Changes in DNase activities in Bacillus subtilis infected with bacteriophage PBS 1.

DNase activities in Bacillus subtilis were fractionated by chromatography on hydroxylapatite. One of the fractions hydrolyzed uracil-containing phage DNA but had no activity on host DNA. This activity on native phage DNA disappeared soon after phage infection, whereas DNase activities on bacterial DNA remained at the same level during phage development. An inhibitor of protein nature was induced by phage infection and this inhibitor was shown to be responsible for the disappearance of the DNase activity on phage DNA. Bacterial DNA in infected cells might be fragmented but is not degraded to acid-soluble oligonucleotides.     

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.     

Primary Adsorption Site of Phage PBS1: the Flagellum of Bacillus subtilis

The adsorption of Bacillus subtilis phage PBS1 was studied, and it was demonstrated that the primary adsorption site for this phage is the flagellum of B. subtilis. The capacity of flagella to function for motility may be lost without the loss of their capacity to adsorb the phage and permit infection. Deoxyribonucleic acid injection by the phage is inhibited by cyanide, suggesting the requirement for cellular energy in the infection process.     

Rifampicin-resistant bacteriophage PBS2 infection and RNA polymerase in Bacillus subtilis

Bacteriophage PBS2 replication is unaffected by rifampicin and other rifamycin derivatives, which are potent inhibitors of Bacillus subtilis RNA synthesis. Extracts of gently-lysed infected cells contain a DNA-dependent RNA polymerase activity which is specific for uracil-containing PBS2 DNA. The PBS2-induced RNA polymerase is insensitive to rifamycin derivatives which inhibit the host's RNA polymerase.     

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.     

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.     

Interaction between bacteriophage PBS1 and clay minerals and transduction of Bacillus subtilis by clay-phage complexes

Bacteriophage PBS1 of Bacillus subtilis was rapidly adsorbed on montmorillonite (M) and kaolinite (K), and adsorption was maximal after 30 min on both clays. There was no correlation between adsorption and the cation exchange capacity of the clays. Studies with sodium metaphosphate (a polyanion that interacts with positively charged sites on clay) indicated that positively charged sites on K were primarily responsible for the adsorption of the phage, whereas other mechanisms appeared to be involved in adsorption of the phage on M. X-ray diffraction and electron microscopic analyses showed that the phage partially intercalated M. Survival of the phage was increased by adsorption on the clays, and adsorbed phage maintained its ability to transduce bacterial cells for at least 30 days (the longest time studied) after the preparation of the clay–phage complexes. Electron microscopic observations indicated that transduction by the clay–phage complexes was primarily the result of the phage detaching from the clays in the presence of host cells.     

Bacteriophage transformation of PBS2 in Bacillus subtilis.

Transformation of temperature-sensitive mutants of bacteriophage PBS2 for Bacillus subtilis was demonstrated. The number of transformants was linearly related to the concentration of DNA within a range of 0.01 to 1 mug/ml. No transformants were obtained when the DNA was pretreated with DNase. PBS2 DNA sheared to approximately 1% of the total chromosome length was centrifuged in Cs2SO4-Hg gradients to fractionate the DNA according to the base composition. Transformation experiments carried out with the fractionated DNA indicated the possibility of determining the base composition of different regions of the phage chromosome.     

Characterization of the Bacillus subtilis bacteriophage PBS2-induced DNA polymerase and its associated exonuclease activity.

The DNA polymerase induced by Bacillus subtilis bacteriophage PBS2 has a Stokes radius of 7.2 in buffers of high ioninc strength, suggesting a molecular weight in the range 145,000 to 195,000. The polypeptide bands observed on gel electrophoresis in dodecyl sulfate have apparent molecular weights of 78,000 and 69,000 (and possibly another 27,000) in equimolar amounts. In buffers of low ionic strength, the enzyme appears to form large aggregates and even precipitates, with about 90% loss of activity. A nuclease activity co-purifies with the PBS2 DNA polymerase and shows similar responses to changes in pH, MgCl2, N-ethylmaleimide, temperature, and dextran sulfate levels. The nuclease produces deoxyribonucleoside 5'monophosphates from denatured DNA containing thymine or uracil. No endonuclease activity is detectable on supercoiled DNA. The inhibition of nuclease activity by added deoxyribonucleoside triphosphates, the DNA-dependent turnover of triphosphates, to free monophosphates during DNA polymerization, the inhibition of nuclease activity by 3'-phosphates on the DNA template-primer, and the pattern of digestion of 5'-[32P]phosphate-labeled DNA all indicate that the PBS2 DNA polymerase-associated hydrolytic activity is a 3' leads to 5'-exonuclease.     

N-Glycosidase activity in extracts of Bacillus subtilis and its inhibition after infection with bacteriophage PBS2.

We have detected in crude extracts of Bacillus subtilis an N-glycosidase activity which catalyzes the release of free uracil from DNA of the subtilis phage PBS2 labeled with [3H]uridine. This DNA contains deoxyuridine instead of thymidine. The enzyme is active in the presence of 1.0 mM EDTA and under these conditions Escherichia coli or T7 DNA labeled with [3H]thymidine is not degraded to labeled acid-soluble products. The activity resembles an N-glycosidase from E. coli which releases free uracil from DNA containing deaminated cytosine residues. Both enzymes in crude extracts are active in the presence of EDTA, do not require dialyzable co-factors, and have the same pH optimum. They differ in that the enzyme from E. coli is more sensitive to heat, sulfhydryl reagents, and salt. The enzyme from B. subtilis is inactive on DNA containing 5-bromouracil or hydroxymethyluracil. Extracts of PBS2-infected B. subtilis lose the N-glycosidase activity within 4 min after infection and contain a factor that inhibits the N-glycosidase activity within 4 min after infection and contain a factor that inhibits the N-glycosidase activity in extracts of uninfected cells in vitro.     

Clay minerals protect bacteriophage PBS1 of Bacillus subtilis against inactivation and loss of transducing ability by UV radiation

The effect of UV radiation on the survival of and transduction by phage PBS1 of Bacillus subtilis, free or adsorbed on the clay minerals montmorillonite (M) and kaolinite (K), was studied. After free or clay-associated phage (approximately 10(7) PFU.mL-1) was irradiated with UV light (254 nm) for 0, 1, 2, 5, 10, and 30 min and then allowed to infect B. subtilis FB300 (thiB4 metA29 argF4 Rfmr), the phage was titered, and Met+ transductants were enumerated on selective media. After 1 min of irradiation, the titer of free and clay-associated phage decreased significantly (approximately 1.6 times for free phage, and approximately 4.9 and 6.8 times for M and K, respectively), whereas the transduction frequency increased significantly (approximately 3 times for free phage and approximately 1.4 and 2.2 times for M and K, respectively). The titer and transduction frequency of clay-associated phage remain essentially constant between 1 and 10 min of irradiation, whereas the titer of free phage decreased by approximately 1 order of magnitude after 5 min of irradiation. When free phage was irradiated for 10 min, the titer and transduction frequency decreased by approximately 2 and 0.5 orders of magnitude, respectively, whereas 30 min of irradiation was necessary to obtain comparable decreases with clay-associated phage. These results indicated that phages are protected to some extent from UV radiation when adsorbed on clay minerals.     

A cytotoxic early gene of Bacillus subtilis bacteriophage SPO1.

Some of the early genes of Bacillus subtilis bacteriophage SPO1 were hypothesized to function in the shutoff of host biosyntheses. Two of these genes, e3 and e22, were cloned and sequenced. E22 showed no similarity to any known protein, while E3, a highly acidic protein, showed significant similarity only to other similarly acidic proteins. Each gene was immediately downstream of a very active early promoter. Each was expressed actively during the first few minutes of infection and was then rapidly shut off and its RNA rapidly degraded. An e3 nonsense mutation severely retarded the degradation of e3 RNA. Expression of a plasmid-borne e3 gene, in either B. subtilis or Escherichia coli, resulted in the inhibition of host DNA, RNA, and protein syntheses and prevented colony formation. However, the e3 nonsense mutation caused no measurable decrease in either burst size or host shutoff during infection and, in fact, caused an increased burst size at high multiplicities of infection. We suggest that e3 is one of several genes involved in host shutoff, that its function is dispensable both for host shutoff and for phage multiplication, and that its shutoff function is not entirely specific to host activities.     

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.     

Functional analysis of the Bacillus subtilis bacteriophage SPP1 pac site.

Encapsidation of the DNA of the virulent Bacillus subtilis phage SPP1 follows a processive unidirectional headful-mechanism and initiates at a unique genomic location (pac). We have cloned a fragment of SPP1 DNA containing the pac site flanked by reporter genes into the chromosome of B. subtilis. Infection of such cells with SPP1 led to highly efficient packaging, initiated at the inserted pac site, of chromosomal DNA. The directionality in the packaging of this DNA was the same as observed with vegetative phage DNA. Mutagenizing the chromosomal pac insert defined an 83 base pair segment containing the pac cleavage site which is sufficient to direct phage specific DNA encapsidation. The packaging recognition signal as defined can also be utilized by the SPP1 related phages 41c, SF6 and rho 15.     

Viral mutation affecting bacteriophage phi 1 development in Bacillus subtilis 168.

Bacillus subtilis bacteriophage phi 1m, a host-range variant, was isolated after mutagenesis of virulent bacteriophage phi 1. Unlike its wild-type antecedent, phi 1m could not form plaques on lawns of B subtilis 168 at 37 C, although it adsorbed to, penetrated, and killed this bacterium. Experiments conducted in liquid medium at 37 C showed that B. subtilis 168 cells allowed reduced levels of phi 1m development at low multiplicities of infection, whereas high multiplicity infections of this strain by the phage were abortive. Certain mutants, derived originally from B. subtilis 168, were observed to be permissive for phi 1m at 37 C; moreover, their permissive phenotype could be duplicated by growing wild-type B. subtilis 168 cells at temperatures above 47 C. Studies on phi 1m and host nucleic acid synthesis under nonpermissive conditions demonstrated that transciption and DNA synthesis proceeded up to 20 min after infection, after which time there was a cessation of all nucleic acid production. These observations are discussed with respect to other abortive bacteriophage infections in B. subtilis.