Penicillin-Binding Proteins in Bacteria

Transcription

1 NTIMICROBIL GENTS ND CHEMOTHERPY, JUly 1980, p /80/ /10$02.00/0 Vol. 18, No. 1 Penicillin-Binding Proteins in Bacteria NFSIK H. GEORGOPPDKOU* ND FUNG Y. LIU The Squibb Institute for Medical Research, Princeton, New Jersey The penicillin-binding proteins (PBPs) of several gram-positive and gramnegative bacteria have been examined. The results indicate that: (i) PBPs are membrane proteins with molecular weights ranging from 40,000 to 120,000. When extracted with Triton X-100 from sonicated cells, they appear to fall into two patterns: one found in rods and the other in spheres. major difference is in the low-molecular-weight component, which is usually the major PBP in bacilli but a minor one in cocci. (ii) There is a wide variation in both the number and the amount of PBPs in different bacteria, and taxonomically related bacteria tend to have similar PBP patterns. These patterns often correlate with the affinity of PBPs for penicillin and other fl-lactam antibiotics. (iii) The low-molecular-weight component usually releases penicillin spontaneously with a half-life of 10 min or less. Most, but not all, PBPs release bound penicillin in the presence of neutral hydroxylamine (0.2 to 0.8 M). 148 It is generally accepted that /3-lactam antibiotics, such as penicillin, kill bacteria by inhibiting cross-linking of bacterial cell wall (32, 34). The exact relationship between inhibition and cell death is not clearly understood, but activation of autolysins probably plays an important role (24, 33).,B-Lactam antibiotics bind covalently to a number of membrane proteins (penicillin-binding proteins [PBPs]) (2). In the case of Escherichia coli, they do not bind to proteins other than PBPs (27). Most of the PBPs have no demonstrable enzymatic activity, but they nevertheless seem to perform important physiological functions. So far, such functions have been assigned to individual PBPs only for E. coli (26). Presumably, at least one of the PBPs is essential for normal growth and, therefore, the interaction of,b-lactam antibiotics with this component is responsible for the initiation of the bactericidal action. These essential PBPs are sometimes referred to as the "killing site" of the antibiotic. Thus far, microbiological and pharmacological studies have suggested different (usually highmolecular-weight) PBPs to be essential in different bacteria (6, 7, 14, 23, 29). That is, the killing site(s) may not be universal. It should be emphasized that studies with PBPs have been limited to a small number of gram-positive and gram-negative organisms. Thus, no generalizations could be made about interaction-inhibition profiles of f8-lactam antibiotics, which in turn might suggest potential killing site(s). The interaction of 8i-lactam antibiotics with their targets has also been studied through the isolation and study of penicillin-sensitive enzymes which are probably among PBPs. Obviously, a primary requirement for such studies is knowledge of enzymatic activity, and until recently, the only PBPs with demonstrable enzymatic activity had been D-la-D-la-carboxypeptidases. With a notable exception (14), these enzymes have not been found to be essential in the organisms studied (1, 15, 22, 29). The only studied PBP with substantial transpeptidase (cross-linking) activity has been recently isolated from Salmonella typhimurium (25). However, inferring from the taxonomically related E. coli (15, 22), we think that this PBP is very unlikely to be a killing site. lthough individual PBPs have been recently isolated by affinity chromatography (16, 25), with the exception of the low-molecular-weight components, they have been found to have no enzymatic activity. Prompted by the lack of a systematic study of bacterial PBPs and using the recently described method for PBP detection (28), we have examined the PBP patterns of several gram-negative and gram-positive bacteria. Such patterns (relative amounts and molecular weight distribution of PBPs present, sensitivity to different,8-lactam antibiotics) may be more significant than the number of PBPs, as the latter depends upon the experimental conditions (concentration and specific activity of penicillin G, incubation time, time of exposure of X-ray film). Thus, based on molecular weights, sensitivity to different classes of f8-lactam antibiotics, and ability to release penicillin in the presence or absence of hydroxylamine, we have been able to distinguish major PBP patterns in bacteria and different subgroups within each major pattern. The possible relevance of these findings to the action of penicillin is discussed.

2 VOL. 18, 1980 PENICILLIN-BINDING PROTEINS IN BCTERI 149 MTERLS ND METHODS Materials. Brain heart infusion (BHI) was obtained from Difco Laboratories (Detroit, Mich.); [8-14C]penicdilin G (50 mci/mmol) was obtained from mersham Corp. (rlington Heights, Ill.); penicillinase and ovalbumin (5x crystallized) were obtained from Calbiochem (La Jolla, Calif.); hydroxylamine hydrochloride, trichloroacetic acid, glycine, bromophenol blue, and 2,5-diphenyloxazole were obtained from Fisher Scientific Co. (Pittsburgh, Pa.); deoxyribonuclease, tris(hydroxymethyl)aminomethane (Tris) base, and Triton X-100 were from Sigma Chemical Co. (St. Louis, Mo.); phosphorylase was from Worthington Biochemicals Corp. (Freehold, N.J.); reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PGE), including SDS, were from Bio-Rad Laboratories (Richmond, Calif.); ribonucleic acid polymerase, bovine serum albumin, and trypsin inhibitor were from Boehringer Mannheim Corp. (New York, N.Y.); X-ray film (XR-5; 12.5 x 17.5 cm) from Eastman Kodak (Rochester, N.Y.). Cephalothin and mecillinam were kindly provided by Eli Lilly & Co. and Leo Pharmaceutical, respectively. Clavulanic acid was a gift of Richard B. Sykes. ll chemicals were reagent grade and were used as supplied. Doubly deionized water was used throughout the study. Organisms. Spirillum itersonii (TCC 11,331) and Spirillum serpens (TCC 11,335) were purchased from the merican Type Culture Collection. Streptomyces R61 was a gift from J. M. Ghuysen (University of Liege) while Staphylococcus aureus H was a gift from J. L. Strominger (Harvard University). ll other organisms were from the Squibb Culture Collection and, unless indicated otherwise, were clinical isolates. Media and culturing methods. Organisms, with the exception of those of Table 1, were grown in BHI medium at 37 C on a New Brunswick gyrotory shaker (280 rpm) as follows. The organism was grown in a 125-ml Erlenmeyer flask containing 25 ml of BHI for 16 h; the culture was then transferred into a 1,000-ml Erlenmeyer flask containing 250 ml of BHI and was grown for 4 h. Cells were harvested by centrifugation, washed once with 30 ml of 0.05 M K-PO4 buffer (ph 7.0) and, if not used immediately, were stored at -20 C. Yields ranged from 1 to 4 g of cells per 250 ml of medium. Preparation of membranes. Cells were suspended in 1 to 2 volumes of 0.05 M Tris-hydrochloride (ph 7.5) containing 10 M MgCl2 and 5 fig of deoxyribonuclease per ml. They were then sonicated in a model W-350 sonicator (Heat Systems-Ultrasonics Inc., Plainview, N.Y.); sonication applied at 50/s pulses) for the times indicated in Tables 2 and 3 with 5-min cooling intervals every 3 min. Sonic extracts were centrifuged at 45,000 x g (Sorvall RC-2B centrifuge; SS-34 rotor) for 30 min. Pellets were washed once with 20 ml of the above buffer and, if not used immediately, were stored at -20 C. Solubilization of membranes. The pellets from the previous step were homogenized in 2 to 3 volumes of 0.05 M K-P04 buffer (ph 7.0) containing 1 M NaCl, 2% Triton X-100, and 1 mm,b-mercaptoethanol. The mixture was incubated at 0 C for 30 min, then centrifuged at 45,000 x g for 30 min. The supematant was used either immediately or was stored at -20 C and used within 1 to 3 days. In the latter case, PBPs were checked (SDS-PGE followed by fluorography) before and after storage to ensure that they remained unchanged. Protein determination. Protein was determined according to Lowry et al. (21; solution contained 1% SDS) with bovine serum albumin as standard. Binding of [14C]penicillin G. pproximately 100 ug of protein of Triton X-100 solubilized membrane was incubated with 0.5,uCi (10 nmol) of ['4C]penicillin at 30 C for 10 min. Four volumes of cold acetone were added, and the suspension was centrifuged at 7,000 x g for 10 min. Supernatants were carefully pipetted, and pellets were air dried. Proteins were separated by SDS-PGE, and those that bound [14C]penicillin G were detected by fluorography. Binding of other,-lactam antibiotics. pprox- TBLE 1. Conditions, other than standard, for growing bacteria Incubation time (h) Organism Medium Temp ( C) Firt Second Remarks culture culture Spirillum itersonii Spirillum serpens Peptone-succinate-salts Static culture cinetobacter calcoaceticus BHI Shaker Staphylococcus epidermidis Yeast-beef broth Shaker Streptococcus lactis BHI Static culture Nocardia lurida Yeast-beef broth Shaker Nocardia rhodochrous Streptomyces griseus Yeast-malt ex Shaker tract Streptomyces R61 Glycerol-casein Shaker (20)

3 150 GEORGOPPDKOU ND LIU imately 100,ug of solubilized membrane protein was incubated with the appropriate amount of fi-lactam antibiotic (freshly made aqueous solution; final concentration: 0, 0.1, 0.5, 2.0, 10, 100,ig/ml) at 30 C for 10 min. ["C]penicillin G (10 nmol) was added, and the incubations were continued for another 10 min. Protein was precipitated with acetone and subjected to SDS-PGE followed by fluorography. Release of bound penicillin. pproximately 100 jig of solubilized membrane protein was incubated with 10 nmol of ['4C]penicillin G at 300C for 10 min. total of 4,000 U of penicillinase was added to destroy unbound penicillin G (in a separate experiment it was found that 4,000 U of penicillinase hydrolyzed 10 nmol of penicillin G in less than 1 min), and the incubation was continued for 10, 20, or 50 min. To study hydroxylamine-induced release, the appropriate amount of neutral hydroxylamine (freshly made aqueous solution of NH20H * HCI, neutralized with NaOH final concentrations: 0, 0.2, 0.4, 0.8 M) was added after 20 min of incubation with penicillinase, and the mixture was incubated for 30 min. Protein was precipitated with acetone and subjected to SDS-PGE followed by fluorography. PGE. Polyacrylamide gels were prepared according to Laemmli (18). Stacking gel and running gel contained 5 and 10% acrylamide, respectively. Molecular weights were determined from mobility versus logarithm of M, curves. The standard proteins used for the construction of such curves were ribonucleic acid polymerase (a subunit = 39,000; 86 subunit = 155,000; 16' subunit = 165,000), phosphorylase (100,000), bovine serum albumin (68,000), ovalbumin (45,000), and trypsin inhibitor (21,500). Fluorography. For fluorography, gels were equilibrated with dimethyl sulfoxide, impregnated with 22.2% (wt/vol) 2,5-diphenyloxazole in dimethyl sulfoxide, soaked in water, dried, and exposed to X-ray film at -50 C (5, 19). Typical time of exposure was 20 days. PBP was determined by visual examination of the developed X-ray film. RESULTS NTIMICROB. GENTS CHEMOTHER. Molecular weights and subcellular locadon ofpbps. The PBPs of a variety of bacteria are shown in Tables 2 and 3. Molecular weights ranged from 40,000 to 120,000. The high-molecular-weight PBPs were invariably found in the membrane fraction (45,000 x g pellet) only, whereas some of the low-molecular-weight PBPs were also found in the soluble fraction (45,000 x g supernatant) as well. Membrane-bound PBPs, solubilized with nonionic detergent Triton X-100 in the presence of NaCl, appeared to retain their ability to bind to penicillin G. Both the total amount of PBPs and the amount of individual PBPs varied widely among bacteria, and taxonomically related bacteria had similar PBP pattems. This was especially true among enterobacteria where PBP pattem divergence closely paralleled taxonomic and evolutionary divergence (8). Interestingly enough, Proteus and Serratia (12) had slightly different patterns from the rest of enterobacteria. Penicillin-binding and release. To further characterize the PBPs in different bacteria, we determined their sensitivity to penicillin G. Solubilized membranes were preincubated with different -amounts of non-radioactive penicillin G and then incubated with [1'C]penicilhin G (see above). s shown in Fig. 1, the low-molecularweight component (36,000 in cinetobacter calcoaceticus) was relatively insensitive to penicillin G (more than 10,ug/ml for complete inhibition). In contrast, some of the 50,000-dalton PBPs of enterobacteria (corresponding to PBP 4 of E. coli) were sensitive to penicillin concentrations of less than 0.1,Ig/ml). The release of bound ['4C]penicillin G from solubilized membranes was studied next because this property can be utilized in the purification of PBPs by affinity chromatography (3). Furthermore, in a few cases studied in some detail (4, 17), such release has been shown to be enzymatic. The low-molecular-weight component of most bacteria (major PBP in rods) spontaneously releases bound penicillin G, usually achieving complete release in less than 20 min; (Fig. 1B shows a typical example). In contrast, some of the high-molecular-weight components do not release penicillin G even in the presence of 0.8 M hydroxylamine (Tables 4 and 5). Streptococcus lactis constitutes an exception; in that organism spontaneous release of bound penicillin G also occurs with the high-molecular-weight components. The lack of penicillin G release might be due to partial inactivation of these PBPs during solubilization of the membranes. Sensitivity to other f8-lactam antibiotics. To gain more information on PBPs of similar electrophoretic mobilities in different bacteria, we examined their sensitivity to cephalothin, clavulanic acid, and mecillinam. Given the 1,000- fold range in sensitivities of PBPs to,b-lactam antibiotics, such studies could be useful without needing to be very precise. The results for cephalothin are shown in Tables 4 and 5, and Fig. 2 shows an example. In gram-negative rods there was almost always a 65,000-dalton PBP that was highly sensitive to mecillinam (less than 0.1 jig/ ml). This PBP corresponds to PBP 2 reported to bind mecillinam and to be involved in the maintenance of cell shape in E. coli (26). The low-molecular-weight component (PBP 5/6 of E. coli, PBP 6 of Bacillus subtilis, etc.) was generally insensitive to cephalothin (more than 100,ug/ml); those of S. lactis (39,000) and Streptococcus faecalis (42,000) were notable exceptions. The sensitivity of other PBPs to cephalo-

4 VoL. 18, 1980 PENICILLIN-BINDING PROTEINS IN BCTERI 151 TBLE 2. PBP in gram-negative bacteria Sonication PBP- mol wta. Sonication PBP- mol Organism wta time (min) (x 103) Organism time (min) (x 103) Enterobacteriaceae Enterobacter cloacae (SC 10,441) Enterobacter faecalis Escherichia coli (SC 10,439) Klebsiella pneumoniae (SC 10,440) Proteus mirabilis (SC 9,571) Proteus vulgaris (TCC 8,427) Salmonella typhimurium (SC 3,821) Serratia marcescens (TCC 4,003) (3-68) (4-62) (3-68) (4-63) (3-66) (4-62) (5-51) (6-45) (4-65) (8-34) Pseudomonadaceae Pseudomonas aeruginosa (SC 11,003) Pseudomonas fluorescens (TCC 13,525) Spirillaceae Spirillum itersonii (TCC 11,331) Spirillum serpens (TCC 11,335) Neisseriaceae cinetobacter calcoaceticus (SC 8,333) Branhamella catarrhalis (SC 10,954) (4-71)b (5-58) (3-62) C 5-37" (6-30) (7-27) b (3-83) (4-78) d a Minor PBPs, observed at the 20,000- to 35,000-dalton range on SDS-PGE were usually not counted as such. They probably represent degradation products of the high-molecular-weight PBPs. b Major PBPs are underlined. Very minor PBPs (<1% of total PBPs) are enclosed in parentheses. c Major PBPs in whole cells but not solubilized membranes; partially soluble (i.e., found mostly in the supernatant after sonication and centrifugation), especially the 47,000-dalton PBP. d lthough the major PBP, it was less than 5% of the 40,000-dalton (major) PBP of a rod.

5 152 GEORGOPPDKOU ND LIU TBLE 3. Penicillin-binding proteins in gram-positive bacteria Sonication PBP- mol wta Sonication PBP- mol wta Organism time (min) (x 103) Organism time (min) (x 103) Bacillaceae Bacillus subtilis (TCC 6,633) Bacillus megaterium (SC 3,515) Nocardiaceae Nocardia lurida (NRRL 2,430) Nocardia rhodochrous (SC 2,318) Streptomycetaceae Streptomyces griseus (NRRL 3,851) Streptomyces R (5-71) 6_44b (2-79) 3-76 (4-56) (4-55) C (1-97) 2-90 (3-79) (4-51) (5-47) d Micrococcaceae Micrococcus luteus (TCC 9,341) Staphylococcus aureus (SC 2,399) Staphylococcus aureus H Staphylococcus epidermidis (SC 9,613) Streptococcaceae Streptococcus faecalis (SC 9,011) Streptococcus lactis (TCC 11,454) (4-53) (2-83) a Minor PBPs, observed in the 20,000- to 35,000-dalton range on SDS-PGE were not counted as such. They probably represent degradation products of the high-molecular-weight PBPs. 'Major PBPs are underlined. Very minor PBPs (<1% of total PBPs) are enclosed in parentheses. c bout one-fifth of regular amount of protein was applied on slap gel (about 20 ug). Therefore, only major PBPs could be detected. d Present in solubilized membranes but not whole cells. thin was variable, less so between related than between unrelated bacteria. In some cases, PBPs in the 90,000 to 100,000 range were highly sensitive to cephalothin (less than 0.1,ug/ml). The sensitivity of PBPs to clavulanic acid was rather unremarkable, ranging from 10,ug/ml to more than 100 jig/ml. DISCUSSION The survey of PBPs in clinically important bacteria was initiated in the hope of finding PBP systems of lower complexity than those already described, from which essential PBPs could be determined and enzymes could be isolated. However, preliminary experiments indicated that, as NTIMICROB. GENTS CHEMOTHER. a rule, fl-lactam binding systems were complex: PBPs usually ranged between five and seven. This was true even in the "higher bacteria" Nocardia and Streptomyces (Table 3). systematic exploration of electrophorectic PBP patterns and patterns of PBP interaction with f- lactam antibiotics was, therefore, undertaken. Such studies seemed to be prerequisite for isolating (by affinity chromatography) individual PBPs and eventually clarifying their mechanism of action. PBP patterns appeared to fall along taxonomical lines; in the case of enterobacteria they closely paralleled phylogeny as well (8). Generally, the low-molecular-weight (35,000 to 45,000)

6 VOL. 18, 1980 PENICILLIN-BINDING PROTEINS IN BCTERI 153 B: I*C..Iis=Y,T,=D,I.94k.1.. C PE MW (K).65 \ FRDYE 1::FRONJT f FIG. 1. Fluorograms of SDS slab gels of solubilized membranes from. calcoaceticus after ['4C]penicillin G binding. () Preceded by a 10-min incubation with 0.1, 0.5, 2.0, 10, and 100 pg of nonradioactive penicillin G per ml; (B) followed by a 10- (a), 20- (b), and 50- (c) min incubation after the addition ofpenicillinase or followed by a 20-min incubation after the addition ofpenicillinase and a 30-min incubation in the presence of 0.2 (d), 0.4 (e), and 0.8 (f) M NH20H. C indicates untreated control ([4C]penicillin G binding only). PBP was the major component in rods (underlined in Tables 2 and 3) and a minor one in spheres. Bacteria are shaped as rods (bacilli), spheres (cocci),, or helices (spirilla), although clinically important bacteria are mainly rods and spheres. For the sake of completeness, two helical bacteria, S. serpens and S. itersonii, were included in the initial survey. Both appeared to have a PBP pattern typical of rods. The correlation between the low-molecular-weight component and cell shape is intriguing. Carboxypeptidase, an enzymatic activity usually associated with this component, is very unlikely to be involved in cell shape (26). It should be noted that the results of the present study rest on the assumptions that (i) PBPs are independent of growth phase (2); (ii) PBPs are bound to membranes and not to cell walls (as are some autolysins [10]); (iii) PBP-binding activity is not substantially altered by solubilization in 2% Triton X-100 and 1 M NaCl..a: The use of solubilized membranes in studies of penicillin G binding and release and binding of other f,-lactam antibiotics provides conditions where permeability barriers between the antibiotic and its target(s) are absent. potential limitation of this type of experiment is that interaction with proteins other than PBPs cannot be studied since binding to proteins is determined from the resulting prevention of ["C]penicillin G binding. Penicillin G, although traditionally considered as the prototype of f8-lactam antibiotics, may not adequately represent all of them. One cannot a priori rule out the possibility that membrane proteins exist which bind f,-lactam antibiotics other than penicillin, but not penicillin itself. With respect to penicillin release, the lowmolecular-weight PBPs (carboxypeptidases) generally release penicillin (spontaneously) with a half-life of less than 1 h. In the few cases where the identity of the release products has been

7 154 GEORGOPPDKOU ND LIU NTIMICROB. GENTS CHEMOTHER Cq m m N m m N C C4 ~~~ o Ā i 0 V-4 doo o o C4 o o ci 6 r i - -f -4..4t L. C) I 0.0 CO F..0 2 I a..... * " Ġ) N.- 2. C * *u U "2: CO ul - cq C i 0co tr C) 0 00 e 400 c ea V o 44 _-C1O cḍ ri o m Q --- C)~I CO, CC CD t ~ _10QC ~ 1 - CN CO v U10C E- to.e L 00( V/ V V ~~66 66V V o In n.0 c0.0q 00 > X cs c LO to _ 0 10 _Cc4CO c 1I0t C) 08 C) c) " 000 _. 0 _ -4 COm v C - H _.0 e.0to T-.22 a) C 00 v co CO) CS O.0.0 _ C CO I" 1c0 ~-4 Cq CO Ul"100 C- ~=@.e ;t ~4 - I

8 VOL. 18, 1980 PENICILLIN-BINDING PROTEINS IN BCTERI 155 TBLE 5. Some properties of PBPs from gram-positive bacteria Complete inhibition of penicillin Complete release of bound Organism Protein no.' G binding by cephalothinb penicillin hydroxylamine in- (g/ml) ducedb (M required) B. subtilis O < B. megaterium > S. aureus <0.2 S. faecalis : S. lactis 1 10 < < <0.2 a PBP numbering corresponds to that of Table 3. b Estimated by visual inspection of X-ray films of slab gel. reported, slow release has been associated with fragmentation of the penicillin molecule (11, 13), and fast release has been associated with opening of the f8-lactam ring to give penicilloic acid (17, 31). Since both processes are enzymatic, the different modes of release may reflect differences in the architecture of the active site. Both types of carboxypeptidases, however, appear to be similar in their insensitivity to cephalothin (Tables 4 and 5). PBP patterns might be of value in correlating the effect of 8i-lactam antibiotics on individual PBPs with that on the organism. For example, the sensitivity of carboxypeptidase to penicillin correlates well with the sensitivity of the organism in the case of Gaffkya homari (14) but not B. subtilis (1). possible explanation for this anomaly might be that in the former organism, but not the latter, carboxypeptidase is in limiting amounts and serves an essential function. Variations in both the total amount and in the amount of individual PBPs that occur among bacteria might contribute to the phenomenon of intrinsic resistance. Correlating f8-lactam binding to a particular PBP with minimum inhibitory concentrations (in the absence of ft-lactamases and permeability barriers) would, therefore, depend not only on whether that particular PBP is essential per se, but also on whether all of the amount present is essential. In other words, the killing site for penicillin G (or for any B8-lactam antibiotic) might be different in different bacteria or in different mutants of a bacterium (29, 30), depending on the affinities and amounts of essential enzymes and proteins which bind to penicillin G. In conclusion, the described bacterial PBP systems can provide a basis for isolating individual components by affmiity chromatography and eventually studying their function. They also provide means for screening novel ft-lactam antibiotics under conditions where permeability barriers are absent. Finally, the differences in the amounts of PBPs among bacteria, observed in solubilized membranes, might indicate the importance of the relative amounts of each particular PBP for the lethal action of,b-lactam antibiotics. fter these studies were completed, a report appeared in the literature on S. faecalis PBPs (9). In that study, in which protoplasts of logphase cells were used, the 42,000-dalton com-

9 156 GEORGOPPDKOU ND LIU NTIMICROB. GENTS CHEMOTHER. MW (K) _/65 \ * _~~~~~~~~~~~~~~.....,o,v,~~~~~~~~~~~~~~~~~~~~~... * X ~~~~~' \ W \ t±w I& e&ei@;=e-4 I + w$ 10J I1O 0] C C:E- CL * ::: :.:. 68 I 'I"--.:,-L;~-.,:::!:-:.1 I I DYE 2 j010 1 tme FRONT FIG. 2. Fluorogram of an SDS slab gel of solubilized membranes from. calcoaceticus after a 10-min incubation with cephalothin (CE), clavulanic acid (CL), and mecillinam (ME) followed by [14C]penicillin G binding. ntibiotic concentrations were 0.1, 0.5, 2.0, 10, and 100 pg/ml. C indicates untreated control (14C]- penicillin G binding only). ponent (carboxypeptidase) appeared to be the major PBP and was suggested to be the killing site. The S. faecalis (SC 9,011) study was, therefore, repeated with protoplasts of log-phase cells. It was found that the 42,000-dalton component was not the major PBP, although it was substantially increased when compared to that from sonicated early stationary-phase cells. It is also unlikely that the 42,000-dalton component is an essential PBP, since it is sensitive to 0.1 jig of cefoxitin per ml, although the organism is not sensitive up to 100lg/ml (N.H.G. and M. Resnick, unpublished data). CKNOWLEDGMENTS We thank M.. Ondetti for his encouragement throughout this study and both him and J. L. Strominger for critical reading of the manuscript. We also thank F. rnow and R. Hugill (Microbiology Department) for supplying most of the cultures used in the study and for their helpful discussions. LITERTURE CITD 1. Bluamberg, P. M., and J. L. Strominger Inactivation of D-alanine carboxypeptidase by penicillins and cephalosporins is not lethal in Bacillus subtilis. Proc. Natl. cad. Sci. U.S.. 68: Blumberg, P. M., and J. L. Strominger Interaction of penicillin with the bacterial cell: penicillin-binding proteins and penicillin-sensitive enzymes. Bacteriol. Rev. 38: Blumberg, P. M., and J. L. Strominger Isolation by covalent affinity chromatography of the penicillin binding components from membranes of Bacillus subtilis. Proc. Natl. cad. Sci. U.S.. 69: Blumberg, P. M., R. R. Yocum, E. Willoughby, and J. L. Strominger Binding of ['4C]penicillin G to the membrane-bound and purified D-alanine carboxypeptidases from Bacillus stearothermophilus and Bacillus subtilis and its release. J. Biol. Chem. 249: Bonner, W. M., and R.. Laskey film detection method for tritium labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46: Buchanan, C. E., and J. L. Strominger ltered penicillin-binding components in penicillin-resistant mutants of Bacillus subtilis. Proc. Natl. cad. Sci. U.S.. 73: Chase,. H., S. T. Shepherd, and P. E. Reynolds Studies on the penicillin-binding components of Bacillus megaterium. FEBS Lett. 76: Cocks, G. T., and. C. Wilson Enzyme evolution in Enterobacteriaceae. J. Bacteriol. 110: Coyette, J., J.-M. Ghuysen, and R. Fontana Solubilization and isolation of the membrane-bound DD-carboxypeptidase of Streptococcus faecalis TCC Eur. J. Biochem. 88: Forsberg, C. W., and J. B. Ward N-cetylmuramyl-L-alaiine amidase of Bacillus licheniformis and its L-form. J. Bacteriol. 110: Frere, J. M., J.-M. Ghuysen, J. Degelaen,. Loffet,

10 VOL. 18, 1980 and H. R. Perkins Fragmentation of benzylpenicillin after interaction with the exocellular DD-carboxypeptidase-transpeptidases of Streptomyces R61 and R39. Nature (London) 258: Grimont, P.., and F. Grimont The genus Serratia. nnu. Rev. Microbiol. 32: Hammarstrom, S., and J. L. Strominger Degradation of penicillin G to phenylacetylglycine by D- alanine carboxypeptidase from Bacillus stearothermophilus. Proc. Natl. cad. Sci. U.S.. 72: Hammes, W. P Biosynthesis of peptidoglycan in Gaffkya homari: the mode of action of penicillin G and mecillinam. Eur. J. Biochem. 70: Iwaya, M., and J. L. Strominger Simultaneous deletion of D-alanine carboxypeptidase IB-C and penicillin-binding component IV in a mutant of Escherichia coli K12. Proc. Natl. cad. Sci. U.S.. 74: Kleppe, G., and J. L. Strominger Studies on the high molecular weight penicillin-binding proteins of Bacillus subtilis. J. Biol. Chem. 254: Kozarich, J. W., and J. L. Strominger membrane enzyme from Staphylococcus aureus which catalyzes transpeptidase, carboxypeptidase and penicillinase activities. J. Biol. Chem. 253: Laemmli, U. K Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: Laskey, R.., and. D. Mills Quantitative film detection of 3H and '4C in polyacrylamide gels by fluorogriphy. Eur. J. Biochem. 56: Leyh-]louille, M., J. Coyette, J.-M. Ghuysen, J. Idjak, H. R. Perkins, and M. Nieto Penicillin-sensitive DD-carb'- ypeptidase from Streptomyces strain R61. Biochemistry 10: Lowry, 0. H., N. J. Rosebrough,. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Matsuhashi, M., Y. Takagaki, I. N. Muryama, S. Tamaki, Y. Nishimura, H. Suzuki, U. Ogino, and Y. Hirota Mutants of Escherichia coli lacking in highly penicillin sensitive D-alanine carboxypeptidase activity. Proc. Natl. cad. Sci. U.S.. 74: Reynolds, P. E., S. T. Shepherd, and H.. Chase Identification of the binding protein which may PENICILLIN-BINDING PROTEINS IN BCTERI 157 be the target of penicillin action in Bacillus megaterium. Nature (London) 271: Rogers, H. J Killing of staphylococci by penicillins. Nature (London) 213: Shepherd, S. T., H.. Chase, and P. E. Reynolds The separation and properties of two penicillinbinding proteins from Salnonella typhimurium. Eur. J. Biochem. 78: Spratt, B. G Distinct penicillin-binding proteins involved in the division, elongation and shape of Escherichia coli K 12. Proc. Natl. cad. Sci. U.S.. 72: Spratt, B. G Properties of the penicillin-binding proteins of Escherichia coli K 12. Eur. J. Biochem. 72: Spratt, B. G., and. B. Pardee Penicillin-binding proteins and cell-shape in E. coli. Nature (London) 254: Suzuki, H., Y. Nishimura, and Y. Hirota On the process of cellular division in Escherichia coli: a series of mutants of E. coli altered in the penicillin-binding proteins. Proc. Natl. cad. Sci. U.S.. 75: Tamaki, S., J. Nakagawa, I. N. Maruyama, and M. Matsuhashi Supersensitivity to,b-lactam antibiotics in Escherichia coli caused by D-alanine carboxypeptidase I mutation. gric. Biol. Chem. 42: Tamura, T., Y. Imae, and J. L. Strominger Purification to homogeneity and properties of two D- alanine carboxypeptidases I from Escherichia coli. J. Biol. Chem. 251: Tipper, D. J., and J. L. Strominger Mechanism of action of penicillins: a proposal based on their structural similarity to acyl-d-alanyl-d-alanine. Proc. Natl. cad. Sci. U.S.. 64: Tomasz,., and S. Waks Mechanism of action of penicillin: triggering of the pneumococcal autolytic enzyme by inhibitors of cell-wall synthesis. Proc. Natl. cad. Sci. U.S.. 72: Wise, E. M., Jr., and J. T. Park Penicillin: its basic site of action as an inhibitor of a peptide crosslinking reaction to cell wall mucopeptide synthesis. Proc. Natl. cad. Sci. U.S.. 54:75-88.