Identification of a carotenoid-binding protein in the cytoplasmic membrane from the heterotrophic cyanobacterium Synechocystis sp. strain PCC6714.

We isolated a carotenoid-binding protein from the cytoplasmic membrane of the cyanobacterium Synechocystis sp. strain PCC6714. The polypeptide demonstrated a characteristic mobility shift when electrophoresed in lithium dodecyl sulfate-polyacrylamide gels. The protein migrated with an apparent molecular mass of 35 kilodaltons when solubilized at 0 degrees C, but after solubilization at 70 degrees C, the protein migrated as a 45-kilodalton species. The carotenoid-binding protein accumulated only in autotrophically grown cells; cytoplasmic membranes prepared from photoheterotrophically grown cells lacked this component.     

Cell wall proteins of Candida albicans

Proteins were solubilized from cell wall fractions of Candida albicans and separated by polyacrylamide gel electrophoresis. Cell walls were isolated from 25 and 37 degrees C growing and stationary phase yeast cultures and from germ tubes. The 42 protein bands detected by dye binding were observed in all wall extracts, regardless of the temperature, growth state, or morphology of the culture. The carbohydrate content of most bands was below the detectable limit of the periodic acid Schiff reagent. The protein complement revealed by autoradiography of radiolabeled proteins was half that detected by staining. Two bands showed greater intensity from cultures grown at 37 degrees C. The radio-labeled pattern was similar with both [35S]methionine-and [14C]leucine-labeled proteins and either pulse- or continuous-labeled proteins.     

Extraction and properties of hemagglutinin from cell wall fragments of Fusobacterium nucleatum.

To study the hemagglutinin of Fusobacterium nucleatum, methods were sought to solubilize and purify this component. When cells of F. nucleatum were ruptured by passage through a French press, the fragments lost virtually all ability to agglutinate human erythrocytes. Extraction of the fragments with 2% Triton X-100 for 30 min at 22 degrees C restored hemagglutinating activity (HA). Hemagglutination by these fragments could be inhibited by arginine, as can hemagglutination by intact bacteria. Treatment of active cell wall fragments with pronase and 2% Triton X-100-EDTA at 37 degrees C or with pronase and 0.1% Triton X-100-EDTA at pH 10.0 allowed recovery of solubilized HA. The former HA was inhibited by arginine (arg+) whereas the latter was not (arg-). Fractionation of the arg+ extract by preparative isoelectric focusing showed that HA was recovered from the gel sections having a pH between 4.5 and 5.5. Hemagglutination by this preparation was still arg+. Chromatography of this hemagglutinin on DEAE-Sephadex increased the specific activity to high levels with a loss of inhibition by arginine. A fraction from the DEAE-Sephadex column containing 10,700 HA units per mg of protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Solubilization at 22 degrees C before electrophoresis revealed three Coomassie blue-staining bands which migrated with apparent molecular weights of about 21,000, 38,000 and 60,000. When the same DEAE fraction was boiled in sodium dodecyl sulfate, electrophoresis revealed only one band with an apparent molecular weight of 21,000.     

Molecular cloning of a cell wall exo-beta-1,3-glucanase from Saccharomyces cerevisiae.

A major protein of Saccharomyces cerevisiae cell walls is a 29-kilodalton glycoprotein which shows lectinlike binding to beta-1,3-glucan and chitin. It was solubilized by heating isolated cell walls at 90 degrees C and purified to homogeneity by running two high-pressure liquid chromatography columns. With the sequence information of the N terminus and seven peptides, two oligonucleotides were synthesized and the gene was cloned. Its sequence is similar to those of two plant beta-glucanases, and the protein was shown to possess beta-1,3-exoglucanase activity with laminarin as substrate. Haploid yeast cells contained one copy of the gene (BGL2). Gene disruption did not result in a phenotype.     

Characterization and reassembly of a regular array in the cell wall of Clostridium difficile GAI 4131

The cell wall of Clostridium difficile GAI 4131 was revealed by electron microscopy to have an outer layer composed of a nearly square array and contained the two major proteins with molecular weights of 38 kDa and 42 kDa. The properties and reassembly of the two major proteins into the regular array were investigated. When the isolated cell walls were treated with hydrophobic bond-disrupting agents or a chelating agent specific for Ca2+, the two major proteins were effectively removed and the regularly arranged outer layer disappeared. The amino acid composition of the two major proteins differed from each other. The two major proteins also gave different peptide maps from each other upon proteolysis with Staphylococcus aureus V8 protease. The major proteins solubilized from the isolated cell walls with 8 M urea or 4 M guanidine hydrochloride could be reassembled into open-ended cylinders possessing the native regular pattern by dialysis against neutral buffer containing 5 mM CaCl2. The reassembled cylinders purified by centrifugation on a Percoll density gradient were composed of almost equal amounts of the 38 kDa and 42 kDa proteins and freed from the other proteins. These results suggest that the regular array in the outer cell wall layer is constructed from the two major cell wall proteins and requires Ca2+ for its assembly.     

The Chlamydomonas cell wall: characterization of the wall framework

The cell wall of the biflagellate alga Chlamydomonas reinhardtii is a multilayered, extracellular matrix composed of carbohydrates and 20-25 polypeptides. To learn more about the forces responsible for the integrity of this cellulose-deficient cell wall, we have begun studies to identify and characterize the framework of the wall and to determine the effects of the cell wall-degrading enzyme, lysin, on framework structure and protein composition. In these studies we used walls released into the medium by mating gametes. When isolated shed walls are degraded by exogenously added lysin, no changes are detected in the charge or molecular weight of the 20-25 wall proteins and glycoproteins when analyzed on one- and two-dimensional polyacrylamide gels, which suggests that degradation of these shed walls is due either to cleavage of peptide bonds very near the ends of polypeptides or that degradation occurs via a mechanism other than proteolysis. Incubation of walls with Sarkosyl-urea solutions removes most of the proteins and yields thin structures that appear to be the frameworks of the walls. Analysis by polyacrylamide gel electrophoresis shows that the frameworks are highly enriched in a polypeptide of Mr 100,000. Treatment of frameworks with lysin leads to their degradation, which indicates that this part of the wall is a substrate for the enzyme. Although lysin converts the Mr 100,000 polypeptide from an insoluble to a soluble form, there is no detectable change in Mr of the framework protein. Solubilization in the absence of lysin requires treatment with SDS and dithiothreitol at 100 degrees C. These results suggest that the Chlamydomonas cell wall is composed of two separate domains: one containing approximately 20 proteins held together by noncovalent interactions and a second domain, containing only a few proteins, which constitutes the framework of the wall. The result that shed walls can be solubilized by boiling in SDS-dithiothreitol indicates that disulfide linkages are critical for wall integrity. Using an alternative method for isolating walls from mechanically disrupted gametes, we have also shown that a wall-shaped portion of these unshed walls is insoluble under the same conditions in which shed walls are soluble. One interpretation of these results is that wall release during mating and the wall degradation that follows may involve distinct biochemical events.     

Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity.

A beta-glucanase (Z-glucanase) from Zymolyase was freed from a protease (Z-protease) by affinity chromatography on alpha 2-macroglobulin-Sepharose columns and used to solubilize proteins from isolated cell walls of Saccharomyces cerevisiae. The cell wall proteins were labeled with 125I and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The bulk of the labeled material had very low mobility. Its mannoprotein nature was demonstrated by precipitation with specific antibodies and by conversion to a band with an average molecular weight of 94,000 after incubation with endo-beta-N-acetylglucosaminidase. The intact mannoproteins were hydrolyzed by Z-protease, but were resistant to the enzyme when the carbohydrate was first removed by endo-beta-N-acetylglucosaminidase. In intact cells, lysis of cell walls by Z-glucanase required a previous incubation with z-protease, which led to solubilization of most of the 125I-labeled proteins. Other proteases that did not attack the cell wall mannoproteins were unable to substitute for Z-protease. The specific effect of Z-protease is consistent with the notion that mannoproteins form a surface layer of the cell wall that penetrates the wall to some depth and shields glucans from attack by Z-glucanase. Mannoproteins, however, do not appear to cover the inner face of the cell wall, because isolated cell walls, in contrast to intact cells, were completely solubilized by Z-glucanase in the absence of protease. The function of mannoproteins in determining cell wall porosity was highlighted by the finding that horseradish peroxidase (Mr, 40,000) causes lysis of cells that had been treated with Z-protease. Depletion of mannoproteins by Z-protease also resulted in the disappearance of a darkly stained surface layer of the cell wall, as observed by electron microscopy. Other agents that facilitate cell lysis by Z-glucanase, such as 2-mercaptoethanol, digitonin, and high concentrations of salts, caused little or no solubilization of mannoprotein. We assume that they perturb and loosen the structure of the mannoprotein network, thereby increasing its porosity. The implications of our results for the construction of the yeast cell wall and the anchoring of mannoprotein to the cell are discussed.     

Modifiable chromatophore proteins in photosynthetic bacteria.

The chromatophores of Chromatium vinosum, as well as six other photosynthetic bacteria, contained two or more proteins which were insoluble when heated in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (beta-ME). When the chromatophores were dissolved at room temperature in SDS-beta-ME, these proteins were present in the SDS-polyacrylamide gel electrophoresis profiles, but when the samples were dissolved at 100 degrees C, they were absent or considerably diminished. When one-dimensional gels of chromatophores solubilized at room temperature were soaked in the SDS-beta-ME solution and heated to 100 degrees C and the gels were run in a second dimension, the proteins became immobilized in the original first-dimension gel, where they could be detected by staining. The two major proteins so affected in C. vinosum had apparent molecular weights of 28,000 and 21,000. The chromatophores of several other photosynthetic bacteria also contained predominant proteins between 30,000 and 19,000 molecular weight, which became insoluble when heated in the presence of SDS and beta-ME. In at least two of the species examined, these appeared to be reaction center proteins. The conditions causing the proteins to become insoluble were complex and involved temperature, SDS concentration, and the presence of sulfhydryl reagents. The chromatophores of four of the Chromatiaceae species and two strains of one of the Rhodospirillaceae species examined had a protein-pigment complex that was visible in SDS-polyacrylamide gel profiles of samples dissolved at room temperature but was absent in samples dissolved at 100 degrees C.     

Cell wall composition of the aquatic fungusBlastocladiella emersonii

Cell walls fromBlastocladiella emersonii were isolated by repeated washing and centrifugation. Purity and uniformity of cell wall preparations were assessed by light and electron microscopy and chemical reproducibility. Electron microscopy showed the cell walls to consist of an inner microfibrillar network and an outer amorphous layer. Analyses by X-ray and infrared spectroscopy were consistent with chitin as the major wall component. Gross chemical analysis indicated that the cell walls were composed of 74.7% amino sugar (as anhydroN-acetylhexosamine), 10.7% neutral sugar (as anhydro hexose), 10.6% protein, and 4.2% lipid. Analysis of the neutral sugars showed that isolated cell walls contain 1.5% mannose, 3.0% galactose, and 3.0% glucose. Isolated cell walls were fractionated using a hot sodium dodecyl sulfate (SDS) extraction followed by either Pronase digestion or hot KOH extraction. The hot SDS extract was found to contain two polymer types, galactose- and/or glucose-containing polymers and glycoprotein. However, the residue from the hot SDS extraction still contained most of the neutral sugars and protein present in the isolated walls. Both Pronase digestion and the hot potassium hydroxide extraction removed all of the neutral sugars except glucose. The cell wall fractionation results indicate that the major wall component is microfibrillar chitin. The results further suggest that the SDS-solubilized glycoproteins and neutral sugar polymers may represent an outer amorphous layer.     

Antigenic cell wall mannoproteins in Candida albicans isolates and in other Candida species.

Polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs), raised against mannoprotein components from Candida albicans ATCC 26555 (serotype A) blastoconidia and mycelial cell walls, were used to investigate antigenic similarities among wall mannoproteins from other C. albicans serotype A and B strains, and from C. tropicalis and C. guilliermondii. Radioactively labelled walls isolated from cells grown at either 28 degrees C or 37 degrees C were digested with a beta-glucanase complex (Zymolyase 20T) to release cell-wall-bound mannoproteins. Numerous molecular species with different electrophoretic mobilities were released from the various isolates. Differences appeared to be related to both the organism and the growth temperature. Among the major protein components solubilized were mannoproteins larger than 100 kDa (high molecular mass mannoproteins), heterogeneous in size in most cases. Antigenic homology was detected among the cell wall high molecular mass mannoproteins of the two C. albicans serotype A isolates, whereas significant qualitative and quantitative differences were detected between serotype A and serotype B cell-wall-bound antigenic profiles. Moreover, C. tropicalis and C. guilliermondii wall antigenic determinants were not recognized by the preparations of pAbs and mAbs raised against C. albicans walls. A mannoprotein with a molecular mass of 33-34 kDa was present in the enzymic wall digests of all the organisms studied. When probed with pAbs raised against the protein moiety of the 33 kDa cell wall mannoprotein of Saccharomyces cerevisiae, antigenic cross-reactivity was observed in all cases except C. tropicalis. There appear to be significant antigenic differences between the mannoproteins of different isolates of C. albicans, and between those of C. albicans and other Candida species.     

Reconstitution of model membranes from phospholipid and outer membrane proteins of Proteus mirabilis. Role of proteins in the formation of hydrophilic pores and protection of membranes against detergents.

Outer membrane proteins extracted from isolated cell walls of Proteus mirabilis were able to combine the cell wall phospholipids in a model membrane system. The presence of outer membrane proteins in vesicular model membranes mediated the release of previously entrapped [14C]sucrose while [3H]inulin was retained. Incorporation of lipopolysaccharide from the same cell walls was not required for the formation of such selectively permeable membranes. Three major outer membrane proteins of apparent molecular weights 39000, 36000 and 17000 were isolated using acetic acid and sodium deoxycholate solution as solvents and avoiding the strongly denaturing sodium dodecyl sulfate. The isolated proteins were assayed for their ability to form hydrophilic pores in reconstituted membranes. The trypsin-sensitive 39000-Mr protein and the peptidoglycan-associated 36000-Mr protein were equally effective in this function whereas the 17000-Mr protein mediated little penetration of low molecular weight solute. The 39000-Mr and 36000-Mr proteins also protected reconstituted membrane vesicles from disruption by detergent while 17000-Mr protein was ineffective in this regard.     

Subunit composition of goblet-shaped particles from the cell wall of Flexibacter polymorphus

Submicroscopic goblet-shaped particles ( goblets ) were released from the cell envelope of the marine gliding bacterium Flexibacter polymorphus when treated with the nonionic detergent Triton X-100 followed by sonication. The goblets were purified by cesium chloride density gradient centrifugation and exhibited an equilibrium buoyant density of 1.30 g/mL at 23 degrees C. They were composed of protein and a small amount of carbohydrate (approximately 3.4% by weight). Aqueous suspensions exhibited an absorption maximum in the ultraviolet at a wavelength of 276 nm and a smaller shoulder at 281 nm. Phospholipids were not detected in purified preparations of goblets , though they are known to be prominent constituents of the intact membranes of this microbe. Polyacrylamide gel electrophoresis of goblets solubilized in sodium dodecyl sulfate and 2-mercaptoethanol indicated four major polypeptide components ranging in molecular weight from 13 000 to 80 000. This number of different protein subunits corroborates earlier ultrastructural observations indicating a multisubunit composition.     

Immunochemical characterization of cell wall protein antigen purified from the cell wall autolysate of Clostridium botulinum type A.

The cell wall protein antigen was solubilized from the isolated cell walls of Clostridium botulinum type A by autolysis and purified by diethylaminoethyl-cellulose column chromatography followed by gel filtration on Sephadex G-150. The two fractions showed a high degree of the serological activity and produced a main fused precipitin line in immunodiffusion tests against the homologous antiserum. The fact that antigenic fractions contained various kinds of amino acids but no detectable amounts of amino sugars or carbohydrates suggests that the antigens were principally composed of proteins. The protein antigen possessed multiple antigenic components in immunoelectrophoresis. As serological activity, the antigen was heat-stable and resistant to tryptic digestion but sensitive to the actions of pronase, nagarse or pepsin. The protein antigen appeared to be responsible for the common antigenicity among the proteolytic strains of C. botulinum.     

Isolation of inclusion bodies from vegetative Clostridium perfringens: partial purification of a 47 kDa inclusion protein.

A refractile inclusion body produced by vegetative cells of Clostridium perfringens at temperatures above 40 degrees C was isolated and partially characterized. The inclusion was composed of protein and could be solubilized by sodium dodecyl sulphate plus either dithiothreitol or beta-mercaptoethanol. The solubilized inclusion showed no antigenic relationship with Cl. perfringens enterotoxin. One major band with an apparent MW of 47 kDa was demonstrated after polyacrylamide gel electrophoresis of the solubilized inclusion. Both enterotoxin-positive and enterotoxin-negative strains produced the inclusion body. No effect on the morphology of several eucaryotic cell lines was observed when solubilized or intact inclusion was added to the cell cultures.     

Incorporation of specific wall proteins during yeast and mycelial protoplast regeneration in Candida albicans

The kinectics of incorporation of two precursor mannoproteins into the regenerating cell wall of Candida albicans protoplasts have been followed at 28°C and 37°C using two monoclonal antibodies specific for protein epitopes (MAb 1B12 and 4C12) as probes. Both molecules were secreted from the beginning of the regeneration process, and their incorporation was retarded significantly. Analysis of the secreted materials by Western immunoblotting with MAb 1B12 allowed the identification of two closely migrating bands at apparent Mr higher than 170 kDa and significant amounts of a highly polydisperse material of even greater molecular mass. Some of these mannoproteinaceous species carried both N- and O-glycosidically linked mannose residues, as deduced from their drop in apparent Mr when synthesized in the presence of tunicamycin and by their reactivity with Concanavalin A. Following secretion, the molecules reacting with MAb 1B12 were incorporated into the regenerating walls by covalent binding. Then, when the regenerating walls by covalent binding. Then, when the antigen molecules were solubilized from partially regenerated walls, their mobility differed when regeneration took place at 28°C (blastoconidia) or 37°C (mycelial cells).Abbreviations used IIF indirect immunofluorescence - MAb monoclonal antibody - PBS phosphate-buffed saline - PMSF phenyl methyl sulfonyl fluoride - SDS sodium dodecyl sulfate     

Partial characterization of cell wall from a flocculent strain ofKluyveromyces marxianus

Summary Cell walls from aKluyveromyces marxianus either non flocculent or flocculent strain were isolated and analysed for protein, carbohydrates and phosphate content. Alkaline extract of proteins were analysed by SDS-PAGE. The results revealed a higher protein content in the cell walls from the flocculent strain. Electrophoresis of the cell wall proteins of the flocculent strain showed an extra peptide with an apparent molecular weight of 37 KDa which is absent fom non-flocculent cells. The involvement of this protein in cell adhesion during flocculation is discussed.     

Protein-bound choline is released from the pneumococcal autolytic enzyme during adsorption of the enzyme to cell wall particles.

The inactive precursor form of the pneumococcal autolytic enzyme cloned in Escherichia coli was isolated by affinity chromatography on Sepharose-linked choline. The enzyme was recovered in an electrophoretically pure and activated form by elution from the affinity column with radioactive choline solution. When radioactive choline was used for elutions, the enzyme protein isolated contained protein-bound choline, at approximately 1 mol of choline per mol of enzyme protein, indicating the presence of a single choline recognition site. Radioactive choline remained bound to the enzyme protein during dialysis, precipitation by trichloroacetic acid or ammonium sulfate, and during gel filtration, but not during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation of the choline-labeled autolysin with pneumococcal cell walls at 0 degrees C resulted in the adsorption of the enzyme to the wall particles and a simultaneous release of free choline from the enzyme protein. It is suggested that the choline molecules that became bound to the enzyme protein during the activation of autolysin are expelled from the choline-binding site and replaced by choline residues from the wall teichoic acid as the autolysin molecules adsorb to their insoluble substrate before the onset of enzymatic wall hydrolysis.     

Demonstration and preliminary characterization of a regular array in the cell wall of Clostridium difficile

Abstract The presence of a regular array (RA) was demonstrated on the outer layer of the cell wall in Clostridium difficile GAI0714 by electron microscopy. The RA was composed of squarely arranged subunits with a center-to-center spacing of about 8.2 nm. The outer wall layer carrying the RA was isolated from the wall fragments of early log-phase cells by autolysis. The outer wall layer was composed of two main proteins with apparent M _rs of about 45 000 and 32 000 upon sodiumdodecylsul-fate-polyacrylamide gel electrophoresis (SDS-PAGE). Similar RAs were also present in the cell walls of the other 9 strains of C. difficile . These strains were divided into two groups on the basis of the wall protein composition: one containing M _r 45 000–47 000 and 32 000 proteins and the other containing M _r 42 000 and 38 000 proteins.     

Characterization of the cell wall and outer membrane of Rhodopseudomonas capsulata.

Sucrose density gradient centrifugation of cell envelopes of chemotrophically grown cells of Rhodopseudomonas capsulata St. Louis (= ATCC 23782) resulted in the separation of a cytoplasmic membrane from a cell wall fraction (buoyant densities, 1.139 and 1.215 g/cm3, respectively). The cell wall fractions (untreated or Triton extracted) contained peptidoglycan- and lipopolysaccharide-specific components. Their neutral sugar content, mainly rhamnose and galactose, was high (250 and 100 micrograms/mg [dry weight] of material) due to a non-lipopolysaccharide polymer. The fatty acid content was low (less than or equal to 60 micrograms/mg [dry weight] of material), and half of it was contributed by lipopolysaccharide (3-OH-C10:0, C12:1, and 3-oxo-C14:0). The predominant other fatty acid was C18:1. An outer membrane fraction, obtained by lysozyme treatment of the Triton-extracted cell wall, showed essentially the same chemical composition except for almost complete removal of peptidoglycan. Saline extraction (0.9% NaCl, 37 degrees C, 2 h) removed a lipopolysaccharide-protein(-phospholipid?) complex from whole cells of R. capsulata St. Louis. The polypeptide patterns of the cell wall and outer membrane as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis comprised 20 to 25 different polypeptides (most of them very faint) and were dominated by a single, heat-modifiable major protein (Mr 69,000 after solubilization below 60 degrees C; Mr 33,000 at temperatures above 70 degrees C).     

An oat coleoptile wall protein that induces wall extension in vitro and that is antigenically related to a similar protein from cucumber hypocotyls

Plant cell walls expand considerably during cell enlargement, but the biochemical reactions leading to wall expansion are unknown. McQueen-Mason et al. (1992, Plant Cell 4, 1425) recently identified two proteins from cucumber (Cucumis sativus L.) that induced extension in walls isolated from dicotyledons, but were relatively ineffective on grass coleoptile walls. Here we report the identification and partial characterization of an oat (Avena sativa L.) coleoptile wall protein with similar properties. The oat protein has an apparent molecular mass of 29 kDa as revealed by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis. Activity was optimal between pH 4.5 and 5.0, which makes it a suitable candidate for acid growth responses of plant cell walls. The oat protein induced extension in walls from oat coleoptiles, cucumber hypocotyls and pea (Pisum sativum L.) epicotyls and was specifically recognized by an antibody raised against the 29-kDa wall-extension-inducing protein from cucumber hypocotyls. Contrary to the situation in cucumber walls, the acid-extension response in heat-inactivated oat walls was only partially restored by oat or cucumber wall-extension proteins. Our results show that an antigenically conserved protein in the walls of cucumber and oat seedlings is able to mediate a form of acid-induced wall extension. This implies that dicotyledons and grasses share a common biochemical mechanism for at least part of acid-induced wall extensions, despite the significant differences in wall composition between these two classes of plants.Abbreviations ConA concanavalin A - CM carboxymethyl - DEAE diethylaminoethyl - DTT dithiothreitol - Ex29 29-kDa expansin