Approaching the physiological functions of penicillin-binding proteins in Escherichia coli

A rigid shell of peptidoglycan encases and shapes bacteria and is constructed and maintained by a diverse set of enzymes, among which are the penicillin-binding proteins (PBPs). Although a great deal has been learned about how these proteins synthesize and modify peptidoglycan, the physiological functions of the multitude of bacterial PBPs remain enigmatic. We approached this problem by combining PBP mutations in a comprehensive manner and screening for effects on biochemical processes involving the passage of proteins or nucleic acids across the cell wall. The results indicate that the PBPs or their peptidoglycan product do have significant biological functions, including roles in determination of cell shape, in phage resistance, in induction of capsule synthesis, and in regulation of autolysis.     

Physiological functions of D-alanine carboxypeptidases in Escherichia coli

Bacterial cell shape is, in part, mediated by the peptidoglycan (murein) sacculus. Penicillin-binding proteins (PBPs) catalyze the final stages of murein biogenesis and are the targets of beta-lactam antibiotics. Several low molecular mass PBPs including PBP4, PBP5, PBP6 and DacD seem to possess DD-carboxypeptidase (DD-CPase) activity, but these proteins are dispensable for survival in laboratory culture. The physiological functions of DD-CPases in vivo are unresolved and it is unclear why bacteria retain these seemingly non-essential and enzymatically redundant enzymes. However, PBP5 clearly contributes to maintenance of cell shape in some PBP mutant backgrounds. In this review, we focus on recent findings concerning the physiological functions of the DD-CPases in vivo, identify gaps in the current knowledge of these proteins and suggest some possible courses for future study that might help reconcile current models of bacterial cell 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.     

A mother cell-specific class B penicillin-binding protein, PBP4b, in Bacillus subtilis

The Bacillus subtilis genome encodes 16 penicillin-binding proteins (PBPs), some of which are involved in synthesis of the spore peptidoglycan. The pbpI (yrrR) gene encodes a class B PBP, PBP4b, and is transcribed in the mother cell by RNA polymerase containing sigma(E). Loss of PBP4b, alone and in combination with other sporulation-specific PBPs, had no effect on spore peptidoglycan structure.     

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.     

The Rcs phosphorelay is a cell envelope stress response activated by peptidoglycan stress and contributes to intrinsic antibiotic resistance

Gram-negative bacteria possess stress responses to maintain the integrity of the cell envelope. Stress sensors monitor outer membrane permeability, envelope protein folding, and energization of the inner membrane. The systems used by gram-negative bacteria to sense and combat stress resulting from disruption of the peptidoglycan layer are not well characterized. The peptidoglycan layer is a single molecule that completely surrounds the cell and ensures its structural integrity. During cell growth, new peptidoglycan subunits are incorporated into the peptidoglycan layer by a series of enzymes called the penicillin-binding proteins (PBPs). To explore how gram-negative bacteria respond to peptidoglycan stress, global gene expression analysis was used to identify Escherichia coli stress responses activated following inhibition of specific PBPs by the β-lactam antibiotics amdinocillin (mecillinam) and cefsulodin. Inhibition of PBPs with different roles in peptidoglycan synthesis has different consequences for cell morphology and viability, suggesting that not all perturbations to the peptidoglycan layer generate equivalent stresses. We demonstrate that inhibition of different PBPs resulted in both shared and unique stress responses. The regulation of capsular synthesis (Rcs) phosphorelay was activated by inhibition of all PBPs tested. Furthermore, we show that activation of the Rcs phosphorelay increased survival in the presence of these antibiotics, independently of capsule synthesis. Both activation of the phosphorelay and survival required signal transduction via the outer membrane lipoprotein RcsF and the response regulator RcsB. We propose that the Rcs pathway responds to peptidoglycan damage and contributes to the intrinsic resistance of E. coli to β-lactam antibiotics.     

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.     

Escherichia coli low-molecular-weight penicillin-binding proteins help orient septal FtsZ, and their absence leads to asymmetric cell division and branching

Escherichia coli cells lacking low-molecular-weight penicillin-binding proteins (LMW PBPs) exhibit morphological alterations that also appear when the septal protein FtsZ is mislocalized, suggesting that peptidoglycan modification and division may work together to produce cell shape. We found that in strains lacking PBP5 and other LMW PBPs, higher FtsZ concentrations increased the frequency of branched cells and incorrectly oriented Z rings by 10- to 15-fold. Invagination of these rings produced improperly oriented septa, which in turn gave rise to asymmetric cell poles that eventually elongated into branches. Branches always originated from the remnants of abnormal septation events, cementing the relationship between aberrant cell division and branch formation. In the absence of PBP5, PBP6 and DacD localized to nascent septa, suggesting that these PBPs can partially substitute for the loss of PBP5. We propose that branching begins when mislocalized FtsZ triggers the insertion of inert peptidoglycan at unusual positions during cell division. Only later, after normal cell wall elongation separates the patches, do branches become visible. Thus, a relationship between the LMW PBPs and cytoplasmic FtsZ ultimately affects cell division and overall shape.     

Discrete and overlapping functions of peptidoglycan synthases in growth,cell division and virulence of Listeria monocytogenes

Upon ingestion of contaminated food, Listeria monocytogenes can cause serious infections in humans that are normally treated with β‐lactam antibiotics. These target Listeria's five high molecular weight penicillin‐binding proteins (HMW PBPs), which are required for peptidoglycan biosynthesis. The two bi‐functional class A HMW PBPs PBP A1 and PBP A2 have transglycosylase and transpeptidase domains catalyzing glycan chain polymerization and peptide cross‐linking, respectively, whereas the three class B HMW PBPs B1, B2 and B3 are monofunctional transpeptidases. The precise roles of these PBPs in the cell cycle are unknown. Here we show that green fluorescent protein (GFP)‐PBP fusions localized either at the septum, the lateral wall or both, suggesting distinct and overlapping functions. Genetic data confirmed this view: PBP A1 and PBP A2 could not be inactivated simultaneously, and a conditional double mutant strain is largely inducer dependent. PBP B1 is required for rod‐shape and PBP B2 for cross‐wall biosynthesis and viability, whereas PBP B3 is dispensable for growth and cell division. PBP B1 depletion dramatically increased β‐lactam susceptibilities and stimulated spontaneous autolysis but had no effect on peptidoglycan cross‐linkage. Our in vitro virulence assays indicated that the complete set of all HMW PBPs is required for maximal virulence.     

Rod shape determination by the Bacillus subtilis class B penicillin-binding proteins encoded by pbpA and pbpH

The peptidoglycan cell wall determines the shape and structural integrity of a bacterial cell. Class B penicillin-binding proteins (PBPs) carry a transpeptidase activity that cross-links peptidoglycan strands via their peptide side chains, and some of these proteins are directly involved in cell shape determination. No Bacillus subtilis PBP with a clear role in rod shape maintenance has been identified. However, previous studies showed that during outgrowth of pbpA mutant spores, the cells grew in an ovoid shape for several hours before they recovered and took on a normal rod shape. It was postulated that another PBP, expressed later during outgrowth, was able to compensate for the lack of the pbpA product, PBP2a, and to guide the formation of a rod shape. The B. subtilis pbpH (ykuA) gene product is predicted to be a class B PBP with greatest sequence similarity to PBP2a. We found that a pbpH-lacZ fusion was expressed at very low levels in early log phase and increased in late log phase. A pbpH null mutant was indistinguishable from the wild-type, but a pbpA pbpH double mutant was nonviable. When pbpH was placed under the control of an inducible promoter in a pbpA mutant, viability was dependent on pbpH expression. Growth of this strain in the absence of inducer resulted in conversion of the cells from rods to ovoid/round shapes and lysis. We conclude that PBP2a and PbpH play redundant roles in formation of a rod-shaped peptidoglycan cell wall.     

Penicillin binding protein 5 affects cell diameter, contour, and morphology of Escherichia coli

Although general physiological functions have been ascribed to the high-molecular-weight penicillin binding proteins (PBPs) of Escherichia coli, the low-molecular-weight PBPs have no well-defined biological roles. When we examined the morphology of a set of E. coli mutants lacking multiple PBPs, we observed that strains expressing active PBP 5 produced cells of normal shape, while mutants lacking PBP 5 produced cells with altered diameters, contours, and topological features. These morphological effects were visible in untreated cells, but the defects were exacerbated in cells forced to filament by inactivation of PBP 3 or FtsZ. After filamentation, cellular diameter varied erratically along the length of individual filaments and many filaments exhibited extensive branching. Also, in general, the mean diameter of cells lacking PBP 5 was significantly increased compared to that of cells from isogenic strains expressing active PBP 5. Expression of cloned PBP 5 reversed the effects observed in DeltadacA mutants. Although deletion of PBP 5 was required for these phenotypes, the absence of additional PBPs magnified the effects. The greatest morphological alterations required that at least three PBPs in addition to PBP 5 be deleted from a single strain. In the extreme cases in which six or seven PBPs were deleted from a single mutant, cells and cell filaments expressing PBP 5 retained a normal morphology but cells and filaments lacking PBP 5 were aberrant. In no case did mutation of another PBP produce the same drastic morphological effects. We conclude that among the low-molecular-weight PBPs, PBP 5 plays a principle role in determining cell diameter, surface uniformity, and overall topology of the peptidoglycan sacculus.     

d-Amino Acid Probes for Penicillin Binding Protein-based Bacterial Surface Labeling

Peptidoglycan is an essential and highly conserved mesh structure that surrounds bacterial cells. It plays a critical role in retaining a defined cell shape, and, in the case of pathogenic Gram-positive bacteria, it lies at the interface between bacterial cells and the host organism. Intriguingly, bacteria can metabolically incorporate unnatural d-amino acids into the peptidoglycan stem peptide directly from the surrounding medium, a process mediated by penicillin binding proteins (PBPs). Metabolic peptidoglycan remodeling via unnatural d-amino acids has provided unique insights into peptidoglycan biosynthesis of live bacteria and has also served as the basis of a synthetic immunology strategy with potential therapeutic implications. A striking feature of this process is the vast promiscuity displayed by PBPs in tolerating entirely unnatural side chains. However, the chemical space and physical features of this side chain promiscuity have not been determined systematically. In this report, we designed and synthesized a library of variants displaying diverse side chains to comprehensively establish the tolerability of unnatural d-amino acids by PBPs in both Gram-positive and Gram-negative organisms. In addition, nine Bacillus subtilis PBP-null mutants were evaluated with the goal of identifying a potential primary PBP responsible for unnatural d-amino acid incorporation and gaining insights into the temporal control of PBP activity. We empirically established the scope of physical parameters that govern the metabolic incorporation of unnatural d-amino acids into bacterial peptidoglycan.     

Bridging cell wall biosynthesis and bacterial morphogenesis

The bacterial cell wall is a complex three-dimensional structure that protects the cell from environmental stress and ensures its shape. The biosynthesis of its main component, the peptidoglycan, involves the coordination of activities of proteins present in the cytoplasm, the membrane, and the periplasm, some of which also interact with the bacterial cytoskeleton. The sheer complexity of the cell wall elongation process, which is the main focus of this review, has created a significant challenge for the study of the macromolecular interactions that regulate peptidoglycan biosynthesis. The availability of new structural and biochemical data on a number of components of peptidoglycan assembly machineries, including a complex between MreB and RodZ as well as structures of penicillin-binding proteins (PBPs) from a number of pathogenic species, now provide novel insight into the underpinnings of an intricate molecular machinery.     

Bacterial Cell Wall Synthesis: New Insights from Localization Studies

In order to maintain shape and withstand intracellular pressure, most bacteria are surrounded by a cell wall that consists mainly of the cross-linked polymer peptidoglycan (PG). The importance of PG for the maintenance of bacterial cell shape is underscored by the fact that, for various bacteria, several mutations affecting PG synthesis are associated with cell shape defects. In recent years, the application of fluorescence microscopy to the field of PG synthesis has led to an enormous increase in data on the relationship between cell wall synthesis and bacterial cell shape. First, a novel staining method enabled the visualization of PG precursor incorporation in live cells. Second, penicillin-binding proteins (PBPs), which mediate the final stages of PG synthesis, have been localized in various model organisms by means of immunofluorescence microscopy or green fluorescent protein fusions. In this review, we integrate the knowledge on the last stages of PG synthesis obtained in previous studies with the new data available on localization of PG synthesis and PBPs, in both rod-shaped and coccoid cells. We discuss a model in which, at least for a subset of PBPs, the presence of substrate is a major factor in determining PBP localization.     

Bacterial cell wall synthesis: new insights from localization studies.

In order to maintain shape and withstand intracellular pressure, most bacteria are surrounded by a cell wall that consists mainly of the cross-linked polymer peptidoglycan (PG). The importance of PG for the maintenance of bacterial cell shape is underscored by the fact that, for various bacteria, several mutations affecting PG synthesis are associated with cell shape defects. In recent years, the application of fluorescence microscopy to the field of PG synthesis has led to an enormous increase in data on the relationship between cell wall synthesis and bacterial cell shape. First, a novel staining method enabled the visualization of PG precursor incorporation in live cells. Second, penicillin-binding proteins (PBPs), which mediate the final stages of PG synthesis, have been localized in various model organisms by means of immunofluorescence microscopy or green fluorescent protein fusions. In this review, we integrate the knowledge on the last stages of PG synthesis obtained in previous studies with the new data available on localization of PG synthesis and PBPs, in both rod-shaped and coccoid cells. We discuss a model in which, at least for a subset of PBPs, the presence of substrate is a major factor in determining PBP localization.     

Growth and division of Streptococcus pneumoniae: localization of the high molecular weight penicillin-binding proteins during the cell cycle

The bacterial peptidoglycan, the main component of the cell wall, is synthesized by the penicillin-binding proteins (PBPs). We used immunofluorescence microscopy to determine the cellular localization of all the high molecular weight PBPs of the human pathogen Streptococcus pneumoniae, for a wild type and for several PBP-deficient strains. Progression through the cell cycle was investigated by the simultaneous labelling of DNA and the FtsZ protein. Our main findings are: (i) the temporal dissociation of cell wall synthesis, inferred by the localization of PBP2x and PBP1a, from the constriction of the FtsZ-ring; (ii) the localization of PBP2b and PBP2a at duplicated equatorial sites indicating the existence of peripheral peptidoglycan synthesis, which implies a similarity between the mechanism of cell division in bacilli and streptococci; (iii) the abnormal localization of some class A PBPs in PBP-defective mutants which may explain the apparent redundancy of these proteins in S. pneumoniae.     

Mutations affecting penicillin-binding proteins 2a, 2b and 3 in Bacillus subtilis alter cell shape and peptidoglycan metabolism

Bacillus subtilis mutants with altered penicillin-binding proteins (PBPs), or altered expression of PBPs, were isolated by screening for changes in susceptibility to beta-lactam antibiotics. Mutations affecting only PBPs 2a, 2b and 3 were isolated. Cell shape and peptidoglycan metabolism were examined in representative mutants. Cells of a PBP 2a mutant (UB8521) were usually twisted whereas PBP 2b (UB8524) and 3 (UB8525) mutants produced helices, particularly after growth at 41 degrees C. The PBP 2a mutant (UB8521) had a higher peptidoglycan synthetic activity than its parent strain whereas the opposite applied to the PBP 2b mutant UB8524. The PBP 3 mutant (UB8525) had a similar peptidoglycan synthetic activity to that of the parent strain when grown at 37 degrees C, but 40% higher activity after growth at 41 degrees C. The PBP 2a mutant (UB8521) exhibited the same wall thickening activity as the parent, but the PBP 2b and 3 mutants (UB8524 and UB8525) were partially defective in this respect. The changes in the susceptibility of PBP 2a, 2b and 3 mutants to beta-lactam antibiotics imply that these PBPs are killing targets, consistent with the fact that these PBPs are also important for shape determination and peptidoglycan synthesis.     

Restoration of vegetative penicillin-binding proteins during germination and outgrowth of Bacillus subtilis spores: relationship of individual proteins to specific cell cycle events.

The order in which the vegetative penicillin-binding proteins (PBPs) are first synthesized and the rate of their return to normal levels during germination and outgrowth of Bacillus subtilis spores were determined. The rate of synthesis of most of the PBPs was much faster than that of the majority of other membrane proteins, which is consistent with the involvement of PBPs in biosynthesis of the rapidly expanding peptidoglycan. The pattern of PBP changes that occurred during the cell cycle, including sporulation, suggests a likely role for PBP 2A in cell elongation and a unique requirement for PBP 2B during both symmetric and asymmetric septum formation. PBP 3 is the only PBP that appears to be equally necessary for vegetative and cortical peptidoglycan synthesis.     

Peptidoglycan synthesis by Enterococcus faecalis penicillin binding protein 5

Low-affinity penicillin binding proteins (PBPs) are a particular class of proteins involved in β-lactam antibiotic resistance of enterococci. The activity of these PBPs is just sufficient to allow the cells to survive in the presence of high concentrations of β-lactams that cause saturation (and inhibition) of the other PBPs. For this reason, the low-affinity PBPs are thought to be multifunctional enzymes capable of catalyzing the entire peptidoglycan synthesis. To test the validity of this claim, we analyzed the muropeptide composition by reversed-phase high-performance liquid chromatography of the peptidoglycan synthesized by PBP5 (the low-affinity PBP) of Enterococcus faecalis, in comparison with the peptidoglycan produced normally by the concerted action of the usual PBPs (namely PBPs 1, 2, and 3). Cross-linked peptidoglycan was produced. The main difference consisted in the lack of oligomers higher than trimers, thus suggesting that this oligomer cannot be used as an acceptor/donor by the transpeptidase component of PBP5. The lack of higher oligomers had little impact on total cross-linking because of the increase observed in the dimer family. This increase was distributed among the various members of the dimer family with the result that minor dimer components figured among the prevalent ones in cells in which peptidoglycan was synthesized by PBP5. This also suggests that E. faecalis PBP5 is capable of catalyzing the synthesis of a peptidoglycan that is less precise and refined than usual, and for this reason PBP5 can be considered an enzyme endowed with poor specificity for substrates, as may be expected on the basis of its survival function. Received: 18 March 1998 / Accepted: 26 May 1998