Structural organization of Bacillus subtilis phage phi29. A model

Phage phi29 is a nonisometric virus producing several types of morphological variants in normal infections. The study of these variants by electron microscopy, and their comparison with those from T-even phages, suggest that the capsid of phage phi29 is a prolate icosahedron. Phage phi29 capsid consists of a major protein, p8, and an additional protein, p8.5, making up the fibers. We have determined the number of subunits of each structural protein per viral particle taking into account the phage molecular weight (between 28 and 29.6 x 10(6)), the molecular weight of each structural protein, and the mass percentage of each protein with respect to the total protein mass of the phage. These values, together with the results obtained from chemical crosslinking of the structural proteins on the phage, suggest that the capsid contains protein p8 dimers clustered in trimers.     

Replication of recombinant phi 29 DNA molecules in Bacillus subtilis protoplasts

Recombinant phi 29 DNA molecules of different sizes and containing terminal protein at one or both ends, or without terminal protein, were prepared and their replication in Bacillus subtilis protoplasts was studied. Only phi 29 DNA molecules containing terminal protein at both ends replicate in vivo. The replication of symmetric DNA recombinant molecules (dimers) gives rise to displaced strands which by self-annealing create monomers with the two DNA strands covalently linked. Viral proteins p2, p3, and p6 are essential for replication of phi 29 DNA molecules in this system. Protein p17 is not essential, but stimulates the efficiency of replication. This stimulation depends on the host used.     

Structure of phage phi 29 connector protein assembled in vivo

The protein p10 that forms the connector of phage phi 29, has been produced in Escherichia coli harboring a plasmid that carried the gene coding for this protein. The connector protein is assembled in a 13.4-S oligomer that has an apparent molecular weight of 460,000, suggesting that it is a dodecamer. The purified oligomers have been studied by electron microscopy of the isolated particles as well as by image-processing techniques (Fourier and rotational filtering) of artificially induced two-dimensional aggregates. The results show that the purified p10 is assembled in a circular structure with a hole in its center and 12 morphological units in the periphery. Both the morphology and the dimensions of this p10 oligomer are very similar to those of the upper neck collar extracted from phi 29 viral particles. The results strongly suggest the close relationship between the p10 oligomers assembled in E. coli and the ones produced in phi 29 infected Bacillis subtilis.     

Proteins induced in Bacillus subtilis infected with bacteriophage phi29

Bacteriophage phi29 induces, in UV-irradiated B. subtilis, the synthesis of 12 non-structural proteins as well as the 7 structural polypeptides which form the viral particle. The molecular weight of the nonstructural polypeptides amounts to 180,000 daltons; if all these proteins are phage-coded, this corresponds to about 30% of the information content of phi29 DNA. The phage-induced polypeptides can be classified as early and late proteins, according with the time of their appearance after infection. All the structural proteins as well as three nonstructural proteins are late proteins. The remaining nonstructural proteins appear early after infection.     

Signals in the phi 29 DNA-terminal protein template for the initiation of phage phi 29 DNA replication

The protein-free terminal fragments HindIII B and L, from the left and right ends of phi 29 DNA, respectively, but not internal fragments of similar size, were active as templates in the formation of the p3-dAMP initiation complex in an in vitro system containing purified phi 29 terminal protein p3 and DNA polymerase p2, although the activity was lower than that obtained with the phi 29 DNA-p3 complex. These results indicate the existence of specific sequences at the ends of phi 29 DNA that allow the initiation of phi 29 DNA replication. The template activity of the protein-free terminal fragments was size dependent. The protein-free single strands of the HindIII L fragment were much less active than the corresponding double-stranded fragment. Terminal protein-DNA complexes of phages PZA and phi 15, with a terminal protein closely related to the phi 29 protein p3, were more active as templates in the initiation reaction with the purified phi 29 proteins than the corresponding protein-free DNAs, as it happens in the case of phi 29. However, the terminal protein-DNA complexes of phages Nf, B103, and GA-1, with a terminal protein less related or unrelated to the phi 29 protein p3, were essentially inactive and became active after removal of the parental terminal protein. These results strongly suggest that the parental terminal protein is the major signal in the template for the initiation of phi 29 DNA replication.     

The protein covalently linked to the 5' termini of the DNA of Bacillus subtilis phage phi 29 is involved in the initiation of DNA replication

Bacillus subtilis was infected at 30° with θ29 mutants ts in each of four cistrons involved in phage DNA synthesis and, after a short pulse, the infected bacteria were shifted up to 42°C. Analysis of the labeled DNA, at different times after the shift-up, by alkaline sucrose gradient centrifugation showed that unit-length DNA was formed after infection with mutant ts3(132), suggesting a role of protein p3 in the initiation of replication. A similar result was obtained after ts2(98) infection. On the contrary, mutant ts5(17) did not produce unit-length phage DNA even at very late times after the shift-up, consistent with the possibility that this mutation affects an elongation (or maturation) process. Mutant ts6(1360) gave rise to unit-length DNA late after the shift-up suggesting that the mutation might affect an elongation or maturation process but that either it is leaky or that its function may be slowly replaced by a bacterial function. Infection of B. subtilis, under restrictive conditions, with sus mutants in cistron 3 produced unit-length θ29 DNA, both in neutral and alkaline sucrose gradients, suggesting that the parental protein itself present in the sus3 mutants is able to initiate the viral replication.     

Lethal effects of 32P decay on transfecting activity of Bacillus subtilis phage phi e DNA.

Disintegration of ³²P present in the DNA of Bacillus subtilis phage φe (a phage containing double-strand DNA) results in the loss of viability of intact phage as well as transfecting activity of isolated DNA. Only $\text{1}\text{12}$ of the ³²P disintegrations per phage DNA equivalent inactivates the intact phage while nearly every disintegration inactivates the transfecting DNA. This result provides evidence for a single-strand intermediate in the transfection of B. subtilis by φe DNA.     

A genetic study of temperature-sensitive mutants of the Bacillus subtilis bacteriophage phi 29

Temperature-sensitive mutants of the Bacillus subtilis bacteriophage ∅29, which grow at 37° but not at 45° were obtained by nitrous acid mutagenesis of free phage or by intracellular induction with nitrosoguanidine. Qualitative and quantitative complementation tests were used to group 32 mutants into 13 cistrons. Representative mutants from each of the functional groups were crossed with mutants of every other group. The genetic map of ∅29 derived from these data is linear and has a total length of 10 recombination units. Thus, 13 of the estimated 15–20 genes carried by ∅29 have been marked.     

Effect of aphidicolin and nucleotide analogs on the phage phi 29 DNA polymerase

The drugs aphidicolin and the nucleotide analogs butylanilino dATP, butylphenyl dGTP, and butylphenyl rGTP inhibited the protein-primed replication of phi 29 DNA-protein p3 in the presence of purified terminal protein p3 and phi 29 DNA polymerase p2. The effect of aphidicolin was mainly on the polymerization reaction by decreasing the rate of elongation. The nucleotide analogs inhibited both the formation of the p3-dAMP initiation complex and its further elongation, the latter being also due to a decrease in the elongation rate. When assayed with the phi 29 DNA polymerase as the only protein, all the drugs inhibited polymerization on activated DNA as well as the 3'----5' exonuclease activity of the polymerase, indicating that the target of the drugs is the phi 29 DNA polymerase itself.     

EcoRI cleavage of DNA from Bacillus subtilis phage SPO1.

The hydroxymethyluracil-containing DNA of B. subtilis phage SPO1 is cleaved only with low efficiency by restriction nuclease EcoRI. The efficiency is greatly increased by EcoRI¹ conditions, but even under these conditions, the efficiency is less than with thymine-containing DNA. In contrast with their effect on thymine-containing DNA, EcoRI¹ conditions do not appear to increase the number of sites on the SPO1 genome at which cleavage takes place. We describe the fragments produced by a (nearly) limit digest of SPO1 DNA with EcoRI¹ and assign most of the known SPO1 cistrons to specific fragments.     

Incorporation of thymine into phi e DNA during transfection of Bacillus subtilis.

Phage φe, whose DNA contains hydroxymethyl uracil in place of thymine, excludes thymine from its DNA by inducing enzymes that degrade TTP and block thymidylate synthetase. During transfection of Bacillus subtilis with φe DNA, however, progeny phage can be labeled with [³H]thymine at an average of 1000 thymine residues per phage, a level eightfold higher than that found after normal phage infection. This observation supports a transfection model in which bacterial polymerases, using thymine, repair gaps in phage DNA molecules created by the renaturation of single-strand fragments of phage DNA taken up by competent cells.     

放线菌噬菌体重组酶Sre在大肠杆菌内高效介导位点特异性重组

目的证明放线菌噬菌体重组酶Sre在大肠杆菌细胞内介导高效的位点特异性重组。方法在大肠杆菌内构建分子内重组分析系统。质粒pBZP含有方向相同的attB和attP位点，分别位于LacZ基因的两侧。将pBZP电击导入含有sre基因的大肠杆菌细胞。结果重组的发生导致LacZ基因的切除，使含有X—Gal成分的培养基上细菌菌落从蓝色变成白色。整合后DNA经酶切、PCR和DNA测序证实。发生100％重组率的attB和attP最小片段分别是50bp和47bp。结论在大肠杆菌宿主环境中放线菌噬菌体重组酶sre高效催化位点特异性重组。本分子内重组分析系统是一个简便而有效的方法。     

On the trapping of phage genomes in spores of Bacillus subtilis 168. Reciprocal exclusion of phages phi29 and phie during outgrowth of spores.

The trapping of phages φ29 and φe by spores of Bacillus subtilis 168 has been studied. The optimal time and the efficiency of trapping were different for each phage. The efficiency of φ29 was maximum (0.01-0.02) when infection occurred at stage I of sporulation; that of φe (0.5–1) 1.5 to 2 hr later. A single phage genome was sufficient to infect a spore and there is neither cooperation nor competition between genomes in the trapping event. Viral DNA synthesis is not required for trapping. A single spore may trap two viral genomes, either from the same phage or from the two unrelated phages, φ29 and φe. During outgrowth, spores carrying both genomes, φ29 and φe, produced particles of one or the other phage, but never of both. Normally φ29 multiplied but φe developed when expression of φ29 was impaired by a lethal mutation in cistron 2. However in doubly infected exponentially growing cells φe dominated and prevented reproduction of φ29.     

Characterization of a versatile in vitro DNA-packaging system based on hybrid lambda/phi 29 proheads.

We have studied the assembly of bacteriophage lambda head proteins on the phage phi 29 connector to produce in vitro chimeric proheads, whose ability to package different types of DNA depends on the physical integrity of the phi 29 connector. Terminal protein-free phi 29 as well as nonviral DNAs have been shown to be efficiently packaged by this hybrid system. An RNA, that can be provided by any of the extracts used in the complementation mixture, was required for DNA packaging, both by the hybrid system as well as by the homologous lambda system. The DNA-packaging activity of RNase-treated proheads can be restored by adding a mixture of ribosomal RNAs. There is also a requirement for a minimal length of DNA to be stably packaged. The packaging protein p16 of phi 29 can replace the lambda terminase complex in the in vitro packaging system, both with the chimeric as well as genuine lambda proheads.     

Strand and nucleotide-dependent ATPase activity of gp16 of bacterial virus phi29 DNA packaging motor

Similar to the assembly of other dsDNA viruses, bacterial virus phi29 uses a motor to translocate its DNA into a procapsid, with the aid of protein gp16 that binds to pRNA 5′/3′ helical region. To investigate the mechanism of the motor action, the kinetics of the ATPase activity of gp16 was evaluated as a function of DNA structure (ss- or ds-stranded) or chemistry (purine or pyrimidine). The k_cat and K_m in the absence of DNA was 0.016 s^− 1 and 351.0 μM, respectively, suggesting that gp16 itself is a slow-ATPase with a low affinity for substrate. The affinity of gp16 for ATP was greatly boosted by the presence of DNA or pRNA, but the ATPase rate was strongly affected by DNA structure and chemistry. The order of ATPase stimulation is poly d(pyrimidine) > dsDNA > poly d(purine), which agreed with the order of the DNA binding to gp16, as revealed by single molecule fluorescence microscopy. Interestingly, the stimulation degree by phi29 pRNA was similar to that of poly d(pyrimidine). The results suggest that pRNA accelerates gp16 ATPase activity more significantly than genomic dsDNA, albeit both pRNA and genomic DNA are involved in the contact with gp16 during DNA packaging.     

Physical map of bacteriophage phi29 DNA

Restriction endonuclease EcoRI cleaves linear phi29 DNA at four points yielding five fragments (A-E) of molecular weights 6.0 x 10(6), 3.8 x 10(6), 1.3 x 10(6), 0.6 x 10(6), and 0.3 x 10(6). The relative order of the fragments was shown to be A, B, E, D, C by analysis of the EcoRI partial digestion products and from the study of the fragments obtained by treatment of protein-containing DNA with EcoRI and trypsin. The EcoRI cleavage map of phi29 DNA has been ordered relative to the genetic map by marker rescue experiments.