Essential Penicillin-binding Proteins Research Articles

β-Lactam Resistance in ESKAPE Pathogens Mediated Through Modifications in Penicillin-Binding Proteins: An Overview.

Bacteria acquire β-lactam resistance through a multitude of mechanisms among which production of β-lactamases (enzymes that hydrolyze β-lactams) is the most common, especially in Gram-negatives. Structural changes in the high-molecular-weight, essential penicillin-binding proteins (PBPs) are widespread in Gram-positives and increasingly reported in Gram-negatives. PBP-mediated resistance is largely achieved by accumulation of mutation(s) resulting in reduced binding affinities of β-lactams. Herein, we discuss PBP-mediated resistance among ESKAPE pathogens that cause diverse hospital- and community-acquired infections globally.

Transposon mutagenesis in Mycobacterium abscessus identifies an essential penicillin-binding protein involved in septal peptidoglycan synthesis and antibiotic sensitivity.

Mycobacterium abscessus (Mab) is a rapidly growing non-tuberculous mycobacterium (NTM) that causes a wide range of infections. Treatment of Mab infections is difficult because the bacterium is intrinsically resistant to many classes of antibiotics. Developing new and effective treatments against Mab requires a better understanding of the unique vulnerabilities that can be targeted for future drug development. To achieve this, we identified essential genes in Mab by conducting transposon sequencing (TnSeq) on the reference Mab strain ATCC 19977. We generated ~51,000 unique transposon mutants and used this high-density library to identify 362 essential genes for in vitro growth. To investigate species-specific vulnerabilities in Mab, we further characterized MAB_3167c, a predicted penicillin-binding protein and hypothetical lipoprotein (PBP-lipo) that is essential in Mab and non-essential in Mycobacterium tuberculosis (Mtb). We found that PBP-lipo primarily localizes to the subpolar region and later to the septum as cells prepare to divide. Depletion of Mab PBP-lipo causes cells to elongate, develop ectopic branches, and form multiple septa. Knockdown of PBP-lipo along with PbpB, DacB1, and a carboxypeptidase, MAB_0519 lead to synergistic growth arrest. In contrast, these genetic interactions were absent in the Mtb model organism, Mycobacterium smegmatis, indicating that the PBP-lipo homologs in the two species exist in distinct genetic networks. Finally, repressing PBP-lipo sensitized the reference strain and 11 Mab clinical isolates to several classes of antibiotics, including the β-lactams, ampicillin, and amoxicillin by greater than 128-fold. Altogether, this study presents PBP-lipo as a key enzyme to study Mab-specific processes in cell wall synthesis and importantly positions PBP-lipo as an attractive drug target to treat Mab infections.

New Insights into Beta-Lactam Resistance of Streptococcus pneumoniae: Serine Protease HtrA Degrades Altered Penicillin-Binding Protein 2x.

Reduced amounts of the essential penicillin-binding protein 2x (PBP2x) were detected in two cefotaxime-resistant Streptococcus pneumoniae laboratory mutants C405 and C606. These mutants contain two or four mutations in the penicillin-binding domain of PBP2x, respectively. The transcription of the pbp2x gene was not affected in both mutants; thus, the reduced PBP2x amounts were likely due to post-transcriptional regulation. The mutants carry a mutation in the histidine protein kinase gene ciaH, resulting in enhanced gene expression mediated by the cognate response regulator CiaR. Deletion of htrA, encoding a serine protease regulated by CiaR, or inactivation of HtrA proteolytic activity showed that HtrA is indeed responsible for PBP2x degradation in both mutants, and that this affects β-lactam resistance. Depletion of the PBP2xC405 in different genetic backgrounds confirmed that HtrA degrades PBP2xC405. A GFP-PBP2xC405 fusion protein still localized at the septum in the absence of HtrA. The complementation studies in HtrA deletion strains showed that HtrA can be overexpressed in pneumococcal cells to specific levels, depending on the genetic background. Quantitative Western blotting revealed that the PBP2x amount in C405 strain was less than 20% compared to parental strain, suggesting that PBP2x is an abundant protein in S. pneumoniae R6 strain.

Upregulation of PBP1B and LpoB in cysB Mutants Confers Mecillinam (Amdinocillin) Resistance in Escherichia coli.

Mecillinam (amdinocillin) is a β-lactam antibiotic that inhibits the essential penicillin-binding protein 2 (PBP2). In clinical isolates of Escherichia coli from urinary tract infections, inactivation of the cysB gene (which encodes the main regulator of cysteine biosynthesis, CysB) is the major cause of resistance. How a nonfunctional CysB protein confers resistance is unknown, however, and in this study we wanted to examine the mechanism of resistance. Results show that cysB mutations cause a gene regulatory response that changes the expression of ∼450 genes. Among the proteins that show increased levels are the PBP1B, LpoB, and FtsZ proteins, which are known to be involved in peptidoglycan biosynthesis. Artificial overexpression of either PBP1B or LpoB in a wild-type E. coli strain conferred mecillinam resistance; conversely, inactivation of either the mrcB gene (which encodes PBP1B) or the lpoB gene (which encodes the PBP1B activator LpoB) made cysB mutants susceptible. These results show that expression of the proteins PBP1B and LpoB is both necessary and sufficient to confer mecillinam resistance. The addition of reducing agents to a cysB mutant converted it to full susceptibility, with associated downregulation of PBP1B, LpoB, and FtsZ. We propose a model in which cysB mutants confer mecillinam resistance by inducing a response that causes upregulation of the PBP1B and LpoB proteins. The higher levels of these two proteins can then rescue cells with mecillinam-inhibited PBP2. Our results also show how resistance can be modulated by external conditions such as reducing agents.

Allostery, Recognition of Nascent Peptidoglycan, and Cross-linking of the Cell Wall by the Essential Penicillin-Binding Protein 2x of Streptococcus pneumoniae.

Transpeptidases, members of the penicillin-binding protein (PBP) families, catalyze cross-linking of the bacterial cell wall. This transformation is critical for the survival of bacteria, and it is the target of inhibition by β-lactam antibiotics. We report herein our structural insights into catalysis by the essential PBP2x of Streptococcus pneumoniae by disclosing a total of four X-ray structures, two computational models based on the crystal structures, and molecular-dynamics simulations. The X-ray structures are for the apo PBP2x, the enzyme modified covalently in the active site by oxacillin (a penicillin antibiotic), the enzyme modified by oxacillin in the presence of a synthetic tetrasaccharide surrogate for the cell-wall peptidoglycan, and a noncovalent complex of cefepime (a cephalosporin antibiotic) bound to the active site. A prerequisite for catalysis by transpeptidases, including PBP2x, is the molecular recognition of nascent peptidoglycan strands, which harbor pentapeptide stems. We disclose that the recognition of nascent peptidoglycan by PBP2x takes place by complexation of one pentapeptide stem at an allosteric site located in the PASTA domains of this enzyme. This binding predisposes the third pentapeptide stem in the same nascent peptidoglycan strand to penetration into the active site for the turnover events. The complexation of the two pentapeptide stems in the same peptidoglycan strand is a recognition motif for the nascent peptidoglycan, critical for the cell-wall cross-linking reaction.

Novel Electrophilic Scaffold for Imaging of Essential Penicillin-Binding Proteins in Streptococcus pneumoniae.

Peptidoglycan (PG) is a mesh-like heteropolymer made up of glycan chains cross-linked by short peptides and is the major scaffold of eubacterial cell walls, determining cell shape, size, and chaining. This structure, which is required for growth and survival, is located outside of the cytoplasmic membrane of bacterial cells, making it highly accessible to antibiotics. Penicillin-binding proteins (PBPs) are essential for construction of PG and perform transglycosylase activities to generate the glycan strands and transpeptidation to cross-link the appended peptides. The β-lactam antibiotics, which are among the most clinically effective antibiotics for the treatment of bacterial infections, inhibit PBP transpeptidation, ultimately leading to cell lysis. Despite this importance, the discrete functions of individual PBP homologues have been difficult to determine. These major gaps in understanding of PBP activation and macromolecular interactions largely result from a lack of tools to assess the functional state of specific PBPs in bacterial cells. We have identified β-lactones as a privileged scaffold for the generation of PBP-selective probes and utilized these compounds for imaging of the essential proteins, PBP2x and PBP2b, in Streptococcus pneumoniae. We demonstrated that while PBP2b activity is restricted to a ring surrounding the division sites, PBP2x activity is present both at the septal center and at the surrounding ring. These spatially separate regions of PBP2x activity could not be detected by previous activity-based approaches, which highlights a critical strength of our PBP-selective imaging strategy.

Lipid II overproduction allows direct assay of transpeptidase inhibition by β-lactams.

Peptidoglycan is an essential crosslinked polymer that surrounds bacteria and protects them from osmotic lysis. Beta-lactam antibiotics target the final stages of peptidoglycan biosynthesis by inhibiting the transpeptidases that crosslink glycan strands to complete cell wall assembly. Characterization of transpeptidases and their inhibition by beta-lactams has been hampered by lack of access to substrate. We describe a general approach to accumulate Lipid II in bacteria and to obtain large quantities of this cell wall precursor. We demonstrate utility by isolating Staphylococcus aureus Lipid II and reconstituting the synthesis of crosslinked peptidoglycan by the essential penicillin-binding protein 2, PBP2, which catalyzes both glycan polymerization and transpeptidation. We also show that we can compare the potencies of different beta-lactams by directly monitoring transpeptidase inhibition. The methods reported here will enable a better understanding of cell wall biosynthesis and facilitate studies of next-generation transpeptidase inhibitors.

Transpeptidase activity of penicillin-binding protein SpoVD in peptidoglycan synthesis conditionally depends on the disulfide reductase StoA.

Endospore cortex peptidoglycan synthesis is not required for bacterial growth but essential for endospore heat resistance. It therefore constitutes an amenable system for research on peptidoglycan biogenesis. The Bacillus subtilis sporulation-specific class B penicillin-binding protein (PBP) SpoVD and many homologous PBPs contain two conserved cysteine residues of unknown function in the transpeptidase domain - one as residue x in the SxN catalytic site motif and the other in a flexible loop near the catalytic site. A disulfide bond between these residues blocks the function of SpoVD in cortex synthesis. With a combination of experiments with purified proteins and B. subtilis mutant cells, it was shown that in active SpoVD the two cysteine residues most probably interact by hydrogen bonding and that this is important for peptidoglycan synthesis in vivo. It was furthermore demonstrated that the sporulation-specific thiol-disulfide oxidoreductase StoA reduces SpoVD and that requirement of StoA for cortex synthesis can be suppressed by two completely different types of structural alterations in SpoVD. It is concluded that StoA plays a critical role mainly during maturation of SpoVD in the forespore outer membrane. The findings advance our understanding of essential PBPs and redox control of extra-cytoplasmic protein disulfides in bacterial cells.

From "An Enzyme Able to Destroy Penicillin" to Carbapenemases: 70 Years of Beta-lactamase Misbehaviour.

As early as 1940, Abraham and Chain described "an enzyme able to destroy penicillin". In the late 1940's, penicillin-resistant strains of Staphylococcus aureus were found to be a clinical problem. They produced a penicillinase that could hydrolyze the amide bond in the β-lactam ring. Later, an enzyme mediated by an R-factor was isolated from Enterobacteriaceae. Methicillin and cephalosporins, both very poor substrates of the S. aureus enzyme, were found to be sensitive to this new enzyme. Third generation cephalosporins appeared to solve the problem, but further enzymes were selected that exhibited extended spectra and could for instance hydrolyze cefotaxime and/or ceftazidime. The discovery of carbapenems constituted a major advance for our antimicrobial arsenal: they inactivated most of the essential penicillin binding proteins effectively and escaped the activity of nearly all known -β lactamases. However, the metallo-β-lactamases, which had not been recognised as a major danger before 1990, were found to act as effective carbapenemases and started to spread in a worrying way. Moreover, carbapenem-hydrolyzing enzymes were found in each of the 3 classes of active-site serine β-lactamases.

Streptococcus pneumoniae PBP2x mid‐cell localization requires the C‐terminal PASTA domains and is essential for cell shape maintenance

The transpeptidase activity of the essential penicillin-binding protein 2x (PBP2x) of Streptococcus pneumoniae is believed to be important for murein biosynthesis required for cell division. To study the molecular mechanism driving localization of PBP2x in live cells, we constructed a set of N-terminal GFP-PBP2x fusions under the control of a zinc-inducible promoter. The ectopic fusion protein localized at mid-cell. Cells showed no growth defects even in the absence of the genomic pbp2x, demonstrating that GFP-PBP2x is functional. Depletion of GFP-PBP2x resulted in severe morphological alterations, confirming the essentiality of PBP2x and demonstrating that PBP2x is required for cell division and not for cell elongation. A genetically or antibiotic inactivated GFP-PBP2x still localized at septal sites. Remarkably, the same was true for a GFP-PBP2x derivative containing a deletion of the central transpeptidase domain, although only in the absence of the protease/chaperone HtrA. Thus localization is independent of the catalytic transpeptidase domain but requires the C-terminal PASTA domains, identifying HtrA as targeting GFP-PBP2x derivatives. Finally, PBP2x was positioned at the septum similar to PBP1a and the PASTA domain containing StkP protein, confirming that PBP2x is a key element of the divisome complex.

The Staphylococcus aureus Membrane Protein SA2056 Interacts with Peptidoglycan Synthesis Enzymes.

The yet uncharacterized membrane protein SA2056 belongs to the ubiquitous RND (Resistance-Nodulation-cell Division) family of transmembrane efflux transporters. The sa2056 gene is located downstream of femX, the gene encoding the essential, non-ribosomal peptidyl-transferase adding the first glycine in the staphylococcal cell wall pentaglycine interpeptide. Due to its proximity to and weak co-transcription with femX, we assumed that sa2056 may somehow be involved in peptidoglycan synthesis. Specific antibodies against SA2056 showed that this protein is expressed during growth and present in the membrane fraction of cell preparations. Using a bacterial two hybrid system, SA2056 was shown to interact (i) with itself, (ii) with FemB, which adds glycines 4 and 5 to the peptidoglycan interpeptide and (iii) with the essential penicillin binding proteins, PBP1 and PBP2, required for cell division and incorporation of the peptidoglycan into the cell wall. Unexpectedly, deletion of sa2056 led to no phenotype regarding growth, antibiotic resistances or cell morphology; nor did sa2056 deletion in combination with femB inactivation alter β-lactam and lysostaphin sensitivity and resistance, respectively, pointing to possible redundancy in the cell wall synthesis pathway. These results suggest an accessory role of SA2056 in S. aureus peptidoglycan synthesis, broadening the range of biological functions of RND proteins.

Pan-β-Lactam Resistance Development in Pseudomonas aeruginosa Clinical Strains: Molecular Mechanisms, Penicillin-Binding Protein Profiles, and Binding Affinities

We investigated the mechanisms leading to Pseudomonas aeruginosa pan-β-lactam resistance (PBLR) development during the treatment of nosocomial infections, with a particular focus on the modification of penicillin-binding protein (PBP) profiles and imipenem, ceftazidime, and ceftolozane (former CXA-101) PBP binding affinities. For this purpose, six clonally related pairs of sequential susceptible-PBLR isolates were studied. The presence of oprD, ampD, and dacB mutations was explored by PCR followed by sequencing and the expression of ampC and efflux pump genes by real-time reverse transcription-PCR. The fluorescent penicillin Bocillin FL was used to determine PBP profiles in membrane preparations from all pairs, and 50% inhibitory concentrations (IC(50)s) of ceftolozane, ceftazidime, and imipenem were analyzed in 3 of them. Although a certain increase was noted (0 to 5 2-fold dilutions), the MICs of ceftolozane were ≤4 μg/ml in all PBLR isolates. All 6 PBLR isolates lacked OprD and overexpressed ampC and one or several efflux pumps, particularly mexB and/or mexY. Additionally, 5 of them showed modified PBP profiles, including a modified pattern (n = 1) or diminished expression (n = 1) of PBP1a and a lack of PBP4 expression (n = 4), which correlated with AmpC overexpression driven by dacB mutation. Analysis of the essential PBP IC(50)s revealed significant variation of PBP1a/b binding affinities, both within each susceptible-PBLR pair and across the different pairs. Moreover, despite the absence of significant differences in gene expression or sequence, a clear tendency toward increased PBP2 (imipenem) and PBP3 (ceftazidime, ceftolozane, imipenem) IC(50)s was noted in PBLR isolates. Thus, our results suggest that in addition to AmpC, efflux pumps, and OprD, the modification of PBP patterns appears to play a role in the in vivo emergence of PBLR strains, which still conserve certain susceptibility to the new antipseudomonal cephalosporin ceftolozane.

Lipoprotein Cofactors Located in the Outer Membrane Activate Bacterial Cell Wall Polymerases

Most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by polysaccharide polymerases called penicillin-binding proteins (PBPs). Because they are the targets of penicillin and related antibiotics, the structure and biochemical functions of the PBPs have been extensively studied. Despite this, we still know surprisingly little about how these enzymes build the PG layer invivo. Here, we identify the Escherichia coli outer-membrane lipoproteins LpoA and LpoB as essential PBP cofactors. We show that LpoA and LpoB form specific trans-envelope complexes with their cognate PBP and are critical for PBP function invivo. We further show that LpoB promotes PG synthesis by its partner PBP invitro and that it likely does so by stimulating glycan chain polymerization. Overall, our results indicate that PBP accessory proteins play a central role in PG biogenesis, and like the PBPs they work with, these factors are attractive targets for antibiotic development.

Affinity of the New Cephalosporin CXA-101 to Penicillin-Binding Proteins of Pseudomonas aeruginosa

CXA-101, previously designated FR264205, is a new antipseudomonal cephalosporin. The objective of this study was to determine the penicillin-binding protein (PBP) inhibition profile of CXA-101 compared to that of ceftazidime (PBP3 inhibitor) and imipenem (PBP2 inhibitor). Killing kinetics, the induction of AmpC expression, and associated changes on cell morphology were also investigated. The MICs for CXA-101, ceftazidime, and imipenem were 0.5, 1, and 1 microg/ml, respectively. Killing curves revealed that CXA-101 shows a concentration-independent bactericidal activity, with concentrations of 1x the MIC (0.5 microg/ml) producing a > 3-log reduction in bacterial load after 8 h of incubation. Live-dead staining showed that concentrations of CXA-101 as low as 0.5x the MIC stopped bacterial septation and induced an intense filamentation, which is consistent with the documented high affinity of PBP3. CXA-101 was found to be a potent PBP3 inhibitor and showed affinities > or = 2-fold higher than those of ceftazidime for all of the essential PBPs (1b, 1c, 2, and 3). Compared to imipenem, in addition to the obvious inverse PBP2/PBP3 affinities, CXA-101 showed a significantly higher affinity for PBP1b but a lower affinity for PBP1c. Furthermore, CXA-101, like ceftazidime and in contrast to imipenem, was found to be a very weak inducer of AmpC expression, consistent with the low PBP4 affinity documented.

Binding of Ceftobiprole and Comparators to the Penicillin-Binding Proteins of Escherichia coli , Pseudomonas aeruginosa , Staphylococcus aureus , and Streptococcus pneumoniae

Ceftobiprole exhibited tight binding to PBP2a in methicillin-resistant Staphylococcus aureus, PBP2x in penicillin-resistant Streptococcus pneumoniae, and PBP3 and other essential penicillin-binding proteins in methicillin-susceptible S. aureus, Escherichia coli, and Pseudomonas aeruginosa. Ceftobiprole also bound well to PBP2 in the latter organisms, contributing to the broad-spectrum antibacterial activity against gram-negative and gram-positive bacteria.

Cell wall modifications during osmotic stress in Lactobacillus casei

To study the modification of the cell wall of Lactobacillus casei ATCC 393 grown in high salt conditions. Differences in the overall structure of cell wall between growth in high salt (MRS + 1 mol l(-1) NaCl; N condition) and control (MRS; C condition) conditions were determined by transmission electronic microscopy and analytical procedures. Lactobacillus casei cells grown in N condition were significantly larger than cells grown under unstressed C condition. Increased sensitivity to mutanolysin and antibiotics with target in the cell wall was observed in N condition. Purified cell wall also showed the increased sensitivity to lysis by mutanolysin. Analysis of peptidoglycan (PG) from stressed cells showed that modification was at the structural level in accordance with a decreased PG cross-link involving penicillin-binding proteins (PBP). Nine PBP were first described in this species and these proteins were expressed in low percentages or presented a modified pattern of saturation with penicillin G (Pen G) during growth in high salt. Three of the essential PBP were fully saturated in N condition at lower Pen G concentrations than in C condition, suggesting differences in functionality in vivo. The results show that growth in high salt modified the structural properties of the cell wall. Advances in understanding the adaptation to high osmolarity, in particular those involving sensitivity to lysis of lactic acid bacteria.

Balance between Two Transpeptidation Mechanisms Determines the Expression of β-Lactam Resistance in Enterococcus faecium

The d,d-transpeptidase activity of high molecular weight penicillin-binding proteins (PBPs) is essential to maintain cell wall integrity as it catalyzes the final cross-linking step of bacterial peptidoglycan synthesis. We investigated a novel beta-lactam resistance mechanism involving by-pass of the essential PBPs by l,d-transpeptidation in Enterococcus faecium. Determination of the peptidoglycan structure by reverse phase high performance liquid chromatography coupled to mass spectrometry revealed that stepwise selection for ampicillin resistance led to the gradual replacement of the usual cross-links generated by the PBPs (d-Ala(4) --> d-Asx-Lys(3)) by cross-links resulting from l,d-transpeptidation (l-Lys(3) --> d-Asx-Lys(3)). This was associated with no modification of the level of production of the PBPs or of their affinity for beta-lactams, indicating that altered PBP activity was not required for ampicillin resistance. A beta-lactam-insensitive l,d-transpeptidase was detected in membrane preparations of the parental susceptible strain. Acquisition of resistance was not because of variation of this activity. Instead, selection led to production of a beta-lactam-insensitive d,d-carboxypeptidase that cleaved the C-terminal d-Ala residue of pentapeptide stems in vitro and caused massive accumulation of cytoplasmic precursors containing a tetrapeptide stem in vivo. The parallel dramatic increase in the proportion of l-Lys(3) --> d-Asx-Lys(3) cross-links showed that the enzyme was activating the resistance pathway by generating the substrate for the l,d-transpeptidase.

In vitro and in vivo properties of Ro 63-9141, a novel broad-spectrum cephalosporin with activity against methicillin-resistant staphylococci.

Ro 63-9141 is a new member of the pyrrolidinone-3-ylidenemethyl cephem series of cephalosporins. Its antibacterial spectrum was evaluated against significant gram-positive and gram-negative pathogens in comparison with those of reference drugs, including cefotaxime, cefepime, meropenem, and ciprofloxacin. Ro 63-9141 showed high antibacterial in vitro activity against gram-positive bacteria except ampicillin-resistant enterococci, particularly vancomycin-resistant strains of Enterococcus faecium. Its MIC at which 90% of the isolates tested were inhibited (MIC(90)) for methicillin-resistant Staphylococcus aureus (MRSA) was 4 microg/ml. Ro 63-9141 was bactericidal against MRSA. Development of resistance to the new compound in MRSA was not observed. Ro 63-9141 was more potent than cefotaxime against penicillin-resistant Streptococcus pneumoniae (MIC(90) = 2 microg/ml). It was active against ceftazidime-susceptible strains of Pseudomonas aeruginosa and against Enterobacteriaceae except Proteus vulgaris and some isolates producing extended-spectrum beta-lactamases. The basis for the antibacterial spectrum of Ro 63-9141 lies in its affinity to essential penicillin-binding proteins, including PBP 2' of MRSA, and its stability towards beta-lactamases. The in vivo findings were in accordance with the in vitro susceptibilities of the pathogens. These data suggest the potential utility of Ro 63-9141 for the therapy of infections caused by susceptible pathogens, including MRSA. Since insufficient solubility of Ro 63-9141 itself precludes parenteral administration in humans, a water-soluble prodrug, Ro 65-5788, is considered for development.

Two-step procedure for purification and separation of the essential penicillin-binding proteins PBP 1A and 1Bs of Escherichia coli

The penicillin-binding proteins PBP 1A and 1Bs are the essential murein polymerases of Escherichia coli. Purification of these membrane-bound bifunctional transglycosylase-transpeptidases was a major obstacle in studying the details of both enzymatic reactions. Here we describe a simple, highly specific affinity chromatography method that takes advantage of the availability of the specific inhibitor of the transglycosylase site moenomycin A in order to enrich PBP 1A and 1Bs in one step from crude membrane preparations. Separation of PBP 1A from PBP 1Bs is achieved in a second step employing cation exchange chromatography yielding enzymatically active native murein polymerases.

Two-step procedure for purification and separation of the essential penicillin-binding proteins PBP 1A and 1Bs of Escherichia coli.

The penicillin-binding proteins PBP 1A and 1Bs are the essential murein polymerases of Escherichia coli. Purification of these membrane-bound bifunctional transglycosylase-transpeptidases was a major obstacle in studying the details of both enzymatic reactions. Here we describe a simple, highly specific affinity chromatography method that takes advantage of the availability of the specific inhibitor of the transglycosylase site moenomycin A in order to enrich PBP 1A and 1Bs in one step from crude membrane preparations. Separation of PBP 1A from PBP 1Bs is achieved in a second step employing cation exchange chromatography yielding enzymatically active native murein polymerases.