Copillaria hepatica: Fine structure of egg shell

The structure of the Capillaria hepatica egg shell was studied with the electron microscope and correlated with light microscope histochemical observations. The shell is composed of fibrous and nonfibrous components, both of which stain for protein. The fibrous component, the major portion of the shell, consists of submicroscopic fibers. The nonfibrous component is located in the outer region of the shell but is not always visible; when present it has a reticulated appearance in electron micrographs. The fibrous component is divided into outer and inner regions. The outer region is composed of radially arranged pillars which are connected at their outer surface by a beam-like network and are anchored at the base to a compact inner region. The inner region consists of a series of concentrically arranged lamellae above which is located a nonlaminated region where the pillar bases originate. At each polar end of the shell is a single opening plugged with a material which contains acid mucopolysaccharide. The fine structure of the body of the plug is unresolvable with the electron microscope; its outer surface is impregnated with electron dense particles. Externally the shell is covered by a 250 Å thick continuous membrane which is in close opposition to the surrounding host tissue.     

Electron microscopy of a eukaryotic single-stranded DNA binding protein-DNA complex

The single-stranded DNA binding protein of Ustilago maydis decreases the contour length of φX174 DNA. When DNA complexes were prepared with subsaturating amounts of the protein, its distribution on the DNA was markedly non-random, indicating a high degree of co-operativity in its binding to single-stranded DNA. The analagous Escherichia coli, Salmonella typhimurium and bacteriophage T7 binding proteins also reduced DNA contour lengths to a similar extent, whereas the bacteriophage T4 gene 32 protein, as shown previously, increased the contour length. Despite the fact that the U. maydis protein efficiently denatures poly[d(A-T) · d(A-T)], it appears to initiate denaturation of native bacteriophage λ DNA rather inefficiently.     

Assembly of bacteriophage lambda heads: Protein processing and its genetic control in petit λ assembly

Petit bacteriophage λ is a hollow λ head precursor which is found in λ-infected lysates, including lysates of phage λ carrying mutations in head genes. Wild-type petit λ has a protein composition similar to heads, except that it is missing pD ⁴, a major component of heads. About 95% of the mass of petit λ is pE, the major structural protein of heads, and in addition it has proteins pB, h3, X1, and X2. Tryptic fingerprint analysis shows that h3 is a proteolytic cleavage product of pB, and previous experiments have shown that X1 and X2 are protein fusion products, closely related to each other and containing amino acid sequences of both pC and pE. Petit lambdas derived from infection by phages defective in genes A or D are indistinguishable from wild-type petit λ. B⁻, C⁻, or groE⁻ defective petit lambdas show differences from wild-type in protein composition and in extent of protein processing. On the basis of the properties of mutant petit lambdas it is concluded that: (1) the protein processing reactions (cleavage of pB; fusion of pC with pE) occur on the petit λ structure; (2) cleavage of pB requires the functioning of genes C and groE but not A or D; (3) fusion of pC and pE requires gene groE but not A, B or D; (4) pNu3 participates directly in petit λ assembly but is lost from the structure by the time assembly is complete.Physical studies of petit λ show that wild-type, A⁻, B⁻ and D⁻ petit lambdas sediment at 150 S, while C⁻ and groE⁻ petit lambdas sediment at 190 S. Purified petit λ of either class has an ultraviolet absorption spectrum characteristic of pure protein.     

The Rz1 gene product of bacteriophage lambda is a lipoprotein localized in the outer membrane of Escherichia coli

The Rz1 gene of bacteriophage λ is located within the Rz lysis gene. It codes for the 6.5-kDa prolipoprotein (Rz1) which undergoes N-terminal signal sequence cleavage and post-translational lipid modification of the N-terminal Cys of the mature protein. Globomycin, the antibiotic which inhibits bacterial signal peptidase II, specific for prolipoproteins containing diacylglyceryl cysteine [Hayashi and Wu, J. Bioenerg. Biomembr. 22 (1990) 451–471] inhibits the N-terminal sequence cleavage of the Rz1 precursor. The mature protein is rich in Pro, which constitutes 25% of its amino acids (aa). Using a computer-predicted, synthetic, 15-aa antigenic determinant of Rz1 polyclonal anti-Rz[46–60] antibodies, were obtained, and employed to localize Rz1 in bacterial fractions. In induced Escherichia coli λ lysogens Rz1 was found almost exclusively in the outer membrane (OM). In a strain overproducing Rz1 from the pSB54 plasmid, it was distributed in all the fractions. OM, fraction A and inner membrane (IM). Expression of Rz1 from the pSB54 caused enlargement of fraction A, corresponding to the adhesion sites of OM and IM. Such an enlargement was previously observed in induced λ lysogens, shortly before the onset of lysis.     

Structural changes during the transformation of bacteriophage T4 polyheads: characterization of the initial and final states by freeze-drying and shadowing Fab-fragment-labelled preparations.

The maturation of the head of bacteriophage T4 requires a cleavage of the major capsid protein subunit, P23, and results in a transformation of the unstable prehead shell to the chemically resistant shell of the mature virion. We have studied this transformation by comparing class I and class III polyheads, which have P23 lattices which correspond to the prehead and mature head, respectively. The inner and outer surface topographies of these structures were determined from optically filtered images of freeze-dried and shadowed preparations. Individual antigenic sites were localized on the polyhead surfaces by labelling them with Fab fragments obtained from antisera raised against polyheads and against sheets composed of a fragment of the P23 molecule. We find that the transformation involves a structural change in the surface lattice which eliminates protrusions on the inside surface and produces new protrusions on the outer surface. Changes in antigenicity include at least one site which disappears from the outer surface, the unmasking of a site which appears on the outer surface, and the movement of at least one site from the inside surface to the outside during the transformation. We discuss the mechanism of the transformation in terms of the changes in tertiary and quaternary structure of the subunits required to account for the observed changes in the polyhead structure and antigenicity.     

Structure and assembly of bacteriophage PRD1, an Escherichia coli virus with a membrane

This article describes the structure and assembly of bacteriophage PRD1, a lipid-containing virus able to infect Escherichia coli. This phage, with an approximate diameter of 65 nm, is composed of an outer protein shell surrounding a lipid-protein membrane which, in turn, encloses the nucleic acid. The phage genome consists of a single linear dsDNA molecule of about 15 kb that has a protein covalently linked to each of its 5′ ends. This protein is used as a primer in DNA replication. During assembly membrane proteins are inserted into the host cytoplasmic membrane while major capsid protein multimers are found in the cytoplasm. Capsid multimers, assisted by two nonstructural assembly factors, are capable of translocating the virus-specific membrane resulting in the formation of cytoplasmic empty particles. Subsequent DNA packaging leads to the formation of infectious virus.     

Assembly of the head of bacteriophage P22: X-ray diffraction from heads,proheads and related structures

We have used electron microscopy and small-angle X-ray diffraction to study the three principal structures found in the head assembly pathway of Salmonella phage P22. These structures are, in order of their appearance in the pathway: proheads, unstable filled heads (which lose their DNA and become empty heads upon isolation), and phage. In addition, we can convert proheads to an empty head-like form (the empty prohead) in vitro by treating them with 0.8% sodium dodecyl sulfate at room temperature.We have shown that proheads are composed of a shell of coat protein with a radius of 256 Å, containing within it a thick shell or a solid ball (outer radius 215 Å) of a second protein, the scaffolding protein, which does not appear in phage. The three other structures studied are all about 10% larger than proheads, having outer shells with radii of about 285 Å. Empty heads and empty proheads appear identical by small-angle X-ray diffraction to a resolution of 25 Å, both being shells about 40 Å thick. Phage appear to be made up of a protein shell identical to that in empty heads and empty proheads, within which is packed the DNA.Some details of the DNA packing are also revealed by the diffraction pattern of phage. The inter-helix distance is about 28 Å, and the hydration is about 1.5 g of water per g of DNA. Certain aspects of the pattern suggest that the DNA interacts in a specific mariner with the coat protein subunits on the inside edge of the protein shell.Thus, the prohead-to-head transformation involves, in addition to the loss of an internal scaffold and its replacement by DNA, a structural transition in the outer shell. Diffraction from features of the surface organization in these structures indicates that the clustering of the coat protein does not change radically during the expansion. The fact that the expansion occurs in vitro during the formation of empty proheads shows that it is due to the bonding properties of the coat protein alone, although it could be triggered in vivo by DNA -protein interactions. The significance of the structural transition is discussed in terms of its possible role in the control of head assembly and DNA packaging.     

Proposed alpha-helical super-secondary structure associated with protein-dna recognition

Knowledge of the three-dimensional structure of the bacteriophage λ Cro repressor, combined with an analysis of amino acid sequences and DNA coding sequences for this and other proteins that recognize and bind specific base sequences of double-helical DNA, suggests that a portion of the structure of the Cro repressor that is involved in DNA binding also occurs in the Cro protein from bacteriophage 434, the cII protein from bacteriophage λ, the Salmonella phage P22 c2 repressor and the cI repressor from bacteriophage λ. This α-helical super-secondary structure may be a common structural motif in proteins that bind double-helical DNA in a base sequence-specific manner.     

Suppressor mutations that restore export of a protein with a defective signal sequence

A selection procedure is described that should allow the genetic identification of cellular components involved in the process of protein localization in Escherichia coli. This procedure makes use of mutations that alter the signal sequence of the λ receptor protein (product of the lamB gene), and prevent export of this protein to its normal outer membrane location. Several suppressor mutations have been identified that restore export of the mutant λ receptor protein. Mapping experiments show that the suppressor phenotype is the result of mutations in any of at least three different chromosomal loci. One class of suppressor mutations, the class containing the largest number of independent isolates, maps in the major ribosomal gene cluster, suggesting that the suppressor phenotype is the consequence of an altered ribosomal protein. This class of suppressors phenotypically suppresses all known export-defective mutations, internal to the signal sequence region of the lamB gene. These results suggest that ribosomes play an important role in the export of λ receptor to the outer membrane.     

Structure of the scaffold in bacteriophage lambda preheads removal of the scaffold leads to a change of the prehead shell

Small angle X-ray scattering was performed on unprocessed and processed preheads, intermediates in the morphogenesis of bacteriophage λ heads. Unprocessed preheads possess an internal structure (scaffold), necessary for efficient assembly of closed shells. Processed preheads, formed after removal of the scaffold, are able to pack and cut the viral DNA in vitro. Our data show that the scaffold fills out the inside of the shell in an almost (but not completely) homogeneous fashion; structures of the scaffold with the bulk of the mass in a small core inside the shell can be excluded. Unprocessed preheads are larger than processed ones. A change in shell architecture takes place upon transition from unprocessed to processed prehead; the shell becomes roughened up. Shrinking of the shell as well as roughening up can be triggered by accidental partial degradation of the scaffold. The lattice constant of type A polyheads is in agreement with the lattice constant derived from our icosahedral models of the shell, indicating a close relationship between processed preheads and type A polyheads. This observation, together with the type of subunit clustering found, leads us to propose a simple model for the interaction of prehead shell and protein pD, which stabilizes phage DNA after packaging.     

Structure and assembly of bacteriophage PRD1, and Escherichia coli virus with a membrane

This article describes the structure and assembly of bacteriophage PRD1, a lipid-containing virus able to infect Escherichia coli. This phage, with an approximate diameter of 65 nm, is composed of an outer protein shell surrounding a lipid-protein membrane which, in turn, encloses the nucleic acid. The phage genome consists of a single linear dsDNA molecule of about 15 kb that has a protein covalently linked to each of its 5' ends. This protein is used as a primer in DNA replication. During assembly membrane proteins are inserted into the host cytoplasmic membrane while major capsid protein multimers are found in the cytoplasm. Capsid multimers, assisted by two nonstructural assembly factors, are capable of translocating the virus-specific membrane resulting in the formation of cytoplasmic empty particles. Subsequent DNA packaging leads to the formation of infections virus.     

The bacteriophage lambda gpNu3 scaffolding protein is an intrinsically disordered and biologically functional procapsid assembly catalyst

Procapsid assembly is a process whereby hundreds of copies of a major capsid protein assemble into an icosahedral protein shell into which the viral genome is packaged. The essential features of procapsid assembly are conserved in both eukaryotic and prokaryotic complex double-stranded DNA viruses. Typically, a portal protein nucleates the co-polymerization of an internal scaffolding protein and the major capsid protein into an icosahedral capsid shell. The scaffolding proteins are essential to procapsid assembly. Here, we describe the solution-based biophysical and functional characterization of the bacteriophage lambda (λ) scaffolding protein gpNu3. The purified protein possesses significant α-helical structure and appears to be partially disordered. Thermally induced denaturation studies indicate that secondary structures are lost in a cooperative, apparent two-state transition (T_m = 40.6 ± 0.3 °C) and that unfolding is, at least in part, reversible. Analysis of the purified protein by size-exclusion chromatography suggests that gpNu3 is highly asymmetric, which contributes to an abnormally large Stokes radius. The size-exclusion chromatography data further indicate that the protein self-associates in a concentration-dependent manner. This was confirmed by analytical ultracentrifugation studies, which reveal a monomer-dimer equilibrium (K_d,app ~ 50 μM) and an asymmetric protein structure at biologically relevant concentrations. Purified gpNu3 promotes the polymerization of gpE, the λ major capsid protein, into virus-like particles that possess a native-like procapsid morphology. The relevance of this work with respect to procapsid assembly in the complex double-stranded DNA viruses is discussed.     

Number and size distribution of the DNA chains in Escherichia coli.

The essential replication protein encoded by gene O of bacteriophage λ (O-λ) is one of the major polypeptides produced in vitro in a DNA-dependent protein synthesizing system with λ DNA as template (Yates et al., 1977). We have used this system to identify the proteins encoded by lambdoid phages φ80 and 82 and equivalent in function to O-λ. The O protein of each phage type differs slightly in polypeptide molecular weight. Hybrid λ-φ80 and λ-82 phages derived by recombination within gene O direct synthesis of hybrid O proteins with the aminoterminal segment characteristic of one parent, and the carboxyl-terminal segment characteristic of the other. Differences in structure among O-λ, O-80 and O-λ82 segregate together with specificity determinants for interactions between the O protein and the control site ori, and between the O protein and the product of replication gene P. The coding region for the O protein includes ori.     

A Naturally Occurring Novel Allele of Escherichia coli Outer Membrane Protein A Reduces Sensitivity to Bacteriophage

A novel Escherichia coli outer membrane protein A (OmpA) was discovered through a proteomic investigation of cell surface proteins. DNA polymorphisms were localized to regions encoding the protein's surface-exposed loops which are known phage receptor sites. Bacteriophage sensitivity testing indicated an association between bacteriophage resistance and isolates having the novel ompA allele.     

Structure of the shell and tertiary membranes of eggs of softshell turtles (Trionyx spiniferus)

Eggs of the turtle Trionyx spiniferus are rigid, calcareous spheres averaging 2.5 cm in diameter. The eggshell is morphologically very similar to avian eggshells. The outer crystalline layer is composed of roughly columnar aggregates, or shell units, of calcium carbonate in the aragonite form. Each shell unit tapers to a somewhat conical tip at its base. Interior to the crystalline layer are two tertiary egg membranes: the outer shell membrane and the inner shell membrane. The outer shell membrane is firmly attached to the inner surface of the shell, and the two membranes are in contact except at the air cell, where the inner shell membrane separates from the outer shell membrane. Both membranes are multi-layered, with the inner shell membrane exhibiting a more fibrous structure than the outer shell membrane. Numerous pores are found in the eggshell, and these generally occur at the intersection of four or more shell units.     

The role of the Escherichia coli λ receptor in the transport of maltose and maltodextrins

The λ receptor is a peptidoglycan-associated integral protein that spans the outer membrane. Beside its function in phage λ adsorption it participates in transport. The latter function can be summarized as follows: (1) Receptor allows the nonspecific permeation of small molecules other than maltose and maltodextrins (in close analogy to a molecular sieve). Here the only criterion for selectivity is size and it has the properties of an unspecific pore. In this respect, it is similar to the outer membrane proteins Ia, Ib, and Ic, the porins. (2) It is a binding protein for maltodextrins. Binding affinity is low but increases by a factor of 500 as the chain length of the maltodextrins increases. In contrast, the affinity of the periplasmic maltose-binding protein for maltose and maltodextrins is similarly high (in the μM range). (3) In the in vitro system of liposomes, the λ receptor facilitates specifically the diffusion of maltodextrins that exceed the size limit given by its porin function. This clearly demonstrates that the λ receptor alone is able to specifically overcome the permeability barrier of the outer membrane for maltodextrins. (4) From the genetic and kinetic analysis of maltose and maltodextrin transport, it can be concluded that the λ receptor interacts with the periplasmic maltose-binding protein. (5) Electron microscopic studies indicate a location for the maltose-binding protein in the outer cell envelope. This location is dependent on the presence of the λ receptor.     

DNA packaging steps in bacteriophage lambda head assembly

Petit λ is an empty spherical shell of protein which appears wherever λ grows. If phage DNA and petit λ are added to a cell-free extract of induced lysogenic bacteria, then phage particles are formed that contain the DNA and protein from the petit λ. Petit λ is transformed, without dissociation, into a phage head by addition of DNA and more phage proteins.The products of ten genes, nine phage and one host, are required for λ head assembly. Among these, the products of four phage genes, E, B, C, and Nu3 and of the host gene groE are involved in the synthesis of petit λ, consequently these proteins are dispensable for head assembly in extracts to which petit λ has been added. The products of genes A and D allow DNA to combine with petit λ to form a head that has normal morphology. In an extract, DNA can react with A product and petit λ to become partially DNAase-resistant, as if an unstable DNA-filled intermediate were formed. ATP and spermidine are needed at this stage. This intermediate is subsequently stabilized by addition of D product. The data suggest a pathway for head assembly.     

Construction and analysis of a genetically tuneable lytic phage display system

The Bacteriophage λ capsid protein gpD has been used extensively for fusion polypeptides that can be expressed from plasmids in Escherichia coli and remain soluble. In this study, a genetically controlled dual expression system for the display of enhanced green fluorescent protein (eGFP) was developed and characterized. Wild-type D protein (gpD) expression is encoded by λ Dam15 infecting phage particles, which can only produce a functional gpD protein when translated in amber suppressor strains of E. coli in the absence of complementing gpD from a plasmid. However, the isogenic suppressors vary dramatically in their ability to restore functional packaging to λDam15, imparting the first dimension of decorative control. In combination, the D-fusion protein, gpD::eGFP, was supplied in trans from a multicopy temperature-inducible expression plasmid, influencing D::eGFP expression and hence the availability of gpD::eGFP to complement for the Dam15 mutation and decorate viable phage progeny. Despite being the worst suppressor, maximal incorporation of gpD::eGFP into the λDam15 phage capsid was imparted by the SupD strain, conferring a gpDQ68S substitution, induced for plasmid expression of pD::eGFP. Differences in size, fluorescence and absolute protein decoration between phage preparations could be achieved by varying the temperature of and the suppressor host carrying the pD::eGFP plasmid. The effective preparation with these two variables provides a simple means by which to manage fusion decoration on the surface of phage λ.     

Irregular calcareous sheets preserved in a conch of the Cretaceous ammonoid Hypophylloceras from Hokkaido,Japan

The mantle tissue is essential for understanding the diverse ecology and shell morphology of ammonoid cephalopods. Here, we report on irregular calcareous sheets in a well-preserved shell of a Late Cretaceous phylloceratid ammonoid Hypophylloceras subramosum from Hokkaido, Japan, and their significance for repairing the conch through the mantle inside the body chamber. The sheets are composed of nacreous layers arranged parallel to the irregularly distorted outer whorl surface. The nacreous sheets formed earlier are unevenly distributed and attached to the outer shell wall locally, whereas the last formed sheet covers a wide area of the outer shell wall. The absence of any interruption of ribbing around the irregular area suggests that these sheets were secreted inside the body chamber from the inner mantle. Gross morphological and X-ray computed tomography observations revealed that the spacing of septal formation was not affected by this event. The complex structure of the irregular sheets suggests a highly flexible mantle inside the body chamber.