Rifampin inhibition of bacteriophage phiX174 parental replicative-form DNA synthesis in an Escherichia coli dnaC mutant

L B Dumas, C A Miller, M L Bayne
1975 Journal of Virology  
The Escherichia coli dnaC protein is not absolutely required in vivo for bacteriophage 4X174 parental replicative-form synthesis (Kranias and Dumas, 1974) . However, when rifampin is present at a concentration that inhibits DNA-dependent RNA polymerase, OX174 parental replicative-form synthesis is dependent on the dnaC protein activity. We conclude that E. coli DNA-dependent RNA polymerase can substitute for the dnaC protein in OX174 parental replicative-form DNA synthesis, presumably in its
more » ... tiation. The implications of this result with respect to the in vitro synthesis of the complementary strand of OX174 DNA are discussed. In the first stage of the replication of bacteriophage OX174 DNA in its host, Escherichia coli, a complementary DNA strand is synthesized on the infecting single-stranded DNA template (parental replicative-form [RF] synthesis). This stage is catalyzed by preexisting host cell enzymes; i.e., it requires no phage-induced protein synthesis (11). The initiation of the synthesis of the complementary DNA strand requires RNA synthesis (8) and is resistant to rifampin (8, 10), a specific inhibitor of the host cell DNAdependent RNA polymerase (12, 15). In cell-free extracts of E. coli, the rifampinresistant synthesis of the complementary OX174 DNA strand requires the participation of the protein product of the host cell dnaC,gene (7, 16) . DNA-dependent RNA polymerase-catalyzed synthesis of an RNA primer can obviate the requirement for the dnaC protein (13, 16). The E. coli dnaC protein is required for the initiation of cycles of chromosome replication in vivo (1, 9), as well as for the replication of the double-stranded replicative form of 4X174 DNA (5). However, OX174 parental RF synthesis occurs with near-normal efficiency in a dnaC-defective mutant of E. coli, suggesting that the dnaC protein is not absolutely required for the initation of this synthesis in vivo (5). Since the synthesis of the OX174 complementary DNA strand in vitro requires the dnaC protein only when DNA-dependent RNA polymerase activity is inhibited, we asked whether the same might be true in the cell. We present evidence here showing that the host cell dnaC protein is indeed required for the synthesis of the OX174 complementary DNA strand in vivo when rifampin is present at a concentration that inhibits DNA-dependent RNA polymerase. MATERIALS AND METHODS Bacteria and phage. The mutant host strain LD332 is a nitrosoguanidine-induced temperaturesensitive mutant of H502 (uvrA -, thyA -, endIl) isolated in our laboratory. The temperature-sensitive locus lies within the region of the chromosome carried by F-prime factor 101 and cotransduces with dra-1 at a frequency of 0.29. These data indicate that this marker maps at the dnaC locus (14) . This temperature-sensitive mutant has the same properties as the 4X174-sensitive dnaC mutant previously described (5), except that it is less leaky for DNA synthesis at the nonpermissive temperature. Host strain LD3321 is a spontaneous temperatureinsensitive revertant of LD332. Host strain LD3322 is a spontaneous rifampin-resistant mutant of LD332. 4OX174 am3 is a lysis-defective gene E mutant. Infection and lysis. A 100-ml culture of host bacteria was grown on TPGA medium (3) supplemented with 2 tsg of thymine per ml at 30 C to a cell density of 5 x 10' cells/ml. The cells were collected by centrifugation, resuspended in starvation buffer (2), and treated with 100 ,g of mitomycin C per ml for 20 min. The cells were again collected by centrifugation and resuspended in 100 ml of TPGA medium supplemented with 1 ;g of thymine per ml. Twenty-five-milliliter portions were incubated at 30 and 41 C with aeration for 30 min. Rifampin was added to 200 jg/ml to one of the cultures at 25 min after resuspension. At the end of the 30-min preincubation (zero time), 32P-labeled OX174 am3 was added to each culture of LD332 and LD3321 at a multiplicity of infection of 0.2, and to each culture of LD3322 at a multiplicity of infection of 3. The specific activity of the phage was 10-' counts/min per PFU. [JH]Thymidine (100 MCi) was added to each culture at the same time. After 20 575 on May 4, 2020 by guest
doi:10.1128/jvi.16.3.575-580.1975 fatcat:waauplqxwnebnat7ydjqg5d3mi