ANTiMICROBiAL AGENTS AND CHEMOTHERAPY, Aug. 1978, P. 257-266 0066-4804/78/0014-0257$02.00/0 Copyright ©) 1978 American Society for Microbiology
Vol. 14, No. 2
Printed in U.S.A.
Inhibition of Cell Division of Escherichia coli by a New Synthetic Penicillin, Piperacillin KYOKO IIDA,' * SEIICHI HIRATA,2 SEIICHI NAKAMUTA,3 AND MASAATSU KOIKE' Department of Microbiology, Saga Medical School, Saga, Japan,' and Department ofMicrobiology, School
of Dentistr9 and Department of Urology, School of Medicine,3 Kyushu University, Fukuoka, Japan Received for publication 9 March 1978
The mechanism of the action of piperacillin against Escherichia coli was investigated. This drug converted cells to filaments, but did not show lytic action in a range of concentrations below 25 ,ig/ml. In some of the fiaments, stretched constrictions with various diameters were observed. Addition of piperacillin to a synchronous culture inhibited cell division immediately at any stage of the cell cycle. The results of morphological examination of synchronous cultures show that the percentage of filaments with a stretched constriction corresponds to that of normally septated cells before addition of the drug. Furthermore, peptidoglycan synthesis and cross-linking were not inhibited by this drug. It is likely that this drug inhibits only septum formation, but not the growth of wall, and that stretched constrictions are a result of longitudinal growth of septation caused by the drug. Examination of affinity of the drug to penicillin-binding proteins shows that protein 3 is the most sensitive, proteins 2 and 7 are moderately so, and protein 1 is sensitive only to high concentrations of the drug. It has been known that some penicillin-sensitive enzymes, e.g., D-alanine carboxypeptidase, transpeptidase, and endopeptidase, are involved in the terminal stages of peptidoglycan biosynthesis (10, 16, 26). In Escherichia coli, different penicillins or different concentrations of the penicillin show various morphological effects, e.g., filament formation, bulge formation, spheroplast formation, and osmotically stable ovoid-cell formation. The mechanism of these morphological changes could not be sufficiently explained by the inhibitory effect of penicillin on the peptidoglycan metabolism alone (3, 6, 7, 14, 17). Recently, seven penicillin-binding proteins from the cytoplasmic membrane of E. coli were identified as radioactive gel electrophoretic bands using benzyl-[14C]penicillin. A role of each of the proteins in the mode of action of fi-lactam antibiotics was detected by competition of binding affinity of radioactive benzylpenicillin with other fl-lactam antibiotics. The role was further detected by comparing the biological properties of the mutant strains having altered penicillinbinding protqins with those of the parent strains (19). However, it still remains unsolved as to which of the penicillin-binding proteins corresponds to the penicillin-sensitive enzymes of peptidoglycan metabolism, and as to how penicillin kills bacteria. A semisynthetic penicillin, sodium 6-[D(-)-a-(4-ethyl-2,3-dioxo-1-piperazinylcarbonyl-amino)-a-phenylacetamido] penicillanate (piperacillin [28], a derivative of ampi-
cillin), was developed by Toyama Chemical Co., Tokyo, Japan. We were interested, therefore, in investigating the relationship between morphological effects caused by piperacillin and its biochemical effects. The purpose of this experiment was to clarify the mode of action of penicillin. It has been demonstrated that cell division in E. coli B/r is proceeded by septation, including ingrowth of a distinct septum composed of the cytoplasmic membrane and the peptidoglycan layer (5). For the purpose of examining the precise effect of piperacillin on division, E. coli B/r was mainly used in this experiment. MATERIALS AND METHODS Organisms. The bacteria employed in this work were E. coli B/r, furnished by S. Kondo, Osaka University, Japan, and E. coli W7, which requires lysine and meso-2,6-diaminopimelic acid. The latter strain was kindly supplied by Y. Hirota, National Institute of Genetics, Misima, Japan. Media. Lennox broth (11) was used as the enriched medium. The minimal medium was a modified M9 medium (9). These media were solidified with 1.5% agar. Chemicals. Sodium 6-[D(-)-a-(4-ethyl-2,3-dioxo-
1-piperazinylcarbonyl-amino)-a-phenylacetamido] penicillanate (piperacillin, also known as T-1220) and ampicillin were supplied by Toyama Chemical Co., Tokyo, Japan. 3H-labeled diaminopimelic acid [3H]DAP; specific activity, 669 mCi/mmol) and benzyl[I4C]penicillin (specific activity, 55 mCi/mmol) were purchased from the Radiochemical Center, Amer-
sham, England. 257
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Electron microscopy. Logarithmically growing cells of E. coli B/r treated with or without piperacillin were doubly fixed by 0.25% glutaraldehyde-5% acrolein mixture in 0.05 M cacodylate buffer solution (pH 7.0) for 3 to 6 h at room temperature and then by 1% OS04 in the same buffer for 1 h at room temperature (5). Fixed cells were divided into two aliquots; one was subjected to scanning electron microscopy and the other to thin sectioning. The technique for scanning electron microscopy was essentially that of Anderson (1). Droplets of the fixed cell suspension were sandwiched between the Formvar membrane and collodion membrane and were dehydrated through an alcohol series. Then the specimens were immersed in amylacetate and were dried by the critical-point method. Platinum-palladium was evaporated on the surface of the specimens, and they were examined by a JEM 100C electron microscope equipped with a scanning display device, type ASID-4D. For transparent electron microscopy, fixed cells were stained by 0.5% uranyl acetate. After dehydration through an alcohol series, the specimens were embedded in Epon 812 (13). Thin sections were made with a Reichert OmU2 ultramicrotome and then were stained by 0.4% lead citrate (29). The materials were examined with a JEM 100B electron microscope. Synchronous culture. A modified method of Mitchison and Vincent (15) was used to synchronize E. coli B/r growing in M9 medium. Logarithmically growing cells (about 3 x 108 cells per ml) in 50 ml of M9 medium were harvested by centrifugation. The pellet was suspended in 3 ml of M9 medium, 0.5 ml of which was layered onto 10 ml of a 2 to 12% linear gradient of sucrose dissolved in the medium. Then the pellet was centrifuged at 800 x g for 9 min at room temperature. The topmost fraction of the turbid band, containing about 5% of the total cells, was removed, immediately inoculated into 20 ml of prewarmed M9 medium, and then incubated with reciprocal shaking at 370C. For counting the number of cells, 0.2 ml of the culture was removed at 5-min intervals and was mixed with 0.2 ml of 10% formaldehyde solution. The number of cells was determined with a Coulter Counter model ZB using a 30-rum aperture. In this experiment, survival was equivalent to the number of cells counted with a Coulter Counter. Appearance of the cells at 0, 20, 30, and 35 min after synchronization was observed by a scanning electron microscope. For measuring the effect of piperacillin on the synchronous culture, the drug was added to each culture at 0, 10, 20, 30, 35, and 50 min after synchronization, and the number of cells of each culture was read at 5-min intervals for 120 min. After the addition of the drug at 0, 20, 30, and 35 min, each culture was incubated for 120 min and then was prepared for a scanning electron microscope. Incorporation of radioactive tracers. Cell wall synthesis was measured by the incorporation of [3H]DAP into E. coli W7. Exponentially growing cells in M9 medium supplemented with (per ml) 20 ,ug of lysine, 5 jig of DAP, and 0.4 uCi of [3H]DAP were exposed to various concentrations of piperacillin for 90 min at 37°C. Cold trichloroacetic acid-insoluble fraction was collected on a filter (Whatman, GF/C) and was counted in a liquid scintillation system. The scin-
ANTIMICROB. AGENTS CHEMOTHER.
tillation fluid was made up of 4 g of 2,5-diphenyloxide (Packard Instrument Co.) per liter of toluene. Transpeptidase assay. This assay was made according to the method of Matsuhashi et al. (14). A 0.1ml sample of overnight culture of E. coli W7 in M9 medium containing lysine and DAP was inoculated into 1.9 ml of the medium containing 1 ,iCi of [3H]DAP per ml. The culture was then incubated for 2.5 h with shaking at 32°C with or without piperacillin. The cells were treated with trichloroacetic acid, trypsin, and lysozyme. Finally, peptidoglycan dimer and monomer were separated by paper chromatography in isobutyric acid-I N NH3 (5:3); they were cut out and then counted in a liquid scintillation system. Detection of PBPs. Penicillin-binding proteins (PBPs) of E. coli B/r were detectea according to the Spratt method (20). Logarithmically growing cells in 10 liters of L-broth were harvested and resuspended in 200 ml of 50 mM sodium phosphate buffer (pH 7.0). The cells were then broken by an RIBI cell fractionator at 20,000 lb/in2. After unbroken cells were removed by differential centrifugation, the cell wall and membrane complex was sonically disrupted twice with a 30-s pulse. The complex was centrifuged at 100,000 x g for 40 min at 4°C, washed twice in the same buffer, and stored in a Revco -80°C freezer as envelope fraction containing cytoplasmic membrane, outer membrane, and peptidoglycan. Protein was determined by the method of Lowry et al. (12). A 20-pl volume of benzyl-[14C]penicillin (50 ,uCi/ml) was added to 200 pl of the envelope (about 10 mg of protein per ml), and the mixture was incubated at 30°C for 10 min. The binding was terminated by the simultaneous addition of 5 p1 of nonradioactive benzylpenicillin (120 mg/mi) and 10 ,ul of 20% sodium lauroyl sarcosinate solution. After the inner membrane was solubilized by standing for 20 min at room temperature, the outer membrane and peptidoglycan were removed by centrifugation at 100,000 x g for 40 min at 10°C. After the addition of 20 pl of 2-mercaptoethanol to 100 jil of the supernatant membrane, the mixture was heated for 3 min in a boiling water bath; 60 pi was then loaded into the gel slot of a 10% sodium dodecyl sulfate-polyacrylamide slab gel. Electrophoresis was performed at a constant current of 20 mA. The gels were prepared for fluorography as described by Bonner and Laskey (4), dried under vacuum, and placed on Kodak Nonscreen X-ray film at -80°C. After the envelope was incubated with each drug for 10 min at 30°C, benzyl-[14C]penicillin was added to it. The competition of binding to each of the proteins between the drug and benzyl-['4C] penicillin was then measured.
RESULTS Effects of piperacillin on the growth of E. coli B/r. Sensitivities of E. colt B/r to piperacillin and ampicillin were examined. Logarithmically growing cells in L-broth were exposed to various concentrations of piperacillin and ampicillin, and the survivals were determined by the plating method. When the inoculum size was as large as 3.6 x 108 cells per ml, cell growth was completely inhibited by a range of 0.5 to 25 jLg of
INHIBITION OF CELL DIVISION BY PIPERACILLIN
VOL. 14, 1978
piperacillin per ml; at more than 50 jug/ml, cell death mildly occurred. Ampicillin at 1 ,ug/ml completely inhibited cell growth, and at more than 2.5 ,tg/ml, cell death drastically occurred (Fig. 1A). In the case of small inoculum size such as 4.7 x 106 cells per ml, similar results were obtained (Fig. 1B). Kinetics of piperacillin and ampicillin on the growth of E. coli B/r were
0 .1
259
examined by measuring the optical density and survival of the culture at 15-min intervals after addition of these drugs (fig. 2). Cell division was inhibited soon after the addition of 10 ,ig of piperacillin per ml, but the optical density of the culture continued to increase. After a 120-min exposure to the drug, a droplet of the culture was examined under the phase microscope. Un-
0.5 1 2.5 5 10 25 50100
jg/ml of drugs
FIG. 1. Sensitivity of E. coli B/r to piperacillin and ampicillin. Logarithmically growing cells were incubated with shaking at 37°C for 120 min with various concentrations of piperacillin or ampicillin. The survivors were then counted by plating on L-agarplates. The original inoculum of cells was 3.6 x 108 cells per ml (A) or 4.7 x 106 cells per ml (B) as indicated by arrows. Symbols: O, piperacillin; E, ampicillin. 200
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120
180
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120
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FIG. 2. Effect of piperacillin and ampicillin on the growth of E. coli Blr. Symbols: 0, control (without drug); *, 10 pg ofpiperacillin per ml; 100 pg ofpiperacillin per ml; A, 1 pg of ampicillin per ml; A, 2.5 pg of ampicillin per ml.
260
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der these conditions, the cells formed filaments. Above 50 jig of the drug per ml, the cells began to lyse slowly. On the other hand, division of the cells treated with 1 jig of ampicillin per ml was inhibited soon after addition of the drug, but cell death occurred after a 120-min exposure. Above 2.5 ,ug of ampicilin per ml, cell death by lysis occurred rapidly. These results show that E. coli B/r is susceptible to both drugs, but the killing effect of piperacillin is not as strong as that of ampicillin in the cells of E. coli B/r, since there was a range of division inhibition doses in piperacillin. Morphological effects of piperacillin on E. coli B/r. Effects of piperacillin on the surface and shape of E. coli B/r were examined by a scanning electron microscope, and those on the intracellular structures were examined by a transparent electron microscope after thin sectioning. Figure 3a is a scanning electron micrograph of a normally growing cell. This cell shows a wavy surface, and septal constriction is observed in the middle. Septated cells were detected in about 28% of the total cell population. In a section of the normally growing cell, a distinct septum composed of cytoplasmic membrane and peptidoglycan was recognized (Fig. 3b). After an exposure to 10 jig of piperacillin per ml for 90 min, cells were converted to filaments, in 26% of which stretched constrictions with various diameters were observed in the middle of the filaments (Fig. 4). The percentage of the filaments with a stretched constriction is almost equal to that of septated cells in normally growing cells. In thin sections, a majority of the filaments have no sign of septum formation in the wall and membrane, but the remainder have stretched constrictions whose wall and membrane are joined with a constrictively growing septal wall (Fig. 5a, b). From these results, it may be considered that the stretched constrictions are produced by modification of septal constrictions by piperacillin. On the other hand, the filaments induced by a 90-min expsoure to 1 jig of ampicillin per ml contain three types of appearance: 12% of the filaments with a bulge, 16% with a stretched constriction, and 72% without any alteration. In the case of 2 jig of ampicillin per ml, about 48% of the filaments formed bulges and about 10% of them lysed (Fig. 6). Effects of piperacillin on synchronized culture. The action of piperacillin on cell divi-
ANTIMICROB. AGENTS CHEMOTHER.
sion was examined by measuring the number of cells in synchronous cultures of E. coli B/r growing in M9 medium and was also observed by scanning electron microscopy. Under these conditions, the doubling time was about 50 min, and a good synchronous culture was obtained for two generations. When 0.5 or 5 jig of piperacillin per ml was added to cells at various points in time after synchronization, no further division took place in any sample after the addition (Fig. 7). Similar results have been shown in synchronous cultures treated with sublethal doses of ampicillin (5). These experimental results indicate that piperacillin inhibits cell division immediately at any stage of the cell cycle. The filaments induced by piperacillin show two types of appearance, one with a stretched constriction and the other without it. It is likely that the morphological difference is due to the different stages of cell division cycle at which the drug was added. Numbers of the septated cells in the synchronous culture at 0, 20, 30, and 35 min were morphologically examined by a scanning electron microscope. The same morphological examination was done with respect to numbers of filaments with a stretched constriction, which was produced by a 120-min exposure to piperacillin added after each of the durations above (Table 1). Septated cells were rarely observed at the initiation of the synchronous culture, and their numbers rapidly increased at 30 to 35 min after synchronization. After each of the durations, the percentage of the filaments with the piperacillininduced stretched constriction corresponded to that of the septated cells. Addition of the drug at 0 min after synchronization caused 99.7% of the cells to form filaments without constriction; in contrast, an addition at 35 min induced 51% of the cells to form filaments with a stretched constriction. The foregoing implies that piperacillin inhibits the septum formation at any stage; in cells before septation, the drug inhibits the initiation of growth of the septum, and during septation the drug inhibits the growing of the septum immediately, which results in a stretched constriction. Effects of piperacillin on cell wall synthesis. Cell wall synthesis was measured by incorporation of [3H]DAP into acid-insoluble fraction of E. coli W7 as mentioned in Materials and Methods. Various concentrations of the drug were added to each culture. After incubation for
FIG. 3. Scanning (a) and transparent (b) electron micrographs of a normally growing cell ofE. coli B/r. In a section (b), a distinct septum formation was recognized. Unless otherwise stated, bars indicate 100 nm. FIG. 4. Scanning electron micrographs of the filaments induced by treatment of 10 pg ofpiperacillin per ml for 90 min. Stretched constrictions with various diameters were observed in the middle of the filaments (a, b). Bars indicate 1 jim. (c) Magnification of the portion of stretched constriction. FIG. 5. Thin sections of stretched constrictions of E. coli B/r filaments induced by piperacillin. Constrictively growing septal wall and membrane continue to form the wall and membrane of the stretched part (a).
VOL. 14, 1978
INHIBITION OF CELL DIVISION BY PIPERACILLIN
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FIG. 6. Scanning electron micrograph offilaments with a bulge induced by treatment with 2 jg ofampicillin per ml.
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min at 37°C, incorporation of [3H]DAP was counted. As shown in Fig. 8, in piperacillintreated cells, DAP incorporation was not inhibited at concentrations as high as 100 /Lg/ml. This result shows piperacillin has no effect on peptidoglycan synthesis. In the case of ampicillin, on the other hand, DAP incorporation starts to be inhibited at 1 yg/ml and continues to decrease rapidly. This decrease, however, may be largely due to ampicillin-induced cell lysis. To examine the effects of piperacillin on cross90
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linking of peptidoglycan in vivo, the activity of transpeptidase, which is one of the catalytic enzymes in this reaction, was measured by use of [3H]DAP as a tracer. According to the method as described in Materials and Methods, the dimer/monomer ratio after treatment with various concentrations of piperacillin was almost the same as the control (Table 2). It indicates that piperacillin has no effect on cross-linking of peptidoglycan. The data given above show that this is effective in the inhibition of septum
120 (90IN2)drug formation, but has
60 60 ( M I NS )
FIG. 7. Effects of piperacillin (0.5 ug/ml) on cell division in synchronous culture of E. coli B/r. At different cell ages the drug was added to the culture (arrows), and at 5-min intervals the number of cells was determined.
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peripheral wall. As a result, filaments are formed. Effects of piperacillin on the penicillin-
binding proteins. To find the target protein of this drug, its competition with benzyl-["4C] pen-
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VOL. 14, 1978
icillin was tested according to the Spratt method using slab gel electrophoresis as described in Materials and Methods. The results are shown in Fig. 9. The pattem of seven PBPs with molecular weights of 91,000 (PBP 1) to 32,000 (PBP 7) was obtained from the inner membrane of E. coli B/r, but proteins 5 and 6 were not fully resolved on this gel. Prebinding of 0.05 ,ug of piperacillin per ml completely blocked the subsequent binding of benzyl-['4C]penicillin of PBP 3. Furthermore, 0.75 ,ug of the drug per ml blocked the binding of benzyl-['4C]penicillin to PBPs 2 and 7. Above the concentration of 10 ,ug/ml, the drug inhibited binding to PBP 1. This
pattern with respect to PBPs 1, 2, and 3 resembles the one obtained by prebinding of ampicillin (Fig. 10) (18). Piperacillin is different from ampicillin in its morphological effects in that the former causes filaments without bulges to form while the latter causes ones with a bulge to appear.
DISCUSSION Antibacterial effects of piperacillin, a derivative of ampicillin, on E. coli B/r clearly differs from those of ampicillin: (i) piperacillin does not cause lysis in a range of concentrations below 50 jig/ml and shows mild lytic activity above this level, whereas ampicillin shows rapid lytic action in low concentrations (above 2.0 jug/ml) though the action of 1 jig/ml is mild; (ii) at sublethal concentrations, piperacillin blocks septation and induces the cells to form filaments which have no bulge, whereas ampicillin at sublethal concentrations up to and including 1 jig/ml causes a characteristic bulge in the filaments which is a fore-stage of cell lysis (17). In some of the filaments induced by piperacillin, stretched constrictions with various diameters are observable at the middle of the filaments by a scanning electron microscope. The number of stretched constrictions in the filaments is almost equal to that of septal constrictions in the normally growing cells. This fact indicates hypothetically that piperacillin inhibits septation, resulting in fila-
TABLE 1. Effects of piperacillin on the synchronous culture of E. coli B/ra Time (min)
% Filaments with a % Separated celLs stretched constriction'
0.3 0 0.5 6.9 7.0 20 30 36.0 35.9 35 54.3 51.1 a About 300 cells were examined at each time by a scanning electron microscope, and the number of septated cells or filaments with a stretched constriction was estimated. b Filaments with a stretched constriction were estimated after 120 min of exposure to piperacillin (0.5 ig/ml), which was added at the times shown in the table.
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FIG. 8. Dose response of piperacillin on the peptidoglycan synthesis of E. coli W7. Cells in M9 medium
containing lysine, DAP and /H]DAP were exposed to various concentrations ofpiperacillin (0) or ampicillin (-) for 90 min at 37°C. Cold acid-insoluble fraction was collected and counted.
264
ANTIMICROB. AGENTS CHEMOTHER.
IIDA ET AL.
ment formation, and, more specifically, that addition of the drug to septum-forming cells leads to the filaments with a stretched constriction. The hypothesis is strongly supported by the results obtained from examinations using the TABLE 2. Assay of transpeptidase in E. coli W7a Counts per min Dimer
Monomer
Ratio: dimer/monomer
11,620
13,190
0.88
Addition (,ug/ml)
None
Piperacillin 0.84 10 6,900 8,170 0.79 25 5,700 7,200 a Cells were incubated in M9 medium containing lysine, DAP, and [3H]DAP with or without the drug for 2.5 h and then treated with trichloroacetic acid, trypsin, and lysozyme. Peptidoglycan dimer and monomer were separated by paper chromatography and counted.
synchronous culture of E. coli B/r. The results obtained from measuring numbers of cells indicate that cessation of cell division was abruptly caused by addition of piperacillin at any stage of the cell cycle; the addition results in the formation of filaments without bulge. Scanning electron microscopy shows that there is a good correlation between the population of septated cells at each stage of the synchronous culture and that of the filaments with a stretched constriction induced by piperacillin added at the respective stage. In thin sections of the stretched constriction, the constrictively growing septal wall and membrane continue to form the wall and membrane of the stretched part without breakage. On the other hand, it was clarifled that the cell wall peptidoglycan is synthesized continuously in the existence of piperacillin. It is likely that the drug has no effect on the longitudinal growth of peripheral wall, nor any effect on the
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VOL. 14, 1978
INHIBITION OF CELL DIVISION BY PIPERACILLIN
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FIG. 10. Competition of piperacillin and ampicillin for benzyl-[4C]penicillin binding. Membranes were preincubated with a range of concentrations ofpiperacillin or ampicillin, and the binding proteins remaining accessible to benzylpenicillin were detected by the addition of benzyl-[4C]penicillin. The levels of benzyl['4C]penicillin bound to each of PBPs 1, 2, and 3 were measured by microdensitometry of the X-ray film. Symbols: A, protein 1; Q, protein 2; U, protein 3.
synthesis of wall precursors. Both growth and synthesis may be mediated by piperacillin-insensitive enzymes. But piperacillin has a decisive effect on the enzymes involved in septation that seem to be sensitive to the drug. Consequently, the cells are elongated in filaments without any alteration of the cell structure. The stretched constriction might be due to the longitudinal growth of the wall at the site of interruption of septation by contact with the drug. Recently, six to seven PBPs were identified in the cytoplasmic membrane of E. coli by Spratt, who developed a convenient method for detection of PBPs by fluorography after fractionation of sodium dodecyl sulfate-polyacrylamide slab gel (20). At the present time, the enzymic properties of these PBPs are only partially known. It has been shown that PBPs 5 and 6 correspond to D-alanine carboxypeptidase IA (24, 25), but the enzymic properties of the others are unknown. In our experiment, PBP 3 was the most sensitive to piperacillin, with PBPs 2 and 7 being moderately so. It has been shown that PBP 3 is the target at which penicillins bind to inhibit cell division and cause filament formation (18, 21). PBP 2 is demonstrated to be the target at which mecillinam binds to convert the cells to osmotically stable ovoid cells. PBP 2 is also shown to be essential for the maintenance of cell shape (23). Furthermore, bulge formation induced by ampicillin and its closely related derivatives is considered to be due to the combined effects of the inhibition of PBPs 2 and 3 (19). Piperacillin, which induces filaments without bulge, binds to PBPs 2 and 3. Therefore, it is unlikely that bulge formation is due only to the combined effects of the inhibition of PBPs 2 and
3, but it is likely that bulge formation is due to catalysis by other enzymes (murein hydrolases) which are not PBPs. The killing effect of penicillins seems to be due to cell lysis by activation of murein hydrolases when wall growth is disturbed by the drug (17, 27). However, it has not been established that any murein hydrolase is involved in penicillin's killing action. Moreover, there are many kinds of murein hydrolases. Carboxypeptidase IA corresponds to PBPs 5 and 6; carboxypeptidase IB does not correspond to any PBP (25). Carboxypeptidase II (2), which is most active during septation, and endopeptidase (8), whose activity increases just before cell septation, have not been clarified in their relation to PBPs yet. PBP IB has been proposed to be the target at which penicillins bind to inhibit cell elongation, but no evidence has as yet been obtained for the role of this protein in cell elongation (18, 22). In our experiments, PBPs IA and IB were sensitive to high concentrations of piperacillin; however, cells treated with 100 jig of the drug per ml still elongated and gradually lysed. If more than 100 ,ig of the drug per ml was added to the cells, the drug might inhibit cell elongation. Further study of the functions of PBPs in connection with murein hydrolases will be required to resolve the exact mechanism of the action of penicillin. ACKNOWLEDGMENTS We thank S. Kondo and Y. Hirota for generously donating strains of E. coli used in this study, and A. Takade for excellent technical assistance with the scanning electron microscopy.
LITERATURE CITED 1. Anderson, T. F. 1956. Electron microscopy of microorganisms, p. 177-240. In G. Oster and A. W. Polister
266 2.
3.
4. 5.
6.
7.
8. 9.
10.
11.
12. 13. 14.
15. 16.
ANTIMICROB. AGENTS CHEMOTHER.
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(ed.), Physical techniques in biological research, vol. 3. Academic Press Inc., New York. Beck, B. D., and J. T. Park. 1976. Activity of three murein hydrolases during the cell division cycle of Escherichia coli K-12 as measured in toluene-treated cells. J. Bacteriol. 126:1250-1260. Blumberg, P. M., and J. L. Strominger. 1974. Interaction of penicillin with the bacterial cell; penicillin-binding proteins and penicillin-sensitive enzymes. Bacteriol. Rev. 38:291-335. Bonner, W. M., and R. A. Laskey. 1974. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83-88. Burdett, L. D. J., and R. G. E. Murray. 1974. Septum formation in Escherichia coli: characterization of septal structure and the effects of antibiotics on cell division. J. Bacteriol. 119:303-324. Greenwood, D., and F. O'Grady. 1973. Comparison of the responses of Escherichia coli and Proteus mirabilis to seven /8-lactam antibiotics. J. Infect. Dis. 128:211-222. Greenwood, D., and F. O'Grady. 1973. The two sites of penicillin action in Escherichia coli. J. Infect. Dis. 128:791-794. Hakenbeck, R., and W. Messer. 1977. Activity of murein hydrolases in synchronized cultures of Escherichia coli. J. Bacteriol. 129:1239-1244. lida, K., and M. Koike. 1977. Effects of dihydroxymethyl-furatrizine on cell division of Escherichia coli. Microbiol. Immunol. 21:481-493. Izaki, K., M. Matsuhashi, and J. L. Strominger. 1966. Glycopeptide transpeptidase and D-alanine carboxypeptidase: penicillin-sensitive enzymic reactions. Proc. Natl. Acad. Sci. U.S.A. 55:656-663. Lennox, E. S. 1955. Transduction of linked genetic characters of the host by bacteriophage P 1. Virology 1:190-206. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Luft, J. H. 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9:409-414. Matsuhashi, S., T. Kamiryo, P. M. Blumberg, P. Linnett, E. Willoughby, and J. L. Strominger. 1974. Mechanism of action and development of resistance to a new amidino penicillin. J. Bacteriol. 117:578-587. Mitchison, J. M., and W. S. Vincent. 1965. Preparation of synchronous cultures by sedimentation. Nature (London) 205:987-989. Ngyen-Disteche, M., J. L. Pollock, J. M. Ghysen, J. Puig, P. Reynolds, H. R. Perkins, J. Coyette, and M. R. J. Salton. 1974. Sensitivity to ampicillin and
17. 18.
19.
20.
21. 22.
23. 24.
25.
26.
27.
28.
29.
cephalothin of enzymes involved in wall peptide crosslinking in Escherichia coli K12 strain 44. Eur. J. Biochem. 41:457-463. Schwarz, U., A. Asmus, and H. Frank. 1969. Autolytic enzymes and cell division of Escherichia coli. J. Mol. Biol. 41:419-429. Spratt, B. G. 1975. Distinct penicillin-binding proteins involved in the division, elongation, and shape of Escherichia coli K12. Proc. Natl. Acad. Sci. U.S.A. 72:2999-3003. Spratt, B. G. 1977. Penicillin-binding proteins of Escherichia coli: general properties and characterization of mutants, p. 182-190. In D. Schlessinger (ed.), Microbiology-1977. American Society for Microbiology, Washington, D.C. Spratt, B. G. 1977. Properties of the penicillin-binding proteins of Escherichia coli K12. Eur. J. Biochem. 72:341-352. Spratt, B. G. 1977. Temperature-sensitive cell division mutants of Escherichia coli with thermolabile penicillin-binding proteins. J. Bacteriol. 131:293-305. Spratt, B. G., V. Jobanputra, and U. Schwarz. 1977. Mutants of Escherichia coli which lack a component of penicillin-binding protein IA are viable. FEBS Lett. 79:374-378. Spratt, B. G., and A. B. Pardee. 1975. Penicillin-binding proteins and cell shape in E. coli. Nature (London) 254:516-517. Spratt, B. G., and J. L. Strominger. 1976. Identification of the major penicillin-binding proteins of Escherichia coli as D-alanine carboxypeptidase IA. J. Bacteriol. 127:660-663. Tamura, T., Y. Imae, and J. L. Strominger. 1976. Purification to homogeneity and properties of two Dalanine carboxypeptidases I from Escherichia coli. J. Biol. Chem. 251:414-423. Tipper, D. J., and J. L. Strominger. 1965. Mechanism of action of penicillins: a proposal based on their structure similarity to acyl-D-alanyl-D-alanine. Proc. Natl. Acad. Sci. U.S.A. 54:1131-1141. Tomasz, A., and S. Waks. 1975. Mechanism of action of penicillin: triggering of the pneumococcal autolytic enzyme by inhibitions of cell wall synthesis. Proc. Natl. Acad. Sci. U.S.A. 72:4162-4166. Ueo, K., Y. Hayashi, T. Yasuda, H. Taki, M. Tai, Y. Watafnabe, I. Saikawa, and S. Mitsuhashi. 1977. In vitro and in vivo antibacterial activity of T-1220, a new semisynthetic pericillin. Antimicrob. Agents Chemother. 12:455-460. Vanable, J. H., and R. Coggeshall. 1965. A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25:407-408.
Vol. 14, No. 2
Printed in U.S.A.
Inhibition of Cell Division of Escherichia coli by a New Synthetic Penicillin, Piperacillin KYOKO IIDA,' * SEIICHI HIRATA,2 SEIICHI NAKAMUTA,3 AND MASAATSU KOIKE' Department of Microbiology, Saga Medical School, Saga, Japan,' and Department ofMicrobiology, School
of Dentistr9 and Department of Urology, School of Medicine,3 Kyushu University, Fukuoka, Japan Received for publication 9 March 1978
The mechanism of the action of piperacillin against Escherichia coli was investigated. This drug converted cells to filaments, but did not show lytic action in a range of concentrations below 25 ,ig/ml. In some of the fiaments, stretched constrictions with various diameters were observed. Addition of piperacillin to a synchronous culture inhibited cell division immediately at any stage of the cell cycle. The results of morphological examination of synchronous cultures show that the percentage of filaments with a stretched constriction corresponds to that of normally septated cells before addition of the drug. Furthermore, peptidoglycan synthesis and cross-linking were not inhibited by this drug. It is likely that this drug inhibits only septum formation, but not the growth of wall, and that stretched constrictions are a result of longitudinal growth of septation caused by the drug. Examination of affinity of the drug to penicillin-binding proteins shows that protein 3 is the most sensitive, proteins 2 and 7 are moderately so, and protein 1 is sensitive only to high concentrations of the drug. It has been known that some penicillin-sensitive enzymes, e.g., D-alanine carboxypeptidase, transpeptidase, and endopeptidase, are involved in the terminal stages of peptidoglycan biosynthesis (10, 16, 26). In Escherichia coli, different penicillins or different concentrations of the penicillin show various morphological effects, e.g., filament formation, bulge formation, spheroplast formation, and osmotically stable ovoid-cell formation. The mechanism of these morphological changes could not be sufficiently explained by the inhibitory effect of penicillin on the peptidoglycan metabolism alone (3, 6, 7, 14, 17). Recently, seven penicillin-binding proteins from the cytoplasmic membrane of E. coli were identified as radioactive gel electrophoretic bands using benzyl-[14C]penicillin. A role of each of the proteins in the mode of action of fi-lactam antibiotics was detected by competition of binding affinity of radioactive benzylpenicillin with other fl-lactam antibiotics. The role was further detected by comparing the biological properties of the mutant strains having altered penicillinbinding protqins with those of the parent strains (19). However, it still remains unsolved as to which of the penicillin-binding proteins corresponds to the penicillin-sensitive enzymes of peptidoglycan metabolism, and as to how penicillin kills bacteria. A semisynthetic penicillin, sodium 6-[D(-)-a-(4-ethyl-2,3-dioxo-1-piperazinylcarbonyl-amino)-a-phenylacetamido] penicillanate (piperacillin [28], a derivative of ampi-
cillin), was developed by Toyama Chemical Co., Tokyo, Japan. We were interested, therefore, in investigating the relationship between morphological effects caused by piperacillin and its biochemical effects. The purpose of this experiment was to clarify the mode of action of penicillin. It has been demonstrated that cell division in E. coli B/r is proceeded by septation, including ingrowth of a distinct septum composed of the cytoplasmic membrane and the peptidoglycan layer (5). For the purpose of examining the precise effect of piperacillin on division, E. coli B/r was mainly used in this experiment. MATERIALS AND METHODS Organisms. The bacteria employed in this work were E. coli B/r, furnished by S. Kondo, Osaka University, Japan, and E. coli W7, which requires lysine and meso-2,6-diaminopimelic acid. The latter strain was kindly supplied by Y. Hirota, National Institute of Genetics, Misima, Japan. Media. Lennox broth (11) was used as the enriched medium. The minimal medium was a modified M9 medium (9). These media were solidified with 1.5% agar. Chemicals. Sodium 6-[D(-)-a-(4-ethyl-2,3-dioxo-
1-piperazinylcarbonyl-amino)-a-phenylacetamido] penicillanate (piperacillin, also known as T-1220) and ampicillin were supplied by Toyama Chemical Co., Tokyo, Japan. 3H-labeled diaminopimelic acid [3H]DAP; specific activity, 669 mCi/mmol) and benzyl[I4C]penicillin (specific activity, 55 mCi/mmol) were purchased from the Radiochemical Center, Amer-
sham, England. 257
258
IIDA ET AL.
Electron microscopy. Logarithmically growing cells of E. coli B/r treated with or without piperacillin were doubly fixed by 0.25% glutaraldehyde-5% acrolein mixture in 0.05 M cacodylate buffer solution (pH 7.0) for 3 to 6 h at room temperature and then by 1% OS04 in the same buffer for 1 h at room temperature (5). Fixed cells were divided into two aliquots; one was subjected to scanning electron microscopy and the other to thin sectioning. The technique for scanning electron microscopy was essentially that of Anderson (1). Droplets of the fixed cell suspension were sandwiched between the Formvar membrane and collodion membrane and were dehydrated through an alcohol series. Then the specimens were immersed in amylacetate and were dried by the critical-point method. Platinum-palladium was evaporated on the surface of the specimens, and they were examined by a JEM 100C electron microscope equipped with a scanning display device, type ASID-4D. For transparent electron microscopy, fixed cells were stained by 0.5% uranyl acetate. After dehydration through an alcohol series, the specimens were embedded in Epon 812 (13). Thin sections were made with a Reichert OmU2 ultramicrotome and then were stained by 0.4% lead citrate (29). The materials were examined with a JEM 100B electron microscope. Synchronous culture. A modified method of Mitchison and Vincent (15) was used to synchronize E. coli B/r growing in M9 medium. Logarithmically growing cells (about 3 x 108 cells per ml) in 50 ml of M9 medium were harvested by centrifugation. The pellet was suspended in 3 ml of M9 medium, 0.5 ml of which was layered onto 10 ml of a 2 to 12% linear gradient of sucrose dissolved in the medium. Then the pellet was centrifuged at 800 x g for 9 min at room temperature. The topmost fraction of the turbid band, containing about 5% of the total cells, was removed, immediately inoculated into 20 ml of prewarmed M9 medium, and then incubated with reciprocal shaking at 370C. For counting the number of cells, 0.2 ml of the culture was removed at 5-min intervals and was mixed with 0.2 ml of 10% formaldehyde solution. The number of cells was determined with a Coulter Counter model ZB using a 30-rum aperture. In this experiment, survival was equivalent to the number of cells counted with a Coulter Counter. Appearance of the cells at 0, 20, 30, and 35 min after synchronization was observed by a scanning electron microscope. For measuring the effect of piperacillin on the synchronous culture, the drug was added to each culture at 0, 10, 20, 30, 35, and 50 min after synchronization, and the number of cells of each culture was read at 5-min intervals for 120 min. After the addition of the drug at 0, 20, 30, and 35 min, each culture was incubated for 120 min and then was prepared for a scanning electron microscope. Incorporation of radioactive tracers. Cell wall synthesis was measured by the incorporation of [3H]DAP into E. coli W7. Exponentially growing cells in M9 medium supplemented with (per ml) 20 ,ug of lysine, 5 jig of DAP, and 0.4 uCi of [3H]DAP were exposed to various concentrations of piperacillin for 90 min at 37°C. Cold trichloroacetic acid-insoluble fraction was collected on a filter (Whatman, GF/C) and was counted in a liquid scintillation system. The scin-
ANTIMICROB. AGENTS CHEMOTHER.
tillation fluid was made up of 4 g of 2,5-diphenyloxide (Packard Instrument Co.) per liter of toluene. Transpeptidase assay. This assay was made according to the method of Matsuhashi et al. (14). A 0.1ml sample of overnight culture of E. coli W7 in M9 medium containing lysine and DAP was inoculated into 1.9 ml of the medium containing 1 ,iCi of [3H]DAP per ml. The culture was then incubated for 2.5 h with shaking at 32°C with or without piperacillin. The cells were treated with trichloroacetic acid, trypsin, and lysozyme. Finally, peptidoglycan dimer and monomer were separated by paper chromatography in isobutyric acid-I N NH3 (5:3); they were cut out and then counted in a liquid scintillation system. Detection of PBPs. Penicillin-binding proteins (PBPs) of E. coli B/r were detectea according to the Spratt method (20). Logarithmically growing cells in 10 liters of L-broth were harvested and resuspended in 200 ml of 50 mM sodium phosphate buffer (pH 7.0). The cells were then broken by an RIBI cell fractionator at 20,000 lb/in2. After unbroken cells were removed by differential centrifugation, the cell wall and membrane complex was sonically disrupted twice with a 30-s pulse. The complex was centrifuged at 100,000 x g for 40 min at 4°C, washed twice in the same buffer, and stored in a Revco -80°C freezer as envelope fraction containing cytoplasmic membrane, outer membrane, and peptidoglycan. Protein was determined by the method of Lowry et al. (12). A 20-pl volume of benzyl-[14C]penicillin (50 ,uCi/ml) was added to 200 pl of the envelope (about 10 mg of protein per ml), and the mixture was incubated at 30°C for 10 min. The binding was terminated by the simultaneous addition of 5 p1 of nonradioactive benzylpenicillin (120 mg/mi) and 10 ,ul of 20% sodium lauroyl sarcosinate solution. After the inner membrane was solubilized by standing for 20 min at room temperature, the outer membrane and peptidoglycan were removed by centrifugation at 100,000 x g for 40 min at 10°C. After the addition of 20 pl of 2-mercaptoethanol to 100 jil of the supernatant membrane, the mixture was heated for 3 min in a boiling water bath; 60 pi was then loaded into the gel slot of a 10% sodium dodecyl sulfate-polyacrylamide slab gel. Electrophoresis was performed at a constant current of 20 mA. The gels were prepared for fluorography as described by Bonner and Laskey (4), dried under vacuum, and placed on Kodak Nonscreen X-ray film at -80°C. After the envelope was incubated with each drug for 10 min at 30°C, benzyl-[14C]penicillin was added to it. The competition of binding to each of the proteins between the drug and benzyl-['4C] penicillin was then measured.
RESULTS Effects of piperacillin on the growth of E. coli B/r. Sensitivities of E. colt B/r to piperacillin and ampicillin were examined. Logarithmically growing cells in L-broth were exposed to various concentrations of piperacillin and ampicillin, and the survivals were determined by the plating method. When the inoculum size was as large as 3.6 x 108 cells per ml, cell growth was completely inhibited by a range of 0.5 to 25 jLg of
INHIBITION OF CELL DIVISION BY PIPERACILLIN
VOL. 14, 1978
piperacillin per ml; at more than 50 jug/ml, cell death mildly occurred. Ampicillin at 1 ,ug/ml completely inhibited cell growth, and at more than 2.5 ,tg/ml, cell death drastically occurred (Fig. 1A). In the case of small inoculum size such as 4.7 x 106 cells per ml, similar results were obtained (Fig. 1B). Kinetics of piperacillin and ampicillin on the growth of E. coli B/r were
0 .1
259
examined by measuring the optical density and survival of the culture at 15-min intervals after addition of these drugs (fig. 2). Cell division was inhibited soon after the addition of 10 ,ig of piperacillin per ml, but the optical density of the culture continued to increase. After a 120-min exposure to the drug, a droplet of the culture was examined under the phase microscope. Un-
0.5 1 2.5 5 10 25 50100
jg/ml of drugs
FIG. 1. Sensitivity of E. coli B/r to piperacillin and ampicillin. Logarithmically growing cells were incubated with shaking at 37°C for 120 min with various concentrations of piperacillin or ampicillin. The survivors were then counted by plating on L-agarplates. The original inoculum of cells was 3.6 x 108 cells per ml (A) or 4.7 x 106 cells per ml (B) as indicated by arrows. Symbols: O, piperacillin; E, ampicillin. 200
t
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>
I
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60
120
180
0 min
60
150
120
min
FIG. 2. Effect of piperacillin and ampicillin on the growth of E. coli Blr. Symbols: 0, control (without drug); *, 10 pg ofpiperacillin per ml; 100 pg ofpiperacillin per ml; A, 1 pg of ampicillin per ml; A, 2.5 pg of ampicillin per ml.
260
IIDA ET AL.
der these conditions, the cells formed filaments. Above 50 jig of the drug per ml, the cells began to lyse slowly. On the other hand, division of the cells treated with 1 jig of ampicillin per ml was inhibited soon after addition of the drug, but cell death occurred after a 120-min exposure. Above 2.5 ,ug of ampicilin per ml, cell death by lysis occurred rapidly. These results show that E. coli B/r is susceptible to both drugs, but the killing effect of piperacillin is not as strong as that of ampicillin in the cells of E. coli B/r, since there was a range of division inhibition doses in piperacillin. Morphological effects of piperacillin on E. coli B/r. Effects of piperacillin on the surface and shape of E. coli B/r were examined by a scanning electron microscope, and those on the intracellular structures were examined by a transparent electron microscope after thin sectioning. Figure 3a is a scanning electron micrograph of a normally growing cell. This cell shows a wavy surface, and septal constriction is observed in the middle. Septated cells were detected in about 28% of the total cell population. In a section of the normally growing cell, a distinct septum composed of cytoplasmic membrane and peptidoglycan was recognized (Fig. 3b). After an exposure to 10 jig of piperacillin per ml for 90 min, cells were converted to filaments, in 26% of which stretched constrictions with various diameters were observed in the middle of the filaments (Fig. 4). The percentage of the filaments with a stretched constriction is almost equal to that of septated cells in normally growing cells. In thin sections, a majority of the filaments have no sign of septum formation in the wall and membrane, but the remainder have stretched constrictions whose wall and membrane are joined with a constrictively growing septal wall (Fig. 5a, b). From these results, it may be considered that the stretched constrictions are produced by modification of septal constrictions by piperacillin. On the other hand, the filaments induced by a 90-min expsoure to 1 jig of ampicillin per ml contain three types of appearance: 12% of the filaments with a bulge, 16% with a stretched constriction, and 72% without any alteration. In the case of 2 jig of ampicillin per ml, about 48% of the filaments formed bulges and about 10% of them lysed (Fig. 6). Effects of piperacillin on synchronized culture. The action of piperacillin on cell divi-
ANTIMICROB. AGENTS CHEMOTHER.
sion was examined by measuring the number of cells in synchronous cultures of E. coli B/r growing in M9 medium and was also observed by scanning electron microscopy. Under these conditions, the doubling time was about 50 min, and a good synchronous culture was obtained for two generations. When 0.5 or 5 jig of piperacillin per ml was added to cells at various points in time after synchronization, no further division took place in any sample after the addition (Fig. 7). Similar results have been shown in synchronous cultures treated with sublethal doses of ampicillin (5). These experimental results indicate that piperacillin inhibits cell division immediately at any stage of the cell cycle. The filaments induced by piperacillin show two types of appearance, one with a stretched constriction and the other without it. It is likely that the morphological difference is due to the different stages of cell division cycle at which the drug was added. Numbers of the septated cells in the synchronous culture at 0, 20, 30, and 35 min were morphologically examined by a scanning electron microscope. The same morphological examination was done with respect to numbers of filaments with a stretched constriction, which was produced by a 120-min exposure to piperacillin added after each of the durations above (Table 1). Septated cells were rarely observed at the initiation of the synchronous culture, and their numbers rapidly increased at 30 to 35 min after synchronization. After each of the durations, the percentage of the filaments with the piperacillininduced stretched constriction corresponded to that of the septated cells. Addition of the drug at 0 min after synchronization caused 99.7% of the cells to form filaments without constriction; in contrast, an addition at 35 min induced 51% of the cells to form filaments with a stretched constriction. The foregoing implies that piperacillin inhibits the septum formation at any stage; in cells before septation, the drug inhibits the initiation of growth of the septum, and during septation the drug inhibits the growing of the septum immediately, which results in a stretched constriction. Effects of piperacillin on cell wall synthesis. Cell wall synthesis was measured by incorporation of [3H]DAP into acid-insoluble fraction of E. coli W7 as mentioned in Materials and Methods. Various concentrations of the drug were added to each culture. After incubation for
FIG. 3. Scanning (a) and transparent (b) electron micrographs of a normally growing cell ofE. coli B/r. In a section (b), a distinct septum formation was recognized. Unless otherwise stated, bars indicate 100 nm. FIG. 4. Scanning electron micrographs of the filaments induced by treatment of 10 pg ofpiperacillin per ml for 90 min. Stretched constrictions with various diameters were observed in the middle of the filaments (a, b). Bars indicate 1 jim. (c) Magnification of the portion of stretched constriction. FIG. 5. Thin sections of stretched constrictions of E. coli B/r filaments induced by piperacillin. Constrictively growing septal wall and membrane continue to form the wall and membrane of the stretched part (a).
VOL. 14, 1978
INHIBITION OF CELL DIVISION BY PIPERACILLIN
261
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ANTIMICROB. AGENTS CHEMOTHER.
FIG. 6. Scanning electron micrograph offilaments with a bulge induced by treatment with 2 jg ofampicillin per ml.
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min at 37°C, incorporation of [3H]DAP was counted. As shown in Fig. 8, in piperacillintreated cells, DAP incorporation was not inhibited at concentrations as high as 100 /Lg/ml. This result shows piperacillin has no effect on peptidoglycan synthesis. In the case of ampicillin, on the other hand, DAP incorporation starts to be inhibited at 1 yg/ml and continues to decrease rapidly. This decrease, however, may be largely due to ampicillin-induced cell lysis. To examine the effects of piperacillin on cross90
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linking of peptidoglycan in vivo, the activity of transpeptidase, which is one of the catalytic enzymes in this reaction, was measured by use of [3H]DAP as a tracer. According to the method as described in Materials and Methods, the dimer/monomer ratio after treatment with various concentrations of piperacillin was almost the same as the control (Table 2). It indicates that piperacillin has no effect on cross-linking of peptidoglycan. The data given above show that this is effective in the inhibition of septum
120 (90IN2)drug formation, but has
60 60 ( M I NS )
FIG. 7. Effects of piperacillin (0.5 ug/ml) on cell division in synchronous culture of E. coli B/r. At different cell ages the drug was added to the culture (arrows), and at 5-min intervals the number of cells was determined.
no
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the growth of
peripheral wall. As a result, filaments are formed. Effects of piperacillin on the penicillin-
binding proteins. To find the target protein of this drug, its competition with benzyl-["4C] pen-
INHIBITION OF CELL DIVISION BY PIPERACILLIN
VOL. 14, 1978
icillin was tested according to the Spratt method using slab gel electrophoresis as described in Materials and Methods. The results are shown in Fig. 9. The pattem of seven PBPs with molecular weights of 91,000 (PBP 1) to 32,000 (PBP 7) was obtained from the inner membrane of E. coli B/r, but proteins 5 and 6 were not fully resolved on this gel. Prebinding of 0.05 ,ug of piperacillin per ml completely blocked the subsequent binding of benzyl-['4C]penicillin of PBP 3. Furthermore, 0.75 ,ug of the drug per ml blocked the binding of benzyl-['4C]penicillin to PBPs 2 and 7. Above the concentration of 10 ,ug/ml, the drug inhibited binding to PBP 1. This
pattern with respect to PBPs 1, 2, and 3 resembles the one obtained by prebinding of ampicillin (Fig. 10) (18). Piperacillin is different from ampicillin in its morphological effects in that the former causes filaments without bulges to form while the latter causes ones with a bulge to appear.
DISCUSSION Antibacterial effects of piperacillin, a derivative of ampicillin, on E. coli B/r clearly differs from those of ampicillin: (i) piperacillin does not cause lysis in a range of concentrations below 50 jig/ml and shows mild lytic activity above this level, whereas ampicillin shows rapid lytic action in low concentrations (above 2.0 jug/ml) though the action of 1 jig/ml is mild; (ii) at sublethal concentrations, piperacillin blocks septation and induces the cells to form filaments which have no bulge, whereas ampicillin at sublethal concentrations up to and including 1 jig/ml causes a characteristic bulge in the filaments which is a fore-stage of cell lysis (17). In some of the filaments induced by piperacillin, stretched constrictions with various diameters are observable at the middle of the filaments by a scanning electron microscope. The number of stretched constrictions in the filaments is almost equal to that of septal constrictions in the normally growing cells. This fact indicates hypothetically that piperacillin inhibits septation, resulting in fila-
TABLE 1. Effects of piperacillin on the synchronous culture of E. coli B/ra Time (min)
% Filaments with a % Separated celLs stretched constriction'
0.3 0 0.5 6.9 7.0 20 30 36.0 35.9 35 54.3 51.1 a About 300 cells were examined at each time by a scanning electron microscope, and the number of septated cells or filaments with a stretched constriction was estimated. b Filaments with a stretched constriction were estimated after 120 min of exposure to piperacillin (0.5 ig/ml), which was added at the times shown in the table.
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FIG. 8. Dose response of piperacillin on the peptidoglycan synthesis of E. coli W7. Cells in M9 medium
containing lysine, DAP and /H]DAP were exposed to various concentrations ofpiperacillin (0) or ampicillin (-) for 90 min at 37°C. Cold acid-insoluble fraction was collected and counted.
264
ANTIMICROB. AGENTS CHEMOTHER.
IIDA ET AL.
ment formation, and, more specifically, that addition of the drug to septum-forming cells leads to the filaments with a stretched constriction. The hypothesis is strongly supported by the results obtained from examinations using the TABLE 2. Assay of transpeptidase in E. coli W7a Counts per min Dimer
Monomer
Ratio: dimer/monomer
11,620
13,190
0.88
Addition (,ug/ml)
None
Piperacillin 0.84 10 6,900 8,170 0.79 25 5,700 7,200 a Cells were incubated in M9 medium containing lysine, DAP, and [3H]DAP with or without the drug for 2.5 h and then treated with trichloroacetic acid, trypsin, and lysozyme. Peptidoglycan dimer and monomer were separated by paper chromatography and counted.
synchronous culture of E. coli B/r. The results obtained from measuring numbers of cells indicate that cessation of cell division was abruptly caused by addition of piperacillin at any stage of the cell cycle; the addition results in the formation of filaments without bulge. Scanning electron microscopy shows that there is a good correlation between the population of septated cells at each stage of the synchronous culture and that of the filaments with a stretched constriction induced by piperacillin added at the respective stage. In thin sections of the stretched constriction, the constrictively growing septal wall and membrane continue to form the wall and membrane of the stretched part without breakage. On the other hand, it was clarifled that the cell wall peptidoglycan is synthesized continuously in the existence of piperacillin. It is likely that the drug has no effect on the longitudinal growth of peripheral wall, nor any effect on the
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FIG. 9. Competition of piperacillin for the binding of benzyl-/ 4C]penicillin. According to the method described in the text, various concentrations of the piperacillin were prebound to envelope of E. coli B/r for 10 min, and residual binding of benzyl-['4C]penicillin to each of the PBPs was examined.
VOL. 14, 1978
INHIBITION OF CELL DIVISION BY PIPERACILLIN
265
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5
Piperacillin (pg/ml)
FIG. 10. Competition of piperacillin and ampicillin for benzyl-[4C]penicillin binding. Membranes were preincubated with a range of concentrations ofpiperacillin or ampicillin, and the binding proteins remaining accessible to benzylpenicillin were detected by the addition of benzyl-[4C]penicillin. The levels of benzyl['4C]penicillin bound to each of PBPs 1, 2, and 3 were measured by microdensitometry of the X-ray film. Symbols: A, protein 1; Q, protein 2; U, protein 3.
synthesis of wall precursors. Both growth and synthesis may be mediated by piperacillin-insensitive enzymes. But piperacillin has a decisive effect on the enzymes involved in septation that seem to be sensitive to the drug. Consequently, the cells are elongated in filaments without any alteration of the cell structure. The stretched constriction might be due to the longitudinal growth of the wall at the site of interruption of septation by contact with the drug. Recently, six to seven PBPs were identified in the cytoplasmic membrane of E. coli by Spratt, who developed a convenient method for detection of PBPs by fluorography after fractionation of sodium dodecyl sulfate-polyacrylamide slab gel (20). At the present time, the enzymic properties of these PBPs are only partially known. It has been shown that PBPs 5 and 6 correspond to D-alanine carboxypeptidase IA (24, 25), but the enzymic properties of the others are unknown. In our experiment, PBP 3 was the most sensitive to piperacillin, with PBPs 2 and 7 being moderately so. It has been shown that PBP 3 is the target at which penicillins bind to inhibit cell division and cause filament formation (18, 21). PBP 2 is demonstrated to be the target at which mecillinam binds to convert the cells to osmotically stable ovoid cells. PBP 2 is also shown to be essential for the maintenance of cell shape (23). Furthermore, bulge formation induced by ampicillin and its closely related derivatives is considered to be due to the combined effects of the inhibition of PBPs 2 and 3 (19). Piperacillin, which induces filaments without bulge, binds to PBPs 2 and 3. Therefore, it is unlikely that bulge formation is due only to the combined effects of the inhibition of PBPs 2 and
3, but it is likely that bulge formation is due to catalysis by other enzymes (murein hydrolases) which are not PBPs. The killing effect of penicillins seems to be due to cell lysis by activation of murein hydrolases when wall growth is disturbed by the drug (17, 27). However, it has not been established that any murein hydrolase is involved in penicillin's killing action. Moreover, there are many kinds of murein hydrolases. Carboxypeptidase IA corresponds to PBPs 5 and 6; carboxypeptidase IB does not correspond to any PBP (25). Carboxypeptidase II (2), which is most active during septation, and endopeptidase (8), whose activity increases just before cell septation, have not been clarified in their relation to PBPs yet. PBP IB has been proposed to be the target at which penicillins bind to inhibit cell elongation, but no evidence has as yet been obtained for the role of this protein in cell elongation (18, 22). In our experiments, PBPs IA and IB were sensitive to high concentrations of piperacillin; however, cells treated with 100 jig of the drug per ml still elongated and gradually lysed. If more than 100 ,ig of the drug per ml was added to the cells, the drug might inhibit cell elongation. Further study of the functions of PBPs in connection with murein hydrolases will be required to resolve the exact mechanism of the action of penicillin. ACKNOWLEDGMENTS We thank S. Kondo and Y. Hirota for generously donating strains of E. coli used in this study, and A. Takade for excellent technical assistance with the scanning electron microscopy.
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