Frederick A. Eiserling

Characterization and structural properties of the major biliproteins of Anabaena sp.

AbstractStudies are presented of the biliproteins of Anabaena sp. This filamentous cyanobacterium contains three major biliproteins. Whereas two of these, C-phycocyanin and allophycocyanin, are common to all cyanobacteria, the third, phycoerythrocyanin (λmax∼568nm) has hitherto not been described and its distribution among cyanobacteria appears to be limited. Anabaena variabilis and Anabaena sp. 6411 allophycocyanin, C-phycocyanin, and phycoerythrocyanin were purified to homogeneity and characterized with respect to molecular weight, isoelectric point, absorption spectrum and amino acid composition. The α and β subunits of each of these proteins were also purified to homogeneity and characterized in the same manner. The tetrapyrrole chromophore content was determined for each of the proteins and subunits. The α subunit of phycoerythrocyanin carries a novel phycobiliviolin-like chromophore. This chromophore has not previously been detected in cyanobacterial biliproteins, but has been noted as a prosthetic group of a cryptophytan phycocyanin.Sedimentation equilibrium studies show that at pH 7.0, at protein concentrations of 0.2–0.6 mg/ml, allophycocyanin, C-phycocyanin and phycoerythrocyanin, each exists as a trimeric aggregate, (αβ)3, of molecular weight of approximately 105000. Structural studies of microcrystals of these three biliproteins by electron microscopy and X-ray diffraction reveal a common plan for the construction of higher assembly forms. The major building block appears to be the trimer (αβ)3. It is proposed that this is a dise-like structure about 3.0×12.0 nm. The individual α or β subunits are roughly spherical, 3 nm in diameter. Allophycocyanin trimers stack to form bundles of rods which form long needles. Both phycocyanin and phycoerythrocyanin form double dises (αβ)6 which are visible as ring-shaped structures by electron microscopy. The mode of assembly of the biliproteinstructures in the phycobilisome is, as yet, unknown.

The structural organization of DNA packaged within the heads of T4 wild-type, isometric and giant bacteriophages

We present electron microscopic and X-ray diffraction evidence concerning the structural organization of condensed DNA within a series of T4 bacteriophage with the following head morphologies: prolate (wild-type), isometric and giant (with greatly increased axial ratio). In all cases, the DNA helix segments are locally parallel and 27 A apart. For the giant particles, we show that the DNA forms a large coil whose axis is perpendicular to the axis of the phage tail. This evidence, combined with previous results from a series of isometric bacteriophages (Earnshaw and Harrison, 1977), leads to a model for the organization of condensed DNA that may apply to most dsDNA-containing bacteriophages.

Structural proteins of bacteriophage T4

Abstract The protein composition of purified T4 phage, phage heads and capsids was examined by sodium dodeoyl sulfate-acrylamide gel electrophoresis. The virion contains at least 21 unique proteins which range in molecular weight from 10,000 to 150,000. Both heads and capsids contain eight of these proteins. However, two of the eight proteins are partially released from heads by freeze-thaw or osmotic shock treatment. Thus, they are probably internal proteins. Capsids purified from cells infected with mutants defective in tail assembly can be separated into three fractions by cesium chloride equilibrium centrifugation. About 10% of the capsids have an unusually high density of from 1.35 to 1.40 g/cm 3 . Some of these capsids contain a collar. Below the collar are several 20 by 500 A fibers (whiskers). The two other capsid fractions have densities of about 1.30 and 1.33 g/cm 3 . The capsids in these two fractions lack collars and whiskers. As a result of head assembly the major head structural protein, the gene 23 product, is converted from 55,000 to 46,000 molecular weight. The gene 23 product is not converted in cells infected with any of the head-defective amber mutants in genes 20, 21, 22, 24 and 31.

The structure of form I crystals of d-ribulose-1,5-diphosphate carboxylase☆

Abstract Single crystals of d -ribulose-1,5-diphosphate carboxylase from tobacco leaves, Nicotiana tabacum (variety Turkish Samsun), have been examined by X-ray diffraction, electron microscopy, and optical diffraction. Twelve molecules are loosely packed into a body-centered cubic unit cell, space group I 4 1 32 with cell dimension a = 383 A . The asymmetric unit is one quarter of a molecule, and the minimum molecular symmetry is 222. This symmetry when combined with estimates of the two subunit masses and stoichiometry is compatible with a molecular structure of the composition L 8 S 8 (L is large subunit, S is small). If all bonds between large and small subunits are equivalent, the true molecular symmetry is 422; this symmetry is consistent with molecular images in micrographs.

Studies on the structure, protein composition and assembly of the neck of bacteriophage T4

Abstract We have developed an osmotic shock procedure which disconnects the tail from the head of intact bacteriophage T4, leaving the neck region attached to the tail. Purification of these necked tails permitted detailed structural observations of the neck and the collar/whisker complex attached to it, as well as comparison by gel electrophoresis with tails lacking the neck. Five or six neck proteins were found: N1 (Mr = 52,000; 39 copies/phage) is the product of the wac ‡ gene (Pwac), forms both the collar and six whiskers as a multimeric fibrous protein, and probably assembles onto phage after head to tail joining; N2 (Mr= 35,000; 5 to 6 copies/phage), N3 (Mr= 33,000; 17 copies/phage) identified here as P13, and N6 (Mr= 28,000; 10 to 11 copies/phage) are all assembled in heads prior to tail joining; N4 (Mr= 32,000; 6 to 9 copies/phage) is unusual in that it is present in wac or wac+ phage and necked tails but is absent from purified heads; N5 (Mr =29,000) is probably P14 and like N4 is not found in heads. However, while we find one to two copies of N5 per necked tail, we have not observed it in phage. An aberrant neck structure called the extension assembles on the distal end of the tail connector late (after 33 min, 30 °C) in head-defective, mutant-infected cells. The extension contains five of the six neck proteins (N2 is absent), and blocks head to tail joining in vitro. Mutations in genes 13 and 14, and the double mutant 49:Wac block extension assembly. Other results show that the wac mutant E727J is an amber lesion, and that Pwac can assemble on collarless, wac phage in vitro.

Genomic and Genetic Analysis of Bordetella Bacteriophages Encoding Reverse Transcriptase-Mediated Tropism-Switching Cassettes

Liu et al. recently described a group of related temperate bacteriophages that infect Bordetella subspecies and undergo a unique template-dependent, reverse transcriptase-mediated tropism switching phenomenon (Liu et al., Science 295: 2091-2094, 2002). Tropism switching results from the introduction of single nucleotide substitutions at defined locations in the VR1 (variable region 1) segment of the mtd (major tropism determinant) gene, which determines specificity for receptors on host bacteria. In this report, we describe the complete nucleotide sequences of the 42.5- to 42.7-kb double-stranded DNA genomes of three related phage isolates and characterize two additional regions of variability. Forty-nine coding sequences were identified. Of these coding sequences, bbp36 contained VR2 (variable region 2), which is highly dynamic and consists of a variable number of identical 19-bp repeats separated by one of three 5-bp spacers, and bpm encodes a DNA adenine methylase with unusual site specificity and a homopolymer tract that functions as a hotspot for frameshift mutations. Morphological and sequence analysis suggests that these Bordetella phage are genetic hybrids of P22 and T7 family genomes, lending further support to the idea that regions encoding protein domains, single genes, or blocks of genes are readily exchanged between bacterial and phage genomes. Bordetella bacteriophages are capable of transducing genetic markers in vitro, and by using animal models, we demonstrated that lysogenic conversion can take place in the mouse respiratory tract during infection.

Asymmetrical core structure in phycobilisomes of the cyanobacterium Synechocystis 6701

The light-harvesting complex of cyanobacteria and red algae, the phycobilisome, has two structural domains, the core and the rods. Both contain biliproteins and linker peptides. The core contains the site of attachment to the thylakoid membrane and the energy transfer link between the phycobilisome and chlorophyll. There are also six rod-binding sites in the membrane-distal periphery of the core. The structure of phycobilisomes in the cyanobacterium Synechococcus 6301 was studied by Glazer, who proposed a model for the internal organization of the bicylindrical core. In the construction of that model, it was necessary to make arbitrary decisions between two possible locations for one of the trimeric protein complexes within a core cylinder and between two possible orientations of the basal core cylinders relative to one another. We isolated the tricylindrical cores from an ultraviolet-light-induced mutant of the cyanobacterium Synechocystis 6701 and obtained, by partial dissociation, a unique core substructure that maintained some contacts between the two basal cylinders. From its structure and spectral properties, we conclude that this particle is a central core substructure that resulted from dissociation of the two layers of peripheral trimers in the intact core. The compositions of this particle and the dissociated trimers were inconsistent with the proposed location of one of the trimers in the 6301 core model, but supported the placement of that trimer in the alternative position within the basal core cylinder. Rod-binding sites within the central core substructure were studied by partial dissociation of the short-rod phycobilisomes from another mutant of 6701. This dissociation generated particles that were interpreted as being central core substructures with the two basal rods attached. The appearance of these particles in the electron microscope suggested that both basal rods would be localized towards the same side of the intact core. Such an asymmetrical arrangement of basal rods is supported by previously published edge-views of intact cores with basal rods from strain 6701. These observations suggest a parallel arrangement of the basal cylinders with respect to each other, creating an asymmetrical core. A phycobilisome model was constructed that incorporated core asymmetry. This model predicts the energy transfer pathways from the basal and upper rods to specific trimers in the core.

Bacteriophage-like particles associated with the gene transfer agent of methanococcus voltae PS.

The methanogenic archaeobacterium Methanococcus voltae (strain PS) is known to produce a filterable, DNase-resistant agent (called VTA, for voltae transfer agent), which carries very small fragments (4400 bp) of bacterial DNA and is able to transduce bacterial genes between derivatives of the strain. Examination by electron microscopy of two preparations of VTA that were concentrated and partially purified by different methods showed virus-like particles with isometric heads, about 40 nm in diameter, and with 61 nm long tails. These particles co-sedimented with the minute bacteriophage φX174 in a sucrose density gradient.

Ribulose Bisphosphate Carboxylase: A Two-Layered, Square-Shaped Molecule of Symmetry 422

Electron micrographs and x-ray diffraction patterns of crystals of ribulose bisphosphate carboxylase, probably the most abundant protein on earth, have provided new details of the arrangement of subunits. The eight large subunits and eight small subunits are clustered in two layers, perpendicular to a fourfold axis of symmetry. Viewed down the fourfold axis, the molecule is square-shaped.

Bacteriophage T4 Self-Assembly: Localization of gp3 and Its Role in Determining Tail Length

Gene 3 of bacteriophage T4 participates at a late stage in the T4 tail assembly pathway, but the hypothetical protein product, gp3, has never been identified in extracts of infected cells or in any tail assembly intermediate. In order to overcome this difficulty, we expressed gp3 in a high-efficiency plasmid expression vector and subsequently purified it for further analysis. The N-terminal sequence of the purified protein showed that the initial methionine had been removed. Variant C-terminal amino acid sequences were resolved by determining the cysteine content of the protein. The molecular mass of 20.6 kDa for the pure protein was confirmed by Western blotting, using a specific anti-gp3 serum for which the purified protein was the immunogen. We also demonstrated, for the first time, the physical presence of gp3 in the mature T4 phage particle and localized it to the tail tube. By finding a nonleaky, nonpermissive host for a gene 3 mutant, we could clearly demonstrate a new phenotype: the slow, aberrant elongation of the tail tube in the absence of gp3.