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1.
Rifampicin-resistant bacteriophage PBS2 infection and RNA polymerase in Bacillus subtilis 总被引:2,自引:0,他引:2
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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. 相似文献
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Bacteriophage PBS 1 adsorbs initially on the flagella of its host, Bacillus subtilis (stage I). The phage can adsorb to both active and inactive flagella. Flagellar attachment is nonspecific as PBS 1 was shown to attach to the flagella of Bacillus species other than the normal host B. subtilis. The phage particle then quickly moves down the length of the flagellum to its base, the final adsorption site. Flagellar motion is required for flagellar base attachment (stage II). After proper attachment at the flagellar base, the phage tail sheath contracts sending the tail core through the final adsorption site (stage III). The phage DNA is then injected at this site (stage IV). Stage I adsorption does not cause loss of motility in PBS 1 -- resistant bacilli. The loss of motility observed upon infection of sensitive cells by PBS 1 may be associated with either stage II or stage III of adsorption. 相似文献
4.
Transcriptional specificity of a multisubunit RNA polymerase induced by Bacillus subtilis bacteriophage PBS2. 总被引:3,自引:3,他引:0
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S Clark 《Journal of virology》1978,25(1):224-237
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The structure of Bacillus subtilis bacteriophage PBS 1 总被引:5,自引:0,他引:5
F A Eiserling 《Journal of ultrastructure research》1967,17(3):342-347
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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. 相似文献
7.
N-Glycosidase activity in extracts of Bacillus subtilis and its inhibition after infection with bacteriophage PBS2. 总被引:22,自引:6,他引:16
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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. 相似文献
8.
Metabolism of uracil-containing DNA: degradation of bacteriophage PBS2 DNA in Bacillus subtilis.
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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. 相似文献
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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. 相似文献
10.
Resistance of Bacteriophage PBS2 Infection to 6-(p-Hydroxyphenylazo)-Uracil, an Inhibitor of Bacillus subtilis Deoxyribonucleic Acid Synthesis 总被引:5,自引:4,他引:1
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6-(p-Hydroxyphenylazo)-uracil (HPUra), an inhibitor of semiconservative deoxyribonucleic acid (DNA) synthesis in Bacillus subtilis, does not prevent (but slightly reduces the rate of) replication of the uracil-containing DNA phage PBS2. Our observations are consistent with the hypothesis that all B. subtilis phages which are resistant to HPUra are able to induce a new DNA polymerase activity. 相似文献
11.
Changes in DNase activities in Bacillus subtilis infected with bacteriophage PBS 1. 总被引:7,自引:4,他引:3
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F Tomita 《Journal of virology》1975,15(5):1073-1080
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. 相似文献
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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. 相似文献
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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. 相似文献
14.
Bacteriophage PBS2-Induced Deoxycytidine Triphosphate Deaminase in Bacillus subtilis 总被引:2,自引:2,他引:0
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Alan R. Price 《Journal of virology》1974,14(5):1314-1317
The dCTP deaminase induced by Bacillus subtilis bacteriophage PBS2, whose DNA contains uracil instead of thymine, requires metal ion and thiol activators and has a molecular weight of 125,000. The enzyme displays sigmoidal substrate saturation kinetics and inhibition by dUTP, consistent with the deaminase's proposed role of providing balanced levels of dUTP and dCTP for PBS2 uracil-DNA synthesis. 相似文献
15.
Relationship of Bacillus subtilis DNA polymerase III to bacteriophage PBS2-induced DNA polymerase and to the replication of uracil-containing DNA. 总被引:1,自引:0,他引:1
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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. 相似文献
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Resistance of bacteriophage H1 to restriction and modification by Bacillus subtilis R 总被引:5,自引:1,他引:4
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H1, a 5-hydroxymethyluracil (HMU)-containing Bacillus subtilis bacteriophage, was neither restricted nor modified upon infection of B. subtilis R cells. In vitro, H1 DNA was not restricted by BsuR under standard conditions (200 mM salt), although the expected frequency of -GGCC- cleavage sites was approximately 250. However, four specific sites were cleaved under nonstandard conditions (low salt or high pH) or in the presence of organic solvents, like dimethyl sulfoxide and glycerol. After the substitution of thymine for HMU by DNA cloning in B. subtilis, a BsuR cleavage site was restricted and modified under standard conditions. No additional sites were detected after shotgun-cloning of about 11% of the chromosome. The nucleotide sequence of a cleavage site was found to be 5'. .C-A-Hmu-A-A-C-Hmu-Hmu-Hmu-G-G-C-C-Hmu-A-G-. . .3', which shows the presence of a bona fide BsuR (GGCC) recognition sequence, flanked by (Hmu-A)-rich sequences. The results suggested that the resistance of H1 to restriction and modification by B. subtilis R was due to (i) a strong bias against the GGCC-recognition sequence and (ii) protection of the four remaining GGCC sites as a consequence of HMU-A base pairs flanking the sites. 相似文献
17.
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. 相似文献
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