Characterization, overproduction and purification of the product of gene 1 of Bacillus subtilis phage phi 29

Unit-length phi 29 DNA was not synthesized after restrictive infection of Bacillus subtilis with the phi 29 mutant sus1(629) indicating that the phage phi 29 protein p1 is needed for the viral DNA replication. Sequencing of the ORF-6 of mutant sus1(629) showed that a C in the wild-type (wt) phage had been changed to a T at nt position 19 of the ORF-6, giving rise to a TAA ochre codon, indicating that this ORF corresponds to gene 1. ORF-6 was cloned in plasmid pPLc28 under the control of the pL promoter of phage lambda and, after induction, a protein of about 10 kDa was overproduced, which was absent in the corresponding cells harbouring a recombinant plasmid with the sus1(629) mutation, indicating that the 10-kDa protein is the product of gene 1. In addition, a protein of lower Mr was synthesized after induction of the cells harbouring recombinant plasmids with the wt or the sus1(629) DNA. Both proteins were purified and characterized by N-terminal sequence determination and amino acid analysis. The low-Mr protein, named delta 1, has a size of 6 kDa and corresponds to an internal in-phase initiation event in ORF-6.     

X-ray scattering of the non-isometric Bacillus subtilis phage phi29.

The non-isometric virus φ29 and its empty capsid have enough elements of symmetry so that their size and approximate shape can be determined by low-angle X-ray scattering. The scattering curve of the virus can be simulated by a cylinder of 390 Å diameter and 460 Å height. The protein coat has a thickness of about 30 Å. The DNA is packed tightly and regularly in the phage head.     

A novel DNA polymerase induced by Bacillus subtilis phage phi 29.

A novel DNA polymerase induced by Bacillus subtilis bacteriophage phi 29 has been identified. This polymerase can be separated from host DNA polymerase, by fractionation of extracts prepared from phage infected cells, using phosphocellulose chromatography. The isolated polymerase prefers poly(dA)oligo(dT) as template. The DNA polymerase isolated from the cells infected with a gene 2 temperature sensitive mutant (ts2) showed greater heat-lability than that induced by wild type phi 29. The ts2 DNA polymerase was also thermolabile for its activity in the formation of a covalent complex between phi 29 terminal protein and dAMP, the initiation step of phi 29 DNA replication. These findings indicate that gene 2 is the structural gene for a phi 29 DNA polymerase required for the complex formation step of DNA initiation.     

Relevance of UP elements for three strong Bacillus subtilis phage phi29 promoters

Various Escherichia coli promoters contain, in addition to the classical -35 and -10 hexamers, a third recognition element, named the UP element. Located upstream of the -35 box, UP elements stimulate promoter activity by forming a docking site for the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). Accumulating genetic, biochemical and structural information has provided a detailed picture on the molecular mechanism underlying UP element-dependent promoter stimulation in E.coli. However, far less is known about functional UP elements of Bacillus subtilis promoters. Here we analyse the strong early sigma(A)-RNA polymerase-dependent promoters C2, A2c and A2b of the lytic B.subtilis phage phi29. We demonstrate that the phage promoters contain functional UP elements although their contribution to promoter strength is very different. Moreover, we show that the UP element of the A2b promoter, being critical for its activity, is located further upstream of the -35 box than most E.coli UP elements. The importance of the UP elements for the phage promoters and how they relate to other UP elements are discussed.     

Characterization of a protein covalently linked to the 5' termini of the DNA of Bacillus subtilis phage phi29.

We have isolated a covalent DNA-protein complex from bacteriophage φ29 particles. Polyacrylamide gel electrophoresis and tryptic peptide analysis showed that the protein present in the complex is very similar or identical to p3, an early induced protein essential for viral DNA replication.When the DNA-protein complex is treated with the restriction endonuclease EcoRI, the protein is specifically associated to the two terminal fragments, A and C. The protein is probably linked to the 5′ termini of the DNA since proteinase K-treated DNA is resistant to phosphorylation with polynucleotide kinase, even after treatment with alkaline phosphatase, while it is sensitive to exonuclease III. By electron microscopy the protein is visualized as a dot located at the ends of unit length DNA molecules.Mixed infection of Bacillus subtilis, at 42 °C, with ts² mutants in cistrons 2 and 3 only produces ts 2 progeny. This finding suggests that an inactive protein p3 bound to the DNA of the ts 3 mutant is not replaced by a functional protein and, as a consequence, replication of the ts 3 DNA does not occur.     

Overproduction and purification of the connector protein of Bacillus subtilis phage phi 29.

A phi 29 DNA fragment containing genes 10 and 11, coding for the connector protein and the lower collar protein, respectively, has been cloned in the pBR322 derivative plasmid pKC30 under the control of the PL promoter of phage lambda. Two polypeptides with the electrophoretic mobility of proteins p10 and p11 were labelled with 35S-methionine after heat induction. The proteins were characterized as p10 and p11 by radioimmunoassay and they represented about 10% and 7%, respectively, of the total E. coli protein after 4 hours of induction. These proteins represent less than 1% of the B. subtilis protein in phi 29-infected cells. Protein p10 has been highly purified from the E. coli cells carrying the recombinant plasmid. Antibodies raised against the purified protein p10 reacted with the connector protein produced in phi 29-infected B. subtilis.     

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.     

Protein p56 from the Bacillus subtilis phage phi29 inhibits DNA-binding ability of uracil-DNA glycosylase

Protein p56 (56 amino acids) from the Bacillus subtilis phage ϕ29 inactivates the host uracil-DNA glycosylase (UDG), an enzyme involved in the base excision repair pathway. At present, p56 is the only known example of a UDG inhibitor encoded by a non-uracil containing viral DNA. Using analytical ultracentrifugation methods, we found that protein p56 formed dimers at physiological concentrations. In addition, circular dichroism spectroscopic analyses revealed that protein p56 had a high content of β-strands (around 40%). To understand the mechanism underlying UDG inhibition by p56, we carried out in vitro experiments using the Escherichia coli UDG enzyme. The highly acidic protein p56 was able to compete with DNA for binding to UDG. Moreover, the interaction between p56 and UDG blocked DNA binding by UDG. We also demonstrated that Ugi, a protein that interacts with the DNA-binding domain of UDG, was able to replace protein p56 previously bound to the UDG enzyme. These results suggest that protein p56 could be a novel naturally occurring DNA mimicry.     

Construction and characterization of recombinant phage phi 105 d(Cmrmet) for cloning in Bacillus subtilis

A 1.6 kb fragment of DNA of plasmid pBD64, obtained after partial digestion with HpaII, carrying a chloramphenicol-resistance determinant and a single site for the enzyme Bg/II, was inserted into the genome of defective phage phi 105 d/ys. Two types of phage were subsequently isolated and both transduced cells of Bacillus subtilis to chloramphenicol resistance. One type contained 26 kb and the other 32 kb of DNA. Bacillus subtilis chromosomal DNA fragments generated by cleavage with Bg/II were ligated into the unique Bg/II site within the smaller phage genome. A specialized transducing phage was isolated which carried the metC gene on a 6 kb Bg/II fragment. This phage, denoted phi 105 d(Cmrmet), transduced B. subtilis strain MB79 pheA12 metC3 to Met+ and to chloramphenicol resistance, and the metC3 mutation was complemented in transductants.     

The Bacillus subtilis phage phi 29 protein p16.7, involved in phi 29 DNA replication, is a membrane-localized single-stranded DNA-binding protein.

The functional role of the phi 29-encoded integral membrane protein p16.7 in phage DNA replication was studied using a soluble variant, p16.7A, lacking the N-terminal membrane-spanning domain. Because of the protein-primed mechanism of DNA replication, the bacteriophage phi 29 replication intermediates contain long stretches of single-stranded DNA (ssDNA). Protein p16.7A was found to be an ssDNA-binding protein. In addition, by direct and functional analysis we show that protein p16.7A binds to the stretches of ssDNA of the phi 29 DNA replication intermediates. Properties of protein p16.7A were compared with those of the phi 29-encoded single-stranded DNA-binding protein p5. The results obtained show that both proteins have different, non-overlapping functions. The likely role of p16.7 in attaching phi 29 DNA replication intermediates to the membrane of the infected cell is discussed. Homologues of gene 16.7 are present in phi 29-related phages, suggesting that the proposed role of p16.7 is conserved in this family of phages.     

The missing link in phage lysis of gram-positive bacteria: gene 14 of Bacillus subtilis phage phi 29 encodes the functional homolog of lambda S protein.

In most bacteriophages of gram-negative bacteria, the phage endolysin is released to its murein substrate through a lesion in the inner membrane. The lesion is brought about by a second phage-encoded lysis function. For the first time, we present evidence that the same strategy is elaborated by a phage of a gram-positive bacterium. Thus, there appears to be an evolutionarily conserved lysis pathway for most phages whether their host bacterium is gram negative or gram positive. Phage phi 29 gene 14, the product of which is required for efficient lysis of Bacillus subtilis, was cloned in Escherichia coli. Production of protein 14 in E. coli resulted in cell death, whereas production of protein 14 concomitantly with the phi 29 lysozyme or unrelated murein-degrading enzymes led to lysis, suggesting that membrane-bound protein 14 induces a nonspecific lesion in the cytoplasmic membrane.     

Bacillus subtilis mutants defective in bacteriophage phi 29 head assembly.

Virus assembly mutants of asporogenous Bacillus subtilis defective in bacteriophage phi 29 head assembly were detected by the use of antibodies that reacted strongly with the free dodecameric phi 29 portal vertex composed of gene product 10 (gp10) but weakly with the portal vertex assembled into proheads or phage. Phage adsorption and the synthesis of phage proteins, DNA-gene product 3, and prohead RNA were normal in these mutants, but prohead and phage production was greatly reduced. The assembly defect was transferred to competent B. subtilis by transformation and transduction. PBS1 transduction showed that the vam locus was linked to Tn917 located at 317 degrees on the B. subtilis chromosome.