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.     

Two penicillin binding proteins of Haemophilus influenzae are lost after cells enter stationary phase

Abstract The penicillin binding proteins (PBPs) of 4 representative isolates of Haemophilus influenzae were studied using crude membrane preparations and whole cells grown to the logarithmic and stationary phases of growth. Relative binding, % of total bound, and binding affinities were compared. The PBP patterns were similar for crude membranes and whole cells for all 4 strains tested at each phase of growth. However, PBP 2 was slightly reduced and PBP 4 was markedly reduced with whole-cell labelling in comparison to crude membranes. 8 PBPs were detected in cells labelled during the logarithmic phase of growth, while 6 were detected in stationary phase cells. The pBPs 'lost' in stationary phase (PBPs 4 and 6) with apparent M _r of 62 000 and 45 000, respectively, have a high affinity for ampicillin ( I ₅₀≃ 0.04 μ g/ml). This suggests that these proteins may have an important role in cell growth, and are targets for β-lactam substrates.     

Several distinct localization patterns for penicillin-binding proteins in Bacillus subtilis

Bacterial cell shape is determined by a rigid external cell wall. In most non-coccoid bacteria, this shape is also determined by an internal cytoskeleton formed by the actin homologues MreB and/or Mbl. To gain further insights into the topological control of cell wall synthesis in bacteria, we have constructed green fluorescent protein (GFP) fusions to all 11 penicillin-binding proteins (PBPs) expressed during vegetative growth of Bacillus subtilis. The localization of these fusions was studied in a wild-type background as well as in strains deficient in FtsZ, MreB or Mbl. PBP3 and PBP4a localized specifically to the lateral wall, in distinct foci, whereas PBP1 and PBP2b localized specifically to the septum. All other PBPs localized to both the septum and the lateral cell wall, sometimes with irregular distribution along the lateral wall or a preference for the septum. This suggests that cell wall synthesis is not dispersed but occurs at specific places along the lateral cell wall. The results implicate PBP3, PBP5 and PBP4a, and possibly PBP4, in lateral wall growth. Localization of PBPs to the septum was found to be dependent on FtsZ, but the GFP-PBP fluorescence patterns were not detectably altered in the absence of MreB or Mbl.     

Identification of two penicillin-binding multienzyme complexes in Haemophilus influenzae.

Dansyl-labeled penicillin, reversed-phase chromatography, and peptide mapping have been used to detect, separate, and study penicillin-binding proteins (PBPs) and PBP multienzyme complexes of H. influenzae. The cross-linking of proteins in the multienzyme complex was accomplished with the aid of cyanogen, a salt-bridge specific cross-linking agent. The chromatographic profile of the PBPs clearly showed a dramatic change in the number and identity of peaks after treatment of the bacterial cells with cyanogen. The disappearance of all seven peaks corresponding to the PBPs was accompanied by the emergence of two new peaks with molecular weights between 400 kDa and 600 kDa. The results hint at the existence of two penicillin-binding multienzyme complexes, each containing subunits that interact via salt-bridges. Chromatographic active site peptide mapping of PBPs and PBP complexes was used to determine the identity of PBPs involved in each complex. It is postulated that one multienzyme complex containing PBP 2 may be involved in cell elongation while the other complex containing PBP 3 may be responsible for cell division.     

Eliminating a Set of Four Penicillin Binding Proteins Triggers the Rcs Phosphorelay and Cpx Stress Responses in Escherichia coli

Penicillin binding proteins (PBPs) are responsible for synthesizing and modifying the bacterial cell wall, and in Escherichia coli the loss of several nonessential low-molecular-weight PBPs gives rise to abnormalities in cell shape and division. To determine whether these proteins help connect the flagellar basal body to the peptidoglycan wall, we surveyed a set of PBP mutants and found that motility in an agar migration assay was compromised by the simultaneous absence of four enzymes: PBP4, PBP5, PBP7, and AmpH. A wild-type copy of any one of these restored migration, and complementation depended on the integrity of the PBP active-site serine. However, the migration defect was caused by the absence of flagella instead of improper flagellar assembly. Migration was restored if the flhDC genes were overexpressed or if the rcsB or cpxR genes were deleted. Thus, migration was inhibited because the Rcs and Cpx stress response systems were induced in the absence of these four specific PBPs. Furthermore, in this situation Rcs induction depended on the presence of CpxR. The results imply that small changes in peptidoglycan structure are sufficient to activate these stress responses, suggesting that a specific cell wall fragment may be the signal being sensed. The fact that four PBPs must be inactivated may explain why large perturbations to the envelope are required to induce stress responses.     

Virulence of non-β-lactamase-mediated ampicilin-resistant Haemophilus influenzae

We questioned whether strains of ampicillin-resistant, non-beta-lactamase-producing (AmpR NBLP) Haemophilus influenzae with lower affinity penicillin-binding proteins (PBPs) might have altered virulence. The virulence of resistant transformant strains and the susceptible recipient was compared using infant rats. Following intraperitoneal inoculation, there was a significantly lower mortality rate and incidence and magnitude of bacteremia with two of three transformants compared to the recipient strain. Reduced virulence was not associated with greater bactericidal activity of serum or human neutrophils or faster clearance of the transformant following intravenous injection. Heated rat or human plasma supported exponential growth of the recipient, but not the transformant, suggesting deficient in vivo multiplication. We conclude that H. influenzae with altered PBPs are less virulent in an infant rat model which may be related to differences in in vivo growth.     

Expression and characterization of the ponA (ORF I) gene of Haemophilus influenzae: functional complementation in a heterologous system.

The coding sequence of the Haemophilus influenzae ORF I gene was amplified by PCR and cloned into different Escherichia coli expression vectors. The ORF I-encoded protein was approximately 90 kDa and bound 3H-benzyl-penicillin and 125I-cephradine. This high-molecular-weight penicillin-binding protein (PBP) was also shown to possess transglycosylase activity, indicating that the ORF I product is a bifunctional PBP. The ORF I protein was capable of maintaining the viability of E. coli delta ponA ponB::spcr cells in transcomplementation experiments, establishing the functional relevance of the significant amino acid homology seen between E. coli PBP 1A and 1B and the H. influenzae ORF I product. In addition, the physiological functioning of the H. influenzae ORF I (PBP 1A) product in a heterologous species established the ability of the enzyme not only to recognize the E. coli substrate but also to interact with heterologous cell division proteins. The affinity of the ORF I product for 3H-benzylpenicillin and 125I-cephradine, the MIC of beta-lactams for E. coli delta ponA ponB::spcr expressing the ORF I gene, and the amino acid alignment of the PBP 1 family of high-molecular-weight PBPs group the ORF I protein into the PBP 1A family of high-molecular-weight PBPs.     

Differences in penicillin-binding proteins of Streptococcus pyogenes and two derived, stabilized L forms.

The penicillin-binding proteins (PBPs) of Streptococcus pyogenes and two of its derived, stabilized (i.e., nonreverting) L forms, an osmotically fragile L form and a physiologic isotonic L form, were compared. The numbers of PBPs in the membranes of these organisms were 6, 4, and 2 for the coccus and the osmotically fragile and physiologic isotonic L forms, respectively. Likewise, the relative amounts of total PBPs were 1.00: 1.48:0.32 for this coccus and the osmotically fragile and physiologic isotonic L forms, respectively. The two largest PBPs (PBPs 1 and 2) of the coccus were absent in both L forms, while the smallest PBPs (PBPs 5 and 6) were found in all three membranes. Deacylation (half-life) of three of the four PBPs in the osmotically fragile L form membrane required a significantly longer time than did deacylation of these presumed identical enzymes in the parental coccal membrane. Conversely, there was no such difference between the only two PBPs of the physiologic isotonic L form and the same coccal membrane proteins. Intact cells of all three organisms secreted PBPs and what appeared to be penicilloic acid and a minimal amount of free penicillin. A greater amount of these PBPs was secreted by both L forms than by the coccus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns and ratios of secreted PBPs were identical to those from labeled membrane preparations. These differences are correlated with some of our previous findings and are discussed in terms of inhibition of cell wall synthesis and resulting membrane changes in these two derived, stabilized coccal L forms.     

Endopeptidase penicillin-binding proteins 4 and 7 play auxiliary roles in determining uniform morphology of Escherichia coli

The low-molecular-weight (LMW) penicillin-binding protein, PBP 5, plays a dominant role in determining the uniform cell shape of Escherichia coli. However, the physiological functions of six other LMW PBPs are unknown, even though the existence and enzymatic activities of four of these were established three decades ago. By applying fluorescence-activated cell sorting (FACS) to quantify the cellular dimensions of multiple PBP mutants, we found that the endopeptidases PBP 4 and PBP 7 also influence cell shape in concert with PBP 5. This is the first reported biological function for these two proteins. In addition, the combined loss of three DD-carboxypeptidases, PBPs 5 and 6 and DacD, also impaired cell shape. In contrast to previous reports based on visual inspection alone, FACS analysis revealed aberrant morphology in a mutant lacking only PBP 5, a phenotype not shared by any other strain lacking a single LMW PBP. PBP 5 removes the terminal D-alanine from pentapeptide side chains of muropeptide subunits, and pentapeptides act as donors for cross-linking adjacent side chains. As endopeptidases, PBPs 4 and 7 cleave cross-links in the cell wall. Therefore, overall cell shape may be determined by the existence or location of a specific type of peptide cross-link, with PBP 5 activity influencing how many cross-links are made and PBPs 4 and 7 acting as editing enzymes to remove inappropriate cross-links.     

Changes in peptidoglycan composition and penicillin-binding proteins in slowly growing Escherichia coli.

The composition of peptidoglycan of chemostat-grown cultures of Escherichia coli was investigated as a function of growth rate. As the generation time was lengthened from 0.8 to 13.8 h, there was a decrease in the major monomer (disaccharide tetrapeptide) and dimer (bis-disaccharide tetrapeptide), while disaccharide tripeptide moieties increased to greater than 50% of the total wall. The average chain length became much shorter; lipoprotein density tripled, and the number of unusual diaminopimelyl-diaminopimelic acid crossbridges increased fivefold. As cells grew more slowly, amounts of penicillin-binding proteins (PBPs) 1a-1b complex and 4 decreased, while amounts of PBPs 3 and the 5-6 complex increased. We propose that the chemical composition of E. coli cell walls changes with growth rate in a manner consistent with alterations in the activities of PBPs and cell shape.     

Escherichia coli Mutants Lacking All Possible Combinations of Eight Penicillin Binding Proteins: Viability, Characteristics, and Implications for Peptidoglycan Synthesis

The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology of Escherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two res sites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via lambda phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bp res site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, the dacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the beta-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.     

Role of penicillin-binding proteins in bacterial cell morphogenesis

The penicillin-binding proteins (PBPs) polymerize and modify peptidoglycan, the stress-bearing component of the bacterial cell wall. As part of this process, the PBPs help to create the morphology of the peptidoglycan exoskeleton together with cytoskeleton proteins that regulate septum formation and cell shape. Genetic and microscopic studies reveal clear morphological responsibilities for class A and class B PBPs and suggest that the mechanism of shape determination involves differential protein localization and interactions with specific cell components. In addition, the low molecular weight PBPs, by varying the substrates on which other PBPs act, alter peptidoglycan synthesis or turnover, with profound effects on morphology.     

Peptidoglycan synthesis in the absence of class A penicillin-binding proteins in Bacillus subtilis

Penicillin-binding proteins (PBPs) catalyze the final, essential reactions of peptidoglycan synthesis. Three classes of PBPs catalyze either trans-, endo-, or carboxypeptidase activities on the peptidoglycan peptide side chains. Only the class A high-molecular-weight PBPs have clearly demonstrated glycosyltransferase activities that polymerize the glycan strands, and in some species these proteins have been shown to be essential. The Bacillus subtilis genome sequence contains four genes encoding class A PBPs and no other genes with similarity to their glycosyltransferase domain. A strain lacking all four class A PBPs has been constructed and produces a peptidoglycan wall with only small structural differences from that of the wild type. The growth rate of the quadruple mutant is much lower than those of strains lacking only three of the class A PBPs, and increases in cell length and frequencies of wall abnormalities were noticeable. The viability and wall production of the quadruple-mutant strain indicate that a novel enzyme can perform the glycosyltransferase activity required for peptidoglycan synthesis. This activity was demonstrated in vitro and shown to be sensitive to the glycosyltransferase inhibitor moenomycin. In contrast, the quadruple-mutant strain was resistant to moenomycin in vivo. Exposure of the wild-type strain to moenomycin resulted in production of a phenotype similar to that of the quadruple mutant.     

Contributions of PBP 5 and DD-carboxypeptidase penicillin binding proteins to maintenance of cell shape in Escherichia coli

Escherichia coli has 12 recognized penicillin binding proteins (PBPs), four of which (PBPs 4, 5, and 6 and DacD) have DD-carboxypeptidase activity. Although the enzymology of the DD-carboxypeptidases has been studied extensively, the in vivo functions of these proteins are poorly understood. To explain why E. coli maintains four independent loci encoding enzymes of considerable sequence identity and comparable in vitro activity, it has been proposed that the DD-carboxypeptidases may substitute for one another in vivo. We tested the validity of this equivalent substitution hypothesis by investigating the effects of these proteins on the aberrant morphology of DeltadacA mutants, which produce no PBP 5. Although cloned PBP 5 complemented the morphological phenotype of a DeltadacA mutant lacking a total of seven PBPs, controlled expression of PBP 4, PBP 6, or DacD did not. Also, a truncated PBP 5 protein lacking its amphipathic C-terminal membrane binding sequence did not reverse the morphological defects and was lethal at low levels of expression, implying that membrane anchoring is essential for the proper functioning of PBP 5. By examining a set of mutants from which multiple PBP genes were deleted, we found that significant morphological aberrations required the absence of at least three different PBPs. The greatest defects were observed in cells lacking, at minimum, PBPs 5 and 6 and one of the endopeptidases (either PBP 4 or PBP 7). The results further differentiate the roles of the low-molecular-weight PBPs, suggest a functional significance for the amphipathic membrane anchor of PBP 5 and, when combined with the recently determined crystal structure of PBP 5, suggest possible mechanisms by which these PBPs may contribute to maintenance of a uniform cell shape in E. coli.     

Conformational analysis of bacterial cell wall peptides indicates how particular conformations have influenced the evolution of penicillin-binding proteins,beta-lactam antibiotics and antibiotic resistance mechanisms

Our aim was to use a conformational analysis technique developed for peptides to identify structural relationships between bacterial cell wall peptides and beta-lactam antibiotics that might help to explain their different actions as substrates and inhibitors of penicillin binding proteins (PBPs). The conformational forms of the model cell wall peptide Ac-L-Lys(Ac)-D-Ala-D-Ala are described by just a few backbone torsion combinations: three C-terminal carboxylate regions, with Tor8 (psi(i+1)) ranges of D3 region (50 degrees to 70 degrees ), D6 region (140 degrees to 170 degrees ) and D9 region (-50 degrees to -70 degrees ) are combined with either of two Tor6 (phi(i))-Tor4 (psi(i)) combinations, C4 region (-50 degrees to -80 degrees ) with B8 region (-40 degrees to -70 degrees ) or C11 region (30 degrees to 50 degrees ) with B2 region (30 degrees to 70 degrees ). From these results, and comparisons with conformational analyses of various beta-lactams and Ac-L-Lys(Ac)-D-Ala-D-Lac, it is concluded that molecular recognition of cell wall peptide substrates by PBPs requires conformers with backbone torsion angles of D3C4B8. beta-Lactam antibiotics are constrained compounds with fewer conformational forms; these match well the backbone torsions of cell wall peptides at D3C4, allowing their recognition and acylation by PBPs, whereas their unique Tor4 produces differently orientated CO and N atoms that appear to prevent subsequent deacylation, leading to their action as suicide substrates. The results are also related to the selective pressures involved in evolution of beta-lactamases from PBPs. From analysis of conformers of Ac-L-Lys(Ac)-D-Ala-D-Ala and the vancomycin-resistant analogue Ac-L-Lys(Ac)-D-Ala-D-Lac, it is concluded that vancomycin may recognise D6C11B2 conformers, giving it complementary substrate specificity to PBPs. This approach could have applications in the rational design of antibiotics targeted against PBPs and their substrates.     

Synthesis of peptidoglycan by high molecular weight penicillin-binding proteins of Bacillus subtilis and Bacillus stearothermophilus

The high molecular weight penicillin-binding proteins (PBP(s) ) Bacillus subtilis PBPs 1, 2, and 4 and Bacillus stearothermophilus PBPs 1-4 were shown to catalyze peptidoglycan synthesis from the undecaprenol-containing lipid intermediate substrate in two assay systems. In a filter paper assay system, high levels of substrate polymerization occurred when reaction mixtures were incubated on Whatman 3MM filter paper. The pH optimum for peptidoglycan synthesis was 7.5 for B. subtilis PBPs 1, 2, and 4 and 8.5 for B. stearothermophilus PBPs 1-4. Polymerization was Mg2+-independent and was unaffected by sulfhydryl reagents. Reconstitution with membrane lipids or addition of detergent (optimal concentration, 0.1%) was necessary for synthesis to occur. Bacitracin, penicillin, and cephalothin did not affect polymerization while vancomycin, ristocetin, moenomycin, and macarbomycin were strong inhibitors. In a test tube assay system, optimal synthesis occurred either in the presence of 10% ethylene glycol, 10% glycerol, and 8% methanol or in the presence of 10% N-acetylglucosamine. The products of lysozyme digestion of the synthesized peptidoglycan were analyzed by gel filtration and paper chromatography. B. stearothermophilus PBPs 1-4 synthesized a peptidoglycan product that was 5-7% cross-linked. No evidence for cross-linking was apparent in the peptidoglycan product of B. subtilis PBPs 1, 2, and 4.     

Newly made enzymes determine ongoing cell wall synthesis and the antibacterial effects of cell wall synthesis inhibitors.

Cell wall synthesis can continue with less than the total complement of cell wall synthetic enzymes present in normal growing cells. A method was developed to investigate whether there exists an excess of cell wall-synthesizing enzymes (penicillin-binding proteins [PBPs]) which all remain functional or whether a mixed population of functional and nonfunctional enzymes characterize normal cells. Surprisingly, cells in which less than 10% of the PBPs were functional could grow at a normal rate, as evidenced by increases in viable counts, culture turbidity, and rates of peptidoglycan, protein, and RNA synthesis. This subset of functional enzymes was biosynthetically new. Penicillin-induced lysis occurred contingent on the acylation of this same small fraction of PBPs, the copy number and affinities of which were below the level of detection by current fluorographic assay techniques. We propose that PBPs have a short functional half-life and that cell wall synthesis and bacterial lysis reflect the activity of newly synthesized PBPs.     

Wide variation in the cyanobacterial complement of presumptive penicillin-binding proteins

A genomic analysis of putative penicillin-binding proteins (PBPs) that are involved in the synthesis of the peptidoglycan layer of the cell wall and are encoded in 12 cyanobacterial genomes was performed in order to help elucidate the role(s) of these proteins in peptidoglycan synthesis, especially during cyanobacterial cellular differentiation. The analysis suggested that the minimum set of PBPs needed to assemble the peptidoglycan layer in cyanobacteria probably does not exceed one bifunctional transpeptidase–transglycosylase Class A high-molecular-weight PBP; two Class B high-molecular-weight PBPs, one of them probably involved in cellular elongation and the other in septum formation; and one low-molecular-weight PBP. The low-molecular-weight PBPs of all of the cyanobacteria analyzed are putative endopeptidases and are encoded by fewer genes than in Escherichia coli. We show that in Anabaena sp. strain PCC 7120, predicted proteins All2981 and Alr4579, like Alr5101, are Class A high-molecular-weight PBPs that are required for the functional differentiation of aerobically diazotrophic heterocysts, indicating that some members of this class of PBPs are required specifically for cellular developmental processes.     

Separation of Escherichia coli penicillin-binding proteins into different membrane vesicles by agarose electrophoresis and sizing chromatography.

Membrane vesicles from the envelope of Escherichia coli were separated by electrophoresis through dilute agarose and by sizing chromatography through Sephacryl S-1000. These techniques revealed that proteins were associated with different subsets of vesicles. In particular, dilute agarose electrophoresis clearly separated the inner membrane penicillin-binding proteins (PBPs) into different vesicle groups. Vesicles containing PBPs 4, 6, 7, and 8 migrated rapidly through agarose; vesicles with PBPs 1a, 1b, 2, 3, and 5 eluted later. With the exception of PBP 4, which migrated with PBPs 1 through 5, chromatography through Sephacryl S-1000 was able to distinguish the same two vesicle sets, though the extent of separation was poorer than with agarose. The existence of these associations among vesicles and proteins suggests that there is an organization to the inner membrane of E. coli which is not observed when membrane vesicles are separated solely on the basis of density in sucrose gradients.     

A microtiter plate-based β-lactam binding assay for inhibitors of high-molecular-mass penicillin-binding proteins

High-molecular-mass penicillin-binding proteins (HMM PBPs) are essential for bacterial cell wall biosynthesis and are the lethal targets of β-lactam antibiotics. When purified, HMM PBPs give undetectable or weak enzyme activity. This has impeded efforts to develop assays for HMM PBPs and to develop new inhibitors for HMM PBPs as HMM PBP targeted antibacterial agents. However, even when purified, HMM PBPs retain their ability to bind β-lactams. Here we describe a fluorescently detected microtiter plate-based assay for inhibitor binding to HMM PBPs based on competition with biotin-ampicillin conjugate (BIO-AMP) binding.