In Vitro Synthesis of Cross-linked Murein and Its Attachment to Sacculi by PBP1A from Escherichia coli

Abstract

The penicillin-binding protein (PBP) 1A is a major murein (peptidoglycan) synthase in Escherichia coli. The murein synthesis activity of PBP1A was studied in vitro with radioactive lipid II substrate. PBP1A produced murein glycan strands by transglycosylation and formed peptide cross-links by transpeptidation. Time course experiments revealed that PBP1A, unlike PBP1B, required the presence of polymerized glycan strands carrying monomeric peptides for cross-linking activity. PBP1A was capable of attaching nascent murein synthesized from radioactive lipid II to nonlabeled murein sacculi. The attachment of the new material occurred by transpeptidation reactions in which monomeric triand tetrapeptides in the sacculi were the acceptors. The penicillin-binding protein (PBP) 1A is a major murein (peptidoglycan) synthase in Escherichia coli. The murein synthesis activity of PBP1A was studied in vitro with radioactive lipid II substrate. PBP1A produced murein glycan strands by transglycosylation and formed peptide cross-links by transpeptidation. Time course experiments revealed that PBP1A, unlike PBP1B, required the presence of polymerized glycan strands carrying monomeric peptides for cross-linking activity. PBP1A was capable of attaching nascent murein synthesized from radioactive lipid II to nonlabeled murein sacculi. The attachment of the new material occurred by transpeptidation reactions in which monomeric triand tetrapeptides in the sacculi were the acceptors. Most bacteria have a murein (peptidoglycan) sacculus that forms an uninterrupted net-like structure around the cytoplasmic membrane. The sacculus is essential for the osmotic stability of the cell (1Weidel W. Pelzer H. Adv. Enzymol. 1964; 26: 193-232PubMed Google Scholar) and consists of glycan strands that are cross-linked by short peptides (2Höltje J.V. Microbiol. Mol. Biol. Rev. 1998; 62: 181-203Crossref PubMed Google Scholar). During growth and division, the sacculus is enlarged by the incorporation of lipid II precursor by a yet unknown mechanism, which involves the coordinated action of murein synthases, murein hydrolases, and probably regulatory proteins (3Vollmer W. Höltje J.V. Curr. Opin. Microbiol. 2001; 4: 625-633Crossref PubMed Scopus (82) Google Scholar). Escherichia coli contains six known murein synthases (4Goffin C. Ghuysen J.M. Microbiol. Mol. Biol. Rev. 1998; 62: 1079-1093Crossref PubMed Google Scholar, 5Suzuki H. Nishimura Y. Hirota Y. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 664-668Crossref PubMed Scopus (177) Google Scholar, 6Matsuhashi M. Wachi M. Ishino F. Res. Microbiol. 1990; 141: 89-103Crossref PubMed Scopus (52) Google Scholar, 7Di Berardino M. Dijkstra A. Stuber D. Keck W. Gubler M. FEBS Lett. 1996; 392: 184-188Crossref PubMed Scopus (49) Google Scholar, 8Schiffer G. Höltje J.V. J. Biol. Chem. 1999; 274: 32031-32039Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). However, it is not known what particular reaction these enzymes catalyze during the enlargement of the sacculus and, hence, which precise function they have in cell wall growth. Furthermore, most of the murein synthases from E. coli and other species are poorly or not characterized so far, perhaps because of difficulties in obtaining pure enzyme and lipid II substrate in sufficient quantities. The penicillin-binding proteins (PBPs) 2The abbreviations used are: PBP, penicillin-binding protein; HPLC, high pressure liquid chromatography; TG, transglycosylase; TP, transpeptidase.2The abbreviations used are: PBP, penicillin-binding protein; HPLC, high pressure liquid chromatography; TG, transglycosylase; TP, transpeptidase. PBP1A and PBP1B are the major bifunctional murein synthases in E. coli catalyzing both the oligomerization of the glycan strands and the formation of the peptide cross-links (9Kato J. Suzuki H. Hirota Y. Mol. Gen. Genet. 1984; 196: 449-457Crossref PubMed Scopus (23) Google Scholar, 10Nakagawa J. Matsuhashi M. Biochem. Biophys. Res. Commun. 1982; 105: 1546-1553Crossref PubMed Scopus (42) Google Scholar, 11Rojo F. Ayala J.A. De Pedro M.A. Vazquez D. Eur. J. Biochem. 1984; 144: 571-576Crossref PubMed Scopus (8) Google Scholar). Both PBPs form independent dimers in vivo (12Charpentier X. Chalut C. Remy M.H. Masson J.M. J. Bacteriol. 2002; 184: 3749-3752Crossref PubMed Scopus (28) Google Scholar). PBP1A and PBP1B are not essential for cell growth, but cells lacking both enzymes are not viable, indicating that both have a similar, essential function that cannot be taken over by other murein synthases (5Suzuki H. Nishimura Y. Hirota Y. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 664-668Crossref PubMed Scopus (177) Google Scholar, 13Spratt B.G. Jobanputra V. FEBS Lett. 1977; 79: 374-378Crossref PubMed Scopus (41) Google Scholar). Yet, mutants lacking either PBP1A or PBP1B show particular phenotypes, indicating that these synthases may play distinct roles during cell growth and division. For example, mutants without PBP1B are more sensitive to β-lactam antibiotics than mutants without PBP1A (14Yousif S.Y. Broome-Smith J.K. Spratt B.G. J. Gen. Microbiol. 1985; 131: 2839-2845PubMed Google Scholar). Furthermore, PBP1B-deficient mutants, but not PBP1A-deficient mutants, lose cell integrity upon inactivation of PBP2, PBP3, or the cell division protein FtsQ (15Garcia del Portillo F. de Pedro M.A. J. Bacteriol. 1990; 172: 5863-5870Crossref PubMed Google Scholar). The enzymatic activities of PBP1B with lipid II or artificial substrates have been studied previously (16Terrak M. Ghosh T.K. van Heijenoort J. Van Beeumen J. Lampilas M. Aszodi J. Ayala J.A. Ghuysen J.M. Nguyen-Disteche M. Mol. Microbiol. 1999; 34: 350-364Crossref PubMed Scopus (146) Google Scholar, 17Nakagawa J. Tamaki S. Tomioka S. Matsuhashi M. J. Biol. Chem. 1984; 259: 13937-13946Abstract Full Text PDF PubMed Google Scholar, 18Barrett D.S. Chen L. Litterman N.K. Walker S. Biochemistry. 2004; 43: 12375-12381Crossref PubMed Scopus (31) Google Scholar, 19Bertsche U. Breukink E. Kast T. Vollmer W. J. Biol. Chem. 2005; 280: 38096-38101Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). However, apart from two early studies from Matsuhashi and colleagues (20Ishino F. Mitsui K. Tamaki S. Matsuhashi M. Biochem. Biophys. Res. Commun. 1980; 97: 287-293Crossref PubMed Scopus (93) Google Scholar, 21Tomioka S. Ishino F. Tamaki S. Matsuhashi M. Biochem. Biophys. Res. Commun. 1982; 106: 1175-1182Crossref PubMed Scopus (15) Google Scholar), the activities of PBP1A remain uncharacterized. In this article, we report that PBP1A is capable of producing in vitro a cross-linked murein with glycan strands of (on average) almost 20 disaccharide units and with 18–26% of the peptides being part of cross-links. We have also studied the specificity of the PBP1A-catalyzed transpeptidation reaction. For the first time, we could demonstrate in vitro an attachment of nascent murein to isolated murein sacculi by a murein synthase, namely by PBP1A. E. coli Strains and Growth Conditions—XL1-Blue (Stratagene) and BL21(DE3) (Novagen) were used for cloning and PBP1A overproduction, respectively. All experiments were performed in Luria-Bertani (LB) medium. Kanamycin (50 μg/ml) was added to strains carrying overexpression plasmids. Materials—All chemicals were purchased from Sigma unless indicated otherwise. [14C]GlcNAc-labeled lipid II was prepared as published (22Breukink E. van Heusden H.E. Vollmerhaus P.J. Swiezewska E. Brunner L. Walker S. Heck A.J. de Kruijff B. J. Biol. Chem. 2003; 278: 19898-19903Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar), with modifications described previously (19Bertsche U. Breukink E. Kast T. Vollmer W. J. Biol. Chem. 2005; 280: 38096-38101Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), and had a specific activity of 9180 dpm/nmol. UDP-MurNAc-tripeptide, DP-MurNAc-tetrapeptide, and UDP-MurNAc-pentapeptide were prepared as published (23Kohlrausch U. Höltje J.-V. FEMS Microbiol. Lett. 1991; 62: 253-257Crossref PubMed Scopus (59) Google Scholar). Murein sacculi were prepared from E. coli MC1061 (24Casabadan M.J. Cohen S.N. J. Mol. Biol. 1980; 138: 179-207Crossref PubMed Scopus (1747) Google Scholar) as described (25Glauner B. Höltje J.V. Schwarz U. J. Biol. Chem. 1988; 263: 10088-10095Abstract Full Text PDF PubMed Google Scholar). Cellosyl was kindly provided by Hoechst AG, Frankfurt, Germany. Cloning of a mrcA Expression Plasmid—The mrcA gene encoding for PBP1A (GenBank™ accession no. AAA58193) was amplified by PCR from chromosomal DNA from E. coli wild type MC1061 using the primers 1A_up (5′-GGACGAATTCCCAAATGAAACTAAATGGG-3′) and 1A_down (5′-ATTGGATCCATCAGAACAATTCCTGTGCC-3′). The PCR fragment was digested with EcoRI and BamHI and cloned into the plasmid pJFK118EH (26Bishop R.E. Weiner J.H. FEMS Microbiol. Lett. 1993; 114: 349-354Crossref PubMed Scopus (24) Google Scholar), resulting in pTK1A. This plasmid was used as template in a PCR for the amplification of the mrcA gene with the primers PBP1Ahis_up (5′-GCGGCTAGCATGAAACTAAATGGGAAAT-3′) and PBP1Ahis_down (5′-CCTGAATTCATCAGAACAATTCCTGTGC-3′). After digestion with NheI and EcoRI, the fragment was cloned into the overexpression plasmid pET28a(+) (Novagen), resulting in pTK1Ahis. The correctness of the mrcA sequence in pTK1Ahis was confirmed by sequencing. Site-directed Mutagenesis—For the production of PBP1A-(TG*), with a Glu94 → Gln exchange in the first transglycosylase motif, pTK1Ahis was changed by site-directed mutagenesis using the primers 1A-TG*_up (5′-AGCCTTTATCGCGACACAAGACAGCCGCTTCTACG-3′) and 1A-TG*_-down (5′-TCGTAGAAGCGGCTGTCTTGTGTCGCGATAAAGGC-3′). For the purification of PBP1A(TP*), with a Ser473 → Ala exchange in the first transpeptidase motif, pTK1Ahis was changed by site-directed mutagenesis using the primers 1A-TP*_up (5′-TGCGTCAGGTGGGTGCCAACATCAAACCGTTCC-3′) and 1A-TP*_down (5′-AACGGTTTGATGTTGGCACCCACCTGACGCAGTGC-3′). Mutagenesis was performed according to the protocol of the manufacturer employing the QuikChange site-directed mutagenesis kit (Stratagene). The correct sequences of the inserts in the resulting plasmids, pTK1Ahis(TG*) and pTK1Ahis(TP*), were confirmed. Preparation of Cell Extracts—For the overproduction of the His-tagged form of the PBP1A variants, E. coli BL21(DE3) carrying the appropriate overexpression plasmid was grown in 4 liters of LB medium at 30 °C until an optical density (578 nm) of 0.3 was reached. The cells were induced with 0.05 mm isopropyl 1-thio-β-d-galactopyranoside (Gerbu, Gailberg, Germany). After 90 min of further growth, the cells were chilled on ice for 10 min and harvested by centrifugation (4200 × g, 20 min, 4 °C). The pellet was resuspended in 30 ml of 25 mm Tris/HCl, 10 mm MgCl2, 100 mm NaCl, 0.02% NaN3, pH 7.5. After addition of DNase and 1 mm phenylmethylsulfonyl fluoride, the cells were broken in a French pressure cell at 700 psi. After centrifugation (62,000 × g, 1 h, 4 °C), the pellet was resuspended in 25 mm Tris/HCl, 10 mm MgCl2, 0.02% NaN3, 1 m NaCl, 20% glycerol, pH 7.5, and stirred for 2 h at 6 °C. After another centrifugation (see above), the pellet was resuspended with 20 ml of 25 mm Tris/HCl, 10 mm MgCl2, 0.02% NaN3, 1 m NaCl, 2% Triton X-100, 20% glycerol, pH 7.5 (buffer A), and stirred for 18 h at 6 °C. The supernatant obtained after a third centrifugation (see above) contained the solubilized membrane proteins. Purification of PBP1A Variants—The purification was performed in two chromatography steps. First, 20 ml of membrane extract (see above) was incubated with 2 ml of washed and equilibrated Ni2+-nitrilotriacetic acid Superflow beads (Qiagen) for 2 h at 6°C. The beads were spun down (4200 × g, 30 min, 4 °C) and washed twice with buffer A containing 5 mm imidazole. His-tagged PBP1A variants were eluted with buffer A containing 500 mm imidazole for 30 min at 6 °C. The eluate was dialyzed against 25 mm Tris/HCl, 10 mm MgCl2, 0.02% NaN3, 1 m NaCl, 10% glycerol, pH 7.5, for 4 h. For the removal of the His tag, 16 units of thrombin (restriction grade, Novagen) were added, and dialysis continued for 16–20 h. Then the sample was dialyzed for 1.5 h against 2 liters of buffer I (10 mm sodium acetate, 10 mm MgCl2, 0.02% NaN3, 500 mm NaCl, 10% glycerol, pH 4.8) and for 16 h against buffer I containing 200 mm NaCl. The sample was diluted 1-fold with 10 mm sodium acetate, 10 mm MgCl2, 0.02% NaN3, pH 4.8, and the protein was purified on a 1-ml HiTrap SP HP column (GE Healthcare) in a 20-ml gradient from 100 mm NaCl and 0.2% Triton X-100 to 1 m NaCl and 0.2% Triton X-100. The PBP1A-containing fractions were dialyzed against 25 mm HEPES, 10 mm MgCl2, 0.02% NaN3, 200 mm NaCl, 10% glycerol, pH 7.5, and stored at –74 °C. The recombinant PBP1A obtained after removal of the His tag has an of six and has the was with as described previously M. F. PubMed Scopus (42) Google Scholar). and of PBP1A with II was and in 5 of The reaction was performed in a of in 25 mm Tris/HCl, 10 mm MgCl2, 100 mm NaCl, Triton X-100, pH 7.5 (buffer with PBP1A In the precursor UDP-MurNAc-tripeptide, or UDP-MurNAc-pentapeptide was added 1 In the transpeptidation of PBP1A was with G. The reaction was performed for 1 h at The pH of the was to to for 5 min by a digestion with for 1 h at and for 5 Time a of in buffer lipid II and PBP1A were incubated at 30 °C. and were taken and for 5 The were chilled on and digestion was as described In a glycan strands with monomeric peptide were with in a of for 1 h. The sample was for 10 min, and PBP1A was added by an for 10 min on After the addition of lipid II the course was performed as described of to was for 10 min on ice with murein sacculi from in buffer B. II was added, and the reaction in a of 100 for 1 h at The sacculi were by centrifugation × g, 10 and the pellet was washed twice with 200 of buffer A containing 0.2% Triton X-100. The pellet were digested for 18 h at °C with The of sample two and was by For the reaction was performed in a of 200 PBP1A was incubated for 10 min on ice with or without 1 sacculi II was added, and the reaction was performed for 1 h at 30 °C. After × g, 10 the were washed twice with 200 of buffer A containing 0.2% Triton X-100 and resuspended in 200 of 20 mm sodium pH The pH of the supernatant and of the was to 4.8, and the were digested with for 18 h at °C. were for 5 of the to the were with as described B. Biochem. 1988; 172: PubMed Scopus Google Scholar). The reaction were by according to a published (25Glauner B. Höltje J.V. Schwarz U. J. Biol. Chem. 1988; 263: 10088-10095Abstract Full Text PDF PubMed Google Scholar, B. Biochem. 1988; 172: PubMed Scopus Google Scholar), using a The were by with the of (19Bertsche U. Breukink E. Kast T. Vollmer W. J. Biol. Chem. 2005; 280: 38096-38101Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, B. Biochem. 1988; 172: PubMed Scopus Google Scholar). 1 could be to 2 either by or by with an by with (19Bertsche U. Breukink E. Kast T. Vollmer W. J. Biol. Chem. 2005; 280: 38096-38101Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). PBP1A to the A PBPs and is to the cytoplasmic membrane a Most of the enzyme is in the it contains a transglycosylase and a transpeptidase The of PBP1A is to that of PBP1B The in the transglycosylase and the in the transpeptidase were in both In PBP1B, and are essential for transglycosylation and transpeptidation (16Terrak M. Ghosh T.K. van Heijenoort J. Van Beeumen J. Lampilas M. Aszodi J. Ayala J.A. Ghuysen J.M. Nguyen-Disteche M. Mol. Microbiol. 1999; 34: 350-364Crossref PubMed Scopus (146) Google Scholar). The in PBP1A are Glu94 and Ser473 the enzymatic reactions of PBP1A, we cloned for the purification of PBP1A and of two variants with acid with a Glu94 → Gln exchange and with a Ser473 → Ala Purification the of membrane Ni2+-nitrilotriacetic acid of the tag by and exchange 2 the purified proteins and a β-lactam PBP1A and but not PBP1A(TP*), The activities of the PBP1A variants were studied with radioactive lipid II substrate employing a in vitro murein synthesis 19Bertsche U. Breukink E. Kast T. Vollmer W. J. Biol. Chem. 2005; 280: 38096-38101Abstract Full Text Full Text PDF PubMed Scopus (118) Google for a of lipid II the disaccharide of during the and a of disaccharide was reaction with a glycan strands were with and disaccharide and disaccharide the from glycan and lipid a transpeptidase is the also a the PBP1A glycan strands with an of disaccharide units and formed peptide cross-links In 18 and of the peptides part of cross-links. produced glycan strands with an of disaccharide units and peptide cross-linking as PBP1A that was with G. not glycan strands and not form peptide cross-links. The specificity of the transpeptidation reaction was studied by to the reaction of PBP1A with lipid II a lipid of artificial the murein UDP-MurNAc-tripeptide, or UDP-MurNAc-pentapeptide of the almost cross-linking reactions the two lipid II as by the of the PBP1A could and substrates as in transpeptidation to the formation of transpeptidation and the formation of the 4 could be by the of PBP1A with and 4 was not formed from lipid II and by not we studied the course of the reaction of PBP1A with lipid transglycosylation not occurred the first min of the reaction of of the lipid II and with a of lipid II of PBP1A and min, transglycosylation and transpeptidation reactions until almost lipid II was cross-links were formed at a of PBP1A The of transpeptidation activity that PBP1A the presence of glycan strands carrying monomeric peptides for cross-linking activity. the course was but this in the presence of glycan strands that had been from lipid II by glycan strands with monomeric peptides were the transpeptidation reaction with cross-links being formed with a of PBP1A The course experiments indicated that murein and not lipid II precursor could be the for the PBP1A-catalyzed cross-linking we in a PBP1A was to synthesized murein to isolated sacculi In the of murein the reaction of PBP1A and lipid II was not by centrifugation sample In the presence of of the with the sacculi sample indicating that a of the synthesized murein had been to the sacculi. attachment to sacculi occurred with or with and The PBP1A-catalyzed attachment of new material to sacculi could be with sample that the attachment occurred transpeptidation the of the attachment the reaction were by and the as as the murein was digested with by and The reaction of PBP1A and lipid II was performed in the presence and of murein sacculi. In the of murein sacculi two of the was in the and the produced murein had an glycan of disaccharide with of the peptides being part of cross-links. In the presence of murein sacculi two of the was with the and of the was in the revealed a in the supernatant and in the pellet the of the is in C. The supernatant contained a as the sample without murein sacculi that the glycan was to disaccharide In the the glycan was disaccharide and it contained radioactive cross-linked the and the could be produced by cross-linking reactions new radioactive lipid as and monomeric and tetrapeptides in the sacculi as acceptors. the attachment of new material to sacculi by PBP1A occurred by transpeptidation reactions with monomeric not with cross-linked or acceptors. PBP1A formed in vitro a murein from lipid II from sequence the Glu94 and Ser473 are essential for the the with transpeptidase could catalyze transglycosylation a with transglycosylase both transglycosylase and transpeptidase have been with variants of PBP1B from E. coli (16Terrak M. Ghosh T.K. van Heijenoort J. Van Beeumen J. Lampilas M. Aszodi J. Ayala J.A. Ghuysen J.M. Nguyen-Disteche M. Mol. Microbiol. 1999; 34: 350-364Crossref PubMed Scopus (146) Google Scholar). by occurred in the presence of an reaction produced by in a reaction containing PBP1A(TP*), and lipid II not it is that the transpeptidase of PBP1A is by transglycosylation reactions and that the cross-linking with in the glycan strands produced by the transglycosylase The course experiments of the reaction of PBP1A with lipid II revealed that the cross-linking reaction was and after glycan strands with monomeric had been produced by material was added to PBP1A and lipid formation of cross-links and until of the added peptides part of cross-links. the other 2% of the peptides part of cross-links glycan strands were incubated with PBP1A lipid for min transpeptidation transglycosylation (see above) and, in the presence of glycan strands carrying monomeric of the enzyme to this material the transpeptidase function or the material is a substrate for the transpeptidation reaction both We have obtained that monomeric peptides in high murein function as in PBP1A-catalyzed cross-linking First, a of synthesized murein was to murein and this attachment was by an of the transpeptidation reaction we with and cross-links as that cross-linking reactions had occurred lipid II synthesized glycan as and and tetrapeptides in the sacculi as The cross-linking reactions of PBP1A, with peptides in nascent murein or in murein are in the formation of cross-links nascent murein and sacculi not at the of the formation of cross-links new and in the presence of of the peptides part of cross-links the which was than in the murein produced in the of sacculi In this a of of the from lipid had as in transpeptidation of which with lipid and with and tetrapeptides as acceptors. PBP1A and PBP1B are in in vitro activities in that both artificial and as for transpeptidation reactions (19Bertsche U. Breukink E. Kast T. Vollmer W. J. Biol. Chem. 2005; 280: 38096-38101Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). reactions have been in cells W. B. Höltje J.V. J. Bacteriol. 1985; PubMed Google Scholar, W. Höltje J.-V. J. Bacteriol. PubMed Google Scholar). the other PBP1A and PBP1B are with to The transglycosylation and transpeptidation were for PBP1B produced a murein with almost of the peptides being in cross-links. PBP1A, we a of of the peptides part of cross-links. PBP1B produced a by of a and a A the production of a cross-linked by PBP1A S. Ishino F. Tamaki S. Matsuhashi M. Biochem. Biophys. Res. Commun. 1982; 106: 1175-1182Crossref PubMed Scopus (15) Google Scholar). We have in experiments the formation of a in at 100 is that the in the enzyme and, hence, in the of the enzyme for this both PBP1A and PBP1B required the presence of Triton X-100 for the activities of PBP1A, but not of PBP1B, the of the 0.2% not PBP1B was most as a of purified PBP1A was not in the of PBP1A, course experiments not a of PBP1B for polymerized murein glycan strands with monomeric peptides as for the transpeptidation reaction. In we have that PBP1B a of nascent murein to sacculi in vitro as with PBP1A not is that in the activities of both enzymes for the of mutants lacking either PBP1A or PBP1B (see the For the first time, we could demonstrate in vitro an attachment of nascent murein to a reaction that in cells during the growth of the to in vitro it has been that incorporation of new material in vivo involves the formation of new during cell division, cross-links are formed two new during cell cross-links are also formed new peptides and peptides in the sacculus Proc. Natl. Acad. Sci. U. S. A. 1984; PubMed Scopus Google Scholar, S. J. Bacteriol. 1988; PubMed Scopus Google Scholar, F. J. Bacteriol. PubMed Google Scholar, B. Höltje J.V. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). In PBP1A most new material without the of the sacculi. The enlargement of the sacculus in vivo is a more and a coordinated bifunctional and murein synthases, murein hydrolases, and regulatory proteins. The precise of PBP1A in the enlargement of the sacculus during cell and division to be that PBP1A of nascent murein and to the We for a of the