INDUCTION OF STAPHYLOCOCCAL PENICILLINASE BYBENZYLPENICILLIN: EFFECT OF pH, CONCENTRATIONOF FERROUS ION AND INDUCER, AND DURATION

OF EXPOSURE OF CELLS TO INDUCER

FELIX LEITNER, HELEN M. SWEENEY, T. F. MARTIN, AND SIDNEY COHEN

Department of Microbiology, Michael Reese Hospital and Medical Center, Chicago, Illinois

Received for publication 1 May 1963

ABSTRACT

LEITNER, FELIX (Michael Reese Hospital andMedical Center, Chicago, Ill.), HELEN M.SWEENEY, T. F. MARTIN, AND SIDNEY COHEN.Induction of staphylococcal penicillinase bybenzylpenicillin: effect of pH, concentration offerrous ion and inducer, and duration of exposureof cells to inducer. J. Bacteriol. 86:717-727.1963.-The kinetics of induction of penicillinaseby benzylpenicillin in exponentially multiplyingStaphylococcus aureus strain 55-C-1 were shownto vary with the pH. At pH 7.3 in the absence offree inducer, the rate of increase of penicillinaseactivity rapidly declined and came to a halt. AtpH 5.4 to 5.5 and in the presence of optimal con-centrations of Fe++, the penicillinase activity ofthe induced culture increased linearly with timefor 2.5 or more generations, but the rate of in-crease usually declined eventually. Evidence wasadvanced to support the concept that the acidicpH and optimal Fe++ concentration maintainthe induced formation of enzyme. The inducedincrease in penicillinase activity appeared 3 to 4min after the addition of benzylpenicillin. Thedegree of induction of penicillinase varied withthe duration of exposure of the staphylococci tothe inducer and with the concentration of in-ducer. Maximal induction under our conditionswas attained by exposure for 15 min to benzyl-penicillin at an initial concentration between 0.6and 2 units/ml.

Although the inducibility of staphylococcalpenicillinase has been known for some time(Geronimus and Cohen, 1957; Steinman, 1961;Crompton et al., 1962), more information on thecharacteristics of this system, comparable withthat developed for classical systems such as peni-cillinase in Bacillus cereus and f-galactosidase in

Escherichia coli, has been lacking. In this paper,we present results of studies of the kinetics ofinduction of staphylococcal penicillinase. Wehave found that the kinetics of induction of thisenzyme in exponentially multiplying cultures ofStaphylococcus aureus vary markedly with pHand Fe concentration. At an acidic pH in therange from 5.4 to 5.5 and a favorable Fe con-centration, penicillinase activity increases lin-early with time in the absence of free inducer, ina fashion kinetically similar to but usually notso prolonged as that described by Pollock (1950)for B. cereus. Other aspects of induction in thissystem, including the effects of time of exposureof cells to inducer, are also presented.

MATERIALS AND METHODS

Organisms. S. aureus strain 55-C-1 (Geronimusand Cohen, 1957) was the routine test organism.Drops of overnight broth cultures taken up onporcelain beads and then desiccated over silicagel served as routine inocula. The dried beads,stored at 4 C, were used for periods up to 6months. A few experiments were repeated withseveral other penicillinase-producing strains of S.aureus to test the generality of the observations.These strains were stored and inoculated fromnutrient agar slants.

Media. Cultures were grown in tryptic digestbroth (TD), lot no. 003607 (BBL), unless other-wise stated. Several other lots failed to sustaingrowth at pH 5.5. It was eventually found thatacetic acid added to the tryptic meat digest dur-ing the preparation of the broth was probablyresponsible for the inhibition. Batches of trypticdigest of beef heart prepared by us according toFields (1956) supported good growth at pH 5.5when HCl was substituted for acetic acid.TD lot no. 003607 contained 0.001 M inorganic

phosphate (Leitner and Cohen, 1962). To avoid

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undesired changes in pH during experiments andto improve growth at acidic pH, 0.01 to 0.05 Msodium phosphate was added to the broth pre-pared from this lot as indicated in the text.Broth was sterilized routinely at pH 7.4 in an

autoclave at a pressure of 12 to 14 psi for 15 min.The sterile broth was adjusted to pH 5.4 to 5.5,when desired, by addition of 1 N HCl.

Soluble penicillinase. Soluble staphylococcalpenicillinase was prepared from induced cells ofstrain 55-C-1, possessing penicillinase activityfrom 140 to 210 units/mg of protein. Aqueous sus-pensions of washed organisms (12 to 26 mg ofprotein/ml) were sonically disrupted, in instru-ments made by Raytheon Co. (10 kc, model DF101) or by Branson Instruments, Inc. (modelLS-75). With the Raytheon instrument, the cellswere treated for 1 hr; with the Branson, for 5 minin 30-sec periods alternating with cooling. Celldebris was removed by centrifugation for 30 minat 105,000 X g (average) in a model L Spinco ul-tracentrifuge. The supernatants, containing 580to 3760 units/ml, were divided into 1- to 1.5-mlportions and stored at -20 C.

Induction. Experiments were begun from cul-tures incubated with slow shaking for approxi-mately 16 hr at 37 C in TD containing 0.05 Mphosphate. A portion was diluted sterilely 12- to50-fold (to a cell density of 11 to 19 ,g of protein/ml) with TD of the desired pH, containing suffi-cient phosphate ion to give a final concentrationof 0.01 M. A 50-ml portion of this diluted culturewas incubated at 37 C with vigorous shaking (225rotations/min in a rotatory shaker) in a conicalflask of 125-ml capacity with a 19-mm cuvetteside arm. After two to three generations of ex-ponential growth, a portion of this culture wasdiluted again with the same sterile medium to acell density of about 6 ,ug of bacterial protein/ml.Clean but nonsterile glassware was used for thisdilution and for subsequent manipulations. Re-peated cultures and smears confirmed that nosignificant bacterial contamination took placeunder these circumstances during the remaining2 to 3 hr of the experiments. Culture volumes of50 ml or less were handled in 125-ml flasks, vol-umes of 50 to 150 ml in 300-ml flasks, and vol-umes of 150 to 350 ml in 1-liter flasks. In typicalexperiments, incubation was continued with shak-ing until a cell density of 10 to 11 MAg of protein/mlwas attained, at which time inducer was added.In kinetic studies, this point is defined as zeroreference time. Additions of Fe(NH4)2(SO4)2.

6H20 were usually made 2 min before induction.Growth curves, which were followed through allexperiments, were exponential for the periods ofthe experiments cited, as judged by absorbancy.The generation times of uninduced cultures were24 to 32 min at pH 7.3 to 7.4 and 26 to 38 min atpH 5.4 to 5.5. In induced cultures, the generationtimes were 0 to 6 min longer than in the absenceof inducer.The exposure of cells to free inducer was ter-

minated, when desired, by one of two methods.In the first, free penicillin was removed by Milli-pore filtration followed by washing and resuspen-sion of the organisms with fresh broth at 37 C(Pardee and Prestidge, 1961). Incubation withshaking at 37 C was then resumed. In this manip-ulation, some of the cell crop was not recovered,as judged by absorbancy of the resuspended cells.The reported penicillinase activity per ml of cul-ture is corrected for the fraction lost experi-mentally. Alternatively, the inducer was de-stroyed without interrupting the incubation byaddition of soluble penicillinase (Pollock, 1952).The inactivation of the antibiotic was followedby the loss of its inducing power. Table 1 givesthe results of a typical experiment. Induction bypenicillin was markedly reduced after 30 sec,especially at pH 5.5, and was undetectable, ateither pH, after exposure of the inducer to theadded enzyme for 90 sec. In the actual inductionexperiments, the organisms' own penicillinaseactivity, low at the start, increased rapidly andsoon became comparable with the activity ofthe added enzyme. Under these circumstances,the inducer must have been destroyed appreciablyfaster than is shown in the table.

Penicillinase assay. Samples of cultures wereremoved at appropriate times, mixed with chlor-amphenicol (100 ,ug/ml), and chilled promptly ina Dry Ice-acetone bath. The penicillinase ac-tivity was determined iodometrically according toPerret (1954) by using phosphate buffer at pH5.8, the optimal pH for the staphylococcal en-zyme (Novick, 1962). A manometric assay ofcell-bound penicillinase was employed in some ex-periments, either when the enzymatic activity perml of culture was too low to be measured withoutconcentration or when soluble penicillinase hadbeen added to the culture to terminate inductionmore sharply than was possible by filtration. Inthese cases, samples were taken without chlor-amphenicol and were chilled promptly in a DryIce-acetone bath. The organisms were collected

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INDUCTION OF STAPHYLOCOCCAL PENICILLINASE

TABLE 1. Inactivation of benzylpenicillin by solublestaphylococcal penicillinase*

Cell-bound penicillinaseTime of addition of cells (units/mg of protein)

pH 5.5 pH 7.4

sec

30 19.5 26.660 9.5 12.790 7.9 4.5120 8.0 4.3

ControlsEnzyme + cells 7.7 4.5Penicillin + cells 167 80.0

* Cells of strain 55-C-1 grown for 2.5 generationswith shaking in TD (first shipment of lot no.003607) with 0.01 M added phosphate at pH 5.5and 7.4 were harvested by brief centrifugation atroom temperature and resuspended in water.Two sets of fresh broth of corresponding pHvalues, containing penicillin (2 units/ml) andshaken at 37 C, received soluble penicillinase to afinal concentration of 0.95 units/ml, followed,at the indicated times later, by the correspondinginoculum. (Initial bacterial concentration of theacid and neutral cultures were 8 and 12,g of pro-tein/ml.) In the first control (enzyme + cells),penicillin was omitted. In the second (penicillin +cells), the order of addition was reversed: thecells were added to the penicillin-containing brothfirst, followed by the enzyme 10 min later. Figuresrepresent the specific activities of cell-boundpenicillinase in washed organisms collected 55min (acid cultures) and 35 min (neutral cultures)after inoculation. The corresponding specificactivities of the acid and neutral inocula were4.8 and 2.5 units/mg of protein, respectively.Penicillinase was determined manometrically.

by centrifugation at 4 C. If soluble penicillinasehad been used, they were washed once with cold0.05 M NaHCO3 solution and once with cold 0.017M NaHCO3; otherwise, they were washed onlywith the latter. The cell-bound enzyme activitywas determined manometrically as previouslydescribed (Leitner and Cohen, 1962). It was con-firmed that added soluble enzyme was removedby the washing and did not interfere in this assay.The extracellular enzyme fraction was not meas-ured in this assay, but the results reported werecorrected for it, when desirable, by data from in-dependent experiments on the distribution ofpenicillinase activity between cells and super-natant.One unit of penicillinase was defined as the

amount of enzyme that hydrolyzes 1 ,umole ofpenicillin per hr at 30 C and pH 5.80, the condi-tions of the iodometric assay. Specific activitywas defined as units of penicillinase per mg ofbacterial protein. This unit was virtually identicalwith that based on the manometric assay, per-formed at 37 C and pH 7.46 (Leitner and Cohen,1962). The ratio of manometric to iodometricunits in eight experiments was 1.0 (-t 0.07 SD).Accordingly, results obtained by either methodwere used interchangeably without correction.

Protein determination. The concentration ofstaphylococci was followed by absorbancy at 540m,u in a Coleman junior spectrophotometer. Thesevalues were related to bacterial protein by experi-mentally determined curves (Leitner and Cohen,1962).Iron determination. The iron content of the

broth was determined colorimetrically by theo-phenanthroline method (Schales, 1958) afterdigestion with nitric and perchloric acids.

Chemicals. Sodium benzylpenicillin, a gift ofChas. Pfizer & Co., Inc., is referred to in thispaper as penicillin. Concentrations are given inOxford units.

Chloramphenicol was a gift of Parke, Davis &Co.

Chemicals were reagent grade.RESULTS

Effect of pH on kinetics of induction. The kinet-ics of the increase in penicillinase activity ofcultures of S. aureus 55-C-1 exposed for a limitedtime to free inducer while growing exponentiallyat pH 5.4 and 7.3 are compared in Fig. 1. In theunsupplemented acidic medium after a short lag,the rate of increase of penicillinase activity perml of culture was constant for about one genera-tion (40 min), and then declined progressively(Fig. la). In earlier experiments with othersamples of TD, including the first shipment oflot no. 003607, a constant rate of increase of peni-cillinase activity was maintained for three ormore generations. In trying to reconcile theseconflicting results, we were influenced by ourearlier observations of the striking potentiationby Fe++ of the constitutive synthesis of peni-cillinase in strain 55-C-1 at pH 4.7 (Leitner andCohen, 1962). The TD (lot no. 003607) used inthe experiments shown in Fig. 1 contained 1.75 X10-5 M iron. The addition of 0.5 X 10-5 MFe(NH4)2(SO4)2.6H20 permitted linear increasein penicillinase activity for about 2.5 generations

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GENERATIONS

LLIU)

z

-J

a-

7I-

z

cz--IN.

C)

-U

0

mz

z

C)N

C)

-U

0

mz

30 60 90 120TIME (MIN)

FIG. 1. Kinetics of penicillinase induction in cultures of Staphylococcus aureus growing exponentiallyat pH 5.4 (a) and 7.3 (b). (a) Strain 55-C-I was grown with shaking at 37 C in TD with 0.01 m addedphosphate at pH 5.4 for 3.6 generations. The culture was divided into two portions, one receiving a supple-ment of 0.5 X 10-5 M Fe(NH4) 2(S04) 2-6H20, 2 min before induction. At zero time, both cultures were

induced with penicillin (2 units/ml) at a cell concentration of 11 jig of protein/ml. After 10 min, inductionwas terminated by Millipore filtration. During the subsequent growth period, samples were taken at 10- to15-min intervals for iodometric penicillinase assay. Symbols: * =penicillinase, units/ml in the culturewithout added iron; 0 = penicillinase, units/ml and 0 = penicillinase, units/mg of staphylococcalprotein in the culture supplemented with Fe++. The specific penicillinase activity before induction was 6.6units/mg of staphylococcal protein. (b) Strain 56-C-1 was grown with shaking at 37 C in TD with 0.01 Madded phosphate at pH 7.3 for 3.5 generations. At 2 min before induction, 0.5 X 10- m Fe (NH4) 2(S04) 2-

6H20 was added. At zero time, the culture was induced with penicillin (2 units/ml) at a cell concentrationof 10 Mug of protein/ml. After 10 min, the inducer was removed by Millipore filtration. Samples were takenat 10- to 15-min intervals for iodometric penicillinase assay. Symbols: A = penicillinase, units/ml; A =

penicillinase, units/mg of staphylococcal protein. The specific penicillinase activity before induction was

3.1 units/mg of staphylococcal protein.

(Fig. la), and, at other times, for the durationof the experiments (as long as 3.5 generations).This concentration of iron was without effect onthe growth rate at pH 5.4, but higher concentra-tions reduced the duration of exponential growth.In the medium at pH 7.3 (Fig. lb), after asimilar lag, the rate of increase in penicillinaseactivity was roughly constant for less than onegeneration (18 min), and progressively declinedthereafter, becoming nil after two generations. Atthis pH, additionof 5 X 10-5 M Fe++did not affectgrowth nor alter the kinetics of penicillinase in-duction.

Specific penicillinase activities reached peak

values at 1.5 generations of increase in enzymaticactivity at pH 5.4 (Fig. la) and at 1 genera-tion at pH 7.4 (Fig. lb). The sudden rapiddecline, 100 min after induction, of the specificactivity of the acid-grown culture is the reflectionof the concomitant drop in the rate of increase ofpenicillinase activity of the whole culture. Thedotted line illustrates the curve that the specificpenicillinase activity would have followed if en-zymatic activity per ml of culture had continuedto increase at a constant rate.The two kinetic patterns could be converted

one into the other during the early phases of in-duced enzyme formation by the appropriate

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INDUCTION OF STAPHYLOCOCCAL PENICILLINASE

change in pH after elimination of free penicillin.Thus, the results cannot be ascribed to an effectof pH upon the uptake of inducer by the cells.Figure 2 illustrates the results of neutralization,12 min after induction, of a culture growing atpH 5.4. At 15 min after neutralization, the rate ofincrease of penicillinase activity started to declineprogressively, reaching zero about 70 min afterneutralization. The generation time, 36 min dur-ing the acidic phase of growth, fell to 33 min afterneutralization. The reciprocal procedure at 12,32, and 42 min is shown in Fig. 3. After acidifica-tion at 12 min, linear increase in enzymatic ac-tivity persisted, although at a rate slightly slowerthan that prevailing at the time of acidification.Acidification at 32 min, when the rate of increaseof penicillinase activity in the neutral culture wasabout to decline, caused only a transitory pro-longation of the increase in penicillinase activity.Acidification at 42 min, when penicillinase ac-tivity in the neutral culture was stationary, hadvirtually no effect. In these experiments, genera-tion times after acidification increased only from0 to 2 min.The different kinetics of induction at acid and

neutral pH could not be shown to result from ahigher rate of inactivation of enzyme in the neu-tral medium. This point was tested by stoppingprotein synthesis at varying times after inductionat pH 7.3 by the addition of chloramphenicol andassaying the constancy of the penicillinase ac-tivity of the culture during a further period ofincubation. The results (Fig. 4) indicate a slowloss of activity, amounting to 10 to 13% per hr,a rate of inactivation that obviously cannot ac-count for the difference in induction in the acidand neutral cultures.The cells induced at neutral pH did not contain

unrecognized, cryptic enzyme, for their peni-cillinase activity did not increase appreciablywhen they were broken in a sonic oscillator. Simi-larly, crypticityv could be excluded as an explana-tion for the lower penicillinase activities of cul-tures induced at pH 5.4 without added Fe++.The mechanism of the effect of Fe++ on induc-

tion of penicillinase remains obscure. We wereunable to obtain any evidence suggesting that itfunctioned as a cofactor with penicillinase. Thus,addition of Fe++, up to 10-4 M, to broken-cellpreparations of organisms induced either at pH5.4 and 0.8 X 10-6 MFe or at pH 7.3 and 1.3 X10-5 M Fe did not alter their penicillinase activi-

10-

X2-

N8U)F-

z

30 60 90 120TIME (MIN )

FIG. 2. Kinetics of penicillinase synthesis in anexponential culture of strain 66-C-i induced at pH6.4 and a portion subsequently adjusted to and grownat pH 7.8. The organisms were grown with shakingat 87 C in TD with 0.01 M added phosphate at pH6.4 for four generations. At 2 mmn after the additionof 0.5 X 10-6sX Fe(NH4) z(504)2.-6H20, the culturewas induced with penicillin (2 units/ml) at a cellconcentration of 11 ,usg of protein/ml. At 10 minafter induction, the inducer was removed by Milli-porefiltration. At 12 mmn after induction, the culturewas divtided into two portions, one being adjusted topH 7.8. Samples were removed at indicated timesfor iodometric penicillinase assay. Symbols: 0 =cuxlture at pH 6.4; A\ = culture at pH 7.8; arrowindicates time of neutralization.

ties significantly (Table 2). In the same broken-cell preparations, addition of o-phenanthroline,ax,a'-dipyridyl, or sodium ethylenediaminetetra-acetate did not alter the penicillinase activities.The distribution of penicillinase activity be-

tween cells and broth is shown in Table 3. Afterinduction at pH 5.4, 9 to 13% of the penicillinaseactivity was found in the supernatant 60 to 120mmn after induction. Washing the cells withNaHCO, solution removed little or no enzyme,

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b

I

2

LI-,

cn

z

U)

zwL0L

30 60 90 60 0 30 60 90TIME (MIN)

FIG. 3. Kinetics of penicillinase formation in exponential cultures of Staphylococcus aureus induced atpH 7.8 and portions subsequently acidified and grown at pH 5.4. Three cultures of strain 55-C-1 were grownwith shaking at 37 C in TD with 0.01 m added phosphate at pH 7.8 for 3.5 generations. At 2 min after theaddition of 0.5 X 10- m Fe(NH4)2(SO4)2.6H20, they were induced with penicillin (2 units/ml) at cellconcentrations of 10 tol11 iAg of protein/ml. At 10 min after induction, the inducer was removed by Milliporefiltration. At 12 min (a), 82 min (b), and 42 min (c) after induction, each culture was divided into twoportions, one of each pair being adjusted to pH 5.4. Samples were taken at 10- to 15-min intervals foriodometric penicillinase assay. Symbols: A = cultures at pH 7.8; 0 = cultures at pH 5.4; arrows indi-cate time of acidification.

although this procedure had been shown to re-move added soluble penicillinase. Thus, the cell-bound enzyme did not include significant amountsof adsorbed extracellular enzyme. After inductionat pH 7.3, 13 to 19% of the enzyme was extra-cellular. Here, too, no additional enzyme was re-moved from the cells 80 to 120 min after induc-tion by washing with NaHCO3 solution, butdetectable amounts were removed from cells col-lected at 20 to 60 min.

In contrast to the current observations, extra-cellular penicillinase was not detected in earlierexperiments of shorter duration (Geronimus andCohen, 1957). Novick (1962) and Swallow andSneath (1962) found 15 to 45% of inducedstaphylococcal penicillinase to be extracellular,larger proportions being found several hours afterinduction. The presence of a lower proportion ofinduced extracellular staphylococcal penicillinaseat pH 5.4 than at pH 7.3 resembles an earlierobservation with B. subtilis penicillinase (Pollock,1961 b).

Comparable differences in induction at neutraland acidic pH were obtained with strain 55-C-1growing in Difco Heart Infusion Broth and withthree randomly selected strains of penicillinase-producing staphylococci in TD.Lag period. The pH of the culture medium had

no detectable influence on the lag period. An in-crease in penicillinase activity was noted 3 to 4min after addition of the inducer (Fig. 5). Theseresults resemble those of Pardee and Prestidge(1961), who found a lag period of 3 to 4 min forseveral inducible enzymes in E. coli. Extrapola-tion of the straight-line portion of the plot inFig. 5 intercepts the abscissa between 7 and 8min. This may be compared with the value of 14( i1.7) min found upon induction of the B. cereuspenicillinase system at 35 C (Pollock, 1952). Itshould be noted that in Pollock's experiments onthe lag period, inducer was eliminated after 1 minby the addition of soluble penicillinase, but inours it was not. The possibility has not been ex-cluded that the length of the lag period may be

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12- 5

JI)

Z lO- / 60 90 120

zt|1 4 1 X

,J 6- Yo 120 150

30 60 90 120 150

W 40_

2-

60 90 120 150

TIME (MIN)

FIG. 4. Rate of inactivation of penicillinase atpH 7.3 in an induced culture of strain 55-C-i.The organisms were grown exponentially with shak-ing at 37 C in TD with 0.01 M added phosphate atpH 5.4 for 3.5 generations. At 2 min after the addi-tion of 0.5 X 10-5 M Fe(NH4)2(SO4)2-6H20, theculture was induced with penicillin (2 units/ml) at acell concentration of 11 ,ug of protein/ml. At 5 minafter induction (indicated by arrow), the culturewas divided into two portions, one being adjustedto pH 7.3. Both cultures were sampled for iodometricpenicillinase determination at the times indicatedin the figure (O = culture at pH 5.4; = cultureat pH 7.3). At 60 and 90 min after induction, por-

tions of the neutral culture received chloramphenicol(100 jg/ml). They were incubated with shaking at37 Cfor 60 min, samples for iodometric penicillinasedetermination being taken at 5- to 10-min intervals.Inserts show in detail the penicillinase activitiesof the chloramphenicol-treated cultures.

TABLE 2. Effect of Fe++ and of chelating agents onthe penicillinase activity of sonically disrupted

cells of strain 55-C-i *

Penicillinase(units/ml)

Addition

pH 5.4 pH 7.3

None (unincubated control) 8.90 8.88None (incubated control) 8.56 8.38

Fe(NH4)2(SO4)2-6H20 (10-5 M) 8.64Fe (NH4) 2 (SO4)26H20 (3 X 8.68 8.23

10- 6 M)Fe(NH4) 2(SO4) 26H20 (10-4 M) 8.33 8.19

o-Phenanthroline (5 X 10-4 M) 8.51 8.76o-Phenanthroline (10- 3 M) 8.37 8.74

a,a'-Dipyridyl (5 X 10-4 M) 8.55 8.56a,a'-Dipyridyl (10-3 M) 8.58 8.78

EDTAt (5 X 10-4 M) 8.26EDTA (10-3 M) 8.29 8.25EDTA (3 X 10-3M) 8.29 8.19

* Sonically disrupted cells from cultures in-duced in TD (lot no. 203662) with 0.01 M addedphosphate at pH 5.4 and 7.3 were appropriatelydiluted and incubated for 1 hr at 37 C in 0.01 Mtris(hydroxymethyl)aminomethane buffer (pH7.2) containing the indicated additions. Peni-cillinase was determined iodometrically. The acidculture medium contained 0.8 X 10-5 M iron; theneutral culture medium contained 1.3 X 10-5 Miron. Specific activities of the acid and neutralcultures at the time of harvesting were 41.8 and22.7 units/mg of protein, respectively.

t Sodium ethylenediaminetetraacetate.

affected by the length of time that free inducer ispresent.

Concentration of penicillin and time of exposure.Figure 6 illustrates the feffects of variation of theconcentration of added inducer, and of the timeof exposure of strain 55-C-1 to it, upon inductionof penicillinase. The experiments were conductedat pH 5.5, since the linear relation of enzymeformation to time at this pH made it possible totake the specific activity of the cells at a con-venient time (45 to 55 min after induction) as ameasure of the degree of induction, provided cellconcentration at the time of induction and growthrates were constant, as was the case. Figure 6indicates that, under the conditions of our ex-

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periments, the degree of induction was approxi-mately linearly related to the length of exposureto inducer over a 15-min period. This relationcannot be accounted for by the saturation of newreceptor sites for penicillin, formed at an un-changing rate, since the cells increased by about40% and the degree of induction by about five-fold in the interval from 2 to 15 min after addi-tiQn of penicillin. Similar experiments at pH 7.3with 2 and 4 units of penicillin/ml gave com-parable results.The degree of induction also varied with the

initial concentration of penicillin. Saturation ap-peared to be reached at an initial penicillin con-

TABLE 3. Distribution of penicillinase between cellsand medium in cultures of strain 55-C-i *

Penicillinase (units/ml)

pH Time after Cellsinduction Super-

Toanatant

washed Washed

min

5.4 20 1.87 1.81 1.7240 4.90 4.78 4.62 <0.360 7.75 6.93 6.93 0.7080 10.4 9.08 8.80 1.08100 13.7 12.4 11.6 1.85120 17.1 15.3 14.8 1.94

7.3 20 2.36 2.19 1.8640 4.56 3.85 3.43 0.5860 4.81 3.81 3.44 0.8780 4.68 3.76 3.67 0.85100 4.70 3.72 3.80 0.88120 4.50 3.66 3.79 0.67

* The organisms were grown with shaking forfour generations in TD with 0.01 M added phos-phate at pH 5.4 and 7.3. At 2 min after the additionof 0.5 X 10- 5 M Fe (NH4) 2(SO4) 2 6H20 each culturewas induced with penicillin (2 units/ml) at acell concentration of 11 ,ug/ml. Samples were re-moved at 20-min intervals and divided into threeqets. In two sets, the cells were separated fromthe supernatant by centrifugation at 10,000 X gfor 15 min (acid culture) and 20 min (neutralculture) at 4 C. One set was washed once withcold 0.05 M NaHCO3, and once with cold 0.017M NaHCOO . The organisms of both sets were thenresuspended to the original volume in fresh brothcontaining 100 ,g/ml of chloramphenicol. Peni-cillinase was determined iodometrically in thesupernatants, the washed and unwashed cells,and the whole cultures.

Cl) ~~~~~~~1.5-z

3-w ~~~~~~~~1.0-

zW ~~~~~~0.5

0 5 10 15 0 5 10 15TIME (MIN.) TIME (MIN)

FIG. 5. The lag phase at pH 5.4 (a) and 7.4 (b).(a) Strain 55-C-i, grown exponentially in TD (lotno. 11956 containing 0.01 M phosphate) at pH 5.4for four generations, was induced at zero time withpenicillin (2 units/ml) at a cell concentration of 57jug oj protein/ml. Samples for manometric peni-cillinase assay of cell-bound enzyme were removedat 1- to 2-min intervals. (b) Strain 55-C-1 was grownexponentially in TD with 0.01 M added phosphate atpH 7.4 for 3.5 generations. Induction and assaywere performed as in a.

centration between 0.6 and 2 units/ml. In otherexperiments with slightly larger concentrations oforganisms, 20 units of penicillin/ml gave nogreater induction than did 2 units. The data ofFig. 6 indicate that 0.15 unit of penicillin/ml in-duces penicillinase to approximately half themaximal level attainable by this method. A veryslight degree of induction was detected with aslittle as 0.01 to 0.02 unit/ml.

DISCUSSION

The persistence of induced formation of peni-cillinase, after removal of free inducer, in strain55-C-1 growing at pH 5.4 resembles that found inB. cereus by Pollock (1950) but is less prolonged.In the latter system, penicillinase continues to beformed indefinitely in the absence of free inducerbut, in the staphylococcal system, its rate offormation usually declines within three to fourgenerations. The induced production of peni-cillinase in B. subtilis is also of limited duration(Pollock, 1961a). The interesting feature of thestaphylococcal system is the effect of an increasedhydrogen ion concentration which, in conjunctionwith an appropriate concentration of iron, ap-pears to prolong markedly the formation of en -zyme in the absence of free inducer.

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INDUCTION OF STAPHYLOCOCCAL PENICILLINASE

We have mentioned above the possibility thatthe change in the pattern of penicillinase forma-tion with pH might be due primarily to an in-creased rate of inactivation of enzyme at neutralpH in the presence of an unchanged rate of syn-thesis. Possible mechanisms include enzyme in-hibition, destruction by proteinases, or surfaceinactivation (Novick, 1962). Surface inactivationseems unlikely since the experiments were per-formed in complex broth media, which minimizesurface inactivation. Also, the course of inductionat neutral pH was unaltered in the presence of0.5% gelatin. Inactivation by proteinases seemsunlikely in view of the relatively slight degree ofinactivation of penicillinase in induced culturesat pH 7.3 after treatment with chloramphenicol.It is probable, however, that penicillinase is inac-tivated principally in its extracellular fraction. Ifthis is true and if its excretion is inhibited bychloramphenicol, as was the case with the B. sub-tilis enzyme (Pollock, 1961b), more enzyme mightbe available for inactivation extracellularly afterinduction at pH 7.3 in the absence of chloram-phenicol than is suggested by Fig. 4. But even ifexcretion of penicillinase were doubled withoutchloramphenicol, as was the case with B. subtilis(Pollock, 1961b), and its inactivation were in-creased proportionately, the amount of enzymeinactivated would still be less than 2 units perml per hr, an amount far too small to account forthe difference between the acidic and neutralcultures.An argument against inhibitors, in the sense

of compounds combining stoichiometrically withpenicillinase, may be derived from the resultsgiven in Fig. 3. If the effect of pH on inductionwere due solely to differences in rates of inactiva-tion of penicillinase, one would expect that acidi-fication of an induced neutral culture, after ap-parent enzyme formation had declined or ceased,should restore enzyme formation at a rate inde-pendent of the time of acidification. The data ofFig. 3 indicate that this is not the case. Thus,acidification at 42 min of the neutral inducedculture produced no significant increase in peni-cillinase activity, although, at this time, organ-isms from the sample acidified at 12 min stillexhibited a linear increase in penicillinase ac-tivity. The results would not be changed byreckoning time in bacterial generations, since thegrowth rates of the freshly acidified cultures were

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CONTACT WITH FREE INDUCER(MIN)

FIG. 6. Effect of the time of contact with variousconcentrations of free inducer on penicillinasesynthesis in exponentially growing Staphylococcusaureus. Five cultures of strain 55-C-I were grownwith shaking at 37 C in TD (first shipment of lotno. 003607) with 0.01 m added phosphate at pH 6.5for three to four generations. When the bacterialcultures reached a density of 1 ,ug of cell protein/ml,inducer was added at the following concentrations:0.06 (0), 0.16 (Q), 0.6 (@), 2.0 (O), and 4.0 (U)units of penicillin/ml. Immediately after induction,each culture was divided into six portions and growthwas continued. In five of the daughter cultures, in-duction was terminated with soluble penicillinase (0.94units/ml), the times of addition being 2, 5, 8, 10, and15 min. No soluble penicillinase was added to thesixth culture (marked oo). At 45 and 66 min afterinduction, samples uere removed for the manometricassay of cell-bound penicillinase. The mean of theobtained values (corrected for extracellular enzyme)is plotted against the time of contact of the cellswith free inducer. The penicillinase content of thecells before induction rangedfrom 3.7 to 4.4 units/mgof protein. For the cultures receiving 0.05, 0.15,and 0.6 units of penicillin/ml, recrystallized in-ducer was used.

virtually unchanged from those of the neutralcultures. A comparable argument applies to theresults of acidification at 32 min. Although, inthis case, the change in pH was followed by aslight prolongation of the increase in penicillinaseactivity, this declined at 45 min and soon stoppedaltogether, again much sooner than was the casewith a culture exposed to neutral pH for a shorter

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LEITNER ET AL.

time, i.e., the sample acidified at 12 min. We con-sider that the results of these experiments are bestunderstood on the interpretation that the peni-cilinase-synthesizing capabilities of induced or-ganisms deteriorate more rapidly during growthat neutral than at acidic pH.The effect of Fe++ on induction at acidic pH is

not understood and is being studied further. Fe++stimulated the constitutive formation of peni-cillinase in cells of strain 55-C-1 subjected to a pHof 4.7 (Leitner and Cohen, 1962). In these experi-ments, the specific penicillinase activity of thecells increased as much as 150-fold after exposurefor 180 min to pH 4.7, and evidence was adducedto show that the increase in activity was the re-sult of synthesis of enzyme. It is reasonable toassume that the effect of Fe+ on induction ofpenicillinase at the slightly less acidic pH of 5.4to 5.5 is exerted also on synthetic processes. Thisassumption is supported by our inability to dem-onstrate any other function for the added Fe++,such as that of a cofactor for penicillinase or al-teration in the accessibility of substrate to cell-bound enzyme.The relatively slow union of penicillin and

staphylococci, as measured by the degree of in-duction after varying periods of contact with freeinducer, contrasts with the situation in B. cereuswhere effective union is 50% complete in 1 minand virtually complete in 5 min (Pollock, 1952).Pollock demonstrated that the degree of induc-tion of penicillin was proportional to the amountof penicillin bound to the cells. If his observationmay be extended to S. aureus, our results wouldsuggest that penicillin binding by exponentiallymultiplying strain 55-C-1 continues for at least15 min. This is probably not due to a slow rateof union of penicillin with available sites butrather to the unmasking of previously unavailablesites during growth, in accordance with observa-tions of Cooper (1955) on the binding of labeledpenicillin by a penicillin-sensitive strain of S.aureus.

Other investigators have inferred a slow unionbetween penicillin and penicillinase-producingstaphylococci, based upon the relatively high con-centrations of penicillin they found necessary foroptimal induction (Batchelor et al., 1961). A simi-lar observation was made by Crompton et al.(1962). They suggested that this did not neces-sarily mean that penicillin was intrinsically a poorinducer but rather might mean that high initial

concentrations of penicillin were required to keepthe induction sites saturated in spite of the actionof the penicillinase produced by the large inocu-lum used in their experiments.The delineation in our experiments of optimal

conditions for induction reveals penicillin to be ahighly effective inducer of staphylococcal peni-cillinase. A brief exposure to 0.15 units of peni-cillin/ml induced formation of the enzyme to alevel one-half the maximum attainable at anypenicillin concentration in experiments of thistype. This contrasts with results of others underless favorable conditions, where either muchlarger concentrations of penicillin were requiredfor effective induction (Steinman, 1961; Cromp-ton et al., 1962) or little or no induction was ob-tained (Swallow and Sneath, 1962).

Factors other than those described above ap-pear to play an important part in induction ofstaphylococcal penicillinase. In studies as yet in-complete and not included in this paper, we havefound, as have others (Steinman, 1961; Knox andSmith, 1962), that the concentration of organismsat the time of induction appears to be of greatimportance. Induction at cell densities appreci-ably higher than 11 ,ug of protein/ml yields lowerdegrees of induction than at a density of 11Ag/ml, at both acid and neutral pH. This effectdoes not appear to be due solely to more rapidinactivation of inducer by the larger initial cellmass, since it can be overcome by the addition of10-I or 10-4 M Fe++. Studies of these and otheraspects of induction of staphylococcal penicil-linase are being continued.

ACKNOWLEDGMENT

This research was supported by grant E-2457from the National Institute of Allergy and In-fectious Diseases, U.S. Public Health Service,and grant G-15848 from the National ScienceFoundation.

LITERATURE CITED

BATCHELOR, F. R., J. CAMERON-WOOD, E. B.CHAIN, AND G. N. ROLINSON. 1961. 6-Amino-penicillanic acid. V. 6-Aminopenicillanic acidas a substrate for penicillinase and an inducerof penicillinase formation. Proc. Roy. Soc.(London), Ser. B 154:514-521.

COOPER, P. 1). 1955. The site of action of penicillin:some changes in Staphylococcus aureus cduringthe first two hours growth in penicillin media.J. Gen. Microbiol. 13:22-38.

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CROMPTON, B., M. JAGO, K. CRAWFORD, G. G. F.NEWTON, AND E. P. ABRAHAM. 1962. Be-haviour of some derivatives of 7-aminocepha-losporanic acid and 6-aminopenicillanic acidas substrates, inhibitors and inducers ofpenicillinases. Biochem. J. 83:52-63.

FIELDS, V. J. 1956. Preparation of a basal mediumfrom tryptic digested beef heart muscle.Newsletter, Society of American Bacteriolo-gists 22:8-9.

GERONIMUS, L. H., AND S. COHEN. 1957. Inductionof staphylococcal penicillinase. J. Bacteriol.73:28-34.

KNOX, R., AND J. T. SMITH. 1962. Antibacterialactivity, penicillinase stability and inducingability of different penicillins. J. Gen. Micro-biol. 28:471-479.

LEITNER, F., AND S. COHEN. 1962. Effect of pHand inorganic salts on penicillinase formationin Staphylococcus aureus. J. Bacteriol. 83:314-323.

NOVICK, R. P. 1962. Staphylococcal penicillinaseand the new penicillins. Biochem. J. 83:229-235.

PARDEE, A. B., AND L. S. PRESTIDGE. 1961. Theinitial kinetics of enzyme induction. Biochim.Biophys. Acta 49:77-88.

PERRET, C. J. 1954. lodometric assay of penicilli-nase. Nature 174:1012-1013.

POLLOCK, M. R. 1950. Penicillinase adaptation inB. cereus: adaptive enzyme formation in theabsence of free substrate. Brit. J. Exptl.Pathol. 31:739-753.

POLLOCK, M. R. 1952. Penicillinase adaptation inBacillus cereus: an analysis of three phases inthe response of logarithmically growing cul-tures to induction of penicillinase formationby penicillin. Brit. J. Exptl. Pathol. 33:587-600.

POLLOCK, M. R. 1961a. The measurement of theliberation of penicillinase from Bacillussubtilis. J. Gen. Microbiol. 26:239-253.

POLLOCK, M. R. 1961b. The mechanism of libera-tion of penicillinase from Bacillus subtilis. J.Gen. Microbiol. 26:267-276.

SCHALES, 0. 1958. Iron in serum, p. 69-78. In D.Seligson [ed.], Standard methods of clinicalchemistry, vol. 2. Academic Press, Inc., NewYork.

STEINMAN, H. G. 1961. Factors modifying inducedformation of penicillinase in Staphylococcusaureus. J. Bacteriol. 81:895-902.

SWALLOW, D. L., AND P. H. A. SNEATH. 1962.Studies on staphylococcal penicillinase. J.Gen. Microbiol. 28:461-469.

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