Novel dimeric structure of phage φ29-encoded protein p56: insights into uracil-DNA glycosylase inhibition

Protein p56 encoded by the Bacillus subtilis phage φ29 inhibits the host uracil-DNA glycosylase (UDG) activity. To get insights into the structural basis for this inhibition, the NMR solution structure of p56 has been determined. The inhibitor defines a novel dimeric fold, stabilized by a combination of polar and extensive hydrophobic interactions. Each polypeptide chain contains three stretches of anti-parallel β-sheets and a helical region linked by three short loops. In addition, microcalorimetry titration experiments showed that it forms a tight 2:1 complex with UDG, strongly suggesting that the dimer represents the functional form of the inhibitor. This was further confirmed by the functional analysis of p56 mutants unable to assemble into dimers. We have also shown that the highly anionic region of the inhibitor plays a significant role in the inhibition of UDG. Thus, based on these findings and taking into account previous results that revealed similarities between the association mode of p56 and the phage PBS-1/PBS-2-encoded inhibitor Ugi with UDG, we propose that protein p56 might inhibit the enzyme by mimicking its DNA substrate.     

Synthesis of Bacteriophage φ29 Proteins in Bacillus subtilis

Seventeen bacteriophage phi29 proteins were detected in ultraviolet light-irradiated Bacillus subtilis by autoradiography of polyacrylamide slab gels. The appearance of phi29 proteins occurred either before or concomitantly with viral DNA replication. Viral proteins detected early in the infectious cycle consisted of nine polypeptides ranging from 5,200 daltons to 54,000 daltons. Two of the early proteins were identified as, respectively, the major capsid protein and the protein comprising the filaments which extend from the head of the virus. Late phi29 proteins were composed of eight polypeptides ranging from 14,000 daltons to 95,000 daltons. Only three late proteins were noncapsid proteins. Among the early proteins, six were synthesized at diminishing rates late in the infectious cycle. One of the early proteins (protein 12) lacked histidine, whereas two (proteins 10 and 15) lacked tryptophan. Among the 17 proteins detected, 10 were viral noncapsid proteins. The amount of viral genetic information required to code for the 17 proteins detected in these experiments (81% of the potential genetic information of phi29 DNA) compares favorably with the genetic information detected as mRNA in a previous report, 85% of the potential information on the phi29 chromosome.     

A genetic approach to the identification of functional amino acids in protein p6 of Bacillus subtilis phage ø29

Protein p6 of the Bacillus subtilis phage ø29 is essential for in vivo viral DNA replication. This protein activates the initiation of ø29 DNA replication in vitro by forming a multimeric nucleoprotein complex at the replication origins. The N-terminal region of protein p6 is involved in DNA binding, as shown by in vitro studies with p6 proteins altered by deletions or missense mutations. We report on the development of an in vivo functional assay for protein p6. This assay is based on the ability of protein p6-producing B. subtilis non-suppressor (su ^?) cells to support growth of a ø29 sus6 mutant phage. We have used this trans-complementation assay to investigate the effect on in vivo viral DNA synthesis of missense mutations introduced into the protein p6 N-terminal region. The alteration of lysine to alanine at position 2 resulted in a partially functional protein, whereas the replacement of arginine by alanine at position 6 gave rise to an inactive protein. These results indicate that arginine at position 6 is critical for the in vivo activity of protein p6. Our complementation system provides a useful genetic approach for the identification of functionally important amino acids in protein p6.     

In Situ Lysis of φ29- and SPO1-Infected Bacillus subtilis

An improved method of in situ lysis of bacteriophage-infected Bacillus subtilis was developed and used to study 29 and SPO1 phage structures produced by individual cells.     

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.     

Genetic Study of Suppressor-Sensitive Mutants of the Bacillus subtilis Bacteriophage φ29

With bacteriophage phi29 of Bacillus subtilis 133, suppressor-sensitive (sus) hydroxylamine mutants have been isolated. Intracistronic and intercistronic quantitative complementation placed the mutants in 13 cistrons, and three-factor crosses have been used to assign an unambiguous order for 10 cistrons. Recombination frequencies have been presented for several regions of the genome to facilitate comparison of the sus system with the previously published temperature-sensitive mapping systems.     

Abortive Infection of Sporulating Bacillus subtilis 168 by φ2 Bacteriophage

Bacteriophage phi2 is unable to replicate in Bacillus subtilis 168. Although some phage deoxyribonucleic acid (DNA) synthesis can occur, the DNA made is not biologically active and sedimentation analysis reveals that it is smaller in size than that of mature DNA or DNA isolated from phi2-infected permissive hosts. Messenger ribonucleic acid hybridizable with phi2 DNA is also synthesized in phi2-infected cells of 168. Mutants of 168 which are permissive hosts for phi2 have been isolated. These mutants are defective in sporulation and possess the phenotype of "early sporulation mutants." The majority map in two locations, one near the lys locus opposite the trp locus (spoA locus) and the other tightly linked to a phe locus.     

Isolation and Properties of Bacillus subtilis Strains Lysogenized by a Clear Plaque Mutant of Bacteriophage φ105

A clear plaque mutant of the temperate Bacillus phage phi105 lysogenized a small fraction of infected cells forming an integrated prophage at or near the normal phi105 insertion site. These lysogens exhibited a spontaneous induction rate approximately 1,000-fold lower than wild type and were noninducible (ind(-)) by mitomycin C. Prophage was induced, however, when competent cultures were incubated with transforming DNA. The ind(-) phenotype could not be attributed solely to the clear plaque mutation and appears to involve a cell-specific factor. Lysogenization by the clear plaque mutant, in contrast to wild-type phage, did not cause a marked reduction in transformation efficiency.     

Morphology and Physiology of the Intracellular Development of Bacillus subtilis Bacteriophage φ25

The morphology of the intracellular development of bacteriophage phi25 in Bacillus subtilis 168M has been correlated with nucleic acid synthesis in infected cells. Host deoxyribonucleic acid (DNA) synthesis was shut off by a phage-induced enzyme within 5 min after infection, and another phage-mediated function extensively degraded host DNA at the time of cell lysis. Synthesis of phage DNA in infected cells began within 5 min and continued until late in the rise period. After phage DNA synthesis and coinciding with lysis, much of the unpackaged, newly synthesized phage DNA was degraded. Studies of thin sections of phi25 infected cells suggested that unfilled capsids may be precursors to filled capsids in the packaging process. To assess dependence of capsid formation on phage DNA replication, cells were either treated with mitomycin C and infected with normal phage or infected with ultraviolet-irradiated (99% killed) phi25. Only empty capsids were found in these cells, indicating that capsid production may be independent of the presence of newly synthesized viral DNA.     

Glucosyl Glycerophosphoric Acid and its Polymer Excreted by α-Amylase-forming Bacillus subtilis

α-Amylase formation by washed cell suspensions of Bac. subtilis was found to be accompanied by the excretion of a compound consisting of glucose, glycerol and phosphoric acid. It was excreted as a polymer and a monomer. The former, a kind of teichoic acid, was significantly dominant in quantity when the cells were incubated under the conditions suitable for α-amylase formation. On the other hand, the monomer prevailed when the bacterial cells were under the unfavorable conditions for the enzyme formation.

Both compounds were purified by ion exchange column chromatography. Chemical and enzymatic investigations revealed the following structures: 2-O-α-d-glucopyranosyl-glycerol-3-monophosphoric acid for the monomer, and a polymerized form of the monomer through phosphodiester linkages involving the hydroxyl groups on C3 of the glycerol, for the polymer.     

Analysis of Bacteriophage φ29 Gene Function: Protein Synthesis in Suppressor-Sensitive Mutant Infection of Bacillus subtilis

Phage phi29 suppressor-sensitive (sus) mutants of 14 cistrons have been examined for production of (14)C-labeled viral-specific proteins in restrictive infections of Bacillus subtilis. Proteins specified by four cistrons (H, J, L, and N) have been resolved and identified by sodium dodecyl sulfate gel electrophoresis and autoradiography, and fragments of the normal polypeptides were detected. Mutants of six cistrons (C, D, E, F, I, and M) demonstrated two or more missing bands in the gel profiles, and thus some of these gene products may have regulatory functions. Mutation was detected in at least five genes coding for low-molecular-weight proteins, but a conditionally lethal mutant in only one of these genes has been isolated. Preliminary evidence that a precursor protein is cleaved to generate the neck appendage structural protein and a low-molecular-weight product has been obtained.     

A phage protein that binds φC31 integrase to switch its directionality

The serine integrase, Int, from the Streptomyces phage φC31 mediates the integration and excision of the phage genome into and out of the host chromosome. Integrases usually require a recombination directionality factor (RDF) or Xis to control integration and excision and, as φC31 Int only mediates integration in the absence of other phage proteins, we sought to identify a φC31 RDF. Here we report that the φC31 early protein, gp3 activated attL x attR recombination and inhibited attP x attB recombination. Gp3 binds to Int in solution and when Int is bound to the attachment sites. Kinetic analysis of the excision reaction suggested that gp3 modifies the interactions between Int and the substrates to form an active recombinase. In the presence of gp3, Int assembles an excision synaptic complex and the accumulation of the integration complex is inhibited. The structure of the excision synaptic complex, like that of the hyperactive mutant of Int, IntE449K, appeared to be biased towards one that favours the production of correctly joined products. The functional properties of φC31 gp3 resemble those of the evolutionarily unrelated RDF from phage Bxb1, suggesting that these two RDFs have arisen through convergent evolution.     

Growth of Bacteriophage φ105 and Its Deoxyribonucleic Acid in Radiation-Sensitive Mutants of Bacillus subtilis

Growth of phage phi105 and its deoxyribonucleic acid (DNA) was studied in radiation-sensitive mutants of Bacillus subtilis. The recA gene is required for optimal prophage induction with mitomycin C and for infectivity of prophage DNA. rec B gene is required for marker rescue from mature DNA. The importance of bacterial genes for phage DNA activity seems to depend on phage DNA structure.     

Control of ��-amylase production by Bacillus subtilis

This study proposes two adaptive control algorithms for the fed-batch production of α-amylase. The first one uses online information from hardware measuring glucose. Online information of both biomass and glucose concentrations measured with different frequency is used in the second algorithm. Hardware measuring variables are inputs for software sensors of glucose concentration and (specific) glucose consumption rate. Either of the algorithms do not require any kinetic coefficients. This is a benefit, because the kinetic coefficients can vary during cultivation and between cultivations, leading to low process reproducibility and the non-stationary state of the bioprocess. The results of simulation investigations show good performance of the proposed control schemes.     

Selective Replication of Bacteriophage φ29 Deoxyribonucleic Acid in 6-(p-Hydroxyphenylazo)-Uracil-Treated Bacillus subtilis

Phage phi29 deoxyribonucleic acid (DNA) replicated under conditions where semiconservative DNA production in Bacillus subtilis host cells was blocked with 6-(p-hydroxyphenylazo)-uracil (HPUra). The time of initiation of phi29 DNA replication was not affected by HPUra, and normal quantities of viable phage were produced in the presence of the inhibitor. Studies with conditional lethal mutants of phage phi29 demonstrated the usefulness of HPUra for detection of viral-specific DNA production.     

Gene Expression During the Development of Bacillus subtilis Bacteriophage φ29 II. Resolution of Viral-Specific Ribonucleic Acid Molecules

The ribonucleic acid (RNA) specified by bacteriophage 29 was isolated under conditions which minimized physical and enzymatic degradation, reduced aggregation, and enriched for completed molecules. This RNA was fractionated both by sedimentation through sucrose density gradients and electrophoresis through polyacrylamide gels to measure the size and relative amount of each component. Early RNA consisted of six components of molecular weight 0.75 × 10⁶, 0.44 × 10⁶, 0.37 × 10⁶, 0.25 × 10⁶, 0.09 × 10⁶, and 0.04 × 10⁶, accounting for 35% of the coding capacity of 29 deoxyribonucleic acid (DNA). All of these components except the one at 0.44 × 10⁶ were detected when infection occurred in the presence of chloramphenicol. Synthesis of the major early component (0.75 × 10⁶) ceased shortly after the onset of viral DNA synthesis. The other species of early RNA were synthesized throughout the latent period. Three additional components, 1.75 × 10⁶, 0.93 × 10⁶, and 0.07 × 10⁶, appear at late times. The two large RNAs may be polycistronic messenger RNAs corresponding to the seven viral capsid proteins.