Nucleotide sequence at the site of single-strand DNA breaks in Pseudomonas aeruginosa bacteriophage phi kF77

It is known that DNA molecules from the phage group phi k specific to Pseudomonas aeruginosa possess single-strand breaks (nicks). The sequences around the nicks in the bacteriophage phi kF77 DNA have been determined by various methods. In addition, an EcoRV-HindIII fragment, containing a nick, was cloned into the plasmid pUC9 and sequenced by Maxam-Gilbert technique. The sequence common for all nicks was CCTAohpCTCCGG.     

Localization of canonical single-strand breaks in DNA of the Pseudomonas aeruginosa bacteriophage phi kF77

Bacteriophages phi k of P. aeruginosa were characterized by the presence of T4 DNA-ligase-repaired, single-chain breaks in their genome. A restriction map was constructed for one of these phages (phi kF77) with restriction endonucleases SalI, HindIII, EcoRI, MluI, XbaI and ClaI. phi kF77 DNA was resistant to the cleavage by BamHI, BglII, HpaI, PstI, PvuII and XhoI endonucleases. Single-chain breaks were mapped by means of electron microscopy of partially denatured DNA molecules, electrophoretic studies of denatured DNA and S1-analysis. Four major nicks were thus located which were revealed in 33 to 83% of DNA molecules. On the basis of mutual hybridization of single-strand DNA fragments it was shown that all nicks are located in one of the phi kF77 DNA chains. S1-treated hybrids of 32P-labeled single-strand fragments with intact DNA chain were used for DNA orientation. The physical map of phi kF77 DNA was constructed.     

Dynamic interaction of virulent Pseudomonas aeruginosa bacteriophages with the host bacteria

The population interactions of Pseudomonas aeruginosa virulent bacteriophages phi kF77 and phi mnF82 with host bacterial cells were studied in dynamics under the conditions of continuous cultivation in the chemostat regime with glucose limitation. Two different types of maintaining the bacterium and its specific bacteriophages in the population were detected. When P. aeruginosa was cultivated with phage phi mnF82, such a maintenance was realized due to the successive appearance of bacterial mutants resistant to the phage and of phage mutants overcoming this resistance. When P. aeruginosa was cultivated with phage phi kF77, these were maintained owing to the ability of P. aeruginosa to form unstable phage-resistant variants with the segregation of phage-sensitive cells.     

Physical map of Pseudomonas aeruginosa phage phi kF77 genome. Localization of sites sensitive to restriction endonucleases

A physical map of the P. aeruginosa bacteriophage phi kF77 has been constructed using the restriction endonucleases SalI, HindIII, EcoRI, EcoRV, MuI, XbaI, ClaI. The phi kF77 DNA is resistant to cleavage by the restriction endonucleases BamHI, BglII, HpaI, PstI, PvuII, SmaI, XhoI.     

Distribution of "minor" nicks in bacteriophage T5 DNA.

Bacteriophage T5 DNA can be released from the phage particle in such a way that one end of 5 to 10% of the DNA molecules remains attached to either the phage head or tail. Under partial denaturation conditions, the DNA preferentially denatures in the vicinity of a nick so that the nicks can be located relative to the end that remains attached to the phage head or tail. Two classes of nicks were found. "Major" nicks were those found in more than 20% of the molecules and were located at the same points along the DNA molecule as reported by others. "Minor" nicks were found in 5 to 10% of the molecules and often occurred at specific locations near a "major" nick.     

Molecular structure of hybrid phage phi80hy43

The phage hybrid phi80hy43 derived from a vegetative cross phi80cIhlambda x lambdacIc17 was constructed for discrimination phi80mono- and polylysogens. Molecular structure of this hybrid was established using heteroduplex analysis and restriction endonuclease EcoRI. It is found that the hybrid phi80hy43 represents a phage phi80 containing a foreign piece of DNA between genes cI and 0. The length of this piece of DNA comprises 0.7%, corresponding to the length of cy-cII region of th phage lambda. So it is believed that the hybrid phi80hy43 was formed due to insertion of the lambdacy region with the mutation c17 into phi80hlambda phage genome.     

Translocation of nicked but not gapped DNA by the packaging motor of bacteriophage phi29

The biomolecular mechanism that the double-stranded DNA viruses employ to insert and package their genomic DNA into a preformed procapsid is still elusive. To better characterize this process, we investigated packaging of bacteriophage phi29 DNA with structural alterations. phi29 DNA was modified in vitro by nicking at random sites with DNase I, or at specific sites with nicking enzyme N.BbvC IA. Single-strand gaps were created by expanding site-specific nicks with T4 DNA polymerase. Packaging of modified phi29 DNA was studied in a completely defined in vitro system. Nicked DNA was packaged at full genome length and with the same efficiency as untreated DNA. Nicks were not repaired during packaging. Gapped DNA was packaged only as a fragment corresponding to the DNA between the genome terminus and gap. Thus the phi29 DNA packaging machinery tolerated nicks, but stopped at gaps. The packaging motor did not require a nick-free DNA backbone, but the presence of both DNA strands, for uninterrupted packaging.     

Characterization of Infectious Deoxyribonucleic Acid from Temperate Bacillus subtilis Bacteriophage φ 105

Phenol-extracted, infectious deoxyribonucleic acid (DNA) species from phi105 phage particles, from phi105 lysogenic bacteria, and from induced phi105 lysogenic bacteria were sedimented in sucrose gradients. Infectious DNA from phi105 particles sedimented like the bulk of mature phage DNA in neutral sucrose. Infectivity of prophage DNA was associated with fast-sedimenting material of heterogenous size. Infectious vegetative phage DNA sedimented somewhat faster than mature phage DNA; it was rapidly converted to a poorly infectious form during the infection.     

Growth and DNA synthesis of bacteriophage phi x174 in a dnaP mutant of Escherichia coli.

phi X174am3trD, a temperature-resistant mutant of bacteriophage phi X174am3, exhibited a reduced ability to grow in a dnaP mutant, Escherichia coli KM107, at the restrictive temperature (43 degrees C). Under conditions at which the dnaP gene function was inactivated, the amount and the rate of phi X174am3trD DNA synthesis were reduced. The efficiency of phage attachment to E. coli KM107 at 43 degrees C was the same as to the parental strain, E. coli KD4301, but phage eclipse and phage DNA penetration were inhibited in E. coli KM107 at 43 degrees C. It is suggested that the dnaP gene product, which is necessary for the initiation of host DNA replication, participates in the conversion of attached phages to eclipsed particles and in phage DNA penetration in vivo in normal infection.     

Properties of a DNA repair endonuclease from mouse plasmacytoma cells

The properties of a DNA-repair endonuclease isolated from mouse plasmacytoma cells have been further studied. It acted on ultraviolet-light-irradiated supercoiled DNA, and the requirement for a supercoiled substrate was absolute at ultraviolet light doses below 1.5 kJ m-2. At higher doses relaxed DNA could also serve as a substrate, but the activity on this DNA was due mostly to hydrolysis of ultraviolet-light-induced apurinic/apyrimidinic (AP) sites by the AP-endonuclease activity associated with the enzyme. The latter enzyme activity did not require a supercoiled form of the DNA. The enzyme also introduced nicks in unirradiated d(A-T)n. The nicked ultraviolet-light-irradiated DNA served as a substrate for DNA polymerase I, showing that the nicks contained free 3'-OH ends. Treatment of the nicked ultraviolet-light-irradiated DNA with bacterial alkaline phosphatase followed by T4 polynucleotide kinase, resulted in the phosphorylation of the 5' ends of the nicks, indicating that the nicks possessed a 5'-phosphate group; 5'- and 3'-mononucleotide analyses of the labelled DNA suggested that the enzyme introduced breaks primarily between G and T residues. The enzyme did not act on any specific region on the supercoiled DNA molecule; it produced random nicks in ultraviolet-light-modified phi X 174 replicative form I DNA. Antibodies raised against ultraviolet-light-irradiated DNA inhibited the activity. DNA adducts such as N-acetoxy-2-acetylaminofluorene and psoralen were not recognized by the enzyme. It is suggested that the enzyme has a specificity directed toward helical distortions.     

The Lactococcal Plasmid pNP40 Encodes a Third Bacteriophage Resistance Mechanism, One Which Affects Phage DNA Penetration

The lactococcal plasmid pNP40 mediates insensitivity to (phi)c2 by an early-acting phage resistance mechanism in addition to the previously identified abortive infection system, AbiF, in the Lactococcus lactis subsp. lactis MG1614 background. A second abortive infection determinant on pNP40, AbiE, does not confer resistance to (phi)c2. The early-acting mechanism on pNP40 does not prevent phage adsorption nor does it appear to operate by restriction/modification. Phage DNA was not detected in pNP40-containing cells until 30 min following exposure to (phi)c2 compared with 5 min in a sensitive host; however, electroporation of phage DNA into resistant hosts resulted in the release of phage progeny from a dramatically elevated number of cells compared with conventionally infected hosts. It appears therefore that pNP40 encodes a novel phage resistance mechanism which blocks DNA penetration specifically for (phi)c2.     

Synthesis of phage-specific transfer RNA molecules by vibriophage phi 149

32P-Labelled tRNA was isolated from uninfected and phage phi 149-infected Vibrio cholerae cells. These tRNA preparations were then hybridised with DNA isolated from phage phi 149. Significant hybridisation was observed only with tRNA from phage phi 149-infected cells. This strongly suggests that infection of classical vibrio with phage phi 149 results in the synthesis of phage-specific tRNA molecules.     

Cloning of restriction fragments of DNA from staphylococcal bacteriophage phi 11.

EcoRI fragments of Staphylococcus aureus bacteriophage phi 11 DNA were cloned in vector plasmid pSA2100 in S. aureus. The clones were analyzed in marker rescue experiments with suppressor- and temperature-sensitive mutants of phi 11 to correlate the genetic and physical map. Several mutants could be identified on the physical map, and a clone containing fragment EcoRI-B of phi 11 DNA expressed immunity to phage infection. In addition, it was found that recombinant plasmids containing phi 11 DNA sequences can be transferred by high-frequency transduction after phage phi 11 infection of host cells.     

Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: DNA-gp3 intermediate in in vivo and in vitro assembly

The assembly of phage phi 29 occurs by a single pathway, and DNA-protein (DNA-gp3) has been shown to be an intermediate on the assembly pathway by a highly efficient in vitro complementation. At 30 degrees C, about one-half of the viral DNA synthesized was assembled into mature phage, and the absolute plating efficiency of phi 29 approached unity. DNA packaging at 45 degrees C was comparable to that at 30 degrees C, but the burst size was reduced by one-third. When cells infected with mutant ts3(132) at 30 degrees C to permit DNA synthesis were shifted to 45 degrees C before phage assembly, DNA synthesis ceased and no phage were produced. However, a variable amount of DNA packaging occurred. Superinfection by wild-type phage reinitiated ts3(132) DNA synthesis at 45 degrees C, and if native gp3 was covalently linked to this DNA during superinfection replication, it was effectively packaged and assembled. Treatment of the DNA-gp3 complex with trypsin prevented in vitro maturation of phi 29, although substantial DNA packaging occurred. A functional gp3 linked to the 5' termini of phi 29 DNA is a requirement for effective phage assembly in vivo and in vitro.     

Nucleotide sequence of the right early region of Bacillus phage phi 15 and comparison with related phages: reorganization of gene 17 during evolution

The rightmost 2016 bp of the Bacillus subtilis phage phi 15 genome were sequenced. The nucleotide sequence was compared with the homologous regions of the related phages PZA and phi 29. There are six open reading frames (ORFs) in this region of the phi 15 genome; all of them are present in the PZA and phi 29 genomes. One of the ORFs was assigned to gene 17, which is involved in the replication of the phage DNA. Gene 17 has undergone reorganization during the evolution of this phage family. Comparison of the nucleotide sequence of its mRNA-like strand in phi 15, PZA and phi 29 showed that deletions in its central and 3'-end-proximal parts are tolerated and do not interfere with the gene 17 product function. It seems that the only portion of gene 17 that has to be conserved to encode the functional product is its 5'-end-proximal part.     

Heat Induction of Prophage φ 105 in Bacillus subtilis: Bacteriophage-Induced Bidirectional Replication of the Bacterial Chromosome

A mutant of Bacillus subtilis, dna-1, which cannot initiate new rounds of DNA replication (obtained from N. Sueoka) was lysogenized with wild-type phi 105 and with the heat-inducible mutant phi 105 cts23. Bacteria were incubated at the permissive temperature in the presence of chloramphenicol and then shifted to the nonpermissive temperature where induction of phi 105 cts23 occurs. DNA made after the shift was labeled with a density label, and the distribution of bacterial and phage markers in replicated and unreplicated DNA was determined. Similar experiments were performed with nonlysogenic dna-1 infected with phage phi 105 cts23 after the temperature shift. The results show that after induction of phi 105 cts23 prophage, bacterial markers on either side of the prophage replicate at an increased rate compared to more distant markers. No selective stimulation of bacterial DNA synthesis was observed on infection or after shifting bacteria lysogenic for noninducible phage to the higher temperature. Attempts to suppress the initiation mutation dna-1 by phage phi 105 were unsuccessful.     

Cleavage of single-stranded DNA by the A and A* proteins of bacteriophage phi X174.

The purified A protein and A* protein of bacteriophage phi X174 have been tested for endonuclease activity on single stranded viral phi X174 DNA. The A protein (55.000 daltons) nicks single-stranded DNA in the same way and at the same place as it does superhelical RFI DNA, at the origin of DNA replication. The A* protein (37.000 daltons) can cleave the single-stranded viral DNA at many different sites. It has however a strong preference for the origin of replication. Both proteins generate 3'OH ends and blocked 5' termini at the nick site.     

Phi SC623, a temperate actinophage of Streptomyces coelicolor Müller, and its relatives phi SC347 and phi SC681

Three species-specific, temperate actinophages of Streptomyces coelicolor Müller, phi SC623, phi SC347 and phi SC681, were compared with respect to host range, virion structure, antiserum cross-inactivation, DNA-restriction pattern, DNA hybridization, and DNA base composition. The restriction map of phi SC623 (57 kb) was established with eight restriction enzymes; the homologies of this phage with phi SC347 and phi SC681 suggested that it might be a hybrid phage composed of approximately equal parts homologous to one of the other two phages. No homology was detected between phi SC623 and R4, a temperate, wide-host-range phage which can also lysogenize S. coelicolor Müller.     

Location of the Bacillus subtilis temperate bacteriophage phi 105 attP attachment site.

Chromosomal DNAs of lysogens of phi 105 and phi 105 DI:1t were digested with restriction enzymes EcoRI and HpaI and were probed with nick-translated mature phi 105 DNA. Altered bacteriophage-specific bands in the lysogens were detected, indicating that the phage integrates into the host chromosome at a single site, probably via a Campbell-type circular intermediate. The phage attachment site is centrally located in the phage genome and lies between the phage immunity region and the nonessential deletable region of phi 105.     

Glycosylation of a Streptomyces coelicolor A3(2) cell envelope protein is required for infection by bacteriophage phi C31

Mutants of Streptomyces coelicolor A3(2) J1929 (Delta pglY) were isolated that were resistant to the Streptomyces temperate phage phi C31. These strains could be transfected with phi C31 DNA, but could not act as infective centres after exposure to phage. Thus, it was concluded that infection was blocked at the adsorption/DNA injection step. The mutants fell into three classes. Class I mutants were complemented by a gene, SCE87.05, isolated from the cosmid library of S. coelicolor A3(2). The product of SCE87.05 had good overall homology to a Mycobacterium tuberculosis hypothetical protein and regions with similarity to dolichol phosphate-D-mannose:protein O-D-mannosyltransferases. Concanavalin A (ConA) inhibited phi C31 infection of S. coelicolor J1929, and this could be partially reversed by the addition of the sugar, alpha-D-methyl-pyranoside. Moreover, glycosylated proteins from J1929, but not from the class I mutant DT1017, were detected using ConA as a probe in Western blots. Class I and II mutants were sensitive to phi C31hc, a previously isolated phage exhibiting an extended host range phenotype, conferred by h. A phage with the same phenotype, phi DT4002, was isolated independently, and a missense mutation was found in a putative tail gene. It is proposed that the phi C31 receptor is a cell wall glycoprotein, and that the phi C31h mutation compensates for the lack of glycosylation of the receptor.