeared somewhat erratic; how-ever, further examination revealed a definite pat-tern. In several separate cultures inoculated withE. coli B6, the culture first achieved a chemostatsteady state where the ,B-galactosidase level was0.9 to 1.4 SEU. When the dilution rate waschanged and a new steady state was reached, theenzyme level remained at 0.9 to 1.4 SEU, andonly at steady states where the dilution rate ex-ceeded 0.9 hr-' did the enzyme level diminish.After 30 to 50 generations (depending on dilutionrate) of continuous culture on the mixed sub-strates, a dramatic rise in culture ,B-galactosidaselevel was observed, and, at steady states followingthis, the enzyme level was a monotonically de-creasing function of dilution rate, reaching in ex-cess of 13.5 SEU at a dilution rate of 0.1 hr-'.The increase in enzyme level observed after 30

to 50 generations could have been due to selectionof a constitutive mutant (14, 34) or to selection ofa mutant resistant to control of ,B-galactosidase.To distinguish between these possibilities, sam-ples of culture taken toward the later stages of arun were plated out, and colonies were selectedand tested for resistance to catabolite repressionby use of the CR agar (1% glucose, 0.2% lactose)technique of Loomis and Magasanik (22); totest for constitutivity, a similar technique andNutrient Agar were used. All isolates werestrongly constitutive but still subject to cataboliterepression of j3-galactosidase synthesis. One ofthese was chosen for further studies and wasdesignated E. coli B6b2.The selection of constitutive mutants in con-

tinuous cultures fed lactose alone is well known,as mentioned above. The selective pressure forsuch mutants is, however, much greater for acontinuous culture fed mixed glucose and lactose,since the mutation affords the culture the advan-tage of increased availability of the substratelactose at high dilution rates (an advantage lesspronounced for cultures fed lactose alone). The

selection of the constitutive mutant B6b2, andthe consequent increase of the culture ,3-galactosi-dase level, is shown in Fig. 1 for chemostatgrowth on 0.5 g of glucose per liter and 0.5 g oflactose per liter at a dilution rate of 0.51 hr-1.Feeding mixed glucose and lactose to a chemostatculture increases the rate of selection of constitu-tive mutants over rates possible when lactose isfed alone.When a chemostat culture was inoculated with

the constitutive B6b2 and fed basal medium con-taining 0.5 g of glucose per liter and 0.5 g oflactose per liter, the /3-galactosidase level was higheven at the first steady state (Fig. 2), and thedecrease ofenzyme level with growth rate was seenimmediately.

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Both glucose and lactose were simultaneouslyand completely taken up by continuous culturesof inducible E. coli B6 or constitutive mutant E.coli B6b2 below a dilution rate of 0.65 hr-I (Fig.3). The inducible culture (low numbers) con-tinued to utilize glucose up to the wash-outgrowth rate (0.9 hr-1), but increasing amounts oflactose appeared in the effluent at growth ratesabove 0.65 hr-1. The constitutive culture (highnumbers), however, utilized both glucose andlactose equally well up to the wash-out growthrate (0.9 hr'1). Slow permeation of inducer hasbeen suggested (3) as the reason lactose is utilizedonly after a delay of many generations by a con-tinuous culture fed mixed glucose and lactose.The above evidence indicates that it is selectionof a constitutive mutant, rather than slow inducerpermeation, which permits the eventual lactoseutilization.

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Continuous allture on single sugars. The levelof ,3-galactosidase at different steady-state growthrates is shown in Fig. 4a and 4b for continuouscultures fed 0.5 g of lactose per liter or 0.5 g ofglucose per liter, respectively. Lactose run A22was inoculated with constitutive B6b2, and lactoseruns B12 and C12 represent inducible B6. Theseruns (B12 and C12) were carried out with inoc-ulum obtained from B6 continuous culture onglucose alone (runs Bil and Cl 1, respectively),where no pressure for constitutive selectionexisted, and it was apparent that no such selectionhad occurred. Glucose-fed cultures Bll and Cllwere inoculated with inducible B6, and run B13was inoculated with a mutant obtained from alactose-fed chemostat culture (a continuation ofrun B12, not shown).

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FIG. 4. (a) Specific ,-galactosidase level in contin-uous cultures of E. coli B6 (lower curve; runs B12,C12) and E. coli B6b2 (upper curve; run A22) fed 0.5g of lactose per liter. (b) Specific fl-galactosidase levelin continuous cultures of E. coli B6 (lower curve; runsBll, Cli) and a mutant B6 strain (upper curve; runB13) fed 0.5 g ofglucoseper liter.

538 J. BACTERIOL.

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B6 grown on glucose alone produced almostno ,B-galactosidase regardless of dilution rate,whereas in run B13 a moderate enzyme level wasproduced which decreased with increasing dilu-tion rate (Fig. 4b). The mutant selected duringcontinuous growth on lactose and used in run B13appears to be only partially constitutive, notidentical to B6b2 which produced a specificenzyme level 10 times greater in cultures fedmixed substrates (Fig. 2).

Control of lactose unlization in continuous cul-tures fed mixed glucose and lactose. When a cul-ture is fed a mixture of glucose and lactose, glu-cose may control the production of the lacenzymes and utilization of lactose by severaldifferent mechanisms. (i) By virtue of the rapidflow of glucose into the catabolite pools and aresulting large steady-state pool size, productionof the lac enzymes may be diminished by catabo-lite repression. This repression, by reducing thelevel of permease for lactose, restricts the influxof the inducer lactose and further reduces enzymeproduction. (ii) Glucose may competitively in-hibit permeation of lactose and further reduceinduction of the lac operon. Such competitionmay be at the permease level or at the transporterlevel (16, 17, 19). (iii) Finally, a high-level ca-tabolite pool may inhibit preformed lac enzymesand interfere with lactose utilization. Duringcontinuous culture on mixed glucose and lactose,constitutive mutants were rapidly selected, andthese mutants displaced the inducible strain,resulting in a high ,B-galactosidase level which de-creased with increasing growth rate. Fully con-stitutive cells are not subject to controls based onexclusion of inducer, and catabolite repressionemerges as the dominant control on the rate ofsynthesis of,B-galactosidase.

@-Galactosidase synthesis and lactose utilizationin batch culture. During chemostat culture on amixture of glucose and lactose, control exerted onlactose utilization appeared to be primarily bycatabolite repression of the lac enzymes. To deter-mine whether this domination of control by re-pression was due to the physiological peculiaritiesof the chemostat steady state, i.e., energy-sourcelimitation and low external substrate concentra-tion, an examination was made of the batch-culture growth on several substrates of E. coliB6 and the constitutive strain B6b2. The results ofbatch-culture experiments are summarized inTables 1 and 2.The specific growth rate of E. coli B6 was the

same on lactose as on glucose, indicating thattransport or hydrolysis of lactose did not limit.This strain gave diauxic growth on mixed glucoseand lactose with a lag of 15 min (data not shown).

TABLE 1. Batch growth of E. coli B6 and B6b2 onsingle substrates

p-Galactosidase(SEU)

Genera-Strain Medium tion After Aftertime Afe Afr

(hr) approxi- approxi-mately 1 mately 4genera- genera-tion tionsa

B6 Glucose (1 g/liter) 0.75 0.0023 0Lactose (1 g/liter) 0.75 1.8 2.7Glycerol (2 g/liter) 1.0 0.015 0.009Glycerol (2 g/liter) 1.1 5.4 9.0+5X 10-4MIPTG

Glucose (1 g/liter) 0.79 2.7 2.7+5X 10-4MIPTG

Lactose (1 g/liter) 0.70 2.7 5.4+5X 10-4MIPTG

B6b2 Glucose (1 g/liter) 0.78 3.2 4.1Lactose (1 g/liter) 0.85 3.2 6.3Glycerol (2 g/liter) 1.05 8.1 9.9

aSubstrate exhausted.

The enzyme level was about 0.09 to 0.18 SEUduring the glucose growth phase and rose rapidlyto 1.8 to 2.7 SEU when glucose had been ex-hausted. Lactose was not utilized until glucose hadbeen almost completely consumed and j-galac-tosidase was induced.The strong (B-galactosidase inducer isopropyl-

thiogalactoside (IPTG) was used to compare therelative effects of induction and catabolite repres-sion. When B6 was grown on either glucose orlactose and induced with 5 X 10-4 M IPTG, ap-proximately 2.7 ,B-galactosidase SEU were pro-duced during experimental growth. When lactosewas exhausted, ,B-galactosidase synthesis was im-mediately derepressed, and the enzyme level roseto 5.4 SEU; no such immediate derepression wasobserved after glucose exhaustion. Uninduced B6produced about 0.01 SEU when grown on glyc-erol, but induction with 5 X 10-4 M IPTG al-lowed production of 5.4 to 6.3 SEU, and a dere-pression to 9.0 SEU was observed after exhaus-tion of the glycerol.

If the mutant B6b2 isolated from continuousculture is fully constitutive, no external inducerneed permeate the cell to effect full induction, andonly catabolite repression or possible cataboliteinhibition could control the rate of lac enzymesynthesis or enzyme activity. Tables 1 and 2 sum-marize the results obtained during growth of B6b2on various substrates.B6b2 cells grown on glucose contained about

3.6 ,8-galactosidase SEU compared with 9.0SEU for cells grown on the weaker catabolite

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TABLE 2. Batch growth of E. coli B6 and B6b2 on glucose + lactose

Generation time (hr) 8-Galactosidase (SEU)

Strain Medium After ap- After ap-First growth Second After 1 proximately proximately

phase growth phase generation 2 genera- 4 genera-tions tionea

B6 Glucose (0.5 g/liter) + lactose 0.76 0.88 0.09-0.18 1-1.8 2.7(0.5 g/liter)

Glucose (0.5 g/liter) + lactose 0.70 1.3 3.2 2.7 7.2(0.5 g/liter) + 5 X 10-4 M IPTG

B6b2 Glucose (0.5 g/liter) + lactose 0.80 -b 3.6 - 6.3(0.5 g/liter)

,Both glucose and lactose exhausted.bNot determined.

repressor, glycerol. During growth on lactose,the f3-galactosidase level was approximately thesame as on glucose until lactose was exhausted,at which time enzyme synthesis was derepressedand a level of 5.4 to 6.3 SEU resulted. Theseresults are almost identical to those obtainedduring growth of B6 on the same substrates with5 X 10-4 M IPTG as inducer.Many workers (e.g., 4, 21, 25; D. Brown and J.

Monod, Federation Proc. 20:222, 1961) havepreviously reported the effect of complementaryinteractions (similar to those described above) ofinduction and catabolite repression on the rate ofsynthesis of,B-galactosidase. Utilization of thesubstrate lactose depends, however, not only onthe level of the lac enzymes but also on the activityof these enzymes and on the ability of lactose topermeate the cell. The diauxic growth of B6 onglucose and lactose could have been due to ca-tabolite repression, exclusion of the apo-inducerlactose from the cell, or a combination of bothmechanisms. Glucose may exclude an inducingsubstrate by competing either for a nonspecificpermease (1, 2, 13) or at the "transporter" level(16, 17, 19). Two ways of testing this are to inducewith IPTG, which can permeate by a system im-mune to glucose competition (1, 13), or to use theconstitutive B6b2.

Several groups (5, 6, 23, 31) have indicated thatstrongly induced or constitutive cells do not showdiauxie on glucose and lactose. This was examinedby growing B6 induced with 5 X 10-4 M IPTG andB6b2 on glucose and lactose (Table 2). In bothcases, both sugars were utilized simultaneouslyuntil exhausted, with no break in the growth curve(data not shown) and with the B3-galactosidaselevel remaining at 3.2 to 3.6 SEU. Diauxie with-out lag was observed, however, in both casesafter the exhaustion of both glucose and lactose.In this secondary growth phase, which continued

for one generation of B6 and slightly less for B6b2(not shown), accumulated catabolites were con-sumed at a lower growth rate and ,B-galactosidasesynthesis was derepressed to 6.3 to 7.2 SEU.These findings support the concept that strong

catabolite repression alone is insufficient to pre-vent lactose utilization, and control of lactosepermeation must also be exerted. The fact thatlactose was not utilized during the first diauxicleg of B6 growth on glucose and lactose, but wassimultaneously utilized with glucose by IPTG-induced or constitutive cells, indicates that for E.coli B6 transport-level controls act by excludinglactose as an apo-inducer rather than as a sub-strate. While induction and catabolite repressionmust be considered as independent controls of,B-galactosidase synthesis, they are complementaryand can be visualized as two resistances in seriesregulating the rate of lac transmission, with in-duction control as the larger resistance and ca-tabolite repression as the "trim" or fine control.

Continuous culture of E. coli B6 on mixtures ofglucose and L-aspartic acid. L-Aspartic acid canbe catabolized by E. coli via the deaminase aspar-tase, or it can be incorporated directly into theprotein biosynthetic pathways via the asparticand glutamic "families" of amino acids (38).Historically, the glucose effect was first elucidatedwith respect to the catabolic amino acid deami-nases such as aspartase (9, 30).A mixture of 0.5 g of glucose per liter and 0.5 g

of L-aspartic acid per liter was fed to continuouscultures of E. coli B6, and steady states wereachieved at different growth rates. The level ofthe catabolic enzyme aspartase (Fig. 5) fell offlinearly with increasing growth rate between 0.25and 0.9 hr-1, in much the same way that ,B-galac-tosidase decreased in mixed glucose and lactosecultures (Fig. 2), but the rate of change and theabsolute changes were considerably smaller. As

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tic acid (0.5 glliter).

the growth rate was raised above 0.9 hr'1, thespecific enzyme level rapidly dropped off until itwas completely repressed. This pattern suggeststhat the rate of generation of catabolite co-repres-sors increased linearly with growth rate, andcatabolite repression of aspartase became moresevere by a mechanism similar to that whichcauses 3-galactosidase production to decrease athigher growth rates.

Aspartic acid can be anabolically utilized evenwhile catabolic incorporation is totally repressed,as shown by the dependence on growth rate of theunutilized glucose and L-aspartic acid concentra-tions in the effluent from continuous culture(Fig. 6). Both substrates were partly assimilatedat all growth rates studied, but the fraction ofresidual L-aspartic acid increased with increasinggrowth rate. The greater catabolite repression ofaspartase at higher growth rates caused the de-crease in L-aspartase utilization, but more thanhalf of the L-aspartic acid fed was utilized atgrowth rates significantly higher than the batchgrowth rate of E. coli B6 when L-aspartic acid wasthe sole carbon source (0.28 hr-'). Although therate of generation of catabolite co-repressors fromglucose increased with growth rate in the carbon-limited continuous culture, the cells growing onglucose at a high growth rate could still anaboli-cally incorporate L-aspartic acid into protein.

DISCUSSIONSelection of constitutive mutants and control

mechanisms. Continuous culture of E. coli B6on a mixture of glucose and lactose resulted inthe rapid selection of constitutive mutants whichdisplaced the inducible wild type. Although con-stitutive mutants can be selected in chemostatcultures fed lactose alone (14, 34), the selectivepressure is greater in the mixed substrate culture,since the mutant cells are resistant to controls

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FIG. 6. Effluent glucose and aspartic acid in contin-uous cultures of E. coli B6 fed glucose (0.5 glliter) +aspartic acid (0.5 glliter). Rwns 21, 22, and 32 arereplicates begun from batch E. coli B6 inoculwn.

exerted by glucose on the catabolism of lactose,which becomes available to the mutants. Glucosecan control lactose catabolism in inducible cellsboth by catabolite repression and by exclusion ofthe apo-inducer and substrate lactose. Cataboliteinhibition is another possible mechanism, but noreliable evidence supports the existence of thiscontrol for the lac system.

T'he mutant B6b2 appears to be fully constitu-tive and therefore not subject to control of ,B-galactosidase level by inducer exclusion. Duringcontinuous culture on glucose and lactose, thismutant produced an enzyme level which de-creased with increasing dilution rate.

Current models of catabolite repression postu-late the activation by a catabolite co-repressor ofa genetic apo-repressor which can turn off trans-scription of the lac operon through a specificoperator gene. The co-repressor may be a carboncatabolite (25, 28) or an energy-rich intermediate

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(8, 35, 36). In a carbon-limited continuous cul-ture, the level of co-repressors increases as thecells grow faster, resulting in stronger repressionand diminishing enzyme level.The constitutive mutant B6b2 showed no

glucose/lactose diauxie when grown on these sub-strates in batch culture, indicating that for thisstrain catabolite repression alone is not a suffi-ciently powerful control to reduce the B-galactosi-dase level to the point where lactose utilization isprevented. Preferential utilization of glucose didnot occur when inducible E. coli B6 was grown incontinuous culture at moderate growth rates on amixture of glucose and lactose. Under these con-ditions, the external glucose concentration wastoo low (Fig. 3) to cause competitive exclusion oflactose, and the level of catabolite repression wasnot strong enough to prevent synthesis of 0.9SEU, which was sufficient to assimilate the lactosefed. At higher growth rates (above 0.8 hr-'),where the level of catabolite co-repressors is pos-tulated to increase greatly, the enzyme leveldropped to essentially zero and lactose passedthrough the chemostat unutilized. Chemostatcultures of constitutive B6b2 fed glucose andlactose produced ,3-galactosidase levels of 1.8SEU at high growth rates (e.g., 0.9 hr-1) whererepression was most severe, and thus were able tocompletely consume lactose up to wash-out.

Glucose/lactose diauxie occurred in batchculture only when both catabolite repression andmechanisms which exclude the inducer acted. Theexclusion of inducer by competition at thepermease or transporter level is a more powerfulcontrol mechanism than catabolite repression;the latter appears to act as a fine control on therate of enzyme synthesis.

Function of control mecaniss. Control mech-anisms serve to match the rate of catabolism ofavailable substrates to the rate of anabolism pos-sible under existing growing conditions. Comple-mentary controls such as induction and cataboliterepression enable a more precise regulation of therates of cellular metabolism. If the regulatorycontrols on catabolism become overloaded, i.e.,unable to reduce the catabolic capacity of the cellto match the biosynthetic capacity, metabolicintermediates accumulate within the cell andeventually spill out into the medium. Excretionof gluconic acid, 2-ketogluconic acid, acetate,pyruvate, and other catabolites by cells growingunder fully aerobic conditions has been amplydocumented (8, 27, 32). Excretion of intermediatecatabolites has been recently demonstrated incarbon-limited continuous cultures fed mixed sub-strates (S. K. Chian, Ph.D. Thesis, MassachusettsInstitute of Technology, Cambridge, 1967; R. I.Mateles and S. K. Chian, submitted for publica-

lion). In this situation, excreted catabolites areremoved in the culture effluent and therefore arenot utilized in the single-stage chemostat at highgrowth rates.Chemostat growth on mixed glucose and L-

aspartic acid followed a pattern in some wayssimilar to growth on glucose and lactose but withseveral distinct differences resulting from the dis-similarity of lactose and L-aspartic acid metabo-lism. Catabolite repression of both f3-galactosidaseand aspartase became more severe with increasinggrowth rate, and the specific enzyme levels de-creased with increasing growth rate. However,the ability of L-aspartic acid to be anabolicallyincorporated allowed substantial assimilation ofthe amino acid even when the catabolic enzymeaspartase was completely absent, whereas thetotal absence off,-galactosidase resulted in thetermination of lactose utilization.

ACKNOWLEDGMENT

This investigation was supported by grant NsG-496 from theNational Aeronautics and Space Administration.

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