Genome organization of Sp beta c2 bacteriophage carrying the thyP3 gene

Thymine auxotrophs of Bacillus subtilis strains lysogenic for temperate bacteriophage SP beta c2 were transformed to prototrophy by DNA from related phage phi 3T. During transformation, the phi 3T-encoded thymidylate synthetase gene, thyP3, became integrated into the extreme right end of the SP beta c2 prophage near the bacterial citK gene. Upon heat induction, the transformed B. subtilis cells released SP beta c2T phages that could lysogenize thymine auxotrophs and convert them to prototrophy. Comparison of restriction endonuclease fragments of DNAs from SP beta c2 and SP beta c2T phages revealed that the latter contained a large region of deletion and substitution near the center of the chromosome. This region included the phage attachment site on the SP beta c2 genome.     

Restriction enzyme analysis of Bacillus subtilis bacteriophage phi 105 DNA

The recognition sites on phi 105 DNA for the restriction endonucleases EcoRI, Bg/II, SmaI, KpnI, SstI, SalI, XhoI, NcoI, PstI, HindIII, ClaI, EcoRV and MluI have been mapped. The sites for EcoRI are shown to be different from those published earlier. The DNA from phi 105 contains no recognition sites for the endonucleases BamHI and XbaI.     

Genome homology and divergence in the SP beta-related bacteriophages of Bacillus subtilis

Chromosomal organization in related temperate Bacillus subtilis bacteriophages SP beta, phi 3T, rho 11, Z, and E was compared. DNA-DNA hybridization studies done in conjunction with available restriction fragment maps of SP beta, phi 3T, and rho 11 demonstrated that DNA homology between these three phages extended over most of their respective genomes, although each contained unique chromosomal segments, phi 3T, rho 11, Z, and E, but not SP beta, possessed apparently homologous structural genes (thyP) for thymidylate synthetase. DNA from all thyP-containing phages transformed thymine auxotrophs of B. subtilis SP beta lysogens to prototrophy. This transformation commonly involved incorporation of the thyP gene into SP beta prophage within a region corresponding to the middle of the viral chromosome. Chimeric plasmids containing the thyP gene from phi 3T or cloned fragments of SP beta DNA were used in DNA-DNA hybridization studies to locate the thymidylate synthetase gene near the center of the phi 3T chromosome, and to demonstrate that the organization of this region resembled the analogous portion of the SP beta genome. Profiles of virion structural proteins from the five phages were also very similar, further suggesting functional homology between these viruses. However, despite these evidences of relatedness, populations of fragments generated by digesting SP beta, phi 3T, rho 11, Z, and E DNA with restriction enzymes were quite dissimilar.     

Physical map of the Rhodobacter sphaeroides bacteriophage phi RsG1 genome and location of the prophage on the host chromosome.

We constructed a physical map of the 50-kilobase-pair (kb) DNA of the temperate Rhodobacter sphaeroides bacteriophage phi RsG1, with the relative positions of the cleavage sites for the nine restriction endonucleases KpnI, HindIII, XbaI, ClaI, BclI, EcoRV, EcoRI, BglII, and BamHI indicated. Using biotinylated phi RsG1 DNA as a probe in hybridization studies, we detected homologies with virus DNA and fragments of restriction endonuclease-digested host chromosomal DNA but not with plasmid DNA. This indicates that the prophage is integrated into the host chromosome. In addition, the use of specific probes such as the 10.4-kb BglII A fragment and the 2.65-kb BamHI H fragment allowed the determination of the position of phage attachment site (attP).     

Restriction map of permuted DNA of Erwinia carotovora temperate bacteriophage 59

The cyclic permutation and terminal redundancy were found in the genomes of Erwinia carotovora temperate bacteriophage 59 by electron microscopic studies. The headful mechanism for bacteriophage DNA cleavage and packaging during the phage morphogenesis was confirmed by the restriction analysis technique. Restriction map of the bacteriophage 59 DNA was constructed for restriction endonucleases BamHI, Bg1II, Eco31, Sa1I, SmaI, EcoRI.     

Structure and restriction enzyme maps of the circularly permuted DNA of staphylococcal bacteriophage phi 11.

One restriction enzyme map of Staphylococcus aureus bacteriophage phi 11 DNA was established by reciprocal double digestions with the enzymes EcoRI, HaeII, and KpnI. The sequential order of the EcoRI fragments was thereafter established by a novel approach involving blotting of DNA partially cleaved with EcoRI and the probing the blots with nick-translated terminal fragments. A circular map of the phi 11 DNA was established, and the phage genome was circularly permuted based on the failure to end label mature viral DNA, restriction maps of replicating DNA, and finally, homoduplex analysis in the electron microscope. A restriction enzyme map of the prophage form of phi 11 DNA was obtained by analysis of chromosomal DNA from a lysogenic strain.     

Integration of the bacteriophage phi 3T-coded thymidylate synthetase gene into the Bacillus subtilis chromosome

Transformation of Bacillus subtilis 168 Thy- auxotrophs with phi 3T deoxyribonucleic acid (DNA) to thymine independence was found to involve site-specific recombination of phi 3T DNA sequences with their homologous counterparts in the bacterial chromosome. During the transformation, the phage phi 3T-encoded thymidylate synthetase gene, thyP3, was shown to integrate at two genetically distinct sites in the B. Subtilis 168 chromosome. The first site was identified to be in the bacterial thymidylate synthetase gene, thyA. The second site was in a prophage (SPB) known to be carried in the host genome. The frequency of the integration of the thyP3 gene at each of the two loci and some of the parameters affecting this frequency were studied. The common origin of the thyP3 and thyA genes and their molecular evolution are also reported.     

Temperate Bacillus subtilis bacteriophage phi 3T: chromosomal attachment site and comparison with temperate bacteriophages phi 105 and SPO2.

The temperate Bacillus subtilis bacteriophage phi 3T contains within its genome a locus, designated thyP3, that encodes for a protein with thymidylate synthetase activity. Bacteriophage phi 3T is different from the two previously characterized temperate phages, phi 105 and SPO2, in: heteroimmunity, response to bacteriophage antisera, endonuclease digestion pattern, induction in the presence of 6-(p-hydroxyphenylazo)-uracil, and effect on the lytic cycle of bacteriophage phi 1. The mean burst size of phi 3T is 56. The dose response curve with bacteriophage phi 3T DNA is linear for transfection and transformation to the Thy+ phenotype. The inserted prophage has been mapped by PBS1 transduction; it is between chromosomal markers ilvA8 and gltA in the terminus of the chromosome. Thus thyP3 maps at a site separate from, but between, the bacterial markers thyA and thyB when thyP3 is in the prophage state.     

New temperate bacteriophage for Bacillus subtilis, rho 11.

A new temperate bacteriophage, rho11, isolated by J. Hoch, has been characterized. This new phage is very similar to the temperate phage phi3T in size (380 nm), host range, homoimmunity, DNA buoyant density (1.694 g/ml), antigenicity, and molecular weight (around 6.0 X 10(7)) as determined in gels. Like phi3T, rho11 converts thymine auxotrophs to prototrophy at high frequency (250 out of 250 tested). Phage rho11 differs from phi3T in plaque morphology and in the endonuclease R-EcoRI digest pattern. Sixteen of the 20 rho11 DNA fragments have migration patterns corresponding to those of the 21 fragments of phi3T. The close similarities yet clear differences between these phages suggest that the two phages have a common ancestor.     

Evidence for circular permutation of the prophage genome of Bacillus subtilis bacteriophage phi 105.

Analysis of DNA extracted from Bacillus subtilis lysogenic for bacteriophage phi 105 was performed by restriction endonuclease digestion and Southern hybridization using mature phi 105 DNA as a probe. The data revealed that the phi 105 prophage is circularly permuted. Digests using the enzymes EcoRI, SmaI, PstI, and HindIII localized the bacteriophage attachment site (att) to a region 63.4 to 65.7% from the left end of the mature bacteriophage genome. The phi 105 att site-containing SmaI C, PstI J, and HindIII L fragments were not present in digests of phi 105 prophage DNA. phi 105-homologous "junction" fragments were visualized by probing digests of prophage DNA with the purified PstI J fragment isolated from the mature bacteriophage genome. The excision of the phi 105 prophage was detected by observing the appearance of the mature PstI J fragment and the concomitant disappearance of a junction fragment during the course of prophage induction.     

Cloning of bacteriophage T5 DNA fragments in the plasmid pBR322. Analysis of recombinant plasmids by the method of bonding with RNA-polymerase from Escherichia coli on nitrocellulose filters

DNA of bacteriophage T5 was hydrolyzed with restriction endonucleases HindIII and BamHI, and subjected to the combined hydrolysis with BamHI+EcoRI and BamHI+ +HindIII. Fragments obtained were cloned in the plasmid pBR322. About 17% of T5 genome were recovered in recombinant plasmids. Cloned fragments were localized on the physical map of the phage by restriction analysis and Southern hybridization. With the aim of direct cloning of T5 promoters, PstI/HindIII fragments were inserted into pBR322 followed by selection of recombinants on ApsTCr phenotype. Binding of BsuRI and AluI fragments of hybrid plasmids with E. coli RNA polymerase was studied by nitrocellulose filter assay. The fragments, which were capable to form heparin resistant complexes were identified.     

Distribution of bacteriophage phi 3T homologous deoxyribonucleic acid sequences in Bacillus subtilis 168, related bacteriophages, and other Bacillus species.

The Bacillus subtilis 168 chromosome was found to share extensive homology with the genome of bacteriophage phi 3T. At least three different regions of the bacterial genome hydridized to ribonucleic acid complementary to phi 3T deoxyribonucleic acid (DNA). The thymidylate synthetase gene, thyA, of B. subtilis and the sequences adjacent to it were shown to be homologous to the region in the phi 3T DNA containing the phage-encoded thymidylate synthetase gene, thyP3. SP beta, a temperate bacteriophage known to be integrated into the B. subtilis 168 chromosome, was demonstrated to be closely related to phi 3T. Other regions of the bacterial genome were also found to hybridize to the phi 3T probe. The nature and location of these sequences in the bacterial and phage chromosomes were not identified. It was shown however, that they were not homologous to either the thyP3 gene or the DNA surrounding the thyP3 gene. The chromosomes of other Bacillus species were also screened for the presence of phi 3T homologous sequences, and the thyP3 gene was localized in the linear genomes of phages phi 3T and rho 11 by heteroduplex mapping. It is suggested that the presence of sequences of phage origin in the B. subtilis 168 chromosome might contribute to the restructuring and evolution of the viral and bacterial DNAs.     

Characterization of the genome of Pseudomonas aeruginosa bacteriophage phi PLS27 with particular reference to the ends of the DNA.

The DNA of Pseudomonas aeruginosa rough-specific bacteriophage phi PLS27 was studied. The genome size as determined by summing the sizes of restriction fragments was 42.7 kilobase pairs. Of particular interest was the fact that the DNA was insensitive to certain common restriction endonucleases including EcoRI, BamHI, and HindIII. The ends of the phage DNA were cloned and sequenced, revealing direct repeats of 318 nucleotides. The left end of the genome when cloned into the promoter selection vector pKK232-8 exhibited promoter activity in Escherichia coli. Two promoters bearing greater than 70% sequence homology to the plasmid pNM74 TOL operon and PAK pilin promoters were identified.     

A restriction map of cauliflower mosaic virus DNA (strain PV 147). Mapping of the cleavage sites of HhaI, SacI, AvaI, PvuII, PstI, XbaI, EcoRI, Bg/II, HincII, HpaII and HindII + III.

The virion-extracted DNA (Mr5 x 10(6)) of cauliflower mosaic virus (CaMV) has three single-stranded interruptions. The mapping of this DNA using eleven restriction endonucleases (HhaI, SacI, AvaI, PvuII, PstI, XbaI, EcoRI, Bg/II, HincII, HpaII and HindII + III) is reported here. The existence of the three single-stranded breaks complicates the identification and the molecular weight determination of fragments produced by HpaII, HindIII and HindII + III. Indeed the electrophoretic mobility of some fragments in which a single-stranded discontinuity is located is modified, and the fluorescence of ethidium bromide complexed with these fragments is reduced as compared to that observed for the other fragments existing in a molar ratio. These drawbacks were overcome by performing experiments of nick-translation of CaMV DNA with Escherichia coli DNA polymerase I. FRom the data it follows that the CaMV DNA molecule bears bears 1 site for HhaI and SacI, 2 for AvaI and PvuII, 3 for PstI, 4 for XbaI, 5 for EcoRI, 6 for Bg/II and HincII, 11 for HpaII and 15 for HindII + III. The corresponding fragments have all been ordered and precisely located providing a suitable map for further investigations connected with the study of the fine structure and the function of the CaMV genome.     

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.     

Transformation of Bacillus subtilis and Escherichia coli by a hybrid plasmid pCD1.

The gene thyP3 from Bacillus subtilis bacteriophage phi 3T was cloned in the plasmid pMB9. The resulting chimeric plasmid, pCD1, is effective in transforming both Escherichia coli and Bacillus subtilis to thymine prototrophy. The activity of the thyP3 gene product, thymidylate synthetase, was assayed and found to be 9 times greater in a transformed strain of Escherichia coli than in a phi 3T lysogen of Bacillus subtilis. The physical location of restriction sites has been determined for two related plasmids pCD1 and pCD2. Hybridization studies clearly indicate that the plasmid gene responsible for Thy+ transformation is the gene from the bacteriophage phi 3T. The lack of restriction in this transformation process is consistent with our previous studies using bacterial DNA in heterospecific exchanges indicating that the nucleotide sequence surrounding the gene is the dominant factor in determining interspecific transformation.     

Physical map of bacteriophage BF23 DNA: restriction enzyme analysis.

Cleavage maps of bacteriophage BF23 DNA have been constructed for the restriction endonucleases SalI (3 fragments), BamHI (5 fragments), EcoRI, (8 fragments), BalI (13 fragments), and HpaI (49 fragments, 32 of which have been ordered). The maps were determined by (i) analysis of deletion mutants, (ii) digestion with two endonucleases, (iii) digestion of isolated fragments with a second enzyme, (iv) analysis of partial digests, and (v) digestion after treatment with lambda exonuclease.     

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.     

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.     

Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: oriented and quantized in vitro packaging of DNA protein gp3.

The assembly of phage phi 29 occurs by a single pathway, and the DNA protein (DNA-gp3) of "packaging intermediates" can be obtained after DNase I interruption of in vitro complementation. A broad spectrum of DNA molecules of variable length was isolated from DNase I-treated proheads. Restriction endonuclease EcoRI digestion and electrophoretic analysis of these DNA molecules suggested that DNA-gp3 packaging was oriented with respect to the physical map and was a complex process. Proteinase K-treated exogenous DNA was not packaged. When exogenous DNA-gp3 was predigested with the restriction endonucleases BstEII. EcoRI, HpaI, and HpaII, the left-end fragments, ranging in size from 8 to 0.9 megadaltons, were selectively and efficiently packaged. During in vivo and in vitro assembly, DNA-gp3 is packaged into proheads, the "core-scaffolding" protein gp7 exits from the particles, and the DNA-filled heads assume the angular morphology of phage phi 29. The packaging of a 4.1-megadalton DNA-gp3 left-end fragment (one third of the genome) resulted in the exit of gp7 and the transition to angularity.