Structure of Bacillus subtilis bacteriophage phi 29 and the length of phi 29 deoxyribonucleic acid

Anderson, D. L. (University of Minnesota, Minneapolis), D. D. Hickman, and B. E. Reilly. Structure of Bacillus subtilis bacteriophage phi29 and the length of phi29 deoxyribonucleic acid. J. Bacteriol. 91:2081-2089. 1966-Bacillus subtilis bacteriophage phi29 were negatively stained with phosphotungstic acid. The head of phi29 has a hexagonal outline with a flattened base, and is about 315 A wide and 415 A in length. The virus has an intricate tail about 325 A in length. Twelve spindle-shaped appendages are attached to the lower of two collars which comprise the proximal portion of the tail. The distal 130 A of the tail axis has a diameter of about 60 A and is larger in diameter than the axis of the upper portion of the tail. Comparison of electron microscopic counts of phi29 with plaque-forming units indicated that about 50% of the microscopic entities were infective. Phenol-extracted phi29 deoxyribonucleic acid (DNA) molecules were prepared for electron microscopy by the cytochrome c film technique of Kleinschmidt et al. Measurement of contour lengths of DNA molecules from three preparations gave skewed distributions of lengths with observed modal class values ranging from 5.7 to 5.9 mu. Assuming that phi29 DNA is a double helix in the B form, the corresponding molecular weights would be 10.9 x 10(6) to 11.3 x 10(6) daltons. The largest DNA molecules would have a volume of 1.9 x 10(7) A(3) which is about 25% greater than the estimated 1.4 x 10(7) A(3) internal volume of the phage head.     

The three-dimensional structure of a DNA translocating machine at 10 A resolution.

BACKGROUND: Head-tail connectors are viral substructures that are very important in the viral morphogenetic cycle, having roles in the formation of the precursor capsid (prohead), DNA packaging, tail binding to the mature head and in the infection process. Structural information on the connector would, therefore, help us to understand how this structure is related to a multiplicity of functions. RESULTS: Recombinant bacteriophage phi29 connectors have been crystallized in two-dimensional aggregates. An average projection image and a three-dimensional map have been obtained at 8 A and 10 A resolution, respectively, from untilted and tilted images of vitrified specimens of the two-dimensional crystals. The average projection image reveals a central mass surrounding a channel with 12 appendages protruding from the central mass. The three-dimensional map reveals a wide domain surrounded by 12 appendages that interact with the prohead vertex, and a narrow domain that interacts with the bacteriophage tail. At the junction of the two domains, 12 smaller appendages are visualized. A channel runs along the axis of the connector structure and is sufficiently wide to allow a double-stranded DNA molecule to pass through. CONCLUSIONS: The propeller-like structure of the phi29 connector strengthens the notion of the connector rotating during DNA packaging. The groove formed by the two lanes of large and small appendages may act as a rail to prevent the liberation of the connector from the prohead vertex during rotation.     

DNA poised for release in bacteriophage phi29

We present here the first asymmetric, three-dimensional reconstruction of a tailed dsDNA virus, the mature bacteriophage phi29, at subnanometer resolution. This structure reveals the rich detail of the asymmetric interactions and conformational dynamics of the phi29 protein and DNA components, and provides novel insight into the mechanics of virus assembly. For example, the dodecameric head-tail connector protein undergoes significant rearrangement upon assembly into the virion. Specific interactions occur between the tightly packed dsDNA and the proteins of the head and tail. Of particular interest and novelty, an approximately 60A diameter toroid of dsDNA was observed in the connector-lower collar cavity. The extreme deformation that occurs over a small stretch of DNA is likely a consequence of the high pressure of the packaged genome. This toroid structure may help retain the DNA inside the capsid prior to its injection into the bacterial host.     

Order of assembly of the lower collar and the tail proteins of Bacillus subtilis bacteriophage phi 29.

Extracts obtained after restrictive infection of Bacillus subtilis with mutants in cistron 11 of bacteriophage phi 29 are complemented in vitro by extract donors of the lower collar protein (p11). Purified 11- heads, containing the major capsid protein (p8), the fiber protein (p8.5), the upper collar protein (p10), and the virus DNA, can be also complemented in vitro to produce infective virus. This result suggests that 11- heads are intermediates in phage phi 29 morphogenesis. The order of assembly of the lower collar protein p11 and the tail protein p9 was determined in vitro in two complementation steps. The results obtained indicate that the lower collar protein is assembled before the tail protein.     

Genetic map of the Staphylococcal bacteriophage phi11.

Ten sus mutants of the staphylococcal bacteriophage phi 11, each a representative from a different complementation group, have been used in three-factor cross experiments. The results of these crosses indicate a circular genetic map for phi 11. Functional studies of the mutants have been limited to electron microscopic examinations of lysates after prophage induction (or infection). One gene is an early gene, five genes are concerned with tail formation, and three are concerned with head formation. The tenth gene is possibly a head gene. The contribution by phi 11 to the genomic content of the plasmid-phage hybrid phi 11 de has been investigated. Phi 11 de contains most of the late genes and appears to be missing a continuous phi 11 segment that includes the early gene flanked by two late genes.     

Antigenic Properties of Bacteriophage φ29 Structural Proteins

Serological methods and electron microscopy were used to study the structural proteins of the small Bacillus subtilis bacteriophage phi29. This virus has a large number of fibers attached at both ends of its prolate head. A complex neck assembly is comprised of 12 symmetrically arranged appendages as the outer component. Head fibers, neck appendages, and the head surface bind anti-phi29 antibodies. Immune sera absorbed with defective lysates of suppressor-sensitive (sus) mutants have been used to determine the genetic control of neck appendages production. Studies on the serum-blocking power of lysates defective in different tail components showed that appendages contain the main serum-blocking protein. This finding suggests an essential role of the neck appendages in phage adsorption or DNA injection.     

Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: preliminary isolation and characterization of intermediate particles of the assembly pathway.

Three classes of particles have been identified in restrictive phi 29 suppressor-sensitive (sus) mutant infections of Bacillus subtilis, including DNA-containing heads or phage, prohead, and empty heads. Pulse-chase labeling experiments indicate that the prohead, the first particle assembled in 14-infected cells, is converted to DNA-filled heads and phi 29. In addition to the proteins Hd, P10, and F found in mature phi 29, the prohead contains a "core" protein P7 that exits as the prohead matures and appears to recycle during subsequent rounds of prohead assembly. Prohead-like structures accumulate in UV-irradiated cells and are present in restrictive infections with sus mutants of cistrons 9 and 16. Empty heads are observed only when infection results in the formation of DNA-containing particles; this and other evidence indicates that the empty heads are probably not true intermediates. Phage phi 29 assembly apparently occurs by a single pathway in which neck and tail components interact to stabilize the completed DNA-containing head.     

Genomic diversity of Burkholderia pseudomallei clinical isolates: subtractive hybridization reveals a Burkholderia mallei-specific prophage in B. pseudomallei 1026b

Burkholderia pseudomallei is the etiologic agent of the disease melioidosis and is a category B biological threat agent. The genomic sequence of B. pseudomallei K96243 was recently determined, but little is known about the overall genetic diversity of this species. Suppression subtractive hybridization was employed to assess the genetic variability between two distinct clinical isolates of B. pseudomallei, 1026b and K96243. Numerous mobile genetic elements, including a temperate bacteriophage designated phi1026b, were identified among the 1026b-specific suppression subtractive hybridization products. Bacteriophage phi1026b was spontaneously produced by 1026b, and it had a restricted host range, infecting only Burkholderia mallei. It possessed a noncontractile tail, an isometric head, and a linear 54,865-bp genome. The mosaic nature of the phi1026b genome was revealed by comparison with bacteriophage phiE125, a B. mallei-specific bacteriophage produced by Burkholderia thailandensis. The phi1026b genes for DNA packaging, tail morphogenesis, host lysis, integration, and DNA replication were nearly identical to the corresponding genes in phiE125. On the other hand, phi1026b genes involved in head morphogenesis were similar to head morphogenesis genes encoded by Pseudomonas putida and Pseudomonas aeruginosa bacteriophages. Consistent with this observation, immunogold electron microscopy demonstrated that polyclonal antiserum against phiE125 reacted with the tail of phi1026b but not with the head. The results presented here suggest that B. pseudomallei strains are genetically heterogeneous and that bacteriophages are major contributors to the genomic diversity of this species. The bacteriophage characterized in this study may be a useful diagnostic tool for differentiating B. pseudomallei and B. mallei, two closely related biological threat agents.     

Composition and mass of the bacteriophage phi29 prohead and virion

The protein composition of the Bacillus subtilis bacteriophage phi29 prohead and virion was determined by combustion of gel bands of (3)H-labeled proteins. Copy numbers of individual proteins were calculated relative to the 12 copies of the head-tail connector protein. The mean numbers of copies of the major capsid protein in the prohead and virion were 241 and 218, respectively, approaching the 235 copies determined previously by cryoelectron microscopy. The mean numbers of copies of the dimeric head fiber on the prohead and virion were 24 and 31, respectively, demonstrating partial occupancy of the 55 fiber binding sites. Measured copies of neck and tail proteins in the virion included 11 of the lower collar, 58 of the appendage, and 9 of the tail; if the true copies of these proteins are 12, 60, and 9, respectively, the entire neck and tail of phi29 has quasi-sixfold symmetry. The mass of the fiberless prohead with pRNA was about 14.2 MDa, and the mass of the prohead determined by scanning transmission electron microscopy was consistent with the biochemical data. The mass of the fiberless virion containing the 12.8-MDa DNA genome was about 30.4 MDa. A full complement of dimeric fibers on the prohead or virion would increase the mass of the particle by about 3.2 MDa. The data complement studies relating the structure of phi29 components to dynamic functions in morphogenesis and infection.     

Transformation of a Bacillus subtilis L-form with bacteriophage deoxyribonucleic acid.

A stable L-form, sal-1, of Bacillus subtilis was transformed with deoxyribonucleic acid (DNA) from bacteriophages phi 25 and phi 29 to determine whether exogenous DNA can be introduced into this organism. The viral transformation (transfection) was successful with the use of polyethylene glycol. In the presence of the fusogen, bacteriophage phi 25 DNA initiated a single cycle of infection. When compared with transfection of competent cells of Bacillus subtilis, the appearance of viral particles was delayed and their production occurred over a longer time period. L-form cells were best able to support intracellular replication of phi 25 viral particles when in balanced growth in a rich medium. The addition of polyethylene glycol also induced infection of sal-1 with whole bacteriophage phi 25 particles which could not otherwise infect the L-form and enhanced infection by intact phi 29 particles. Primary recombination was shown to be required for polyethylene glycol-mediated phi 25 transfection, but not phi 29 transfection or for whole bacteriophage phi 25 infection mediated by polyethylene glycol. Successful transfection of sal-1 suggests that the L-form may be amenable to genetic modification with exogenous DNA.     

Isolation and characteristics of the bacteriophage from the vaccine strain Tohama phase I

Some properties of bacteriophage phi T isolated from the vaccine strain Bordetella pertussis Tohama phase I and propagated in Bordetella parapertussis 504 cells are presented. Phage phi T belongs to the IV group in accordance with Tikhonenko classification. The diameter of head and length of noncontractile tail sheath are 49.5 +/- 0.5 and 145 +/- 7 nm, respectively. Diameter of the tail sheath is 3.2 +/- 0.6 nm. Molecular mass of the phage DNA is 37 +/- 3 kb. Population of phi T phage is polymorphous and consists of particles the genomes or which vary from each other by the "insert" located 6.8 +/- 0.6 kb from the end of molecule. The blot hybridization has demonstrated that the bacteriophage genome is not inserted into the chromosome of the lysogenic strain. Autonomous location of the phage genome in the host cell is suggested. The temperature and hydrogen ions concentration effects on bacteriophage phi T stability were studied. The conditions for phage suspension storage are described.     

Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: mapping and functional analysis of the head fiber gene.

A set of mutants of Bacillus subtilis bacteriophage phi29 unable to synthesize the head fiber protein has been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Infectious phage are produced during restrictive infection. We have focused on mutant sus 8.5(900) because the mutation is suppressible by both the su(+3) and su(+44) hosts, and it can be mapped by three- and four-factor crosses. After restrictive infection with mutant sus 8.5(900), a fragment about 70% of the size of the normal fiber is produced as well as particles that are fast-sedimenting in sucrose gradients relative to phi29(+). These particles have the buoyant density of particles with the fibers removed and have the absolute plating efficiency of phi29(+). Fiber protein is absent from prohead as well as virion. A second set of mutants produces fiber protein with a slightly altered electrophoretic mobility. This type of fiber protein is either present or absent on both prohead and virion. A third class of mutants, typified by 914, produces a "normal" fiber, but a major head protein of altered electrophoretic mobility. After infection by this mutant, the fiber is absent from both prohead and virion, and the biological and physical properties of the 914(-) particle are similar to those of particles produced after infection of the su(-) host by sus8.5(900). These observations suggest that the head fiber is not an essential component of the prohead or virion and that the assembly process is efficient in the absence of fiber protein. Three- and four-factor genetic crosses have established the order sus8(769)-8(914)-sus8.5(900)-sus9(756) and indicate that cistrons 8 and 8.5 code for the major head protein and head fiber protein, respectively.     

RNA-mediated specificity of DNA packaging into hybrid lambda/phi 29 proheads.

A small RNA (pRNA, 174 nt) is known to be essential for DNA packaging in bacteriophage phi 29. However, in an in vitro DNA packaging system based on hybrid lambda/phi 29 proheads (made up of head proteins from phage lambda and connectors from phage phi 29), the specificity of DNA packaging is lost, and different RNA molecules fulfil the requirements for DNA packaging, albeit with less efficiency than phi 29 pRNA. Competition assays with RNAs from different sources have shown that phi 29 connectors bind preferentially pRNA. An increase in the efficiency of phi 29 DNA packaging into hybrid proheads induced by phi 29 pRNA is observed because, when phi 29 pRNA is incubated with hybrid proheads, phi 29 DNA is packaged more efficiently than other DNAs of similar length. Furthermore, when hybrid proheads carrying phi 29 pRNA are incubated with a mixture of DNAs from different sources, phi 29 DNA is selectively packaged, thus indicating that phi 29 pRNA determines the specificity of DNA packaging.     

Morphogenesis of bacteriophage phi29 of Bacillus subtilis: cleavage and assembly of the neck appendage protein.

Each of the 12 neck appendages of the Bacillus subtilis bacteriophage phi29 consists of a single protein molecule with a molecular weight of about 75,000, and on the mature virion the appendages are assembled to the lower of two collars. The appendage protein is cleaved from a precursor protein, P(J), with a molecular weight of about 88,000. This cleavage is independent of neck assembly, occurring during infection by mutants that cannot synthesize the proteins of the upper and lower collars of the neck. The cleaved form of the appendage protein is efficiently complemented in vitro to particles lacking appendages. Thus, cleavage of the appendage precursor protein apparently does not occur in situ on the maturing virus.     

Detailed architecture of a DNA translocating machine: the high-resolution structure of the bacteriophage phi29 connector particle.

The three-dimensional crystal structure of the bacteriophage phi29 connector has been solved and refined to 2.1A resolution. This 422 kDa oligomeric protein connects the head of the phage to its tail and translocates the DNA into the prohead during packaging. Each monomer has an elongated shape and is composed of a central, mainly alpha-helical domain that includes a three-helix bundle, a distal alpha/beta domain and a proximal six-stranded SH3-like domain. The protomers assemble into a 12-mer, propeller-like, super-structure with a 35 A wide central channel. The surface of the channel is mainly electronegative, but it includes two lysine rings 20 A apart. On the external surface of the particle a hydrophobic belt extends to the concave area below the SH3-like domain, which forms a crown that retains the particle in the head. The lipophilic belt contacts the non-matching symmetry vertex of the capsid and forms a bearing for the connector rotation. The structure suggests a translocation mechanism in which the longitudinal displacement of the DNA along its axis is coupled to connector spinning.     

Structure of Bacillus subtilis Bacteriophage φ25 and φ25 Deoxyribonucleic Acid

The structure of Bacillus subtilis bacteriophage phi25 and phi25 deoxyribonucleic acid (DNA) were studied by electron microscopy. The head of phi25 is a regular polyhedron measuring 75 nm in diameter. The uncontracted tail of phi25 is 130 nm in length and includes a large, complex tail plate. Phage phi25 DNA is double-stranded and has a molecular weight of approximately 100 million as determined by electron microscopic length measurements and analytical band sedimentation in CsCl. The complementary strands of phi25 DNA contain numerous random interruptions. Chemical analysis of phi25 DNA demonstrated that 5-hydroxymethyluracil replaces thymine and that the DNA has a mole per cent (guanine plus cytosine) of 42.     

A set of expression plasmids for the synthesis of fused and unfused polypeptides in Escherichia coli

A set of plasmid expression vectors for cloning of DNA fragments containing open reading frames has been obtained. The plasmids carry the strong leftward promoter of bacteriophage lambda and the translation initiation signals from either the gene ner of bacteriophage Mu or the gene 4 of bacteriophage phi 29. The vectors could overexpress the cloned sequences as fusion peptides at the N terminus with the N-terminal segment of the phi 29 protein p4 or at the C terminus with the Escherichia coli beta-galactosidase from its 8th residue, or both. Alternatively, the cloned sequences could be directed to overproduce proteins in an unfused form. DNA fragments of the hemagglutinin gene from human influenza A virus, have been cloned in one of the plasmid vectors and some potential antigenic determinants have been characterized using monoclonal antibodies.     

Effect of Elevated Temperature on Deoxyribonucleic Acid Synthesis in Bacteriophage φ29-Infected Bacillus amyloliquefaciens

Deoxyribonucleic acid (DNA) synthesis in bacteriophage phi29-infected Bacillus amyloliquefaciens was studied at 37 and 45 C. Infectious intracellular particles appear at the same time at both temperatures, but the average burst size is reduced 45 to 50% at 45 C. There is a transient inhibition of cellular mass increase at 45 C which is not observed at the lower temperature. In addition, the rate of host DNA synthesis is reduced and the onset of viral-specific DNA replication is delayed for 6 to 9 min at 45 C. These findings allowed us to screen phage phi29 mutants which are sensitive to growth at 45 C for their ability to synthesize phi29 DNA in the absence of host DNA replication. We obtained mutants which make no viral DNA, reduced levels of DNA, or normal quantities of DNA under nonpermissive conditions. Pulse-labeled viral DNA which sediments more rapidly than mature phi29 DNA molecules was observed after gentle cell lysis and zone sedimentation. This DNA is not a precursor of normally sedimenting phi29 DNA and apparently consists of mature phi29 DNA molecules aggregated with large pieces of bacterial DNA.