SITE OF RADIOPENICILLIN30) P. D. COOPER [VOL. 20 (11) by: (a) an identical uptake curve with a andRowleyandtheir colleagues, showing that radiopenicillin preparation containing less

SITE OF ACTION OF RADIOPENICILLIN

P. D. COOPERWright-Fleming Institute of Microbiology, St. Mary's Hospital Medical School, Paddington, London,

W.C. 2, England

CONTENTSI. The Penicillin-Binding Component (PBC) as the Initial Site of Action of Penicillin ...... .... 28

The Types of Binding Possible and the Binding Found.................................. 28Purity of the Radiopenicillin Preparations ............................................ 29Correlations Between Penicillin Binding and Its Antibacterial Action................... 30

II. Permeability of the Bacterial Cell to Penicillin........................................... 31III. The Chemical Properties of the Penicillin-Binding Component....................... ..... 32IV. The Nature of the Reaction Between the Penicillin Molecule and PBC.33V. The Nature of the Process First Damaged by Penicillin in Sensitive Bacteria. 35

Location in the Osmotic Barrier as a Possibility ....................................... 35Consideration of Early Effects of Penicillin ............................................ 36Increase of Penicillin Uptake when Growth Occurs .................................... 37Reactions that Are Not the Function of PBC..................................8........38

VI. Hypothetical Time-Table of Metabolic Events Following Addition of Penicillin.38VII. The Reasons for Penicillin Resistance ................................................... 40

Possession of the Means To Destroy Penicillin........................................ 40Differences in Rate or Amount of Penicillin Binding ................................... 42Apparent Independence of PBC........................................................ 44

VIII. Mechanism of Synergism................................................................ 45IX. Conclusion............................................................................. 45

A full description of the "mode of action" of the association between drug and cell, forof a drug should include all the changes which which the most sensitive and convenient assaythe drug brings about from its first contact with is radioisotopic labeling of the drug. This shouldthe susceptible cell. Perhaps from the difficult give information which applies directly to thenature of this task, implying as it does a full initial damage. It still appears difficult to intro-knowledge of what is normal also, and from the duce enough radioactive atoms into the moleculeobvious importance of the initial step in the to detect the small amounts of the more highlyseries of changes, most interest is usually at- active antibiotics bound by bacteria, but for-tached to the nature of this initial damage. tunately penicillin, unique in many other ways,However, the bacterial cell must contain very is an exception in this case also.many individual metabolic systems, and without The following review discusses the uses andsome guide the chances of stumbling upon the limitations of a rational approach to pencillinvery reaction which is first damaged seem mode of action derived from the binding ofrather remote., Thus, although chemical struc- radioactive penicillin by bacterial cells. It in-ture may provide an excellent guess, there is a cludes an examination of the likelihood thatneed for a rational approach to the general penicillin binding is related to its site of action,problem of antibacterial action. This need has together with fresh information and interpreta-been felt for some years (1) and has been filled tions of older data afforded by the propertiesto some extent by the concept of analogue of the several penicillin-binding componentscompetition, but unfortunately this is difficult to discovered.apply to most of the more potent antibacterialsubstances. This is perhaps because such a con- I. THE PENICILLIN-BINDING COMPONENT AS THEcept does not include the several types of inter- INITIAL SITE OF ACTION OF PENICILLINaction theoretically possible between drug and Types of Binding Possible and thek Binding Foundreceptor site, or else that we have not yet dis-covered the competing metabolite. A more It seems to be self evident that to affect ageneral approach may be the quantitative study living cell some molecules of a drug must be

28

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1956] SITE OF ACTION OF RADIOPENICILLIN 29

able to reach, to interact with, and thus to control solution without cells under the samederange a certain cellular system in a manner conditions.detectable as a direct association between drug The shape of the irreversible uptake curve,molecules and cell. Such a binding site can be of Rowley et al. (figure 1) suggested that thetermed the site of action of the drug and may cells contained a definite trace component, which,represent the site controlling the reaction first for convenience, has been termed the penicillin-disorganized by the drug. An alternative is that binding component (PBC). Any componentthe drug changes the cell's environment in some which similarly binds penicillin has in this reviewway. With penicillin such an alternative must been termed PBC, although it is recognized thatinvolve some quantitative relation between in different organisms it may differ chemicallypenicillin and a medium constituent, so that or even have quite different functions. In theory,any changes in medium should produce much for any particular organism there may be asgreater differences in penicillin sensitivity in a many types of binding sites as there are mole-given organism than have in fact been found cules bound (200-750 molecules/cell), but, in(2, 3). Thus this possibility seems most unlikely. general, the sites appeared to be similar in thatTo return to the general case, the binding of chemical agents either completely prevented

drug by the cell may be freely reversible in the binding or had no effect. This similarity, togethersense that bound drug molecules can be rapidly with the small amounts bound, suggested thatremoved by washing or by equilibration with the site of action was not multiple, although thefreshly added drug, or the bound molecules may possibility of several irreversible binding sites,be removed so slowly that the combination only one of which was connected with penicillinappears to be irreversible. The measurement of action, has been suggested (12, 12a, b) to accountbinding by the site of action may be made for the lack of correlation between resistance anddifficult by a large amount of both reversible penicillin bound in organisms trained to re-and irreversible binding by other cellular com- sistance.ponents, which has negligible biological effect.It is also possible that binding by these othersiPesuTht of t aeRadinpenicillin arationis necessary to produce the characteristic bio- That "PBC" was binding penicillin and notlogical effects, i.e., the site of action is multiple. radioactive impurities was regarded as provedEven a single site of action may produce many Units aPencillin"metabolic defects, one originating from another Bound/m Dry Wt Bacteriain the form of a chain.

This seemed to be true with penicillin, and to Acid lioctivoted Penicillinobtain information about the initial stage in theseries Cooper and Rowley (4) and Maass and -3Johnson (5, 6) independently examined theuptake of penicillin by bacteria, using penicillinlabeled with S35 by biosynthesis. They confirmed / Hoan earlier suspicion (7) that the amount of O epenicillin bound either reversibly or irreversiblywas very small, and Rowley et al. (8) demon-strated that little increase in irreversible peni- Old RadIo-penIcIllin Freshly tractedcillin binding occurred above very low concen- *trations. Later use of purer radiopenicillinsolutions (11) showed that saturation of these Radlo-peniciubinding sites was practically complete at 0.1 Inactvated By Peolcillinaseu/ml. Pasinsky and Kastorskaya (9) also found 0 0.25 0.5 0.75 1.0that a similar amount (about 1000 molecules/ Units Penicillin In Suspending Fluidcell) was lost overnight from abroth supernatant Figure 1. Uptake of purified radiopenicillin

cll) ctwalothovernight from abrothis coesupenat and various degradation products by Staphylococ-i contact with cells at 37 C, but thi corresponds CU8 aureus. Abscissa legend refers to units/ml.with the amount which can be calculated from Originally published in The Journal of Generalthe stability curves (10) to be inactivated in a Microbiology (11).


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30) P. D. COOPER [VOL. 20

(11) by: (a) an identical uptake curve with a and Rowley and their colleagues, showing thatradiopenicillin preparation containing less than naturally occurring penicillin-resistant strains5 per cent of its 535 in impurities, so that the S35 were able to bind penicillin to an extent vary-bound was more than the total amount of im- ing with their sensitivity, although artificiallypurity present; (b) rapid prevention of all uptake trained strains were not. Thus, with naturalbypretrain. thece.ls with0..A /l.ordinary resistors the final uptake was always of thebyptatingtencells with u/mi ordiar same order when the penicillin was at equi-

crystallie penicillin; (c) destruction of the effective (LD99.9) titers.horizontal portion at low concentrations by b. At low extracellular concentrations sensi-inactivating the drug beforehand with the specific tive cells bound penicillin so that the intra-enzyme penicillinase (figure 1). Pollock and cellular concentration was greater than thePerret (13) distinguish between a "specific" extracellular (8). This concentration of peni-penicillin binding, similar to that obtained using cillin by the sensitive cell occurred only overpurified radiopenicillin and a "nonspecific" the minimal inhibitory range (0.01-0.1 u/ml)binding, which may be due to nonpenicillin S35 (8) and, because of the low collision frequency,impurities. However, other workers (5, 6, 12, the rate of penicillin binding was sufficientlyslow in this range to be of the same order as12a, 14, 15, 16) do not appear to consider the the rate of rangerialbgrowthe s ordertedeffetofsmalamuntsof nnpencilin S1

th rat ofbacterial growth (6). This promptedeffect of small amounts of nonpenicilin SC the interesting hypothesis that the minimalimpurities. These can be strongly bound by inhibitory titer was the result of a balancecells and be present in considerable amounts even between rate of binding at this concentrationin "crystalline" radiopenicillin preparations and rate of PBC resynthesis, rather than of a(11), giving uptake curves with a marked upward removal of penicillin from the cell by dis-slope on sensitive organisms (figure 1). This sociation of the PBC-penicillin complex at lowupward slope is sufficient to mask "specific" concentrations.binding of 535 completely at higher concentra- It will be mentioned later (p. 37) that PBCtions, and can be due to penicilloic or penillic appears to be made available to penicillin bytwo co-existent mechanisms: (i) normal syn-acids, and possibly others of the many s t- thesis = rate bacterial growth in mg/ml/min Xinsoluble penicillin degradation products. It is PBC content in u/g; (ii) "turnover" = totalessential to be sure that "penicillin" binding is rate of exposure less the total rate of synthesis.in fact due to penicillin when considering the It will be seen that the rate of binding hasbiological effects of the drug, and a good test of only to exceed slightly the rate of synthesis,this appears to be the horizontal nature of the rather than total exposure, for the cell to beuptake curve with certain organisms, or the lack doomed by slow annihilation of its free PBC.of effect on the uptake curve of absorbing the Under certain conditions (18) normal syn-penicillin preparation with a small amount of thesis was 0.2 u/g/30 min. It is therefore per-themeasuring its uptake. haps further evidence for the hypothesis oft Maass and Johnson that the rate of binding at

Correlations Between Penicillin Binding and Its the minimum inhibitory concentration (0.02Antibacterial Action u/ml) by the same organism under slightly

different conditions (19) was observed to beFrom the point of view of the possible initial 0.6 u/g/30 min, which is of a similar order to

site of action the irreversible PBC proved inter- the rate of PBC synthesis actually observed.esting because of its several properties which c. Pretreatment with crystalline penicillinfitted in well with known aspects of pencillin prevented all uptake but cetyltrimethylam-action. These were: monium bromide, Aerosol "OT" and Tween 80

had no effect (8, 20). These detergents, togethera. PBC occurred in penicillin-sensitive with penicillin, failed to remove bound peni-

strains, but resistant strains previously se- cillin. Thus binding by PBC was not just alected as free from penicillinase were either nonspecific surface adsorption, but was specificwithout PBC or sheltered it from penicillin at to the penicillin molecule, an essential observa-low penicillin concentrations. There was a tion if PBC is to be the site of action of peni-correlation between penicillin bound and cillin.sensitivity (8). This was not the experience of d. The penicillin bound increased on growthMaass and Johnson (5), but Eagle (12, 15) con- in the presence of penicillin (6, 8, 18, 21), andfirmed and extended the work of both Johnson it is known that penicillin is lethal only under


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conditions of growth. The implications of this irreversible binding (by PBC) can be allowed for.finding are discussed in section V. In disagree- The fact that the sonic cell-extracts sedimentingment with these observations, any increase of between 27,000 and 144,000 X G bind penicillinbinding on growth was regarded as within to the same extent as intact cell (15) does notexperimental error by Eagle (15).

e. At 0.1 u/mi the rate of binding of peni- necessarily mean that the cell is permeable to

cillin by PBC became almost instantaneous, so penicillin. In fact, as this fraction would notthat PBC was rapidly saturated above 0.1 include the particulate fraction which containsu/ml but required a measurable time to reach 75% of the PBC (23), and should thereforesaturation below this level. The rate of killing bind less penicillin than intact cells, this isby penicillin also increased up to 0.1 u/ml (17) evidence similar to section b below that the cellbut stayed constant above this titer until the is impermeable to penicillin. As PBC appears torate began to drop at high concentrations. reside at or near the surface of the cell (23),This optimal concentration (0.1 u/ml) is again penicillin need not be able to penetrate the intactthe lowest at which production of fresh viableunits is stopped immediately by adding peni- cell completely nm order to reach PBC, and incillin (18, 22). fact complete permeability of the cell with little

f. The uptake by PBC is sufficiently small to reversible binding is unexpected in view of theallow the well-known lack of effect of inoculum following experimental findings:size on penicillin activity.

a. Similar cells are not freely permeable toThere is thus a considerable amount of evidence smaller negatively charged ions such as gluta-which strongly suggests that PBC, the irrever- mate, aspartate and phosphate (24, 25, 26).sible penicillin-binding component, is in fact the b. The unfractionated cytoplasmic contentsinitial site of action of penicillin, although it liberated on rupture reversibly bind veryseems a particularly difficult connection to prove much larger amounts of penicillin than do in-in spite of the fact that it seems to be the only tact cells (23). This high binding is to be ex-association of penicillin with the cell having the pected from the proteins present (27), althoughnec.ssarypsLit was not found in the fraction sedimentingnecessary properties. isttle work has been done between 27,000 and 144,000 X G (15). Although

on possible reversible binding of penicillin, and peniiln is ano surface atv (8.) Athleas. . ~penicillin is not surface active (28) at least

indeed evidence that it exists is rather indirect, some reversible association with the proteinsas shown in the next section. of the cell surface as well as the interior might

be anticipated from the work of Klotz et al.II. PERMSEABIITY OF THIE BAC'TERIAELL,TO ~ (27). If the intact cell were freely permeable to

PENICII.IJN penicillin, this would lead to the expectationIf it could be shown that penicillin cannot that reversible binding should be much higher

penetrate sensitive cells, then the search for the than was actually observed, although it isinitial reaction disorganized by penicillin could possible that many binding sites are onlybe confined to the part of the cell external to the exposed when the cellular proteins are disar-osmotic barrier. On the other hand, if it can be ranged by rupture.shownthatpeniilli canpeneratesenstive

c. The cell contains some reserve PRO whichshown that penicillin can penetrate sensitive is not made available to penicillin until aftercells, then impermeability becomes a possible rupture in a fashion which does not suggestmechanism of resistance. For these reasons it is enzymic synthesis (23). Thus some mechanismproposed to discuss this question a little more to protect this reserve PBC from penicillinfully than would otherwise be necessary. must be postulated.

Experiments involving the addition of peni- d. The qualitatively different effect of peni-cillin in known amounts to thick cell suspensions cillin at high concentrations (17) implies either(5) suggest that the cell is freely permeable to greatly increased binding at high titers, or apenicillin, and these results have been confirmed greatly increased rate of penetration of peni-

cillin in order to reach sites not previouslyi our laboratory using a Cowan's type II strai available. Neither of these concepts can beof staphylococcus. However, this type of experi- reconciled with the idea of little reversible orment involves the assumption that even at very irreversible binding (even at high concentra-high concentrations negligible amounts of peni- tion) accompanied by rapid and completecillin are reversibly bound by the cell. The permeability (even at low concentration).


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32 P. D. COOPER [VOL. 20

On the other hand, it is rather a coincidence to penicillin in that PBC is somehow not availablethat the amount presumed to be reversibly for reaction with penicillin (31). With yeast, atbound is just enough to cancel out the increase least, the question of impermeability was notin concentration to be expected from the exclu- obscured by any significant reversible binding.sion of penicillin from an impermeable cell in the Considerable amounts of S3I bound by Escherichiathick-suspension experiments mentioned. If the coli K12 from high concentrations of radiopeni-volume impenetrable by penicillin is the same cillin could be removed by washing, so that peni-as that impenetrable by phosphate [about 5 cillin was assumed to penetrate K12 and to beml/g dry wt for the Staphylococcus aureus inactivated intracellularly by that organism (15),(Micrococcus pyogenes var. aureus)] used by and it seemed likely that mammalian cells wereCooper (18), then the reversible binding of permeable to penicillin (32). Nevertheless, thesepenicillin by this organism cannot be more than data do not permit a strict decision as to whether0.5 u/g at 0.1 u/ml and is directly proportional penetration or reversible adsorption accounts forto concentration. This, like the irreversible the penicillin which can be eluted.binding, would be small enough to render inocu-lum size of no effect on penicillin activity. Also, III. THE CHEMICNIPROPERTIES OF THE PENICILLIN-as pointed out by Maass and Johnson (5), some BINDING COMPONENTevidence of saturation might be expected at The most likely interpretation of the observa-10,000 u/ml, although it is not necessarily true to tions that certain bacteria apparently bindsay that the amount of penicillin which can be penicillin specifically is that these cells containadsorbed outside the osmotic barrier must be a limited amount of a substance, 'PBC', whichlimited to a single monomolecular layer, as the should be definable in chemical terms. It haspart of the cell outside the osmotic barrier may been already noted that the experimental evi-have considerable depth (26, 29) with many dence is in favor of this being the site of actionpotential reversible penicillin binding sites. It is of penicillin. Therefore, it should greatly help usinteresting that this depth [about 20 mjs for to understand the mode of action of penicillin ifS. aureus, 261 is the same as the thickness of the we knew the chemical nature of PBC; an essen-cell wall liberated on mechanical rupture (30), tial stage in its purification must be its isolationand may be identifiable with the space occupied from the cell in a soluble form. There are likely toby the cell wall (29). be difficulties in its purification, as it may be a

The question of permeability is therefore still large molecule rather than an easily removableopen, but it would seem likely that the intact cell cofactor, thus involving removal of a very highdoes bind a little penicillin reversibly but is not proportion of chemically similar impurities, orreadily permeable to penicillin. If so, PBC either it may be the result of the conjunction of tworesides at the external interface of the osmotic large molecules between which the penicillinbarrier or within that small metabolic zone which happens to fit. Any attempt to separate the twomay exist outside the osmotic barrier but within large molecules would then naturally lead to thethe confines of the cell wall (29). disappearance of PBC. Nevertheless, severalOne must ask if such reversible binding, rather cell-free preparations have been obtained by

than PBC, causes the lethal effect. This is difficult mechanical cell rupture which can bind peni-to investigate experimentally but, as has been cillin in a similar manner to intact cells, and canalready seen in Section I, many properties of PBC thus be presumed to contain PBC (15, 16, 21,fit in well with the observed effects of penicillin. 23, 31).The reversible binding, if it exists, could show no Little success has been obtained in character-marked change at a particular concentration such izing this component, and nothing is known withas the minimal inhibitory titer. Other obvious certainty of its chemical nature. Daniel andrelations between reversible binding and lethal Johnson (16) find that the 'specific' binding isaction of penicillin have not been sought. It is not destroyed by trypsin and Cooper (19) findsinteresting that Saccharomyces cerevisiae cells that it is not destroyed by trypsin, lecithinase, orwere impermeable to penicillin and did not bind ribonuclease. Cooper (23) has shown that, withthe drug reversibly or irreversibly (5), and the particular staphylococcus he used, the PBCBaciUus cereus spores appear to be impermeable is concentrated in a small particulate fraction


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which contains about 10 per cent phospholipid Iv. TE NATURE OF THE REACTION BETWEEN THEand 10 per cent other lipid, the remainder (pro- PENICILLIN MOLECULE AND PBCtein plus a polyglycerophosphate complex orPGP) being material very similar to the cell wall ph ocl, by whi PBCns bound bpenicil isin composition (39). This PBC-lpid-protein- . . .POP complex was not fractionated by several important for the design of other antibacterial

mild techniques, and both the lipid and PRO agents that may function similarly to penicillinand yet may pnetrate the cell more easily or beappear to be strongly bound, producing a close y y pe y

correlation between lipid P, PRO and penicillin unharmed by penidillinase. Also such knowledge35 which had been attached before rupture. should contribute information on the constitution

Solvent treatments which had no lipid-phosphate of the penicillin binding component and on theextracting effect did not remove PBC, but PBC penicillin mode of action.was not found in any fraction after extraction The curve relating penicillin uptake by sensi-procedures which were sufficiently drastic to tive bacteria to concentration in the suspendingremove phospholipid-so that PBC appeared fluid (figure 1) is that to be expected from ato be destroyed by these procedures. However, rapid reaction irreversibly saturating a com-cells grown in radiopenicillin, washed, and ponent present in small amount. The binding oftreated with 90 per cent phenol lost their lipid penicillin has been mentioned already to be verybut not their S36, suggesting that PBC may be firm indeed, as penicillin S35 cannot be removed,more closely associated with the protein-PGP once attached, by fresh penicillin, copious wash-moiety than is the lipid. Eagle (15) found ing or treatment with anionic, cationic orpenicillin binding by the large-molecular material neutral detergents (5, 8, 15, 20). Pretreatmentsedimenting at 144,000 X G but binding was to with detergents does not affect binding (exceptthe same extent as intact cells, showing no con- in the special case of phenol), but binding iscentration of PBC in terms of dry weight. This completely prevented by small amounts ofmaterial had previously been centrifuged at crystalline penicillin added beforehand. Thus, as27,000 X G, when the cell walls and the PBC- penicillin is also only feebly surface active (28),lipid-containing particles would have been re- binding does not appear to be by adsorption at amoved, leaving only the soluble portion of the surface in similar fashion to detergents. Anlipid particles with a large amount of soluble alternative mechanism would be by van derprotein. Considerable 'nonspecific' binding was Waals' attractive forces similar to those pre-suggested by the rate of increase of uptake with sumed to bind antibody to antigen or haptene,concentration. but most antigen-antibody combinations have anPBC appeared to be rather unstable in the appreciable degree of reversibility.

intact cell or in the cell-free state, being rapidly It is perhaps more likely with a reactivedestroyed by acid and moderate heat (16, 19). molecule such as penicillin that the PBC-peni-Even at the optimum pH and 2 C, 25 to 50 per cillin complex is formed by chemical reaction.cent was lost overnight. It can be seen that this If so, unlike a physical type of binding, thecharacter of instability, when coupled with the penicillin once fixed has changed chemically andvery low solubility of the only soluble preparation is no longer penicillin. The correlation betweenobtained (the lipid-containing particles), renders amounts bound of 5" and 014 labeled in the po-further chemical purification difficult, and a more sition indicated below shows, however, that theefficient and stable solvent system would seem to molecule, even if altered, is not split betweenbe a prime requirement. the S and C14 atoms (15).

S Me

R-C140-NH-CH-CH C

MeCO-N ~CH-COONa


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A clue to the nature of the binding reaction may plex extracted the S16 as penicilloic acid (35a), abe gained from a consideration of the parts of result to be expected if penicillin is bound intactthe penicillin molecule which can be altered or or by acylation. Penicilloic acid itself is onlydispensed with without gross loss of activity. slightly bound (11), so that one would not expectThis shows that: (a) The nature of the R radical the bound S16 to be in this state before alkaliis in general unimportant, as many different treatment.substituents can be used without loss of activity. It may be argued that if the fl-lactam ring isThe a-amino residue in cephalosporin N is an the functional group, then the rest of the mole-exception to this (33). (b) The carboxyl group cule should not be necessary, but a reactiveand thus the negative charge on the molecule are molecule such as penicillin would probably neednot vital, as penicillin amide (34) is at least 15 some means of specifically limiting its reactivity,per cent as active as penicillin against sensitive such as by steric hindrance, so that at highspecies. This has been confirmed in our labora- dilution the penicillin is not wasted by acylationtory. As the activity is more resistant to peni- of sites that are not essential for the cell. Anothercillinase than penicillin itself, the active form of function of the thiazolidine ring is likely to be tothe amide is not free penicillin, as may be the activate the f-lactam system, as other substancescase with the diethylaminoethyl ester (35). (c) containing the P-lactam are much less reactiveAny penicillin derivative not possessing both the chemically as well as biologically. It is possibleB-lactam or thiazolidine rings intact are bio- too that PBC is a substance which is extremelylogically inactive. This supports the findings (16) easy to acylate, perhaps undergoing reversiblethat several penicillin derivatives (penicilloic, acylation in the bacterium, and penicillin repre-penilloic, penillic, penicillenic and penillonic sents a compound of intermediate acylatingacids, desthiopenicillin, cysteinepenicillin and ability, a compound that is without the wastefulpenicillamine which all lack one or other of these reactivity of acetyl chloride, but is more reactivestructures) do not prevent penicillin uptake by than the j3-lactam ring alone. A search for athemselves binding PBC. Thus the important similarly intermediate type of acylating agentpart of the molecule appears to the central part, that is simpler chemically than penicillin wouldand it is probably significant that practically all represent a new approach to a synthetic peni-the reactions rapidly undergone by penicillin at cillin and may reveal a substance which is37 C and neutral pH are acylations performed by cheaper, less susceptible to penicillinase or morethe splitting of the P-lactam bond, e.g.: capable of penetrating the resistant bacterial cell

R-CO.N*CH-Csf%"~ R- H-% c~s but still able to bind PBC fairly specifically.If penicillin is in fact bound by acylation, then

CO-N-CH*COON@ co N OO the likely receptor groups are -OH, -SH or4. (SEt -NH2. The facts that penicillin reacts preferen-

tially with the -NH2 group of cysteine rather+ than the -SH (36) and that pretreatment of the

of HO-H COOH cell with formalin has no effect on penicillinbinding (21) do not necessarily rule out -SH or

+ -NH2 as receptors. Daniel and Johnson (16)or H&NCHCOOH C04HjCOOH have reported the interesting finding that their

ASH CHISH PBC may have to be in an oxidized state to

As the thiazolidine ring appears to be rather bind penicillin. Perhaps the 50 per cent of theunreactive chemically, the implication of these total PBC which is not available to penicillin infindings is that penicillin acylates its receptor intact resting cells (23) is in the reduced formPBC by a reaction which splits the f-lactam rather than sheltered within the cell, and thering. This is supported by the finding that pre- increase of bound penicillin during growth (18) istreatment of the cell in strongly buffered suspen- due to the progressive oxidation of this group insion with a small amount of acetic anhydride or the presence of penicillin as part of a routineacetyl chloride, but not acetic acid, completely oxidation-reduction cycle. Thus the increase inprevented penicillin uptake (8). Also a mild available PBC in cell-free preparations on stand-alkaline treatment of the radiopenicillin-cell com- ing (19) may be due to its oxidation in air.


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19561 SITE OF ACTION OF RADIOPENICILLIN 35

The finding that penicillin is more active at initial effect of penicillin. This may be because thepH values slightly lower than 7 has led to the cell requires the product of this reaction, orsuggestion (37) that penicillin competed with the because the precursor to the product accumulateshydroxyl ion for its site of action. That this is not and is toxic (41).the case is shown by the irreversible nature of thepenicillin binding, the activity of penicillin Locatin in tie Osmotic Barrier as a Possibilityamide (34), and the lack of effect of pH on the A clue to the location in the cell of the peni-amount of penicillin finally bound (19), although cillin site of action may be the presence of PBCthe rate at which penicillin was bound was in the particulate lipo-protein fraction liberatedsufficiently increased at a lower pH to account on cell rupture (23). Of the soluble material sofor a proportion of the increase in activity. liberated in distilled water, only this fractionThe rather small effect of pH on rate of with little other dry weight was retained in the

penicillin uptake suggests that penicillin may cell wall fraction when formalin was added to thereact with the cell in the ionized (lipid insoluble) medium before rupture began. This observationform, an idea which is not supported, however, by suggests its proximity to the cell wall in thethe findings that the penicillins, [e.g., most intact cell. Mitchell and Moyle (39) believedazopenicillins (38), the natural penicillins and from chemical assays that it may represent thecephalosporin N (33)] are active roughly in the lipid layer observed cytologically under the cellsame order as their lipid solubility. Perhaps wall. Also, penicillin uptake was prevented in alipid solubility assists penetration into the cell. manner which was closely parallel with osmoticIt is interesting to calculate in this respect that, barrier destruction by pretreating the cell or theat pH 6.7 and 0.02 u/ml, the volume occupied lipid particle fraction with dilute solutions of theby each cell would normally contain 25 penicillin neutral lipid-soluble detergent, phenol, whereasmolecules, but only 1 in 25,000 of these would be lipid insoluble detergents had no effect on peni-un-ionized. The reacting molecules have to find cillin uptake (20).positions at or near the cell's surface, but covering This association between PBC and cell wallonly 10 of that surface. It is evident that, even lipo-protein suggested that penicillin may ini-for the ionized molecule, the chances of effective tially involve some function of the osmoticcontact are small. At pH 6.7 and 0.02 u/ml the barrier, generally believed to have lipoidal prop-penicillin binding rate is of the order of 100 erties and necessarily lying close to the cellmolecules/cell/hr (19). surface. As the osmotic barrier controls many

functions, some generalized damage to it couldv.T

ENATCRELOF TIE PROCESSFIRSTEBAMAEDA account for the many changes induced by peni-BY PENICILLIN IN SENSITIVE BA(CERIA cillin.

If PBC is the site of action of penicillin, then it It is curious that, if one excepts cells that areis probable from the small amounts in bacteria resistant because of penicillinase production and(estimations of the molecules of penicillin bound/ the penicillin-impermeable yeasts (see p. 32),cell vary from 80 to 1600) (5, 13, 15, 18), that there is nearly always a close correlation betweenPBC is part of some catalyst, whether enzymic ability to retain the basic dye in the gram stainingor not, whose activity is changed or halted by the procedure and penicillin sensitivity. There is alsobinding of penicillin. The amounts of penicillin a correlation between gram positivity and, (a)bound are similar to the amounts in bacteria of content of a polyglycerophosphate ester (45a,vitamins (40) such as pyridoxin (2100 to 6600 49) which is bound partly to cell wall protein andmolecules/cell) and folic acid (180 to 1200/cell). partly to the underlying lipo-protein membraneA possible alternative is that the PBC-penicillin (39) and (b) lipid P content. This correlation ledcomplex is itself the toxic substance and blocks Mitchell and Moyle (45a) to suggest that a cell-some other process, but this isnot considered here, wall lipid-polyglycerophosphate-protein com-as too little is known of this complex to help ponent (lipid particle fraction?) may be a com-us study a second unknown. For the purpose of mon structural feature of all gram positivediscussion, then, it is assumed that PBC plays an bacteria. There is also a difference in the osmoticactive and vital part in the cell's economy, and character of gram positive and gram negativedamage to the reaction governed by PBC is the cells which presents itself as a greater rate of low


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of small molecular components in distilled water than liberated by cell lysis (42), the final stagessuspensions of the gram negatives (45a), but there of penicillinase synthesis should proceed at theis no correlation with nucleic acid content. It is outer rather than the inner surface of thepossible but not yet well demonstrated that the osmotic barrier, as otherwise one has thedifference in osmotic character may also include paradox of an osmotic barrier which is per-active transport mechanisms for certain solutes meable to proteins but not to amino acids.

a26v44te Thus the close connection of PBC with peni-(26, 4.h cillinase synthesis in Bacillu8 cereus (see sec-These observations have suggested (45a) two tion VIa) suggests that PBC also functions at

co-existing mechanisms for gram positivity: (a) the external surface. On the other hand, Pol-an ionic mechanism predominating in gram lock (31) suggested that penicillinase synthesisstaining without a mordant, which would explain was induced inside the cell as PBC was in-the differences in pH at which the basic dye is cluded in spores formed within the cell wall.eluted from positives and negatives ("iso-electric However, PBC appears to reside in the lipidpoints"), (b) a mechanism superimposed by use material near to but easily detached from theof a mordant which appears to retain more cell wall (23) and this probably is included inofya mrant which aea so ,. the spore. Thus PBC could be beneath thedyethan the ionic mechanisms, so that differ- structural cell wall yet at the same time atentiation is sharper, and which is somehow the external osmotic surface of the cell, as thisinvolved with the cell wall. This mechanism may is deep to the cell wall surface (26).be a greater retention by the cell wall of grampositives of the poorly soluble dye-iodine com- Conideration of Early Effects of Penicillinplex, as ruptured cell preparations retain very Cooper (18) examined the effect of penicillinlittle dye. In considering the coincidental correla- on certain osmotic barrier functions. His resultstions mentioned, the reviewer finds it difficult to suggest that there may be in fact a serious dis-escape the belief that the staining difference turbance of active osmotic properties, as at leastnoticed by Gram happens to reflect a fundamen- one and perhaps all of the transport mechanismstal difference in cellular physiology related to the had abruptly ceased to function before manycell wall which includes the normal function of other of the principal activities of the cell, suchPBC. Some effects of penicillin on cell wall as glucose oxidation and fermentation, protein oractivities are discussed in reference 23, as well as peptide and nucleic acid synthesis. The cessationelsewhere in this review. of glutamate assimilation (43) at a similar timeHowever, although this concept provided would also lend support to this idea, but it is not

some guide for further research, the multi-func- at present as likely as it had seemed earlier thattional nature of the structure called 'the osmotic glutamic acid assimilation is an active function ofbarrier' itself means that there are many possible the osmotic barrier (44). The accumulation ofcharacters which could be inactivated by peni- acid-soluble nucleotides noticed by earliercillin. Such possibilities include: workers (45, 46, 47) was suggested (18) to be due

to a secondary acid-lability in the RNA, per-a. Synthesis of its own material, perhaps aused by osmotic barrier malfunctionincluding lipid phosphates, and of the over- hapscIlaying cell wall. Its intact condition is neces- rather than to a direct interference with RNAsary to retain those solutes already present in synthesis, as extraction with dilute phenol inthe cell. place of trichloroacetic acid revealed no accumu-

b. Transport into the cell of metabolites lation of nucleotides. Similar nucleotides accumu-which cannot penetrate freely. late in resting cultures and cultures treated with

c. Metabolism at the outer surface of the other antibiotics (47a) which also suggests thatcell of substances which do not penetrate. this may be simply a manifestation of unbalanced

d. Since the osmotic barrier must also have growth.an inner surface, this structure may have Unfortunately, none of these disturbancesmetabolic activities inside the cell which may occurred sufficiently soon to be incriminated asnot be recognized as functions of an osmotic

t .obarrier. It would therefore be interesting to the ste of action of penicillin. The literature

know whether or not the cell is permeable to reports many metabolic defects which have beenpenicillin (see section II). An argument against suggested, with varying degrees of caution, asthis is that as penicillinase is secreted rather the site of action of penicillin. However, since


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the ultimate death of the cell will stop very many / cell notreactions, it is important to place the defects in (.1) valaebie to penicillinsome sequence, in which, by definition, the site Sl te to)of action is the first. Not many early defects of Externalpenicillin have been noticed, but of these the mediumaccumulation of ribonucleotides (45), drop in (surface charge (48), and drop in cell-wall poly- (D 0000glycerophosphate (49) all appeared to start at PBConce. If these are not all related, then in this ®organism and under these conditions the se- ©quence of defects is very rapid indeed. Thedifferent strains and conditions used by Cooper Normal Untreated Cell(18) permitted some separation of metabolicdisorders induced within an hour by penicillin,iparticularly uptake of P and metals, but none ofthese occurred at once. The experiments usedpenicillin at 0.1 u/ml, the lowest concentrationin which saturation of PBC is complete within 2/min, the bactericidal action is most rapid (17), Ilia

and production of fresh viable units is stopped Vimmediately (22), so that the initial lesion should orbe evident practically at once.

Thus, although, (a) a correlation exists between W i) e

penicillin sensitivity and some cell wall properties Penicillin Treated Cellrelated to gram positivity, (b) PBC resides in or Figure S. Hypothesis to explain the increaseclose to the osmotic barrier, and (c) many of the of penicillin uptake on growth in presence of theearliest changes appear connected with active drug. This diagram is, of course, somewhat mech-cell wall functions, yet there is a time lag before anistic, and the disappearance of PBC into thethe earliest change appears, so that the location part of cell not available to penicillin may be aof the initial lesion is still not known. The intact change of chemical rather than physical state.nature of the osmotic barrier was not affected occurred as part of a routine cycle while PBC wasuntil considerably after these changes, nor was metabolizing its normal substrate S (figure 2),synthesis of lipid phosphate or the 'lipid particle' and as soon as PBC was released from its dutiesfraction. with S it was immediately bound by the peni-Increase of Penicillin Uptake when Growth Occur8 cillin present. Thus the rate of penicillin binding

above that due to total synthesis of PBC ("re-Rowley and his colleagues (8, 18, 21) and serve" + "free") may be a measure of the rateMaass and Johnson (6), but not Eagle (15), found of turnover of PBC. It was calculated on thisthat the penicillin bound progressively increased basis that PBC metabolizes 3000 molecules of Swhen growth occurred in the presence of peni- for every cell produced, or about 10 moleculescillin to a value about double that of resting per hour per molecule of penicillin bound. Suchcells. This was shown (18) to be due to the ex- a turnover rate is rather small compared withposure of a reserve of PBC equal in amount to the many enzymes but is of similar order to certain'free' PBC which was not normally available to trace reactions such as synthesis of pantothenatepenicillin (23), rather than to an increased rate of in Pseudomonas aerugtnosa (40). The figure ofsynthesis of PBC. It could not be due to an 3000 molecules/cell could probably be up to 10increased rate of reaction between penicillin and times larger, but even so a very specific analyticalPBC per se, as this was already very fast (com- method would be needed to detect the quantita-plete in 2 to 3 min) compared with rate of binding tive change brought about by addition of peni-observed (complete in 30 min). Thus the rate cillin.Thus it should be of little avail, for example,of PBC exposure was the rate-limiting step. It was to look for a change in over-all rate of nucleicsuggested that this exposure of reserve PBC acid synthesis.


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Reactions that Are Not the Function of PBC the cell surface, and which is completely andThe very small "turnover rate" of PBC, the very rapidly stopped by very low concentration

effectiveness of penicillin in the absence of air of penicillin.or all organic matter other than glucose, and thelikelihood that, above 0.1 u/ml, the use of PBC Th EITICFL TIME ABLETOF O Lis completely stopped very soon after the peni- EVENTS FOILOWING ADION OFcillin is added, were suggested (18) as indications PENICILLINof what are probably not the functions of PBC, Penicillin causes many defects in the cell'snamely: (a) gross assimilation of 02, Ca++, Na+, metabolism, any one of which-for example,M&+ K+, P04-, Fe+-++, or Co++, organic blockage in phosphate or glutamate uptake,substances or other substances contributing to the shortage of Mg, Nat or KE, damage to RNAdry weight; and (b) synthesis of the major part and protein synthesis, inactivation of free PBCof lipid phosphates and the lipid particle fraction, or inability to synthesize more PBC-is suffi-and cell wall polyglycerophosphate, proteins, ciently serious to merit the title "cause of death."nucleic acids or intermediates. However, PBC Thus it is of little value to nominate as the causemay assimilate or synthesize a small fraction of of death any one stage of the chain of defectsthese substances, or assimilate a trace substance induced by penicillin except perhaps the first, andrequired in amounts similar to that of cobalt it is more desirable to attempt the considerable(about 104 atoms/cell). Alternatively, the sug- task of enumerating the defects in the order ofgestion that PBC may be capable of reversible their occurrence. This is clearly impossible to dooxidation-reduction mentioned in section IV in detail, but as has been already pointed out,may mean that PBC is concerned with the some such scheme is necessary to describe fullyenergy source of a very small reaction, but this is the "mode of action" of any antibiotic.presumably not at the stage of oxygen utilization. The various properties of the penicillin-bindingIt may be that the suggested simultaneous failure component which have been outlined above,of a number of active functions of the osmoticbarrersdutotheinital readownof ome together with observations in the literature on thebarrierisdue to the initial.breakdownofse effects of penicillin on bacterial growth, suggest acommon factor such as an energy supply localized e ets

in the osmotic barrier. Further progress is sequence for the events which are induced byhindered by the following obstacles: (a) isolation penicillin in those fully sensitive cells inhibited byand chemical definition of PBC and demonstra- less than 0.1 u/ml, and it may be helpful totion of some catalytic activity to the exclusion of summarize this inferred sequence as a timetableothers, and (b) demonstration of a very small starting with addition of penicillin to a growingcatalytic activity, requiring a very sensitive and culture. Inferences followed by a query haveselective assay method, which probably occurs at little or no direct supporting evidence.

Inferred Event Supporting Evidence

Time: O0- minutes

a. Above 0.1 u/ml and below those concentrations a. Examination of rate of penicillin binding atwhere secondary effects may be expected, peni- different penicillin concentrations.cillin reacts with and firmly fixes all free PBCat once (displacing its normal substrate S?).Metabolism of S ceases at once when all freePBC is fixed?

b. At the minimal inhibitory level (e.g., 0.02 u/ml) b. Firmness of binding penicillin (5, 6, 8) elimi-the lower rate of inactivation of PBC is just nates dissociation of PBC-penicillin complexequal to its rate of resynthesis. When the rate as cause of minimal inhibitory level. Rates ofof inactivation of PBC exceeds the rate of syn- binding and of growth are comparable atthesis, the rate of utilization of S declines? minimal inhibitory level; calculated equiva-

lence of penicillin binding at 0.02 u/ml andPBC synthesis (see paragraph b on p 30).

c. Production of fresh viable units as measured by c. Equivalence of lowest concentration (0.1 u/ml)


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viable counts ceases when all free PBC fixed where all PBC is fixed within 2 min (6, 19)(i.e., when metabolism of S also ceases?). and lowest concentration where viable unit

production is stopped at once (18, 22).d. Other metabolism is unaffected for the time d. Numerous references discussed in section V.

being.

Time: 8-30 minutes

a. This is a period of bacteriostasis. a. No deaths occur if penicillin is removed in thistime (50).

b. Above 0.1 u/ml, additional PBC is exposed and b. Doubling of bound penicillin in 30 min if drugis fixed at once by penicillin (on being released present continuously during growth, coupledfrom its duties with S?). with loss of "reserve" internal PBC (18).

c. If penicillin is removed before the last of the re- c. No lag in resumption of growth if penicillinserve PBC is freed (i.e. before 30 min) (18), then removed within 30 min (22).this residual PBC enables growth to begin againat once.

d. Less than the optimum amount of PBC is then d. Rate of resumed growth is slower (22).immediately available to the cell.

Time: 30-60 minutes

a. Bactericidal action commences. Removal of a. Viable counts begin to decrease in exponentiallast of reserve PBC allows cells to die in a ran- curve if penicillin still present at this time (50).dom fashion.

b. Helvolic acid prevents exposure of reserve PBC, b. Irreversible damage to the cells is completelyor prevents consumption of nutrients. prevented if growth is stopped within 5 min by

helvolic acid, but after 30 min no protectionis afforded (51).

c. Although PBC is still synthesized and exposed c. Penicillin bound per unit volume of culture stillthe penicillin still present immobilizes it before increases, but the rate steadily diminishes (18).it can be used (thus depriving the cell of theessential substrate S which is ultimately neces-sary for PBC synthesis?).

d. The absence of S causes loss of ability to assimi- d. The cell ceases to accumulate gross dry weight,late essential nutrilites (?) and perhaps other Na+, Mg++, K+, phosphate (18) and glutamatedamage (?) and this, in the consumption of pre- (43) during this period, although other metab-existing nutrilites by the continued metabolism has continued.olism of the cell, wastes the cell's ability to syn-thesize fresh PBC; therefore death is due tocontinued metabolism in the absence of usablePBC.

e. If penicillin is removed in this period (as ready- e. After 30-min contact, there is a lag beforemade PBC and S stockpile increasingly de- growth commences which is longer the longerpleted?) an interval is required to synthesize the time spent in contact with penicillin (22).enough PBC to build up enough S.

f. By definition of PBC as essential to the cell, f. Removal of penicillin at any stage preventswhen PBC synthesis ceases the cell is nonviable. further cells dying (22).A very small residual rate of PBC synthesiswould suffice to rescue the survivors.

g. The osmotic barrier shows some damage to g. Uptake of Nat, Mg I, K+, phosphate and dryits active functions while many other metabolic weight (18) and glutamate (43) cease, cell wallactivities continue. material (49) and cell protein (52) are no longer

synthesized, although nucleic acid and peptidesyntheses continue. Total cobalt and ironuptake and total lipid P synthesis are un-affected (18).


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Time: 60-75minutes;Nucleic acid and peptide syntheses slow. Many (18, 51, 52)

reactions can be expected to be affected by short-age of metals, phosphate and glutamate andperhaps other nutrilites.

Time: 75 minutesThe osmotic barrier begins to lose its osmotic The cell swells and loses solutes, and large mo-

properties and the dissolution of the cell com- lecular weight phosphate decreases (18).mences.

On this hypothesis there appear to be two that many other defects commence at levelsreasons for penicillin requiring active growth to 100 to 1000 times the minimal concentrationbe bactericidal: (a) the reserve PBC has to be (see section VII).removed by continued exposure to penicillinduring growth, and (b) the cell must also lose its VII. THE REASONS FOR PENICILLIN RESISTANCEability to resynthesize the PBC by continued Demerec (53) has shown that the training ofmetabolic activity in a deficiency of many sub- organisms to penicillin resistance results in astances either by exhaustion of an essential series of small increases of resistance rather thancomponent (e.g., metals or phosphate) or by one large one, and when sensitive pneumococciaccumulation of a toxic product. When this are transformed by deoxyribonucleic acid fromhappens, the cell cannot again obtain PBC, and resistant pneumococci a degree of resistance issince by definition PBC is essential, it can from acquired which is intermediate between that ofthis moment be regarded as nonviable. Growing the sensitive and resistant organisms employedin a deficient medium without penicillin would (54). Strains which have changed their penicillinnot be expected to be bactericidal, as the reserve resistance by a mechanism which is very likelyPBC would not be affected, and would be present to be but a one-step mutation have neverthelessto rescue the cell when transferred to a full changed simultaneously in other characters,medium. particularly growth requirements (55, 56).

It will be recognized that this approximate These findings can be interpreted most simplytime-table is very incomplete and contains many as indicating that pencillin resistance is controlledpresumptions, and that the experimental findings by several genic factors. If penicillin sensitivitydo not necessarily exclude alternative hypoth- is caused by possession of a vital component,eses. Nevertheless this sequence is suggested PBC, which the penicillin molecule must reachas one of the simplest explanations of most of the and react with, it can be shown that an organismknown data. Some qualifications are necessary, may be resistant to penicillin in many wayshowever, in that most experiments have to be which are not mutually exclusive. Some of theseperformed on very large populations. It is not ways may each vary quantitatively giving manyknown, for example, whether 50 per cent of the possible degrees of penicillin resistance, and evenresting cells take up 1.8u/g of penicillin and 50 per individual cells may differ in their penicillincent none (so that the doubling of uptake on sensitivity (57). It is therefore clear that thegrowth represents exposure of PBC in half the mechanism of resistance of any particular straincells only), or whether all take up 0.9 u/g, al- may be complex, and should be consideredthough this may not matter for the present individually on its merits.argument. It is possible also that the sequence of The information provided by a study of theevents differs somewhat in different organisms penicillin binding component on the differentand events may follow each other more rapidly causes of penicillin resistance is discussed in thein some, as the onset of swelling occurred at once following section.with one organism (45) but not for an hourwith another (18). It seems likely, though, that Possess of the Means to Destroy Peniiinthe initial site of action is very similar in allsen- This means is thought of as a type of enzymesitive species provided that the penicillin is collectively termed penicillinase, which in one

only acting at minimal levels. It has been shown extracellular state has been shown to hydrolyse


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penicillin to penicilloic acid (58). It occurs both inhibitory concentration (1.0 u/ml with lowextracellularly and intracellularly in many types inocula where little penicillinase is produced).of organisms, and it is possible that the various Thus, the specific PBC of the B. cereus isforms are not chemically identical and form identical, as far as we can see, with that of S.different degradation products (59). As it is easy aureus, with the exception of its connection withto see how an otherwise sensitive organism is penicillinase adaptation. On the basis of thenevertheless not killed if the drug can be elim- probability also discussed in section I that PBCinated faster than its site of action is inactivated, in S. aureus is the site of action of penicillin, itit is not proposed to discuss this mechanism seems likely that the PBC of the sensitive B.further. cereus also performs the same function because ofThere is, however, an extremely interesting this similarity. One is led to wonder how it is

connection observed by Pollock between the that inactivation of this component leads in onepenicillin-binding component and the means by case only to death of the cell but in the otherwhich the cell can increase its ability to destroy also provokes a vigorous protective reaction.penicillin. This connection is important with As Pollock (61) has pointed out, it is easy toregard to penicillin's mode of action as well as to speculate on the mechanism of penicillinasethe more general topic of protein synthesis. adaptation. One possibility, the simplicity ofPenicillinase synthesis can be strongly stimulated which merits consideration, however, is anin Bacillus cereus by contacting the cell with analogy between penicillinase and diphtheriapenicillin at 0 C and removing the excess by toxin. In Corynebacterium diphtheriae the contin-thorough washing before incubation (42). This ued metabolism of the cell in a deficiency of ironstimulation is likely to be caused by some results in an accumulation of a substance whichpenicillin product which is still attached to the happens to be toxic to animals and which hascell when incubation begins, as there has been been suggested to be bound normally in thelittle chance for irreversible damage such as iron-sufficient cell in the form of an iron-por-disturbance of RNA synthesis [which should phyrin complex (62). In B. cereus the analogueaffect protein synthesis (60)] during the period of iron would be the product S of the normalat 0 C. It is possible that some penicillin molecules function of PBC, and the analogue of diphtheriawhich happen to be reversibly bound cause some toxin would be the penicillinase-precursor (ratherdamage which remains after the reversibly bound than penicillinase itself for reasons to be discussedpenicillin has been washed away, but stimulation in the next paragraph but one). The continuedof penicillinase synthesis was marked at 0.0008 metabolism of a B. cereus culture in the absenceu/ml, at which concentration reversibly bound of a given amount Y of PBC per ml of culturepenicillin should be very small (if Staphylococcus (the result of binding a given amount Y ofaureus and Bacillus cereus are comparable, penicillin) should result in the culture becomingreversible binding should be less than two relatively deficient in S at a constant rate kYmolecules/cell). proportional to the amount of PBC inactivated.The stimulation appears to be due to the There is evidence that inactivation of some PBC

penicillin irreversibly bound as there is a close does not result in a stimulation of its rate ofcorrelation between the rate of penicillinase synthesis (18). Thus, if the rate of PBC functionproduction and the amount of penicillin irrever- is normally geared to rate of production of somesibly and "specifically" bound, both being max- other substance (for example, so that S can com-imal at and above 1.0 u/ml (13). It is possible bine with it), this substance will be produced inthat the "nonspecifically" bound S35 from the excess at the same rate kY. This deficiency willpenicillin preparation is responsible, but there become less important as it is diluted out by cellwas no correlation between the amount of this growth.and enzyme synthesis (the question of whether The rate of penicillinase production per ml ofthe "nonspecific" S35 binding does represent culture noted after "priming" with penicillin atpenicillin is discussed in section I). Like other 0 C remained constant during growth and wassensitive organisms, the concentration at which proportional to the PBC fixed (13), so that thethe pencillin irreversibly and specifically bound rate/g DW dropped by dilution with cell matter.is maximal corresponds also with the minimal This is in contrast to the exponential rate of


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increase of many other adaptive enzymes. The is less than before anaerobiosis. This scheme mayPBC-radiopenicillin complex remained firmly also help us to understand why PBC and penicil-bound to cell material throughout several linase, apparently unrelated substances, shouldgenerations (6, 13), so that the amount of S35/g both react with penicillin. Either S or its pre-DW also dropped only by dilution with cell cursor (presumably sharing with penicillin somematter. property such as acylating activity) can reactThese facts prompted the suggestion that the with both PBC and penicillinase precursor,

PBC/penicillin complex was actually the catalyst which is in turn likely to be very similar toconcerned, which implies that PBC still retains penicillinase.some enzymic activity after binding with Whether or not one of these schemes is thepenicillin. The analogy with diphtheria toxin is correct one, it would appear that a study of thesimply the other alternative that binding with properties and normal functions of penicillinasepenicillin inactivates PBC completely, and ap- and its precursors may well throw light on thepears simpler and is equally supported by the initial reaction disorganized by penicillin. Itexperimental evidence. Such an alternative would be interesting to see if the correlationimplies that penicillinase is the substance which between rate of penicillinase synthesis andis normally united with the product of PBC, but amount of PBC bound extended even to thethis is not supported by the latent phase no- 'reserve' of PBC such as was found in S. aureusticed between addition of penicillin and increase of and which was only exposed during growth in therate of penicillinase synthesis (61). The latent presence of penicillin (18). This might be achievedphase, together with the "rebound" [transient by adding penicillin continuously during growthincrease in rate of synthesis (63)] and a shortening at a rate greater than its destruction by peni-of the latent phase, both observed after a short cillinase. Also the effect on penicillinase synthesisperiod of anaerobiosis, has been satisfactorily of inactivation of free PBC by other means,accounted for by postulating the accumulation of such as reaction with buffered acetyl chloridea precursor whose synthesis was not affected by (8), should permit a decision between the twoanaerobiosis, while the synthesis of penicillinase alternatives discussed above, i.e., as to whetheritself was stopped abruptly by the absence of simple inactivation of PBC or the presence of aoxygen. penicillin "template" is necessary for penicillinaseThe function of PBC is unaffected by anaero- stimulation. It was mentioned in section IV that

biosis in S. aureus (18), and one may therefore the action likely to be undergone by penicillinsuggest the following scheme for B. cereus: in binding PBC would change its chemical

facultativelyanaerobic obligatory

Earlier precursors penicillinase - penicillinaseprecursor aerobic (secreted)

facultativelyanaerobic

S precursor S S-(penicillinase-precursor) complex, a normalPBC product retained by cell.

That penicillinase or its precursor plays a part in constitution so that it was no longer penicillin,normal cell function is suggested by its occurrence but this does not necessarily mean that itsin cultures grown in the absence of penicillin molecular shape was so changed that it was(59). incapable of acting as a template for penicillinaseIn the relative rates of production of P1C, S synthesis.

or penicillinase-precursor under anaerobic condi-tions or in the exposure of "reserve" PBC may DiBfferecesin Rate or Amount of Penicillinlie a clue to the phenomenon of "reversion," Bingwhere, if anaerobiosis is applied much before 40 An examination of the penicillin uptake overmin after addition of penicillin, the rate of a relatively short time by resistant organismssynthesis of penicillinase on repassage of oxygen which did not produce penicillinase and at


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19561 SITE OF ACTION OF RADIOPENICILLIN 43

concentrations well below their minimal inhibi- absolute amounts of binding does not appear totory level (8) revealed negligible binding of have been examined experimentally for resistantpenicillin. The results of Maass and Johnson (5) strains by any of the authors quoted, but it iswere inconclusive with resistant organisms, some interesting that the data of Reynolds et al.binding more penicillin than sensitive forms and (64) and Pollock and Perret (13) show that thesome less. However, by increasing the penicillin rates of penicillin uptake by two organisms thatconcentration to equally effective (LDg9.9) levels, are relatively more resistant (StreptococcusEagle (15) found that the amount bound by faecalis, 8 u/ml, Bacillus cereus, 1 u/ml) arenaturally occurring resistant strains was rela- markedly slower at any given concentration thantively constant despite wide variations in their the rate of uptake on a sensitive Staphylococcussensitivities. Thus it appears that in these cases aureus, which is practically instantaneous abovepenicillin resistance is quantitatively correlated 0.1 u/ml. This has also been found with awith different "reactivities" of penicillin with staphylococcus just inhibited by 1 u/ml (Cooper,PBC. This was not so with strains which had unpublished). There seem to be three differentbeen selectively trained to resistance (12), where, ways in which the rate of reaction of penicillinin a manner similar to the experience of Maass with PBC could be slowed in resistant strains:and Johnson, the resistant strain could bind less (a) the PBC itself may be less reactive chemicallyor more or the same amount of penicillin as the so that the proportion of effective to total con-parent sensitive strain. In one trial Eagle's tacts between penicillin and PBC becomes less;strain of Diplococcus pneumoniae progressively (b) steric hindrance becomes more marked,bound less per gram over a period of about a year reducing the total number of contacts; (c) if thewithout change in sensitivity (12). This was sensitive cell is in fact freely penetrated byalso found with a staphylococcus by Cooper (18), penicillin, which cannot yet be decided, and ifand occurred simultaneously with an increase in PBC resides internally, then resistance could becell volume so that the number of molecules achieved by a decrease in the permeability of thebound per cell was roughly constant. cell wall to penicillin. This might come about for

However, it is not clear whether the "reactivi- a variety of reasons, one of which could conceiv-ties" with penicillin of the strains described by ably be a decrease in the proportion of lecithinEagle truly refer to the final amount which is in the cell wall phospholipids, as lecithin iscapable of being bound at a given concentration penetrated by penicillin, while cephalin andor to the rate at which binding occurs. If the cardiolipin are not (28). However, penicillincells were contacted for a longer time, the amount binding by sonic extracts of resistant cells is thebound at subinhibitory concentrations might be same as that of intact cells, suggesting that theresignificantly greater. It may be that the change is no change in permeability but rather that thein rate of binding with concentration is more reactivity of the site is affected (12).important than the final amount bound in any These ways taken together would permit ancorrelation between "reactivity" and sensitivity. infinitely large possible number of small differ-Maass and Johnson (6) and Cooper (19) have ences in penicillin resistance. They do not exhaustshown that the rate of penicillin binding increases the list, however, for some strains may be able towith penicillin concentration over the inhibitory synthesize PBC faster than others giving a highrange up to a level where it becomes too rapid to uptake in u/g DW, yet these would be moremeasure. The low binding by Eagle's resistant resistant to the drug, since their deficiency couldstrains at low concentrations, compared with be made good faster. Also, strains selected forsensitive strains, would lead to the expectation resistance may have the same uptake in u/g asthat at least in naturally resistant strains the the parents, yet their rate of synthesis of PBCrate of binding is considerably slowed, as the may be stimulated by penicillin or they may becontent of PBC and the rates of synthesis of less dependent on the product of PBC, makingPBC (rate of cell growth multiplied by content of them apparently more resistant. On the otherPBC expressed as penicillin bound per gram hand, the cell may alter in size, yet the amountwhen uptake is saturated) were all very simi- of PBC required by each cell may remain con-lar. Any other effect of concentration would in- stant, so that the uptake may vary withoutvolve reversible binding. change in sensitivity.The important question of rates rather than In view of the rather confusing number of


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theoretical possibilities it might perhaps be specific type of fixation by any particular guineaexpected that little correlation between rate or pig tissue or subcellular particle.amount of penicillin bound and sensitivity If a cell is independent of PBC, then whyshould exist. This somewhat pessimistic forecast should it be inhibited at all by penicillin at anyrenders all the more interesting the several concentration less than that which will interfereobservations that such a correlation does in fact with the osmotic properties of the medium? Theoccur in naturally occurring resistant strains, answer to this is probably that the type ofalthough not necessarily existing for strains inhibition exerted by penicillin changes qualita-selected for resistance. tively at higher concentrations, becoming

bacteriostatic rather than bactericidal (17),suggesting that growth is inhibited by inter-

If the cell does not possess PBC or is not ference with difference sites in the cell. Thus, inaffected by its loss, then the concept of PBC as comparing work on the "site of action" of peni-the site of action of penicillin will mean that cillin one must bear in mind the concentrationsthis cell will be resistant to penicillin. ]XIost of penicillin at which it was studied. Eagle (41)bacteria possessed PBC to some extent, but it makes the interesting suggestion that theseemed possible at one time that the penicillin- bactericidal action at lower concentrations couldresistant yeasts may be independent of PBC, be due to accumulation of a toxic precursor,since they were incapable of binding penicillin normally metabolized by PBC, so that the con-(5, 19). 'More recently Daniel and Johnson at siderable slowing of killing at higher concentra-Wisconsin (16) showed that a soluble extract of tions is due to slowing of the rate of synthesisruptured yeast cells could bind penicillin in a of this precursor. However, as he pointed out,specific manner similar to that of sensitive there may equally be a slowing of the rate ofbacteria, so yeasts do in fact possess a PBC, exhaustion of the cell's limited reserve of metabo-but Cooper (19) found that, in contrast to lites by inhibition at higher drug titers of, e.g.,staphylococcus, the "lipid particles" of yeast nucleic acid synthesis. K+, Na+, Mg++ or phos-obtained on mechanical rupture were incapable phate could fill the role of metabolites exhausted.of binding penicillin. It is not known whether the Several metabolic effects of penicillin have beenspecific binding of one yeast used by Daniel and noted only at concentrations considerably higherJohnson is related to the lipid particles. Yeast than the minimal inhibitiory titers (66, 67, 68, 69).cells were impermeable to penicillin (5), so that A reaction undergone by resting cells which isperhaps PBC is contained within the yeast cell inhibited by high concentrations is glutamateand sheltered from penicillin rather than at or exchange (66) and this is also 10 per cent in-near the osmotic surface as in staphylococci. hibited at very low concentrations (0.01 u/ml).

Practically no "specific" binding of penicillin However, the inhibition is not complete even atoccurs with mammalian cells such as occurs with 1000 u/ml. This inhibition only changes by 2 topenicillin sensitive bacteria (32). It therefore 5 per cent over the minimal growth inhibitionseems likely that the lack of toxicity of penicillin range of the test organism, in contrast to thefor such cells is accounted for by their lack of sharp change in binding by PBC at about thesePBC. It would be interesting, however, to see concentrations, suggesting that this inhibitionif penicillin is bound specifically by a small is not the result of fixation by PBC. The typefraction of the cell material such as the subcellular of inhibition curve suggests some kind of rever-particles (e.g., mitochondria). Such a small sible binding.binding may be masked by the large amount of It could be inferred, from the observation thatinert material present. The mitochondria are penicillin uptake does not increase in restingnot freely permeable to all ions (65) and in fact cells (18), that PBC does not turn over in theseem to possess an osmotic barrier in some ways resting state, and therefore perhaps any reactionanalogous to that of bacteria, so that it may be which can proceed in the resting state is unlikelynecessary to rupture them in order to allow the to be the initial reaction disorganized by peni-penicillin to penetrate. It would be interesting cillin. If it is conceded that the increase ofalso to see whether the relative toxicity of peni- penicillin bound during growth does in fact repre-cillin for guinea pigs can be correlated with a sent turn-over of PBC, then in order to prove


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any identity between the reaction governed by to be very complex; we are not sure how toPBC and any reaction inhibited by penicillin it approach the question of synergism, and theshould, at least, be shown that PBC can also function of the reaction initially disorganized byturn over under any "resting" condition under penicillin and chemical nature of its site of actionwhich this reaction can normally be demonstrated. are still unknown. It can be seen that muchAlso, as contact in the resting state binds only further work needs to be done, and new tech-50% of the total PBC, then it would be ex- niques are needed in which radiopenicillin itselfpected that resting contact followed by wash- may play little part.ing should halve rather than eliminate the rateof the reaction under test on subsequent growth. ACKNOWLEDGMENTS

I am indebted to Prof. R. Cruickshank and theVIII. MECHANISM OF SYNERGISM late Sir Alexander Fleming, F.R.S., and to Dr.

Two hypotheses have been considered likely to D. Rowley for his initial introduction to theaccount for synergism between antibiotics (70): problem and his invaluable support and advice(a) one drug may increase the ability of the other since.to reach its receptor site, perhaps by increasingthe permeability of the cell wall; (b) alternative REFERENCESmetabolic pathways may be simultaneously 1. FILDES, P. 1940 A rational approach toblocked. research in chemotherapy. Lancet, i,

In the synergism which occurs between peni- 955-957.cillin and streptomycin, possibility (a) above may 2. HUNTER, T. H., AND BAKER, K. T. 1949result in an increased rate or final level of peni- The action of penicillin on Bacillus subtiliscillin uptake if it is the streptomycin which has growing in the absence of amino acids.the effect on permeability to penicillin (64). Science, 110, 423-425.

eeaf l penicillin 3. FRIEDEN, E. H., AND FRAZIER, C. N. 1947Unfrtu atey th rat an fialevlo The effects of various agents upon the sen-

uptake on Streptococcus faecalis seemed to be sTivity ofSaphylococcutsaupeu to pen-completely unaffected by streptomycin under cillin. Arch. Biochem. and Biophys., 15,resting or growing conditions, although syner- 265-278.gism in the form of an increased rate of killing 4. COOPER, P. D., AND ROWLEY, D. 1949 In-was clearly demonstrated when growth occurred. vestigations with radioactive penicillin.In view of the effects of penicillin on cell wall Nature, 163, 480-481.functions mentioned in section V, it would be 5. MAASS, E. A., AND JOHNSON, M. J. 1949interesting to try the reciprocal experiment of Penicillin uptake by bacterial cells. J.measuring streptomycin uptake in the presence Bacteriol., 57, 415-422.of penicillin. 6.MAASS, E. A. AND JOHNSON, M. J. 1949

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penicillin and governs the reaction first dis- 1942 Activity of penicillin in vitro. Proc.Organized it is evident that use of radiopenicillin Soc. Exptl. Biol. Med., 50, 277-280.appears to explain many previous experimental 8. ROWLEY, D., COOPER, P. D., ROBERTS, P.findings and may guide future ones. In general, W., AND SMITH, E. L. 1950 The site ofthe results of the four groups of workers concerned action of penicillin. 1. Uptake of peni-have agreed well, and illustrate the value of cillin on bacteria. Biochem. J., 46, 157-several independent approaches. Beyond that, 161.the results up to the present are a little disap- 9. PASINSKY, A., AND KASTORSKAYA, T. 1947pointing in that they represent only an introduc- Physico-chemical mechanism of penicillin

. ~~~action. Biochimia, 12, 465-476. (Trans-tion to the problem and often the actual happen- lated title).ings can only be inferred rather than proven. 10. BRODERSEN, R. 1947 Stability of penicillinRadiopenicillin as a "rational approach" is G in aqueous solution as a function of hy-only half the tool which is needed. The possible drogen-ion concentration and temperature.mechanisms of penicillin resistance have proved Acta. Pharmacol. Toxicol., 3, 345-363.


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11. COOPER, P. D., CLOWES, R. C., AND ROWLEY, 22. PARKER, R. F., AND MARSH, H. C. 1946D. 1954 A note on the use of radioactive The action of penicillin on Staphylococcus.penicillin. J. Gen. Microbiol., 10, 246-249. J. Bacteriol., 51, 181-186.

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12b. EAGLE, HE LEVY, M., AND FLEISCHMAN, R. of Streptococcus faecalis. J. Gen. Micro-1955 The binding of penicillin in relation biol., 1, 53-76.to its cytoxic action. IV. The amounts 25. GALE, E. F., AND VAN HALTEREN, M. B.bound by bacteria at ineffective, growth 1951 The effect of certain amino-acids oninhibitory, bactericidal and maximally ef- the accumulation of free glutamic acid byfective concentrations. J. Bacteriol., 69, Staphylococcus aureus: extracellular peptide167-172. formation. Biochem. J., 50, 34-43.

13. POLLOCK, M. R., AND PERRET, C. J. 1951 26. MITCHELL, P. 1953 Transport of phosphateRelation between fixation of penicillin across the surface of Micrococcus pyogenes:sulphur and penicillinase adaptation in B. Nature of the cell inorganic phosphate.cereus. Brit. J. Exptl. Pathol., 32, 387- J. Gen. Microbiol., 9, 273-287.396. 27. KLOTZ, I. M., URQUHART, J. M., AND WEBER,

14. EAGLE, H. 1953 The binding of penicillin W. W. 1950 Penicillin-protein complexes.in relation to its cytotoxic action. In Arch Biochem., 26, 420-435.Symposium on growth inhibition and chemo- 28. FEW, A. V., AND SCHULMAN, J. H. 1953 Ontherapy, pp. 3-9. 6th Intern. Congr. Mi- the surface chemistry of sodium penicillincrobiol., Rome. G. Biochim. et Biophys. Acta, 10, 302-

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17. EAGLE, H., AND MUSSELMAN, A. D. 1948 land.The rate of bactericidal action of peni- 31. POLLOCK, M. R. 1953 Penicillinase adapta-cillin in vitro as a function of its concentration and fixation of penicillin sulphur bytion, and its paradoxically reduced activity Bacillus cereus spores. J. Gen. Microbiol.,at high concentrations against certain or- 8, 186-197.ganisms. J. Exptl. Med., 88, 99-131. 32. EAGLE, H. 1954 The binding of penicillin

18. COOPER, P. D. 1955 The site of action of by mammalian cells in tissue culture (HeLapenicillin: some changes in Staphylococcus and L strains). J. Exptl. Med., 100, 117-aureus during the first two hours' growth in 124.penicillin media. J. Gen. Microbiol., 13, 33. ABRAHAM, E. P., CRAWFORD, K., NEWTON, G.22-38. G. F., BURTON, H. S., AND HALE, C. W. 1953

19. COOPER, P. D. 1955 The site of action of Cephalosporin N: a new type of penicillin.penicillin: some properties of the pencillin- Nature, 171, 343.binding component of Staphylococcus 34. COOPER, D. E., AND BINKLEY, S. B. 1948aureus. J. Gen. Microbiol., 12, 100-106. Penicillin amide. J. Am. Chem. Soc., 70,

20. COOPER, P. D. 1954 The site of action of 3966-3967.penicillin. III. Effect of surface-active 35. NIELSON, T., AND ROHOLT, K. 1952 Benzylsubstances on penicillin uptake by Staph- penicillin diethylaminoethyl ester. J.ylococcus aureus. Biochim. et Biophys. Pharm. and Pharmacol., 4, 266-267.Acta, 13, 433-438. 35a. SCHEPARTZ, S. A., AND JOHNSON, M. J.

21. FEW, A. V., COOPER, P. D., AND ROWLEY, D. 1956 The nature of the binding of peni-1952 Reaction of penicillin with the staph- cillin by bacterial cells. J. Bacteriol., 71,ylococcal cell wall. Nature, 169, 283-284. 84-90.


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36. WINTERSTEINER, O., STAVELY, H. E., Edited by W. D. McELROY and B. GLASS.DUTCHER, J. D., AND SPIELMAN, M. A. The Johns Hopkins Press, Baltimore, Md.1949 The inactivation of penicillin by 47a. STROMINGER, J. F. 1953 Biochemical effectscysteine and related compounds. In The of penicillin on nucleotide and nucleic acidchemistry of penicillin. pp. 209-221. Edited metabolism in Staphylococcus aureus andby H. T. CLARKE, J. R. JOHNSON, and R. Lactobacillus helveticus. Fed. Proc. 12, 277.ROBINSON. Princeton University Press, 48. MCQUILLEN, K. 1951 Effect of penicillin onPrinceton, N. J. electrophoretic mobility of Staphylococcus

37. ABRAHAM, E. P., AND DUTHIE, E. S. 1946 aureus. Biochim. et Biophys. Acta, 6,Effect of pH of the medium on activity of 534-547.streptomycin and penicillin and other 49. MITCHELL, P., AND MOYLE, J. 1950 OC-chemotherapeutic substances. Lancet, i, currence of a phosphoric ester in certain455-459. bacteria: its relation to Gram staining and

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39. MITCHELL, P., AND MOYLE, JENNIFER. 1951 58,475-490.The glycerophosphoprotein complex en- 51. CHAIN, E., AND DUTHIE, E. S. 1945 Bac-velope of Micrococcus pyogenes. J. Gen. tericidal and bacteriolytic action of peni-Microbiol., 5, 981-992. cillin on the staphylococcus. Lancet, i,

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41. EAGLE, H. 1951 Further observations on accumulation of free glutamic acid and thethe zone phenomenon in the bactericidial formation of combined glutamate in Staph-action of penicillin. J. Bacteriol., 62, ylococcus aureus. Biochem. J., 48, 298-301.663-668. 53. DEMEREC, M. 1945 Production of staphy-

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43. GALE, E. F., AND TAYLOR, E. S. 1947 The cillin resistance in pneumococci by theaction of penicillin in preventing the as- desoxyribonucleate derived from resistantsimilation of glutamic acid by Staphylococ- cultures. Cold Spring Harbor Symposiacus aureus. J. Gen. Microbiol., 1, 314-326. Quant. Biol., 16, 457-461.

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45. MITCHELL, P., AND MOYLE, J. 1951 Rela- 56. PLOUGH, H. H., AND GRIMM, M. R. 1949tionships between cell growth, surface Reversal to penicillin sensitivity in aproperties and nucleic acid production in cysteine-requiring mutant of Salmonella.normal and penicillin-treated Micrococcus Science, 109, 173-174.pyogenes. J. Gen. Microbiol., 5, 421-438. 57. HUGHES, W. H. 1952 Variation in penicillin

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47. STROMINGER, J. L. 1951 Discussion on paper 59. MANSON, E. E. D., POLLOCK, M. R., ANDby J. M. Buchanan. Symposium on phos- TRIDGELL, E. J. 1954 A comparision ofphorus metabolism, Vol. II, pp. 422-423. the properties of penicillinase produced by


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Bacillus subtilis and Bacillus cereus with tive transport of ions by subcellular par-and without the addition of penicillin. J. ticles. Biochem. J., 57, 37-49.Gen. Microbiol., 11, 493-505. 66. GALE, E. F. 1953 Amino acid incorporation

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SITE OF RADIOPENICILLIN30) P. D. COOPER [VOL. 20 (11) by: (a) an identical uptake curve with a andRowleyandtheir colleagues, showing that radiopenicillin preparation containing less