Purification and characterization of PBP4a, a new low-molecular-weight penicillin-binding protein from Bacillus subtilis

Penicillin-binding protein 4a (PBP4a) from Bacillus subtilis was overproduced and purified to homogeneity. It clearly exhibits DD-carboxypeptidase and thiolesterase activities in vitro. Although highly isologous to the Actinomadura sp. strain R39 DD-peptidase (B. Granier, C. Duez, S. Lepage, S. Englebert, J. Dusart, O. Dideberg, J. van Beeumen, J. M. Frère, and J. M. Ghuysen, Biochem. J. 282:781-788, 1992), which is rapidly inactivated by many beta-lactams, PBP4a is only moderately sensitive to these compounds. The second-order rate constant (k(2)/K) for the acylation of the essential serine by benzylpenicillin is 300,000 M(-1) s(-1) for the Actinomadura sp. strain R39 peptidase, 1,400 M(-1) s(-1) for B. subtilis PBP4a, and 7,000 M(-1) s(-1) for Escherichia coli PBP4, the third member of this class of PBPs. Cephaloridine, however, efficiently inactivates PBP4a (k(2)/K = 46,000 M(-1) s(-1)). PBP4a is also much more thermostable than the R39 enzyme.     

Substrate specificity of low-molecular mass bacterial DD-peptidases

The bacterial DD-peptidases or penicillin-binding proteins (PBPs) catalyze the formation and regulation of cross-links in peptidoglycan biosynthesis. They are classified into two groups, the high-molecular mass (HMM) and low-molecular mass (LMM) enzymes. The latter group, which is subdivided into classes A-C (LMMA, -B, and -C, respectively), is believed to catalyze DD-carboxypeptidase and endopeptidase reactions in vivo. To date, the specificity of their reactions with particular elements of peptidoglycan structure has not, in general, been defined. This paper describes the steady-state kinetics of hydrolysis of a series of specific peptidoglycan-mimetic peptides, representing various elements of stem peptide structure, catalyzed by a range of LMM PBPs (the LMMA enzymes, Escherichia coli PBP5, Neisseria gonorrhoeae PBP4, and Streptococcus pneumoniae PBP3, and the LMMC enzymes, the Actinomadura R39 dd-peptidase, Bacillus subtilis PBP4a, and N. gonorrhoeae PBP3). The R39 enzyme (LMMC), like the previously studied Streptomyces R61 DD-peptidase (LMMB), specifically and rapidly hydrolyzes stem peptide fragments with a free N-terminus. In accord with this result, the crystal structures of the R61 and R39 enzymes display a binding site specific to the stem peptide N-terminus. These are water-soluble enzymes, however, with no known specific function in vivo. On the other hand, soluble versions of the remaining enzymes of those noted above, all of which are likely to be membrane-bound and/or associated in vivo and have been assigned particular roles in cell wall biosynthesis and maintenance, show little or no specificity for peptides containing elements of peptidoglycan structure. Peptidoglycan-mimetic boronate transition-state analogues do inhibit these enzymes but display notable specificity only for the LMMC enzymes, where, unlike peptide substrates, they may be able to effectively induce a specific active site structure. The manner in which LMMA (and HMM) DD-peptidases achieve substrate specificity, both in vitro and in vivo, remains unknown.     

Affinity of exocellular DD-carboxypeptidase/transpeptidase from Saccharopolyspora erythraea PZH TZ-575 to beta-lactam compounds

The DD-carboxypeptidase/transpeptidases (DD-peptidases) involved in bacterial cell wall metabolism, catalyse the attack of C-terminal D-alanyl-D-alanine peptide bond of the peptydoglycan precursor. These enzymes are inactivated by beta-lactam antibiotics. DD-peptidase from Saccharopolyspora erythraea PZH TZ 64-575 was purified by the use of DEAE-cellulose, Sephadex G-100, Q-Sepharose resins and FPLC (Mono Q). After each step the effluent was concentrated by Amicon ultrafiltration. The purified enzyme showed DD-carboxypeptidase specific activity of 50.9 U/mg. The enzyme exhibited high affinity to beta-lactam compounds e.g. cefamandole, cefapirin, cefradin 1.5-2.6 x 10(-8) M. It was used to screen strains from the Culture Collection of the National Institute of Hygiene in Warsaw for the production of DD-peptidase inhibitors.     

dacD, an Escherichia coli gene encoding a novel penicillin-binding protein (PBP6b) with DD-carboxypeptidase activity.

In the course of a study of genes located at min 44 of the Escherichia coli genome, we identified an open reading frame with the capacity to encode a 43-kDa polypeptide whose predicted amino acid sequence is strikingly similar to those of the well-known DD-carboxipeptidases penicillin-binding proteins PBP5 and PBP6. The gene product was shown to bind [3H]benzylpenicillin and to have DD-carboxypeptidase activity on pentapeptide muropeptides in vivo. Therefore, we called the protein PBP6b and the gene dacD. As with other E. coli DD-carboxypeptidases, PBP6b is not essential for cell growth. A quadruple dacA dacB dacC dacD mutant was constructed and shown to grow as well as its isogenic wild-type strain, indicating that the loss of any known PBP-associated DD-carboxypeptidase activity is not deleterious for E. coli. We also identified the homologous gene of dacD in Salmonella typhimurium as one of the components of the previously described phsBCDEF gene cluster.     

Activities and regulation of peptidoglycan synthases

Peptidoglycan (PG) is an essential component in the cell wall of nearly all bacteria, forming a continuous, mesh-like structure, called the sacculus, around the cytoplasmic membrane to protect the cell from bursting by its turgor. Although PG synthases, the penicillin-binding proteins (PBPs), have been studied for 70 years, useful in vitro assays for measuring their activities were established only recently, and these provided the first insights into the regulation of these enzymes. Here, we review the current knowledge on the glycosyltransferase and transpeptidase activities of PG synthases. We provide new data showing that the bifunctional PBP1A and PBP1B from Escherichia coli are active upon reconstitution into the membrane environment of proteoliposomes, and that these enzymes also exhibit DD-carboxypeptidase activity in certain conditions. Both novel features are relevant for their functioning within the cell. We also review recent data on the impact of protein–protein interactions and other factors on the activities of PBPs. As an example, we demonstrate a synergistic effect of multiple protein–protein interactions on the glycosyltransferase activity of PBP1B, by its cognate lipoprotein activator LpoB and the essential cell division protein FtsN.     

Analysis of peptidoglycan structure from vegetative cells of Bacillus subtilis 168 and role of PBP 5 in peptidoglycan maturation.

The composition and fine structure of the vegetative cell wall peptidoglycan from Bacillus subtilis were determined by analysis of its constituent muropeptides. The structures of 39 muropeptides, representing 97% of the total peptidoglycan, were elucidated. About 99% analyzed muropeptides in B. subtilis vegetative cell peptidoglycan have the free carboxylic group of diaminopimelic acid amidated. Anhydromuropeptides and products missing a glucosamine at the nonreducing terminus account for 0.4 and 1.5%, respectively, of the total muropeptides. These two types of muropeptides are suggested to end glycan strands. An unexpected feature of B. subtilis muropeptides was the occurrence of a glycine residue in position 5 of the peptide side chain on monomers or oligomers, which account for 2.7% of the total muropeptides. This amount is, however, dependent on the composition of the growth media. Potential attachment sites for anionic polymers to peptidoglycan occur on dominant muropeptides and account for 2.1% of the total. B. subtilis peptidoglycan is incompletely digested by lysozyme due to de-N-acetylation of glucosamine, which occurs on 17.3% of muropeptides. The cross-linking index of the polymer changes with the growth phase. It is highest in late stationary phase, with a value of 33.2 or 44% per muramic acid residue, as determined by reverse-phase high-pressure liquid chromatography or gel filtration, respectively. Analysis of the muropeptide composition of a dacA (PBP 5) mutant shows a dramatic decrease of muropeptides with tripeptide side chains and an increase or appearance of muropeptides with pentapeptide side chains in monomers or oligomers. The total muropeptides with pentapeptide side chains accounts for almost 82% in the dacA mutant. This major low-molecular-weight PBP (DD-carboxypeptidase) is suggested to play a role in peptidoglycan maturation.     

Comparison of high-performance liquid chromatography and fluorophore-assisted carbohydrate electrophoresis methods for analyzing peptidoglycan composition of Escherichia coli

Currently, reversed-phase high-performance liquid chromatography (HPLC) is the method of choice for determining the types and amounts of muropeptide subunits comprising bacterial peptidoglycan. Although effective and sensitive, the technique does not lend itself to high throughput screening, and its complexity and equipment requirements may dissuade some investigators from pursuing certain types of cell wall experiments. Previously, we showed that muropeptides can be labeled with a fluorescent dye and separated by fluorophore-assisted carbohydrate electrophoresis (FACE), a simple and rapid gel procedure that might serve as a prelude to more intense analysis by HPLC. To validate the utility of FACE, we used both techniques to perform a side-by-side analysis of the peptidoglycan of eight mutants and their Escherichia coli parent strain. FACE and HPLC both detected the seven major muropeptides, which represent more than 95% of the total muropeptides present in this organism. In addition, FACE returned the same relative and quantitative results in 92% of 72 measurements, indicating that the procedure gives an accurate overview of peptidoglycan composition. The results also suggest a possible biochemical activity for the AmpC and AmpH proteins of E. coli, and the use of FACE as an in vitro enzyme assay detected possible substrate preferences for the endopeptidase penicillin binding protein 4.     

Substitution of Alanine at Position 184 with Glutamic Acid in <Emphasis Type="Italic">Escherichia coli</Emphasis> PBP5 Ω-Like Loop Introduces a Moderate Cephalosporinase Activity

Escherichia coli PBP5, a DD-carboxypeptidase (DD-CPase), helps in maintaining cell shape and intrinsic β-lactam resistance. Though PBP5 does not have β-lactamase activity under physiological pH, it has a common but shorter Ω-like loop resembling class A β-lactamases. However, such Ω-like loop lacks the key glutamic acid residue that is present in β-lactamases. It is speculated that β-lactamases and DD-CPases might have undergone divergent evolution leading to distinct enzymes with different substrate specificities and functions indicating the versatility of the Ω-loops. Nonetheless, direct experimental evidence favoring the idea is insufficient. Here, aiming to investigate the effect of introducing a glutamic acid residue in the PBP5 Ω-like loop, we substituted A184 to E to create PBP5_A184E. Expression of PBP5_A184E in E. coli ?PBP5 mutant elevates the β-lactam resistance, especially for cephalosporins. However, like PBP5, PBP5_A184E has the ability to complement the aberrantly shaped E. coli septuple PBP mutant indicating an unaffected in vivo DD-CPase activity. Biochemical and bioinformatics analyses have substantiated the dual enzyme nature of the mutated enzyme possessing both DD-CPase and β-lactamase activities. Therefore, substitution of A184 to E of Ω-like loop alone can introduce the cephalosporinase activity in E. coli PBP5 supporting the phenomenon of a single amino acid polymorphism.     

Multivariate geometrical analysis of catalytic residues in the penicillin-binding proteins

Penicillin-binding proteins (PBPs) are bacterial enzymes involved in the final stages of cell wall biosynthesis, and are targets of the β-lactam antibiotics. They can be subdivided into essential high-molecular-mass (HMM) and non-essential low-molecular-mass (LMM) PBPs, and further divided into subclasses based on sequence homologies. PBPs can catalyze transpeptidase or hydrolase (carboxypeptidase and endopeptidase) reactions. The PBPs are of interest for their role in bacterial cell wall biosynthesis, and as mechanistically interesting enzymes which can catalyze alternative reaction pathways using the same catalytic machinery. A global catalytic residue comparison seemed likely to provide insight into structure-function correlations within the PBPs. More than 90 PBP structures were aligned, and a number (40) of active site geometrical parameters extracted. This dataset was analyzed using both univariate and multivariate statistical methods. Several interesting relationships were observed. (1) Distribution of the dihedral angle for the SXXK-motif Lys side chain (DA_1) was bimodal, and strongly correlated with HMM/transpeptidase vs LMM/hydrolase classification/activity (P<0.001). This structural feature may therefore be associated with the main functional difference between the HMM and LMM PBPs. (2) The distance between the SXXK-motif Lys-NZ atom and the Lys/His-nitrogen atom of the (K/H)T(S)G-motif was highly conserved, suggesting importance for PBP function, and a possibly conserved role in the catalytic mechanism of the PBPs. (3) Principal components-based cluster analysis revealed several distinct clusters, with the HMM Class A and B, LMM Class C, and LMM Class A K15 PBPs forming one "Main" cluster, and demonstrating a globally similar arrangement of catalytic residues within this group.     

Contribution of membrane-binding and enzymatic domains of penicillin binding protein 5 to maintenance of uniform cellular morphology of Escherichia coli

Four low-molecular-weight penicillin binding proteins (LMW PBPs) of Escherichia coli are closely related and have similar DD-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD). However, only one, PBP 5, has a demonstrated physiological function. In its absence, certain mutants of E. coli have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells. To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other DD-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5. Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD. However, complementation was not restored by the putative carboxy-terminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound. Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required. A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal beta-sheet domain of PBP 6. In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional. The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the DD-carboxypeptidase enzymatic core.     

Crystal structure of the Actinomadura R39 DD-peptidase reveals new domains in penicillin-binding proteins

Actinomadura sp. R39 produces an exocellular DD-peptidase/penicillin-binding protein (PBP) whose primary structure is similar to that of Escherichia coli PBP4. It is characterized by a high beta-lactam-binding activity (second order rate constant for the acylation of the active site serine by benzylpenicillin: k2/K = 300 mm(-1) s(-1)). The crystal structure of the DD-peptidase from Actinomadura R39 was solved at a resolution of 1.8 angstroms by single anomalous dispersion at the cobalt resonance wavelength. The structure is composed of three domains: a penicillin-binding domain similar to the penicillin-binding domain of E. coli PBP5 and two domains of unknown function. In most multimodular PBPs, additional domains are generally located at the C or N termini of the penicillin-binding domain. In R39, the other two domains are inserted in the penicillin-binding domain, between the SXXK and SXN motifs, in a manner similar to "Matryoshka dolls." One of these domains is composed of a five-stranded beta-sheet with two helices on one side, and the other domain is a double three-stranded beta-sheet inserted in the previous domain. Additionally, the 2.4-angstroms structure of the acyl-enzyme complex of R39 with nitrocefin reveals the absence of active site conformational change upon binding the beta-lactams.     

Characterization of the bifunctional glycosyltransferase/acyltransferase penicillin-binding protein 4 of Listeria monocytogenes

Multimodular penicillin-binding proteins (PBPs) are essential enzymes responsible for bacterial cell wall peptidoglycan (PG) assembly. Their glycosyltransferase activity catalyzes glycan chain elongation from lipid II substrate (undecaprenyl-pyrophosphoryl-N-acetylglucosamine-N-acetylmuramic acid-pentapeptide), and their transpeptidase activity catalyzes cross-linking between peptides carried by two adjacent glycan chains. Listeria monocytogenes is a food-borne pathogen which exerts its virulence through secreted and cell wall PG-associated virulence factors. This bacterium has five PBPs, including two bifunctional glycosyltransferase/transpeptidase class A PBPs, namely, PBP1 and PBP4. We have expressed and purified the latter and have shown that it binds penicillin and catalyzes in vitro glycan chain polymerization with an efficiency of 1,400 M(-1) s(-1) from Escherichia coli lipid II substrate. PBP4 also catalyzes the aminolysis (d-Ala as acceptor) and hydrolysis of the thiolester donor substrate benzoyl-Gly-thioglycolate, indicating that PBP4 possesses both transpeptidase and carboxypeptidase activities. Disruption of the gene lmo2229 encoding PBP4 in L. monocytogenes EGD did not have any significant effect on growth rate, peptidoglycan composition, cell morphology, or sensitivity to beta-lactam antibiotics but did increase the resistance of the mutant to moenomycin.     

Characterization of a beta -N-acetylglucosaminidase of Escherichia coli and elucidation of its role in muropeptide recycling and beta -lactamase induction

Using the known mapping position the gene encoding a beta-1, 4-N-acetylglucosaminidase needed for the degradation of muropeptides could be identified. nagZ encodes a cytosolic enzyme active on N-actylglucosamyl-beta-1,4-(1,6)-anhydromuramic acid containing muropeptides. These degradation products of the peptidoglycan are formed during the enlargement of the murein sacculus as a consequence of a growth mechanism, which couples the controlled degradation of the cell wall polymer with the insertion of new material. NagZ is needed for the formation of monosaccharides from the released disaccharides during the cytosolic steps of the muropeptide-recycling pathway. The formation of intracellular 1, 6-anhydro-N-acetylmuramyl-peptides is important for the expression control of the inducible beta-lactamases of the AmpC type. A mutant lacking active NagZ cannot establish AmpC mediated beta-lactam resistance. The biochemical characterization of the enzyme showed its activity on different muropeptides and inhibitors of enzyme activity could be identified. This observation might be important for designing inhibitors of NagZ that could prevent the establishment of beta-lactam resistance of Enterobacteria possessing inducible beta-lactamases.     

Correlation between alterations of the penicillin-binding protein 2 and modifications of the peptidoglycan structure in Neisseria meningitidis with reduced susceptibility to penicillin G

Reduced susceptibility to penicillin G in Neisseria meningitidis is directly correlated with alterations in the penA gene, which encodes the penicillin-binding protein 2 (PBP2). Using purified PBP2s from different backgrounds, we confirmed that the reduced susceptibility to penicillin G is associated with a decreased affinity of altered PBP2s for penicillin G. Infrared spectroscopy analysis using isogenic penicillin-susceptible strains and strains with reduced susceptibility to penicillin G suggested that the meningococcal cell wall is also modified in a penA-dependent manner. Moreover, reverse-phase high pressure liquid chromatography and mass spectrometry analysis of these meningococcal strains confirmed the modifications of peptidoglycan components and showed an increase in the peaks corresponding to pentapeptide-containing muropeptides. These results suggest that the D,D-transpeptidase and/or D,D-carboxypeptidase activities of PBP2 are modified by the changes in penA gene.     

Two distinct transpeptidation reactions during murein synthesis in Escherichia coli.

Murein synthesized in ether-permeabilized cells of Escherichia coli deficient in individual penicillin-binding proteins (PBPs) and in the presence of certain beta-lactam antibiotics was analyzed by high-pressure liquid chromatography separation of the muramidase split products. PBP 1b was found to to be the major murein synthesizing activity that was poorly compensated for by PBP 1a. A PBP 2 mutant as well as mecillinam-inhibited cells showed increased activity in the formation of oligomeric muropeptides as well as UDP-muramylpeptidyl-linked muropeptides, the reaction products of transpeptidation, bypassing the lipid intermediate. In contrast, penicillin G and furazlocillin severely inhibited these reactions but stimulated normal dimer production. It is concluded that two distinct transpeptidases exist in E. coli: one, highly sensitive to penicillin G and furazlocillin, catalyzes the formation of hyper-cross-linked muropeptides, and a second one, quite resistant to these antibiotics, synthesizes muropeptide dimers.     

Kinetics of reactions of the Actinomadura R39 DD-peptidase with specific substrates

The Actinomadura R39 DD-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are not, however, well understood. This paper describes the interactions of the R39 enzyme with two peptidoglycan-mimetic substrates 3-(D-cysteinyl)propanoyl-D-alanyl-D-alanine and 3-(D-cysteinyl)propanoyl-D-alanyl-D-thiolactate. A detailed study of the reactions of the former substrate, catalyzed by the enzyme, showed DD-carboxypeptidase, DD-transpeptidase, and DD-endopeptidase activities. These results confirm the specificity of the enzyme for a free D-amino acid at the N-terminus of good substrates and indicated a preference for extended D-amino acid leaving groups. The latter was supported by determination of the structural specificity of amine nucleophiles for the acyl-enzyme generated by reaction of the enzyme with the thiolactate substrate. It was concluded that a specific substrate for this enzyme, and possibly the in vivo substrate, may consist of a partly cross-linked peptidoglycan polymer where a free side chain N-terminal un-cross-linked amino acid serves as the specific acyl group in an endopeptidase reaction. The enzyme is most likely a DD-endopeptidase in vivo. pH-rate profiles for reactions of the enzyme with peptides, the thiolactate named above, and β-lactams indicated the presence of complex proton dissociation pathways with sticky substrates and/or protons. The local structure of the active site may differ significantly for reactions of peptides and β-lactams. Solvent kinetic deuterium isotope effects indicate the presence of classical general acid/base catalysis in both acylation and deacylation; there is no evidence of the low fractionation factor active site hydrogen found previously in class A and C β-lactamases.     

Multiple mechanisms of membrane anchoring of Escherichia coli penicillin-binding proteins

Abstract: The major penicillin-binding proteins (PBPs) of Escherichia coli play vital roles in cell wall biosynthesis and are located in the inner membrane. The high M _r PBPs 1A, 1B, 2 and 3 are essential bifunctional transglycosylases/transpeptidases which are thought to be type II integral inner membrane proteins with their C-terminal enzymatic domains projecting into the periplasm. The low M _r PBP4 is a DD-carboxypeptidase/endopeptidase, whereas PBPs 5 and are DD-carboxypeptidases. All three low M _r , PBPs act in the modification of peptidoglycan to allow expansion of the sacculus and are thought to be periplasmic proteins attached with varying affinities to the inner membrane via C-terminal amphiphilic α-helices. It is possible that the PBPs and other inner membrane proteins form a peptidoglycan synthesizing complex to coordinate their activities.     

Early midcell localization of Escherichia coli PBP4 supports the function of peptidoglycan amidases

Insertion of new material into the Escherichia coli peptidoglycan (PG) sacculus between the cytoplasmic membrane and the outer membrane requires a well-organized balance between synthetic and hydrolytic activities to maintain cell shape and avoid lysis. Since most bacteria carry multiple enzymes carrying the same type of PG hydrolytic activity, we know little about the specific function of given enzymes. Here we show that the DD-carboxy/endopeptidase PBP4 localizes in a PBP1A/LpoA and FtsEX dependent fashion at midcell during septal PG synthesis. Midcell localization of PBP4 requires its non-catalytic domain 3 of unknown function, but not the activity of PBP4 or FtsE. Microscale thermophoresis with isolated proteins shows that PBP4 interacts with NlpI and the FtsEX-interacting protein EnvC, an activator of amidases AmiA and AmiB, which are needed to generate denuded glycan strands to recruit the initiator of septal PG synthesis, FtsN. The domain 3 of PBP4 is needed for the interaction with NlpI and EnvC, but not PBP1A or LpoA. In vivo crosslinking experiments confirm the interaction of PBP4 with PBP1A and LpoA. We propose that the interaction of PBP4 with EnvC, whilst not absolutely necessary for mid-cell recruitment of either protein, coordinates the activities of PBP4 and the amidases, which affects the formation of denuded glycan strands that attract FtsN. Consistent with this model, we found that the divisome assembly at midcell was premature in cells lacking PBP4, illustrating how the complexity of interactions affect the timing of cell division initiation.     

Inhibition of bacterial DD-peptidases (penicillin-binding proteins) in membranes and in vivo by peptidoglycan-mimetic boronic acids

The DD-peptidases or penicillin-binding proteins (PBPs) catalyze the final steps of bacterial peptidoglycan biosynthesis and are inhibited by the β-lactam antibiotics. There is at present a question of whether the active site structure and activity of these enzymes is the same in the solubilized (truncated) DD-peptidase constructs employed in crystallographic and kinetics studies as in membrane-bound holoenzymes. Recent experiments with peptidoglycan-mimetic boronic acids have suggested that these transition state analogue-generating inhibitors may be able to induce reactive conformations of these enzymes and thus inhibit strongly. We have now, therefore, measured the dissociation constants of peptidoglycan-mimetic boronic acids from Escherichia coli and Bacillus subtilis PBPs in membrane preparations and, in the former case, in vivo, by means of competition experiments with the fluorescent penicillin Bocillin Fl. The experiments showed that the boronic acids bound measurably (K(i) < 1 mM) to the low-molecular mass PBPs but not to the high-molecular mass enzymes, both in membrane preparations and in whole cells. In two cases, E. coli PBP2 and PBP5, the dissociation constants obtained were very similar to those obtained with the pure enzymes in homogeneous solution. The boronic acids, therefore, are unable to induce tightly binding conformations of these enzymes in vivo. There is no evidence from these experiments that DD-peptidase inhibitors are more or less effective in vivo than in homogeneous solution.     

The DD-carboxypeptidase activity encoded by pbp4B is not essential for the cell growth of Escherichia coli

The gene (pbp4B) encoding a putative DD-carboxypeptidase has been deleted in Escherichia coli and it is shown to be not essential for cell division. Disruption of the gene in a genetic background where all putative activities of DD-carboxypeptidases and/or DD-endopeptidases had been eliminated indicates that these activities are not required for cell growth in enterobacteria. The penicillin-binding capacity and a low DD-carboxypeptidase activity of PBP4B are demonstrated. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.