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J. Biol. Chem., Vol. 281, Issue 37, 26985-26993, September 15, 2006
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1
From the
Mikrobielle Genetik, Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany and Center of Biomembranes and Lipid Enzymology, Department of Biochemistry of Membranes, Institute for Biomembranes, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands
Received for publication, April 28, 2006 , and in revised form, June 12, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Escherichia coli contains six known murein synthases (4–8). 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)2 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 (9–11). Both PBPs form independent dimers in vivo (12). 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 (5, 13). 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 
-lactam antibiotics than mutants without PBP1A (14). Furthermore, PBP1B-deficient mutants, but not PBP1A-deficient mutants, lose cell integrity upon inactivation of PBP2, PBP3, or the cell division protein FtsQ (15).
The enzymatic activities of PBP1B with lipid II or artificial substrates have been studied previously (16–19). However, apart from two early studies from Matsuhashi and colleagues (20, 21), 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials—All chemicals were purchased from Sigma unless indicated otherwise. [14C]GlcNAc-labeled lipid II was prepared as published (22), with modifications described previously (19), and had a specific activity of 9180 dpm/nmol. UDP-MurNAc-tripeptide, DP-MurNAc-tetrapeptide, and UDP-MurNAc-pentapeptide were prepared as published (23). Murein sacculi were prepared from E. coli MC1061 (24) as described (25). Cellosyl was kindly provided by Hoechst AG, Frankfurt, Germany.
Cloning of a mrcA Expression Plasmid—The mrcA gene encoding for PBP1A (GenBankTM accession no. AAA58193 [GenBank] ) 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 (26), 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.
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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--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 x 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 x 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 x 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 N-terminal extension of six amino acids and has the following sequence, GSHMAS-PBP1A(M1-F858).
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-Lactam Binding (PBP Assay)—-Lactam binding was assayed with biotinylated ampicillin as described previously (27). Transglycosylation and Transpeptidation Reaction of PBP1A with Lipid II—Lipid II (1.2 nmol, 11,000 dpm) was vacuum dried and dissolved in 5 µl of methanol. The reaction was performed in a total volume of 50 µl in 25 mM Tris/HCl, 10 mM MgCl2, 100 mM NaCl, 0.05% Triton X-100, pH 7.5 (buffer B), with 0.5–0.8 µM PBP1A variant. In different experiments, the precursor UDP-MurNAc-tripeptide, UDP-MurNAc-tetrapeptide, or UDP-MurNAc-pentapeptide was added 1 mM. In one experiment, the transpeptidation domain of PBP1A was blocked with 50 µg/ml penicillin G. The reaction was performed for 1 h at 30°C. The pH of the samples was adjusted to 4.8 prior to boiling for 5 min followed by a digestion with 50 µg/ml cellosyl for 1 h at 37°C and boiling for 5 min.
Time Course Experiments—In a total volume of 325 µl in buffer B, lipid II (24 µM) and PBP1A (0.8 µM) were incubated at 30 °C. At different time points (0, 5, 10, 15, 30, and 60 min), 50 µl-aliquots were taken and boiled for 5 min. The samples were chilled on ice, and cellosyl digestion was done as described above.
In a second experiment, glycan strands with monomeric peptide side chains were preformed with PBP1A(TP*) in a total volume of 425 µl for 1 h. The sample was boiled for 10 min, and PBP1A (0.8 µM) was added followed by an incubation for 10 min on ice. After the addition of lipid II (24 µM), the time course experiment was performed as described above.
Attachment of Newly Synthesized Material to Sacculi—PBP1A (0.8 µM) was preincubated for 10 min on ice with 0.5 mg/ml murein sacculi (isolated from MC1061) in buffer B. Lipid II (4.8 µM) was added, and the reaction proceeded in a final volume of 100 µl for 1 h at 30°C. The sacculi were sedimented by centrifugation (14,000 x g, 10 min), and the pellet was washed twice with 200 µl of buffer A containing 0.2% Triton X-100. The pellet samples were digested for 18 h at 37 °C with 35 µg/ml cellosyl. The radioactivity of each sample (digested pellet, two wash fractions, and supernatant) was determined by scintillation counting.
For HPLC analysis, the reaction was performed in a final volume of 200 µl. PBP1A (0.8 µM) was incubated for 10 min on ice with or without 1 mg/ml sacculi (from MC1061). Lipid II (12 µM) was added, and the reaction was performed for 1 h at 30 °C. After sedimentation (14,000 x g, 10 min), the pellets were washed twice with 200 µl of buffer A containing 0.2% Triton X-100 and resuspended in 200 µl of 20 mM sodium phosphate, pH 4.8. The pH of the supernatant and of the washes was adjusted to 4.8, and the samples were digested with 17.5 µg/ml cellosyl for 18 h at 37 °C. Samples were boiled for 5 min.
Analysis of the Reaction Products—Prior to HPLC analysis, the samples were reduced with NaBH4 as described (28). The reaction products were separated by HPLC according to a published method (25, 28), using a radioactivity flow-through detector (Canberra). The separated muropeptides were identified by their retention times with the help of muropeptide standards (19, 28). Compound 1 (Fig. 4C) could be dephosphorylated to compound 2 either by extended boiling or by treatment with an acidic phosphatase followed by reduction with NaBH4 (19).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To study the enzymatic reactions of PBP1A, we cloned pET28a(+)-based expression plasmids for the purification of PBP1A and of two variants with single amino acid exchanges, PBP1A(TG*) with a Glu94 Gln exchange and PBP1A(TP*) with a Ser473 Ala exchange. Purification involved the preparation of membrane fraction, Ni2+-nitrilotriacetic acid affinity chromatography, cleavage of the N-terminal oligo-histidine tag by thrombin, and cation exchange chromatography. Fig. 2 shows the purified proteins and a -lactam binding assay. As expected, PBP1A and PBP1A(TG*), but not PBP1A(TP*), bound biotinylated ampicillin.
The activities of the PBP1A variants were studied with radioactive lipid II substrate employing a recently developed in vitro murein synthesis assay (Fig. 3; see Ref. 19 for a scheme). Briefly, boiling of lipid II yielded the phosphorylated disaccharide pentapeptide (Fig. 4C, compound 1). Because of partial dephosphorylation during the boiling and reduction steps, a small fraction (0–15%) of nonphosphorylated disaccharide pentapeptide was formed. Upon reaction with a transglycosylase, glycan strands were produced. Digestion with cellosyl and boiling yielded nonphosphorylated disaccharide pentapeptide (Fig. 4C, compound 2) and phosphorylated disaccharide pentapeptide, the latter originating from glycan chain ends and nonreacted lipid II. If a transpeptidase is present, the procedure also yields a dimeric (cross-linked) muropeptide, the bisdisaccharide tetrapentapeptide (Fig. 4C, compound 3). PBP1A oligomerized glycan strands with an average length of 19.6 disaccharide units and formed peptide cross-links (Fig. 3). In different experiments, between 18 and 26% of the peptides became part of cross-links. PBP1A(TP*) produced glycan strands with an average of 10.4 disaccharide units and completely lacked peptide cross-linking activity, as did PBP1A that was blocked with penicillin G. PBP1A(TG*) did not produce glycan strands and did not form peptide cross-links.
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Subsequently, we studied the time course of the reaction of PBP1A with lipid II. Strikingly, only transglycosylation (but not transpeptidation) occurred within the first 15 min of the reaction under consumption of 
25% of the available lipid II (Fig. 5A) and with a rate of lipid II consumption of 0.75 mol/(mol PBP1A · min). Between 15 and 60 min, transglycosylation and transpeptidation reactions proceeded until almost all lipid II was consumed. Peptide cross-links were formed at a rate of 0.06 mol/(mol PBP1A · min). The absence of initial transpeptidation activity indicates that PBP1A requires the presence of oligomerized glycan strands carrying monomeric peptides for cross-linking activity. Therefore, the time course experiment was repeated but this time in the presence of oligomerized glycan strands that had been preformed from lipid II by PBP1A(TP*). Indeed, if glycan strands with monomeric peptides were present, the transpeptidation reaction started immediately, with cross-links being formed with a rate of 0.06 mol/(mol PBP1A · min) (Fig. 5B).
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25% of the total radioactivity sedimented together with the sacculi (Fig. 6, sample C), indicating that a fraction of the newly synthesized murein had been covalently attached to the sacculi. No attachment to sacculi occurred with PBP1A(TG*) or with PBP1A(TP*) (Fig. 6, samples D and E). The PBP1A-catalyzed attachment of new material to sacculi could be blocked with penicillin G (Fig. 6, sample F), suggesting that the attachment occurred via transpeptidation reactions.
To determine the structure(s) of the attachment site(s), the reaction products were sedimented by centrifugation, and the supernatant, as well as the sedimented murein sacculi, was digested with cellosyl followed by reduction and HPLC analysis (Fig. 7). The reaction of PBP1A and lipid II was performed in the presence and absence of murein sacculi. In the absence of murein sacculi (Fig. 7A, left two bars), 98.1% of the radioactivity was present in the supernatant, and the produced murein had an average glycan strand length of 18.2 disaccharide units, with 26.4% of the peptides being part of cross-links. In the presence of murein sacculi (Fig. 7A, right two bars), 44% of the total radioactivity was sedimented together with the sacculi, and 56% of the total radioactivity was present in the supernatant. HPLC analysis revealed a different muropeptide composition in the supernatant and in the pellet (Fig. 7B); the quantification of the respective compounds is depicted in C. The supernatant (Fig. 7B, chromatogram III) contained a similar muropeptide profile as the sample without murein sacculi (chromatogram I), except that the average glycan strand length was decreased to 6.1 disaccharide units. In the pellet, the average glycan strand length was >40 disaccharide units, and it contained additional radioactive cross-linked compounds (Fig. 7B, chromatogram IV), the bisdisaccharide tetratripeptide and the bisdisaccharide tetratetrapeptide. These compounds could only be produced by cross-linking reactions between new pentapeptides (from radioactive lipid II) as donors and monomeric tri- and tetrapeptides in the sacculi as acceptors. Thus, the attachment of new material to sacculi by PBP1A occurred by transpeptidation reactions with monomeric (and not with cross-linked dimeric or trimeric) acceptors.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The time course experiments of the reaction of PBP1A with lipid II revealed that the cross-linking reaction was delayed and started only after glycan strands with monomeric pentapeptides had been produced by transglycosylation. If such material was added initially to PBP1A and lipid II, formation of cross-links started immediately and proceeded until 21% of the newly added peptides became part of cross-links. On the other hand, only 2% of the peptides became part of cross-links if oligomerized glycan strands were incubated with PBP1A alone (without lipid II) for 60 min (not shown). Apparently, transpeptidation requires ongoing transglycosylation (see above) and, in addition, the presence of oligomerized glycan strands carrying monomeric peptides. Either binding of the enzyme to this oligomeric material activates the transpeptidase function or the oligomeric material is a substrate for the transpeptidation reaction (or both possibilities). We have obtained evidence that monomeric peptides in high molecular weight murein function as acceptors in PBP1A-catalyzed cross-linking reactions. First, a fraction of newly synthesized murein was covalently attached to murein sacculi, and this attachment was inhibited by penicillin G, an inhibitor of the transpeptidation reaction (Fig. 6). Second, we identified dimeric muropeptides with tetratripeptide and tetratetrapeptide cross-links as products, proving that cross-linking reactions had occurred between pentapeptides (from lipid II and/or newly synthesized glycan strands) as donors and tri- and tetrapeptides in the sacculi as acceptors (Fig. 7). The observed cross-linking reactions of PBP1A, with acceptor peptides in nascent murein or in murein sacculi, are depicted schematically in Fig. 8. Interestingly, the formation of cross-links between nascent murein and sacculi did not take place at the expense of the formation of cross-links between new (pentapeptide) donors and acceptors; in the presence of sacculi, 23% of the peptides became part of tetrapentapeptide cross-links (in the attached material), which was only slightly less than in the murein produced in the absence of sacculi (26%) (Fig. 7). In this experiment, a total of 30% of the pentapeptides (originating from lipid II) had participated as donors in transpeptidation reactions, of which 11.5% reacted with pentapeptides (in lipid II) and 19% with tri- and tetrapeptides (in sacculi) as acceptors.
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For the first time, we could demonstrate in vitro an attachment of nascent murein to sacculi, a reaction that occurs in living cells during the growth of the sacculus. Similar to our in vitro experiments, it has been shown that incorporation of new material in vivo involves the formation of new cross-links; during cell division, cross-links are formed mainly between two new peptides, whereas during cell elongation, cross-links are also formed between new peptides and peptides in the existing sacculus (31–34). In vitro, PBP1A most likely attached new material without increasing the surface of the sacculi. The enlargement of the sacculus in vivo is a more complex process and presumably requires a coordinated interplay between different bifunctional and monofunctional murein synthases, murein hydrolases, and regulatory proteins. The precise role of PBP1A in the enlargement of the sacculus during cell elongation and division remains to be determined. Our results indicate that PBP1A might synthesize patches of nascent murein and attach them to the sacculus.
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FOOTNOTES |
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1 To whom correspondence should be addressed: Microbial Genetics, University of Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany. Tel.: 49-7071-2974635; Fax: 49-7071-295065; E-mail: waldemar.vollmer{at}uni-tuebingen.de.
2 The abbreviations used are: PBP, penicillin-binding protein; HPLC, high pressure liquid chromatography; TG, transglycosylase; TP, transpeptidase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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