Overproduction of inactive variants of the murein synthase PBP1B causes lysis in Escherichia coli

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 (alpha, beta, and gamma) 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.     

Demonstration of molecular interactions between the murein polymerase PBP1B, the lytic transglycosylase MltA, and the scaffolding protein MipA of Escherichia coli

Enlargement of the stress-bearing murein sacculus of bacteria depends on the coordinated interaction of murein synthases and hydrolases. To understand the mechanism of interaction of these two classes of proteins affinity chromatography and surface plasmon resonance (SPR) studies were performed. The membrane-bound lytic transglycosylase MltA when covalently linked to CNBr-activated Sepharose specifically retained the penicillin-binding proteins (PBPs) 1B, 1C, 2, and 3 from a crude Triton X-100 membrane extract of Escherichia coli. In the presence of periplasmic proteins also PBP1A was specifically bound. At least five different non-PBPs showed specificity for MltA-Sepharose. The amino-terminal amino acid sequence of one of these proteins could be obtained, and the corresponding gene was mapped at 40 min on the E. coli genome. This MltA-interacting protein, named MipA, in addition binds to PBP1B, a bifunctional murein transglycosylase/transpeptidase. SPR studies with PBP1B immobilized to ampicillin-coated sensor chips showed an oligomerization of PBP1B that may indicate a dimerization. Simultaneous application of MipA and MltA onto a PBP1B sensor chip surface resulted in the formation of a trimeric complex. The dissociation constant was determined to be about 10(-6) M. The formation of a complex between a murein polymerase (PBP1B) and a murein hydrolase (MltA) in the presence of MipA represents a first step in a reconstitution of the hypothetical murein-synthesizing holoenzyme, postulated to be responsible for controlled growth of the stress-bearing sacculus of E. coli.     

Interaction of monoclonal antibodies with the enzymatic domains of penicillin-binding protein 1b of Escherichia coli.

Monoclonal antibodies (MAbs) against four different antigenic determinants of penicillin-binding protein (PBP) 1b were used to study the transglycosylase and transpeptidase activities of PBP 1b. Enzyme kinetics in the presence of and without the MAbs were determined, and the synthesized murein was analyzed. Two MAbs against the transglycosylase domain of PBP 1b appeared to inhibit this reaction. One MAb inhibited only the transpeptidase reaction, and one inhibited both enzymatic activities of PBP 1b. The latter two MAbs bound to the transpeptidase domain of PBP 1b. The following major conclusions were deduced from the results. (i) Transpeptidation is the rate-limiting step of the reaction cascade, and it is dependent on the product of transglycosylation. (ii) PBP 1b has only one type of transpeptidase activity, i.e., a penta-tetra transpeptidase activity. (iii) PBP 1b is probably a globular protein which has two intimately associated enzymatic domains.     

Cloning and characterization of PBP 1C, a third member of the multimodular class A penicillin-binding proteins of Escherichia coli.

All proteins of Escherichia coli that covalently bind penicillin have been cloned except for the penicillin-binding protein (PBP) 1C. For a detailed understanding of the mode of action of beta-lactam antibiotics, cloning of the gene encoding PBP1C was of major importance. Therefore, the structural gene was identified in the E. coli genomic lambda library of Kohara and subcloned, and PBP1C was characterized biochemically. PBP1C is a close homologue to the bifunctional transpeptidases/transglycosylases PBP1A and PBP1B and likewise shows murein polymerizing activity, which can be blocked by the transglycosylase inhibitor moenomycin. Covalently linked to activated Sepharose, PBP1C specifically retained PBP1B and the transpeptidases PBP2 and -3 in addition to the murein hydrolase MltA. The specific interaction with these proteins suggests that PBP1C is assembled into a multienzyme complex consisting of both murein polymerases and hydrolases. Overexpression of PBP1C does not support growth of a PBP1A(ts)/PBP1B double mutant at the restrictive temperature, and PBP1C does not bind to the same variety of penicillin derivatives as PBPs 1A and 1B. Deletion of PBP1C resulted in an altered mode of murein synthesis. It is suggested that PBP1C functions in vivo as a transglycosylase only.     

Interaction between two murein (peptidoglycan) synthases, PBP3 and PBP1B, in Escherichia coli

The murein (peptidoglycan) sacculus is an essential polymer embedded in the bacterial envelope. The Escherichia coli class B penicillin-binding protein (PBP) 3 is a murein transpeptidase and essential for cell division. In an affinity chromatography experiment, the bifunctional transglycosylase-transpeptidase murein synthase PBP1B was retained by PBP3-sepharose when a membrane fraction of E. coli was applied. The direct protein-protein interaction between purified PBP3 and PBP1B was characterized in vitro by surface plasmon resonance. The interaction was confirmed in vivo employing two different methods: by a bacterial two-hybrid system, and by cross-linking/co-immunoprecipitation. In the bacterial two-hybrid system, a truncated PBP3 comprising the N-terminal 56 amino acids interacted with PBP1B. Both synthases could be cross-linked in vivo in wild-type cells and in cells lacking FtsW or FtsN. PBP1B localized diffusely and in foci at the septation site and also at the side wall. Statistical analysis of the immunofluorescence signals revealed that the localization of PBP1B at the septation site depended on the physical presence of PBP3, but not on the activity of PBP3. These studies have demonstrated, for the first time, a direct interaction between a class B PBP (PBP3) and a class A PBP (PBP1B) in vitro and in vivo, indicating that different murein synthases might act in concert to enlarge the murein sacculus during cell division.     

Extensive re-modelling of the transpeptidase domain of penicillin-binding protein 2B of a penicillin-resistant South African isolate of Streptococcus pneumoniae

Clinical isolates of Streptococcus pneumoniae that have greatly increased levels of resistance to penicillin (greater than 1000-fold) have been reported from South Africa during the last ten years. Penicillin resistance in these strains is entirely due to the development of penicillin-binding proteins (PBPs) with decreased affinity for penicillin. We have cloned and sequenced the coding region for the transpeptidase domain of penicillin-binding protein 2B from three penicillin-sensitive strains of S. pneumoniae and from a penicillin-resistant South African strain. The amino acid sequences of the transpeptidase domains of PBP2B of the three penicillin-sensitive strains were identical and there were only between one and four differences in the nucleotide sequences of their coding regions. The corresponding region of the PBP2B gene from the penicillin-resistant strain differed by 74 nucleotide substitutions which resulted in 17 alterations in the amino acid sequence of PBP2B. The most remarkable alteration that has occurred during the development of the 'penicillin-resistant' form of PBP2B is the substitution of seven consecutive residues in a region that is predicted to form a loop at the bottom of the penicillin-binding site.     

Control of the cell elongation-division cycle by shuttling of PBP1 protein in Bacillus subtilis

The characteristic shape of bacterial cells is mainly determined by the cell wall, the synthesis of which is orchestrated by penicillin-binding proteins (PBPs). Rod-shaped bacteria have two distinct modes of cell wall synthesis, involved in cell elongation and cell division, which are believed to employ different sets of PBPs. A long-held question has been how these different modes of growth are co-ordinated in space and time. We have now identified the cell division protein, EzrA, and a newly discovered protein, GpsB, as key players in the elongation-division cycle of Bacillus subtilis. Mutations in these genes have a synthetic phenotype with defects in both cell division and cell elongation. They also have an unusual bulging phenotype apparently due to a failure in properly completing cell pole maturation. We show that these phenotypes are tightly associated with disturbed localization of the major transglycosylase/transpeptidase of the cell, PBP1. EzrA and GpsB have partially differentiated roles in the localization cycle of PBP1, with EzrA mainly promoting the recruitment of PBP1 to division sites, and GpsB facilitating its removal from the cell pole, after the completion of pole maturation.     

Role of penicillin-binding protein 2 (PBP2) in the antibiotic susceptibility and cell wall cross-linking of Staphylococcus aureus: evidence for the cooperative functioning of PBP2, PBP4, and PBP2A

Ceftizoxime, a beta-lactam antibiotic with high selective affinity for penicillin-binding protein 2 (PBP2) of Staphylococcus aureus, was used to select a spontaneous resistant mutant of S. aureus strain 27s. The stable resistant mutant ZOX3 had an increased ceftizoxime MIC and a decreased affinity of its PBP2 for ceftizoxime and produced peptidoglycan in which the proportion of highly cross-linked muropeptides was reduced. The pbpB gene of ZOX3 carried a single C-to-T nucleotide substitution at nucleotide 1373, causing replacement of a proline with a leucine at amino acid residue 458 of the transpeptidase domain of the protein, close to the SFN conserved motif. Experimental proof that this point mutation was responsible for the drug-resistant phenotype, and also for the decreased PBP2 affinity and reduced cell wall cross-linking, was provided by allelic replacement experiments and site-directed mutagenesis. Disruption of pbpD, the structural gene of PBP4, in either the parental strain or the mutant caused a large decrease in the highly cross-linked muropeptide components of the cell wall and in the mutant caused a massive accumulation of muropeptide monomers as well. Disruption of pbpD also caused increased sensitivity to ceftizoxime in both the parental cells and the ZOX3 mutant, while introduction of the plasmid-borne mecA gene, the genetic determinant of the beta-lactam resistance protein PBP2A, had the opposite effects. The findings provide evidence for the cooperative functioning of two native S. aureus transpeptidases (PBP2 and PBP4) and an acquired transpeptidase (PBP2A) in staphylococcal cell wall biosynthesis and susceptibility to antimicrobial agents.     

Suppression of a deletion mutation in the gene encoding essential PBP2b reveals a new lytic transglycosylase involved in peripheral peptidoglycan synthesis in Streptococcus pneumoniae D39

In ellipsoid‐shaped ovococcus bacteria, such as the pathogen Streptococcus pneumoniae (pneumococcus), side‐wall (peripheral) peptidoglycan (PG) synthesis emanates from midcells and is catalyzed by the essential class B penicillin‐binding protein PBP2b transpeptidase (TP). We report that mutations that inactivate the pneumococcal YceG‐domain protein, Spd_1346 (renamed MltG), remove the requirement for PBP2b. ΔmltG mutants in unencapsulated strains accumulate inactivation mutations of class A PBP1a, which possesses TP and transglycosylase (TG) activities. The ‘synthetic viable’ genetic relationship between Δpbp1a and ΔmltG mutations extends to essential ΔmreCD and ΔrodZ mutations that misregulate peripheral PG synthesis. Remarkably, the single MltG(Y488D) change suppresses the requirement for PBP2b, MreCD, RodZ and RodA. Structural modeling and comparisons, catalytic‐site changes and an interspecies chimera indicate that pneumococcal MltG is the functional homologue of the recently reported MltG endo‐lytic transglycosylase of Escherichia coli. Depletion of pneumococcal MltG or mltG(Y488D) increases sphericity of cells, and MltG localizes with peripheral PG synthesis proteins during division. Finally, growth of Δpbp1a ΔmltG or mltG(Y488D) mutants depends on induction of expression of the WalRK TCS regulon of PG hydrolases. These results fit a model in which MltG releases anchored PG glycan strands synthesized by PBP1a for crosslinking by a PBP2b:RodA complex in peripheral PG synthesis.     

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

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

Two-step procedure for purification and separation of the essential penicillin-binding proteins PBP 1A and 1Bs of Escherichia coli

The penicillin-binding proteins PBP 1A and 1Bs are the essential murein polymerases of Escherichia coli. Purification of these membrane-bound bifunctional transglycosylase-transpeptidases was a major obstacle in studying the details of both enzymatic reactions. Here we describe a simple, highly specific affinity chromatography method that takes advantage of the availability of the specific inhibitor of the transglycosylase site moenomycin A in order to enrich PBP 1A and 1Bs in one step from crude membrane preparations. Separation of PBP 1A from PBP 1Bs is achieved in a second step employing cation exchange chromatography yielding enzymatically active native murein polymerases.     

Functional biosynthesis of cell wall peptidoglycan by polymorphic bifunctional polypeptides. Penicillin-binding protein 1Bs of Escherichia coli with activities of transglycosylase and transpeptidase

Dual enzyme activities for the biosynthesis of peptidoglycan of the cell wall are located in major higher molecular weight penicillin-binding proteins (PBP) of Escherichia coli. Each of these proteins catalyzes the two successive final reactions in the synthesis of cross-linked peptidoglycan from the precursor N-acetylglucosaminyl-N-acetylmuramyl peptide linked to undecaprenol diphosphate; namely, the transglycosylation that extends the glycan chain and the penicillin-sensitive DD-transpeptidation that cross-links the glycan chains through two peptide side chains. Both transglycosylation and transpeptidation catalyzed by PBP-1Bs represent de novo synthesis of cross-linked peptidoglycan. Under appropriate conditions, about 25% cross-linkage was observed during the reaction, the main reaction product supposedly being a regularly cross-linked network of peptidoglycan. The two domains for the transglycosylase and transpeptidase activities were found to be located on a 50-kDa portion of the PBP-1Bs, which are about 90 kDa. Gene recombination experiments indicated that the transglycosylase domain is located upstream, i.e. on the N-terminal side of the transpeptidase domain, suggesting that the gene for these bifunctional peptides may have been formed by fusion of the genes for transglycosylase and transpeptidase that were previously located separately on the chromosome in this order.     

Common antigenic determinants of pneumococcal penicillin-binding protein (PBP) 1a and Streptococcus pyogenes PBP 2

Abstract Antisera raised against penicillin-binding protein (PBP) 1a of Streptococcus pneumoniae reacted with PBP 2 in certain strains of Streptococcus pyogenes . Cross-reactivity could be demonstrated on immunoblots as well as by immunoprecipitation of native solubilised proteins, indicating a similar structural arrangement also in the native form of the two PBPs.     

Kinetic characterization of the glycosyltransferase module of Staphylococcus aureus PBP2

We report the heterologous overexpression and purification of Staphylococcus aureus PBP2 and demonstrate efficient glycan polymerization from lipid II in vitro. S. aureus PBP2 is the first purified gram-positive class A penicillin-binding protein to show good transglycosylase activity. This enables further studies on this important class of enzymes.     

Osmolality-dependent relocation of penicillin-binding protein PBP2 to the division site in Caulobacter crescentus

The synthesis of the peptidoglycan cell wall is carefully regulated in time and space. In nature, this essential process occurs in cells that live in fluctuating environments. Here we show that the spatial distributions of specific cell wall proteins in Caulobacter crescentus are sensitive to small external osmotic upshifts. The penicillin-binding protein PBP2, which is commonly branded as an essential cell elongation-specific transpeptidase, switches its localization from a dispersed, patchy pattern to an accumulation at the FtsZ ring location in response to osmotic upshifts as low as 40 mosmol/kg. This osmolality-dependent relocation to the division apparatus is initiated within less than a minute, while restoration to the patchy localization pattern is dependent on cell growth and takes 1 to 2 generations. Cell wall morphogenetic protein RodA and penicillin-binding protein PBP1a also change their spatial distribution by accumulating at the division site in response to external osmotic upshifts. Consistent with its ecological distribution, C. crescentus displays a narrow range of osmotolerance, with an upper limit of 225 mosmol/kg in minimal medium. Collectively, our findings reveal an unsuspected level of environmental regulation of cell wall protein behavior that is likely linked to an ecological adaptation.     

Regulation of cell wall morphogenesis in Bacillus subtilis by recruitment of PBP1 to the MreB helix

The bacterial actin homologue MreB plays a key role in cell morphogenesis. In Bacillus subtilis MreB is essential under normal growth conditions and mreB mutants are defective in the control of cell diameter. However, the precise role of MreB is still unclear. Analysis of the lethal phenotypic consequences of mreB disruption revealed an unusual bulging phenotype that precedes cell death. A similar phenotype was seen in wild-type cells at very low Mg²⁺ concentrations. We found that inactivation of the major bi-functional penicillin-binding protein (PBP) PBP1 of B. subtilis restored the viability of an mreB null mutant as well as preventing bulging in both mutant and wild-type backgrounds. Bulging was associated with delocalization of PBP1. We show that the normal pattern of localization of PBP1 is dependent on MreB and that the proteins can physically interact using in vivo pull-down and bacterial two-hybrid approaches. Interactions between MreB and several other PBPs were also detected. Our results suggest that MreB filaments associate directly with the peptidoglycan biosynthetic machinery in B. subtilis as part of the mechanism that brings about controlled cell elongation.     

Streptococcus pneumoniae PBP2x mid‐cell localization requires the C‐terminal PASTA domains and is essential for cell shape maintenance

The transpeptidase activity of the essential penicillin‐binding protein 2x (PBP2x) of Streptococcus pneumoniae is believed to be important for murein biosynthesis required for cell division. To study the molecular mechanism driving localization of PBP2x in live cells, we constructed a set of N‐terminal GFP–PBP2x fusions under the control of a zinc‐inducible promoter. The ectopic fusion protein localized at mid‐cell. Cells showed no growth defects even in the absence of the genomic pbp2x, demonstrating that GFP–PBP2x is functional. Depletion of GFP–PBP2x resulted in severe morphological alterations, confirming the essentiality of PBP2x and demonstrating that PBP2x is required for cell division and not for cell elongation. A genetically or antibiotic inactivated GFP–PBP2x still localized at septal sites. Remarkably, the same was true for a GFP–PBP2x derivative containing a deletion of the central transpeptidase domain, although only in the absence of the protease/chaperone HtrA. Thus localization is independent of the catalytic transpeptidase domain but requires the C‐terminal PASTA domains, identifying HtrA as targeting GFP–PBP2x derivatives. Finally, PBP2x was positioned at the septum similar to PBP1a and the PASTA domain containing StkP protein, confirming that PBP2x is a key element of the divisome complex.     

PBP5 complementation of a PBP3 deficiency in Enterococcus hirae

The low susceptibility of enterococci to beta-lactams is due to the activity of the low-affinity penicillin-binding protein 5 (PBP5). One important feature of PBP5 is its ability to substitute for most, if not all, penicillin-binding proteins when they are inhibited. That substitution activity was analyzed in Enterococcus hirae SL2, a mutant whose pbp5 gene was interrupted by the nisRK genes and whose PBP3 synthesis was submitted to nisin induction. Noninduced SL2 cells were unable to divide except when plasmid-borne pbp5 genes were present, provided that the PBP5 active site was functional. Potential protein-protein interaction sites of the PBP5 N-terminal module were mutagenized by site-directed mutagenesis. The T167-L184 region (designated site D) appeared to be an essential intramolecular site needed for the stability of the protein. Mutations made in the two globular domains present in the N-terminal module indicated that they were needed for the suppletive activity. The P197-N209 segment (site E) in one of these domains seemed to be particularly important, as single and double mutations reduced or almost completely abolished, respectively, the action of PBP5.