Overinitiation of chromosome and plasmid replication in a dna Acos mutant of Escherichia coli K12. Evidence for dnaA-dnaB interactions

The dnaAcos mutations are phenotypic suppressors of dnaAts46 that are co-transduced with dnaA, render the cell cold sensitive, and cause an excess of chromosome replication relative to cell mass when the cells are shifted from 42 degrees C to 32 degrees C. We have used pulse labelling and DNA-DNA hybridization to follow the effect of a temperature shift on the replication of the chromosome and of the plasmids pSC101, RTF-Tc, and lambda dv in such strains. After a shift of a dnaAcos strain from 42 degrees C to 32 degrees C (non-permissive temperature), initiation of the chromosome and replication of the plasmid pSC101 are stimulated, while the dnaA-independent plasmid RTF-Tc is not affected. The presence of pSC101 does not affect the level of overinitiation of the chromosome. The presence of lambda dv suppresses the cold sensitivity of dnaAcos mutants and allows the cells to grow at both 32 degrees C and 42 degrees C. The presence of lambda dv suppresses the overinitiation of chromosome and of pSC101 replication at 32 degrees C. Previous reports had shown that these suppressions involve an interaction between the dnaA product and the lambda P protein, which is also known to interact with dnaB. We show here that the mutant prophage P1 bac-crr, which produces high levels of a dnaB analogue, suppresses the dnaAcos phenotype, while wild type P1 does not. These results suggest that initiation involves interactions between the dnaA and dnaB products.     

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.     

Host cell DNA chain initiation protein requirements for replication of bacteriophage G4 replicative-form DNA.

Bacteriophages G4ev1 and G4bs1 are simple temperature-resistant derivatives of wild-type G4 as demonstrated by restriction endonuclease analyses. The rate of replication of the duplex replicative-form DNA of these phages was normal in dnaB and dnaC mutants of the host, whereas the rate was markedly reduced in a dnaG host mutant at the restrictive temperature. We conclude that G4 duplex DNA replication requires the host cell dnaG protein, but not the dnaB and dnaC proteins. The reasons for the differences between our conclusions and those based on previously published data are documented and discussed.     

Prophage Induction by High Temperature in Thermosensitive dna Mutants Lysogenic for Bacteriophage Lambda

High-temperature treatment of thermosensitive dna mutants lysogenic for phage lambda leads to prophage induction and release of phage (at the permissive temperature) in elongation-defective mutants of the genotypes dnaB, dnaE, and dnaG. In initiation-defective mutants no prophage induction occurs at 42 C in mutants of the genotype dnaA, whereas with a dnaC mutant as well as with strain HfrH 252 (map position not yet known) phages are released at 42 C. DNA degradation at the replication fork at 42 C is observed in all dnaB(lambda) mutants tested, but not in mutants of the genotypes dnaE(lambda) and dnaG(lambda). Therefore, degradation of replication fork DNA is not a prerequisite for prophage induction.     

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 bacteriophage G13 DNA in dna mutants of Escherichia coli.

Host functions required for replication of microvirid phage G13 DNA were investigated in vivo, using thermosensitive dna mutants of Escherichia coli. In dna+ bacteria, conversion of viral single-stranded DNA into double-stranded replicative form (stage I synthesis) was resistant to 150 microgram/ml of chloramphenicol or 200 microgram/ml of rifampicin. Although multiplication of G13 phage was severely inhibited at 42--43 degrees C even in dna+ host, considerable amount of parental replicative form was synthesized at 43 degrees C in dna+, dnaA or dnaE bacteria. In dnaB and dnaG mutants, however, synthesis of parental replicative form was severely inhibited at the restrictive temperature. Interestingly enough, stage I replication of G13 DNA was, unlike that of phiX174, dependent on host dnaC(D) function. Moreover, the stage I synthesis of G13 DNA in dnaZ was thermosensitive in nutrient broth but not in Tris/casamino acids/glucose medium. In contrast with the stage I replication, synthesis of G13 progeny replicative form was remarkably thermosensitive even in dna+ or dnA cells.     

Conversion of bacteriophage G4 single-stranded viral DNA to double-stranded replicative form in dna mutants of Escherichia coli.

Host functions involved in synthesis of parental replicative form of bacteriophage G4 were investigated using various replication mutants of Escheria coli. In dna+ bacteria, conversion of single-stranded viral DNA to replicative form DNA was insensitive to 200 microng/ml of rifampicin or 25 microng/ml of chloramphenicol. At high temperature, synthesis of parental replicative form was unaffected in mutants thermosensitive for dnaA, dnaB, dnaC(D), dnaE or dnaH. In dnaG or dnaZ mutants, however, parental replicative from DNA synthesis was clearly thermosensitive at 43 degrees C. Although the host rep product was essential for viral multiplication, the conversion of single stranded to replicative form was independent of the rep function.     

Replication of bacteriophage phiK duplex replicative-form DNA in dnaB and dnaC mutants of Escherichia coli.

We have directly tested the effects of host cell DNA synthesis mutations on bacteriophage phiK replicative-form (RF) DNA replication in vivo. We observed that phiK RF DNA replication continued at normal rates in both dnaB and dnaC mutant hosts under conditions in which the activities of the dnaB and dnaC gene products were shown to be markedly reduced. This suggests that these two host proteins are not essential for normal phiK RF DNA replication. In control experiments we observed markedly reduced rates of phiK RF DNA replication in temperature-sensitive dnaG and dnaE host mutants, indicating that the products of these genes are essential. Thus, the mechanism of DNA chain initiation in vivo on the duplex RF DNA templates of isometric phages such as phiK apparently is different from that on the similar templates of isometric phages such as phiX174. The implications of this difference are discussed in the text.     

Synthesis of bacteriophage phiC DNA in dna mutants of Esherichia coli.

Host dna functions involved in the replication of microvirid phage phiC DNA were investigated in vivo. Although growth of this phage was markedly inhibited even at 35-37 degrees C even in dna+ host, conversion of the infecting single-stranded DNA into the double-stranded parental replicative form (stage I synthesis) occurred normally at 43 degrees C in dna+, dnaA, dnaB, dnaC(D), and dnaE cells. In dnaG mutant, the stage I synthesis was severely inhibited at 43 degrees C but not at 30 degrees C. The stage I replication of phiC DNA was clearly thermosensitive in dnaZ cells incubated in nutrient broth. In Tris-casamino acids-glucose medium, however, the dnaZ mutant sufficiently supported synthesis of the parental replicative form. At 43 degrees C, synthesis of the progeny replicative form DNA (stage II replication) was significantly inhibited even in dna+ cells and was nearly completely blocked in dnaB or dnaC(D) mutant. At 37 degrees C, the stage II replication proceeded normally in dna+ bacteria.     

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.     

Escherichia coli deoxyribonucleic acid synthesis mutants: their effect upon bacteriophage P2 and satellite bacteriophage P4 deoxyribonucleic acid synthesis.

Escherichia coli C strains containing different deoxyribonucleic acid (DNA) synthesis mutations have been tested for their support of the DNA synthesis of bacteriophage P2 and its satellite phage P4. Bacteriophage P2 requires functional dnaB, dnaE, and dnaG E. coli gene products for DNA synthesis, whereas it does not require the products of the dnaA, dnaC, or dnaH genes. In contrast, the satellite virus P4 requires functional dnaE and dnaH gene products for DNA synthesis and does not need the products of the dnaA, dnaB, dnaC, and dnaG genes. Thus the P2 and P4 genomes are replicated differently, even though they are packaged in heads made of the same protein.     

Growth of Salmonella bacteriophage P22 in Escherichia coli dna(Ts) mutants.

Salmonella bacteriophage P22 grows in two deoxyribonucleic acid initiation mutants of Escherichia coli under nonpermissive conditions, dnaA and dnaC. Functional products of genes dnaE, dnaZ, lig, dnaK, and dnaG are indispensable for deoxyribonucleic acid replication of P22. In 11 E. coli dnaB mutants belonging to all phenotypic groups, phage were produced at 42 degrees C.     

Separate roles of Escherichia coli replication proteins in synthesis and partitioning of pSC101 plasmid DNA

We report here that the Escherichia coli replication proteins DnaA, which is required to initiate replication of both the chromosome and plasmid pSC101, and DnaB, the helicase that unwinds strands during DNA replication, have effects on plasmid partitioning that are distinct from their functions in promoting plasmid DNA replication. Temperature-sensitive dnaB mutants cultured under conditions permissive for DNA replication failed to partition plasmids normally, and when cultured under conditions that prevent replication, they showed loss of the entire multicopy pool of plasmid replicons from half of the bacterial population during a single cell division. As was observed previously for DnaA, overexpression of the wild-type DnaB protein conversely stabilized the inheritance of partition-defective plasmids while not increasing plasmid copy number. The identification of dnaA mutations that selectively affected either replication or partitioning further demonstrated the separate roles of DnaA in these functions. The partition-related actions of DnaA were localized to a domain (the cell membrane binding domain) that is physically separate from the DnaA domain that interacts with other host replication proteins. Our results identify bacterial replication proteins that participate in partitioning of the pSC101 plasmid and provide evidence that these proteins mediate plasmid partitioning independently of their role in DNA synthesis.     

Involvement of integration host factor (IHF) in maintenance of plasmid pSC101 in Escherichia coli: mutations in the topA gene allow pSC101 replication in the absence of IHF.

Integration host factor (IHF), encoded by the himA and himD genes, is a histonelike DNA-binding protein that participates in many cellular functions in Escherichia coli, including the maintenance of plasmid pSC101. We have isolated and characterized a chromosomal mutation that compensates for the absence of IHF and allows the maintenance of wild-type pSC101 in him mutants, but does not restore IHF production. The mutation is recessive and was found to affect the gene topA, which encodes topoisomerase I, a protein that relaxes negatively supercoiled DNA and acts in concert with DNA gyrase to regulate levels of DNA supercoiling. A previously characterized topA mutation, topA10, could also compensate for the absence of IHF to allow pSC101 replication. IHF-compensating mutations affecting topA resulted in a large reduction in topoisomerase I activity, and plasmid DNA isolated from such strains was more negatively supercoiled than DNA from wild-type strains. In addition, our experiments show that both pSC101 and pBR322 plasmid DNAs isolated from him mutants were of lower superhelical density than DNA isolated from Him+ strains. A concurrent gyrB gene mutation, which reduces supercoiling, reversed the ability of topA mutations to compensate for a lack of him gene function. Together, these findings indicate that the topological state of the pSC101 plasmid profoundly influences its ability to be maintained in populations of dividing cells and suggest a model to account for the functional interactions of the him, rep, topA, and gyr gene products in pSC101 maintenance.     

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.     

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.     

Identification and characterization of a novel allele of Escherichia coli dnaB helicase that compromises the stability of plasmid P1

Bacteriophage P1 lysogenizes Escherichia coli cells as a plasmid with approximately the same copy number as the copy number of the host chromosome. Faithful inheritance of the plasmids relies upon proper DNA replication, as well as a partition system that actively segregates plasmids to new daughter cells. We genetically screened for E. coli chromosomal mutations that influenced P1 stability and identified a novel temperature-sensitive allele of the dnaB helicase gene (dnaB277) that replaces serine 277 with a leucine residue (DnaB S277L). This allele conferred a severe temperature-sensitive phenotype to the host; dnaB277 cells were not viable at temperatures above 34 degrees C. Shifting dnaB277 cells to 42 degrees C resulted in an immediate reduction in the rate of DNA synthesis and extensive cell filamentation. The dnaB277 allele destabilized P1 plasmids but had no significant influence on the stability of the F low-copy-number plasmid. This observation suggests that there is a specific requirement for DnaB in P1 plasmid maintenance in addition to the general requirement for DnaB as the replicative helicase during elongation.     

Mutations of temperature sensitivity in R plasmid pSC101.

Temperature-sensitive (Ts) mutant plasmids isolated from tetracycline resistance R plasmid pSC101 were investigated for their segregation kinetics and deoxyribonucleic acid (DNA) replication. The results fit well with the hypothesis that multiple copies of a plasmid are distributed to daughter cells in a random fashion and are thus diluted out when a new round of plasmid DNA replication is blocked. When cells harboring type I mutant plasmids were grown at 43 degrees C in the absence of tetracycline, antibiotic-sensitive cells were segregated after a certain lag time. This lag most likely corresponds to a dilution of plasmids existing prior to the temperature shift. The synthesis of plasmid DNA in cells harboring type I mutant plasmids was almost completely blocked at 43 degrees C. It seems that these plasmids have mutations in the gene(s) necessary for plasmid DNA replication. Cells haboring a type II mutant plasmid exhibited neither segregation due to antibiotic sensitivity nor inhibition of plasmid DNA replication throughout cultivation at high temperature. It is likely that the type II mutant plasmid has a temperature-sensitive mutation in the tetracycline resistance gene. Antibiotic-sensitive cells haboring type III mutant plasmids appeared at high frequency after a certain lag time, and the plasmid DNA synthesis was partially suppressed at the nonpermissive temperature. They exhibited also a pleiotrophic phenotype, such as an increase of drug resistance level at 30 degrees C and a decrease in the number of plasmid genomes in a cell.     

Dominant lethal mutations in the dnaB helicase gene of Salmonella typhimurium

A class of dominant lethal mutations in the dnaB (replicative helicase) gene of Salmonella typhimurium is described. The mutated genes, when present on multicopy plasmids, interfered with colony formation by Escherichia coli host strains with a functional chromosomal dnaB gene. The lethal phenotype was expressed specifically in supE (glutamine-inserting) host strains and not in Sup+ strains, because the mutant genes, by design, also possessed an amber mutation derived from a glutamine codon. Mutations located at 11 sites by deletion mapping and DNA sequence analysis varied in the temperature dependence and severity of their lethal effects. None of the mutations complemented a dnaB(Ts) host strain at high temperature (42 degrees C). Therefore, these nonfunctional DnaB proteins must engage some component(s) of the DNA replication machinery and inhibit replication. These mutations are predicted to confer limited, specific defects in either the catalytic activity of DnaB or the ability of DnaB to interact with one of its ligands such as DNA, nucleotide, or another replication protein. The variety of mutant sites and detailed phenotypes represented in this group of mutations may indicate the operation of more than one specific mechanism of lethality.     

Plasmid ColE1 DNA replication in Escherichia coli strains temperature-sensitive for DNA replication

Mutants of the dnaA, dnaC, dnaD, polC, dnaF and dnaG gene loci were tested for their capacity for colicinogenic plasmid E1 (ColE1) replication at a non-permissive temperature. It was found that ColE1 replication was independent of the dnaA gene function and dependent on dnaC, D, F and G. ColE1 replication in the polC mutant E486 continued for several hours but at a greatly reduced rate. No effect was found of the dnaG mutation on thymine-deprivation-induced "priming" of ColE1 replication at the non-permissive temperature. The mutants also were tested for aberrant replication intermediates of plasmid DNA as well as a temperature sensitive supercoiled DNA-protein relaxation complex. RNA-containing supercoils were found to accumulate in a poIC mutant also blocked for protein synthesis.