Penicillin-binding Activity Research Articles

Detection and analysis of Lactobacillus paracasei penicillin-binding proteins revealed the presence of cholate-sensitive penicillin-binding protein 3 and an elongated cell shape in a cholate-sensitive strain.

Penicillin-binding proteins (PBPs) are responsible for peptidoglycan synthesis. By using biotinylated ampicillin, we detected PBPs of Lactobacillus paracasei strains. Ten PBPs were identified, 7 of which had apparent molecular sizes similar to those of Escherichia coli. In the presence of cholate, strain NRIC 0625 showed an elongated shape, and its putative PBP3 showed cholate-sensitive penicillin-binding activity. Furthermore, this strain was highly sensitive to cefalexin, which is known to inhibit cell division by inactivating PBP3. These results suggest that the septum synthetase PBP3 of lactic acid bacteria can be one of the targets of intestinal bile acid.

Expression and Characterization of Penicillin-Binding Proteins in Burkholderia cenocepacia

Putative penicillin-binding proteins (PBPs) were identified in the genome of the Burkholderia cenocepacia strain J2315 based on homology to E. coli PBPs. The three sequences identified as homologs of E. coli PBP1a, BCAL2021, BCAL0274, and BCAM2632, were cloned and expressed as His(6)-tagged fusion proteins in E. coli. The fusion proteins were isolated and shown to bind beta-lactams, indicating these putative PBPs have penicillin-binding activity.

Specificity inversion of Ochrobactrum anthropi D‐aminopeptidase to a D,D‐carboxypeptidase with new penicillin binding activity by directed mutagenesis

The serine penicillin-recognizing proteins have been extensively studied. They show a wide range of substrate specificities accompanied by multidomain features. Their adaptation capacity has resulted in the emergence of pathogenic bacteria resistant to beta-lactam antibiotics. The most divergent enzymatic activities in this protein family are those of the Ochrobactrum anthropi D-aminopeptidase and of the Streptomyces R61 D,D-carboxypeptidase/transpeptidase. With the help of structural data, we have attempted to identify the factors responsible for this opposite specificity. A loop deletion mutant of the Ochrobactrum anthropi D-aminopeptidase lost its original activity in favor of a new penicillin-binding activity. D-aminopeptidase activity of the deletion mutant can be restored by complementation with another deletion mutant corresponding to the noncatalytic domain of the wild-type enzyme. By a second step site-directed mutagenesis, the specificity of the Ochrobactrum anthropi D-aminopeptidase was inverted to a D,D-carboxypeptidase specificity. These results imply a core enzyme with high diversity potential surrounded by specificity modulators. It is the first example of drastic specificity change in the serine penicillin-recognizing proteins. These results open new perspectives in the conception of new enzymes with nonnatural specificities. The structure/specificity relationship in the serine penicillin-recognizing proteins are discussed.

Crystal Structure of the 47-kDa Lipoprotein of Treponema pallidum Reveals a Novel Penicillin-binding Protein

Syphilis is a complex sexually transmitted disease caused by the spirochetal bacterium Treponema pallidum. T. pallidum has remained exquisitely sensitive to penicillin, but the mode of action and lethal targets for beta-lactams are still unknown. We previously identified the T. pallidum 47-kDa lipoprotein (Tp47) as a penicillin-binding protein (PBP). Tp47 contains three hypothetical consensus motifs (SVTK, TEN, and KTG) that typically form the active center of other PBPs. Yet, in this study, mutations of key amino acids within these motifs failed to abolish the penicillin binding activity of Tp47. The crystal structure of Tp47 at a resolution of 1.95 A revealed a fold different from any other known PBP; Tp47 is predominantly beta-sheet, in contrast to the alpha/beta-fold common to other PBPs. It comprises four distinct domains: two complex beta-sheet-containing N-terminal domains and two C-terminal domains that adopt immunoglobulin-like folds. The three hypothetical PBP signature motifs do not come together to form a typical PBP active site. Furthermore, Tp47 is unusual in that it displays beta-lactamase activity (k(cat) for penicillin = 271 +/- 6 s(-1)), a feature that hindered attempts to identify the active site in Tp47 by co-crystallization and mass spectrometric techniques. Taken together, Tp47 does not fit the classical structural and mechanistic paradigms for PBPs, and thus Tp47 appears to represent a new class of PBP.

Characterization of derivatives of the high-molecular-mass penicillin-binding protein (PBP) 1 of Mycobacterium leprae

Mycobacterium leprae has two high-molecular-mass multimodular penicillin-binding proteins (PBPs) of class A, termed PBP1 and PBP1* [Lepage, Dubois, Ghosh, Joris, Mahapatra, Kundu, Basu, Chakrabarti, Cole, Nguyen-Disteche and Ghuysen (1997) J. Bacteriol. 179, 4627–4630]. PBP1-Xaa–β-lactamase fusions generated periplasmic β-lactamase activity when Xaa (the amino acid of PBP1 at the fusion junction) was residue 314, 363, 407, 450 or 480. Truncation of the N-terminal part of the protein up to residue Leu-147 generated a penicillin-binding polypeptide which could still associate with the plasma membrane, whereas [∆M1–R314]PBP1 (PBP1 lacking residues Met-1 to Arg-314) failed to associate with the membrane, suggesting that the region between residues Leu-147 and Arg-314 harbours an additional plasma membrane association site for PBP1. Truncation of the C-terminus up to 42 residues downstream of the KTG (Lys-Thr-Gly) motif also generated a polypeptide that retained penicillin-binding activity. [∆M1–R314]PBP1 could be extracted from inclusion bodies and refolded under appropriate conditions to give a form capable of binding penicillin with the same efficiency as full-length PBP1. This is, to the best of our knowledge, the first report of a soluble derivative of a penicillin-resistant high-molecular-mass PBP of class A that is capable of binding penicillin. A chimaeric PBP in which the penicillin-binding (PB) module of PBP1 was fused at its N-terminal end with the non-penicillin-binding (n-PB) module of PBP1* retained pencillin-binding activity similar to that of PBP1, corroborating the finding that the n-PB module of PBP1 is dispensable for its penicillin-binding activity.

Characterization of derivatives of the high-molecular-mass penicillin-binding protein (PBP) 1 of Mycobacterium leprae

Mycobacterium leprae has two high-molecular-mass multimodular penicillin-binding proteins (PBPs) of class A, termed PBP1 and PBP1* [Lepage, Dubois, Ghosh, Joris, Mahapatra, Kundu, Basu, Chakrabarti, Cole, Nguyen-Disteche and Ghuysen (1997) J. Bacteriol. 179, 4627-4630]. PBP1-Xaa-beta-lactamase fusions generated periplasmic beta-lactamase activity when Xaa (the amino acid of PBP1 at the fusion junction) was residue 314, 363, 407, 450 or 480. Truncation of the N-terminal part of the protein up to residue Leu-147 generated a penicillin-binding polypeptide which could still associate with the plasma membrane, whereas [DeltaM1-R314]PBP1 (PBP1 lacking residues Met-1 to Arg-314) failed to associate with the membrane, suggesting that the region between residues Leu-147 and Arg-314 harbours an additional plasma membrane association site for PBP1. Truncation of the C-terminus up to 42 residues downstream of the KTG (Lys-Thr-Gly) motif also generated a polypeptide that retained penicillin-binding activity. [DeltaM1-R314]PBP1 could be extracted from inclusion bodies and refolded under appropriate conditions to give a form capable of binding penicillin with the same efficiency as full-length PBP1. This is, to the best of our knowledge, the first report of a soluble derivative of a penicillin-resistant high-molecular-mass PBP of class A that is capable of binding penicillin. A chimaeric PBP in which the penicillin-binding (PB) module of PBP1 was fused at its N-terminal end with the non-penicillin-binding (n-PB) module of PBP1* retained pencillin-binding activity similar to that of PBP1, corroborating the finding that the n-PB module of PBP1 is dispensable for its penicillin-binding activity.

Identification of a fmtA-like gene that has similarity to other PBPs and β-lactamases in Staphylococcus aureus

We identified a gene from Staphylococcus aureus, flp ( fmtA-like protein), encoding a protein of 489 amino acid residues with a molecular mass of 56.4 kDa. The deduced amino acid sequence shows similarity to previously characterized penicillin binding proteins (PBPs) and FmtA of S. aureus (one of the factors which affect methicillin resistance). FLP protein has three motifs, which are conserved in PBPs and β-lactamases, suggesting that it might be associated with cell wall synthesis. Recombinant FLP protein, however, lacks penicillin binding activity, and the inactivation of flp in two methicillin-resistant S. aureus strains did not cause a reduction in the methicillin resistance.

Identification of a fmtA-like gene that has similarity to other PBPs and beta-lactamases in Staphylococcus aureus.

We identified a gene from Staphylococcus aureus, flp (fmtA-like protein), encoding a protein of 489 amino acid residues with a molecular mass of 56.4 kDa. The deduced amino acid sequence shows similarity to previously characterized penicillin binding proteins (PBPs) and FmtA of S. aureus (one of the factors which affect methicillin resistance). FLP protein has three motifs, which are conserved in PBPs and beta-lactamases, suggesting that it might be associated with cell wall synthesis. Recombinant FLP protein, however, lacks penicillin binding activity, and the inactivation of flp in two methicillin-resistant S. aureus strains did not cause a reduction in the methicillin resistance.

Characterization of fmtA, a gene that modulates the expression of methicillin resistance in Staphylococcus aureus.

FmtA is a factor which affects the methicillin resistance level in methicillin-resistant Staphylococcus aureus. Since FmtA has two of three conserved motifs which are typically found in penicillin-binding proteins (PBPs) and beta-lactamases, we investigated the penicillin-binding activity of recombinant FmtA and found no such activity. Immunoblotting analysis revealed that FmtA localizes in the membrane fraction. To investigate the function of FmtA, high-pressure liquid chromatography analysis of cell wall muropeptides was performed with an fmtA-inactivated mutant and its parent. The mutant showed a reduced cross-linking and partially reduced amidation of glutamate residues in the peptidoglycan of the mutant. The transcription of fmtA was dose dependently increased by the addition of beta-lactam antibiotics, fosfomycin, and bacitracin, while its transcription was not changed by the addition of vancomycin or tetracycline. These results reveal that Fmt is a membrane-located, non-penicillin-binding protein and that mutation of fmtA affects the cell wall structure, although its precise function is still unknown.

Cloning and Characterization of a Gene, pbpF , Encoding a New Penicillin-Binding Protein, PBP2B, in Staphylococcus aureus

A previously unrecognized penicillin binding protein (PBP) gene, pbpF, was identified in Staphylococcus aureus. This gene encodes a protein of 691 amino acid residues with an estimated molecular mass of 78 kDa. The molecular mass is very close to that of S. aureus PBP2 (81 kDa), and the protein is tentatively named PBP2B. PBP2B has three motifs, SSVK, SSN, and KTG, that can be found in PBPs and beta-lactamases. Recombinant PBP2B (rPBP2B), which lacks a putative signal peptide at the N terminus and has a histidine tag at the C terminus, was expressed in Escherichia coli. The purified rPBP2B was shown to have penicillin binding activity. A protein band was detected from S. aureus membrane fraction by immunoblotting with anti-rPBP2B serum. Also, penicillin binding activity of the protein immunoprecipitated with anti-rPBP2B serum was detected. These results suggest the presence of PBP2B in S. aureus cell membrane that covalently binds penicillin. The internal region of pbpF and PBP2B protein were found in all 12 S. aureus strains tested by PCR and immunoblotting.

AmpC and AmpH, proteins related to the class C beta-lactamases, bind penicillin and contribute to the normal morphology of Escherichia coli.

Two proteins that bind penicillin were observed in Escherichia coli infected with lambda phages 141, 142, 650, and 651 from the Kohara genomic library. These phages carry chromosomal DNA fragments that do not contain any known penicillin binding protein (PBP) genes, indicating that unrecognized gene products were exhibiting penicillin binding activity. The genes encoding these proteins were subcloned, sequenced, and identified. One gene was ampC, which encodes a chromosomal class C beta-lactamase. The second gene was located at about 8.5 min on the E. coli genomic map and is a previously uncharacterized open reading frame, here named ampH, that encodes a protein closely related to the class C beta-lactamases. The predicted AmpH protein is similar in length to AmpC, but there are extensive alterations in the amino acid sequence between the SXXK and YXN motifs of the two proteins. AmpH bound strongly to penicillin G, cefoxitin, and cephalosporin C; was temperature sensitive; and disappeared from cells after overnight incubation in stationary phase. Although closely related to AmpC and other class C beta-lactamases, AmpH showed no beta-lactamase activity toward the substrate nitrocefin. Mutation of the ampC and/or ampH genes in E. coli lacking PBPs 1a and 5 produced morphologically aberrant cells, particularly in cell filaments induced by aztreonam. Thus, these two members of the beta-lactamase family exhibit characteristics similar to those of the classical PBPs, and their absence affects cell morphology. These traits suggest that AmpC and AmpH may play roles in the normal course of peptidoglycan synthesis, remodeling, or recycling.

Topographical and functional investigation of Escherichia coli penicillin-binding protein 1b by alanine stretch scanning mutagenesis.

Penicillin-binding proteins (PBPs) are the targets of beta-lactam antibiotics. We have used a systematic five-alanine substitution method (called ASS [alanine stretch scanning] mutagenesis) to investigate the functional or structural role of various stretches of amino acids in the PBP1b of Escherichia coli. To probe the specific activity of each variant, the antibiotic discs assay was used with strain QCB1 (delta ponB) in the presence of cefaloridine, which totally inhibits the complementing action of PBP1a. This in vivo test has been combined with a quick and efficient in vitro test of the penicillin-binding activity of each of these variants with fluorescent penicillin. This approach has enabled us to show an unexpected role of the N-terminal and C-terminal tails of PBP1b. Moreover, we have established the correct position in PBP1b of the SMN motif that, with the SXXK and the KTG motifs, constitutes the signature of the penicilloyl serine transferases family. Finally, we have shown that the transglycosylase and the transpeptidase domains are separated by an inert linker region, where substitutions and insertions can be made without hindering the in vivo and in vitro activity of the protein.

Inactivation of Escherichia coli penicillin-binding proteins by human neutrophils

Neutrophils use a variety of microbicidal mechanisms in their role as one of the primary arms of the human host defense system. We have previously observed that a cell-free system containing myeloperoxidase (MPO), one of the major components of the neutrophil's oxidative antimicrobial systems, inactivated microbial penicillin-binding proteins (PBPs), which mediate the formation of the peptidoglycan layer of eubacterial cell walls. This is a potentially important mechanism of MPO-mediated bacterial toxicity. Since numerous other microbicidal systems, both oxidative and nonoxidative, are used by whole neutrophils, we investigated the effect of intact neutrophils on Escherichia coli PBPs. Penicillin binding activity was progressively reduced by neutrophil exposure for all PBPs. Loss of penicillin binding activity correlated well with loss of microbial viability for almost all PBPs. Azide-treated neutrophils, MPO-deficient neutrophils, and chronic granulomatous disease neutrophils inactivated E. coli PBPs in a manner similar to that of normal neutrophils, suggesting that MPO-independent, and even oxygen-independent, microbicidal systems are also involved in inactivation of PBPs. PBP inactivation, an antimicrobial strategy used by beta-lactam-producing molds (and now by physicians), may be an important microbicidal mechanism used by human neutrophils.

Site-directed mutagenesis of the mecA gene from a methicillin-resistant strain of Staphylococcus aureus

The mecA-27r gene from Staphylococcus aureus 27r encodes penicillin-binding protein 2a (PBP2a-27r), which causes this strain to be methicillin resistant. Removal or replacement of the N-terminal transmembrane domain had no effect on binding of penicillin, but removal of portions of the putative transglycosylase domain (144, 245, or 341 amino acids after the transmembrane region) destroyed penicillin-binding activity. The SXXK, SXN, and KSG motifs, present in all penicillin-interacting enzymes, were found in the expected linear spatial arrangement within the putative transpeptidase region of PBP2a-27r. Alterations of amino acids in all three of these motifs resulted in elimination of penicillin-binding activity, confirming their roles in the interaction with penicillin.

Active-site residues of the transpeptidase domain of penicillin-binding protein 2 from Escherichia coli: similarity in catalytic mechanism to class A .beta.-lactamases

By means of amino acid sequence alignment with class A beta-lactamases, the residues essential for the catalytic activity of the peptidoglycan transpeptidase of penicillin-binding protein 2 (PBP2) have been predicted to be Lys333, Asp447, and Lys544, in addition to the acylation site residue for the acyl-enzyme mechanism, Ser330. Accordingly, these residues were replaced by site-directed mutagenesis, and the resultant mutants were examined as to penicillin-binding activity and genetic complementation, which represent only the acylation step and the total reaction during transpeptidation, respectively. All the mutants at position 333 showed the complete loss of both the binding and complementation activities. Most of the mutants at position 447 retained the binding activity but lost the complementation activity, the exception being the D447E mutant, which retained both. The binding rates for various penicillins of the D447N mutant, which had lost the complementation activity, were almost identical to those of the wild type. The binding of the mutants at position 544 tended to require a higher penicillin concentration, and that of the K544H mutant required a lower pH. When the roles of the counterpart residues, Lys73, Glu166, and Lys234, in class A beta-lactamases were considered, the results suggested that Lys333 and Asp447 are essential for the acylation and acyl-transfer steps, respectively, and that Lys544 stabilizes the Michaelis complex through its side-chain positive charge.

Membrane topology of penicillin-binding protein 3 of Escherichia coli.

The beta-lactamase fusion vector, pJBS633, has been used to analyse the organization of penicillin-binding protein 3 (PBP3) in the cytoplasmic membrane of Escherichia coli. The fusion junctions in 84 in-frame fusions of the coding region of mature TEM beta-lactamase to random positions within the PBP3 gene were determined. Fusions of beta-lactamase to 61 different positions in PBP3 were obtained. Fusions to positions within the first 31 residues of PBP3 resulted in enzymatically active fusion proteins which could not protect single cells of E. coli from killing by ampicillin, indicating that the beta-lactamase moieties of these fusion proteins were not translocated to the periplasm. However, all fusions that contained greater than or equal to 36 residues of PBP3 provided single cells of E. coli with substantial levels of resistance to ampicillin, indicating that the beta-lactamase moieties of these fusion proteins were translocated to the periplasm. PBP3 therefore appeared to have a simple membrane topology with residues 36 to the carboxy-terminus exposed on the periplasmic side of the cytoplasmic membrane. This topology was confirmed by showing that PBP3 was protected from proteolytic digestion at the cytoplasmic side of the inner membrane but was completely digested by proteolytic attack from the periplasmic side. PBP3 was only inserted in the cytoplasmic membrane at its amino terminus since replacement of its putative lipoprotein signal peptide with a normal signal peptide resulted in a water-soluble, periplasmic form of the enzyme. The periplasmic form of PBP3 retained its penicillin-binding activity and appeared to be truly water-soluble since it fractionated, in the absence of detergents, with the expected molecular weight on Sephadex G-100 and was not retarded by hydrophobic interaction chromatography on Phenyl-Superose.

Identification of the penicillin-binding active site of penicillin-binding protein 2 of Escherichia coli.

We determined the active site of penicillin-binding protein (PBP) 2 of Escherichia coli. A water-soluble form of PBP 2, which was constructed by site-directed mutagenesis, was purified by affinity chromatography, labeled with dansyl-penicillin, and then digested with a combination of proteases. The amino acid composition of the labeled chymotryptic peptide purified by HPLC was identical with that of the amino acid sequence, Ala-Thr-Gln-Gly-Val-Tyr-Pro-Pro-Ala-Ser330-Thr-Val-Lys-Pro (residues 321-334) of PBP 2, which was deduced from the nucleotide sequence of the pbpA gene encoding PBP 2. This amino acid sequence was verified by sequencing the labeled tryptic peptide containing the labeled chymotryptic peptide region. A mutant PBP 2 (thiol-PBP 2), constructed by site-directed mutagenesis to replace Ser330 with Cys, lacked the penicillin-binding activity. These findings provided evidence that Ser330 near the middle of the primary structure of PBP 2 is the penicillin-binding active-site residue, as predicted previously on the basis of the sequence homology. Around this active site, the sequence Ser-Xaa-Xaa-Lys was observed, which is conserved in the active-site regions of all E. coli PBPs so far studied, class A and class C beta-lactamases, and D-Ala carboxypeptidases. The COOH-terminal amino acid of PBP 2 was identified as His633.

A water-soluble form of penicillin-binding protein 2 of Escherichia coli constructed by site-directed mutagenesis

Penicillin-binding protein (PBP) 2 of Escherichia coli is located in the cytoplasmic membrane. The N-terminal hydrophobic segment (31 amino acids, residues 15–45) of PBP2 was removed by a deletion in the PBP2 gene by site-directed mutagenesis, resulting in the production of a water-soluble form of PBP2 (called PBP2*). PBP2* retained the penicillin-binding activity, was localized in the cytoplasm and was overproduced under the control of the lpp-lac promoter. This indicates that the removed hydrophobic segment is an uncleaved signal sequence required for translocation of PBP2 across the cytoplasmic membrane, and also suggests that the segment anchors the protein to the membrane.

Major peptidoglycan transglycosylase activity inStreptococcus pneumoniaethat is not a penicillin-binding protein

As in the Gram-positive cocci Staphylococcus aureus and Micrococcus luteus, which divide in 3 planes, Streptococcus pneumoniae, which divides on 1 fixed plane, possesses peptidoglycan transglycosylase activity that lacks penicillin-binding activity as the sole detectable transglycosylase activity in vitro. The penicillin-binding proteins (PBPs) in this organism did not show transglycosylase activity under the conditions in which the PBPs of Escherichia coli showed both transglycosylase and transpeptidase activities.

Isolation of five penicillin-binding proteins fromStaphylococcus aureus

Five penicillin-binding proteins have been isolated from detergent-solubilized membranes of Staphylococcus aureus and have been separated from other membrane proteins by covalent affinity chromatography on 6-aminopenicillanic acid-Sepharose. The PBPs were resolved by electrophoresis on SDS-polyacrylamide gels and their ability to bind radioactive penicillin assayed after transfer from the polyacrylamide gel onto nitrocellulose membrane filters. Synthesis of a β-lactam antibiotic, propionylampicillin and of [2,3-3H]propionylampicillin of high specific activity greatly facilitated the assay of penicillin-binding activity.