Teiichi Minagawa

Structure and assembly of bacteriophage T3 tails

Abstract Bacteriophage T3 gene products found in the virion tail structure are identified by in vitro complementation and serum blocking activity. On the basis of these measurements, a pathway for the assembly of the T3 tail is proposed. The products of genes 11 and 12 (gp11 and gp12) are assembled on the head to form the tail. The assembly of gp11 and gp12 proceeds cooperatively, so that in the absence of either protein, attachment of the other does not occur. T3 serum blocking activity is due to gp17. Gp17 is assembled onto the tail structure after attachment of gpll and gp12 onto the head. The structure and composition of purified T3 tails have been examined. Purified tails have a sixfold longitudinal axis of symmetry. When viewed along the symmetry axis, tails appear hexagonal with a hole in the center and with bent tail fibers radiating from the apices. Tail fiber proteins are controlled by gene 17. Tails isolated from osmotically shocked T3 phage contain gp11, gp12, and gp17. In addition, isolated tails also contain gp8, a minor head protein, suggesting that gp8 is located at a unique site in the T3 head where the tail attaches.

In vitro packaging of phage T3 DNA

Abstract We have developed an in vitro system for packaging of mature bacteriophage DNA. DNA purified from phage T3, when incubated in a reaction mixture containing binary combinations of extracts prepared from Escherichia coli cells infected with T3 amber mutants of genes involved in DNA replication (genes 3, 4, 5, and 6), is packaged intact and fully conserved into infectious particles. The products of genes 3, 4, 5, 6, and 19 (necessary for DNA maturation in vivo ) are required for packaging in vitro . Other requirements are Mg 2 +, spermidine, and either ATP or GTP. The packaging efficiency decreases with decreasing concentration of exogenous DNA. Exogenous DNA was converted into concatemeric forms in the reaction mixture, depending upon the products of genes 3, 4, 5, and 6. These results suggest that the exogenous DNA proceeds by way of concatemeric intermediates before being packaged. Genetic recombination also occurs in the in vitro packaging system. Recombination is accompanied by physical exchange of sequences between exogenous DNA molecules. ATP is required for in vitro recombination; GTP cannot be substituted for ATP in this reaction.

Overproduction and purification of the products of bacteriophage T3 genes 18 and 19, two genes involved in DNA packaging

The products of gene 18 (gp18) and gene 19 (gp19) of bacteriophage T3 are noncapsid proteins involved in DNA packaging. A restriction fragment containing gene 18 or 19 was cloned into the plasmid vector pNT45 under the control of the inducible leftward promoter (PL) of phage lambda. Induction of transcription of gene 18 or 19 by derepression of the PL promoter led to the synthesis of a high level of gp18 or gp19. By using complementation of T3 DNA packaging in vitro as an assay, gp18 and gp19 were purified to near homogeneity. The overall yields of gp18 and gp19 were 1.4 mg and 0.35 mg, respectively, from 1 g wet wt cells. Addition of gp18 to the in vitro DNA packaging system resulted in increased phage production with increasing amounts of gp18 until 10% of the DNA was packaged into infectious phage particles. In contrast, addition of gp19 to the packaging system initially caused an increase in phage production, but increasing amounts of gp19 inhibited DNA packaging.

Role of gene 8 product in morphogenesis of bacteriophage T3

The product of gene 8 (gp8) of T3 phage is one of the minor head proteins located at the phage head-tail junction. To determine the role of gp8, an amber (8-) and four temperature-sensitive mutants (ts8) were characterized by sedimentation analysis, polyacrylamide gel electrophoresis, and extract complementation. Neither DNA-containing particles nor empty particles were formed in cells infected with 8-. In addition, prohead assembly was greatly reduced. Prohead assembly was also blocked in cells infected with all ts8 mutants at 42 degrees and with some ts8 even at 37 degrees. Proheads containing gpts8 were converted to empty heads when cell lysates were treated with chloroform. The protein compositions of proheads showed that the minor head proteins, gp8, gp15, and gp16, were lost from proheads formed in cells infected with ts8, but these minor proteins were present in proheads formed in cells infected with double mutants of ts8 and 5- or 19-, which are defective in DNA synthesis or DNA maturation, respectively. In vitro complementation experiments suggested that a ts mutation in gene 8 affected not only DNA packaging but also subsequent assembly steps. From these results, it is concluded that gp8 plays multiple roles in T3 phage morphogenesis, including prohead assembly, prohead stabilization, DNA packaging, and subsequent events.

Substrate specificity of gene 49-controlled deoxyribonuclease of bacteriophage T4: special reference to DNA packaging.

Abstract It has been reported that T4 gene 49 controls a nuclease-like activity which attacks very fast sedimenting DNA (VFS-DNA) formed in cells infected with a mutant defective in gene 49 (Frankel, F. R., Batcheler, M. L., and Clark, C. K. (1971) J. Mol. Biol. 62: 439–463). The protein responsible for this activity was partially purified and shown to be an endonuclease. The DNase only attacks T4 DNA having a specific structure; besides VFS-DNA, it cleaves T4 DNA which contains gaps with an internal 5′-phosphorylated terminus. Native, denatured, and nicked T4 DNAs are not susceptible to the enzyme action.

Cloning and sequencing of the genetic right end of bacteriophage T3 DNA.

The genetic right end of phage T3 DNA, from the beginning of gene 17, was cloned and sequenced. Genes 17, 18, and 19 were identified by comparing the sequence with the genetic map and by comparing the calculated and observed molecular weights of gene products. N-terminal amino acid sequence of the gene 17 product (gp17) predicted from the nucleotide sequence was consistent with the data from the analysis of purified gp17. Gene 17.5 was identified as the lysis gene on the basis of the presence of a nonsense codon within an open reading frame in the sequence of DNA from an amber mutant of lysis gene. In addition, five potential genes have been identified. Sequences corresponding to a promoter for phage T7 RNA polymerase (Rosa and Andrews, 1981) and to a class-III promoter for phage T3 RNA polymerase (Sarker et al., 1985) were found. The genomic organization and the nucleotide and deduced amino acid sequences of T3 were compared with those of T7. The genomic organizations of T3 and T7 were identical in this region. The sequence comparisons of T3 and T7 DNA point out the highly conserved sequences in all genes but also heavily varied regions in some genes. From these comparisons, possible implications with regard to structural and functional domains within several genes are discussed.

Mutation in bacteriophage T3 affecting host cell lysis

Abstract Amber mutants of T3 phage defective in cell lysis were isolated. The mutational sites involve a new gene (called lys ) between gene 17 and gene 19. Su − host cells infected with the mutant fail to lyse beyond the normal latent period though lysozyme is normally produced. Progeny phage production continues a little longer in mutant infected cells than in wild-type infected cells.

Purification and characterization of gene 17 product of bacteriophage T3

Tail fiber proteins of bacteriophage T3 are encoded by gene 17. By using in vitro complementation for phage assembly as an assay, the product of gene 17 (gp17) was purified to near homogeneity from cells infected with a double mutant of T3 defective in DNA synthesis and head assembly. The purified gp17 consists of a single polypeptide having a molecular weight of 67,000. Electron microscopy of the purified gp17 showed a fiber structure similar to the tail fiber in a virion. The subunit structure of the purified, native gp17 was analyzed by using a crosslinking agent, dimethyl suberimidate. The results indicate that native gp17 is a trimer of gp17 monomer.

Isolation and characterization of bacteriophage T3/T7 hybrids and their use in studies on molecular basis of DNA-packaging specificity

In vitro DNA-packaging systems of bacteriophages T3 and T7 packaged homologous DNA more efficiently than heterologous DNA. Packaging of phage DNA proceeds by way of concatemeric intermediates (H. Fujisawa, J. Miyazaki, and T. Minagawa (1978), Virology 87, 394-400). The conversion of mature homologous and heterologous DNAs to concatemers was efficient in both the T3- and T7-packaging systems. In vitro complementation experiments indicate that the gene 19 product (gp19) specifies which DNA enters the capsid. To identify DNA regions recognized by the packaging systems, T3/T7 hybrids were constructed and physical maps of the hybrid DNAs were determined by restriction enzyme analysis. By comparing restriction maps and in vitro packaging of hybrid DNAs, it is concluded that the sequence responsible for specificity of DNA packaging is confined within 5% of the ends of the T3 and T7 genomes.

Endonuclease of T4 ghosts

Abstract An endonuclease was found to associate with purified T4 particles. Its activity was low with intact phages but became distinct when phage particles were disrupted by osmotic shock, and more distinct when they were disassembled by guanidine. The enzyme cleaved all DNA species tested so far, including glucosylated and nonglucosylated T4 DNA, T3 DNA and fd DNA. The cleavage was more efficient with doublestranded DNA than with single-stranded DNA. Defective capsids of mutants of genes 4, 10, 14, 16, and 49 had enzyme activity which was comparable to capsids of the wild type, suggesting that the enzyme is located on capsids. The nucleolytic activity was barely detectable, if at all, with four out of five mutants of gene 17.