JOURNAL OF BACTERIOLOGY, Aug. 1986, p. 535-543 Vol. 167, No. 20021-9193/86/080535-09$02.00/0Copyright © 1986, American Society for Microbiology

Newly Made Enzymes Determine Ongoing Cell Wall Synthesis andthe Antibacterial Effects of Cell Wall Synthesis Inhibitors

E. TUOMANEN

The Rockefeller University, New York, New York 10021

Received 21 February 1986/Accepted 28 April 1986

Cell wall synthesis can continue with less than the total complement of cell wall synthetic enzymes present innormal growing cells. A method was developed to investigate whether there exists an excess of cellwall-synthesizing enzymes (penicillin-binding proteins rPBPs]) which all remain functional or whether a mixedpopulation of functional and nonfunctional enzymes charicterize normal cells. Surprisingly, cells in which lessthan 10% of the PBPs were functional could grow at a normal rate, as evidenced by increases in viable counts,culture turbidity, and rates of peptidoglycan, protein, and RNA synthesis. This subset of functional enzymeswas biosynthetically new. Penicillin-induced lysis occurred contingent on the acylation of this same smallfraction of PBPs, the copy number and affinities of which were below the level of detection by currentfluorographic assay techniques. We propose that PBPs have a short functional half-life and that cell wallsynthesis and bacterial lysis reflect the activity of newly synthesized PBPs.

Cell wall synthetic enzymes are integral membrane pro-teins. The fact that these proteins form covalent complexeswith radioactive penicillin allows the quick detection andquantitation of these important membrane proteins withoutneed of purification or other complex strategies that are usedfor thti detection and titration of other protein membraneconiponnts. The availability of such a sensitive and specificassay opens up the possibility of studying the biosynthesis,membrane incorporation, and regulation of this set of proteinmolecules of great biological importance.The catalytic functions of cell wall synthetic enzymes

(penicillin-binding protein [PBPs]) involve a strong topo-graphic element: these enzymes, while anchored in theplasma membrane, catalyze the building and rearrangementof covalent bonds in the macromolecular sheets of cell wallexterior to the plasma membrane. In the bacteria studied inthis report, this incorporation reaction is restricted to alimited number of anatomical areas of the cell suirface, mostcommonly at the cell equator (1, 6). PBPs are minor proteinconstituents of the plasma membrane which have beenestimated to represent about 3,000 molecules per cell inEscherichia coli (17) and about 20,000 molecules in pneumo-cocci (21). However, only 1,000 molecules per cell in E. coliand 2/3 of the total number of PBPs in pneumococci appearto perform essential functions. Additional studies haveshown that apparently normal wall synthesis (and cell mul-tiplication) can continue with less than a full complement ofthese essential PBPs (3, 22), indicating either that theseproteins are present in excess or that only some fraction ofall the proteins estimated by the PBP assay are functional.The purpose of the studies described in this paper was to

test this point. Our experimental strategy exploited condi-tions of penicillin tolerance in which cell wall syntheticenzymes could be fully acylated with penicillin withoutkilling or lysis of the cells. We carefully examined thekinetics of resumption of cell wall synthesis and the appear-ance of functional (i.e., nonacylated) PBPs after removal ofthe excess penicillin from the medium. The findings suggestthat 'the functional half-life of PBPs may be surprisinglyshort.

(This material was presented in part at the InterscienceConference on Antimicrobial Agents and Chemotherapy,Minneapolis, 30 September 1985 [Program Abstr. 25thICAAC, abstr. no. 993, 1985].)

MATERIALS AND METHODSBacterial strains and cultivation conditions. Pneumococcus

sp. strain R6 was grown in a chemically defined, syntheticmedium (A. Tomasz, Bacteriol. Proc., p. 29, 1964) in astationary water bath at 37°C. E. coli W7 (dap lys) wasgrown in a shaking water bath at 37°C in M9 minimal saltsmedium (12) supplemented with 25 ,ug of lysine and 5 ,ug ofdiaminopimelic acid per ml and substituted with 4 mg ofglycerol instead of glucose per ml. The addition of five timesthe MIC of penicillin to cultures of strain R6 (0.05 ,ug/ml) orW7 (25 ,ug/ml) resulted in rapid lysis.Treatment with penicillin under lysis-nonpermissive condi-

tions. The general experimental design involved exposingcells to high enough concentrations of penicillin to achievecomplete acylation of all PBPs under growth conditions thatprevented production of new PBP molecules and which alsocaused penicillin tolerance (i.e., conditions that preventedgrowth, lysis, and killing). Excess penicillin was then re-moved, lysis-permissive conditions were restored, and theresumption of cell wall synthesis and other polymer synthe-ses was followed. All experiments consisted of three phases.In phase I, logarithmically growing cells were transferred tolysis-nonpermissive conditions. A number of different con-ditions were used to induce phenotypic tolerance in thepneumococcal strain R6 and E. coli W7. To prevent lysis ofpneumococcal strain R6 during acylation of PBPs, we used(i) deprivation of the required amino acid leucine, (ii) addi-tion of chloramphenicol at the MIC (1 ,ug/ml), (iii) growth inethanolamine-containing medium (19), or (iv) growth inmedium containing a high concentration (1 mg/ml) of choline(8). The first two conditions halt growth, and the latter twoinhibit the pneumococcal autolytic system. In E. coli, peni-cillin tolerance was produced by transferring the cells to amedium lacking lysine. In both cases amino acid starvationwas achieved by filtering the culture psnd suspending the cells

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in medium lacking the required amino acid. Growth haltedwithin 2 min in each case, and the nongrowing cultures weretolerant to the lytic effects of up to 50 times the MIC ofbenzylpenicillin within 10 min.

In phase II, cultures in lysis-nonpermissive conditionswere exposed to 10 to 50 times the MIC of benzylpenicillinfor 10 to 60 min to acylate the PBPs. In phase III, excesspenicillin was removed and the cultures were reintroducedinto lysis-permissive growth conditions as follows. Penicil-linase (1,000 U/ml; Sigmal Chemical Co., St. Louis, Mo.)was added and the cultures were immediately filtered andsuspended in full growth media, i.e., readdition of 10 ,ug ofleucine per ml for pneumococci or 25 ,ug of lysine per ml forE. coli, chloramphenicol-free medium, or medium contain-ing normal concentrations of choline (10 ,ug/ml) (pneumo-cocci). All cultures were monitored for turbidity (SequoiaTurner spectrophotometer, A620) and CFU on tryptic soyagar containing 5% sheep blood (pneumococci) or supple-mented M9 agar (E. coli).

Rates of protein, RNA, and cell wall synthesis. Cultures ofpneumococcal strain R6 or E. coli W7 were sampled in allthree experimental phases described above. Samples (200,ul) were removed and incubated for 5 min at 37°C with thefollowing additions: S ,uCi and 5 pug of [3H]phenylalanine perml (New England Nuclear Corp., Boston, Mass.) to assessthe rate of protein synthesis; 10 ,uCi and 10 ,ug of [3H]uridineper ml (New England Nuclear) to assess the rate of RNAsynthesis; or, to assess rates of peptidoglycan synthesis, 10,uCi and 10 ,ug of 3H-labeled N-acetylglucosamine per ml forE. coli (Amersham, Inc., Amersham, U.K.) or 10 ,uCi and 5,ug of [3H]choline per ml for the pneumococci (New EnglandNuclear). At the end of pulse-labeling, the samples werefrozen on dry ice and processed for material insoluble in cold(4°C) 5% trichloroacetic acid (TCA) for protein and RNAlabel (15) or hot (100°C) 5% sodium dodecyl sulfate (SDS) orcold 5% TCA for cell wall label (11).

In some experiments, the rate of cell wall degradation(lysis) was also determined. The bacteria were pulsed withcell wall-associated radiolabel (3H-diaminopimelic acid forE. coli or [3H]choline for pneumococci) for two generationsand then chased in nonradioactive medium for two genera-tions prior to phase I. Loss of cell wall-associated radiolabelwas measured during treatment with penicillin in lysis-nonpermissive (phase II) and -permissive (phase III) condi-tions by assessing the decrease in counts insoluble in hotSDS (11).PBPs. The PBP profile was assessed in whole cells as

described previously for pneumococcus and E. coli (5, 23).Triplicate samples were taken from all three phases of theexperiment at the following times: at the beginning of phaseI, to assess the total complement of PBPs per cell; in phaseII, just before acylation of the PBPs with a saturatingconcentration (50 times the MIC) of unlabeled penicillin(i.e., after 15 min of amino acid deprivation, 2 h of chloram-phenicol treatment, or 10 min in high choline concentrations)and also at 5, 10, and 60 min after acylation to assess thedegree of saturation of PBPs. In phase III, samples wereremoved for the titration of PBPs with [3H]penicillin at 0, 5,10, 20, 30, 45, and 60 min after the removal of excess coldpenicillin from the medium to assess the synthesis of newPBPs. All samples were adjusted to the same turbidity andincubated for 10 min at 37°C with 1, 10, or 50 times the MICof [3H]benzylpenicillin (Merck, Sharp and Dohme, Rahway,N.J.; 25 Ci/mmol). The reaction was stopped by addingunlabeled penicillin at 4°C. Samples were processed forSDS-polyacrylamide slab gel electrophoresis as described

previously (5, 17, 23). Fluorograms of the gels were scannedto quantitatively compare band densities (Quikscan; HelenaInstruments, Beaumont, Tex.).The rates of deacylation of PBPs bound under lysis-

permissive and lysis-nonpermissive conditions were com-pared during subsequent incubation in both growing andnongrowing conditions. Fifty times the MIC of [3H]ben-zylpenicillin was added to each culture for 10 min and thenremoved by filtration. The degree of radiolabel remainingwith the PBPs was then determined over the subsequent 120min.The detection limit of the gel autoradiographic assay was

estimated by comparing the band densities of the followingseries of samples. Samples (1 ml) containing 2 x 108, 1 x 10,2 x 107, 1 X 107, or 4 x 106 pneumococci were incubatedwith 5 ,ug of [3H]benzylpenicillin (50 times the MIC) for 10min and processed for gel electrophoresis (23). Proteindeterminations were made on each sample by the method ofLowry et al. (14) with bovine serum albumin containing 1%Sarkosyl as a standard. All cultures were monitored forturbidity and CFU on tryptic soy agar containing 5% sheepblood.

RESULTS

Biosynthetic capacity of pneumococci following acylation ofcell wall synthetic enzymes. A representative experiment forpneumococci is shown in Fig. 1. A culture of the pneumo-coccal strain R6 was transferred to leucine-free medium, andafter 50 min of amino acid starvation (phase I of experimen-tal design) the culture was exposed to 50 times the MICequivalent of penicillin for 10 min to acylate the PBPs (phaseII). A control culture underwent the same treatment exceptwithout penicillin exposure. Next, penicillinase (1,000 U/ml)was added, the culture was washed and suspended in fullmedium, and incubation continued at 37°C (phase III).Bacterial growth rapidly resumed in the experimental culturedespite the fact that cellular PBPs were inhibited (penicillin-bound) (Fig. 1, top). Rates of cell wall, RNA, and proteinsynthesis were measured by pulse-labeling throughout thethree phases of the experiment, as outlined in Materials andMethods. In cells starved of leucine for 50 min, cell wall,RNA, and protein synthesis rates decreased to 36, 59, and57% of prestarvation levels, respectively. Penicillin treat-ment for 10 min during starvation did not alter the basalsynthetic rates. Within 5 min of readdition of leucine, cellwall, RNA, and protein synthesis rates increased to 66, 89,and 81% of prestarvation levels in both penicillin-treated andcontrol cultures. Within 10 min of leucine readdition, allrates equalled or surpassed the prestarvation levels (Fig. 1,bottom).

Figure 2 shows the status of the pneumococcal PBPs in thesame experiment. After acylation of the PBPs with 50 timesthe MIC of nonradioactive penicillin for 10 min, a test with a[3H]penicillin pulse showed no detectable labeling, indicat-ing that all PBPs were fully acylated by the nonradioactivepenicillin (Fig. 2A, lane 0). Upon removal of the excessnonradioactive penicillin and reintroduction of leucine intothe culture, additional samples were removed at 5, 20, 40,and 60 min into phase III and pulsed with [3H]penicillin todetect free (unacylated) PBPs. Faint radioactive bands ap-peared after 5 min; they became more visible after 20 minand reached an intensity approaching that of the preacylatedculture by 60 min. These PBP bands clearly representedprotein molecules newly synthesized in phase III since theirappearance required the removal of the acylating agent and

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FIG. 1. Resumption of growth and macromolecular synthetic rates of pneumococci after exposure to penicillin. (Top) Phase I:pneumococcal strain R6 was grown to mid-logarithmic phase (A) and transferred to leucine-free medium (arrow a). Phase II: 50 times the MICof penicillin was added (arrow p) for 10 min. Phase III: penicillinase was added and the culture was either maintained without leucine (OI) orIysis-permissive conditions were restored by readdition of leucine (A). Viability determinations paralleled culture turbidity results(absorbance units [A]). (Bottom) Rates of synthesis of protein ([3H]phenylalanine, A, A), RNA ([3H]uracil, 0, 0) and cell wall ([3H]choline,U, OI), were determined in pneumococci deprived of leucine for 1 h and treated with penicillin (P) as described for the top panel. Removalof penicillin and readdition of leucine (at time zero) resulted in rapid resumption of biopolymer synthesis in both penicillin-treated cells (opensymbols) and control cells (closed symbols).

the resumption of protein synthesis (compare Fig. 2A with2B). A portion of the acylated culture from which the excesspenicillin was removed but protein synthesis remained in-hibited (continued deprivation of leucine or chloramphenicoltreatment) produced free PBPs (i.e., PBPs that could bedetected by pulsing with [3H]penicillin) only after a moreprolonged period (40 to 60 min) of incubation beyond theremoval of the cold penicillin (Fig. 2B). The faint bands firstdetectable at 40 min in this control must represent PBPs thathave become deacylated (deacylation rate is the same in plusand minus amino acid conditions; see below).

In a second experimental design, pneumococcal strain R6received a high concentration of choline (1 mg/ml) 10 minprior to nonradioactive penicillin (50 times the MIC for 1 h)

to inhibit the triggering of autolytic activity by the antibiotic.Samples for the titration of PBPs were removed just prior tothe addition of high choline concentrations to the medium(Fig. 3, lane Cl), 10 min after choline addition (Fig. 3, laneC2) and after 1 h of treatment with nonradioactive penicillinfollowed by suspension in normal (low-choline) medium(Fig. 3, lane C3). The intense labeling in lanes Cl and C2represents the maximum amount of free PBPs available inthe culture, and the absence of label in lane C3 indicates thatthe nonradioactive penicillin fully acylated all these PBPs.After the removal of the nonradioactive penicillin (andtransfer of the cells to the low-choline medium) the culturepromptly resumed growth. The viable titers over the firsthour of regrowth in phase III increased rapidly from 4.2 x

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FIG. 2. Appearance of biosynthetically new PBPs in growingand nongrowing pneumococci. Pneumococcal strain R6 deprived ofleucine for 15 min was treated with 50 times the MIC of unlabeledpenicillin for 10 min to saturate biosynthetically old PBPs. At timezero penicillinase was added to the culture, which was then split intotwo portions: A received leucine, B was maintained without leucine.Samples of the two cultures were taken at the times indicated andexposed to 50 times the MIC of [3H]penicillin to determine theappearance of free PBPs over time. The full complement of cellularPBPs is comparable to that shown in lane A60 (see Fig. 3, lane Cl).

107 to 1.3 x 108, indicating a doubling time of about 30 min,which closely resembled that of untreated cells (about 40min). Macromolecular syntheses (determined as described inthe legend to Fig. 1) which fell during penicillin treatment tovalues similar to those of amino acid-starved cells resumedpre-penicillin treatment rates by 10 min after transfer togrowth medium (data not shown). During this periodtitration of free PBPs in the culture by pulsing with[3H]penicillin indicated that faint bands first became detect-able after 5 min into phase III. PBP 2 became visible first,followed by PBPs 3 and la at 10 min and finally PBP lb at 40min (Fig. 3).To determine the limit of detection of the fluorographic

assay, PBP titrations were performed on a series of culturesdiluted between 1- and 50-fold. While the concentration ofbacteria routinely used in PBP experiments (2 x 108 cells perml; Fig. 3, lane 5) gave strong, easily measurable bandintensities, the PBPs of 2 x 107 cell equivalents of pneumo-cocci (lane 3) were only barely detectable. Quantitation byscanning densitometry indicated that fluorograms preparedwith the same procedure from cultures at identical densitiesgave comparable PBP labeling from one experiment toanother. Thus, a comparison of the titration in the left panelof Fig. 3 with the band densities in other titrations (e.g.,either the full complement of PBPs in 2 x 108 cells per ml[lanes Cl and C2, Fig. 3] or the same number of cells2~

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resuming growth in phase III [Fig. 2B and 3 at 5 min]) wasmade to estimate the number of free PBPs in each case. Inthe pneumococcal cultures which had resumed normal ratesof wall synthesis in phase III of experiments 1 and 2, lessthan 10% of the total cellular content of PBPs was free (i.e.,functional). This number of new PBPs which carried out thenormal rate of peptidoglycan synthesis that appeared withinminutes of removal of penicillin and transfer to growthmedium was below the limit of detection of the fluorographictechnique.The rebound in growth seen in phase III of the experi-

ments described was also observed in three other experi-mental designs in which phase II consisted of chloramphen-icol treatment, change in cell wall susceptibility to autolysisby substitution of ethanolamine for choline, and finally useof the autolysin-deficient strain Lyt 4-4 (19). In all cases, anormal rate of growth was achieved by 10 min into phase III(data not shown).

Biosynthetic capacity of E. coli following acylation of cellwall synthetic enzymes. To test the general validity of thesefindings, a third set of experiments was performed with E.coli W7 made tolerant by starvation for the essential aminoacid lysine (Fig. 4). As with the pneumococci, a return to therate of growth (increase in culture turbidity and CFU) equalto the normal generation time (45 min) was apparent within10 min of penicillin removal (curve A, T = 48 min; curve B,T = 52 min).Following acylation of the PBPs by penicillin treatment,

the culture was transferred to penicillin-free complete me-dium, and the rates of RNA, protein, and cell wall synthesiswere followed during phase III of the experiment. Forcomparison, cells transferred back to full medium afterlysine starvation but not exposed to penicillin were assayedin a similar manner. Figure 5 depicts the rates of biopolymersynthesis as determined for culture B in Fig. 4. In controlcultures starved of lysine, macromolecular synthesis ratesdecreased in a coordinate manner about 50% for peptidogly-can, about 75% for protein, and about 70% for RNA. Resultswere the same for hot SDS-insoluble (incorporated material)and cold TCA-insoluble (incorporated plus precursor mate-rial) cell wall material, indicating that no accumulation ofnonincorporated cell wall precursors occurred during star-vation. Addition of penicillin to the starved cells did notfurther depress these rates of synthesis over 10 min (Fig. 5)or over 60 min (data not shown). Upon removal of penicillinand readdition of lysine, the rates of synthesis of all threemacromolecular components increased within 5 min to morethan 70% of the prestarvation level in both penicillin-treated

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FIG. 3. Profile of new and old PBPs of pneumococci. (Left) Limit of detection of the gel fluorographic PBP assay was determined byexposing the following serial dilutions of a culture of growing pneumococci to 50 times the MIC of [3H]benzylpenicillin (concentrations ofdilutions were confirmed by CFU counting and protein determination): lane 5, 2 x 108 cells; lane 4, 108 cells; lane 3, 2 x 10' cells; lane 2,107 cells; lane 1, 4 x 106 cells. (Right) PBP patterns for pneumococci were determined with 50 times the MIC of [H]penicillin at the followingtime points: lane Cl, 2 x 108 untreated cells in normal medium; lane C2, 2 x 108 choline-treated (tolerant) cells; lane C3, 2 x 108 tolerant cellsafter 10 min with 50 times the MIC of penicillin. Other lanes: Appearance of newly synthesized PBPs when penicillin was removed from theculture of lane 3 and growth resumed. Samples were taken from growing cells at the times indicated after removal of penicillin.

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FIG. 4. Resumption of normal growth rate in E. coli despiteinhibition of old PBPs. E. coli W7 was grown to mid-logarithmicphase. The bacteria were then transferred to medium lacking theessential amino acid lysine (solid arrowhead). The cells haltedgrowth and became phenotypically tolerant to penicillin. Culturesamples were treated with 50 times the MIC of penicillin (P) foreither 1 h (A) or 15 min (B). Cultures then received penicillinase andreaddition of lysine (open arrowheads) (lysis-permissive conditions)and rapidly resumed growth despite acylation of PBPs synthesizedbefore that time. A, Absorbance units.

and untreated cultures. Importantly, while the cell wallsynthetic rate of cells removed from lysine starvation in-creased to that of unstarved cells approximately 25 min afterlysine readdition, a similar culture treated with penicillin atconcentrations expected to irreversibly acylate the majorityof the cell surface PBPs also reachieved the prestarvationrate of cell wall synthesis by 25 min after lysine readdition.

Evaluation of the PBPs during phase III of the experi-ments in such E. coli cultures by in vivo labeling indicatedthat, as in the analogous experiments with pneumococci, thefluorographic technique could only detect barely visibleradioactive bands in the positions of PBPs 1 through 4 at 20min after transfer of the cells to the full medium free ofpenicillin (data not shown). This indicates that the nearlynormal rates of wall synthesis were catalyzed by a smallfraction of the full complement of PBPs expected to bepresent at this cell concentration of E. coli.

Effect of tolerance-inducing conditions on labeling of PBPs.The results of appropriate control experiments are summa-rized in Table 1. Amino acid-starved and growing cells of E.coli and pneumococci produced virtually identical patternsof PBP labeling in whole cells. In fact, the only decrease inlabeling which could contribute to decreased antibiotic ef-fectiveness was seen in PBP 3 in membrane preparations oflysine-deprived E. coli cells.

Deacylation rates of PBPs were also not altered comparedwith those of growing control cells within the first 60 min ofstarvation for both E. coli and pneumococci. A greater than10% decline in the band densities was seen at the followingtimes in both growing and nongrowing cells which had beensubsequently incubated in growing or nongrowing condi-tions: pneumococcus PBP 1, > 60 min; PBP 2, 45 min; PBP3, >60 min; E. coli PBP 1 through 4, >60 min; and PBP 5/6,5 min.

Loss of viability, bacterial lysis, and acylation of PBPs. Inthe studies described, experimental conditions were soughtthat prevented loss of viability and lysis during the acylationof PBPs by penicillin in phase II of the experiments. Inter-estingly, these conditions not only prevented the irreversibleantibacterial effects of penicillin during phase II; after re-moval of the penicillin from the medium, the pretreatedbacteria could rapidly resume normal growth without furtherneed for lysis-protective conditions. This was observed inboth E. coli and pneumococci. However, there was nopermanent change in the lysis sensitivity of bacteria putthrough the various phases of our experiments since asecond addition of penicillin to E. coli cells in phase IIIcaused rapid lysis (Fig. 6 and 7, line 6). Culture lysisfollowed penicillin addition even at time zero, indicating thatthe acylation of new PBPs could induce lysis. Interestingly,cells pretreated with high concentrations of penicillin tosaturate all PBPs in phase II of the experiments werehypersensitive to penicillin if challenged with a secondexposure to the drug in phase III (Fig. 7). Pneumococci werepretreated with penicillin to fully saturate the PBPs in amedium containing the lysis-protective (high) concentrationof choline. Subsequently, penicillin was removed and thecells were transferred to low-choline medium and thendistributed into three tubes. One of these (tube 3) containedno addition, and this culture rapidly resumed growth. In thetwo other tubes, the cultures received a second challengewith sub-MICs of penicillin (1/10 the MIC in tube 4, 1/2 theMIC in tube 5). These penicillin-pretreated cells were hyper-sensitive to the growth-inhibitory effects of penicillin. Incontrast, 1/2 the MIC of penicillin did not alter the growthcurve of control cells without preacylated PBPs.

DISCUSSION

Earlier studies have shown that bacterial cells can con-tinue cell wall synthesis and growth with less than the totalnormal complement of wall synthetic enzymes, suggestingthat only a subclass of all these proteins detectable by thepenicillin-binding technique is actually essential (3, 22). Weintroduced an experimental design that allowed us to furtherdissect this question. The experimental design involvedpreventing synthesis of new PBPs (by inhibition of proteinsynthesis) and inactivating (acylating) all preexisting bindingproteins with penicillin under conditions that did not causestructural damage to the bacterial cells. We then proceededto carefully measure the rate of resumption of cell wallincorporation that might occur after removal of the excessacylating agent and reintroduction of the cells into full

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FIG. 5. Rates of peptidoglycan, RNA, and protein synthesis in E. coli after exposure to penicillin. Incorporation of radiolabel over 5 minwas determined as described in Materials and Methods over the entire course of growth, lysine deprivation (-130 to 0 min), penicillin (P)treatment, and recovery of growth for the experiment shown in Fig. 4 (line B). No difference in the absolute rates of protein (A, A,[3H]phenylalanine), RNA (0, 0, [3H]uracil), or peptidoglycan (O, E, [3H]N-acetylglucosamine) synthesis or in the time required to reach theprestarved rate was found between cultures receiving 50 times the MIC of penicillin (solid symbols) and control cultures (open symbols).

growth medium. It appeared reasonable to expect either oftwo findings: (i) resumption of wall synthesis could parallelthe recovery by deacylation of a substantial fraction of thecell's PBPs, in an enzymatically active form; or (ii) cells withvirtually all wall synthetic enzymes incapacitated could loseviability and lyse after reintroduction of lysis-permissiveconditions. Neither proved to be the case.

In both the gram-negative E. coli and the gram-positivepneumococcal strains, it was possible to inactivate the fullcomplement of cell wall synthetic enzymes. In the presenceof the inhibitor (penicillin), the rate of cell wall incorporationrapidly declined to a value that depended on the bacterialstrain and presumably represented continued cell incorpora-tion by penicillin-insensitive enzymes (such as transgly-cosylase [16]). However, after removal of the excess peni-cillin from the medium, the rate of cell wall incorporationrebounded, quickly reaching a value found in cultures before

drug addition. In a typical experiment with E. coli, the rateof wall incorporation reached 75% of the pre-drug additionrate within 5 min after the removal of excess inhibitor. Inpneumococci, the normal (i.e., pre-drug addition) syntheticrate was reached within 10 min after the removal of excesspenicillin.A careful evaluation of PBPs in such post-penicillin treat-

ment, growing cells indicated that during the dramatic in-crease in wall synthetic rates less than 10% of the totalcellular content of PBPs was functional (nonacylated). Infact, the number of free PBP molecules present at 10 minafter drug removal in pneumococci were: PBP la/b, about500, PBP 2a/b, about 800 and PBP 3, about 500 molecules,compared with the corresponding values of 6,000, 9,200, and5,300 molecules per cell at the beginninng of the experiments(21). Similarly, less than 10% of the E. coli PBPs were freeduring periods of normal rates of cell wall synthesis. Thus, a

TABLE 1. Relative band density of PBPs in tolerant and nontolerant bacteriaRelative band densitya

E. colib PneumococcicPBP

Whole cells Membranes Leucine deprivedFull medium for 1 h

Full medium Lysine deprived Full medium Lysine deprivedla 1,430 1,230 1,060 1,230

lb 5,520 4,720 2,250 3,130la/b 5,810 5,8102 420 560 540 980d2a/b 5,430 5,3403 2,450 2,190 2,300 1,480e 5,050 4,8904 2,300 2,460 2,370 2,2705/6 5,480 5,280 7,230 6,180

a Arbitrary units.b Titration at 20 times the MIC for 10 min.c Titration at 100 times the MIC for 10 min.d Value more than 120% of controle Value less than 80%'o of control

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FIG. 6. Bacterial lysis following exposure of new PBPs to penicillin. E. coli W7 cells were radiolabeled in the cell wall with[3H]N-acetylglucosamine -- -) and then chased in normal medium ( ). The culture was then transferred to medium lacking lysine (solidarrowhead) (control, *). Fifty times the MIC of penicillin (P) did not induce lysis in the tolerant nongrowing cells (O). Lysine was then addedto the culture (arrow). Culture samples (0) were then treated with two times the MIC of penicillin for various lengths of time after resumptionof growth (open arrowheads). Cell wall degradation paralleled loss of turbidity (absorbance units [A]).

strikingly minor fraction of the total cellular content of PBPsis sufficient for a normal rate of cell wall synthesis.As to the nature of the small fraction of functioning PBPs,

two potential sources exist: deacylation of old PBPs orsynthesis of new PBPs. While both sources could contributeto the pool of functioning PBPs, the evidence suggests thatbiosynthesis of new enzymes is the major source. During the

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FIG. 7. Growth-inhibitory effects of the interaction of sub-MICpenicillin with new PBPs. PBPs of pneumococcal strain R6, tolerantby virtue of high concentrations of choline (+c) in the medium, weresaturated by exposure to 50 times the MIC of penicillin (+p) for 1 h.Control cultures (0, 0) received no penicillin in this phase. Bacteriawere transferred to normal growth medium (-p), and culturesamples were treated immediately with the following concentrationsof penicillin: lines 1 and 3, no drug; line 4, 1/10 the MIC; lines 2 and5, 1/2 the MIC; and line 6, 10 times the MIC. The second dose ofpenicillin to cultures 4 and 5 interacted with nonacylated PBPsresponsible for the growth in culture 3. A, Absorbance units.

10- to 15-min burst in wall synthetic rates after penicillinremoval there was no detectable deacylation of PBPs. Infact, there was no detectable decline in the intensity of labelin the PBPs (with the exception of PBPs 5/6 of E. coli) for upto 40 to 50 min past the time of antibiotic removal. This isconsistent with the known deacylation rates for these orga-nisms: more than 45 min for each of PBPs of pneumococcus(20) and for PBPs 1 through 4 of E. coli (17). In contrast, thefirst detectable labeling of PBPs occurred about 10 min afterthe removal of excess penicillin from the medium. Thesefindings suggest that the rapid resumption of cell wall syn-thesis that followed the removal of excess penicillin from thegrowth medium was catalyzed by new enzymes that becameaccessible to the penicillin probe after removal of excessantibiotic. While it is possible that newly available PBPs mayrepresent enzymes emerging from protected sites onto thecell surface, the appearance of faint radioactive bands in thePBP fluorograms within this time frame most likely repre-sents the production of new PBP molecules (rather thandeacylated old PBPs) since labeling depended on proteinsynthesis.Our data indicate that the cell walls synthesized by the

small fraction of normally detectable PBPs represented thesynthesis of normal wall material. First, wall synthesis wasaccompanied by normal cell growth and division. Secondly,no unusual build-up of cell wall precursors was found priorto the burst of new synthesis. Thirdly, thin-layer chromato-graphic analysis of muramidase digests of glucosamine-labeled peptidoglycan of E. coli produced shortly after theresumption of wall synthesis indicated the presence ofdisaccharide peptide monomers, bis-disaccharide peptidedimers, and oligomers in the ratio characteristic of normallygrowing E. coli cells (data not shown). However, smalldifferences in such a minor fraction of total "average" cellwall mnaterial (i.e., 10 min of synthesis) would be difficult todetect by either high-performance liquid chromatography (9)or thin-layer chromatography (10).The lack of lysis in our experiments was a second surpris-

ing finding. It is a widely accepted notion that the antibac-terial (cytocidal and lytic) effects of penicillin are ultimatelythe consequences of the inhibition (acylation) of (some)PBPs. Yet, in our experimental design, acylation of most ofthe PBPs was not only irrelevant for the resumption of wall

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542 TUOMANEN

synthesis but also had no lethal consequences for the bacte-ria. The fact that penicillin-bound PBPs did not trigger lysiswhen acylation was temporally dissociated from conditionspermitting autolytic activity suggests that PBPs have a finitelife span within which they remain integrated into the eventswhich lead to autolysin activation. That this is a propertyshared by two organisms as disparate as pneumococci and E.coli indicates that such a functional time zone for PBPs maybe a common occurrence in bacteria in general. Both E. coliand pneumococci are known to incorporate new cell wallcomponents into the preexisting cell wall sacculus in definedgrowth zones (1, 6). It has also been shown that bacterialmembranes contain zones which are enriched for certainPBPs (2, 4). The concept of a finite functional life of PBPsmay be related to the topography of new cell wall synthesisas follows. Biosynthetically new PBPs may first appear on

the cell surface in the growth zone. Acylation at this locationwould lead to disruption of cell wall synthesis and uncon-trolled autolytic activity. However, if with age the PBPsmove out of'the growth zone, perhaps in register with theexpanding cell wall sacculus, they could become discon-nected from new cell wall synthesis and potentially from theautolytic pathway. Thus, as suggested by the results shownin Fig. 6, lethality is a function of the acylation of newlyproduced PBP molecules, the same subset of PBPs respon-

sible for ongoing cell wall synthesis.It is important that a nonessential function for old PBPs

cannot be ruled out by our experiments; for instance,modification of cell wall with age or cell wall thickening as

seen in chloramphenicol-treated staphylococci (13). Theobservation that cells pretreated with penicillin and allowedto resume growth are hypersensitive to the growth-inhibitoryactivity of penicillin suggests that in normal cells the "si-lent" majority of cellular PBPs may act as a sink which canbind antibiotic without lethal consequences. In such a sys-tem antibacterial efficacy would be determined by how muchpenicillin reaches new, perhaps less accessible, PBPs. In thiscontext, it is interesting that the findings described may bearon the phenomenon termed the postantibiotic effect (7, 18).Normally, after a dose of antibiotic, the population ofbacteria which survive exhibit a growth lag after the antibi-otic concentration falls below the MIC, i.e., the postanti-biotic effect. This would' parallel closely the findings shownin Fig. 7, in which growth inhibition was the predominantphenotype when new PBPs were treated with less thanoptimal doses of drug in conjunction with acylated old PBPs.In contrast, no postantibiotic effect was observed when oldPBPs alone were acylated (Fig. 1 and 2). In nontolerantnormal cells, old PBPs may have a sparing effect on thelethality of a penicillin dose by competing with new PBPs forthe drug and thereby decreasing, perhaps locally, the overalleffective drug concentration.

If it is presumed that the'relative affinity of a PBP for anantibiotic remains similar whether it is new or old, then PBPprofiles as currently performed may still reflect the relativeaffinities of the actively'synthesizing new enzymes eventhough they actually include a majority of "senescent"enzymes. The difficulty in defining the lethal target ofpenicillin in many bacteria may relate to our current inabilityto detect which new PBP (singly or in combination) isinhibited to what extent at the MIC.

ACKNOWLEDGMENTS

E.T. was supported by a fellowship from the Parker B. FrancisFoundation for Pulmonary Research. This work was supported by

Public Health Service grant RO1-AI-16794 from the National Insti-tutes of Health.The technical assistance of K. Gilbert and extensive discussion

with A. Tomasz are greatly appreciated.

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