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.     

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

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

Unequal distribution of penicillin-binding proteins among inner membrane vesicles of Escherichia coli

Escherichia coli penicillin-binding proteins (PBPs) were associated only with inner membrane vesicles when separated on 30 to 65% or 19 to 49% (wt/wt) sucrose gradients. Fractionation of vesicles through the low-density gradient revealed at least two classes of PBP-inner membrane associations. The first class consisted of PBPs 1 through 4, and the second class consisted of PBPs 5 through 8. These classes were distinguished by the density of vesicles with which they were associated; class 1 PBPs migrated with vesicles of higher density than did class 2 PBPs. Such combinations suggest that PBPs are nonrandomly distributed within the inner membrane, implying potential functional relationships among the PBPs themselves and with particular membrane domains. In addition, in cell lysates and in vesicle fractions, a 60,000-dalton aztreonam-insensitive PBP or protein fragment was observed which could potentially be confused with PBP3.     

Nucleotide sequence of the pbpA gene and characteristics of the deduced amino acid sequence of penicillin-binding protein 2 of Escherichia coli K12

We have determined the nucleotide sequence of the pbpA gene encoding penicillin-binding protein (PBP) 2 of Escherichia coli. The coding region for PBP 2 was 1899 base pairs in length and was preceded by a possible promoter sequence and two open reading frames. The primary structure of PBP 2, deduced from the nucleotide sequence, comprised 633 amino acid residues. The relative molecular mass was calculated to be 70867. The deduced sequence agreed with the NH2-terminal sequence of PBP 2 purified from membranes, suggesting that PBP 2 has no signal peptide. The hydropathy profile suggested that the NH2-terminal hydrophobic region (a stretch of 25 non-ionic amino acids) may anchor PBP 2 in the cytoplasmic membrane as an ectoprotein. There were nine homologous segments in the amino acid sequence of PBP 2 when compared with PBP 3 of E. coli. The active-site serine residue of PBP 2 was predicted to be Ser-330. Around this putative active-site serine residue was found the conserved sequence of Ser-Xaa-Xaa-Lys, which has been identified in all of the other E. coli PBPs so far studied (PBPs 1A, 1B, 3, 5 and 6) and class A and class C beta-lactamases. In the higher-molecular-mass PBPs 1A, 1B, 2 and 3, Ser-Xaa-Xaa-Lys-Pro was conserved. In the putative peptidoglycan transpeptidase domain there were six amino acid residues, which are common only in the PBPs of higher molecular mass.     

Nucleotide sequences of the pbpX genes encoding the penicillin-binding proteins 2x from Streptococcus pneumoniae R6 and a cefotaxime-resistant mutant, C506

Development of penicillin resistance in Streptococcus pneumoniae is due to successive mutations in penicillin-binding proteins (PBPs) which reduce their affinity for beta-lactam antibiotics. PBP2x is one of the high-Mr PBPs which appears to be altered both in resistant clinical isolates, and in cefotaxime-resistant laboratory mutants. In this study, we have sequenced a 2564 base-pair chromosomal fragment from the penicillin-sensitive S. pneumoniae strain R6, which contains the PBP2x gene. Within this fragment, a 2250 base-pair open reading frame was found which coded for a protein having an Mr of 82.35kD, a value which is in good agreement with the Mr of 80-85 kD measured by SDS-gel electrophoresis of the PBP2x protein itself. The N-terminal region resembled an unprocessed signal peptide and was followed by a hydrophobic sequence that may be responsible for membrane attachment of PBP2x. The corresponding nucleotide sequence of the PBP2x gene from C504, a cefotaxime-resistant laboratory mutant obtained after five selection steps, contained three nucleotide substitutions, causing three amino acid alterations within the beta-lactam binding domain of the PBP2x protein. Alterations affecting similar regions of Escherichia coli PBP3 and Neisseria gonorrhoeae PBP2 from beta-lactam-resistant strains are known. The penicillin-binding domain of PBP2x shows highest homology with these two PBPs and S. pneumoniae PBP2b. In contrast, the N-terminal extension of PBP2x has the highest homology with E. coli PBP2 and methicillin-resistant Staphylococcus aureus PBP2'. No significant homology was detected with PBP1a or PBP1b of Escherichia coli, or with the low-Mr PBPs.     

Plasmid segregation into minicells is associated with membrane attachment and independent of plasmid replication.

The role of plasmid replication in the segregation of plasmids into Escherichia coli minicells was investigated with temperature-sensitive replication mutants derived from E. coli plasmids ColE1 and pSC101. For as long as six generations of growth, at permissive or nonpermissive temperatures (when greater than 80% of plasmid replication was inhibited), the same amount of previously 3H-labeled plasmid DNA segregated into minicells. Density gradient separations of wild-type and temperature-sensitive plasmid DNA from both replicons segregated into the minicells showed that about 20 to 25% was stably associated with the minicell membrane at both temperatures. Electron microscopy showed this DNA to consist of circular plasmid molecules attached to the minicell membrane. These combined findings suggest that segregation of plasmids into minicells and their association with the minicell membrane are interrelated and independent of plasmid replication.     

Labeling pattern of major penicillin-binding proteins of Escherichia coli during the division cycle.

Escherichia coli cells were synchronized by the elutriation technique. The pattern of penicillin-binding proteins (PBPs) in synchronously growing cells was determined with an iodinated derivative of ampicillin in intact cells as well as in isolated membranes. This was done under nonsaturating conditions as well as under conditions in which the PBPs were saturated with [125I]ampicillin. No evidence was found for fluctuations in the PBP pattern: the PBPs seem to be present in a constant ratio throughout the division cycle. The E. coli cells exert their control on shape maintenance and cell wall growth apparently not on the level of concentration of PBPs in the cell but rather on activation of existing components.     

R Factor Proteins Synthesized in Escherichia coli Minicells: Incorporation Studies with Different R Factors and Detection of Deoxyribonucleic Acid-Binding Proteins

Analysis of the protein synthesized by Escherichia coli minicells containing R factors demonstrated a variety of low- and high-molecular-weight polypeptides in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Only half of this protein was released into a soluble fraction on lysis of these minicells. The other half remained associated with the minicell envelope. The efficiency of precursor incorporation into protein and the kinds of proteins synthesized changed with the age of the minicells at the time of harvest. About 1 to 2% of the soluble R factor-coded protein bound to calf thymus, E. coli, or R factor DNA-cellulose. Although most of these proteins were excluded from Sephadex G-100 columns, they migrated chiefly as low-molecular-weight-polypeptides (13,000 to 15,000) in SDS-polyacrylamide gels. Additional DNA-binding proteins that appeared to be higher-molecular-weight peptides were noted in extracts from younger minicells. At least one protein, identified as an SDS band, appeared to bind selectively to R factor DNA-cellulose. Minicells with R factors also contained DNA-binding proteins of cell origin, including the core RNA polymerase. No such binding proteins were found in R(-) minicells. These studies suggest that: (i) R factors code for proteins that may be involved in their own DNA metabolism; (ii) R factor DNA-binding proteins may be associated with larger host cell DNA-binding proteins or subunits of larger R factor proteins; and (iii) the age of the minicell influences the extent of protein synthesis and the kinds of proteins synthesized by R factors in minicells.     

Studies of the high molecular weight penicillin-binding proteins of Bacillus subtilis.

Seven or eight penicillin-binding proteins (PBPs) were detected in Bacillus subtilis membranes. By introducing covalent affinity chromatography employing cephalosporins as ligands, milligram amounts of three high molecular weight PBPs (PBP 1 ab, Mr = 120,000; PBP 2b, Mr = 94,000; and PBP 4, Mr = 78,000) were obtained without any contamination of the major PBP 5, the D-alanine carboxypeptidase. Small amounts of pure PBP 2b could be isolated by manipulation of the affinity chromatography conditions. Structural and physical properties of these proteins as well as the generation of one major penicilloyl peptide from each PBP by digestion with pepsin suggest that each PBP is the product of a separate gene. No enzymatic activity could be found in mixtures of these high molecular weight PBPs employing substrates used for the transpeptidase and D-alanine carboxypeptidase assays in particulate membrane fractions.     

Contribution of membrane-binding and enzymatic domains of penicillin binding protein 5 to maintenance of uniform cellular morphology of Escherichia coli

Four low-molecular-weight penicillin binding proteins (LMW PBPs) of Escherichia coli are closely related and have similar DD-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD). However, only one, PBP 5, has a demonstrated physiological function. In its absence, certain mutants of E. coli have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells. To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other DD-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5. Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD. However, complementation was not restored by the putative carboxy-terminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound. Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required. A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal beta-sheet domain of PBP 6. In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional. The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the DD-carboxypeptidase enzymatic core.     

Penicillin-binding proteins of Thiobacillus versutus

The cytoplasmic membrane of Thiobacillus versutus was found to contain at least nine penicillin-binding proteins (PBPs) with apparent molecular weights as judged by sodium dodecyl sulphate polyacrylamide slab gel electrophoresis of 87000 (PBP1), 81000 (PBP2), 68000 (PBP3), 63000 (PBP4), 57000 (PBP5), 40000 (PBP6), 37000 (PBP70, 33000 (PBP8) and 31000 (PBP9). The PBP pattern of T. versutus was thus quite different from that of the Enterobacteria and the Pseudomonads. Also the properties of the PBPs of T. versutus such as affinity for various beta-lactam antibiotics, heat stability and release of bound penicillin were different from similar properties of Escherichia coli, Pseudomonas aeruginosa and other gram-negative bacteria.     

Membrane assembly in Escherichia coli. II. Segregation of preformed and newly formed membrane proteins into cells and minicells

Cells of a minicell producing strain of Escherichia coli were freed of minicells and innoculated into fresh medium for further growth. The protein composition of the minicell membranes formed during periods of further growth reflected that of the total cellular membrane proteins, rather than being representative of the membrane proteins formed during the period of production of the minicells. These data, when viewed together with published observations on minicell morphogenesis, do not support models of membrane assembly where the membrane protein matrix grows preferentially either at the poles or at the equitorial perimeter of this organism with conservation of structure of preformed and newly formed membrane.     

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.     

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.     

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

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

Interaction of beta-lactam antibiotics with penicillin-binding proteins from Bacillus megaterium

The binding properties of 25 beta-lactam antibiotics to Bacillus megaterium membranes have been studied. The affinities of the antibiotics for the penicillin-binding proteins (PBPs) are also reported. We found that PBP 4 has the highest affinity for nearly all the antibiotics studied whereas PBP 5 has the lowest affinity. Both PBP 4 and PBP 5 appear to be dispensable for the maintenance of bacterial growth and survival and appear to be DD-carboxypeptidases. Only the beta-lactam cefmetazol bound preferentially to PBP 5 and has been used to study the inhibition of DD-carboxypeptidase. Comparative studies with beta-lactam that simultaneously result in (a) binding to PBPs 1 and 3, (b) inhibition of cell growth and (c) lysis, stressed the importance of PBPs 1 and 3 for cell growth and survival.     

Expression and purification of a soluble form of penicillin-binding protein 2 from both penicillin-susceptible and penicillin-resistant Neisseria gonorrhoeae.

Resistance to penicillin in non-beta-lactamase-producing strains of Neisseria gonorrhoeae (CMRNG strains) is mediated in part by the production of altered forms of penicillin-binding protein 2 (PBP 2) that have a decreased affinity for penicillin. The reduction in the affinity of PBP 2 is largely due to the insertion of an aspartic acid residue (Asp-345a) into the amino acid sequence of PBP 2. Truncated forms of N. gonorrhoeae PBP 2, which differed only by the insertion of Asp-345a, were constructed by placing the region of the penA genes encoding the periplasmic domain of PBP 2 (amino acids 42-581) into an ATG expression vector. When the recombinant PBP 2 molecules were overexpressed in Escherichia coli, insoluble PBP 2 inclusion bodies, which could be isolated by low-speed centrifugation of cell lysates, were formed. These insoluble aggregates were solubilized and the truncated PBP 2 polypeptides were partially purified by cation-exchange chromatography and gel filtration in the presence of denaturant prior to the refolding of the enzyme in vitro. After renaturation, gel filtration was used to separate monomeric soluble PBP 2 from improperly folded protein aggregates and other protein contaminants. A 4-liter culture of induced E. coli cells yielded 1.4 mg of soluble PBP 2 or PBP 2' (PBP 2 containing the Asp-345a insertion), both of which were estimated to be 99% pure. The affinity of soluble PBP 2' for [3H]penicillin G was decreased fourfold relative to that of soluble PBP 2, and their affinities were found to be identical to the affinities of the full-length PBP 2 enzymes that were previously determined in N. gonorrhoeae membranes. Furthermore, soluble PBP 2 displayed a rank order of affinity for several other beta-lactam antibiotics that was consistent with the rank order of affinities previously reported for the native molecules. On the basis of these results, both of these soluble PBPs should be suitable for crystallization and X-ray crystallographic analysis.     

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

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

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.     

Penicillin-binding proteins of Rhodopseudomonas sphaeroides and their membrane localization.

Cytoplasmic membranes (CM) prepared from both chemotrophic and phototrophic cells of Rhodopseudomonas sphaeroides possess penicillin-binding proteins (PBPs), as demonstrated by binding of [125]furazlocillin to isolated membranes, the subsequent separation of the constituent PBPs by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their detection by autoradiography. The major PBP present in CM from R. sphaeroides corresponds in molecular weight to PBP-5, the predominant PBP present in CM of Escherichia coli. In contrast, the outer membrane of R. sphaeroides shows only low-level furazlocillin-binding activity on a per milligram of protein basis compared with chemotrophic CM. The intracytoplasmic membrane (ICM) derived from phototrophic cells contains less than 5% of the furazlocillin-binding activity of the CM. Based on the specific localization of PBPs in the CM, it is possible to provide quantitative estimates of the extent of CM present in preparations of ICM. This method demonstrates that highly purified preparations of ICM contain less than 5% CM. Additionally, the assay for PBPs demonstrates that during ICM remodeling, which occurs upon a shift from phototrophic to chemotrophic growth, there is no significant insertion of PBPs into the ICM over the first two generations after a shift to chemotrophic growth.