SPRATT, BG

STROMINGER, JL

Journal of Bacteriology

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JOURNAL OF BACTERIOLOGY, JUlY 1976, P. 660-663Copyright © 1976 American Society for MicrobiologyVol. 127, No. 1Printed in U.S.A.Identification of the Major Penicillin-Binding Proteins ofEscherichia coli as -Alanine Carboxypeptidase IABRIAN G. SPRATT' AND JACK L. STROMINGER*Moffett Laboratories, Department ofBiochemical Sciences, Princeton University, Princeton, New Jersey08540, and The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138*Received for publication 14 April 1976Penicillin-binding proteins 5 and 6 of Escherichia coli have been identified asD-alanine carboxypeptidase IA.Beta-lactam antibiotics exert their lethal ef-fects on bacteria by inhibiting enzymes involvedin the terminal stages of peptidoglycan metabo-lism (2). Recently a more precise understand-ing of the interaction of penicillins with bac-terial cells has been achieved through thestudy of penicillin-sensitive enzymes (2, 4, 7)and penicillin-binding proteins (1, 2, 5-8; B.G. Sprott and A. B. Pardee, Fed. Proc. 34:668,1975). Since penicillins are thought generally toacylate the enzymes that they inhibit (2), peni-cillin-binding proteins and penicillin-sensitiveenzymes should in most cases be identical.In Escherichia coli, three enzymatic activi-ties are known to be penicillin sensitive (2): D-alanine carboxypeptidase I, peptidoglycantranspeptidase, and endopeptidase. D-alaninecarboxypeptidase I activities have recentlybeen purified from E. coli, and their propertieshave been described (5, 7, 8).Pure carboxypeptidase IA was a doublet withapparent molecular weights of 32,000 and34,000 on a sodium dodecyl sulfate (SDS)-poly-acrylamide gradient gel (8). The enzyme ismembrane bound, sensitive to beta-lactam an-tibiotics, and binds ['4C]benzylpenicillin re-versibly. It also catalyzes a transpeptidase re-action and is a weak penicillinase (8). Carboxy-peptidase IB is loosely membrane associatedand can be extracted with LiCl; an apparentlyidentical enzyme (carboxypeptidase IC) isfound in the cytoplasm in larger amounts. Thisenzyme is more sensitive to beta-lactam anti-biotics than carboxypeptidase IA; it has endo-peptidase activity but is only a weak transpep-tidase. It fails to bind ['4C]benzylpenicillin, al-though it is a weak penicillinase (8).Since carboxypeptidase IA is membranebound and binds ['4C]benzylpenicillin, it shouldbe detected in studies of the penicillin-bindingPresent address: Department of Genetics, University ofLeicester, Leicester LE1 7RH, England.proteins of the E. coli cell envelope. Six penicil-lin-binding proteins have been detected in theinner membrane of E. coli (5, 6). Binding pro-teins 5 and 6 have several properties whichsuggest that these proteins are identical withcarboxypeptidase IA. (i) The binding proteins(5 and 6) and carboxypeptidase IA are bothmembrane bound. (ii) Both bind['4C]benzylpenicillin and release the boundpenicillin with similar half-times (5 and 15 minat 30 C for binding proteins 5 and 6, respec-tively (B. Spratt, unpublished data) and 5 minat 37 C for carboxypeptidase IA (8). (iii) Bothare doublets on SDS-polyacrylamide gels withmolecular weights between either 32,000 and34,000 (10) or 40,000 and 42,000 (B. C. Sprattand A. B. Pardee, Fed. Proc. 34:668, 1975).Since the reported molecular weights of thecarboxypeptidase IA doublet and binding pro-teins 5 and 6 were rather different, we havecompared their mobility on the same SDS-poly-acrylamide slab gel. ['4C]benzylpenicillin wasbound to purified cell envelopes (prepared fromE. coli KN126-a K-12 strain-as previouslydescribed [5]) and to pure carboxypeptidase IA(prepared by Tamura et al. [8] from E. coliH2134-a K-12 strain), and the proteins werefractionated as described in the legend to Fig. 1.The gel was stained with Coomassie brilliantblue, and an autoradiograph was made. Bothcomponents of the carboxypeptidase IA doubletbound ['4C]benzylpenicillin (Fig. la, slot a),and the mobilities of the doublet were identicalto those of binding proteins 5 and 6 (Fig. la, slotc). The stained gel showed that the carboxypep-tidase 1A doublet (Fig. lb, slot a, b) corre-sponded to two minor bands in the inner mem-brane (Fig. lb, slot c).Figure 2 shows that the apparent subunitmolecular weights of the doublet and bindingproteins 5 and 6 were 40,000 and 42,000 whencompared with eight molecular-weight stand-ards. It is not clear why the molecular weight660NOTES 661a bcd ab cdI234a~ISla lbVOL. 127, 1976662 NOTESFIG. 1. (Slot a) Five microliters of ['4C]benzylpenicillin (31 pg/ml final concentration; 54 mCi/mM,Amersham/Searle) was bound to 50 p1 ofcarboxypeptidase IA (0.5 mg/ml in 0.05M sodium phosphate buffer,pH 7.0; 1% Triton X-100) for 10 min at 30 C. Binding was stopped by the addition of 5pl of unlabeledbenzylpenicillin (120 mg/ml) and 30 p1 ofSDS buffer (0.2 M tris(hydroxymethyl)aminomethane-hydrochlo-ride, pH 6.8; 3% SDS; 30% glycerol; 0.002% bromophenol blue). Twenty microliters was loaded on the gel.(Slot b) Ten microliters ofSDS buffer was added to 20 ,u of carboxypeptidase IA. Twenty microliters wereloaded on the gel. (Slot c) Twenty microliters of ['4C]benzylpenicillin (final concentration, 31 pg/ml) wasbound to 200 p1 ofpurified cell envelopes (20 mg/ml in 0.05 M phosphate buffer, pH 7.0) for 10 min at 30 C.Five microliters of unlabeled benzylpenicillin (120 mg/ml) was added, and the inner membrane wasselectively solubilized with Sarkosyl as described previously (5). One hundred microliters of the innermembrane was added to 50 p1 ofSDS buffer, and 55 p1 was loaded on the gel. (Slot d) Five microliters ofSDSbuffer was added to 100 p1 ofovalbumin (1 mg/ml; Sigma; molecular weight, 43,000), and 10 p1 was loadedon the gel. f3mercaptoethanol (10% final concentration) was added, the samples were heated to 100 C for 2min, and then applied to a 12% SDS-discontinuous polyacrylamide slab gel using the apparatus and buffersystem described by Laemmli and Favre (3). After electrophoresis, the gel was stained with Coomassiebrilliant blue, destained by diffusion, and dried. Electrophoresis is from top to bottom. (a) Autoradiographprepared by exposure of the gel to Ilford industrial G X-ray film for 30 days. (b) Photograph ofthe stained gel.The numbers on the left indicate the penicillin-binding proteins in slot c. Binding protein 1 appears as adoublet on this gel.J . BACTERIOL .NOTES 6631501000- 70xH50j 400 30204 8 12 16MOBILITY (CM)FIG. 2. Ten microliters ofD-alanine carboxypepti-dase IA (300 pg/ml in SDS buffer) was subjected toelectrophoresis on a 12% SDS-polyacrylamide slabgel together with 20 pi of a solution containing thefollowing standard proteins (each protein, 80 pg/ml): (1) ,3galactosidase (molecular weight,130,000), (2) phosphorylase a (molecular weight,100,000), (3) serum albumin (molecular weight,68,000), (4) pyruvate kinase (molecular weight,57,000), (5) ovalbumin (molecular weight, 43,000),(6) glyceraldehyde-3-phosphate dehydrogenase (mo-lecular weight, 36,000), (7) carbonic anhydrase (mo-lecular weight, 29,000), and (8) chymotrypsinogen(molecular weight, 25,700). The mobilities of theproteins are plotted against the log (molecularweight). Arrows show the position of the two com-ponents of D-alanine carboxypeptidase IA.reported here differs from one of the previousestimates (8), although different standard pro-teins and a different gel system were used.D-Alanine carboxypeptidase IA is identicalwith penicillin-binding proteins 5 and 6 in E.coli. It is not clear why the protein forms adoublet on SDS-polyacrylamide gels. The situa-tion in E. coli is similar to that in Bacillussubtilis: the major penicillin-binding proteincorresponds to a n-alanine carboxypeptidase(1). In both organisms the major carboxypepti-dase (IB in the case of E. coli) appears to beunimportant for cell growth (1, 5; M. Iwaya, T.Tamura, and J. L. Strominger, Fed. Proc. 33:1240, 1974). Minor penicillin-sensitive enzymesare probably involved in the lethal effect ofbeta-lactam antibiotics in these organisms (1,2, 5, 6).Supported by Public Health Service grant AI-09152 fromthe National Institute of Allergy and Infectious Diseasesand a grant from the National Science Foundation (GB-29747) to J.L.S., and by Public Health Service grantCA-11595 from the National Cancer Institute to A. B.Pardee. B.G.S. thanks A. B. Pardee for support and en-couragement.LITERATURE CITED1. Blumberg, P. M. 1974. Penicillin-binding componentsof bacterial cells and their relationship to the mecha-nism of penicillin action. Ann. N.Y. Acad. Sci.235:310-325.2. Blumberg, P. M., and J. L. Strominger. Interaction ofpenicillin with the bacterial cell: penicillin-bindingproteins and penicillin-sensitive enzymes. Bacteriol.Rev. 38:291-335.3. Laemmli, U. K., and M. Favre. 1973. Maturation of thehead of bacteriophage T4. 1. DNA packaging events.J. Mol. Biol. 80:575-599.4. Pollock, J. J., M. Nguyen-Disteche, J. M. Ghuysen, J.Coyette, R. Linder, M. R. J. Salton, K. S. Kim, H. R.Perkins, and P. Reynolds. 1974. Fractionation of theDD-carboxypeptidase-transpeptidase activities solu-bilized from the membranes of Escherichia coli K12strain 44. Eur. J. Biochem. 41:439-446.5. Spratt, B. G. 1975. Distinct penicillin-binding proteinsinvolved in the division, elongation, and shape ofEscherichia coli K12. Proc. Natl. Acad. Sci. U.S.A.72:2999-3003.6. Spratt, B. G., and A. B. Pardee. 1975. Penicillin-binding proteins and cell shape in E. coli. Nature(London) 254:516-517.7. Strominger, J. L., E. Willoughby, T. Kamiryo, P. M.Blumberg, and R. R. Yocum. 1974. Penicillin-sensi-tive enzymes and penicillin-binding components inbacterial cells. Ann. N.Y. Acad. Sci., 234:210-224.8. Tamura, T., Y. Imae, and J. L. Strominger. 1976. Puri-fication to homogeneity and properties of two D-ala-nine carboxypeptidases I from Escherichia coli. J.Biol. Chem. 251:414-423.24i I aVOL. 127, 1976II

Identification of major pencillin-binding proteins of Escherichia-Coli as D-alanine carboxypeptidase IA

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Identification of major pencillin-binding proteins of Escherichia-Coli as D-alanine carboxypeptidase IA

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