SP-10 bacteriophage-specific nucleic acid and enzyme synthesis in Bacillus subtilis W23

Abstract

Bacillus subtilis W23 was infected with a clear-plaque variant of SP-10 phage, namely, SP-lOc. Exogenous thymidine was not incorporated into phage DNA (even in the presence of deoxyadenosine), nor was there any transfer of thymidine nucleotides from bacterial to viral DNA. The lytic program was unaffected by concentrations of 5-fluorodeoxyuridine sufficient to reduce bacterial DNA synthesis by >95%. Although these data are consistent with the interpretation that thymidine nucleotides are excluded from phage DNA, formic acid digests of SP-lOc DNA contained what appeared to be the four conventional bases; however, adenine and thymine were not recovered in equimolar yields. DNA-RNA hybridization and hybridization competition experiments were done. Synthesis of host RNA started to wane moments postinfection and stopped completely by 36 min. SP-lOc coded for discrete classes ofearly and late RNA. The possibility of discrete subclasses of early RNA exists. Replication of the bacterial genome appeared to terminate 12 min postinfection. Degradation of the host DNA to acid-soluble material started at 36 min and, by the end of the latent period, >90% of the host chromosome was hydrolyzed. Four apparent phage-coded enzymes have been identified. A di-and triphosphatase degraded dUTP, dUDP, dTTP, and dTDP (and, to a lesser extent, dCDP and dCTP) to the corresponding monophosphates; the enzyme had no apparent activity on dATP and dGTP. SP-lOc also coded for a DNA-dependent DNA polymerase, lysozyme, and a nuclease that degrades native bacterial DNA. Judging from the dependence of enzyme synthesis on the time of addition of rifampin (an inhibitor of the initiation of RNA synthesis), messengers for the di-and triphosphatase, as well as the nuclease, are transcribed from promoters that start to function 6 min postinfection. Promoters for polymerase and lysozyme did not become functional until 8 and 16 min postinfection, respectively. MATERIALS AND METHODS Phage and bacteria. B. subtilis W23 (ATCC 23059) was used as host. SP-lOc is a clear-plaque variant present in ATCC 23059B. SP-lOc was readily inactivated by antiserum prepared against SP-10c and provided by K. Bott (Table 1). The eclipse and latent periods of SP-10c at 37°C were 25 and 55 min, 84

DNA Synthesis and Gene Expression in Bacillus subtilis Infected with Wild-Type and Hypermodification-Defective Bacteriophage SP10

“…At temperatures of < 30°C, mutant (hmd) phage gave bursts that were within 60% of normal, but at higher temperatures the burst size declined sharply (Table 1). For this paper, hmd+ and hmd phage were compared at 37°C since this is the temperature at which most biochemical studies with SP10 were performed (10,29,30,39,40). SPno hmdl 20 54 12 sl x 10-4 sl x 10-4 SP10 hmd2 21 58 12 sl x i-0 sl x i0-4…”

“…DNA-RNA hybridization and hybridization-competition were carried out as described by Gillespie and Spiegelman (14). Enzyme assays and definition of activity units are given elsewhere (29,39). Serum blocking power was determined by the method of DeMars (8).…”

“…Sucrose gradient centrifugational analysis of replicating DNA. Cultures were infected as described previously (29,30). The labeled precursor, typically 32p, (10 ,Ci/ml), was introduced 12 min after infection.…”

Production and expression of dTMP-enriched DNA of bacteriophage SP15

“…Delayed appearance of certain proteins under FUdR-inhibited conditions could reflect delayed transcription or translation or both. To obtain additional information on this point, experiments were done that measured the dependence of protein synthesis on the time of addition of antibiotic inhibitors of transcription (9,21). The antibiotics used were rifampin and streptolydigin, selective inhibitors of RNA chain initiation and elongation, respectively (7,29,37).…”

Changes in macromolecular synthesis in Xanthomonas oryzae infected with bacteriophage XP-12

“…In this case, decreased thymidine incorporation was found despite substantial DNA synthesis, as measured by incorporation of [6-3Hjuridine or 32p into DNA. In addition, it has recently been reported that exogenous thymidine is excluded from B. subtilis phage DNA; however, the properties of SP-10 DNA and of SP-10-infected cells lead to uncertainty as to whether unmodified thymine residues are present in this phage DNA (14). Typically, after infection with other lytic phages containing thymine in their genome, there is an increase in incorporation of [3H]thymidine into DNA (9,21,29), which is partially due to a higher rate of phage DNA synthesis in infected cells than to host DNA synthesis in analogous uninfected cells (16).…”