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.     

Benzylpenicillin-induced filament formation of Clostridium perfringens

Growth of Clostridium perfringens with low concentrations of benzylpenicillin inhibited septum formation and division of the organisms. This resulted in continued growth of the organisms as aseptate filaments. The effect was reversed on removal of the antibiotic. The composition of walls isolated from organisms grown with the antibiotic was similar to that of walls from untreated bacteria. In addition, both contained non-N-acetylated glucosamine residues in their peptidoglycan. No differences were detected in the degree of cross-linkage of peptidoglycan. Clostridium perfringens contains six membrane-associated penicillin-binding proteins (PBPs) which have different affinities for [3H]benzylpenicillin. Concentrations of the antibiotic which were sufficient to cause filamentation of apparently all organisms in a culture caused almost complete saturation of PBPs 3, 4, 5 and 6. At these concentrations there was no measurable interaction with PBPs 1 and 2. Thus interaction of the antibiotic with the lower molecular weight PBPs is correlated with the inhibition of septum formation in C. perfringens.     

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     

Deacylation kinetics analysis of Streptococcus pneumoniae penicillin-binding protein 2x mutants resistant to beta-lactam antibiotics using electrospray ionization- mass spectrometry

Penicillin-binding proteins (PBPs) catalyze the transpeptidase reaction involved in peptidoglycan synthesis and are covalently inhibited by the beta-lactam antibiotics. In a previous work we have focused on acylation efficiency measurements of various Streptococcus pneumoniae PBP2x* mutants to study the molecular determinants of resistance to beta-lactams. In the present paper we have developed a method to improve an accurate determination of the deacylation rate constant using electrospray ionization-mass spectrometry. This method is adaptable to the analysis of deacylation of any beta-lactam. Compared to the fluorographic technique, the ESI-MS method is insensitive to variations in the concentration of functional proteins and is therefore more reliable. We have established that the resistance of PBPs to beta-lactams is mostly due to a decrease of the acylation efficiency with only marginal effects on the deacylation rates.     

Growth of Streptococcus mutans protoplasts is not inhibited by penicillin.

A method is described in which cells of Streptococcus mutans BHT can be converted to spherical, osmotically fragile protoplasts. Exponential-phase cells were suspended in a solution containing 0.5 M melezitose, and their cell walls were hydrolyzed with mutanolysin (M-1 enzyme). When the resultant protoplasts were incubated in a chemically defined growth medium containing 0.5 M NH4Cl, the protoplast suspensions increased in turbidity, protein, ribonucleic acid, and deoxyribonucleic acid in a balanced fashion. In the presence of benzylpenicillin (5 microgram/ml), balanced growth of protoplasts was indistinguishable from untreated controls. This absence of inhibition of protoplast growth in the presence of benzylpenicillin was apparently not due to inactivation of the antibiotic. When exponential-phase cells of S. mutans BHT were first exposed to 5 microgram of benzyl-penicillin per ml for 1 h and then converted to protoplasts, these protoplasts were also able to grow in chemically defined, osmotically stabilized medium. The ability of wall-free protoplasts to grow and to synthesize ribonucleic acid and protein in the presence of a relatively high concentration of benzylpenicillin contrasts with the previously reported rapid inhibition of ribonucleic acid and protein synthesis in intact streptococci. These data suggest that this secondary inhibition of ribonucleic acid and protein synthesis in whole cells is due to factors involved with the continued assembly of an intact, insoluble cell wall rather than with earlier stages of peptidoglycan synthesis.     

Effects of penicillin on macromolecular synthesis and surface growth of a tolerant streptococcus as studied by computer reconstruction methods.

Strains of Streptococcus mutans are very susceptible to growth inhibition by benzylpenicillin, but are tolerant to lysis when exposed to even high concentrations of this drug. These properties enabled this study of S. mutans GS-5 surface growth and peptidoglycan, ribonucleic acid, protein, and deoxyribonucleic acid syntheses in the absence of osmotic stabilization. Inhibition of syntheses of peptidoglycan, ribonucleic acid, and protein was dose dependent. Synthesis of peptidoglycan was most susceptible. Substantial but less severe inhibitions of ribonucleic acid and protein syntheses rapidly followed decreased peptidoglycan synthesis, whereas inhibition of deoxyribonucleic acid synthesis was delayed and minimal. Computer-assisted reconstructions of surface growth zones and poles observed in electron micrographs of replicas were performed and indicated that at low concentrations of benzylpenicillin (0.03 micrograms/ml), growth sites reached abnormally large sizes and surface/volume ratios. The observed shifts in surface/volume ratio were attributed to an inhibition of the normal constrictive division mechanism. The poles of these cells also increased in size over those of the controls, but the relatively smaller change in surface/volume ratio confirmed the visual impression that the shape of the poles was much less altered than the shape of the growth sites. As the concentration of benzylpenicillin used was raised from 0.03 to 2 micrograms/ml, the ability of growth sites and poles to enlarge was restricted in a manner that most closely agreed with the extent of inhibition of peptidoglycan (rather than deoxyribonucleic acid, ribonucleic acid, or protein) synthesis. This correlation suggested that increases in cell size may be regulated by the supply of peptidoglycan precursors.     

Recruitment of penicillin-binding protein PBP2 to the division site of Staphylococcus aureus is dependent on its transpeptidation substrates

Staphylococcus aureus penicillin-binding protein PBP2 is an enzyme involved in the last stages of peptidoglycan assembly and is an important player in the mechanism of methicillin resistance of this pathogen. PBP2 localized to the division site but its recruitment to the forming division septum was prevented after acylation by oxacillin. The presence of the antibiotic did not affect FtsZ ring maintenance nor the localization of externalized peptidoglycan precursors. Delocalization of PBP2 was also observed when its pentapeptide substrate was eliminated by addition of d-cycloserine or blocked by addition of vancomycin. Taken together these observations suggest that PBP2 is recruited to the division site by binding to its substrate, which is localized at that place. In methicillin-resistant S. aureus, addition of oxacillin does not result in delocalization of PBP2 indicating that acylated PBP2 can be maintained in place by functional PBP2A, the central element of this resistance mechanism.     

Cross-linking and O-acetylation of peptidoglycan in Staphylococcus aureus (strains H and MR-1) grown in the presence of sub-growth-inhibitory concentrations of beta-lactam antibiotics

Staphylococcus aureus H was grown for 4 generation times with various sub-growth-inhibitory concentrations of beta-lactam antibiotics specific for particular penicillin-binding proteins (PBPs) - PBP2, clavulanic acid; PBP3, methicillin; PBP4, cefoxitin - and also with the non-specific benzylpenicillin. Isolated cell walls were digested with Chalaropsis muramidase and the resulting peptidoglycan fragments were fractionated by HPLC into disaccharide-peptide monomers and cross-linked dimers, trimers, tetramers and greater oligomers. The pattern of relative fragment concentrations with increasing amounts of drug was roughly the same regardless of the antibiotic used, monomers and dimers increasing while trimers and tetramers changed little and oligomers decreased rapidly. The patterns resembled closely those predicted by the 'random addition' model for multiple cross-link formation and not at all those predicted by the 'monomer addition' model. The O-acetylation of the peptidoglycan remained essentially unaffected under all these conditions. S. aureus MR-1, a constitutive producer of PBP2', gave similar results when treated with methicillin.     

Crystal structure of the Bacillus subtilis penicillin-binding protein 4a, and its complex with a peptidoglycan mimetic peptide

The genome of Bacillus subtilis encodes 16 penicillin-binding proteins (PBPs) involved in the synthesis and/or remodelling of the peptidoglycan during the complex life cycle of this sporulating Gram-positive rod-shaped bacterium. PBP4a (encoded by the dacC gene) is a low-molecular mass PBP clearly exhibiting in vitro DD-carboxypeptidase activity. We have solved the crystal structure of this protein alone and in complex with a peptide (D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine) that mimics the C-terminal end of the Bacillus peptidoglycan stem peptide. PBP4a is composed of three domains: the penicillin-binding domain with a fold similar to the class A beta-lactamase structure and two domains inserted between the conserved motifs 1 and 2 characteristic of the penicillin-recognizing enzymes. The soaking of PBP4a in a solution of D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine resulted in an adduct between PBP4a and a D-alpha-aminopimelyl-epsilon-D-alanine dipeptide and an unbound D-alanine, i.e. the products of acylation of PBP4a by D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine with the release of a D-alanine. The adduct also reveals a binding pocket specific to the diaminopimelic acid, the third residue of the peptidoglycan stem pentapeptide of B. subtilis. This pocket is specific for this class of PBPs.     

Saturation of penicillin-binding protein 1 by β-lactam antibiotics in growing cells of Bacillus licheniformis

With the help of a new highly sensitive method allowing the quantification of free penicillin-binding proteins (PBPs) and of an integrated mathematical model, the progressive saturation of PBP1 by various β-lactam antibiotics in growing cells of Bacillus licheniformis was studied. Although the results confirmed PBP1 as a major lethal target for these compounds, they also underlined several weaknesses in our present understanding of this phenomenon. In growing cells, but not in resting cells, the penicillin target(s) appeared to be somewhat protected from the action of the inactivators. In vitro experiments indicated that amino acids, peptides and depsipeptides mimicking the peptide moiety of the nascent peptidoglycan significantly interfered with the acylation of PBP1 by the antibiotics. In addition, the level of PBP1 saturation at antibiotic concentrations corresponding to the minimum inhibitory concentrations was not constant, suggesting that additional, presently undiscovered, factors might be necessary to account for the experimental observations.     

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.     

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.     

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.     

Direct quantitation of the number of individual penicillin-binding proteins per cell in Escherichia coli.

The penicillin-binding proteins (PBPs) are a set of enzymes that participate in the terminal stages of bacterial peptidoglycan assembly. As their name implies, these proteins also covalently bind and are inhibited by beta-lactam antibiotics. Although many studies have examined the relative binding affinities of a number of beta-lactam antibiotics, a surprisingly small number of studies have addressed the absolute numbers of each of the PBPs present in the bacterial cell. In the present study, the PBP values initially reported in Escherichia coli almost 20 years ago by B. G. Spratt (Eur. J. Biochem. 72:341-352, 1977) were refined. The individual PBPs from a known number of bacteria radiolabeled with [3H]benzylpenicillin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The radioactive bands were located, excised, and quantitatively extracted from the gel slices. The radioactivity was measured by scintillation counting, and the absolute disintegrations per minute were calculated. From the specific activity of the labeled penicillin, the absolute disintegrations per minute, and the CFU per milliliter, a determination of the number of each of the PBPs per cell was made. The measurements were performed on multiple samples to place statistical limits on the numbers obtained. The values for the individual PBPs found in E. coli deviated in several ways from the previously reported observations. Of particular significance is the higher number of molecules of PBP 2 and 3 observed, since these PBPs are known to participate in cell morphogenesis. The PBP content in both rich Luria broth medium and M9 minimal medium was determined, with the slower-growing cells in minimal medium possessing fewer of the individual PBPs per cell.     

Purification and partial characterization of a penicillin-binding protein from Mycobacterium smegmatis.

Penicillin-binding proteins (PBPs), although characterized from several organisms, have so far not been studied in mycobacteria. The present study is the first characterization of a PBP from Mycobacterium smegmatis. The PBP was purified by solubilization of the membranes with Triton X-100 and successive chromatography of the solubilized proteins on ampicillin-linked CH Sepharose 4B and DE-52. The purified PBP (M(r), 49,500) catalyzed a model transpeptidase reaction with the tripeptide acetyl2-L-Lys-D-Ala-D-Ala as the substrate and Gly-Gly as the acceptor. The transpeptidase activity was inhibited by 50% at a benzylpenicillin concentration of 1.8 x 10(-7) M, which was similar to the concentration (1.1 x 10(-7) M) of benzylpenicillin required to saturate to 50% this PBP. Of several antibiotics tested, the concentration of antibiotic required to inhibit [35S]penicillin binding by 90% was found to be the lowest for cefoxitin and Sch 34343.     

Peptidoglycan synthesis in Bacillus licheniformis. The inhibition of cross-linking by benzylpenicillin and cephaloridine in vivo accompanied by the formation of soluble peptidoglycan.

The synthesis of peptidoglycan by an autolysin-deficient beta-lactamase-negative mutant of Bacillus licheniformis was studied in vivo in the absence of protein synthesis. Benzylpenicillin and cephaloridine inhibited the formation of cross-bridges between newly synthesized peptidoglycan and the pre-existing cell wall. This inhibition, detected by measurement of the incorporation of N-acetyl[14C]glucosamine into the glycan fraction of the cell wall, was reversed by treatment with beta-lactamase and washing. Inhibition of D-alanine carboxypeptidase by benzylpenicillin was not reversed under similar conditions. Cells in which the initial penicillin inhibition of transpeptidation had been reversed showed an increased sensitivity to a subsequent addition of the antibiotic. Chemical analysis of peptidoglycan synthesized after reversal of penicillin inhibition revealed the presence of excess of alanine resulting from the continued inhibition of D-alanine carboxypeptidase. When the cell walls were digested to yield muropeptides so that the degree of cross-linking could be measured, the product after reversal of penicillin inhibition contained fewer cross-links than did the control preparation. Cultures treated with benzylpenicillin and cephaloridine continued to synthesize uncross-linked soluble peptidoglycan, which accumulated in the medium. This soluble material was all newly synthesized peptidoglycan and did not result from autolysis of the bacteria. The average chain lengths of the glycan synthesized in vivo and released as soluble peptidoglycan in the presence of both benzylpenicillin and cephaloridine were similar to those found previously in this organism.     

Correlation of penicillin-binding protein composition with different functions of two membranes in Bacillus subtilis forespores.

The distribution of penicillin-binding proteins (PBPs) within different membranes of sporulating cells of Bacillus subtilis was examined in an effort to correlate the location of individual PBPs with their proposed involvement in either cortical or vegetative peptidoglycan synthesis. The PBP composition of forespores was determined by two methods: examination of isolated forespore membranes and assay of the in vivo accessibility of the PBPs to penicillin. In both cases, it was apparent that PBP 5*, the major PBP synthesized during sporulation, was present primarily, but not exclusively, in the forespore. The membranes from mature dormant spores were prepared by either chemically stripping the integument layers of the spores, followed by lysozyme digestion, or lysozyme digestion alone of coat-defective gerE spores. PBP 5* was detected in membranes from unstripped spores but was never found in stripped ones, which suggests that the primary location of this PBP is the outer forespore membrane. This is consistent with a role for PBP 5* exclusively in cortex synthesis. In contrast, vegetative PBPs 1 and 2A were only observed in stripped spore preparations that were greatly enriched for the inner forespore membrane, which supports the proposed requirement for these PBPs early in germination. The apparent presence of PBP 3 in both membranes of the spore reinforces the suggestion that it catalyzes a step common to both cortical and vegetative peptidoglycan synthesis.     

Effect of growth rate on the penicillin-binding proteins of Escherichia coli

Abstract In an Escherichia coli strain, the levels of penicillin-binding proteins (PBPs) 1A plus 1B, both peptidoglycan transglycosylase/transpeptidases, were found to be relatively independent of the imposed growth ratw in chemostat cultures under different nutrient limitation conditions. A considerable increase in levels of PBP 6 was observed as the growth rate was reduced, whilst, in contrast, a decrease was observed in levels of the other PBPs.     

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.     

A new mercury-penicillin V derivative as a probe for ultrastructural localization of penicillin-binding proteins in Escherichia coli.

The precise ultrastructural localization of penicillin-binding protein (PBP)-antibiotic complexes in Escherichia coli JM101, JM101 (pBS96), and JM101(pPH116) was investigated by high-resolution electron microscopy. We used mercury-penicillin V (Hg-pen V) as a heavy-metal-labeled, electron-dense probe for accurately localizing PBPs in situ in single bacterial cells grown to exponential growth phase. Biochemical data derived from susceptibility tests and bacteriolysis experiments revealed no significant differences between Hg-pen V and the parent compound, penicillin V, or between strains. Both antibiotics revealed differences in the binding affinities for PBPs of all strains. Deacylation rates for PBPs were slow despite the relatively low binding affinities of antibiotics. Cells bound most of the Hg-pen V added to cultures, and the antibiotic-PBP complex could readily be seen by electron microscopy of unstained whole mounts as distinct, randomly situated electron-dense particles. Fifty to 60% of the antibiotic was retained by cells during processing for conventional embedding so that thin sections could also be examined. These revealed similar electron-dense particles located predominantly on the plasma membrane and less frequently in the cytoplasm. Particles positioned on the plasma membranes were occasionally shown to protrude into the periplasmic space, thereby reflecting the high resolution of the Hg-pen V probe. Moreover, some particles were observed free in the periplasm, suggesting, for the first time, that a proportion of PBPs may not be restricted to the plasma membrane but may be tightly associated with the peptidoglycan for higher efficiency of peptidoglycan assembly. All controls were devoid of the electron-dense particles. The presence of electron-dense particles in cells of the wild-type JM101, demonstrated that our probe could identify PBPs in naturally occurring strains without inducing PBP overproduction.