The D,D-carboxypeptidase PBP3 organizes the division process of Streptococcus pneumoniae

Bacterial division requires the co-ordination of membrane invagination, driven by the constriction of the FtsZ-ring, and concomitant cell wall synthesis, performed by the high-molecular-weight penicillin-binding proteins (HMW PBPs). Using immunofluorescence techniques, we show in Streptococcus pneumoniae that this co-ordination requires PBP3, a D,D-carboxypeptidase that degrades the substrate of the HMW PBPs. In a mutant deprived of PBP3, the apparent rings of HMW PBPs and that of FtsZ are no longer co-localized. In wild-type cells, PBP3 is absent at the future division site and present over the rest of the cell surface, implying that the localization of the HMW PBPs at mid-cell depends on the availability of their substrate. FtsW, a putative translocase of the substrate of the PBPs, forms an apparent ring that is co-localized with the septal HMW PBPs throughout the cell cycle of wild-type cells. In particular, the constriction of the FtsW-ring occurs after that of the FtsZ-ring, with the same delay as the constriction of the septal PBP-rings. However, in the absence of PBP3, FtsW remains co-localized with FtsZ in contrast to the HMW PBPs. Our work reveals an unexpected complexity in the relationships between the division proteins. The consequences of the absence of PBP3 indicate that the peptidoglycan composition is central to the co-ordination of the division process.     

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.     

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.     

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.     

Glycosyltransferase domain of penicillin-binding protein 2a from Streptococcus pneumoniae is membrane associated

Penicillin-binding proteins (PBPs) are bacterial cytoplasmic membrane proteins that catalyze the final steps of the peptidoglycan synthesis. Resistance to beta-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 beta-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 beta-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.     

Identification of pneumococcal proteins that are functionally linked to penicillin‐binding protein 2b (PBP2b)

The oval shape of pneumococci results from a combination of septal and lateral peptidoglycan synthesis. The septal cross‐wall is synthesized by the divisome, while the elongasome drives cell elongation by inserting new peptidoglycan into the lateral cell wall. Each of these molecular machines contains penicillin‐binding proteins (PBPs), which catalyze the final stages of peptidoglycan synthesis, plus a number of accessory proteins. Much effort has been made to identify these accessory proteins and determine their function. In the present paper we have used a novel approach to identify members of the pneumococcal elongasome that are functionally closely linked to PBP2b. We discovered that cells depleted in PBP2b, a key component of the elongasome, display several distinct phenotypic traits. We searched for proteins that, when depleted or deleted, display the same phenotypic changes. Four proteins, RodA, MreD, DivIVA and Spr0777, were identified by this approach. Together with PBP2b these proteins are essential for the normal function of the elongasome. Furthermore, our findings suggest that DivIVA, which was previously assigned as a divisomal protein, is required to correctly localize the elongasome at the negatively curved membrane region between the septal and lateral cell wall.     

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     

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.     

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.     

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.     

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.     

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.     

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.     

pbpB, a gene coding for a putative penicillin-binding protein, is required for aerobic nitrogen fixation in the cyanobacterium Anabaena sp. strain PCC7120

Transposon mutagenesis of Anabaena sp. strain PCC7120 led to the isolation of a mutant strain, SNa1, which is unable to fix nitrogen aerobically but is perfectly able to grow with combined nitrogen (i.e., nitrate). Reconstruction of the transposon mutation of SNa1 in the wild-type strain reproduced the phenotype of the original mutant. The transposon had inserted within an open reading frame whose translation product shows significant homology with a family of proteins known as high-molecular-weight penicillin-binding proteins (PBPs), which are involved in the synthesis of the peptidoglycan layer of the cell wall. A sequence similarity search allowed us to identify at least 12 putative PBPs in the recently sequenced Anabaena sp. strain PCC7120 genome, which we have named and organized according to predicted molecular size and the Escherichia coli nomenclature for PBPs; based on this nomenclature, we have denoted the gene interrupted in SNal as pbpB and its product as PBP2. The wild-type form of pbpB on a shuttle vector successfully complemented the mutation in SNa1. In vivo expression studies indicated that PBP2 is probably present when both sources of nitrogen, nitrate and N₂, are used. When nitrate is used, the function of PBP2 either is dispensable or may be substituted by other PBPs; however, under nitrogen deprivation, where the differentiation of the heterocyst takes place, the role of PBP2 in the formation and/or maintenance of the peptidoglycan layer is essential.     

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.     

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.     

Fluctuations in nuclear envelope's potential mediate synchronization of early neural activity

Penicillin binding proteins (PBPs) catalyze essential steps in the biosynthesis of peptidoglycan, the main component of the bacterial cell wall. PBPs can harbor two catalytic domains, namely the glycosyltransferase (GT) and transpeptidase (TP) activities, the latter being the target for β-lactam antibiotics. Despite the availability of structural information regarding bi-functional PBPs, little is known regarding the interaction and flexibility between the TP and GT domains. Here, we describe the structural characterization in solution by small angle X-ray scattering (SAXS) of PBP1b, a bi-functional PBP from Streptococcus pneumoniae. The molecule is present in solution as an elongated monomer. Refinement of internal coordinates starting from a homology model yields models in which the two domains are in an extended conformation without any mutual contact compatible with the existence of restricted mobility.     

Role of penicillin-binding protein 1b in competitive stationary-phase survival of Escherichia coli

The penicillin-binding proteins (PBPs) catalyze the synthesis and modification of bacterial cell wall peptidoglycan. Although the biochemical activities of these proteins have been determined in Escherichia coli, the physiological roles of many PBPs remain enigmatic. Previous studies have cast doubt on the individual importance of the majority of PBPs during log phase growth. We show here that PBP1b is vital for competitive survival of E. coli during extended stationary phase, but the other nine PBPs studied are dispensable. Loss of PBP1b leads to the stationary phase-specific competition defective phenotype and causes cells to become more sensitive to osmotic stress. Additionally, we present evidence that this protein, as well as AmpC, may assist in cellular resistance to beta-lactam antibiotics.     

The requirement for pneumococcal MreC and MreD is relieved by inactivation of the gene encoding PBP1a

MreC and MreD, along with the actin homologue MreB, are required to maintain the shape of rod-shaped bacteria. The depletion of MreCD in rod-shaped bacteria leads to the formation of spherical cells and the accumulation of suppressor mutations. Ovococcus bacteria, such as Streptococcus pneumoniae, lack MreB homologues, and the functions of the S. pneumoniae MreCD (MreCD(Spn)) proteins are unknown. mreCD are located upstream from the pcsB cell division gene in most Streptococcus species, but we found that mreCD and pcsB are transcribed independently. Similarly to rod-shaped bacteria, we show that mreCD are essential in the virulent serotype 2 D39 strain of S. pneumoniae, and the depletion of MreCD results in cell rounding and lysis. In contrast, laboratory strain R6 contains suppressors that allow the growth of ΔmreCD mutants, and bypass suppressors accumulate in D39 ΔmreCD mutants. One class of suppressors eliminates the function of class A penicillin binding protein 1a (PBP1a). Unencapsulated Δpbp1a D39 mutants have smaller diameters than their pbp1a(+) parent or Δpbp2a and Δpbp1b mutants, which lack other class A PBPs and do not show the suppression of ΔmreCD mutations. Suppressed ΔmreCD Δpbp1a double mutants form aberrantly shaped cells, some with misplaced peptidoglycan (PG) biosynthesis compared to that of single Δpbp1a mutants. Quantitative Western blotting showed that MreC(Spn) is abundant (≈8,500 dimers per cell), and immunofluorescent microscopy (IFM) located MreCD(Spn) to the equators and septa of dividing cells, similarly to the PBPs and PG pentapeptides indicative of PG synthesis. These combined results are consistent with a model in which MreCD(Spn) direct peripheral PG synthesis and control PBP1a localization or activity.