JOURNAL OF BACTERIOLOGY, June 1978, p. 992-10010021-9193/78/013440992$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 134, No. 3

Printed in U.S.A.

Acetate Kinase Production by Escherichia coli DuringSteady-State and Transient Growth in Continuous Culture

H. MICHAEL KOPLOVEt AND CHARLES L. COONEY*Department ofNutrition and Food Science, Massachuwetts Institute of Technology, Cambridge,

Massachusetts 02139

Received for publication 25 July 1977

The synthesis of acetate kinase by Escherichia coli ATCC 9637 was studiedduring growth in anaerobic continuous cultures under steady-state and transientconditions. During growth in anaerobic, glucose-limited chemostats, acetate ki-nase synthesis was linearly associated with growth. Two types of non-steady-statetransients were studied: the perturbation in one was the addition of glucose. alone,and, in the second, glucose plus Casamino Acids. During the nutritional shift-upin the second case, but not in the first, the instantaneous specific acetate kinaseactivities and specific synthesis rates exceeded pre- and postshift values. Trajec-tory curves demonstrated that the increase in specific activity remained withinthe bounds of values obtainable under steady-state conditions with minimal andCasamino Acids media. Specific synthesis rates, however, greatly exceeded steady-state values. Enzyme yield values on glucose after the transient nutritional shift-up increased up to fivefold. Active protein synthesis is shown to be necessary toachieve the enhanced specific synthesis rates and enzyme yields. The results fromthese transient responses are discussed in terms of a conceptual model formetabolic regulation.

When microorganisms in balanced growth aresubjected to rapid environmental alterationsthat permit growth rates to increase, such as arelease from nutrient limitation or an enrich-ment of the growth medium, the cells oftenrespond by immediately increasing the synthesisrates of rRNA, ribosomal proteins, and RNApolymerase (5, 8, 13). The increase in the ratesofDNA synthesis and "general" or bulk proteinsynthesis occurs considerably later (13, 19).As the rRNA synthesis rate increases, the

total RNA synthesis rate declines, apparentlyindicating a "preferential" binding of RNA po-lymerase to the rRNA and ribosomal proteinoperons and a decrease in the availability ofunbound RNA polymerase. Examples ofoperonswhose transcription rates decline after nutri-tional shift-ups-i.e., the addition ofamino acidsto a minimal medium-include the Lac and Trpoperons (3, 18). This form of regulation is called"metabolic regulation" (6, 18). At some pointduring the transition, the activity of those en-zymes that controlled cellular metabolism at theoriginal growth rate becomes rate limiting andmust be differentially synthesized (6).

In this investigation, the synthesis of acetatekinase, an ATP-synthesizing enzyme, has beenstudied during unbalanced growth in anaerobic

t Present address: Union Carbide Corp., South Charleston,WV 25303.

continuous cultures. The role of acetate kinasein vivo is amphibolic, because it is both a cata-bolic enzyme necessary for growth on acetateand a biosynthetic enzyme that catalyzes theproduction of ATP anaerobically (2, 4, 17). It isfound primarily in facultative anaerobic micro-organisms (16). The role of this enzyme in cen-tral metabolism makes it a useful enzyme tofollow during a transient response to understandbetter the dynamics of microbial response to achanging environment.The transient response of Escherichia coli in

continuous cultures has been studied afterrelease from glucose limitation both with andwithout a concomitant addition of CasaminoAcids. The specific activity, specific synthesisrate, and yield ofacetate kinase were determinedas a function of time during the transient re-sponse.

MATERIALS AND METHODSMicroorganism. E. coli W (ATCC 9637-1) was

stored on minimal agar slants and subcultured period-ically.Medium. The growth medium minus carbon

source, designated MM3, had the following composi-tion (in grams per liter): (NH4)2SO4, 1.0; MgS04, 0.2;KH2PO4, 0.9; Na2HPO4, 1.4; and trace mineral solution,10 ml/liter. The trace mineral solution contained (ingrams per liter): CaCI2-2H20, 0.015; CuSO4 5H20,0.025; FeSO4 7H20, 0.028; Na2MoO4 .2H20, 0.024;

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ACETATE KINASE IN CONTINUOUS CULTURE 993

CoCI2* 6H20, 0.024; MnSO4 7H20, 0.084; andZnSO4* 7H20, 0.14. An antifoam solution of 25% P2000(Dow) in 75% ethanol was added to the medium in aconcentration of 0.35 ml/liter before autoclaving.Inoculum preparation and fermentation start-

up. All inoculum cultures were started from slantsstored in a refrigerator at 4 to 8°C. A loopful of slantculture was transferred to a 250-ml Erlenmeyer shakeflask containing 75 ml of MM3 medium plus 1 g ofglucose per liter, adjusted to pH 7.0. The flask wasincubated on a rotary shaker at 200 rpm in a 37°Cconstant-temperature room. During exponentialgrowth, which occurred in 5 to 8 h, 50 ml of the brothwas used to inoculate an aerated, agitated fermentorcontaining 400 to 700 ml of the desired medium. Con-tinuous flow to the fermentor was started after avisible increase in turbidity occurred (-25 Klett units)at a low flow rate (<100 ml/h). In anaerobic systems,a flow of 95% N2-5% CO2 gas mixture (Medical-Tech-nical Co.) was begun simultaneously.

Fermentor. The fermentor used throughout thecontinuous culture portion of the experiments wasdescribed in detail by H. M. Koplove (Ph.D. thesis,Massachusetts Institute of Technology, Cambridge,Mass., 1977). In brief, it consisted of a 1-liter Pyrexbeaker fitted with a 0.5-inch (ca. 1.26-cm)-thick Teflon(Dupont Chemical Co.) top through which variousstainless steel Swagelock (Crawford Fittings) adaptorswere threaded.A sterilizable Ingold pH electrode was secured by a

0.5-inch female Swagelock connector in which thebottom ferrule was inverted. Stainless steel inlet andexit lines (0.125 inch, ca. 0.318 cm) were provided forfeeding, base addition, gas inlet and exit, and sampling.A length of 0.03125-inch (ca. 0.0794-cm) tubing pro-vided a continuous sample to the acetate kinase anal-ysis apparatus.

These tubes were attached to 0.125-inch siliconerubber tubing (New Brunswick Scientific Co.). Theends of the sampling lines were kept in a 70% ethanolsolution when not in use.The three Teflon baffles in the fermentor were

secured with stainless steel rings. They were 0.6 cmthick and 1.0 cm wide and rested on the bottom of thevessel. They were recessed a short distance from thewall to promote fluid circulation.Continuous culture apparatus. The nutrient

feed was pumped to the fermentor by a Buchler vari-able-speed peristaltic pump. A 20-kg capacity triple-beam balance (Ohaus Scale Corp.) was used to weighthe feed reservoir for daily flow rate calibration.The gas inlet lines consisted of a Brooks rotameter

(Emerson Electric Co.) with a needle valve followedby a 15-cm drying tube packed with glass wool. Duringanaerobiosis, a 95% N2-5% CO2 gas was spargedthrough the fermentor. If air was used for sparging, asmall aquarium pump supplied the air. The sparginggas entered the fermentor just above the magneticstirrer. Exit gas passed through a 70% ethanol mixtureto prevent back-contamination.The pH was monitored with an Ingold electrode

and controlled with a Leeds and Northrup SpeedomaxH recorder-controller. On demand, the controller ac-tivated a pump (Sigmamotor T-8), which pumped a10% NaOH solution into the fermentor.

The fermentor sat in a 5-gallon (ca. 19-liter) waterbath maintained at 37°C with a temperature control-ler-circulator. The vessel was agitated with a magneticstirrer located below the water bath and a 1-inchTeflon-coated magnetic stirring bar inside the fermen-tor.

For aerobic growth, a dissolved-oxygen probe wasalso included in the fermentor. The probe was similarin style to that described by Johnson et al. (9). Aera-tion was supplied to maintain the dissolved oxygenabove 25% of air saturation during aerobiosis.The fermentor and its contents were autoclaved for

45 min at 121°C. During cooling, sterile air was spargedto prevent contamination.Growth measurements. Optical densities of the

culture were determined on a Klett-Summerson col-orimeter with a red filter or a Gilford spectrophotom-eter at 600 nm. Samples were diluted 1:6 in water toreduce the turbidity of the samples below 50 Klettunits. The factor of 290 Klett units per g of dry cellweight, or 3.31 optical density units per g of dry cellweight, was used to convert optical measurements todry cell weight. These values were obtained fromsteady-state continuous culture measurements and arereproducible ±5%.

Glucose assay. Glucose was measured enzymati-cally with a prepackaged kit (Calbiochem)-a methodbased on hexokinase-glucose-6-phosphate dehydro-genase. Samples of fermentation broth were removedand rapidly filtered with a Swinnex apparatus and a0.45-,um filter (Millipore Corp.). The samples werediluted 1:3, and a 20-d sample was added to 1 mil ofglucose Stat-pak reagent solution. The optical densityat 340 nm was measured at equilibrium (-5 min). Acalibration factor of 4.43 g/liter per unit of opticaldensity was calculated for this system.

Acetate kinase assay. A continuous procedure formeasuring acetate kinase activity was developed toanalyze intracellular enzyme activities. The equipmentemployed in the assay consisted of a Branson Sonifier350W (Heat Systems-Ultrasonics) and various Auto-Analyzer (Technicon) components. Full details aregiven by Koplove and Cooney (11).RNA assay. An adaptation of the Schmidt-Thann-

hauser assay described by Koch and Deppe (10) wasused to assay total cellular RNA concentration. Sam-ples were taken from the fermentor and immediatelyadded to an equal volume of ice-cold 0.4 N perchloricacid. The sample was centrifuged at 12,000 x g for 4min at 4°C, washed with cold 0.2 N perchloric acid,and recentrifuged. The remaining pellet was hydro-lyzed in 4 ml of 0.3 N KOH at room temperature for1 h. The remaining polymers were precipitated by anaddition of 2 ml of cold 1.2 N perchloric acid. Thesupernatant was read at 260 nm. Based on the data ofMunro and Fleck (15), the extinction coefficient of 32liters/g-cm was used to calculate RNA concentrations.

Calculations. It was necessary to calculate variousspecific rates (the specific growth rate, the specificglucose uptake rate, and the specific acetate kinasesynthesis rate) and the yield of enzyme and cell massat various times during the transients. The materialbalance equations from which these parameters arecalculated are summarized in Tables 1 and 2.A computer program was developed to facilitate

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994 KOPLOVE AND COONEY

TABLE 1. Material balance equations used to calculate specific rates and yieldsaDetermination

Instantaneous specific growth rate

Instantaneous specific glucose uptake rate

Instantaneous specific acetate kinase synthesis rate

Equation

dxV-= -Fx + VpAxdtdx

x

dsV-=FS. -FS- Vq8xdtdt

= qs

dE-= FE +VqE xdt

-E + DEdt

= qackx

Integration for yieldb

Cell yield, Df'xdt + x,-x(O)v Soa + DS.At- DfSdt - St

Enzyme yield, Df'Edt + E,-E(0)V Soa + DSoAt - DofSdt -St

a D, Dilution rate (h-'); E, volumetric acetate kinase activity (units per liter); F, flow rate (liters per hour);qack, specific acetate kinase synthesis rate (units per milligram of dry cell weight per hour); q8, specific glucoseuptake rate (grams of glucose per grams of dry cell weight per hour); 8,, inlet glucose concentration (grams ofglucose per gram of dry cell weight per hour); S, residual glucose concentration (grams per liter); Soa,concentration of glucose in pulse (grams per liter); t, time (hours); V, volume of fermentation broth (liters); Va,volume of addition (liters); x, dry cell weight (grams per liter); ,u, specific growth rate (h-').

b See Table 2.

TABLE 2. Integration for yield determinationaCompo- Amtadded Amt re- Total re-nent moved maining

Cells 0 Foftxdt Vx,Enzyme 0 FoftEdt VE,Glucose VaSOa + FSoA&t FoftSdt VS,a Nomenclature as in Table 1.

these calculations. The data obtained from the varioustransients included turbidity and glucose measure-ments taken at approximately 20-min intervals and acontinuous trace of optical density at 340 nm from theacetate kinase analyzer. Data were read from thecontinuous acetate kinase trace at 10-min intervalsand were used as volumetric acetate kinase input datafor the computer program.

In calculating the specific acetate kinase synthesisrate, it was necessary to calculate the derivative of the

volumetric acetate kinase activity curve (Table 1).This was done by assuming a linear change duringeach 10-min interval and calculating the slope of theresulting straight line. Because continuous data wereavailable for acetate kinase activity, there was littlescatter in specific acetate kinase synthesis rate values.The same method of calculating derivatives was

attempted for specific growth rate and specific glucoseuptake rate; however, when derivatives were calcu-lated by assuming linear responses during each 10-mininterval, a great amount of noise was generated in theinterpolation. Therefore, the method described byChurchill (3) was employed.The calculation of yield required integration of the

volumetric enzyme activity curve, the glucose concen-tration curve, and the optical density curve (Table 2).Once again, linear curves were assumed for each 10-min interval, and the area of each resulting trapezoidwas computed. Because integration is a self-smoothingprocess, no scatter was encountered with this calcu-lation technique.

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ACETATE KINASE IN CONTINUOUS CULTURE 995

RESULTSTwo series of transients were conducted in

these studies: step increases in glucose concen-tration during anaerobiosis, and step increasesin glucose concentration during anaerobiosiswith a simultaneous addition of Casamino Acids(nutritional enrichment).In several nutritional enrichment experi-

ments, chloramphenicol (CAP) was added eitherbefore or during the transient response in aneffort to determine whether the changes in ace-tate kinase activity required active protein syn-thesis.Transitions induced by glucose addition.

An experimental series was conducted to deter-mine what effects a step increase in glucoseconcentration would have upon the specific ace-tate kinase synthesis rate of anaerobic, glucose-limited chemostats. In all cases, the preshift

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medium reservoir contained 3 g of glucose perliter plus the MM3 medium. At the initiation ofthe perturbation, glucose was added directly tothe fermentor to raise the concentration to 3g/liter; the feed reservoir was switched from theinitial vessel to one containing 6 g of glucose perliter plus salts. Four dilution rates were studied:0.113, 0.183, 0.23, and 0.35 h-'. The pre- andpostshift steady-state culture turbidities arelisted in the figure legends. Figure 1 shows theresults of the experiment conducted at 0.113 h-'.

In all cases, except at the highest dilution rate,the specific acetate kinase activity increasedslightly (<50%) during the transient, whereasthe specific acetate kinase synthesis rate in-creased substantially (>300%) in the chemostatsoperating at D = 0.113, 0.183, and 0.23 h- .As can be seen in the experiment conducted

at D = 0.113 h-', an immediate and temporary

6 Sp. Activity

2

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s~tat Stop 20 60 100 140 ISO 220 Steady state8 20

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TIME (min)FIG. 1. Transient response of an anaerobic, glucose-limited continuous culture after the step addition of

glucose to both the feed medium and the fermentor (D = 0.113 h-'). The pre- and posttransient cultureturbidities were 96 and 213 Klett units, respectively.

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996 KOPLOVE AND COONEY

increase in specific acetate kinase synthesis rateoccurred after the step. The growth rate jumpedinitially to approximately 0.325 h-1, declined,and again increased. The percent RNA (gramsof RNA per grams of dry cells) remained con-stant throughout the transient. Similar resultswere obtained at D = 0.183 and 0.23 h-'. Theperiodic oscillations of specific growth rate andspecific glucose uptake rate are discussed byKoplove and Cooney (manuscript in prepara-tion).At the highest dilution rate, 0.35 h-', the

specific activity, specific acetate kinase synthesisrate, specific growth rate, and percent RNAremained constant at the preshift rate for up to180 min. The only parameter that routinely in-creased during this experiment was the specificglucose uptake rate. The maximum growth rate

2 4

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during the transients did not exceed 0.35 h-'.Transients induced by glucose plus Cas-

amino Acid addition. In the second experi-mental series, the previously described step in-crease in glucose was combined with a simulta-neous addition of Casamino Acid solution toboth the fermentor and feed reservoir. This formof perturbation is often called a nutritional shift-up. The concentration of Casamino Acids wasadjusted from 0 to 1 g/liter in both the fermentorand feed reservoir at the onset of the perturba-tion. Three dilution rates were studied: 0.115,0.23, and 0.4 h-'. The pre- and postshift cultureturbidities are listed in the figure legends. Theresults of the experiment conducted at 0.115 h-'are shown in Fig. 2.

In all cases, the specific acetate kinase activityduring the postshift steady state in the amino

Sp.Ac,y

S.A.

qAck

qAt140 160 220 St Stbto* 20

-10

qs 5!

qs

TIME (.4,)FIG. 2. Transient response of an anaerobic, glucose-limited continuous culture after the step addition of

glucoseplus Casamino Acids to both the feed medium and the fermentor (D - 0.115h-'). Thepre- andpostshiftculture turbidities were 96 and 192 Kktt units, respectively.

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ACETATE KINASE IN CONTINUOUS CULTURE 997

acid-containing medium exceeded the preshiftsteady-state specific activity in the miniimal me-dium. Instantaneous specific activity values dur-ing the transient response exceeded the postshiftspecific activity values during all three nutri-tional enrichment experiments; the maximumincrease was observed at the lowest dilution rate(Fig. 2). The specific acetate kinase synthesisrate also exceeded pre- and postshift synthesisrates by as much as an order of magnitudeduring these experiments. The period of in-creased specific synthesis rate lasted for 100 mior longer.At the lowest dilution rate, 0.115 h-', an initial

increase in specific glucose uptake rate was fol-lowed by a peak in specific growth rate; bothrates then declined. The specific growth ratethen increased steadily from the local minimumat 50 min to a maximum of 0.52 h-' at 130 min.The RNA concentration reached a maximum of16% at 80 min.From these experiments, we see that nutri-

tional shift-ups increased the specific acetatekinase synthesis rate and sustained the increasefor longer periods than those observed in per-turbations without Casamino Acids. The maxi-mum value of instantaneous acetate kinase spe-cific activity exceeded both preshift and post-shift specific activities.Trajectory analysis of transient re-

sponse. During unsteady-state growth, such asa transient response, the acetate kinase specificactivity and synthesis rate and the observedcellular growth rate will change with time; therelative change in each variable will, in general,be different. During the course ofan experiment,data are taken as functions of time and areusually plotted as such. However, to determinethe relative change of one variable as a functionof another, such as the specific activity versusthe instantaneous growth rate, it is necessary toplot one variable versus another rather thanversus time. Connecting the points in time se-quence yields a trajectory plot, with time as animplicit parameter.

Trajectory analyses are similar to conven-tional phase-plane diagrams, in which systemderivatives are plotted against one another. Atraditional use of phase-plane analysis has beento elucidate oscillatory relationships (1).The trajectory diagrams show the steady-

state specific activity or synthesis rate as a func-tion of steady-state growth rate. These steady-state curves are not trajectories themselves, butare analogous to variables of state in thermo-dynamics: the system will proceed from onesteady state to another, depending upon envi-ronmental conditions. The path of approach isthe trajectory curve.

Figure 3 shows the specific activity trajectoryfrom the nutritional enrichment experiments.The two bold lines are the steady-state profiles:the lower line is the steady-state profile in theminimal medium; the upper line is the steady-state profile in the enriched medium. The curveis divided into three regions: region II is boundedby the steady-state profiles; if the trajectorycurves remain in this region, then the specificactivity during the transient does not exceed thespecific activity obtainable in a one-state chem-ostat at steady state. In region I, the specificactivity exceeds the activity obtainable insteady-state cultures and, in region III, thegrowth rate has increased without a concomitantincrease in acetate kinase activity. For the nu-tritional enrichment, the trajectory curves re-main, for the most part, in region II.The specific synthesis rate trajectories, on the

other hand, track primarily in region I (Fig. 4)and exceed specific synthesis rates obtainable inone-stage steady-state chemostat cultures.During the perturbation in which only glucose

was added to a minimal medium chemostat, thespecific acetate kinase activity increased onlyslightly. Consequently, the specific activity inthe trajectory either remained coupled with, ordecreased below, the steady state profile. Thespecific synthesis rate trajectories for these ex-periments tracked mostly in the region boundedby the steady states, with some excursion intoregion III. The specific synthesis rates duringnutritional shift-up, however, greatly exceededthe specific synthesis rates obtainable withoutCasamino Acid additions.Enzyme and cell yield during transient

response. During the transient responses, spe-cific glucose uptake rate, specific growth rate,and specific acetate kinase synthesis rate allchange as a function of time. To determinewhether the glucose taken up by the microor-ganism is more efficiently converted to cell massand acetate kinase during the transient than atsteady state, the yield of cell mass and enzymeactivity must be determined. Yield of enzyme isdefined as the units of enzyme produced pergram of glucose consumed. The material bal-ances and methods used to calculate yields werediscussed above. These yields are cumulativerather than instantaneous; they may be consid-ered as the enzyme yields obtainable from areactor system consisting of a well-mixed chem-ostat followed by a plug-flow reactor with aresidence time equivalent to the total timeelapsed after the perturbation.Data are presented in Fig. 5 for enzyme and

cell yields after the glucose step-up at dilutionrates of 0.113, 0.183, 0.23, and 0.35 h-1. Neitherthe enzyme yield nor cell mass shows an in-

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998 KOPLOVE AND COONEY

00aE6)

0

CPcn

Z~

4-

0.r_

C.

C.

8

4

2

J. BACTERIOL.

0 0.1 0.2 0.3 0.4 0.5 0.6

Specific Growth Rate (h-1 )FIG. 3. Trajectory curves of instantaneous specific activity of acetate kinase versus specific growth rate

during the transient response after nutritional enrichment of anaerobic, glucose-linited continuous cultures.Symbols: -----,D= 0.115h-'; -----, D = 0.23h;-,D= 0.4 h-.

I

CP

0

c3

0

Co

.O-

cr

0.

0 0.1 0.2 0.3 0.4 0.5 0.6Specific Growth Rate (0h)

FIG. 4. Trajectory curves of instantaneous specific synthesis rate of acetate kinase versus specific growthrate during transient responses after nutritional shift-up of anaerobic, glucose-limited continuous cultures.-,D= 0.115h-'; ----- D= 0.23h'; ,D 0.4 h-'.

101

s6eady State6 Enridd Mediumw

Steady StateMinimal Medium

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ACETATE KINASE IN CONTINUOUS CULTURE

crease, and, if anything, they decrease duringthe transient period. A small increase in enzymeyield was observed during the transient at adilution rate of 0.23 h-' (Fig. 5).

Nutritional shift-up produced an entirely dif-ferent result (Fig. 6). In all cases (e.g., D = 0.115,0.23, and 0.4 h-1), the yield of cell mass initiallydecreased and then increased to the new steady-state level. The yield of enzyme, however, over-shot both the preshift and postshift steadystates. At the lowest dilution rate, D = 0.115 h-',there was an overshoot ofmore than 500% abovethe steady-state yield.CAP additions. In an attempt to determine

whether the increase in the acetate kinase activ-ity during the transient response was due toactual de novo enzyme synthesis or to activationof existing acetate kinase, experiments were con-ducted in which CAP, an antibiotic that stopsthe transfer of peptide chains during the trans-lation step of protein synthesis, was added tochemostat cultures during the transient period.Sufficient CAP was added to raise the brothconcentration to 50,tg/ml.

In one experiment, the CAP was added ap-

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STEADY...AIr . ThANSIENT

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TIME AFTER PERTURBATIONFIG. 5. Enzyme and cellularyield during transient

response afterglucose addition to anaerobic, glucose-limited continuous cultures.

NUTRITIONAL ENRICHMENTS

4t8~auE

E 0I

2--*E

CM

a E

-Jk J

zw

0 40 80 120 160TIME AFTER PERTURBATION (min)

FIG. 6. Enzyme and cellular yields during tran-sient responses after glucose and Casamino Acidadditions to anaerobic, glucose-limited continuouscultures.

proximately 5 min after the perturbation to ter-minate protein synthesis before acetate kinaseactivity increased. The specific acetate kinasesynthesis rate was zero throughout the experi-ment. When CAP was added during the periodof increased acetate kinase synthesis (55 min),the specific synthesis rate fell rapidly and be-came negative immediately after the CAP ad-dition, thus indicating some destruction of activ-ity (Fig. 7). The growth rate decreased dramat-ically but remained greater than zero for over100 min. In both experiments, glucose continuedto be consumed for at least 2 h.

DISCUSSIONAcetate kinase production was observed to be

a linear function of growth rate in anaerobic,glucose-limited chemostats (Fig. 3) and to beindependent of growth rate in aerobic, glucose-limited chemostats (unpublished data). This ob-servation is in agreement with the presumedmetabolic role of acetate kinase in anaerobicATP biosynthesis. Because the objective of thisinvestigation was to study the response of anATP-biosynthesizing enzyme, a growth-associ-ated steady-state activity (Fig. 3) was desirable(6,7).The experiments in which CAP was added to

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1000 KOPLOVE AND COONEY

- aIar E%._.

_

-

X .}._E*i2 S

Uf

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SI

c 7

&15

0

a; 2

Ui 3

c*Q

-a0

10

64 -- Sp.Ac*tivt

2I I J

SteOdy 20 60 100 140 ISO 220CAP

qs

2 I I

I20 .9

15 3

10 o0

at

TIME (sui)

FIG. 7. Effect of CAP addition on the transient response of an anaerobic, glucose-limited continuouscultureperturbed by a nutritional shift-up with glucose plus Casamino Acids (50 pg ofCAPper ml was addedat 55 min).

the cultures were perforned to determinewhether active protein synthesis was required toobserve the traient responses in enzyme activ-ity. From these experiments, we can assert (i)that active protein synthesis is required to ob-serve the increase in synthesis rate, (ii) that a

protein synthesis inhibitor will quickly stop theincrease in acetate kinase activity, and (iii) thatenzyme inactivation or destruction may play aregulatory role in enzyme activity.The results of the glucose addition experi-

ments are in general agreement with the resultsof other investigators (6, 10, 13, 14). Under someconditions, microorgani are capable of im-mediately increasing their growth rates to ap-proximately 0.3 h-1 if the growth-limiting nutri-ent is added to the chemostats at a dilution rateof less than 0.3 h-'.

In comparing the specific acetate kinase activ-ities and synthesis rates in the glucose additionexperiments with the nutritional shift-up exper-iments, the following general observations canbe made: (i) the specific activity during the

nutritional shift-up can considerably exceed thetransient specific activity observed after glucoseaddition; (ii) the trajectory curves of the specificacetate kinase synthesis rate in the nutritionalenrichment experiments (Fig. 4) greatly exceedthe synthesis rate trajectory curves of the glu-cose addition experiments; (iii) the specific activ-ity trajectories remain bounded, for the mostpart, by steady-state profiles (Fig. 3); (iv) higherenzyme yields can be observed after nutritionalshift-up than are observed at pre- and postshiftsteady-state conditions.We propose that, during the tmnsient period

after the addition of glucose to glucose-limitedculture, the response of the culture is limited bythe rate of biosynthesis; it may be an ATPlimitation, an amino acid limitation, or any otherbiosynthetic limitation.The addition of Casamino Acids, on the other

hand, causes multiple repressions of amino acidbiosynthetic operons. A mutiple repressionwould have several effects: (i) it would reducethe portion of the genome that must be tran-

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ACETATE KINASE IN CONTINUOUS CULTURE 1001

scribed; (ii) it would decrease the amount ofATP utilized for amino acid biosynthesis so thatATP could be utilized more efficiently for nu-cleic acid and protein synthesis; and (iii) if anygrowth rate limitation occurs in amino acid path-ways, as Koch and Deppe (10) and Harvey (6)have suggested, it is overcome by the additionof amino acids.

Parts (i) and (ii) of the above statement implythat the amount of polymerase and the energyper "open" operon immediately increase afterthe nutritional shift-up. If the acetate kinaseoperon is indeed metabolically regulated in themanner described by Rose and Yanofsky (18), it,like rRNA and ribosomal protein genes, wouldbenefit from this increase in transcriptional po-tential. In addition, this effect may be amplified,because the increased acetate kinase activitymay act to increase the size of the ATP pool.We also noticed an increase in enzyme yield

during the transient. If the availability of energyper operon increases during this transient, andifany amino acid limitations to protein synthesisare overcome by a surge in the intracellularamino acid pools, it is conceivable that preferredprotein synthesis (such as ribosomal proteinsand acetate kinase) occurs during this period.As sufficient acetate kinase becomes available

to meet biosynthetic demands, the transcriptionrate of this ATP-producing enzyme decreases tothe rate characteristic of balanced growth (thederivative of specific activity returns to zero),and the yield of enzyme on glucose decreases tobalanced growth yields.

Therefore, the observations we have maderegarding synthesis rates and yields are consist-ent with the molecular events reported in morefundamental studies. Keeping in mind that tran-scriptional and translational reactions competefor a common substrate and enzyme supply, ifone reduces the number of competing reactionsby supplying metabolic end products, then thesesubstrates and enzymes will be more efficientlydirected toward enzyme synthesis.

ACKNOWLEDGMENT8

We acknowledge the support of Public Health ServiceTraining Grant 5T01E500063 ESTC and the National ScienceFoundation grant GI34284.The technical assistance of David Millstone during a por-

tion of this work is also gratefully acknowledged.

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