NOVEL Escherichia coli dnaB mutant: direct involvement of the dnaB252 gene product in the synthesis of an origin-ribonucleic acid species during initiaion of a round of deoxyribonucleic acid replication.

The initiation process of deoxyribonucleic acid (DNA) replication in Escherichia coli has been studied using the thermoreversible dna initiation mutant E. coli HfrHl65/120/6 dna-252. This dna mutation was incorrectly classed as a dnaA mutation. Biochemical and genetic evidence suggests that the dna-252 mutant is a novel dnaB mutant, possessing phenotypic properties which distinguish it from other dnaB mutants. Sensitivity of reinitiation in the dna-252 mutant to specific inhibitors of protein, ribonucleic acid (RNA), and DNA synthesis was studied. Reinitiation is shown to be sensitive to rifampin and streptolydigin but not to cholramphenicol. Thus, the dna-252 gene product appears to be required during the initiation process for a step occurring either before or during synthesis of an RNA species (origin-RNA). Using reversible inhibition of RNA synthesis by streptolydigin of a streptolydigin-sensitive derivative of the dna-252 mutant, the dna-252 gene product is shown to be directly involved in the synthesis of an orgin-RNA species. These results are included in a schematic model presented in the accompanying paper of the temporal sequence of events occurring during the initiation process.     

Reinitiation of deoxyribonucleic acid synthesis by deoxyribonucleic acid initiation mutants of Escherichia coli: role of ribonucleic acid synthesis, protein synthesis, and cell division.

The dnaA and dnaC genes are thought to code for two proteins required for the initiation of chromosomal deoxyribonucleic acid replication in Escherichia coli. When a strain carrying a mutation in either of these genes is shifted from a permissive to a restrictive temperature, chromosome replication ceases after a period of residual synthesis. When the strains are reincubated at the permissive temperature, replication again resumes after a short lag. This reinitiation does not require either protein synthesis (as measured by resistance to chloramphenicol) or ribonucleic acid synthesis (as measured by resistance to rifampin). Thus, if there is a requirement for the synthesis of a specific ribonucleic acid to initiate deoxyribonucleic acid replication, this ribonucleic acid can be synthesized prior to the time of initiation and is relatively stable. Furthermore, the synthesis of this hypothetical ribonucleic acid does not require either the dnaA of dnaC gene products. The buildup at the restrictive temperature of the potential to reinitiate deoxyribonucleic acid synthesis at the permissive temperature shows rather complex kinetics the buildup roughly parallels the rate of mass increase of the culture for at least the first mass doubling at the restrictive temperature. At later times there appears to be a gradual loss of initiation potential despite a continued increase in mass. Under optimal conditions the increase in initiation potential can equal, but not exceed, the increase in cell division at the restrictive temperature. These results are most easily interpreted according to models that postulate a relationship between the initiation of deoxyribonucleic acid synthesis and the processes leading to cell division.     

Bacteriophage G4 DNA synthesis in temperature-sensitive dna mutants of Escherichia coli.

The synthesis of bacteriophage G4 DNA was examined in temperature-sensitive dna mutants under permissive and nonpermissive conditions. The infecting single-stranded G4 DNA was converted to the parental replicative form (RF) at the nonpermissive temperature in infected cells containing a temperature sensitive mutation in the dnaA, dnaB, dnaC, dnaE, or dnaG gene. The presence of 30 mug of chloramphenicol or 200 mug of rifampin per ml had no effect on parental RF synthesis in these mutants. Replication of G4 double-stranded RF DNA occurred at a normal rate in dnaAts cells at the nonpermissive temperature, but the rate was greatly reduced in cells containing a temperature-sensitive mutation in the dnaB, dnaC, dnaE, or dnaG gene. RF DNA replicated at normal rates in revertants of these dna temperature-sensitive host cells. The simplest interpretation of these observations is that none of the dna gene products tested is essential for the synthesis of the complementary DNA strand on the infecting single-stranded G4 DNA, whereas the dnaB, dnaC, dnaE, (DNA polymerase III), and dnaG gene products are all essential for replication of the double-stranded G4 RF DNA. The alternate possibility that one or more of the gene products are actually essential for G4 parental RF synthesis, even though this synthesis is not defective in the mutant hosts, is also discussed.     

Replication of the Bacteriocinogenic Plasmid Clo DF13 in Thermosensitive Escherichia coli Mutants Defective in Initiation or Elongation of Deoxyribonucleic Acid Replication

The replication of the bacteriocinogenic plasmid Clo DF13 has been studied in the seven temperature-sensitive Escherichia coli mutants defective in deoxyribonucleic acid (DNA) replication (dnaA-dnaG). Experiments with dna initiation mutants revealed that the replication of the Clo DF13 plasmid depends to a great extent on the host-determined dnaC (dnaD) gene product, but depends slightly on the dnaA gene product. The synthesis of Clo DF13 plasmid DNA also requires the dnaF and dnaG gene products, which are involved in the elongation of chromosomal DNA replication. In contrast, the Clo DF13 plasmid is able to replicate in the dnaB and dnaE elongation mutants at the restrictive temperature. When de novo protein synthesis is inhibited by chloramphenicol in wild-type cells, the Clo DF13 plasmid continues to replicate for at least 12 h, long after chromosomal DNA synthesis has ceased, resulting in an accumulation of Clo DF13 DNA molecules of about 500 copies per cell. After 3 h of chloramphenicol treatment, the Clo DF13 plasmid replicates at a rate approximately five times the rate in the absence of chloramphenicol. Inhibition of protein synthesis by chloramphenicol does not influence the level of Clo DF13 DNA synthesis at the restrictive temperature in the dna mutants, except for the dnaA mutant. Chloramphenicol abolishes the inhibition of Clo DF13 DNA synthesis in the dnaA mutant at the nonpermissive temperature. Under these conditions, Clo DF13 DNA synthesis was slightly stimulated in the first 30 min after the temperature shift, and continued for more than 3 h at an almost uninhibited level.     

Initiation of chromosome replication in dnaA and dnaC mutants of Escherichia coli B/r F.

Regulatory aspects of chromosome replication were investigated in dnaA5 and dnaC2 mutants of the Escherichia coli B/r F. When cultures growing at 25 degrees C were shifted to 41 degrees C for extended periods and then returned to 25 degrees C, the subsequent synchronous initiations of chromosome replication were spaced at fixed intervals. When chloramphenicol was added coincident with the temperature downshift, the extend of chromosome replication in the dnaA mutant was greater than that in the dnaC mutant, but the time intervals between initiations were the same in both mutants. Furthermore, the time interval between the first two initiation events was unaffected by alterations in the rate of rifampin-sensitive RNA synthesis or cell mass increase. In the dnaC2 mutant, the capacities for both initiations were achieved in the absence of extensive DNA replication at 25 degrees C as long as protein synthesis was permitted, but the cells did not progress toward the second initiation at 25 degrees C when both protein synthesis and DNA replication were prevented. Cells of the dnaA5 mutant did not achieve the capacity for the second initiation event in the absence of extensive chromosome replication, although delayed initiation may have taken place. A plausible hypothesis to explain the data is that the minimum interval is determined by the time required for formation of a supercoiled, membrane-attached structure in the vicinity of oriC which is required for initiation of DNA synthesis.     

Initation of DNA replication in Escherichia coli. I. Characteristics of the initation process in dna mutants

Summary When a culture of E. coli strain carrying a temperature-sensitive DNA initiation mutation, dna-167 or dnaC2, is exposed to a nonpermissive temperature for a certain period of time, and then transferred back to a permissive temperature, DNA synthesis is resumed even in the presence of chloramphenicol. This shows that thermolabile components coded by either of these mutated genes can be reactivated after return to permissive temperatures, and consequently initiation of a new replication cycle can occur in the absence of concomitant protein synthesis in both strains. The reinitiation of replication occurring after lowering the temperature is sensitive to rifampicin in the dna-167 cells, but not in the dnaC2 mutant. The capacity for initiating a new round of replication is very labile in the dna-167 mutant, but not in the dnaC2 mutant, when a culture of the mutant is maintained at a nonpermissive temperature in the presence of rifampicin. Mechanisms of blocking of the initiation process with these mutants are discussed.After a prolonged exposure of an early-exponential phase culture to high temperatures, reinitiation of DNA replication never exceeds a doubling in both strains, when the temperature is lowered in the presence of chloramphenicol. However, after an exposure of a late-exponential phase culture to a nonpermissive temperature, more than one round of replication occurs in both strains even in the presence of chloramphenicol.     

Bacteriophage phiX174 single-stranded viral DNA synthesis in temperature-sensitive dnaB and dna C mutants of Escherichia coli.

We asked if phiX174 single-stranded DNA synthesis could reinitiate at the nonpermissive temperature in dnaB and dnaC temperature-sensitive host mutants. The rates of single-stranded DNA synthesis were measured after the removal of chlorampheicol that had been added at various times after infection to specifically stop this stage of phiX174 DNA synthesis. Reinitiation was not defective in either mutant host. Our data suggested that the reinitiation of the single-stranded stage of phiX174 DNA synthesis in these experiments was analogous to the normal initiation of this stage of phiX174 DNA synthesis in infections without chloramphenicol. Assuming this to be the case, we conclude that the host cell dnaB and dnaC proteins are not essential for the normal initiation of the single-stranded synthesis stage of phiX174 DNA synthesis. In related experiments we observed that in the dnaC mutant host at the permissive temperature, phiX174 replicative form DNA synthesis continued at its initial rate even during the single-stranded DNA synthesis stage. This indicates that these two stages of phiX174 DNA synthesis are not necessarily mutually exclusive.     

Replication of ColE1 plasmid deoxyribonucleic acid in a thermosensitive dnaA mutant of Escherichia coli.

The replication of ColE1 deoxyribonucleic acid (DNA) took place at the restrictive temperature in a dnaA mutant, dnaA167(Ts). It proceeded at a constant rate at 42 degrees C for at least 3 h. The replication was insensitive to rifampin, which blocked replication at the permissive temperature or in the presence of chloramphenicol, even at the restrictive temperature. A linear DNA strand of ColE1 longer than unit genome size was synthesized. The structure of the replicating molecules observed by electron microscopy was mostly sigma shaped, composed of a circle of a unit genome length with a double-stranded tail. These observations suggest that the replication of ColE1 DNA proceeds via a rolling-circle type of structure in the absence of dnaA function.     

Mode of initiation of constitutive stable DNA replication in RNase H-defective mutants of Escherichia coli K-12.

The alternative pathway of DNA replication in rnh mutants of Escherichia coli can be continuously initiated in the presence of chloramphenicol, giving rise to constitutive stable DNA replication (cSDR). We conducted a physiological analysis of cSDR in rnh-224 mutants in the presence or absence of the normal DNA replication system. The following results were obtained. cSDR allowed the cells to grow in the absence of the normal replication system at a 30 to 40% reduced growth rate and with an approximately twofold-decreased DNA content. cSDR initiation was random with respect to time in the cell cycle as well as choice of origins. cSDR initiation continued to increase exponentially for more than one doubling time when protein synthesis was inhibited by chloramphenicol. cSDR initiation was inhibited during amino acid starvation in stringent (relA+) but not in relaxed (relA1) strains, indicating its sensitivity to ppGpp. cSDR initiation was rifampin sensitive, demonstrating that RNA polymerase was involved. cSDR functioned in dnaA+ rnh-224 strains parallel to the normal oriC+ dnaA+-dependent chromosome replication system.     

Replication of G4 progeny double-stranded DNA in dna mutants of Escherichia coli.

Host functions required for replication of progeny double-stranded DNA of bacteriophage G4 were examined by using metabolic inhibitors and Escherichia coli dna mutants. In dna+ bacteria, synthesis of the progeny replicative form (RF) was relatively resistant to 30 microgram/ml of chloramphenicol, but considerably sensitive to 200 microgram/ml of rifampicin. The RF replication was severely inhibited by 50 microgram/ml of mitomycin C, 50 microgram/ml of nalidixic acid, or 200 microgram/ml of novobiocin. At 41 degrees C, synthesis of G4 progeny RF was distinctly affected in a dnaC(D) mutant and in a dnaG host. The progeny RF replication was prevented at 42 degrees C in a dnaE strain as well as in a dnaB mutant. In a dnaZ strain, the synthetic rate of the progeny RF was markedly reduced at 42 degrees C. At 43 degrees C, the rate of G4 progeny RF synthesis was reduced even in dna+ or dnaA bacteria, but significant amounts of the progeny RF were still synthesized in these hosts at the high temperature. In addition to five dna gene products, host rep function was essential for the RF replication.     

Interactions of DNA replication factors in vivo as detected by introduction of suppressor alleles of dnaA into other temperature-sensitive dna mutants.

Suppressor mutations located within dnaA can suppress the temperature sensitivity of a dnaZ polymerization mutant, indicating in vivo interaction of the products of these genes. The suppressor allele of dnaA [designated dnaA(SUZ, Cs)] could not be introduced, even at the permissive temperature, by transduction into temperature-sensitive (Ts) dnaC or dnaG recipients; it was transduced into dnaB(Ts) and dnaE(Ts) strains but at very low frequency. Recipient cells which were dnaA+ dnaE(Ts) were killed by the incoming dnaA(SUZ, Cs) allele, and it is presumed that combinations of dnaA(SUZ, Cs) with dnaB(Ts), dnaC(Ts), or dnaG(Ts) are lethal also. In one specific case, the lethality required the presence of three alleles: the incoming dnaA suppressor mutation, the resident dnaA+ gene, and the dnaB(Ts) gene. This was shown by the fact that dnaB(Ts) could readily be introduced into a dnaA(SUZ, Cs) dnaB+ recipient. That is, in the absence of dnaA+, the dnaA suppressor and dnaB(Ts) double mutant was stable. One model to explain these results proposes that the dnaA protein functions not only in initiation but also in the replication complex which contains multiple copies of dnaA and other replication factors.     

Influence of Protein and Ribonucleic Acid Synthesis on the Replication of the Bacteriocinogenic Factor Clo DF13 in Escherichia coli Cells and Minicells

The influence of ribonucleic acid (RNA) and protein synthesis on the replication of the cloacinogenic factor Clo DF13 was studied in Escherichia coli cells and minicells. In chromosomeless minicells harboring the Clo DF13 factor, Clo DF13 deoxyribonucleic acid (DNA) synthesis is slightly stimulated after inhibition of protein synthesis by chloramphenicol or puromycin and continues for more than 8 h. When minicells were treated with rifampin, a specific inhibitor of DNA-dependent RNA polymerase, Clo DF13 RNA and DNA synthesis appeared to stop abruptly. In cells, the Clo DF13 factor continues to replicate during treatment with chloramphenicol long after chromosomal DNA synthesis ceases. When rifampin was included during chloramphenicol treatment of cells, synthesis of Clo DF13 plasmid DNA was blocked completely. Isolated, supercoiled Clo DF13 DNA, synthesized in cells or minicells in the presence of chloramphenicol, appeared to be sensitive to ribonuclease and alkali treatment. These treatments convert a relatively large portion of the covalently closed Clo DF13 DNA to the open circular form, whereas supercoiled Clo DF13 DNA, isolated from non-chloramphenicol-treated cells or minicells, is not significantly affected by these treatments. These results indicate that RNA synthesis and specifically Clo DF13 RNA synthesis are involved in Clo DF13 DNA replication and that the covalently closed Clo DF13 DNA, synthesized in the presence of chloramphenicol, contains one or more RNA sequences. De novo synthesis of chromosomal and Clo DF13-specific proteins is not required for the replication of the Clo DF13 factor. Supercoiled Clo DF13 DNA, isolated from a polA107 (Clo DF13) strain which lacks the 5' --> 3' exonucleolytic activity of DNA polymerase I, is insensitive to ribonuclease or alkali treatment, indicating that in this mutant the RNA sequences are still removed from the RNA-DNA hybrid.     

Rescue of abortive T7 gene 2 mutant phage infection by rifampin.

Infection of Escherichia coli with T7 gene 2 mutant phage was abortive; concatemeric phage DNA was synthesized but was not packaged into the phage head, resulting in an accumulation of DNA species shorter in size than the phage genome, concomitant with an accumulation of phage head-related structures. Appearance of concatemeric T7 DNA in gene 2 mutant phage infection during onset of T7 DNA replication indicates that the product of gene 2 was required for proper processing or packaging of concatemer DNA rather than for the synthesis of T7 progeny DNA or concatemer formation. This abortive infection by gene 2 mutant phage could be rescued by rifampin. If rifampin was added at the onset of T7 DNA replication, concatemeric DNA molecules were properly packaged into phage heads, as evidenced by the production of infectious progeny phage. Since the gene 2 product acts as a specific inhibitor of E. coli RNA polymerase by preventing the enzyme from binding T7 DNA, uninhibited E. coli RNA polymerase in gene 2 mutant phage-infected cells interacts with concatemeric T7 DNA and perturbs proper DNA processing unless another inhibitor of the enzyme (rifampin) was added. Therefore, the involvement of gene 2 protein in T7 DNA processing may be due to its single function as the specific inhibitor of the host E. coli RNA polymerase.     

Mechanism of Action of Rifampin on Mycobacterium smegmatis

Deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase (EC 2.7.7.6) isolated from a rifampin-sensitive strain of Mycobacterium smegmatis was 90% inhibited by 1 mug of rifampin per ml; enzyme from a rifampin-resistant mutant was not affected by this concentration of antibiotic. Inhibition of phenylalanine-1-(14)C incorporation by rifampin in growing cultures was complete about 6 min after addition of antibiotic. Under the same conditions, uracil-2-(14)c incorporated was blocked after 1.5 to 2 min. Rifampin kills M. smegmatis very slowly. When rifampin-inhibited cultures were transferred to a rifampin-free medium, there was a partial resumption of uracil-2-(14)C incorporation, even in the presence of chloramphenicol. We conclude that a primary event in the inhibition of M. smegmatis by rifampin is the block of DNA-dependent RNA polymerase.     

Effect of dna mutations on the replication of plasmid pSC101 in Escherichia coli K-12.

Escherichia coli strains with mutations in genes dnaB, dnaC, and dnaG were tested for their capacity to replicate pSC101 deoxyribonucleic acid (DNA) at a nonpermissive temperature. Only a small amount of radioactive thymine was incorporated into pSC101 DNA in the dna mutants at 42 degrees C, whereas active incorporation into plasmid DNA took place in wild-type strains under the same conditions. The effects of the dnaB and dnaC mutations were greater on plasmid DNA synthesis than on host chromosomal DNA synthesis, suggesting that these gene products are directly involved in the process of pSC101 DNA replication. In dnaG mutants, both plasmid and chromosomal DNA synthesis were blocked soon after the shift to high temperature; although the extent of inhibition of the plasmid DNA synthesis was greater during the early period of temperature shift to 42 degrees C as compared with that of the host DNA synthesis, during the later period it was less. It was found that the number of copies of pSC101 per chromosome in dnaA and dnaC strains, grown at 30 degrees C, was considerably lower than that in wildtype strains, suggesting that the replication of pSC101 in these mutant strains was partially suppressed even under the permissive conditions. No correlation was found between the number of plasmid copies and the tetracycline resistance level of the host bacterium.     

Characterization of an improved in vitro DNA replication system for Escherichia coli plasmids.

A modified in vitro replication system has been characterized and used to catalogue the host proteins required for the replication of plasmid RSF1030. These extracts differ from systems described previously in that endogenous DNA is removed. Replication in vitro therefore requires an exogenouos RSF1030. Synthesis in the in vitro system faithfully mimics in vivo replication with respect to the products synthesized, effects of specific inhibitors, and requirements for RNA polymerase and DNA polymerase I. In addition, we find that proteins encoded by dnaB, dnaC, dnaG, dnaI, dnaP and polC (DNA polymerase III), are required for in vitro plasmid synthesis. The product of dnaA is not required. Extracts prepared from E. coli mutants deficient in in vitro replication can be complemented by addition of purified proteins or of extracts carrying the wild type protein.     

dnaA acts before dnaC in the initiation of DNA replication

We constructed a double mutant of Escherichia coli K-12 carrying dnaA(Ts) and dnaC(Cs) lesions. In this mutant DNA synthesis proeceeds normally at 32 degrees C and initiation is inhibited at both 41 and 20 degrees C. By shifting this culture grown at 32 degrees C to the two restrictive temperatures in different time sequences and assaying protein and DNA synthesis of cells growing at different temperatures, we found that dnaA and dnaC genes work independently with dnaA acting before dnaC. While preparing special strains for this work, we also showed that the order of genes in the neighborhood of dnaA is dnaA-tnaA-phoS-ilv.