Vol. 174, No. 14

July 1992, p. 4689-4700 0021-9193/92/144689-12$02.00/0

JOURNAL OF BACTERIOLOGY,

Copyright © 1992, American Society for Microbiology

A New Mercury-Penicillin V Derivative as a Probe for Ultrastructural Localization of Penicillin-Binding Proteins in Escherichia coli TERRY R.

PAUL,`* NOREEN G. HALLIGAN,2 LARRY C. BLASZCZAK,2 TOM R. PARR, JR.,2

AND TERRY J. BEVERIDGE' Department of Microbiology, College of Biological Sciences, University of Guelph, Guelph, Ontario, Canada NIG 2W1,7 and Lilly Research Laboratories, Eli Lill & Co., Lilly Corporate Center, Indianapolis, Indiana 46285

Received 21 January 1992/Accepted 28 April 1992

The precise ultrastructural localization of penicillin-binding protein (PBP)-antibiotic complexes in Escherichia coli JM101, JM1O1(pBS96), and JM1O1(pPH116) was investigated by high-resolution electron microscopy. We used mercury-penicillin V (Hg-pen V) as a heavy-metal-labeled, electron-dense probe for accurately localizing PBPs in situ in single bacterial cells grown to exponential growth phase. Biochemical data derived from susceptibility tests and bacteriolysis experiments revealed no significant differences between Hg-pen V and the parent compound, penicillin V, or between strains. Both antibiotics revealed differences in the binding affinities for PBPs of all strains. Deacylation rates for PBPs were slow despite the relatively low binding affinities of antibiotics. Cells bound most of the Hg-pen V added to cultures, and the antibiotic-PBP complex could readily be seen by electron microscopy of unstained whole mounts as distinct, randomly situated electron-dense particles. Fifty to 60%6 of the antibiotic was retained by cells during processing for conventional embedding so that thin sections could also be examined. These revealed similar electron-dense particles located predominantly on the plasma membrane and less frequently in the cytoplasm. Particles positioned on the plasma membranes were occasionally shown to protrude into the periplasmic space, thereby reflecting the high resolution of the Hg-pen V probe. Moreover, some particles were observed free in the periplasm, suggesting, for the first time, that a proportion of PBPs may not be restricted to the plasma membrane but may be tightly associated with the peptidoglycan for higher efficiency of peptidoglycan assembly. All controls were devoid of the electron-dense particles. The presence of electron-dense particles in cells of the wild-type JM101, demonstrated that our probe could identify PBPs in naturally occurring strains without inducing PBP overproduction.

Penicillin-binding proteins (PBPs) are a group of distinct enzymes that are responsible for catalyzing the terminal stages of peptidoglycan synthesis in virtually all eubacteria. PBPs control wall-mediated processes which are necessary for maintenance of cell morphology, wall elongation, and cell division (34). In Escherichia coli, seven of these PBPs have been characterized at the genetic and biochemical levels; however, only four, the higher-Mr PBPs, are indispensably involved in murein synthesis (24, 27). The precise physiological function of individual PBPs remains to be resolved. PBPs la and lb are essential for growth and for synthesis of peptidoglycan in the cylindrical portion of the cell, whereas PBPs 2 and 3 are involved in maintenance of the rod shape (24) and new wall synthesis at sites of cell division (26), respectively. The lower-Mr PBPs include PBP 4, which participates in the incorporation of new material into the existing murein sacculus, and PBPs 5 and 6, which regulate the number of pentapeptide side chains of peptidoglycan (27). PBPs 5 and 6 of E. coli are anchored to the membrane by carboxy-terminal sequences so that the bulk of the proteins are exposed to the periplasm (20, 21). Similarly, PBPs lb (28), 2 (17), and 3 (11) are believed to be periplasmic-exposed enzymes that are attached to the membrane through amino-terminal residues. Binding of 1-lactam antibiotics to one or more PBP species within a cell disrupts the delicate equilibrium of enzyme action within the growing *

wall, resulting in the inhibition of bacterial growth and induction of characteristic abnormal cell morphologies or frequently lysis (16, 26, 30). The localization of membrane proteins has usually relied upon cell disruption, membrane fractionation, and gradient sedimentation (23, 25, 29) to isolate various envelope fractions so that PBPs can be separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and compared with the various cell fractions. These methods have designated PBPs to reside primarily in the plasma membrane (25, 29), but in some cases the outer membrane (23) and intermembrane (1) fractions were also implicated. These findings may be explained by the detachment of PBPs from the plasma membrane because of the disruptive forces employed in membrane isolation, as well as plasma membrane contamination of other membrane fractions during separation procedures. Such studies on the structural and functional properties of bacterial macromolecules in isolation from their cellular domains may not provide an accurate perception of in situ activity, especially when cellular regulatory mechanisms are involved. Therefore, greater efforts to examine these components in situ are required. Conventional electron microscopic techniques so far have had limited success in the localization of membrane proteins. However, immunogold labeling has been shown to be a valuable technique for revealing the cellular distribution of murein hydrolases (13, 33) and has also localized thioredoxin (2) and PBP lb (3) on the plasma membrane, in the cytoplasm in close proximity to the plasma membrane, and on

Corresponding author. 4689

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PAUL ET AL.

membrane adhesion sites of E. coli. While this approach has offered a valuable insight into the ultrastructural localization of membrane proteins, the antigenic integrity of target proteins and their epitopes is difficult to maintain during the often harsh chemical treatments encountered during conventional embedding. Given that it is possible by electron microscopy (EM) to achieve a high resolution of biological specimens in the order of 1.0 to 1.5 nm, bacteria can be discerned at the macromolecular level and their membrane organization and internal structure are capable of being resolved. Guided by this principle, we introduce a novel approach for the identification and localization of PBPs in bacteria. We have studied the potential of EM to monitor the progress of a labeled antibiotic as it enters a single bacterial cell and interacts with its target sites. Antibiotic molecules tend to be indistinguishable from biological tissue when examined by EM, therefore it was necessary to chemically synthesize p-(chloromercuric)phenoxyacetamido penicillin (Hg-pen V) so that the mercury is covalently bonded to the side chain well away from the f-lactam nucleus to increase its electron density and promote visualization. Phenoxyacetamido penicillin (penicillin V) was chosen because of its chemical stability and broad PBP activity. By using Hg-pen V as our probe and high-resolution EM, we show that PBPs can be accurately distinguished in situ within single bacterial cells. MATERIALS AND METHODS Bacterial strains and culture conditions. The following E. coli strains were used: parental strain JM101 ATCC 33876 with the genotype (pro-lac) F- lacIlqZM15traDl (18), strain JM101 carrying plasmid pBS96 containing the PBP lb gene that codes for constitutive overproduction of PBP lb (7), and strain JM101 carrying plasmid pPH116 containing the PBP 3 gene that -codes for overproduction of PBP 3 following induction-with isopropyl-,-D-thiogalactopyranoside (IPTG) (8). Strains JM101(pBS96) and JM101(pPH116) were kindly suppliedc-by B. G. Spratt, University of Sussex, Brighton, England. All strains were cultivated under agitation (180 rpm) in Luria broth (LB), pH 7.0, at 37°C with the following modifications: strain JM101(pBS96) was grown in LB supplemented with kanamycin (25 ,ug/ml), strain JM101(pPH116) was grown in LB supplemented with kanamycin and 1 mM IPTG to induce plasmid amplification, and strain JM101 was grown in unmodified LB. Susceptibility testing. MICs of P-lactam antibiotics were determined by the tube dilution method by using MuellerHinton broth (Difco) supplemented with kanamycin, kanamycin and IPTG, or no antibiotic at all for the growth of strains JM101(pBS96), JM101(pPH116), and JM101, respectively. The MIC was defined as the lowest concentration of antibiotic which inhibited visible growth after 18 h of incubation at 37°C. All determinations were performed in triplicate and repeated on at least 10 separate occasions. Membrane preparation. Cells were grown to mid-exponential growth phase in LB medium (optical density at 600 nm, 0.6; path length, 1 cm), harvested by centrifugation (6,000 x g, 10 min), washed twice in 0.2 M sodium phosphate buffer, pH 7.0, then passed three times through a cold French pressure cell operating at 2.61 Pa, and then ultracentrifuged as described by Spratt (25) to obtain Sarkosyl (NL-30)soluble inner membrane fractions. Analysis of PBPs. Binding of ,B-lactams to membranes was performed by using the method of Spratt (25) with modifi-

J. BACTERIOL.

cations (22). A 100-,ul volume of membrane preparation (5 mg of protein per ml) was incubated with 10 ,ul of p-['"I] phenoxyacetamido penicillin (IPV) (specific activity, 37.3 Ci/mmol) for 10 min at 30°C (5). A stock solution containing 40 ,ug of IPV per ml was prepared and diluted for experimental use to give 15 to 20 p,g/ml. The binding reaction was terminated by the addition of 5 pl of cold penicillin V (120 mg/ml) and 10 PI of 20% (vol/vol) Sarkosyl (NL-30) and incubated for 20 min at room temperature. Solubilized inner membranes were separated by centrifugation (100,000 x g for 1 h at 4°C) and a 100-pl volume added to 50 ,ul of electrophoresis sample buffer. Proteins were separated by SDS-PAGE as described by Lugtenberg et al. (14) by using a 10% polyacrylamide gel. Dried gels were exposed to Kodak XAR-5 film with an intensifying screen (Dupont) at -80°C for 1 to 7 days. Competition experiments were performed to study the PBP binding affinities of unlabeled P-lactam antibiotics (25). A 100-,ul volume of bacterial membrane preparation was preincubated with 10 pl of buffer or dilutions of unlabeled j-lactam antibiotics for 10 min at 30°C before the addition of IPV, and then the samples were treated as described above. To determine the release of 13-lactam antibiotics from PBP-,B-lactam complexes, membranes were labeled with IPV (20 p,g/ml) for 10 min at 30°C, then an excess (120 mg/ml) of cold penicillin V or Hg-pen V was added, and samples were removed at intervals during incubation and mixed with SDS-PAGE solubilizing buffer. The relative band intensities on the fluorograms were determined by using a scanning densitometer with peak integration (Bio-Rad Laboratories Ltd., Mississauga, Ontario, Canada). Detection of Hg-pen V in bacterial cells. Metal contamination was minimized by using plasticware leached overnight in 7 N HN03 and then rinsed with high-resistance (-5 MfQ) deionized distilled water. Hg-pen V was added to midexponential-growth-phase cultures of E. coli agitated (180 rpm) for 90 min at 37°C. Cells were harvested by centrifugation (6,000 x g, 10 min), and then the culture supernatant was removed, filter sterilized (0.22-,um pore size; Millipore), and stored at 4°C prior to elemental analysis. Bacterial pellets were washed four times in 0.1 M HEPES (N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid; Research Organics, Inc.) buffer, pH 7.2, at 4°C. Each wash was carried out with 30 ml of buffer and retained for analysis. After washing, bacterial pellets were lyophilized and then digested with 1 ml of concentrated nitric acid for 18 h at room temperature. Analyses of solubilized cells, culture supernatants, and HEPES washes for Hg-pen V were performed with an inductively coupled plasma atomic emission spectrometer (ICP-AES) (LECO Instruments Ltd., Mississauga, Ontario, Canada) (6) by using mercury as an index of the Hg-pen V concentration. Mercury standards (LECO) were used to calibrate the spectrometer. To determine the amount of Hg-pen V retained by bacterial cells during conventional embedding, cultures were treated with Hg-pen V as described previously and then aliquots of cells were removed, washed with HEPES buffer to remove the unbound drug, and processed for conventional embedding. Samples of cells were collected after each processing stage and prepared for ICP-AES analysis as described above. Chemical reagents. IPV was prepared by iododestannylation of sodium p-(trimethylstannyl)phenoxyacetamido penicillin (Tin-penicillin V) by using a variation of the standard chloramine-T method (15) as described by Preston et al. (22). Penicillin V and Hg-pen V (Fig. 1) were synthesized at Eli

LOCALIZATION OF PBPs WITH MERCURY-PENICILLIN V

VOL. 174, 1992

B

A H H N~~

0s

-94

COO-

H

234-

-67 -43

phenoxyacetamido penicillin (penicillin V)

ClHg

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H N °

4691

2

3

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COO< H"'COO -

p-(chloromercuric)-phenoxyacetamido penicillin

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(Hg-pen V) FIG. 1. Chemical structures of ,B-lactam antibiotics.

-30 9

Lilly & Co. Hg-pen V was prepared by electrophilic transmetallation of Tin-penicillin V by using mercuric chloride and sodium acetate in acetone at room temperature (5). The heterogeneous mixture was stirred for 2 h, and the product was isolated by suction filtration and washed with acetone, ethyl acetate, and ether. The resulting penicillin V derivative revealed spectral profiles consistent with the assigned structure. [125I]Na (specific activity, 37.3 Ci/mmol) was obtained from ICN Biomedicals, Inc., Montreal, Quebec, Canada. EM. Cells were harvested by centrifugation (6,000 x g, 5 min) and washed twice in 0.1 M HEPES buffer (pH 7.2), and the pellet was suspended in 4% (vol/vol) glutaraldehyde (Marivac Ltd) in 0.1 M HEPES. Following 90 min of fixation at room temperature, cells were washed five times in 0.1 M HEPES buffer and postfixed for 2 h at room temperature in 2% (wt/vol) aqueous osmium tetroxide (Fisher). Cells were washed five times in 0.1 M HEPES buffer and then five times in 0.05 M HEPES buffer and fixed for 2 h in 2% (wt/vol) aqueous uranyl acetate (Fisher). After washing five times in distilled water, cells were pelleted, enrobed in 2% (wt/vol) Noble agar (Difco), and cut to form blocks 3 mm in length and 1 mm in diameter. Enrobed samples were dehydrated through a graded acetone series of 25, 50, 75, and 95% and three times in 100% acetone for 15 min each. Blocks were infiltrated overnight at room temperature in 100% acetoneEpon 812 resin (1:1), embedded in fresh resin, and polymerized at 60°C for 36 h. Bacteria prepared by the conventional embedding method were thin sectioned on a Reichert-Jung Ultracut E microtome and mounted on Formvar carbon-coated copper grids. Sections were poststained with uranyl acetate and then with lead citrate before examination. EM was performed on a Philips EM 300 electron microscope equipped with a liquid nitrogen cold trap at an operating voltage of 60 kV. RESULTS Binding of antibiotics by PBPs. The MICs of penicillin V or Hg-pen V for E. coli JM1O1(pBS96), JM1O1(pPH116), and the parent strain JM101 were identical, i.e., 32 ,ug/ml. PBP profiles revealed several characteristic PBPs with apparent molecular masses of 92, 72, 64, 47, 36, and 33 kDa (Fig. 2). PBP lb exists in three molecular forms, a, ,1, and y, in order of decreasing Mrs (19, 29). Its relative abundance in profiles of JM101 and JM1O1(pBS96) was evident by its labeling

10

11

13

12

14

15

16 -94

1b-

234-

-67 -43

56-

-30

17

15

19

ZU

21

22

2

24

FIG. 2. Competition of penicillin V (A) and Hg-pen V (B) for binding of IPV to PBPs of E. coli JM101 (top panels), JM101(pBS96) (middle panels), and JM101(pPH116) (bottom panels). Membranes were incubated with increasing concentrations of unlabeled ,3-lactam antibiotics prior to the addition of IPV. Labeled proteins were separated by SDS-PAGE and visualized by autoradiography. The final concentration of the competing 1-lactam antibiotics were 0 (lanes 1, 5, 9, 13, 17, and 21); 32 (lanes 2, 6, 10, 14, 18, and 22); 160 (lanes 3, 7, 11, 15, 19, and 23), and 320 (lanes 4, 8, 12, 16, 20, and 24) ±Lg/ml. Numbers to the left of each panel indicate the major PBPs visualized with IPV. Molecular mass (in kilodaltons) markers are located to the right of each panel. Exposure times were 5 to 7 days.

intensity and the size of the band, which probably is a composite of at least two of the polypeptide components (Fig. 2). PBP lb also masked visualization of PBP la, since shorter exposure times revealed two distinct, although weak bands representing PBPs la and lb (data not shown). PBPs 5 and 6 were not visualized by IPV in strains JM101 or JM1O1(pBS96). In profiles of JM1O1(pPH116), PBP 3 was the most abundant protein, as demonstrated by its relative labeling intensity (Fig. 2). IPV completely saturated all of the PBPs at 15 ,ug/ml within 10 min. The release of bound IPV from IPV-PBP complexes revealed that only 5 to 10% IPV was dissociated from PBPs lb and 2 during a 90-min period. Deacylation rates for PBP 3 were dependent on the strain and antibiotic type. Less than 15% of bound IPV was removed from PBP 3 of JM1O1(pBS96) and JM1O1(pPH116) for Hg-pen V and penicillin V, respectively. However, no more than 20% of IPV was released from PBP 3 under all other conditions in 120 min, despite the relatively poor affinity of this protein for both antibiotics. Deacylation rates for PBP 4 were relatively rapid for all strains and antibiotics.

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TABLE 1. Affinities of PBPs of E. coli strains for penicillin V and Hg-Pen V IC50 (Lg/ml)a

JM101

lb 2 3 4 5 6

Hg-pen V

Penicillin V

PBP

8.4 ND ND 6.2 ND ND

JM101(pBS96)

8.8 6.0

7.6" >50 ND ND

JM101(pPH116)

2.2 ND 4.3 ND 20 ND

JM101

9.6 ND 7,4b 40 ND ND

JM101(pBS96)

8.2 6.4

7.6" >50 ND ND

JM101(pPH116)

5.4 3.2 40 3.5 30 4.3

a Values are concentrations of ,B-lactam antibiotics required for 50% inhibition of binding of IPV to PBPs. ND, not detected (band intensity below the detection limit of the densitometer). I Values obtained from gels exposed for 24 to 48 h to distinguish PBPs 3 and 4.

To estimate the affinities of E. coli PBPs for penicillin, we incubated aliquots of membrane fractions with unlabeled penicillin V or Hg-pen V before labeling with IPV. Quantitation of IPV bound to each PBP was determined by densitometry, and drug concentrations producing 50% saturation of each PBP were calculated (Table 1). In general, both antibiotics revealed differences in binding affinities for PBPs between strains (Table 1). In strain JMlO1(pPH116), Hg-pen V possessed affinity for PBPs lb, 2, 4, and 6, whereas penicillin V bound PBPs lb and 3. In strain JM101, Hg-pen V bound PBPs lb and 3 and penicillin V bound PBPs lb and 4. Both antibiotics had affinities for PBPs lb, 2, and 3 of strain JM101(pBS96). 1-Lactam-induced lysis in E. coli. The bactericidal activities of penicillin V and Hg-pen V were determined for each strain. Generally, there was no significant difference in bactericidal activity between antibiotics or between strains (Fig. 3). Progressive lysis was apparent after 1 h of exposure to 5 or lOx MIC of both antibiotics. Penicillin induces bacteriolysis by disrupting the fine control of endogenous autolytic enzymes (32). Further evidence to support the belief that de novo protein synthesis was required for Hg-pen V-induced lysis was derived from experiments to study the effects of protein synthesis inhibition by chloramphenicol. Figure 4 shows the effects of chloramphenicol on penicillin V- and Hg-pen V-induced lysis on E. coli. The addition of 160 p,g of Hg-pen V per ml caused rapid cell lysis, whereas 130 p,g of chloramphenicol per ml inhibited growth. The simultaneous addition of chloramphenicol and Hg-pen V resulted in lysis to a magnitude similar to that with Hg-pen V alone. The complete inhibition of lysis was observed when chloramphenicol was added 1 h prior to the addition of Hg-pen V. Similar results were obtained when penicillin V-induced lysis of E. coli strains was compared. Detection of Hg-pen V in E. coli. To detect Hg-pen V by its mercury substituent and to determine quantitatively the antibiotic concentration bound to cells, we employed a highly sensitive analytical technique, ICP-AES. As little as 10 to 15 ng of mercury per ml can be detected by this system, which derives information from atomic spectra generated from the atomization of sample elements to form free atoms or ions (6). The percentage of Hg-pen V bound to cells is shown in Table 2. More than 94% of Hg-pen V added to cultures was bound to cells of all strains following extensive washing. Only trace amounts of antibiotic were found in culture supernatants, and no antibiotic was detected in successive washes. E. coli JM101, JM101(pBS96), and JM101(pPH116) bound 4.49, 4.38, and 4.64 ,ug of Hg-pen V per mg (dry weight) of cells, respectively.

ICP-AES was also used to monitor the amount of Hg-pen V retained by cells (by detecting the Hg substituent) during processing for conventional plastic embeddings. Table 3 shows the percentage of antibiotic retained by cells during different stages of conventional embedding. Generally, strains retained 70 to 80% of antibiotic added during fixation in glutaraldehyde, osmium tetroxide, or uranyl acetate. During dehydration, infiltration, and embedding in Epon, 50 to 60% of Hg-pen V remained bound by cells. It is likely that the mercury extracted during processing represented Hg-pen V-PBP complexes removed from the cell envelope. Examination of unstained whole mounts and thin sections of antibiotic-treated E. coli. To visualize penicillin in single bacterial cells, we relied on the relative atomic mass of the mercury atom in Hg-pen V to impart sufficient electron density in order to distinguish it from the cellular mass. Unstained, intact cells from cultures of strains JM101, JM101(pBS96), and JM101(pPH116) grown in the presence of penicillin V, Hg-pen V, and no antibiotic were examined by EM. Cells were cultivated in media containing antibiotic concentrations at 1, 5, and 1Ox MICs. Aliquots of culture were removed at 30-min intervals for 3 h, washed in HEPES buffer to remove unbound antibiotic, and prepared for whole mounts. Figures 5 to 13 show representative cells of E. coli cultures grown in the presence or absence of antibiotics. The most-distinct feature of antibiotic-treated cells was the progressive nature of lysis and the increasing presence of electron-dense particles within the cells. Bacteria underwent extensive lysis over time and with increasing concentrations of penicillin V or Hg-pen V. Lysis was evident by the leakage of cytoplasmic material and distortion of cell morphology (Fig. 6, 9, 10, 12, and 13). Electron-dense particles, 7.1 to 28.0 nm in diameter, were observed in 20 to 40% of Hg-pen V-treated cells of the overproducing strains, JM101(pBS96) (Fig. 10) and JM101(pPH116) (Fig. 13), as well as in the wild-type strain, JM101 (Fig. 7). Particles were apparent in cells exposed to all antibiotic concentrations studied. However, the number of particles per cell increased with the antibiotic concentration up to 5x MIC; thereafter, no further increase was observed. No distinct pattern of distribution was observed over each rod-shaped cell, but often clusters of five or more particles were randomly distributed in cells exposed to 5 or lOx MIC (Fig. 13). Particles were not observed in the external milieu but were always cell associated. No electrondense particles were present in penicillin V-treated or untreated bacteria (Fig. 5, 6, 8, 9, 11, and 12). For the examination of thin sections, all strains were grown under conditions described previously for the prepa-

VOL. 174, 1992

LOCALIZATION OF PBPs WITH MERCURY-PENICILLIN V

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FIG. 3. Killing kinetics of E. coli JM101 (top panels), JM101(pBS96) (middle panels), and JM101(pPH116) (bottom panels) during exposure to penicillin V (A; solid symbols) or Hg-pen V (B; open symbols) at lx MIC, (circles) 5x MIC (triangles), lOx MIC (diamonds), or no antibiotic (squares).

ration of unstained whole mounts, except that aliquots of culture removed at appropiate intervals were chemically fixed and embedded by a conventional protocol. Thin sections of Hg-pen V-treated cells of all strains revealed distinct electron-dense particles, 5.5 to 8.3 nm in diameter (Fig. 14 to 17). All particles were at cell peripheries, and most were located on the plasma membrane or free in the periplasm (Fig. 16) but rarely in the cytoplasm. The extreme high resolution of the mercury probe suggested that the outer face

of the membrane was preferred and that some PBPs extended from the membrane into the periplasmic space (Fig. 15, righthand arrowhead). Since a thin section possesses only a small fraction of the total cell mass, the number of particles per section was low, ranging from one to four, and was dependent on cell alignment within the section. Some lysed or partially lysed cells also demonstrated electrondense particles in close apposition to the plasma membrane, despite the loss of cytoplasmic constituents (data not

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B

A

TABLE 3. Percentage of Hg-pen V retained by E. coli cells during various processing stages (treatments) of conventional embedding

3-

% Hg-pen V added to the following 1-

strain:

Treatment of cells

JM101 JM101(pBS96) JM101(pPH116)

0.1 0.5

1

1.5

2

3

2.5

3.

3-

I1 11-

1.

0

G

l

Washing Glutaraldehyde fixing Osmium tetroxide fixing Uranyl acetate fixing Dehydration in % acetone: 25 50 75 95 100 100 100 Infiltration in acetone-Epon Embedding in Epon

97 88 86 80

97 73 79 75

98 84 80 76

65 61 63 60 55 51 52 50 51

62 64 64 53 60 58 62 61 54

69 66 64

58 61 55 51 50 49

0.1

0.5

1

1.5

2

2.5

0.5

3

1

1.5

2

2-5

3

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0.1 o

0.5

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1.5

2

2.5

3

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1

1.5

2

2.5

3

Time (h) FIG. 4. Effect of chloramphenicol on penicillin V (A)- or Hg-pen Time (h)

V (B)-induced lysis of E. coli JM101 (top panels), JM101(pBS96) (middle panels), and JM101(pPH116) (bottom panels). Chloramphenicol (130 ,ug/ml) and penicillin V (160 ,g/ml) or chloramphenicol (130 ,ug/ml) and Hg-pen V (160 pLg/ml) were added to exponentialgrowth-phase cultures at different times. Data are given for untreated cells (- [A] and O [B]); cells treated with penicillin V (A) or Hg-pen V (A) added at 0 min; cells with CAP added alone (O [A] and * [B]) and with penicillin V (V) or Hg-pen V (V) at 0 min; cells with CAP added at 0 min followed by penicillin V (*) or Hg-pen V (O) 30 min later; and cells with CAP added at 0 min followed by penicillin V (+) or Hg-pen V (x) 60 min later. OD6w, optical density at 600 nm.

shown). Penicillin V-treated and untreated bacteria devoid of electron-dense particles.

were

DISCUSSION

PBPs are associated with plasma membranes of eubacterial cells (25, 29). The localization of PBPs has usually been TABLE 2. Percentage of Hg-Pen V bound by E. coli JM101, JM101(pBS96), and JM101(pPH116) by using ICP-AES Sample

Cells Culture supernatant

Mean (SD) % Hg-pen V added

JM101 94 (5) 6 (0.5)

JM101(pBS96)

96 (2) 4 (0.7)

JM1O1(pPH116) 98 (4) 2 (0.3)

demonstrated by employing disruptive forces upon cells to yield membrane fractions which are solubilized in detergent solutions and separated by SDS-PAGE to reveal the membrane proteins (23, 25, 29). Procedures which employ large, heterogenous cell populations and perturb their individual integrity may not represent an accurate picture of structural and functional relationships of membrane proteins in situ, particularly when influenced by regulatory mechanisms which include topological restraints of enzyme distribution in the cell envelope. A significant departure from studies on isolated membranes to localize PBPs was established by the ultrastructural work of Bayer et al. (3). These workers demonstrated by the use of immunogold labeling that PBP lb was localized predominantly on the plasma membrane. Labeling of PBP lb sites was also found in the cytoplasm and on membrane adhesion sites of E. coli KN126 and in an overproducing strain containing plasmid pHK231. Despite the virtues of immunogold labeling, a few shortcomings which are difficult to resolve still exist. For example, the effects of steric hindrance generated by the size of the gold particle and by the molecules surrounding the target protein could influence the frequency of successful labeling. Given that an antibody will have access only to the exposed surface of a section and not to its inner regions, any attempts to quantitate labeling sites would be grossly underestimated (10). Moreover, the antigenic integrity of target proteins and their epitopes may have been altered from their native configuration during chemical fixation and embedding, thereby altering their antibody-binding capacity (4, 10). Our approach is different to that of immunogold labeling and circumvents many of its problems. By directly incorporating a heavy metal into an antibiotic, we managed to construct a very small, target-specific probe. We used EM to trace the progress of Hg-pen V as it entered single bacterial cells and reacted with its target sites, the PBPs. A mercurypenicillin derivative was synthesized, and a mercury atom covalently attached to the side chain of the ,B-lactam ring of penicillin V (Fig. 1). Since ,3-lactam antibiotics are covalently bound to PBPs forming a stable, inactive acylated complex, we believed this stable complex could be readily identified at the ultrastructural level through the electron density imparted by the mercury signature; this would be

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FIG. 5-7. Unstained whole mounts of E. coli JM101 prepared from cells grown in the absence of antibiotics (Fig. 5) or in the presence of lOx MIC of penicillin V (Fig. 6) or Hg-pen V (Fig. 7) for 3 h. Note the presence of electron-dense particles (arrowheads) in Fig. 7. Bar = 200 nm.

independent of PBP denaturation during EM processing. Electron-dense particles would then represent the actual location of PBPs. Before attempting labeling with Hg-pen V, it was necessary to obtain and compare MICs, binding efficiencies, and kinetical data with its parent compound, penicillin V. In general, these biochemical studies demonstrated few differences between Hg-pen V and penicillin V in E. coli. For example, PBPs possessed different affinities for both ,B-lactam antibiotics in each strain. Both antibiotics competitively inhibited the overproducing PBP lb in strain JM1Ol(pBS96); however, only penicillin V inhibited the overproducing PBP 3 in strain JMlO1(pPH116). We cannot explain why Hg-pen

V failed to inhibit PBP 3 in this strain. These differences would not affect the magnitude of Hg-pen V binding to cells because differences in PBP binding affinities could not be distinguished at the EM level with the Hg-pen V probe. The binding of ,-lactam antibiotics to PBP lb or both PBPs la and lb leads to rapid cell lysis in E. coli (31). Even though PBPs of our E. coli strains possessed relatively low binding affinities for Hg-pen V and penicillin V (Fig. 2; Table 1), the binding was sufficient to inhibit bacterial growth and induce lysis (Fig. 3) in all strains studied. Furthermore, experinfents with chloramphenicol demonstrated that de novo protein synthesis was essential for Hg-pen V- or penicillin V-induced lysis in E. coli. The binding of antibiotics to the

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FIG. 8-10. Unstained whole mounts of E. coli JM101(pBS96) prepared from cells grown in the absence of antibiotics (Fig. 8; bar = 200 nm), in the presence of lOx MIC of penicillin V (Fig. 9; bar = 200 nm) or Hg-pen V (Fig. 10; bar = 100 nm) for 3 h. Note the presence of electron-dense particles (arrowheads) in Fig. 10.

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FIG. 11-13. Unstained whole mounts of E. coli JM101(pPH116) prepared from cells grown in the absence of antibiotics (Fig. 11; bar = 200 nm) or in the presence of lOx MIC penicillin V (Fig. 12; bar = 200 nm) or Hg-pen V (Fig.13; bar = 100 nm) for 3 h. Note the presence of clusters of particles (small arrowheads) as well as single particles (large arrowheads) in Fig. 13.

bacterial cell wall is an essential prerequisite for its subsequent antimicrobial action (9). Accordingly, we demonstrated that Hg-pen V was strongly associated with exponential-growth-phase cells of all strains, despite extensive

washing, and was largely retained by cells throughout conventional processing for EM. EM examination of unstained exponential-growth-phase whole cells revealed the presence of electron-dense particles

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in 20 to 40% of the cell population of each strain. Particles were always cell associated, and control cells did not possess any particles. These findings together with the analytical data strongly suggest that the electron-dense particles contain Hg-pen V. We believe that a single electron-dense particle comprises numerous Hg-pen V-PBP complexes and that low copy numbers of PBPs within a single cell would not be readily visualized by our probe. Particle distribution did not correlate with a specific cell growth event such as cell constriction or cell elongation in any of our strains; instead, particles were randomly organized in single cells. Particles were also observed in cells of the wild-type strain, JM101, which demonstrated that our Hg-pen V probe can resolve PBPs in a naturally occurring strain without inducing PBP overproduction. Interestingly, clusters comprising at least five individual particles were often observed in a single location on a cell. These cells were not chemically fixed or negatively stained for EM; they were simply layered onto EM grids as whole mounts, and it is difficult to imagine that the clustering can be an artifact induced by drying. Particles embedded in membranes can move because of the lateralmotion attributes of bilayer phospholipids, but this is usually random, and, intuitively, because of the large plasma membrane expanses devoid of PBPs, this should lead to particle separation and not clustering. It is possible that clusters of single particles could comprise a single PBP species or multiple PBP species in close interaction to allow higher efficiency for de novo peptidoglycan incorporation. Complexes of some PBP lb molecules on plasma membranes of E. coli have been reported previously by den Blaauwen and

Nanninga (11). To determine the precise intracellular location of PBPs in a single cell, we examined thin sections of Hg-pen V-treated cells. It is not possible to retain all the cellular substance during the processing for conventional embeddings (12), and this is one of the problems of probing thin sections when immunogold is used; antigens can be extracted or denatured during the processing. Recognizing this, we relied on the ability of Hg-pen V to form a highly stable covalent bond with its associated PBP and then used glutaraldehyde to add additional covalent stability to the antibiotic-protein complex. Antigenicity need not be retained, but it does not matter, since the probe is already combined. For these reasons, we feel that thin sections possessing electron-dense particles of Hg-pen V show an accurate picture of antibioticPBP distribution within the cell envelope. Thin sections of Hg-pen V-treated cells revealed distinct electron-dense particles located predominantly on the plasma membranes and rarely in the cytoplasm. The precise protrusion of some plasma membrane-bound particles into the periplasmic space attests to a higher resolution accomplished with the Hg-pen V probe over other techniques. Our images provide ultrastructural evidence to support the fact that PBPs of E. coli are anchored to the periplasmic face of the plasma membrane so that most of the protein is exposed in the periplasmic space (11, 16, 20, 21, 27). Some particles were also observed free within the periplasm, which suggests that PBP distribution within this region is not exclusive to the plasma membrane and may be tightly associated with the

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peptidoglycan (33) in order to catalyze more efficiently cross-linking and insertion of new wall material into the growing multilayered peptidoglycan. Since no particles were located on the outer membrane, we believe it is unlikely that PBPs found in the periplasm were being transported to the outer membrane, as has been suggested previously (3), thereby supporting the distribution of PBPs in the periplasm. We have shown that PBPs can be identified in situ at the ultrastructural level by EM. Our probe endowed a highresolution image of the precise region of Hg-pen V-PBP complexes within a single bacterial cell. One of the questions that may now be addressed through our technique is whether PBPs are under spatial or temporal regulation or both during cell growth. Other heavy metal-labeled 1-lactam antibiotics which have better binding affinity for single PBPs could also be used to see whether they have specific cellular locations during specific stages of cell growth and division; some may be restricted to division sites or newly forming poles, whereas others could be distributed along the sides of each cell. ACKNOWLEDGMENTS This research was supported by a Medical Research Council of Canada University-Industry grant to T.J.B. The NSERC Guelph Regional STEM facility is partially supported by an infrastructure grant from the National Sciences and Engineering Research Council of Canada. We thank our industrial partners, Eli Lilly & Co. and Eli Lilly Canada, Inc., for their financial and in-kind support throughout this research and B. Spratt for his kind donation of bacterial strains. We thank Graham Spiers for performing the ICP-AES analysis. The microscopy was performed in the NSERC Guelph Regional STEM Facility located in the Department of Microbiology, the College of Biological Sciences, University of Guelph. REFERENCES 1. Barbas, J. A., J. Diaz, A. Rodriguez-Tebar, and D. Vaquez. 1986. Specific location of penicillin-binding proteins within the cell envelope of Escherichia coli. J. Bacteriol. 165:269-275. 2. Bayer, M. H., S. Haberer, and M. E. Bayer. 1988. Distribution of membrane-associated proteins: photo-cross-linking, postembedding-immunolabeling, p. 320-331. In P. Actor, L. DaneoMoore, M. L. Higgins, M. R. J. Salton, and G. D. Shockman (ed.), Antibiotic inhibition of bacterial cell surface assembly and function. American Society for Microbiology, Washington, D.C. 3. Bayer, M. H., W. Keck, and M. E. Bayer. 1990. Localization of penicillin-binding protein lb in Escherichia coli: immunoelectron microscopy and immunotransfer studies. J. Bacteriol. 172:125-135. 4. Bendayan, M., A. Nanci, and F. W. K. Kan. 1987. Effect of tissue processing on colloidal gold cytochemistry. J. Histochem. Cytochem. 35:983-996. 5. Blaszczak, L. C., N. G. Halligan, and D. E. Seitz. 1989. Radioiododestannylation. Convenient synthesis of a stable penicillin derivative for rapid penicillin binding protein (PBP) assay. J. Labelled Compd. Radiopharm. 27:401-406. 6. Boumans, P. J. W. M. 1987. Inductively coupled plasma emission spectroscopy. 1. Methodology, instrumentation and performance. John Wiley & Sons, Inc., New York. 7. Broome-Smith, J. K., A. Edelman, S. Yousif, and B. G. Spratt. 1985. The nucleotide sequences of the pon A and pon B genes

FIG. 14-17. Thin sections of cell envelopes of E. coli JM101 (Fig. 14), JM101(pBS96) (Fig. 15), and JM101(pPH116) (Fig. 16 and 17) grown in 10 x MIC of Hg-pen V for 45 min. The large particle (arrowhead, Fig. 16) located free within the periplasm probably comprises several small electron-dense particles that have congealed together to form a single mass. Arrowheads in Fig. 14, 15, and 17 indicate the location of smaller electron-dense particles. Bar = 100 nm.

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