Penicillin-binding proteins (PBPs), which catalyze the biosynthesis of the peptidoglycan chain of the bacterial cell wall, are the major molecular target of bacterial antibiotics. Here, we present the crystal structures of the bifunctional peptidoglycan glycosyltransferase (GT)/transpeptidase (TP) PBP4 from Listeria monocytogenes in the apo-form and covalently linked to two β-lactam antibiotics, ampicillin and carbenicillin. The orientation of the TP domain with respect to the GT domain is distinct from that observed in the previously reported structures of bifunctional PBPs, suggesting interdomain flexibility. In this structure, the active site of the GT domain is occluded by the close apposition of the linker domain, which supports the hypothesis that interdomain flexibility is related to the regulation of GT activity. The acylated structures reveal the mode of action of β-lactam antibiotics toward the class A PBP4 from the human pathogen L. monocytogenes. Ampicillin and carbenicillin can access the active site and be acylated without requiring a structural rearrangement. In addition, the active site of the TP domain in the apo-form is occupied by the tartrate molecule via extensive hydrogen bond interactions with the catalytically important residues; thus, derivatives of the tartrate molecule may be useful in the search for new antibiotics to inhibit PBPs.
Penicillin-binding protein 5 (PBP5) is one of the most abundant PBPs in Pseudomonas aeruginosa. Although its main function is that of a cell wall dd-carboxypeptidase, it possesses sufficient β-lactamase activity to contribute to the ability of P. aeruginosa to resist the antibiotic activity of the β-lactams. The study of these dual activities is important for understanding the mechanisms of antibiotic resistance by P. aeruginosa, an important human pathogen, and to the understanding of the evolution of β-lactamase activity from the PBP enzymes. We purified a soluble version of P. aeruginosa PBP5 (designated Pa sPBP5) by deletion of its C-terminal membrane anchor. Under in vitro conditions, Pa sPBP5 demonstrates both dd-carboxypeptidase and expanded-spectrum β-lactamase activities. Its crystal structure at a 2.05-Å resolution shows features closely resembling those of the class A β-lactamases, including a shortened loop spanning residues 74 to 78 near the active site and with respect to the conformations adopted by two active-site residues, Ser101 and Lys203. These features are absent in the related PBP5 of Escherichia coli. A comparison of the two Pa sPBP5 monomers in the asymmetric unit, together with molecular dynamics simulations, revealed an active-site flexibility that may explain its carbapenemase activity, a function that is absent in the E. coli PBP5 enzyme. Our functional and structural characterizations underscore the versatility of this PBP5 in contributing to the β-lactam resistance of P. aeruginosa while highlighting how broader β-lactamase activity may be encoded in the structural folds shared by the PBP and serine β-lactamase classes.
Resistance to β-lactam antibiotics in Streptococcus pneumoniae is due to alteration of penicillin-binding proteins (PBPs). S. pneumoniae PBP 1a belongs to the class A high-molecular-mass PBPs, which harbor transpeptidase (TP) and glycosyltransferase (GT) activities. The GT active site represents a new potential target for the generation of novel nonpenicillin antibiotics. The 683-amino-acid extracellular region of PBP 1a (PBP 1a*) was expressed in Escherichia coli as a GST fusion protein. The GST-PBP 1a* soluble protein was purified, and its domain organization was revealed by limited proteolysis. A protease-resistant fragment spanning Ser 264 to Arg 653 exhibited a reactivity profile against both β-lactams and substrate analogues similar to that of the parent protein. This protein fragment represents the TP domain. The GT domain (Ser 37 to Lys 263) was expressed as a recombinant GST fusion protein. Protection by moenomycin of the GT domain against trypsin degradation was interpreted as an interaction between the GT domain and the moenomycin.
Penicillin-binding protein 1B (PBP1B) of Escherichia coli is a bifunctional murein synthase containing both a transpeptidase domain and a transglycosylase domain. The protein is present in three forms (α, β, and γ) which differ in the length of their N-terminal cytoplasmic region. Expression plasmids allowing the production of native PBP1B or of PBP1B variants with an inactive transpeptidase or transglycosylase domain or both were constructed. The inactive domains contained a single amino acid exchange in an essential active-site residue. Overproduction of the inactive PBP1B variants, but not of the active proteins, caused lysis of wild-type cells. The cells became tolerant to lysis by inactive PBP1B at a pH of 5.0, which is similar to the known tolerance for penicillin-induced lysis under acid pH conditions. Lysis was also reduced in mutant strains lacking several murein hydrolases. In particular, a strain devoid of activity of all known lytic transglycosylases was virtually tolerant, indicating that mainly the lytic transglycosylases are responsible for the observed lysis effect. A possible structural interaction between PBP1B and murein hydrolases in vivo by the formation of a multienzyme complex is discussed.
Penicillin binding proteins (PBPs) are membrane-associated proteins that catalyze the final step of murein biosynthesis. These proteins function as either transpeptidases or carboxypeptidases and in a few cases demonstrate transglycosylase activity. Both transpeptidase and carboxypeptidase activities of PBPs occur at the d-Ala-d-Ala terminus of a murein precursor containing a disaccharide pentapeptide comprising N-acetylglucosamine and N-acetyl-muramic acid-l-Ala-d-Glu-l-Lys-d-Ala-d-Ala. β-Lactam antibiotics inhibit these enzymes by competing with the pentapeptide precursor for binding to the active site of the enzyme. Here we describe the crystal structure, biochemical characteristics, and expression profile of PBP4, a low-molecular-mass PBP from Staphylococcus aureus strain COL. The crystal structures of PBP4-antibiotic complexes reported here were determined by molecular replacement, using the atomic coordinates deposited by the New York Structural Genomics Consortium. While the pbp4 gene is not essential for the viability of S. aureus, the knockout phenotype of this gene is characterized by a marked reduction in cross-linked muropeptide and increased vancomycin resistance. Unlike other PBPs, we note that expression of PBP4 was not substantially altered under different experimental conditions, nor did it change across representative hospital- or community-associated strains of S. aureus that were examined. In vitro data on purified recombinant S. aureus PBP4 suggest that it is a β-lactamase and is not trapped as an acyl intermediate with β-lactam antibiotics. Put together, the expression analysis and biochemical features of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.
Myeloperoxidase (MPO), H2O2, and chloride comprise a potent antimicrobial system believed to contribute to the antimicrobial functions of neutrophils and monocytes. The mechanisms of microbicidal action are complex and not fully defined. This report describes the MPO-mediated inactivation, in Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, of a class of cytoplasmic membrane enzymes (penicillin-binding proteins, PBPs) found in all eubacteria, that covalently bind beta-lactam antibiotics to their active sites with loss of enzymatic activity. Inactivation of "essential" PBPs, including PBP1-PBP3 of E. coli, leads to unbalanced bacterial growth and cell death. MPO treatment of bacteria was associated with loss of penicillin binding by PBPs, strongly suggesting PBP inactivation. In E. coli, PBP inactivation was most rapid with PBP3, where the rate of decline in binding activity approximated but did not equal loss of viability. Changes in E. coli morphology (elongation), observed just before bacteriolysis, were consistent with early predominant inactivation of PBP3. We conclude that inactivation of essential PBPs is sufficient to account for an important fraction of MPO-mediated bacterial action. This feature of MPO action interestingly recapitulates an antibacterial strategy evolved by beta-lactam-producing molds that must compete with bacteria for limited ecologic niches.
Cefditoren is the active form of cefditoren pivoxil, an oral cephalosporin antibiotic used for the treatment of respiratory tract infections and otitis media caused by bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus pyogenes, Klebsiella pneumoniae, and methicillin-susceptible strains of Staphylococcus aureus. β-Lactam antibiotics, including cefditoren, target penicillin-binding proteins (PBPs), which are membrane-associated enzymes that play essential roles in the peptidoglycan biosynthetic process. To envision the binding of cefditoren to PBPs, we determined the crystal structure of a trypsin-digested form of PBP 2X from S. pneumoniae strain R6 complexed with cefditoren. There are two PBP 2X molecules (designated molecules 1 and 2) per asymmetric unit. The structure reveals that the orientation of Trp374 in each molecule changes in a different way upon the formation of the complex, but each forms a hydrophobic pocket. The methylthiazole group of the C-3 side chain of cefditoren fits into this binding pocket, which consists of residues His394, Trp374, and Thr526 in molecule 1 and residues His394, Asp375, and Thr526 in molecule 2. The formation of the complex is also accompanied by an induced-fit conformational change of the enzyme in the pocket to which the C-7 side chain of cefditoren binds. These features likely play a role in the high level of activity of cefditoren against S. pneumoniae.
PBPA from Mycobacterium tuberculosis (Mtb) is a class B-like penicillin-binding protein (PBP) that is not essential for cell growth in Mtb, but is important for proper cell division in M. smegmatis. We have determined the crystal structure of PBPA at 2.05 Å resolution, the first published structure of a PBP from this important pathogen. Compared to other PBPs, PBPA has a relatively small N-terminal domain and conservation of a cluster of charged residues within this domain suggests that PBPA is more related to Class B PBPs than previously inferred from sequence analysis. The C-terminal domain is a typical transpeptidase fold and contains the three conserved active site motifs that characterize penicillin-interacting enzymes. Whilst the arrangement of the SxxK and KTG motifs is similar to that observed in other PBPs, the SxN motif is markedly displaced away from the active site, such that its serine (Ser281) is not involved in hydrogen bonding with residues of the other two motifs. A disulphide bridge between Cys282 (the “x” of the SxN motif) and Cys266, which resides on an adjacent loop, may be responsible for this unusual conformation. Another interesting feature of the structure is a relatively long connection between β5 and α11, which restricts the space available in the active site of PBPA, and suggests that conformational changes would be required to accommodate peptide substrate or β-lactam antibiotics during acylation. Finally, the structure shows that one of the two threonines postulated to be targets for phosphorylation is inaccessible (Thr362), whereas the other (Thr437) is well placed on a surface loop near the active site.
penicillin-binding proteins; tuberculosis; peptidoglycan; X-ray crystallography
It was suggested previously that the primary structure of penicillin-binding protein 4 (PBP4) is new and unique among proteins that interact with penicillin. Our proposal that PBP4 carries an additional domain, located between the active-site fingerprints SXXK and SXN, was investigated by mutational deletion analysis. A clustered set of internal deletions was created in this region by exonuclease treatment of the dacB coding DNA, starting from two internal restriction sites. PBP4 mutants carrying internal deletions were selected by screening for immunoreactive forms of PBP4 with reduced molecular weight that were still active with respect to penicillin binding. DNA sequencing revealed 24 distinct PBP4 mutants with internal deletions ranging from 37 to 113 amino acids. The amino- and carboxy-terminal end points of the deletions were not randomly distributed but tended to cluster in certain areas. Overproduction of the individual mutated forms of PBP4 resulted in accumulation of the major portion of the proteins in the particulate cell fraction. The yield of soluble and active mutated forms of the protein was reduced from below 1% to 79% of the level obtained for the native protein. The deletions that were introduced had minor effects on the deacylation rate of bound benzylpenicillin. Two pairs of cysteine residues (Cys-139-Cys-153 and Cys-197-Cys-214) that are located in the deletable region may form disulfide bridges.
Because the specificity profile of the membrane anchor-free G57-V577 penicillin-binding protein 3 (PBP3) of Escherichia coli for a large series of beta-lactam antibiotics is similar to that of the full-size membrane-bound PBP, the truncated PBP is expected to adopt the native folded conformation. The truncated PBP3 functions as a thiolesterase. In aqueous media and in the presence of millimolar concentrations of a properly structured amino compound, it catalyzes the aminolysis of the thiolester until completion, suggesting that the penicillin-binding module of PBP3 is designed to catalyze transpeptidation reactions. In contrast, the truncated PBP3 is devoid of glycan polymerization activity on the E. coli lipid II intermediate, suggesting that the non-penicillin-binding module of PBP3 is not a transglycosylase.
Extensive use of β-lactam antibiotics has led to the selection of pathogenic streptococci resistant to β-lactams due to modifications of the penicillin-binding proteins (PBPs). PBP2b from Streptococcus pneumoniae is a monofunctional (class B) high-molecular-weight PBP catalyzing the transpeptidation between adjacent stem peptides of peptidoglycan. The transpeptidase domain of PBP2b isolated from seven clinical resistant (CR) strains contains 7 to 44 amino acid changes over the sequence of PBP2b from the R6 β-lactam-sensitive strain. We show that the extracellular soluble domains of recombinant PBP2b proteins (PBP2b*) originating from these CR strains have an in vitro affinity for penicillin G that is reduced by up to 99% from that of the R6 strain. The Thr446Ala mutation is always observed in CR strains and is close to the key conserved motif (S443SN). The Thr446Ala mutation in R6 PBP2b* displays a 60% reduction in penicillin G affinity in vitro compared to that for the wild-type protein. A recombinant R6 strain expressing the R6 PBP2b Thr446Ala mutation is twofold less sensitive to piperacillin than the parental S. pneumoniae strain. Analysis of the Thr446Ala mutation in the context of the PBP2b CR sequences revealed that its influence depends upon the presence of other unidentified mutations.
Penicillin-binding proteins (PBPs) catalyze the essential reactions in the biosynthesis of cell wall peptidoglycan from glycopeptide precursors. β-Lactam antibiotics normally interfere with this process by reacting covalently with the active site serine to form a stable acyl-enzyme. The design of novel β-lactams active against penicillin-susceptible and penicillin-resistant organisms will require a better understanding of the molecular details of this reaction. To that end, we compared the affinities of different β-lactam antibiotics to a modified soluble form of a resistant Enterococcus faecium PBP5 (Δ1-36 rPBP5). The soluble protein, Δ1-36 rPBP5, was expressed in Escherichia coli and purified, and the NH2-terminal protein sequence was verified by amino acid sequencing. Using β-lactams with different R1 side chains, we show that azlocillin has greater affinity for Δ1-36 rPBP5 than piperacillin and ampicillin (apparent Ki = 7 ± 0.3 μM, compared to 36 ± 3 and 51 ± 10 μM, respectively). Azlocillin also exhibits the most rapid acylation rate (apparent k2 = 15 ± 4 M−1 s−1). Meropenem demonstrates an affinity for Δ1-36 rPBP5 comparable to that of ampicillin (apparent Ki = 51 ± 15 μM) but is slower at acylating (apparent k2 = 0.14 ± 0.02 M−1 s−1). This characterization defines important structure-activity relationships for this clinically relevant type II transpeptidase, shows that the rate of formation of the acyl-enzyme is an essential factor determining the efficacy of a β-lactam, and suggests that the specific side chain interactions of β-lactams could be modified to improve inactivation of resistant PBPs.
Penicillin-binding proteins (PBPs) are well known and validated targets for antibacterial therapy. The most important clinically used inhibitors of PBPs β-lactams inhibit transpeptidase activity of PBPs by forming a covalent penicilloyl-enzyme complex that blocks the normal transpeptidation reaction; this finally results in bacterial death. In some resistant bacteria the resistance is acquired by active-site distortion of PBPs, which lowers their acylation efficiency for β-lactams. To address this problem we focused our attention to discovery of novel noncovalent inhibitors of PBPs.
Our in-house bank of compounds was screened for inhibition of three PBPs from resistant bacteria: PBP2a from Methicillin-resistant Staphylococcus aureus (MRSA), PBP2x from Streptococcus pneumoniae strain 5204, and PBP5fm from Enterococcus faecium strain D63r. Initial hit inhibitor obtained by screening was then used as a starting point for computational similarity searching for structurally related compounds and several new noncovalent inhibitors were discovered. Two compounds had promising inhibitory activities of both PBP2a and PBP2x 5204, and good in-vitro antibacterial activities against a panel of Gram-positive bacterial strains.
We found new noncovalent inhibitors of PBPs which represent important starting points for development of more potent inhibitors of PBPs that can target penicillin-resistant bacteria.
Penicillin-binding proteins (PBPs) in representatives of two Streptococcus pneumoniae clonal groups that are prevalent in Poland, Poland23F-16 and Poland6B-20, were investigated by PBP profile analysis, antibody reactivity pattern analysis, and DNA sequence analysis of the transpeptidase (TP) domain-encoding regions of the pbp2x, pbp2b, and pbp1a genes. The isolates differed in their MICs of β-lactam antibiotics. The majority of the 6B isolates were intermediately susceptible to penicillin (penicillin MICs, 0.12 to 0.5 μg/ml), whereas all 23F isolates were penicillin resistant (MICs, ≥2 μg/ml). The 6B isolates investigated had the same sequence type (ST), determined by multilocus sequence typing, as the Poland6B-20 reference strain (ST315), but in the 23F group, isolates with three distinct single-locus variants (SLVs) in the ddl gene (ST173, ST272, and ST1506) were included. None of the isolates showed an identical PBP profile after labeling with Bocillin FL and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and only one pair of 6B isolates and one pair of 23F isolates (ST173 and ST272) each contained an identical combination of PBP 2x, PBP 2b, and PBP 1a TP domains. Some 23F isolates contained PBP 3 with an apparently higher electrophoretic mobility, and this feature also did not correlate with their STs. The data document a highly variable pool of PBP genes as a result of multiple gene transfer and recombination events within and between different clonal groups.
The emergence of beta-lactam-resistant strains of Pseudomonas aeruginosa in a cystic fibrosis patient treated with high-dose tobramycin and piperacillin was studied. Two serotypes, M and K, were present before treatment and persisted, with changes in their beta-lactam resistance spectra, during treatment. The resistance was correlated with changes in the penicillin-binding proteins (PBPs) in both serotypes. In the low-level-resistant serotype K organism, PBP-3 either was absent or had lost the ability to bind [14C]penicillin G. Tow serotypes M strains, one with low- and one with high-level resistance to several antipseudomonal beta-lactam antibiotics, were isolated at progressively later stages of therapy. Several differences were noted between the PBP patterns of the resistant M and the susceptible M strains. The affinity for [14C]penicillin G was reduced in both resistant strains. PBP bands, with the exception of PBP-6 in the most resistant M type, were barely or not detectable at a [14C]penicillin G concentration of 39 microgram/ml. The graduated decrease in affinity for [14c]penicillin G was correlated with increasing beta-lactam resistance and with an increase in the quantity of the protein corresponding to PBP-6. The emergence of the low-level-resistant strains midway through, and of the highly resistant strain in the final stages of, the reported treatment strongly suggested that the resistance resulted from mutation in those strains present before treatment selected for by the high-dose piperacillin treatment.
The mechanism of action of a new antipseudomonal penicillin, PC-904, was studied with respect to its binding affinities to penicillin-binding proteins (PBPs) and its inhibitory activities on cross-linking enzymes of peptidoglycan synthesis in vitro. PC-904 showed especially high affinity (compared with that of penicillin G) to Escherichia coli PBP-3. It also had high affinities to PBP-2 and -1Bs and low affinities to PBP-1A, -4, -5, and -6. Similar results were obtained with Pseudomonas aeruginosa, in which this antibiotic showed very high affinity (compared with that of penicillin G) to PBP-3, -1A (presumably corresponding to E. coli PBP-1Bs), and -2; there was especially high affinity to PBP-3 and much less affinity to PBP-1B (presumably corresponding to E. coli PBP-1A). These results are compatible with morphological observations that at concentrations near its minimal inhibitory concentration or less, this antibiotic induced the formation of filamentous cells of E. coli and P. aeruginosa. At higher concentrations or after prolonged incubation, it induced lysis of the cells. The remarkably high affinity of PC-904 to pseudomonal PBP-3, -1A, and -2 may partly explain the potent antipseudomonal activity of this antibiotic. In E. coli, the concentration of PC-904 required to inhibit the cross-linking reaction in enzymatic peptidoglycan synthesis, presumably carried out by PBP-1Bs, was as low as the inhibitory concentrations of penicillin G, ampicillin, and carbenicillin.
Resistance to methicillin by Staphylococcus aureus is a persistent clinical problem worldwide. A mechanism for resistance has been proposed in which methicillin resistant Staphylococcus aureus (MRSA) isolates acquired a new protein called β-lactam inducible penicillin binding protein (PBP-2′). The PBP-2′ functions by substituting other penicillin binding proteins which have been inhibited by β-lactam antibiotics. Presently, there is no structural and regulatory information on PBP-2′ protein. We conducted a complete structural and functional regulatory analysis of PBP-2′ protein. Our analysis revealed that the PBP-2′ is very stable with more hydrophilic amino acids expressing antigenic sites. PBP-2′ has three striking regulatory points constituted by first penicillin binding site at Ser25, second penicillin binding site at Ser405, and finally a single metallic ligand binding site at Glu657 which binds to Zn2+ ions. This report highlights structural features of PBP-2′ that can serve as targets for developing new chemotherapeutic agents and conducting site direct mutagenesis experiments.
To understand the biochemical basis of resistance of bacteria to beta-lactam antibiotics, we purified a penicillin-resistant penicillin-binding protein 2x (R-PBP2x) and a penicillin-sensitive PBP2x (S-PBP2x) enzyme of Streptococcus pneumoniae and characterized their transpeptidase activities, using a thioester analog of stem peptides as a substrate. A comparison of the k(cat)/Km values for the two purified enzymes (3,400 M(-1) s(-1) for S-PBP2x and 11.2 M(-1) s(-1) for R-PBP2x) suggests that they are significantly different kinetically. Implications of this finding are discussed. We also found that the two purified enzymes did not possess a detectable level of beta-lactam hydrolytic activity. Finally, we show that the expression levels of both PBP2x enzymes were similar during different growth phases.
Biapenem is a parenteral carbapenem antibiotic that exhibits wide-ranging antibacterial activity, remarkable chemical stability, and extensive stability against human renal dehydropeptidase-I. Tebipenem is the active form of tebipenem pivoxil, a novel oral carbapenem antibiotic that has a high level of bioavailability in humans, in addition to the above-mentioned features. β-lactam antibiotics, including carbapenems, target penicillin-binding proteins (PBPs), which are membrane-associated enzymes that play essential roles in peptidoglycan biosynthesis. To envisage the binding of carbapenems to PBPs, we determined the crystal structures of the trypsin-digested forms of both PBP 2X and PBP 1A from Streptococcus pneumoniae strain R6, each complexed with biapenem or tebipenem. The structures of the complexes revealed that the carbapenem C-2 side chains form hydrophobic interactions with Trp374 and Thr526 of PBP 2X and with Trp411 and Thr543 of PBP 1A. The Trp and Thr residues are conserved in PBP 2B. These results suggest that interactions between the C-2 side chains of carbapenems and the conserved Trp and Thr residues in PBPs play important roles in the binding of carbapenems to PBPs.
Penicillin-binding proteins (PBPs) are bacterial cytoplasmic membrane proteins that catalyze the final steps of the peptidoglycan synthesis. Resistance to β-lactams in Streptococcus pneumoniae is caused by low-affinity PBPs. S. pneumoniae PBP 2a belongs to the class A high-molecular-mass PBPs having both glycosyltransferase (GT) and transpeptide (TP) activities. Structural and functional studies of both domains are required to unravel the mechanisms of resistance, a prerequisite for the development of novel antibiotics. The extracellular region of S. pneumoniae PBP 2a has been expressed (PBP 2a*) in Escherichia coli as a glutathione S-transferase fusion protein. The acylation kinetic parameters of PBP 2a* for β-lactams were determined by stopped-flow fluorometry. The acylation efficiency toward benzylpenicillin was much lower than that toward cefotaxime, a result suggesting that PBP 2a participates in resistance to cefotaxime and other β-lactams, but not in resistance to benzylpenicillin. The TP domain was purified following limited proteolysis. PBP 2a* required detergents for solubility and interacted with lipid vesicles, while the TP domain was water soluble. We propose that PBP 2a* interacts with the cytoplasmic membrane in a region distinct from its transmembrane anchor region, which is located between Lys 78 and Ser 156 of the GT domain.
The crystallization of PBP4 from L. monocytogenes is reported.
Penicillin-binding proteins (PBPs), which catalyze peptidoglycan synthesis, have been extensively studied as a well established target of antimicrobial agents, including β-lactam derivatives. However, remarkable resistance to β-lactams has developed among pathogenic bacteria since the clinical use of penicillin began. Recently, the glycosyltransferase (GT) domain of class A PBPs has been proposed as an attractive target for antibiotic development as moenomycin-bound GT-domain structures have been determined. In this study, a class A PBP4 from Listeria monocytogenes was overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected to 2.1 Å resolution using synchrotron radiation. The crystal belonged to the primitive orthorhombic space group P21212, with unit-cell parameters a = 84.6, b = 127.8, c = 54.9 Å. The structural information will contribute to the further development of moenomycin-derived antibiotics possessing broad-spectrum activity.
penicillin-binding proteins; Listeria monocytogenes
The pbpB gene, which encodes penicillin-binding protein (PBP) 2B of Bacillus subtilis, has been cloned, sequenced, mapped, and mutagenized. The sequence of PBP 2B places it among the class B high-molecular-weight PBPs. It appears to contain three functional domains: an N-terminal domain homologous to the corresponding domain of other class B PBPs, a penicillin-binding domain, and a lengthy carboxy extension. The PBP has a noncleaved signal sequence at its N terminus that presumably serves as its anchor in the cell membrane. Previous studies led to the hypothesis that PBP 2B is required for both vegetative cell division and sporulation septation. Its sequence, map site, and mutant phenotype support this hypothesis. PBP 2B is homologous to PBP 3, the cell division protein encoded by pbpB of Escherichia coli. Moreover, both pbpB genes are located in the same relative position within a cluster of cell division and cell wall genes on their respective chromosomes. However, immediately adjacent to the B. subtilis pbpB gene is spoVD, which appears to be a sporulation-specific homolog of pbpB. Inactivation of SpoVD blocked synthesis of the cortical peptidoglycan in the spore, whereas carboxy truncation of PBP 2B caused cells to grow as filaments. Thus, it appears that a gene duplication has occurred in B. subtilis and that one PBP has evolved to serve a common role in septation during both vegetative growth and sporulation, whereas the other PBP serves a specialized role in sporulation.
By using a broad-host-range vector, pUCP27, the Pseudomonas aeruginosa and Escherichia coli pbpB genes, which encode penicillin-binding protein 3 (PBP3), were separately overexpressed in a P. aeruginosa strain, PAO4089, that is deficient in producing chromosomal beta-lactamase. Susceptibility studies indicated that overproduction of the P. aeruginosa PBP3 in PAO4089 resulted in twofold-increased resistance to aztreonam, fourfold-increased resistance to cefepime and cefsulodin, and eightfold-increased resistance to ceftazidime, whereas overproduction of the P. aeruginosa PBP3 in PAO4089 did not affect susceptibility to PBP1-targeted cephaloridine or PBP2-targeted imipenem. Similar results were obtained with PAO4089 overproducing E. coli PBP3, with the exception that there was no influence on the MICs or minimal bactericidal concentrations of cefsulodin and cefepime, which have very low affinities for E. coli PBP3. These data are consistent with the conclusion that PBP3 has to achieve a certain level of saturation, with beta-lactams targeted to this protein, to result in cell inhibition or death.
A mutant of Pseudomonas aeruginosa strain PAO503 was isolated after ethane-methane-sulfonate mutagenesis and selection of ticarcillin. The mutant, PCC17, displayed reduced affinity for [14C] penicillin G at all of its penicillin-binding proteins as well as a general increase in resistance to all the beta-lactam antibiotics tested. The mutation designated pbpA has been mapped by FP-2-mediated conjugation and was located distal to the proA locus and 33% linked to it. The two loci were not cotransducible with phage F116L. PCC17 and exconjugants produced from it had similar phenotypes, displayed the reduced affinity for [14C] penicillin G, had similar resistance profiles, and had an increased amount of protein corresponding to penicillin-binding protein 6. On back mutation the pbpA locus reverted to the PAO503 phenotype.
Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of β-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 (ponA, pbpF, and pbpZ). Among mutants with single or double deletions, only JH2-2 ΔponA ΔpbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of β-lactams formed a subset of the class A PBPs of E. faecalis, and heterospecific complementation was observed with an ortholog from E. faecium. Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.