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.     

Membrane invagination in Rhodobacter sphaeroides is initiated at curved regions of the cytoplasmic membrane,then forms both budded and fully detached spherical vesicles

The purple phototrophic bacteria synthesize an extensive system of intracytoplasmic membranes (ICM) in order to increase the surface area for absorbing and utilizing solar energy. Rhodobacter sphaeroides cells contain curved membrane invaginations. In order to study the biogenesis of ICM in this bacterium mature (ICM) and precursor (upper pigmented band – UPB) membranes were purified and compared at the single membrane level using electron, atomic force and fluorescence microscopy, revealing fundamental differences in their morphology, protein organization and function. Cryo‐electron tomography demonstrates the complexity of the ICM of Rba. sphaeroides. Some ICM vesicles have no connection with other structures, others are found nearer to the cytoplasmic membrane (CM), often forming interconnected structures that retain a connection to the CM, and possibly having access to the periplasmic space. Near‐spherical single invaginations are also observed, still attached to the CM by a ‘neck’. Small indents of the CM are also seen, which are proposed to give rise to the UPB precursor membranes upon cell disruption. ‘Free‐living’ ICM vesicles, which possess all the machinery for converting light energy into ATP, can be regarded as bacterial membrane organelles.     

Disappearence of the intracytoplasmic membranes inRhodopseudomonas sphaeroides

The photosynthetic bacterium,Rhodopseudomonas sphaeroides, can be grown phototrophically (light, anaerobiosis), of chemotrophically (dark, aerobiosis). In the first case, it contains intracytoplasmic membranes with photosynthetic pigments. When shifted from phototrophy to chemotrophy these membranes disappear in an unknown fashion. In the present experiment, samples were taken for electron microscopy, cell density and bacteriochlorophyll determinations after shift from phototrophy to chemotrophy. The density of intracytoplasmic vesicles was measured on micrographs. During the first 2h growth is very slow and the ultrastructure remains unaltered. As growth resumes, the vesicles disappear at a rate which implies that they are not incorportated into the cytoplasmic membrane, nor actively digested, but remain intact and become increasingly diluted in the cytoplasm as the culture grows. The size of the vesicles was estimated to about 500 Å. The number of vesicles in phototrophically grown cells was calculated to about 575 per cell, and after 6h chemotrophic growth to about 100. The areas of the cytoplasmic and intracytoplasmic membranes are roughly calculated.Abbreviations Bchl bacteriochlorophyll - CM cytoplasmic membranes - ICM intracytoplasmic membranes     

Topographical distribution of penicillin-binding proteins in the Escherichia coli membrane.

The penicillin-binding proteins (PBPs) found in the membranes of Escherichia coli X925 minicells (primarily cell ends or septa) were compared with those found in rod-shaped cells (primarily sidewalls) in an effort to determine whether certain PBPs are unevenly distributed over the bacterial cell membrane. The seven major PBPs of E. coli were all present in minicell membranes. PBP 1B was altered in minicells, however, appearing as two bands on sodium dodecyl sulfate-polyacrylamide gels rather than the usual three. PBP 2, which is needed for longitudinal growth of the cell but not for septum formation, was significantly reduced in minicell membranes. This observation is consistent with the fact that minicells contain very little sidewall material and raises the possibility that the specialized function of PBP 2 may be determined or regulated by its uneven topographical distribution in the membrane. None of the PBPs appeared to be selectively enriched in minicell membranes.     

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.     

Membranes of Rhodopseudomonas sphaeroides. V. Identification of bacteriochlorophyll alpha-depleted cytoplasmic membrane in phototrophically grown cells.

The separation of membrane fragments was investigated in extracts of phototropically grown Rhodopseudomonas sphaeroides to determine if the plasma membrane contains discrete regions. A highly purified fraction of bacteriochlorophyll alpha-deficient membrane fragments was isolated by differential centrifugation, chromatography on Sepharose 2B, reaggregation, and isopycnic sedimentation on sucrose gradients. Significant levels of b- and c-type cytochromes and succinate dehydrogenase were demonstrated in the isolated membrane fragments and their appearance in electron micrographs, their polypeptide profile in dodecyl sulfate-polyacrylamide gel electrophoresis, and overall chemical composition were essentially identical to a similar fraction isolated from aerobically grown cells. Their polypeptide profiles were distinct from those of the intracytoplasmic chromatophore and outer membranes, and on the basis of bacteriochlorophyll content the phototrophic fraction was contaminated with chromatophores by less than 9%. The membrane fragments contained no diaminopimelic acid or glucosamine. It is condluded that the membrane fragments isolated from phototrophically growing Rp. sphaeroides have arisen from photosynthetic pigment-depleted regions of the plasma membrane structurally and functionally differentiated from the intracytoplasmic chromatophore membrane. These regions represent conserved chemotrophic cytoplasmic membrane whose synthesis continues under photoheterotrophic conditions.     

Characterization of cell-envelope fractions of chemotrophically and phototrophically grown Rhodospirillum tenue

Envelope preparations of chemotrophically and phototrophically grown Rhodospirillum tenue were isolated and characterized on the basis of their contents and composition in phospholipids and fatty acids, as well as on the basis of their enzyme activities, absorption spectra and polypeptide patterns. Both preparations were similar in their contents in phospholipids and fatty acids. Their total fatty acids were characterized by a rather high percentage of saturated fatty acids. The activities of the respiratory reactions were considerably higher in chemotrophic than in phototrophic membranes. This is true especially for activities of the terminal oxidase which were over 20 times greater. The affinities of the corresponding enzymes to their respective substrates (K _m ) differed with differences in the culture conditions. Under chemotrophic conditions the K _m values for the NADH-dependent reactions were lower than those values under phototrophic conditions, whereas the K _m values for the succinate dependent reactions were higher. The low temperature (77°K) near infrared spectrum of the phototrophic membrane showed a peak at 875 nm which was not detectable in the chemotrophic membrane. The polypeptide patterns, in the presence of sodium dodecyl sulfate, of both preparations were quite similar except for the presence of two low molecular weight proteins (M. Wt. 12,000 and 10,000) in the phototrophic membrane which were absent in the chemotrophic membrane. Both envelope preparations were further fractionated into enriched cytoplasmic membrane and outer membrane fractions which were identified on the basis of their contents in 2-keto-3-deoxyoctonate, ubiquinone 8 and bacteriochlorophyll (in the case of the phototrophic membrane) and their enzyme activities. The buoyant densities of the corresponding fractions from both envelope preparations were found to be equal. The data presented in this paper demonstrate that envelope preparations of chemotrophically and phototrophically grown R. tenue are similar in their contents in 2-keto-3-deoxyoctonate, ubiquinone 8, phospholipids and fatty acids, yet differ significantly in their spectra, protein patterns and enzyme activities.Abbreviations BChl bacteriochlorophyll - UQ_s ubiquinone 8 - KDO 2-keto-3-deoxyoctonate - PG phosphatidyl glycerol - PE phosphatidyl ethanolamine - DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulfate - SDS sodium dodecyl sulfate     

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.     

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.     

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.     

Induction of the photosynthetic membranes of Rhodopseudomonas sphaeroides: biochemical and morphological studies

Cells of Rhodopseudomonas sphaeroides grown in a 25% O2 atmosphere were rapidly subjected to total anaerobiosis in the presence of light to study the progression of events associated with the de novo synthesis of the inducible intracytoplasmic membrane (ICM). This abrupt change in physiological conditions resulted in the immediate cessation of cell growth and whole cell protein, DNA, and phospholipid accumulation. Detectable cell growth and whole cell protein accumulation resumed ca. 12 h later. Bulk phospholipid accumulation paralleled cell growth, but the synthesis of individual phospholipid species during the adaptation period suggested the existence of a specific regulatory site in phospholipid synthesis at the level of the phosphatidylethanolamine methyltransferase system. Freeze-fracture electron microscopy showed that aerobic cells contain small indentations within the cell membrane that appear to be converted into discrete ICM invaginations within 1 h after the imposition of anaerobiosis. Microscopic examination also revealed a series of morphological changes in ICM structure and organization during the lag period before the initiation of photosynthetic growth. Bacteriochlorophyll synthesis and the formation of the two light-harvesting bacteriochlorophyll-protein complexes of R. sphaeroides (B800-850 and B875) occurred coordinately within 2 h after the shift to anaerobic conditions. Using antibodies prepared against various ICM-specific polypeptides, the synthesis of reaction center proteins and the polypeptides associated with the B800-850 complex was monitored. The reaction center H polypeptide was immunochemically detected at low levels in the cell membrane of aerobic cells, which contained no detectable ICM or bacteriochlorophyll. The results are discussed in terms of the oxygen-dependent regulation of gene expression in R. sphaeroides and the possible role of the reaction center H polypeptide and the cell membrane indentations in the site-specific assembly of ICM pigment-protein complexes during the de novo synthesis of the ICM.     

Streptococcus faecium mutants that are temperature sensitive for cell growth and show alterations in penicillin-binding proteins.

The penicillin-binding proteins (PBPs) of 209 cell division (or growth) temperature-sensitive mutants of Streptococcus faecium were analyzed in this study. A total of nine strains showed either constitutive or temperature-sensitive conditional damage in the PBPs. Analysis of these nine strains yielded the following results: one carried a PBP 1 constitutively showing a lower molecular weight; one constitutively lacked PBP 2; two lacked PBP 3 at 42 degrees C, but not at 30 degrees C; one was normal at 30 degrees C but at 42 degrees C lacked PBP 3 and overproduced PBP 5; two were normal at 42 degrees C and lacked PBP 5 at 30 degrees C; one constitutively lacked PBP 5; and one carried a PBP 6 constitutively split in two bands. The mutant lacking PBP 3 and overproducing PBP 5 continued to grow at 42 degrees C for 150 min and then lysed. Revertants selected for growth capability at 42 degrees C from the mutants altered in PBPs 5 and 6 maintained the same PBP alterations, while those isolated from the strains with altered PBP 1 or lacking PBP 2 or PBP 3 showed a normal PBP pattern. Penicillin-resistant derivatives were isolated at 30 degrees C from the mutants lacking PBP 2 and from that lacking PBP 3. All these derivatives continued to show the same PBP damage as the parents, but overproduced PBP 5 and grew at 42 degrees C. These findings indicate that high-molecular-weight, but not low-molecular-weight, PBPs are essential for cell growth in S. faecium. This is in complete agreement with previous findings obtained with a different experimental system. On the basis of both previous and present data it is suggested that PBPs 1, 2, and 3 appear necessary for cell growth at optimal temperature (and at maximal rate), but not for cell growth at a submaximal one (or at a reduced rate), and an overproduced PBP 5 is capable of taking over the function of PBPs 1, 2, and 3.     

Inhibition of bacterial DD-peptidases (penicillin-binding proteins) in membranes and in vivo by peptidoglycan-mimetic boronic acids

The DD-peptidases or penicillin-binding proteins (PBPs) catalyze the final steps of bacterial peptidoglycan biosynthesis and are inhibited by the β-lactam antibiotics. There is at present a question of whether the active site structure and activity of these enzymes is the same in the solubilized (truncated) DD-peptidase constructs employed in crystallographic and kinetics studies as in membrane-bound holoenzymes. Recent experiments with peptidoglycan-mimetic boronic acids have suggested that these transition state analogue-generating inhibitors may be able to induce reactive conformations of these enzymes and thus inhibit strongly. We have now, therefore, measured the dissociation constants of peptidoglycan-mimetic boronic acids from Escherichia coli and Bacillus subtilis PBPs in membrane preparations and, in the former case, in vivo, by means of competition experiments with the fluorescent penicillin Bocillin Fl. The experiments showed that the boronic acids bound measurably (K(i) < 1 mM) to the low-molecular mass PBPs but not to the high-molecular mass enzymes, both in membrane preparations and in whole cells. In two cases, E. coli PBP2 and PBP5, the dissociation constants obtained were very similar to those obtained with the pure enzymes in homogeneous solution. The boronic acids, therefore, are unable to induce tightly binding conformations of these enzymes in vivo. There is no evidence from these experiments that DD-peptidase inhibitors are more or less effective in vivo than in homogeneous solution.     

Investigations into the mechanisms used by the C-terminal anchors of Escherichia coli penicillin-binding proteins 4, 5, 6 and 6b for membrane interaction.

Escherichia coli low molecular mass penicillin-binding proteins (PBPs) include PBP4, PBP5, PBP6 and PBP6b. Evidence suggests that these proteins interact with the inner membrane via C-terminal amphiphilic alpha-helices. Nonetheless, the membrane interactive mechanisms utilized by the C-terminal anchors of PBP4 and PBP6b show differences to those utilized by PBP5 and PBP6. Here, hydrophobic moment-based analyses have predicted that, in contrast to the PBP4 and PBP6b C-termini, those of PBP5 and PBP6 are candidates to form oblique orientated alpha-helices. Consistent with these predictions, Fourier transform infrared spectroscopy (FTIR) has shown that peptide homologs of the PBP4 and PBP5 C-terminal regions, P4 and P5, respectively, both possessed the ability to adopt alpha-helical structure in the presence of lipid. However, whereas P4 appeared to show a preference for interaction with the surface regions of dimyristoylglycerophosphoethanolamine and dimyristoylglycerophosphoglycerol membranes, P5 appeared to show deep penetration of both these latter membranes and dimyristoylglycerophosphocholine membranes. Based on these results, we have suggested that in contrast to the membrane anchoring of the PBP4 and PBP6b C-terminal alpha-helices, the PBP5 and PBP6 C-terminal alpha-helices may possess hydrophobicity gradients and penetrate membranes in an oblique orientation.     

Immunochemical relationship of the major outer membrane protein of Rhodopseudomonas sphaeroides 2.4.1 to proteins of other photosynthetic bacteria.

Immunoblots of sodium dodecyl sulfate-polyacrylamide gels derived from outer membrane preparations of various strains of Rhodopseudomonas sphaeroides revealed polypeptides which cross-reacted with antibody directed against the major outer membrane protein of R. sphaeroides 2.4.1. Immunochemical quantitation of the major outer membrane protein of strain 2.4.1 showed approximately 5.5 x 10(4) molecules per cell whether cells were grown chemoheterotrophically or photoheterotrophically. Rhodospirillum rubrum outer membranes contained a cross-reactive protein, whereas the outer membranes derived from Rhodopseudomonas capsulata and Paracoccus denitrificans showed no cross-reaction with the antibody prepared against the major outer membrane protein from R. sphaeroides 2.4.1.     

Isolation and characterization of transposon Tn5 mutants of Rhodobacter sphaeroides deficient in both nitrate and chlorate reduction.

Abstract The wild-type strain Rhodobacter sphaeroides DSM 158 is a nitrate-reducing bacterium with a periplasmic nitrate reductase. Addition of chlorate to the culture medium causes a stimulation of the phototrophic growth, indicating that this strain is able to use chlorate as an ancillary oxidant. Several mutant strains of R. sphaeroides deficient in nitrate reductase activity were obtained by transposon Tn5 mutagenesis. Mutant strain NR45 exhibited high constitutive nitrate and chlorate reductase activities and phototrophic growth was also increased by the presence of chlorate. In contrast, the stimulation of growth by chlorate was not observed in mutant strains NR8 and NR13, in which transposon Tn5 insertion causes the simultaneous loss of both nitrate and chlorate reductase activities. Tn5 insertion probably does not affect molybdenum metabolism since NR8 and NR13 mutants exhibit both xanthine dehydrogenase and nitrogenase activities. These results that a single enzyme could reduce both nitrate and chlorate in R. sphaeroides DSM 158.     

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.     

A mecillinam-sensitive peptidoglycan crosslinking reaction in Escherichia coli

The amidinopenicillin, mecillinam, induces the formation of spherical cells of Escherichia coli by inactivation of penicillin-binding protein 2 (PBP2). A mecillinam-sensitive peptidoglycan crosslinking reaction has been demonstrated in particulate membrane preparations from this organism. The activity was detected in membranes that contained elevated levels of PBP2 and in which crosslinking reactions due to all other PBPs had been inactivated with the cephamycin antibiotic, cefmetazole. The particulate membrane preparation catalyzed synthesis of peptidoglycan that was up to 20% crosslinked from nucleotide precursors. Crosslinkage of the peptidoglycan was inhibited 50% by 0.2 μg mecillinam per ml but was not inhibited by much higher concentrations of cephamycins, which have very low affinity for PBP2. The crosslinking reaction appears to be due to the transpeptidase activity of PBP2, which is implicated in the mechanism of cell shape determination, and is the killing target for mecillinam.     

Construction, characterization, and complementation of Rhodospirillum rubrum puf region mutants.

Rhodospirillum rubrum is a facultatively phototrophic bacterium that, under certain growth conditions, forms an intracytoplasmic chromatophore membrane (ICM) housing the photochemical apparatus. The puf operon of R. rubrum encodes protein subunits of the photochemical reaction center and the B880 light-harvesting antenna complex. Mutant strains of R. rubrum were constructed by interposon mutagenesis through which a kanamycin resistance gene cartridge was inserted into restriction sites and in place of restriction fragments of the puf region. Southern blot analysis demonstrated that the defective copies of puf sequences had replaced their normal chromosomal counterparts through homologous recombination. The phenotypes of the mutant strains were evaluated on the basis of puf gene expression, spectral analysis, pigment content of membranes, and electron-microscopic examination of thin sections of cells grown under semi-aerobic and dark anaerobic conditions. Alterations of the puf region affect phototrophic competence and the formation of the ICM. The latter result implies an obligatory role for puf gene products in ICM formation in R. rubrum. One mutant with a deletion in puf structural genes was complemented in trans to the wild-type phenotype. Other mutants could be restored to the wild-type phenotype only by recombination.     

Insertion of Epicatechin Gallate into the Cytoplasmic Membrane of Methicillin-resistant Staphylococcus aureus Disrupts Penicillin-binding Protein (PBP) 2a-mediated ��-Lactam Resistance by Delocalizing PBP2

Epicatechin gallate (ECg) sensitizes methicillin-resistant Staphylococcus aureus (MRSA) to oxacillin and other β-lactam agents; it also reduces the secretion of virulence-associated proteins, prevents biofilm formation, and induces gross morphological changes in MRSA cells without compromising the growth rate. MRSA is resistant to oxacillin because of the presence of penicillin-binding protein 2a (PBP2a), which allows peptidoglycan synthesis to continue after oxacillin-mediated acylation of native PBPs. We show that ECg binds predominantly to the cytoplasmic membrane (CM), initially decreasing the fluidity of the bilayer, and induces changes in gene expression indicative of an attempt to preserve and repair a compromised cell wall. On further incubation, the CM is reorganized; the amount of lysylphosphatidylglycerol is markedly reduced, with a concomitant increase in phosphatidylglycerol, and the proportion of branched chain fatty acids increases, resulting in a more fluid structure. We found no evidence that ECg modulates the enzymatic activity of PBP2a through direct binding to the protein but determined that PBP2 is delocalized from the FtsZ-anchored cell wall biosynthetic machinery at the septal division site following intercalation into the CM. We argue that many features of the ECg-induced phenotype can be explained by changes in the fluid dynamics of the CM.