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

Despite the fact that mature SPlOc DNA contains dTMP, the acid-soluble fraction of infected cells contained no dTTP during the interval of phage replication. However, infected cells contained normal cellular levels of dATP, dGTP, and dCTP. Upon infection of deoxythymidine-starved Bacillus subtilis M160 (a deoxythymidine-requiring mutant of B. subtilis W23), mature phage DNA with a nornal dTMP content was made. SPlOc codes for an enzyme that seems to catalyze the tetrahydrofolate-dependent transfer of 1-carbon fragments to the 5 position of dUMP. The transfer of 1-carbon fragments is not accompanied by oxidation of tetrahydrofolate to dihydrofolate, implying that the enzyme in question is not a dTMP synthetase. It is proposed that dTMP in mature SPlOc DNA is derived by the postreplicational modification of some other nucleotide and not by the direct incorporation of dTTP into DNA.

Normal DNA of Bacillus subtilis phage SP15 contains approximately equimolar quantities of dTMP and a hypernodified nucleotide, 5-dihydroxypentyl-dUMP (DHPdUMP). Deoxythymidine (dThd) rescue of phage DNA synthesis in 5-fluorodeoxyuridine (FUdR)-inhibited cultures resulted in the synthesis of SP15 DNA containing enhanced levels of dTMP and correspondingly reduced levels of DHPdUMP. This rescued system was used to probe possible roles of DHPdUMP in phage development. The results suggested that normal levels of DHPdUMP were not required for proper transcription of phage DNA, but normal amounts of DHPdUMP were indispensable for phage assembly and/or DNA maturation. The amount of exogenous dThd required to rescue phage DNA synthesis in FUdR-inhibited cultures was 20-fold higher than the concentration required to rescue cellular replication, whereas the same low concentrations of dThd sufficed to rescue viral and bacterial DNA syntheses in aminopterininhibited cultures. Normal SP15 DNA was made in rescued, aminopterin-inhibited cultures. We suggest that FUdR (but not aminopterin) partially suppresses biosynthesis of the hypermodified nucleotide and that there is a barrier to replacement of DHPdUMP by dTMP; therefore, exceptionally large amounts of dThd must be salvaged in FUdR-inhibited cultures to force replacement of the unusual nucleotide by dTMP. Phage and bacteria. B. subtilis W23 is ATCC 23059. SP15 was obtained from M. Mandel in November 1975. 753

Bacillus subtilis W23 was infected with bacteriophage SP-15. Two waves of phage-specific RNA synthesis were observed. Wave I was prereplicative, and wave II was coincident with replication of the viral genome. To determine the temporal appearance of general classes of phage-coded messengers and proteins, we studied the dependence of lysozyme synthesis, phage production, and DNA synthesis on time of addition of transcriptional and translational inhibitors. Lysozyme synthesis started to become refractile to a variety of transcriptional inhibitors (rifampin, streptolydigin, and actinomycin D) between 20 and 22 min postinfection and was completely refractile by 30 min. Nevertheless, functional