JOURNAL OF BACTERIOLOGY, Apr. 1969, p. 131-142Copyright @ 1969 American Society for Microbiology

Vol. 98, No. 1Printed in U.S.A.

Effect of Growth Rate on Histidine Catabolism andHistidase Synthesis in Aerobacter aerogenes'

DONALD E. JENSEN2 AND FREDERICK C. NEIDHARDTDepartment of Biological Sciences, Purdue University, Lafayette, Indiana 47907

Received for publication 22 January 1969

A study was made of how the catabolism of a carbon and energy source is affectedby the biosynthetic demands of growing bacterial cells. Cultures of Aerobacter aero-

genes in L-histidine medium were grown in a chemostat at rates determined by thesupply of either sulfate or a required amino acid, L-arginine. It was discovered thatthe rate at which these cells grow under a biosynthetic restriction determines boththe rate and the pattern of histidine degradation. (i) Histidine catabolism is partiallycoupled to the growth rate. This coupling is achieved by catabolite repression ofhistidase (histidine ammonia lyase; EC 4.3.1.3.), and also by a slightly decreasedin vivo function of this enzyme at low growth rates. (ii) The looseness of the cou-

pling results in a direct relationship between growth rate and growth yield, andpossibly is correlated with an altered pattern of carbon flow from histidine. (iii)Sudden decreases in growth rate cause total repression of histidase synthesis forsubstantial periods of time. (iv) Sudden release of biosynthetic restriction leadsrapidly to an increase in the functioning of the cells' complement of histidase, anincrease in the rate of synthesis of this enzyme, and an increase in the growth yieldfrom histidine.

When growing bacterial cells are harvested andsuspended in a nongrowth medium, they continueto metabolize their energy-yielding substrate at anappreciable rate. This common observation leadsnaturally to the view that the coupling betweencatabolism and anabolism in these cells is a veryloose one.Gunsalus and Shuster (2) and Senez (16) have

reviewed this notion and have cited other lines ofevidence that catabolism is not restricted by therate of cellular synthesis. Not only can restingcells metabolize substrates vigorously, but thereis also evidence that, during certain conditions ofbalanced growth, catabolism can proceed at arate disproportionate to growth. In Desulfovibriodesulfuricans, substitution of molecular nitrogenfor ammonia as the nitrogen source causes amarked reduction in growth rate with no parallelreduction in the rate of degradation of the majorcarbon and energy source (5). Similarly, substitu-tion of nitrate for ammonia in Aerobacter aer-ogenes has been reported to reduce the growth

These studies are taken from a thesis submitted by D. E. J.in partial fuifilment of the requirements for a Ph.D. degree atPurdue University, 1966. Part of this work was presented at theAnnual Meeting of the American Society for Microbiology,Cleveland, Ohio, 1963.

2Present address: Miami Valley Laboratories, Proctor andGamble Co., Cincinnati, Ohio 45239.

rate without a parallel decrease in catabolism(13). Rosenberger and Elsden (14) grew Strepto-coccus faecalis anaerobically in a rich medium in achemostat with tryptophan limiting the growth.They found that the rate of substrate consumptionper unit weight of cells was constant at all growthrates. More recently, another possible instance ofuncoupling between catabolism and growth hasbeen seen in Clostridium pasteurianum in whichsucrose consumption fails to be diminished whenthe growth rate is lowered by restricting thesupply of NH3 (1).At first glance, there would seem to be no

difficulty in accepting this view, and the absenceof controls to integrate catabolism with biosyn-thesis may appear of little consequence. Aftersome reflection, however, this view must bemodified. First, it stands in sharp contrast to thefineness with which the bacterial cell adjusts therates of individual biosynthetic pathways andintegrates them with the polymerization reactionsof growth. Second, substrate degradation in theface of restricted growth requires that adenosinediphosphate (ADP), nicotinamide adenine dinu-cleotide, and other cofactors be continuouslyregenerated. This regeneration must be carefullycontrolled so that adenosine triphosphate (ATP)and reduced cofactors are not similarly squan-dered when conditions permit rapid growth. In

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other words, an "uncontrolled" rate of substrateconsumption would be possible only if certainother catabolic processes were extremely sensi-tively controlled.

Finally, there is a third cause for concern. Thetheory of catabolite repression (8) rests on thepremise that the cell does make a regulatoryresponse to an imbalance between catabolism andbiosynthesis. Any substrate which is degraded byinducible, catabolite-repressible enzymes should,during steady-state growth, be metabolized at arate which is commensurate with the growth rateof the cells, for catabolite repression shouldrestrict the synthesis of the appropriate catabolicenzymes. Thus, when the growth rate of a cultureis reduced by limiting the rate of supply of somegrowth factor, continued rapid substrate degrada-tion will lead to high intracellular levels ofrepressing metabolites. Adjustments in cellularmetabolism must occur to lower the levels ofthese metabolites during subsequent slow growth.Such adjustments could conceivably be of twosorts: (i) a reduction in the rate of substratedegradation to couple it to cell growth rate byeither lowered synthesis or lowered activity of thecatabolic enzymes; or (ii) an expansion (byenzyme synthesis) of some metabolic route toprevent the accumulation or to dispose of excessmetabolites.

In view of these implications, we have exploredthe metabolism of L-histidine by A. aerogenes.The metabolism of this substrate is initiated byan inducible set of enzymes that are subject tocatabolite repression (9). The cells grow suffi-ciently rapidly in minimal medium on this sub-strate to permit their growth rate to be manipu-lated over an eightfold range in a chemostat. Theprincipal finding in our study is that L-histidinemetabolism is affected by the growth rate of thecells.A summary of some preliminary experiments

in this study has appeared (11).

MATERIALS AND METHODSOrganism. All experiments were performed with

A. aerogenes LC1. This strain was derived from strainXXXV, the wild strain used in this laboratory, bysequential selection for two blocks in the L-argininebiosynthetic pathway, one before and one afterL-citrulline. The double block enabled continuouscultures to be maintained on limiting L-arginine forperiods as long as 1 week without overgrowth ofrevertant cells.

Media. A minimal medium was used in all experi-ments; it consisted of basal salt solution P (Na2HPO4.7H20, 1.34%; KH2PO4, 1.36%; CaC12, 0.0011%;MgSO4.7H20, 0.0246%; all w/v) supplemented with(NH4)2SO4 (0.2%, w/v) and with L-histidine as themajor carbon and energy source at concentrations

that depended on the particular experiment. Thegrowth factor, L-arginine, was added to give a con-centration of 40 jug per ml in media to be used forbatch culture or for sulfate-limited cultures, and 6 to9 ;Lg per ml in media to be used for L-arginine-limitedchemostat cultures. To construct a sulfate-limitedmedium, the MgSO4 and (NH4)2SO4 of the minimalmedium were replaced by an equivalent amount ofMgCl2 and NH4CI, respectively, and sufficient(NH4)2SO4 was added to give a sulfur concentrationof 1.6 ;g per ml.

Growth measurements. Batch cultures were grownaerobically in Erlenmeyer flasks filled to one-tenththeir capacity. They were agitated on a rotary-actionshaker at 37 C. Growth was followed by measuringthe optical density of the culture at 420 nm in a Zeissspectrophotometer, PMQII. Growth rates were ex-pressed as the specific growth rate constant, k, calcu-lated by the relation:

k = lnC2 - InCt2 - tl

(1)

where Cl and C2 are the optical densities of the cultureat times t1 and t2, respectively, and t is expressed inhours. An optical density of 1.00 at 420 nm with a1-cm light path corresponded to 80,g of protein and150,ug of bacterial dry weight per ml.

Chemostat cultures were grown at 37 C in glasschemostats of 50-, 150-, or 250-ml growth chambercapacity. The apparatus is described in detail else-where (D. Jensen, Ph.D. Thesis, Purdue Univ.,Lafayette, Ind., 1966). In essence, it consists of awater-jacketed growth chamber with a constant liquidvolume maintained by an overflow tube; mixing andaeration are provided by a rapid stream of warmedmoist air bubbling through a sintered-glass sparger atthe bottom of the growth chamber; the steady addi-tion of fresh medium is accomplished by a fingerpump operating on a tube from the medium reservoir.Flow rates were constant to within 3% over manydays of operation. Cell density varied with growth ratewithin the small limits expected theoretically and wasconstant at any given growth rate to within 5%.Temperature variation in the growth chamber wasless than 0.1 degree C. Measurement of the ribonucleicacid (RNA)-protein ratio of the cells at various flowrates gave values indicative of the correspondinggrowth rate of the organism. During steady-stateoperation, the specific growth rate constant, k, of achemostat culture is simply equal to the dilution rate,d,

k = d = v/V (2)where v is the flow rate in milliliters per hour and Vis the volume of the growth chamber in milliliters.During steady-state operation, there is no change inthe optical density of the effluent culture. Should thecells grow, from time t1 to time t2, at a rate which isfaster than the dilution rate, the optical density willincrease; conversely, should the cells be unable togrow as fast as the contents of the growth chamberare diluted, the optical density will fall. To a closeapproximation, the specific growth rate during such a

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period of non-steady-state operation can be estimatedfrom the following equation:

k=+ nC2lInCl(3t2 -tl

where, as before, d is the dilution rate in hr-1, andC1 and C2 are the optical densities of the culture attimes t1 and t2, respectively, expressed in hours.

Measurement of cellular consumption of L-histidine.Samples were removed from batch cultures at specifiedtimes and quickly brought to 0 C. Cells and mediumwere separated by centrifugation at 12,000 X g for10 min. If necessary, the supernatant medium wasstored at -20 C. The amount of L-histidine wasassayed as described below. The amount of L-histidine(in milligrams per milliliter) removed from themedium was then plotted against the cell dry weight(milligrams per milliliter) formed. During balancedgrowth, this plot yields a straight line, the slope ofwhich is the milligrams of L-histidine used to produce1 mg of cell dry weight. This value can be convertedto the specific rate of L-histidine metabolism by multi-plying by k to yield milligrams of L-histidine consumedper hour by 1 mg of cells.

Similarly, L-histidine consumption was measured insteady-state chemostat cultures. Samples of effluentculture were collected in tubes held at 0 C, and cellsand medium were separated by centrifugation. Por-tions of the medium were assayed for L-histidine, andthe specific rate of L-histidine consumption was calcu-lated by the following equation:

(hin - h0t) v

m

where VH is the rate of L-histidine consumption(milligrams of L-histidine per hour per milligram cells),hin and hout represent the steady-state concentrationof L-histidine (milligrams per milliliter) in the inputmedium and the outgoing culture, respectively, v isthe flow rate (milliliters per hour), and m is the totaldry weight (milligrams) of cells in the growth cham-ber. Since m is equal to the total volume of thegrowth chamber, V (milliliters), multiplied by theconcentration of cells, C (mg per ml), equation 4 canbe written as

VH= (hin - hout)v-

(hin - hout)k (5)CV C

provided that steady-state conditions have beenachieved.

Preparation of extracts. Samples (15 to 25 ml) ofcultures containing approximately 100 Ag of totalprotein per ml were collected at 0 C. The cells wereharvested by centrifugation at 0 C, washed twice withsodium pyrophosphate buffer (10-2 M, pH 9.0), andfinally suspended in 2 ml of the same buffer. The cellswere then disrupted by sonic treatment for 2 min witha 20-kv Sonifier (Branson Instruments, Inc., Stan-ford, Conn.), and the resulting mixture was centri-fuged at 12,000 X g for 20 min. The supernatant

liquid was decanted and assayed immediately for totalprotein and for enzyme activity.Enzyme assay. Histidine ammonia lyase (EC

4.3.1.3), commonly called histidase, was assayed bymeasuring the formation of urocanate from L-histi-dine. Reaction mixtures (3 ml) contained sodiumpyrophosphate (10-2 M, pH 9.2) and crude extract(50 to 150 ,ug of protein). The reaction was run at25 C, and was initiated by the addition of L-histidine.The course of the reaction was followed by monitoringthe increase in absorbance at 277 nm in a recordingspectrophotometer (Gilford Instruments, Oberlin,Ohio). One unit of histidase was defined as that activ-ity which converts 1 ,mole of L-histidine to urocanateper hr under the conditions of the assay, assumingthat an increase in absorbance of 1.0 was equivalentto the formation of 5.55 X 10-2 ,umoles of urocanateper ml.

Differential rate of enzyme formation. Duringsteady-state growth, the differential rate of formationof any enzyme is numerically equal to its specificactivity (total units per milligram of protein), whetherthe culture is unrestricted or growing in a chemostat.In some chemostat experiments, it was desirable toestimate the differential rate of histidase formationduring non-steady-state conditions. This estimationwas made by first using equation 3 to calculate theoverall growth rate (k) of the culture between ti andt2, the time interval in question. Next, the growthincrement, C2/C1, of a hypothetical, equivalent batchculture was calculated from the relationship given inequation 1: InC2/Ci = k(t2 - t1). Knowing thespecific activity (units per milligram of protein) ofhistidase at t1 and t2, it was then possible to calculatethe amount of enzyme, ei and e2, associated with thetotal protein at t1 and t2, respectively, in the hypo-thetical batch culture. A plot of e against C then en-abled one to compare the slope of this function be-tween Cl and C2 with the known (steady-state) slopebetween the origin and Cl .

Chemical determination. L-Histidine was assayedby the method of Jorpes (4) in which diazotized sul-fanilic acid reacts with the imidazole group of L-histi-dine to form a colored product.

Protein was measured by the method of Folin (7)and RNA by the orcinol method (15).

Manometric analysis. In one experiment, theamounts of CO2 evolved and 02 taken up by restingcells were measured by standard manometric tech-niques (17). Details are listed in the Results section.

Metabolism of 14C-L-histidine. To trace the fate ofL-histidine carbon during growth at different bio-synthetically restricted rates (results shown in Tables3 and 4), the chemostat apparatus was modified byadding tubing to the exit port so that the effluent gas(air that had bubbled through the culture in thegrowth chamber) was led through two consecutive2-liter flasks half full of 10% KOH to trap CO2.A T tube in this line adjacent to the exit from thegrowth chamber permitted the collection of effluentculture. For radioactivity measurements of CO2 pro-duced by the culture, the effluent gas was passedthrough a KOH solution (5 N) for either 0.5 or 1.0hr. The chemostat was run with sulfate limitation,

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first at k = 0.210, and later at k = 0.714. As soon assteady-state growth was reached at each flow rate,10 gas samples and 10 culture samples were taken. TheCO2 in the gas samples was precipitated by theaddition of BaC12, and the precipitate was collectedon membrane filters. One portion of the culture wasfiltered to permit the separate counting of cells andfiltrate. The radioactivity in a portion of the unfilteredculture was determined separately. The filtered me-dium was assayed colorimetrically for L-histidine.Separate samples were taken to assay the cells forhistidase. All samples were counted for 10 min in 15ml of the scintillation fluid described by Marr et al.(10), by use of a Packard scintillation counter, and inall cases multiple samples of different sizes weremeasured to detect any self-absorption.To measure the distribution of L-histidine carbon

in resting-cell suspensions (results appearing inTables 3 and 4), cells were prepared by growth in asulfur-restricted chemostat (k = 0.19) and by growthin unrestricted batch culture (k = 0.85). Both popula-tions of cells were centrifuged, washed twice, andsuspended in 42 ml of basal medium lacking both sul-fur and L-argmiine, and containing 14C-L-histidine(1.50 mg/ml; 2.9 pc per mmole). The suspensionswere placed in separate closed glass chambers fittedwith a center well containing 10 ml of KOH (10%,w/v). They were incubated at 37 C for 8 hr (slowcells) or 2.5 hr (fast cells). Duplicate samples of eachincubation mixture were removed at the start and endof the experiment to establish the optical density,radioactivity, histidine content, RNA-protein ratio,and histidase level of the cultures. At the end of theincubation, 1 ml of the KOH solution was placedinto 20 ml of 1.0 M BaCh2 to precipitate BaCO8.Samples of cells on membrane filters, filtered medium,and BaCO3 precipitates were counted in a scintillationcounter as described above. In all cases, multiplesamples of different sizes (e.g., 0.25 and 0.50 ml offiltered medium) were counted to detect self-absorp-tion.

RESULTSRate of L-histidine metabolism during steady-

state growth. In a large number of separateexperiments, cultures of A. aerogenes LC1 weregrown in the chemostat at 37 C with either sulfateor L-arginine limiting. In each experiment, theculture was maintained at a particular growthrate for many generations to ensure that steadystate growth had been established. Chemicalassay to determine the concentration of L-histidinein the incoming medium and the outflowingculture (after removal of the cells) was performed.Samples were collected at intervals throughout along time period until a steady-state level ofhistidine concentration had been reached. Thecell density and the flow rate of the chemostatwere similarly monitored. It was then possible tocalculate the specific rate of histidine disappear-ance in the manner described under Materialsand Methods.

Some representative data from these studiesare presented in Table 1 to show the actualmagnitude of the measured parameters and toclarify the calculation of the specific rate of histi-dine catabolism. A summary of all of the meas-urements is presented in Fig. 1, in which thespecific rate of L-histidine catabolism is plottedas a function of growth rate for sulfur-limited andfor L-arginine-limited cultures. Data from threeexperiments with batch (unrestricted) cultures areshown in the same figure.The results provide a clear demonstration that

the rate of L-histidine catabolism in these cells isset at different levels at different growth rates.There appears to be a nearly linear relationship,indicating that catabolism of L-histidine is ap-proximately 80% coupled to the biosyntheticdemands of the cells over the whole range ofpossible growth rates, whether sulfate or L-argi-nine is the rate-limiting nutrilite.

Level of histidase during steady-state growth.The partial coupling of histidine catabolism togrowth rate raises the question of what restrictssubstrate degradation during biosynthetically re-stricted growth. It was reasoned that cataboliterepression should be responsible for this coupling,and so the level of the first enzyme in the inducibleseries that degrades L-histidine was assayed incells grown in the chemostat at different growthrates with either sulfate or L-arginine restriction.

Cells of strain LC1 from chemostat cultures atdifferent growth rates were harvested and assayedfor histidase as described in Materials andMethods. At each flow rate, growth was continueduntil steady-state conditions had been achieved.The measured levels of histidase are plotted as afunction of growth rate in Fig. 2. It is clear that anearly linear relationship exists between thesteady-state level of histidase and the growth rateof A. aerogenes. It is equally clear, however, thata given reduction in growth rate leads to onlyhalf the reduction in histidase level that wouldresult from a tight coupling. Extrapolation of thehistidase level to zero growth rate suggests thatcatabolite repression cannot force the histidaselevel below 10 units per mg of protein duringsteady-state growth.

Unequal functioning of histidase at differentgrowth rates. The reduction in histidase level isnot only less than the reduction in growth rate, itis also slightly less than the reduction in the rateof histidine catabolism. To illustrate the extentof this disparity, the data of Fig. 1 and 2 arereplotted in Fig. 3 so that the rate of L-histidinecatabolism is shown as a function of the histidaselevel. Only those experiments in which bothparameters were measured on the same culturesare included in Fig. 3. The interrupted line is

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VOL. 98, 1969 REGULATION OF HISTIDINE CATABOLISM IN AEROBACTER

TABLE 1. Rate of L-histidine metabolism during biosynthetically restricted growtha

Growth rate Cell density L-Histidine L-Histidine L-Histidine Rate ofExpt no. Limiting nutrient (k) (C)e inflow outflow used (kin - metabolism

hrl mg/ml mg/mi mg/mi mg/mi mg/hr*mg

1 L-Arginine 0.226 0.191 1.48 0.545 0.935 1.110.230 0.198 1.48 0.540 0.940 1.100.509 0.184 1.48 0.840 0.640 1.780.655 0.166 1.48 1.00 0.480 1.90

2 Sulfur 0.208 0.246 3.69 2.23 1.46 1.230.315 0.230 3.69 2.54 1.15 1.57

3 Sulfur 0.584 0.162 1.44 0.822 0.618 2.230.710 0.160 1.44 0.922 0.518 2.30

aThe symbols used in this table are defined in Materials and Methods in reference to equation 5,which has been used to calculate VH, the rate of L-histidine metabolism. The data presented in thistable are a small sampling of the measurements depicted in Fig. 1.

J%.

, II-IZ9 l_

2 cn

- 0I-

.2 .6GROWTH RATE (K)

FIG. 1. Metabolism of L-histidine as a function ofgrowth rate. The specific rate of L-histidine metabolism(expressed as milligrams of L-histidine degraded perhour per milligram, dry weight, of cells) is plotted as a

function of the specific growth rate constant, k (ex-pressed in hr-1), for many cultures ofA. aerogenes LCIin steady-state growth at 37 C in a chemostat withsulfur limitation (0) or with L-arginine limitation (A),or in batch culture (0). The solid line was drawn bythe method of least squares as a line of regression ofyon x.

drawn from the origin and through coordinatescharacteristic of a batch, unrestricted culture; itshows the relationship between histidine metabo-lism and histidase level that would be expected if,at all growth rates, the cellular complement ofhistidase were functioning as actively as duringbatch, unrestricted growth. The solid line, drawnby the method of least squares through the ex-perimental points, deviates from the former curveto such an extent that at low growth rates half ofthe histidase activity in the cell seems not to be

functioning. The points chosen for display inFig. 3 show a rather wide scatter, but the linedrawn through them is in fact superimposableover the line that results from an algebraic com-bination of the curves of Fig. 1 and 2.Growth yield from L-bistidine at different

growth rates. Even though not all of the histidasepresent in slow-growing cells is functioning at therate at which it functions during rapid growth,the fact still remains that histidine catabolismduring slow growth seems to exceed the biosyn-

z

- 240a:

(0

28

C)

0 .2 4 .6GROWTH RATE (K)

.8

Fio. 2. Histidase level as a function of growth rate.The specific activity of histidase (expressed as units of

enzyme per milligram of total protein) is plotted as a

function of the specific growth rate constant, k (ex-pressed in hr-1), for many cultures of A. aerogenes'LCIin steady-state growth at 37 C in a chemostat withsulfur limitation (0) or with L-arginine limitation (A),or in batch culture (0). Histidase was assayed in cell-free extracts. The solid line was drawn by the methodof least squares as a line of regression ofy on x.

I II

2A~~~~~~~~~~~~~~~I2.4 a

-~ ~ ao

1.6 ° o/

ot /

0 SULFUR LIMITATION0.8 A ARGININE LIMITATION -

z o °~~BATCH

QO0 I I I III

0~~~~~~

CO~~~~~~~~O

O SULFUR LIMITATIONA ARGININE LIMITATIONO BATCH

I

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.8

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JENSEN AND NEIDHARDT

IV)-i-

9-jOco(0(

0 ARGININE LIMITATIONa SULFUR LIMITATION /

2.4 0 BATCH

A0A11

1.6 /

0.8 - // 0/ 0°

/ ///

O.0 ,0 8 16 24HISTIDASE (UNITS/MG PROTEIN)

FIG. 3. Metabolism of L-histidine as a function ofhistidase level during growth at different rates. Thespecific rate of L-histidine metabolism (expressed asmilligrams of L-histidine degraded per hour per milli-gram, dry weight, of cells) is plotted as a function ofhistidase level (expressed as units of enzyme per milli-gram of protein) for several cultures of A. aerogenesLCI in steady-state growth in a chemostat with sulfurlimitation (A) or with L-arginine limitation (0), or inbatch culture (0). The data include all cultures shownin Fig. I and 2 for which both histidase level and sub-strate metabolism were measured. The solid line wasdrawn by the method of least squares as a line of re-gression of y on x. The interrupted line is drawn fromthe origin to the coordinates characteristic of the batchculture.

thetic requirements of the cell. (Were histidinecatabolism to be reduced proportionately to thegrowth rate, then the data of Table 1 would havebeen described by a straight line passing throughthe origin.) During unrestricted growth, for ex-ample, the cells grow at k = 0.85 hr-1 by metabo-lizing L-histidine at a rate of 2.6 mg per hr per mgof cells (Fig. 1); at one-fifth this growth rate(k = 0.17 hr-') the cells might be expected togrow by metabolizing this substrate at a rate of0.52 mg per hr per mg of cells. In fact, referenceto Fig. 1 shows that their actual rate of L-histidinemetabolism at this growth rate is 0.96 mg per hrper mg of cells-nearly double the expected value.A part of this "excess" metabolism might berelated to a cellular requirement for maintenanceenergy, i.e., for the operation of those processesthat must occur at rates independent of the growthrate. To discover the contribution of such proc-esses, a culture of LC1 was grown in a chemostatwith the carbon and energy source, L-histidine,as the limiting factor. The flow of medium wasadjusted to give first a dilution rate (=k) of0.144 hr-', and then one of 0.025 hr-1. Continu-ous operation at the former rate for 60 hr resultedin a steady-state value of 0.78 for the optical

density of the culture; 84 hr at the slower rate ledto a steady-state optical density of 0.68 (Table 2).This small difference in culture density duringsubstrate limitation indicates that the amount ofhistidine metabolized strictly for maintenance is asmall fraction of the total histidine metabolism,even at these low growth rates. Therefore, it mustbe concluded that most of the "excess" rate ofL-histidine disappearance observed at low growthrates really does represent an overall imbalancebetween substrate utilization and biosyntheticdemands.

Pattern of L-histidine metabolism at differentgrowth rates. This imbalance during steady-stategrowth raises a serious question about the primarypostulate of the theory of catabolite repression,according to which an imbalance should causefurther repression of the histidase series of en-zymes until a balance between catabolism andanabolism is achieved. It was therefore importantto verify this imbalance and was of some interestto examine the fate of the metabolized L-histidine.To do this, uniformly labeled '4C-L-histidine wasprovided to two cultures in steady-state growth

rABLE 2. Cell density and histidase levelat low dilution rates in an L-histidine

restricted chemostata

Time

hrOc60

Od814253852607284

Dilution rate(d)

hr'

0.1440.144

0.0250.0250.0250.0250.0250.0250.0250.0250.025

Optical density Histidase levelbOptical) (units per mg(420nm) of protein)

0.800.78

0.780.760.720.720.670.710.710.680.68

34.1

32.9

32.7

31.7

a A culture of A. aerogenes LC1 was grown in achemostat in a medium containing excess sulfurand L-arginine (40,ug/ml), but limiting L-histidine(500 ug/ml).bThe histidase level was determined in extracts

of cells from the effluent culture collected overseveral hours of growth and held at 0 C. As acontrol, a portion of the culture growing at a kof 0.025 was inoculated into normal batch mediumand grown for several generations, after whichtheir histidase level was found to be 23.2 units permg of protein.

c An arbitrary time after steady state had beenreached at a dilution rate of 0.144 hr-1.dThe time at which the dilution rate was

changed from 0.144 hr-1 to 0.025 hr-1.

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in a sulfate-limited chemostat at growth rates of0.2 and 0.7 hr-'. The flow of carbon from sub-strate into cells, medium, and volatile materialwas measured by procedures described in Ma-terials and Methods. The results are displayed inTables 3 and 4.

In rapid growth (k = 0.71 hr-'), the cellsconsumed 6.57 ,umoles of L-histidine carbon perhr per mg of cells. Of this, 44% was assimilated ascell carbon, and 56% was excreted as formamide(17%, calculated), CO2 and other volatile ma-terial (33%), and unknown nonvolatile com-pounds (6%). During slow growth (k = 0.21hr-1), the same mass of cells consumed L-histidinecarbon at the rate of 3.71 ,umoles per hr. Theyassimilated only 20% of this carbon, the remain-ing 80% being excreted as formamide (17%,calculated), CO2 and other volatile material(42%), and unknown nonvolatile compounds

(21 %). Overall, these results are consistent withthe previous measurements; also, they provide anindication that metabolic by-products may beexcreted in different proportions during fast andslow growth.

It was of interest to learn whether these differ-ences would be preserved after the cells had beenprepared by growth at two different rates andthen transferred to identical "resting" conditions.Two kinds of experiments were performed. In thefirst, the resting-cell suspensions were presentedwith uniformly labeled '4C-L-histidine, and thefate of the L-histidine carbon was examined as inthe chemostat experiments. The results are shownin Tables 3 and 4. There was, of course, very littleassimilation of carbon by either the fast- or theslow-grown cells. The suspensions of cells pre-served, more or less unchanged, their relativerates of histidine degradation during growth (6.1

TABLE 3. Catabolism of "4C-L-histidine by resting and growing cultures of cells previouslygrown at a fast or slow rate

Initial or inflowmedium,8 L-histidine

Amt(mg/ml)

Growing-cell experiment0.210 1.70 81,1700.714 1.70 81,170

Resting-cell experiment0.190 1.50 29,6000.850 1.50 30,400

Final or outflow materialsb

CeUse

Counts per Amt Counts per

min per ml (mg/ml) min perml

0.1540.142

0.2760.246

9,1169,265

1,8301,558

Mediumd

L-Histidine

Amt(mg/ml)

0.7381.24

0.0700.190

Countsper minper ml

35,20059,400

1,3802,840

Form-amide(countsper minper ml)

7,6113,628

4,7004,420

Other(countsper minper ml)

9,4911,354

9,87014,520

Gase

co,

(countsper minper ml)

1,9404,612

6,1404,160

Non-recovered(countsper minper ml)

2,145547

5,6802,900

a For the growth experiment, the figures represent the chemical concentration (measured colorimetri-cally) and the radioactivity (uniformly labeled 14C-L-histidine) of the medium flowing into the chemo-stat. For the resting, or nongrowth experiment, they represent the values at the start of incubationof the cell suspensions.

b For the growth experiment, the figures describe the material flowing out of the chemostat; for thenongrowth experiment, they describe the contents of the incubation mixture at the end of the experi-ment-8.0 hr in the case of the slow cells, and 2.5 hr in the case of the fast cells.

¢ The dry weight is calculated from the optical density of the culture; the radioactivity was measuredby filtering the cells from the medium.

d The radioactivity of the filtered medium was assumed to consist of (i) unmetabolized L-histidine,(ii) formamide, and (iii) other nonvolatile by-products of L-histidine metabolism. The radioactivityassociated with L-histidine was calculated from the L-histidine remaining (measured colorimetrically)and its specific activity; that associated with formamide was calculated as one-sixth of the radioactivityof the L-histidine consumed (12); the radioactivity of other by-products in the medium was then cal-culated by subtraction.

e In the growing-cell experiment, samples of the gas that had been used to aerate the growth chamberwere bubbled through KOH solutions for 30- and 60-min periods; the results shown here are the averageof all such samples and are expressed as counts per minute of CO2 produced by 1 ml of culture in 1

hour. In the resting-cell experiment, all of the CO2 produced was trapped in the KOH in the center well;the results shown are expressed as counts per minute of CO2 produced by 1 ml of the culture in 8 hrby the slow cells and in 2.75 hr by the fast cells. The "nonrecovered" counts represent radioactivitywhich is presumed to be associated with volatile material either not trapped by KOH or not precipitatedby BaCl,.

Growth rateto prepare

cells, k (hr1)

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JENSEN AND NEIDHARDT

TABLE 4. Comparison of distribution of carbonduring metabolism of "4C-L-histidine by

cells grown at fast or slow ratesa

Percentage distribution ofmetabolized carbon

Medium GasType of Incubation condition

cells _

c 0 0

u~ rZ4 u

Slow Slow growth 20 17 21 20 22Nongrowth 6 17 35 22 20

Fast Fast growth 44 17 6 30 3Nongrowth 6 17 52 15 10

a The percentage distributions of metabolizedL-histidine carbon atoms shown in this table werecalculated from the data of Table 3.

b Calculated as one-sixth of the metabolizedcarbons.

c These figures represent unrecovered radio-activity presumed lost in the gas phase.

Amoles of carbon per hr for the fast cells and2.0 Amoles for the slow cells). The cells that hadbeen prepared by slow growth converted rela-tively more of the histidine carbon to CO2 andother volatile compounds than did the cellsprepared by rapid growth.

The second type of experiment consisted simplyof a classical analysis of respiration and substrateutilization in a Warburg apparatus. Two suspen-sions of LC1 cells were prepared, one by growthat k = 0.85 hr-1 in unrestricted histidine mediumand the other by growth at k = 0.19 hr-1 inhistidine medium in a sulfur-restricted chemostat.The histidase content of the cells and their ratesof histidine utilization, of 02 uptake, and ofCO2 production were then measured as describedin Table 5 and Materials and Methods. Theresults, in general, confirm the isotope experiment,although the fraction of carbon converted to CO2was higher in both slow (35%) and fast (23%)cells than was seen in the isotope experiment.rhe reason for this is unknown, but may possiblybe poorer aeration in the large vessel used in theisotope experiment, or poorer recovery of CO2produced. Nevertheless, the results do demon-strate that slow-grown cells metabolize histidinemore slowly and may possibly oxidize it morecompletely than do fast-grown cells.

This hint that L-histidine may be metabolized ina different way during biosynthetically restrictedgrowth than during batch growth suggests anexplanation for the failure of catabolite repressionto effect a strict coordinacy between histidasesynthesis and growth rate. A steady-state condi-tion may be reached in which potentially re-pressing catabolites are converted into nonre-pressing by-products, such as CO2 .

TABLE 5. Manometric measurements of L-histidine metabolism in suspensions of cellsgrown at fast and slow rates

Rates of metabolismcHistidaseb level (jumoles per hr per mg of cells)

Preparation& of cells (units per mg VC_2_V__C02_of protein)

VH VCo2 VO2

Slow grown (k =0.20)............. 13.1 4.33 9.09 10.2 2.1 42Fast grown (k = 0.85) .............. 22.3 9.78 13.7 15.3 1.4 28

a A chemostat culture of A. aerogenes LC1 was prepared in steady-state growth at a k of 0.19 hr-'with sulfur limitation. One portion was then grown in normal media at a k of 0.85 for several genera-tions. The two cultures were then harvested; the cells were collected and washed by centrifugation, andwere then suspended in basal salts medium (lacking both L-arginine and sulfur). Portions (3 ml) of thesuspensions were placed in Warburg vessels, a known quantity of L-histidine was added, and manometricmeasurement was begun. The suspension of slow cells contained 0.109 mg of cells per ml; the suspensionof fast, 0.106 mg of cells per ml.

b Portions of each cell suspension were used to prepare extracts for the measurement of histidase.c At the conclusion of the manometric measurements, the quantity of L-histidine remaining in each

flask was measured colorimetrically, and the consumption of substrate was used to calculate VH, thespecific rate of L-histidine metabolism. From the manometric data, Vco, and Vo2, the specific rates of CO2evolution and of 02 uptake, respectively, were calculated.

d The ratio, VCO2/VH, is a measure of the number of micromoles of CO2 produced per micromole ofL-histidine metabolized.

L The assumption was made that a molecule of L-histidine has five carbon atoms that can possibly beconverted into CO2 (the sixth appearing as formamide); then, from the ratio Vco2/VH, the CO2 formedwas expressed as a percentage of that which could be formed by complete metabolic combustion.

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VOL. 98, 1969 REGULATION OF HISTIDINE CATABOLISM IN AEROBACTER

Histidase synthesis following a decrease ingrowth rate. If fast and slow cells differ in theirenzymatic capacity to handle excess catabolites,then cells that have been growing rapidly shouldexhibit greater catabolite repression immediatelyafter a shift down in growth rate than later, afterthe alternate pathway has been developed. Toexamine this possibility, cells of strain LC1 weregrown in a chemostat in L-histidine minimalmedium with L-arginine as the limiting factor.Initially the culture was grown at k = 0.66 hr-1,which is 80% of the maximum possible growthrate. After steady-state conditions were achieved,the dilution rate was abruptly changed from0.66 hr-1 to 0.13 hr-1, and samples of the effluentculture were collected at 30-min intervals. Theoptical density, and the total protein content ofthese samples were measured, and then the cellswere assayed for their histidase content (Fig. 4).The very slight increase in optical density andtotal protein per milliliter of culture is a normalresponse of chemostat cultures to decreased flowrate. From the upper panel of the figure, it canbe seen that the histidase content of the cellsemerging from the chemostat began to drop im-mediately after growth was slowed. The physicalnature of a chemostat is such that the time courseof transient responses is not easily followed;

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TIME (HOURS)

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FIG. 4. Growth and histidase formation in responseto a sudden imposition of greater biosynthetic restric-tion. A culture of A. aerogenes LCI was prepared insteady-state growth at 37 C in a chemostat with L-

arginine limitation at a growth rate constant, k, of0.66 hr-1. At zero time, the dilution rate was changedfrom 0.66 hr'I or 0.13 hr-1. The upper portion of thefigure depicts the histidase level (units of enzyme permilligram of total protein), as measured in cell freeextracts, plotted as a function of time; the solid linewas drawn as a visual approximation of the experi-mental points, and the interrupted line shows the ex-pected result if total repression of histidase had occurredat the time of the shift. The lower portion of the figuredepicts the optical density (0) and the protein content(@) of the culture. The ordinate scales are exponential.

samples cannot be removed at discrete times fromthe growth vessel, but rather must be collected byaccumulation of effluent culture over a finite timeperiod (in the present case, 15 min). Nevertheless,it can be judged from Fig. 4 that the histidase levelapproaches its new steady-state value at a ratesuggesting that no new enzyme is made for 5 hr.For a full generation at slow growth rates,

therefore, biosynthetic restriction is capable ofeffecting a total repression of histidase formation.Closely similar results were obtained when sulfaterather than L-arginine restrction was used tolimit growth.Growth and histidase synthesis following release

of a biosynthetic restriction. It appears that thefull effect of catabolite repression on histidaseformation is not maintained during slow growth,presumably because there is both a reduction inthe rate of L-histidine degradation per unit ofhistidase activity and a quantitative or qualitativechange in the by-products produced from L-histi-dine. It was of interest, therefore, to learn howthe cells would respond to a sudden release ofbiosynthetic restriction after steady-state growthat a slow rate had been established. A culture ofstrain LC1 in steady-state growth at k = 0.13 hr-1was prepared in a chemostat on limiting L-argi-nine. To begin the experiment, the dilution ratewas abruptly increased fivefold from 0.13 to0.66 hr-'. The optical density and total proteincontent of the effluent culture were monitored,and cell samples accumulated every 10 min wereassayed for histidase. Many such experimentswere performed, some with L-arginine limitingand some with sulfate limiting the growth. Typicalresults are shown in Fig. 5. The upper paneldepicts the histidase level in the cells; it is clearthat the sudden removal of the L-arginine restric-tion led to a rapid derepression of this enzyme.From the information given in Fig. 5, it can becalculated (see Materials and Methods) that thedifferential rate of histidase formation during thefirst 30 min after the shift is 33 units/mg ofprotein-a value in close agreement with thatobserved during substrate-restricted growth (Ta-ble 2).

But it is the growth response of the cells thatprovides the most interesting result of this experi-ment. From the lower panel of Fig. 5, it can beseen that the cell mass (as measured both byoptical density and by total protein) per milliliterof culture began to decrease as soon as thedilution rate was stepped-up. This decrease indi-cates that the cells were, for almost 2 hr, unableto grow as fast as the dilution rate (0.66 hr-').Their actual rate of growth, calculated as de-scribed in Materials and Methods, was 0.40 hr-'during the first 30 min after the shift. Since this

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JENSEN AND NEIDHARDT

101.0 aa 10

b

.7,_ _.07

- ~~~~~aA.146 -.346o .146 - .420 * PROTEIN

.4 . I_ .040 40 80

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FIG. 5. Growth and histidase formation in responseto a sudden large increase in feeding rate. Details werethe same asfor Fig. 4, except that at zero time the dilu-tion rate was changed from 0.13 hr-1 to 0.66 hr-1. Theordinate scales are exponential.

rate is established almost instantaneously afterthe shift, i.e., before a significant increase inhistidase level has occurred, the cells must quicklyhave been able to make three times more efficientuse of the histidase they already possessed at theslow growth rate.

It was tempting, therefore, to calculate howfast the cells could have grown immediately afterremoval of the biosynthetic restriction if (i) theirhistidase complement were to function as activelyas it does during rapid (unrestricted) growth andalso (ii) they were to begin converting L-histidineto cell material with the efficiency characteristicof rapid growth. In batch culture, these cellscontain 24 units of histidase per mg of protein,they metabolize L-histidine at a rate of 2.7 mg perhr per mg of cells, and they grow at a rate of0.85 hr-' (see Fig. 1 and 3). In the shift-up experi-ment just described, the cells contained 14 units ofhistidase at the time of the shift up; they should,therefore, have been able to metabolize L-histidineat a rate of 1.58 mg per hr per mg of cells and togrow at a rate of 0.49 hr-1. The agreement be-tween this growth rate and that actually achieved(0.40 hr-') strongly suggests that our assumptionof full functioning of histidase and conversion ofL-histidine to protoplasm at batch rate efficiencyis very nearly correct. Four additional shift-upexperiments were performed. In all of them, thecells were able to triple their growth rate in-stantaneously, and to achieve a value close to butnever exceeding the theoretical maximum basedon their histidase content (results not shown).Finally, experiments were performed in which thedilution rate of the chemostat was stepped upabout 2.5-fold-a value that should be within thecapacity of the cells' growth response. As shownin Fig. 6, such shifts resulted in little or no

FIG. 6. Growth and histidase formation in response

to a small increase in feeding rate. Details were thesame as for Fig. 4, except that at zero time the dilutionrate was changedfrom 0.15 to 0.35 hr-1 in the case ofone culture (marked "a"), and histidase (0), opticaldensity (A) and total protein (0) were measured; inthe case of another culture (marked "b"), the dilutionrate was changedfrom 0.15 to 0.42 hr-1, and the opticaldensity (0) was measured. The ordinate scales areexponential.

perceptible decrease in cell density, confirmingthe fact that growth can be accelerated fromk = 0.15 to k = 0.42 by nearly instantaneousmetabolic adjustments.

DISCUSSIONRestriction of growth in Aerobacter clearly does

affect substrate catabolism. Let us trace whathappens to the metabolism of L-histidine as wetake a culture of A. aerogenes in steady-state fastgrowth and decrease its growth rate by means ofsome biosynthetic restriction. First, upon impos-ing the restriction there will be no more than a10% reduction in the specific rate of L-histidinebreakdown (deduced by comparing VH valuesfrom Table 1 with Table 5); in other words,catabolism will greatly exceed anabolism duringthis transient phase. The formation of histidaseand presumably other catabolite-sensitive en-zymes will be totally arrested, and continuedgrowth at the new slow rate will dilute out thecells' complement of histidase (cf. Fig. 4). Thisgradual reduction in the level of histidase [andprobably at least that of the next enzyme in thepathway as well (9) ] gradually reduces the specificrate of L-histidine metabolism.At the same time, two other processes seem also

to come into play. One of these contributes furtherto the reduction in L-histidine metabolism bysomehow slightly restricting the operation of thehistidase pathway (cf. Fig. 3). Since histidaserequires no coenzymes and catalyzes a virtuallyirreversible reaction (9), it is difficult to envisagehow the rate of L-histidine metabolism could be

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VOL. 98, 1969 REGULATION OF HISTIDINE CATABOLISM IN AEROBACTER

inhibited except by mechanisms that affect theactivity of this enzyme directly (e.g., by end-product inhibition) or indirectly (e.g., by re-stricted entry of L-histidine into the cells). Wehave not examined the histidase of this organismfor possible allosteric interactions, but the equiva-lent enzyme in Pseudomonas aeruginosa has asigmoid substrate saturation curve, and is par-tially inactive in vivo under certain growth condi-tions (6). In P. putida, there is evidence thatsuccinate inhibits urocanase, the second enzymein the pathway, and that urocanate inhibitshistidase, leading to a sequential negative feed-back inhibition (3).The third process that seems to occur as the

cells adjust to the slower rate of growth is analteration in the capacity of the cell to dispose ofexcess catabolites. After about a generation ofgrowth with complete catabolite repression ofhistidase, synthesis of this enzyme resumes at arelaxed rate, i.e., at a rate that leads to no morethan a 50% coupling of histidase formation togrowth rate. We interpret this relaxation as beingthe result of an expanded capacity of the cell toconvert repressing catabolites into inert by-prod-ucts (e.g., ethyl alcohol, CO2, lactate, etc.),thereby maintaining the histidase level at a highervalue than actually required for growth at thenew rate. Indirectly, this relaxation also is re-sponsible for the decreased growth yield. We haveno solid information about the nature of thispresumed adjustment in catabolite metabolism,nor of the biochemical entities involved. It isunnecessary that such adjustment cause a qualita-tive change in the nature of the by-productsexcreted by the cell, since the adjustment mightentail simply a coordinate expansion of pathwaysalready used at high growth rate. Nevertheless,evidence for a qualitative change would be wel-come, because it would make more convincingthe argument that the internal level of catabolitesis thereby kept lower during steady-state slowgrowth than during the period of adjustment justafter the shift. Our finding that cells adjusted toslow growth seem to excrete a larger portion oftheir carbon as volatile material provides a pre-liminary indication that such a difference mayexist, but more precise analyses are necessarybefore this can be interpreted.

Finally, a new steady state is reached in which,compared with rapid growth, (i) the cells consumeless L-histidine per hr per mg of cells, (ii) theyproduce less histidase per mg of protein, (iii) thehistidase they possess functions at a slightly loweroverall rate in vivo, and (iv) a different pattern ofby-products may possibly be produced from thesubstrate.To complete our description of how growth

affects catabolism, consider next what happens

when the restriction on biosynthesis is removedand the cells are permitted to return to their batchgrowth rate. The cells almost instantaneouslyincrease their rate of histidase formation, as wouldbe predicted by the release of catabolite repres-sion, but long before the level of enzyme has beensignificantly elevated the cells achieve a near-tripling in growth rate. This ability to grow fasterwith the existing level of histidase must mean thatboth the restriction on L-histidine metabolismand the diversion of carbon to excreted by-products can be quickly reversed.

This behavior seems complex, but upon reflec-tion one can see some physiological sense in it.Consider how catabolism should be controlled.There are two requirements that must be met:(i) cells should be spared from synthesizing largequantities of unnecessary enzymes, but at thesame time (ii) each of the three major products ofcatabolism (intermediary metabolites, energy inthe form of ATP, and reducing power in the formof reduced nicotinamide adenine dinucleotide andreduced nicotinamide adenine dinucleotide phos-phate) must be supplied at an appropriate rateto the biosynthetic machinery of the cell.The former requirement can, of course, be met

by catabolite repression, which prevents the in-duction of unneeded enzyme species and which,as we have seen here, must be potentially capableof maintaining the minimal level of catabolicenzyme consistent with a given rate of cellularbiosynthesis, since the rate of histidase formationis reduced to near zero after a shift down.The latter requirement poses the usual problem

of how to control branching pathways that havemultiple end products. The problem can beparticularly acute in this case, for the relativedemand for carbon compounds and ATP mustvary enormously under different growth condi-tions. In a medium rich in the end products ofbiosynthesis (e.g., amino acids, nucleotides, andvitamins) intermediary metabolites will not bedrained off into biosynthetic pathways at anappreciable rate. And yet the energy demands forgrowth in such a medium are considerable becauseof the large energy requirement for macromolecu-lular synthesis. Therefore, the potential accumula-tion of repressing catabolites must be prevented,or a disastrous curtailment of energy productionwill result. One way to solve this problem wouldbe to bypass catabolite repression by convertingall products of catabolism into such (presumablynonrepressing) by-products as CO2, lactate, for-mate, ethyl alcohol, and acetate. Excretion ofthese compounds would then permit the level ofcatabolic enzymes, and therefore the rate ofcatabolism, to remain high enough to satisfy theenergy demands of the cell. Therefore, the secondrequirement for any general control on catabolism

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JENSEN AND NEIDHARDT

could be met by the existence of pathways thatwould be induced by high levels of intermediarycatabolites and that would dispose of thesecatabolites. The full effect of catabolite repressionwould then be evident only during the transientperiod required to form or expand the newpathway.The results of Rosenberger and Elsden (14) are

consistent with this picture. Their results wereobtained with a rich, anaerobic medium in whichglucose was almost quantitatively converted tolactic acid at each growth rate. In a mediumwhich supplies all or most of the monomericsubunits of protoplasm, no catabolites are usedfor biosynthesis, irrespective of the growth rate.Therefore, as Rosenberger and Elsden found, therate of glucose degradation may well be inde-pendent of growth rate in this situation, forwhatever effect catabolite repression has in thisorganism should already have been present duringrapid growth. Such a result implies, of course,that ADP is regenerated by direct or indirectadenosine triphosphatase systems which them-selves must be under rather stringent (and in-teresting) control.When the growth of a cell is forced to a lower

rate by a biosynthetic restriction, the regulatorysignal seen by the cell is, at least in part, the sameas if a supplement rich in building blocks hadbeen added to the culture-a transiently highconcentration of catabolites. Our results suggestthat the response of the cell may be similar in thesetwo conditions: first, a period of growth duringwhich these catabolites both repress substrate-degrading enzymes and induce catabolite-degrad-ing enzymes, and then steady-state growth inwhich a balance is achieved, the substrate-de-grading enzymes being partially repressed.

Mention was made in the introduction ofseveral instances (1, 4, 12, 15) in which continuedrapid glucose catabolism has been observed whennitrate or molecular nitrogen was substituted forammonia as the nitrogen source, even though thegrowth rate was reduced. In at least one of thesecases, that of A. aerogenes growing with nitrate,we have been able to demonstrate (11) that theincreased glucose catabolism is still sensitive tochanges in the growth rate. In the other cases, lesscan be said with certainty, but we would suggestthat catabolite repression might operate transi-ently in these organisms, as it does in Aerobacter,and then be bypassed even more completely byexpansion of a metabolic route to dispose ofexcess metabolites.

In summary, this study of L-histidine catabo-lism in A. aerogenes has confirmed the premisethat cells make a regulatory response to an im-balance between catabolism and biosynthesis. In

this organism, the response consists chiefly ofcatabolite repression, which is severe at first andthen becomes relaxed, possibly by the expansionof pathways that convert catabolite repressorsinto inert by-products. In addition, at very lowgrowth rates the L-histidine-degrading enzymesappear to function somewhat less rapidly than athigh growth rates.

ACKNOWLEDGMENT

This investigation was supported by Public Health Servicegrant GM-08437 from the National Institute of General MedicalSciences. In addition, D. E. J. received a traineeship supportedby grant GM-1195 from the National Institutes of Health.

LITERATURE CITED

1. Daesch, G., and L. E. Mortenson. 1968. Sucrose catabolismin Clostridium pasteurianum and its relation to N2 fixation.J. Bacteriol. 96:346-351.

2. Gunsalus, L. C., and C. W. Shuster. 1961. Energy-yieldingmetabolism in bacteria, p. 1-59. In L. C. Gunsalus andR. Y. Stanier (ed.), The bacteria, vol. 2. AcademicPress Inc., New York.

3. Hug, D. H., D. Roth, and J. Hunter. 1968. Regulation ofhistidine catabolism by succinate in Pseudomonas putida.J. Bacteriol. 96:396-402.

4. Jorpes, E. 1932. The colorimetric determination of histidine.Biochem. J. 26:1507-1511.

5. LeGall, J., and J. C. Senez. 1960. Influence de la fixation del'azote sur la croissance de Desulfovibrio desulfuricans.Compt. Rend. 250:404-406.

6. Lessie, T. G., and F. C. Neidhardt. 1967. Formation and oper-ation of the histidine-degrading pathway in Pseudomonasaeruginosa. J. Bacteriol. 93:1800-1810.

7. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Ran-dall. 1951. Protein measurement with the Folin phenolreagent. J. Biol. Chem. 193:265-275.

8. Magasanik, B. 1961. Catabolite repression. Cold Spring Har-bor Symp. Quant. Biol. 26:249-254.

9. Magasanik, B., P. Lund, F. C. Neidhardt, and D. T. Schwartz.1965. Induction and repression of the histidine degradingenzymes in Aerobacter aerogenes. J. Biol. Chem. 240:4320-4324.

10. Marr, A. G., E. H. Nilson, and D. J. Clark. 1963. The main-tenance requirement in Escherichla coil. Ann. N.Y. Acad.Sci. 102:536-548.

11. Neidhardt, F. C. 1965. Role of enzyme repression in the regu-lation of catabolism in bacteria, p. 329-336. In Mechanismesde regulation des activites cellulaires chez les micro-organismes. Colloq. Int. Centre Nat. Rech. Sci. No. 124.

12. Neidhardt, F. C., and B. Magasanik. 1957. Reversal of theglucose inhibition of histidase biosynthesis in Aerobacteraerogenes. J. Bacteriol. 73:253-259.

13. Pichinoty, F. 1960. Reduction assimilative du nitrate par lescultures aerobies d'Aerobacter aerogenes. Influence de lanutrition azot6e sur la croissance. Folia Microbiol. (Prague)5:165-170.

14. Rosenberger, R. F., and S. R. Elsden. 1960. The yields ofStreptococcus faecalis grown in continuous culture. J. Gen.Microbiol. 22:726-739.

15. Schneider, W. C. 1945. Phosphorus compounds in animaltissues. I. Extraction and estimation of desoxypentosenucleic acid and of pentose nucleic acid. J. Biol. Chem. 161:293-303.

16. Senez, J. C. 1962. Some considerations on the energetics ofbacterial growth. Bacteriol. Rev. 26:95-107.

17. Umbreit, W. W., R. H. Burris, and J. F. Stauffer. 1964.Manometric techniques, 4th ed., Burgess Publishing Co.,Minneapolis, Minn.

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