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.     

Bacteriophage SPO1 structure and morphogenesis. II. Head structure and DNA size.

The capsid of bacteriophage SPO1 is icosahedral, and the subunit arrangement on the 87-nm-diameter head suggests the triangulation number T = 16. The major capsid protein (45,700 daltons) is cleaved from a 47,700-dalton precursor. Tubular heads (polyheads) are produced by mutations in genes 5 and 8 and contain cores as well as capped ends. The lattice constant of these structures is 13.4 nm; diameter is 109.5 nm. The size of the double-stranded SPO1 DNA (containing 5' hydroxymethyl uracil in place of thymine) was measured by sedimentation analysis and electron microscopy and has a molecular weight of 86 X 10(6) (about 140 kilobase pairs), which is smaller than several previously reported values.     

Regular surface layer of Azotobacter vinelandii.

Washing Azotobacter vinelandii UW1 with Burk buffer or heating cells at 42 degrees C exposed a regular surface layer which was effectively visualized by freeze-etch electron microscopy. This layer was composed of tetragonally arranged subunits separated by a center-to-center spacing of approximately 10 nm. Cells washed with distilled water to remove an acidic major outer membrane protein with a molecular weight of 65,000 did not possess the regular surface layer. This protein, designated the S protein, specifically reattached to the surface of distilled-water-washed cells in the presence of the divalent calcium, magnesium, strontium, or beryllium cations. All of these cations except beryllium supported reassembly of the S protein into a regular tetragonal array. Although the surface localization of the S protein has been demonstrated, radioiodination of exposed envelope proteins in whole cells did not confirm this. The labeling behavior of the S protein could be explained on the basis of varying accessibilities of different tyrosine residues to iodination.     

The S-layer of Caulobacter crescentus: three-dimensional image reconstruction and structure analysis by electron microscopy.

The regular surface protein structure (S-layer) of Caulobacter crescentus was analyzed by electron microscopy and three-dimensional image reconstruction to a resolution of 2 nm. Projections showed that the S-layer is an array of ring structures, each composed of six subunits that are arranged on a lattice with p6 symmetry. Three-dimensional reconstructions showed that the ring subunits were approximately rod-shaped structures and were perpendicular to the plane of the array, with a linker arm emanating from approximately the middle of the rod, accounting for the connections between the rings. The calculated subunit mass was ca. 100 kDa, very close to the size of RsaA (the protein known to be at least the predominant species in the S-layer) predicted from the DNA sequence of the rsaA gene. The core region of the rings creates an open pore 2.5 to 3.5 nm in diameter. The size of the gaps between the neighboring unit cells is in the same range, suggesting a uniform porosity predicted to exclude molecules larger than ca. 17 kDa. Attempts to remove membrane material from S-layer preparations with detergents revealed that the structure spontaneously rearranged into a mirror-image double layer. Negative-stain and thin-section electron microscopy examination of colonies of C. crescentus strains with a mutation in a surface molecule involved in the attachment of the S-layer showed that shed RsaA protein organized into large sheets. The sheets in turn organized into stacks that tended to accumulate near the upper surface of the colony. Image reconstruction indicated that these sheets were also precise mirror-image double layers, and thickness measurements obtained from thin sections were consistent with this finding. The sheets were absent when these mutant strains were grown without calcium, supporting other data that calcium is involved in attachment of the S-layer to a surface molecule and perhaps in subunit-subunit interactions. We propose that when the membrane is removed from S-layer fragments by detergents or the attachment-related surface molecule is absent, the attachment sites of the S-layer align precisely to form a double layer via a calcium interaction.     

Chloroplast membranes of the green alga Acetabularia mediterranea. II. Topography of the chloroplast membrane

The localization of the chlorophyll-protein complexes inside the thylakoid membrane of Acetabularia mediterranea was determined by fractionating the chloroplast membrane with EDTA and Triton X-100, by using pronase treatment, and by labeling the surface-exposed proteins with 125I. The effects of the various treatments were established by electrophoresis of the solubilized membrane fractions and electron microscopy. After EDTA and pronase treatment, the membrane structure was still intact. Only the two chlorophyll-protein complexes of 67,000 and 152,000 daltons and an additional polypeptides were found in the membrane before the EDTA and pronase treatment. The 125,000 dalton complex seems to be buried inside the lipid layer. The 23,000 dalton subunit of the 67,000 dalton complex is largely exposed to the surface of the EDTA-insoluble membrane and only the chlorophyll-binding subunit of 21,500 daltons is buried inside the lipid layer.     

S-layer-coated liposomes as a versatile system for entrapping and binding target molecules

In the present study, unilamellar liposomes coated with the crystalline bacterial cell surface layer (S-layer) protein of Bacillus stearothermophilus PV72/p2 were used as matrix for defined binding of functional molecules via the avidin- or streptavidin-biotin bridge. The liposomes were composed of dipalmitoyl phosphatidylcholine, cholesterol and hexadecylamine in a molar ratio of 10:5:4 and they had an average size of 180 nm. For introducing specific functions into the S-layer lattice without affecting substances encapsulated within the liposomes, crosslinking and activation reagents had to be identified which did not penetrate the liposomal membrane. Among different reagents, a hydrophilic dialdehyde generated by periodate cleavage of raffinose and a sulfo-succinimide activated dicarboxylic acid were found to be impermeable for the liposomal membrane. Both reagents completely crosslinked the S-layer lattice without interfering with its regular structure. Biotinylation of S-layer-coated liposomes was achieved by coupling p-diazobenzoyl biocytin which preferably reacts with the phenolic residue of tyrosine or with the imidazole ring of histidine. By applying this method, two biotin residues accessible for subsequent avidin binding were introduced per S-layer subunit. As visualized by labeling with biotinylated ferritin, an ordered monomolecular layer of streptavidin was formed on the surface of the S-layer-coated liposomes. As a second model system, biotinylated anti-human IgG was attached via the streptavidin bridge to the biotinylated S-layer-coated liposomes. The biological activity of the bound anti-human IgG was confirmed by ELISA.     

Structure of Colletochlorin D from Colletotrichum nicotianae

Complete isolation of Fraction 1 protein from alfalfa leaves was achieved by a combination of ammonium sulfate fractionation and gel filtration. Analytical ultracentrifugation gave a S₂₀°,w value of 18.0. Judging from the CD spectrum the protein contains a large amount of β-form as well as tobacco F-l protein. Electron micrographs showed closely similar appearances for the two F-l proteins. The F-l protein (from alfalfa leaves) was separated to large subunits (53,000 daltons) and small subunits (14,000 daltons) on SDS gel electrophoresis. Further the amino acid composition of the large subunit was found similar to those of tobacco and spinach, but considerably different from them in small subunits.     

Isolation and characterization of gas vesicles from Microcyclus aquaticus

Intact gas vesicles of Microcyclus aquaticus S1 were isolated by using centrifugally accelerated flotation of vesicles and molecular sieve chromatography. Isolated gas vesicles were cylindrical organelles with biconical ends and measured 250×100 nm. The gas vesicle membrane was composed almost entirely of protein; neither lipid nor carbohydrate was detected, although one mole of phosphate per mole of protein was found. Amino acid analysis indicated that the protein contained 54.6% hydrophobic amino acid residues, lacked sulfur-containing amino acids, and had a low aromatic amino acid content. The protein subunit composition of the vesicles was determined by gel electrophoresis in (i) 0.1% sodium dodecyl sulfate at pH 9.0 and (ii) 5 M urea at pH 2.0. The membrane appeared to consist of one protein subunit of MW 50 000 daltons. Charge isomers of this subunit were not detected on urea gels. Antiserum prepared against purified gas vesicles of M. aquaticus S1 cross-reacted with the gas vesicles of all other gas vacuolate strains of M. aquaticus, as well as those of Prosthecomicrobium pneumaticum, Nostoc muscorum, and Anabaena flos-aquae, indicating that the gas vesicles of these widely divergent organisms have some antigenic determinants in common.Abbreviations SDS sodium dodecyl sulfate - MW molecular weight - Tris tris(hydroxymethyl)aminomethane - EDTA disodium ethylenediaminetetraacetic acid - BSA bovine serum albumin - TCA trichloroacetic acid - P _c pressure necessary to collapse gas vesicles     

Analysis of the subunit structure of protochlorophyllide holochrome by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

The subunit structures of protochlorophyllide holochrome (PCH) and chlorophyllide holochrome (CH) were studied by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. PCH from leaves of dark-grown (Phaseolus vulgaris var. red kidney) is a polymeric pigment-protein complex of approximately 600,000 daltons. It is composed of 12 to 14 polypeptides of 45,000 daltons, when examined prior to and immediately following photoconversion. The protochlorophyllide or chlorophyllide pigment molecules are associated with these polypeptides. Subsequent to photoconversion, the absorption maximum of newly formed chlorophyllide shifts from 678 nm to 674 nm upon standing in darkness. Following the 678 to 674 spectral shift, the chlorophyllide is associated with a polypeptide with a molecular weight of 16,000 daltons. In addition, sucrose gradient centrifugation of PCH and CH under nondenaturing conditions indicates that during the course of the dark spectroscopic shift, the 600,000 dalton CH undergoes dissociation into a small chlorophyllide protein. The dissociation of CH, the change in the molecular weight of the chlorophyllide polypeptide from 45,000 to 16,000 daltons, as well as the dark spectroscopic shift are temperature-dependent and blocked below 0 C. It was also found that each holochrome molecule of 600,000 daltons contains at least four protochlorophyllide pigment molecules.     

Changes in the Composition and Size Distribution of Soymilk Protein Particles by Heating

The protein particles in soymilk were fractionated in size by differential centrifugation. Particles of more than 100 nm in diameter (LSP) constituted 40% of the total protein in raw soymilk, 70% of the protein components being 11S globulin. LSP was not formed in the presence of 2-mercaptoethanol and sodium ascorbate. LSP was decreased by heating, and particles of 100–40 nm in diameter (MSP) were increased. The formation of MSP was not due to any degradation of LSP but to the combination of supernatant proteins of less than 40 nm in diameter with each other. MSP formed by heating contained the β subunit of 7S and the basic subunit of 11S as main components. The particles of more than 40 nm in diameter (LSP + MSP) constituted 50% of the total protein in both raw soymilk and soymilk.     

Novel structural patterns in divalent cation-depleted surface layers of Aeromonas salmonicida

The fish pathogen Aeromonas salmonicida possesses a regular surface layer (or A-layer) which is an important virulence determinant. The A-protein, a single bilobed protein organized in a p4 lattice of M₄C₄ arrangement with two morphological domains, comprises this layer. The role of divalent cations in the A-layer structure was studied to better understand A-protein subunit interactions affecting structural flexibility and function. Divalent cation bridges were found to be involved in the integrity of the A-layer. Two novel A-layer patterns were formed as the result of growth under calcium limitation or by chelation of divalent cations with EDTA or EGTA, thereby constituting the first reported case of formation of distinct regular arrays upon divalent cation depletion. Furthermore, under these conditions A-protein was sometimes released as tetrameric units, rather than in monomeric form. The formation of the two novel patterns is best explained by a sequence of structural rearrangements, following disruption of only one of the two A-layer morphological units, that is, those held together by divalent cation bridges. The free tetrameric units represent four A-protein subunits clustered around the unaffected four-fold axis.     

Molecular and Enzymatic Properties of Pyridoxamine (Pyridoxine) 5′-Phosphate Oxidase from Baker’s Yeast

Pyridoxamine (pyridoxine) 5′-phosphate oxidase purified from baker’s yeast was found to have a molecular weight of ca, 55,000 daltons based on polyacrylamide gel electrophoresis. The size of the enzyme subunit was analyzed by gel electrophoresis in the presence of sodium dodecylsulfate. This showed that the enzyme was composed of two nonidentical subunits with a molecular weight of 27,000 and 25,000 daltons. Fluorescence titration of the apoenzyme with FMN suggested that the holoenzyme contained one mol of FMN per mol of the enzyme. The K_m value of FMN for apoenzyme was calculated to be ca. 16 nm on both activities of pyridoxamine 5′-phosphate oxidase and pyridoxine 5′-phosphate oxidase.     

Ultrastructural Architecture and Organization of the Spore Envelope during Development in Thelohania bracteata (Strickland, 1913) after Freeze-Etching*

An ultrastructural study was made of the spore envelope during development in the microsporidan, Thelohania bracteata. The frozen-etched outer (convex) face of the relatively thin spore coat in the earliest immature stage of development has a granular structure in regular array. The inner (concave) face bears particles as well as depressions arranged in a net-like pattern. The mature spore coat has a substructure of numerous microfibers, ～8 nm in diameter, arranged in a matrix and forming thin layers which run parallel to the spore surface. The mature spore coat possesses both outer and inner limiting layers. The outer (convex) face of the outer limiting layer is granular. The convex face of inner limiting layer bears many particles as well as many long, narrow depressions. The concave face of the inner limiting layer carries many stud-like projections, ～40 nm long and 30 nm high, which are complementary to the depressions observed on the convex face. In addition, the concave face has subunits ～15 nm in diameter, apparently arranged in a hexagonal pattern with a center to center distance of ～18 nm. The change in size of these projections, depressions, and subunits presumably is related to spore maturation.     

Purification and immunological characterization of superoxide dismutase of the onion maggot,Delia antiqua

Cytosolic superoxide dismutase (SOD) of the onion maggot, Delia antiqua, was purified to apparent homogeneity by ammonium sulfate fractionation followed by anion exchange, hydrophobic interaction, and gel filtration chromatographies. Native molecular mass was estimated as 32,000 daltons. SDS-PAGE revealed only one subunit of 16,000 daltons, indicating that SOD is a homodimer. Isoelectric focusing revealed 3 charge isomers of pls 5.3, 5.5, and 5.7. The specific activity of purified SOD was 4,250 U/mg protein. A monoclonal antibody (MAb, aSOD2B7) raised against Delia SOD recognized only SOD of the same genus, but another MAb (aSOD1H11) recognized SOD of Drosophila melanogaster as well. © 1995 Wiley-Liss, Inc.     

Characterization of a soybean leaf protein that is related to the seed lectin and is increased with pod removal

Levels of several polypeptides in addition to the vegetative storage protein (VSP) increase in soybean leaves following depodding. Two of these polypeptides interact specifically with antibodies raised against the seed lectins of Phaseolus vulgaris and soybean. The two polypeptides, which had apparent molecular masses of 29,000 daltons and 33,000 daltons, were present in the sink-deprived plants but not in control podded plants and were the subunit polypeptides of a glycoprotein designated lectin-related protein (LRP). Soybean LRP was purified to near homogeneity by a combination of ammonium sulfate precipitation and gel filtration. Dialysis of the resuspended ammonium sulfate precipitate caused LRP to reprecipitate, and LRP was soluble only in the presence of molar NaCl. The native relative molecular mass of LRP was 119,000 daltons, a size consistent with a tetrameric organization of the two polypeptides. LRP precipitated during dialysis in association with a 28,000 dalton polypeptide. The protein coprecipitating with LRP was identified as the dimer of the 28,000 dalton subunit of VSP, one of three native isomeric forms of VSP occurring in leaves of depodded plants. Although the specific association between LRP and VSP was intriguing, an in vivo interaction between LRP and VSP was doubtful. LRP was shown to be immunologically similar to soybean agglutinin but did not have detectable hemagglutinating activity. LRP also was shown to be made up of polypeptides distinct from soybean agglutinin.     

Phosphorylation of yeast DNA-dependent RNA polymerases in vivo and in vitro. Isolation of enzymes and identification of phosphorylated subunits.

Yeast DNA-dependent RNA polymerases I, II, and III are phosphorylated in vivo. Yeast cells were grown continuously in 32Pi and the RNA polymerases were isolated by a new procedure which allows the simultaneous purification of these enzymes from small quantities (35 to 60 g) of cells. Each of the RNA polymerases was phosphorylated. The following phosphorylated polymerase polypeptides were identified: polymerase I subunits of 185,000, 44,000, 36,000, 24,000, and 20,000 daltons; a polymerase II subunit of 24,000 daltons; and polymerase III subunits of 24,000 and 20,000 daltons. The incorporated 32P was acid-stable but base-labile. Phosphoserine and phosphothreonine were identified after partial acid hydrolysis of purified [32P]polymerase I. A yeast protein kinase that co-purifies with polymerase I during part of the isolation procedure was partially purified and characterized. This protein kinase phosphorylates the subunits of the purified polymerases that are phosphorylated in vivo and, in addition, a polymerase I subunit of 48,000 daltons and a polymerase II subunit of 33,500 daltons. Phosphorylation of the purified enzymes with this protein kinase had no substantial effect on polymerase activity in simple assays using native yeast DNA as a template. Preincubation of purified polymerase I with acid or alkaline phosphatase also had no detectable effect on polymerase activity.     

THE CRYSTALLINE CELL WALL OF TETRASELMIS CONVOLUTAE (CHLOROPHYTA): A FREEZE FRACTURE ANALYSIS1

A freeze fracture analysis of the cell wall of Tetraselmis convolutae (Parke et Manton) revealed the existence of a crystalline median layer consisting of regular repeating subunits of 27 nm. These circular subunits lie in curved, interlocking, longitudinal rows with some irregular discontinuities appearing in the subunit pattern of the crystalline lattice. A comparison with the cell walls of other green algal flagellates is presented, revealing similarities and suggesting evolutionary patterns.     

Regular Array in the Cell Wall of Lactobacillus fermenti as Revealed by Freeze-Etching and Negative Staining1

Freeze-etching of Lactobacillus fermenti F-4 (NCTC 7230) revealed that the outer layer of the cell wall was composed of a regular array in which parallel lines ran obliquely to the longitudinal axis of the cell with an average distance between the centers of about 9.6 nm and were intersected by thinner lines with an average periodicity of approximately 6.2 nm at an angle of about 75°. Occasionally the direction of the striation was discontinuously shifted near one end of the cell. Beneath the regular array the middle cell wall layer packed with granules and the smooth inner cell wall layer were discernible and the mesosomes were also visible in the cytoplasm. When the ultrastructure of isolated outer cell wall fragments was examined by negative staining, the regular array appeared to be composed of subunits, about 3.6 nm in diameter, which were arranged in a tetragonal pattern. The tetragonal array consisted of the subunits in rows in two directions at an angle of about 75° to each other. The average spacing between the rows was about 9.3 nm in one direction and 5.5 nm in the other direction.     

Simulation of protein diffusion: a sensitive probe of protein–solvent interactions

Aqueous solutions of Candida antarctica lipase B (CALB) were simulated considering three different water models (SPC/E, TIP3P, TIP4P) by a series of molecular dynamics (MD) simulations of three different box sizes (L = 9, 14, and 19 nm) to determine the diffusion coefficient, the water viscosity and the protein density. The protein–water systems were equilibrated for 500 ns, followed by 100 ns production runs which were analysed. The diffusional properties of CALB were characterized by the Stokes radius (R_S), which was derived from the diffusion coefficient and the viscosity. R_S was compared to the geometric radius (R_G) of CALB, which was derived from the protein density. R_S and R_G differed by 0.27 nm for SPC/E and by 0.40 and 0.39 nm for TIP3P and TIP4P, respectively, which characterizes the thickness of the diffusive hydration layer on the protein surface. The simulated hydration layer of CALB resulted in agreement with those experimentally determined for other seven different proteins of comparable size. By avoiding the most common pitfalls, protein diffusion can be reliably simulated: simulating different box sizes to account for the finite size effect, equilibrating the protein–water system sufficiently, and using the complete production run for the determination of the diffusion coefficient.     

Ribosomal RNA synthesis in mitochondria of Neurospora crassa

Ribosomal RNA synthesis in Neurospora crassa mitochondria has been investigated by continuous labeling with [5-³H]uracil and pulse-chase experiments. A short-lived 32 S mitochondrial RNA was detected, along with two other short-lived components; one slightly larger than large subunit ribosomal RNA, and the other slightly larger than small subunit ribosomal RNA. The experiments give support to the possibility that 32 S RNA is the precursor of large and small subunit ribosomal RNA's. Both mature ribosomal RNA's compete with 32 S RNA in hybridization to mitochondrial DNA. Quantitative results from such hybridization-competition experiments along with measurements of electrophoretic mobility have been used to construct a molecular size model for synthesis of mitochondrial ribosomal RNA's. The large molecular weight precursor (32 S) of both ribosomal RNA's appears to be 2.4 × 10⁶ daltons in size. Maturation to large subunit RNA (1.28 × 10⁶ daltons) is assumed to involve an intermediate ~1.6 × 10⁶ daltons in size, while cleavage to form small subunit RNA (0.72 × 10⁶ daltons) presumably involves a 0.9 × 10⁶ dalton intermediate. In the maturation process ~22% of the precursor molecule is lost. As is the case for ribosomal RNA's, the mitochondrial precursor RNA has a strikingly low G + C content.