Inhibition of bacteriophage PBS2 replication in Bacillus subtilis by phleomycin.

Phleomycin is an effective inhibitor of the replication of Bacillus subtilis bacteriophage PBS2, whose DNA contains uracil instead of thymine. Phleomycin does not affect the induction of the known phage enzymes involved in deoxyribonucleotide metabolism. But phage DNA synthesis is severely inhibited by phleomycin, and late virion protein synthesis is eliminated. These effects appear to result from a phleomycin-induced degradation of the parental phage DNA. Similar inhibitory and degradative effects on DNA are seen in phleomyinc-treated, uninfected cells. This system is unaffected by the related antibiotic, bleomycin.     

DNA replication during development of competence in Bacillus subtilis

Summary A new technique for studying DNA synthesis in competent cells of Bacillus subtilis has been developed.During competence development, the transformable cells are probably synthetizing DNA with a duplication time of approximately 90 min at 30° C; only when the maximum of competence is reached, does synthesis stop.     

Relationship of Bacillus subtilis DNA polymerase III to bacteriophage PBS2-induced DNA polymerase and to the replication of uracil-containing DNA.

In vivo studies of PBS2 phage replication in a temperature-sensitive Bacillus subtilis DNA polymerase III (Pol III) mutant and a temperature-resistant revertant of this mutant have suggested the possible involvement of Pol III in PBS2 DNA synthesis. Previous results with 6-(p-hydroxyphenylazo)-uracil (HPUra), a specific inhibitor of Pol III and DNA replication in uninfected cells, suggest that Pol III is not involved in phage DNA replication, due to its resistance to this drug. Experiments were designed to examine possible explanations for this apparent contradiction. First, assays of the host Pol III and the phage-induced DNA polymerase activities in extracts indicated that a labile Pol III did not result in a labile phage-induced enzyme, suggesting that this new polymerase is not a modified HPUra-resistant form of Pol III. Indeed the purified phage-induced enzyme was resistant to the active, reduced form of HPUra under all assay conditions tested. Since in vitro Pol III was capable of replicating the uracil-containing DNA found in this phage, the sensitivity of the purified enzyme to reduced HPUra was examined using phage DNA as template-primer and dUTP as substrate; these new substrates did not affect the sensitivity of the host enzyme to the drug.     

Metabolism of uracil-containing DNA: degradation of bacteriophage PBS2 DNA in Bacillus subtilis.

When Bacillus subtilis is infected by the uracil-containing DNA phage PBS2, the parental DNA labeled with radioactive uracil and cytosine remains acid insoluble. If the synthesis of the phage-induced uracil-DNA N-glycosidase inhibitor is prevented, the parental DNA is completely degraded to acid-soluble products beginning at about 6 min after infection. The host N-glycosidase probably initiates the degradation pathway, with nucleases being responsible for the remaining degradation of the DNA.     

Plasmid replication in DNA Ts mutants of Bacillus subtilis.

In an attempt to increase our understanding of plasmid replication in Bacillus subtilis we determined the effect of various dna Ts mutations [Gass, K. B., and Cozzarelli, N. R. (1973). J. Biol. Chem. 248, 7688–7700; Gross, J. D., Karamata, D., and Hempstead, P. G. (1968). Cold Spring Harbor Symp. Quant. Biol.33, 307–312; Karamata, D., and Gross, J. D. (1970). Mol. Gen. Genet.108, 277–287] on pUB110 replication. pUB110 is a kanamycin resistance plasmid originally isolated in Staphylococcus aureus and introduced into B. subtilis by transformation. At temperatures nonpermissive for chromosomal DNA synthesis dnaA13, dnaB19, dnaC6, dnaC30, dnaD23, dnaE20, and dnaI102 permit replication of the plasmid. In several cases this “amplification” continues until approximately equal amounts of plasmid and chromosomal DNA are present. dnaG34, dnaH151, dnaF133, mut-1, and polC26 affect both pUB110 and host DNA synthesis at nonpermissive temperatures. The last three mutations are known to affect the activity of DNA polymerase III (PolIII). When polC26 is incubated at a nonpermissive temperature, there is an accumulation of plasmid DNA with a density on EtBr-CsCl gradients intermediate between that of covalently closed circular (CCC) and open circular DNA. pUB110 can replicate in a strain which is deficient in DNA polymerase I (PolI). Finally, chloramphenicol (Cm) inhibits the replication of pUB110 as well as of chromosomal DNA.     

DNA packaging by the Bacillus subtilis defective bacteriophage PBSX.

Defective bacteriophage PBSX, a resident of all Bacillus subtilis 168 chromosomes, packages fragments of DNA from all portions of the host chromosome when induced by mitomycin C. In this study, the physical process for DNA packaging of both chromosomal and plasmid DNAs was examined. Discrete 13-kilobase (kb) lengths of DNA were packaged by wild-type phage, and the process was DNase I resistant and probably occurred by a head-filling mechanism. Genetically engineered isogenic host strains having a chloramphenicol resistance determinant integrated as a genetic flag at two different regions of the chromosome were used to monitor the packaging of specific chromosomal regions. No dramatic selectivity for these regions could be documented. If the wild-type strain 168 contains autonomously replicating plasmids, especially pC194, the mitomycin C induces an increase in size of resident plasmid DNA, which is then packaged as 13-kb pieces into phage heads. In strain RB1144, which lacks substantial portions of the PBSX resident phage region, mitomycin C treatment did not affect the structure of resident plasmids. Induction of PBSX started rolling circle replication on plasmids, which then became packaged as 13-kb fragments. This alteration or cannibalization of plasmid replication resulting from mitomycin C treatment requires for its function some DNA within the prophage deletion of strain RB1144.     

DNA of Bacillus subtilis bacteriophage SPP1: physical mapping and localization of the origin of replication.

The genome of Bacillus subtilis bacteriophage SPP1, a linear, 28.5-megadalton DNA duplex, was mapped by analysis with the restriction endonucleases endo R.Sal I, Sma I, Xba I, Bgl I, Bgl II, and EcoRI. The SPP1 genome, like that of the Salmonella typhimurium phage, P22, was found to be a terminally repetitious, circularly permuted molecule. 6-(p-Hydroxyphenylazo)uracil, a selective, reversible inhibitor of SPP1 DNA synthesis, was exploited to synchronize the initiation of genome replication and to selectively label the site of its initiation with radioactive thymidine. Restriction endonuclease analysis of the distribution of the label located the origin of replicative synthesis at an area approximately 0.2 genome length from one molecular terminus.     

Plasmid replication stimulates DNA recombination in Bacillus subtilis

The effects of plasmid replication on the frequency of homologous recombination have been investigated. For that purpose Bacillus subtilis strains that carry in their chromosome directly repeated DNA sequences, and an integrated copy of plasmid pE194 either proximal or distal to the repeats, were constructed. The repeat consists either of 3.9 X 10(3) base pBR322 sequences or 2.1 X 10(3) base B. subtilis chromosomal sequences. As plasmid pE194 is naturally thermosensitive for replication, the activity of the replicon could be regulated. Recombination between the repeated sequences was infrequent (about 10(-4) per generation) when the integrated plasmid did not replicate. It was 20 to 450 times higher when the plasmid was allowed to replicate, provided that the repeats were in the proximity of the plasmid. These results show that plasmid replication stimulates DNA recombination.     

The DNA polymerase-encoding gene of Bacillus subtilis bacteriophage SPO1.

The bacteriophage SPO1 DNA polymerase-encoding gene, which contains a self-splicing intron, has been sequenced and its amino acid (aa) sequence has been deduced. The aa sequence of SPO1 DNA polymerase shows a high degree of similarity with that of DNA polymerase I from Escherichia coli (Po1I). Alignment with the sequences of Po1I, and the phi 29 and SPO1 DNA polymerases indicate that the aa residues that have been implicated in 3'----5' exonuclease activities are conserved.     

Revised restriction maps of Bacillus subtilis bacteriophage phi 105 DNA.

Physical mapping of Bacillus subtilis temperate phage phi 105 DNA was carried out by using restriction endonucleases EcoRI, SmaI, and KpnI, and a new revised EcoRI cleavage map is presented. In addition, the EcoRI cleavage maps of six specialized transducing phages carrying sporulation genes of B. subtilis were revised.     

Bacillus subtilis bacteriophage SPP1-encoded gene 34.1 product is a recombination-dependent DNA replication protein

SPP1-encoded replication and recombination proteins, involved in the early steps of the initiation of concatemeric DNA synthesis, have been analyzed. Dimeric G34.1P exonuclease degrades, with a 5' to 3' polarity and in a Mg2+-dependent reaction, preferentially linear double-stranded (ds) DNA rather than single-stranded (ss) DNA. Binding of the replisome organizer, G38P, to its cognate sites (oriDNA) halts the 5' to 3' exonucleolytic activity of G34.1P on dsDNA. The G35P recombinase increases the affinity of G34.1P for dsDNA, and stimulates G34.1P activity on dsDNA, but not on ssDNA. Then, filamented G35P promotes limited strand exchange with a homologous sequence. The ssDNA binding protein, G36P, protects ssDNA from the G34.1P exonuclease activity and stimulates G35P-catalyzed strand exchange. The data presented suggest a model for the role of G34.1P during initiation of sigma replication: G38P bound to oriDNA might halt replication fork progression, and G35P, G34.1P and G36P in concert might lead to the re-establishment of a unidirectional recombination-dependent replication that accounts for the direction of DNA packaging.     

Bacillus subtilis bacteriophage PBS2-induced DNA polymerase. Its purification and assay characteristics.

The DNA polymerase induced by Bacillus subtilis bacteriophage PBS2 (whose DNA contains uracil instead of thymine) has been purified and characterized for its specificity. The enzyme requires a high ionic strength for optimal stability and activity and is sensitive to various anions and to sulfhdryl reagents. Both dUTP and dTTP are incorporated efficiently as substrates and are competitive inhibitors at the same active site. The apparent Km and Ki values are about 6 micrometers for dTTP and 15 micrometers for dUTP, when denatured, uracil-containing B. subtilis or salmon sperm DNA (3.9 micrometers for dUTP and 2.6 micrometers for dTTP). The PBS2 enzyme works best on denatured DNA, on double-stranded DNA activated by DNase to produce gaps, or on primed homopolymeric DNA. Using denatured DNA preparations of average molecular weight 6.2 million, the apparent Km values are 270 micrograms/ml for B. subtilis DNA and 360 micrograms/ml for PBS2 DNA; the Vmax value for denatured PBS2 DNA containing uracil is 7-fold greater than that for denatured B. subtilis DNA containing thymine. However, lower molecular weight DNAs have 10-fold lower apparent Km values and show similar Vmax values for both B. subtilis and PBS2 DNAs. Thus, the PBS2 phage-induced DNA polymerase (which likely replicates only uracil-containing phage DNA using dUTP in vivo) has little selectivity for uracil- versus thymine-containing deoxyribonucleotides or DNA in vitro.     

Cloning of DNA segments of phage 2C, which allows autonomous plasmid replication in Bacillus subtilis

The chromosome of Bacillus subtilis phage 2C is a 100-MDa double-stranded DNA molecule, containing hydroxymethyluracil in place of thymine and carrying redundant ends each encompassing 10% of the genome. 2C DNA was cleaved with EcoRI and HindIII, and cloned in the shuttle plasmids pSC 540 and pCP 115, both containing segments originating from B. subtilis and Escherichia coli plasmids. These chimaerical plasmids, carrying the chloramphenicol resistance gene, were unable to replicate in B. subtilis; this ability was restored, however, after the insertion of viral DNA segments. Physical maps of the recombinant plasmids were made; a large deletion of the E. coli-derived segment of pSC 540 was observed (which paralleled a loss of replication in this host), whereas addition of 2C DNA segments in pCP 115 was not accompanied by deletion (replication in E. coli was conserved in this case). Cloned viral segments mapped mostly, but not exclusively, within the redundant ends of 2C DNA. It is suggested that the thirteen recombinant clones carried the replication origin region of phage 2C DNA, and that these sequences originated within or close to the redundant extremities of the viral chromosome.