Replication of small plasmids in extracts of Escherichia coli: involvement of the dnaB and dnaC protein in the replication of early replicative intermediates.

Summary The role of E. coli dnaB and dnaC protein in the replication of plasmid ColE1 and RSF1030 DNA was investigated in a soluble in vitro system (Staudenbauer, 1976a). Extracts from dnaB and dnaC mutants which are phenotypically DNA initiationor DNA elongation-defective were examined for their replicative capacity. It was found that all mutants tested are deficient in the synthesis of supercoiled plasmid DNA. Deficient extracts of dnaB mutants could be partially complemented by purified dnaB wild type protein but required for full complementation dnaC wild type protein as well. The dnaB wild type protein could be replaced by a P1dnaB analog (ban) protein complexed with a dnaB ts protein. Deficient extracts of dnaC mutants were complemented by purified dnaC wild type protein alone.The in vitro plasmid replication cycle had been separated into an early and late stage (Staudenbauer, 1977). Analysis by CsCl velocity centrifugation of the plasmid DNA synthesized in mutant extracts indicates that the early stage, namely the synthesis of early replicative intermediates, proceeds in all dnaB and dnaC mutants tested. However, replication of the early intermediates during the late stage depends on both the dnaB and dnaC protein. These conclusions were confirmed using inhibitors of DNA synthesis.     

Analysis of the dnaB function of Escherichia coli K12 and the dnaB-like function of P1 prophage

The dnaB function of Escherichia coli K12 was studied with a series of isogenic strains differing from each other only by a mutation in the dnaB gene. The strains showed different phenotypes depending on the particular dnaB mutation they carry. A clear example is provided by a strain carrying dnaB266 mutation which turned out to be an amber mutation. When the mutation was suppressed by different suppressors, the strains showed different phenotypes. Thus, dnaB proteins which differ from each other by only one amino acid at the mutation site give different phenotypes. Mutation dnaB266 is lethal to the host when not suppressed. Hence the dnaB protein is essential for bacterial growth.Three P1 mutants, P1mcb-4, P1mcb-5 and P1mcb-8, were isolated which converted the temperature-sensitive bacterial growth of dnaB266-supE to resistant growth. Lysogenization with P1mcb allowed growth of dnaB266su⁻ strain which was absolutely defective in the bacterial dnaB function, indicating that the dnaB-like function of P1 prophage can substitute for the bacterial dnaB function. However, lysogenization by P1mcb did not support the growth of λ and λπ phages on dnaB 266su⁻. While P1mcb-4 and P1mcb-5 prophages altered the phenotypes of other dnaB strains to permit the growth of bacterial and λ phage at 32 °C and 42 °C, P1mcb-8 prophage supports the growth of λ phages and bacteria at 42 °C but not λ phage growth on groP-bacteria at 32 °C. The alteration of phenotypes of the P1mcb lysogens varied depending on the dnaB mutations they carried. Mutual interaction between the bacterial dnaB protein and the phage dnaB-like protein which results in different phenotypes of lysogens is suggested.     

ThegrpD55 locus ofEscherichia coli appears to be an allele ofdnaB

Attempts to characterize thegrpD55 mutation ofEscherichia coli have led us to conclude that the gene had been assigned an incorrect map position. The mutation was found to cotransduce withmalF3089:: Tn10 (at ～91.5 min) and adnaB-expressing plasmid was able to complement fully thegrpD55 defect in λ replication. These studies strongly suggest thatgrpD55 is an allele ofdnaB and is localized near 92 min on theE. coli linkage map.     

The isolation and characterization of escherichia coli dnaB::Tn10 insertion mutations

Summary Exploitation of the ability of the ban protein encoded by phage P1 to compensate for dnaB-defective host mutations, allowed the isolation of dnaB::Tn10 insertion mutations. The presence of P1bac prophage was required for survival of dnaB::Tn10 mutants, and such lysogens were cryosensitive. The insertions were shown to map in dnaB by transduction and this was confirmed by complementation analysis. The dnaB::Tn10 (P1bac) strains were non-permissive for growth but did support the growth of -dnaB ⁺specialized transducing phage. No antigenically active dnaB product could be detected by immunologic assays using either of two methods. In addition, it was shown that the observed cryosensitivity of P1bac suppression was a direct result of reversible inactivation of the ban protein at low temperature.     

Recombinant oxyntomodulin

An artificial gene encoding oxyntomodulin was obtained using chemical and enzymatic methods and cloned into Escherichia coli. A recombinant plasmid was constructed containing a hybrid oxyntomodulin gene and Ssp dnaB intein from Synechocystis sp. The expression of the resulting hybrid gene in E. coli, its properties, and the conditions of its autocatalytic cleavage to oxyntomodulin were studied.     

A dnaB analog specified by bacteriophage P1

Bacteriophage P1 is shown to determine a product that can substitute in DNA replication for the protein specified by cistron dnaB of Escherichia coli. The viral dnaB analog (ban) is repressed in the wild-type P1 prophage and expressed constitutively in plaque-forming mutants, P1bac, described here. A particular P1bac prophage allows lysogens of dnaBts bacteria to survive as colony-formers at temperatures that arrest DNA synthesis in the non-lysogens. The P1bac prophage furthermore permits construction of an otherwise inviable strain bearing the unsuppressed amber mutation dnaB266.P1bac prophages also suppress the groP character which is associated with certain dnaB mutations. The subclass of dnaB mutations called groP are those which prevent the growth of bacteriophage λ⁺ at temperatures permissive for bacterial DNA synthesis, but allow the growth of certain λ mutants (λπ); π mutations have been mapped in gene P. Thus, λ⁺ is enabled to grow in groP hosts by the presence of P1bac-1 prophage. When dnaB protein is absent, however, as in the case of the unsuppressed amber mutant, the ban protein furnished by the P1bac prophage does not support λ growth. Therefore, in the groP(P1bac-1) lysogens both the dnaB and ban products are needed for λ growth, suggesting interactions between these E. coli and P1 proteins or their subunits.Mutations (termed ban) that prevent the expression of the dnaB analog determined by P1 have been obtained. P1bac-1ban-1, unlike P1bac-1, fails to replicate in dnaBts hosts at temperatures non-permissive for bacterial DNA synthesis. Thus, the dnaB protein and its P1-determined analog can interchangeably fulfill an essential role in the replication of both the E. coli and P1 replicons. At permissive temperatures the lysogenization of certain dnaBts strains by P1bac-1ban-1 is very inefficient, probably as a result of negative complementation.Mutations bac-1 and ban-1 are closely linked on the P1 chromosome and their order relative to several amber mutations has been determined. Dominance studies of the alleles in transient diploids show that the ban-1 mutation is recessive to ban⁺. The bac-1 mutation, on the other hand, behaves in dominance tests as a DNA site mutation that permits constitutive expression in cis of the operon to which the ban gene belongs.     

DNA replication in dnaB mutants of Escherichia coli: Gene product interaction and synthesis of 4 S pieces

The DNA produced by several dnaB mutants has been examined both in vivo and in vitro. The alleles chosen for study had previously been shown to differ over a wide range in the apparent severity of their effects on DNA replication.Comparison of DNA replication between dnaB heteroallelic diploids and the constituent haploid strains indicates interaction between the dnaB products in the heteroallelic diploids. The data are consistent with a functional multimeric aggregate of dnaB gene products that is at least a tetramer.Alkaline sucrose gradient profiles of pulse-labeled DNA, synthesized by some of the mutants in vivo and by mutant lysates in vitro, exhibit a peak at about 4 S. The 4 S DNA is most apparent in those mutants in which replication is most severely restricted by temperature.This 4 S material can be chased in vitro into DNA larger than Okazaki pieces, and density transfer experiments indicate that these pieces are formed at the replication fork. Conversion of the 4 S material to large DNA is not altered by inhibition of polynucleotide ligase either by the presence of the lig-4 polA1 mutations in vivo or by the addition of nicotinamide mononucleotide in vitro. The in vitro observations suggest that 4 S pieces are formed on only one side of the replication fork.     

A mutation, bsu, that suppresses temperature-sensitive dnaB function in Escherichia coli K12

Summary A mutation of Escherichia coli K12 that suppresses certain temperature-sensitive dnaB mutations was identified. The suppressor, named bsu maps very near the dnaG mutations. The bsu mutation in dnaB bacteria appears to be dominant over the wildtype allele, and suppresses specifically the temperature-sensitive dnaB mutations which are revertible phenotypically when salt is present. The observed specificity in suppression suggests that the product of bsu alone cannot substitute for the defective dnaB gene products. These findings suggest stronly that gene products of bsu and dnaB interact with each other in the process of DNA replication in E. coli.     

Transposon 10 promoted deletions and inversions in the transfer genes of R100-1

Summary The replication of the ColE1 plasmid was studied in extracts from E. coli dnaG mutants. It was found that the synthesis of the complementary strands of ColE1 DNA can be carried out in these extracts in two consecutive steps: (1) synthesis of the leading L strand independent of the dnaG function, and (2) synthesis of the lagging H strand depending upon addition of wild-type dnaG protein. In contrast to L strand synthesis, the latter reaction is insensitive to rifampicin and novobiocin. Both synthetic pathways are however blocked by antiserum directed against dnaB protein. This indicates an additional role of the dnaB protein in duplex DNA replication besides assisting the dnaG protein in the priming of lagging strand synthesis. The T7 gene-4 protein acting in conjunction with T7 DNA polymerase can substitute for both the function of the dnaB and dnaG protein. It is concluded that plasmid replication proceeds by a semi-discontinuous mechanism.     

Trashing of Single-Stranded DNA Generated during Processing of Arrested Replication Fork in E. coli

We analyzed formation of single-stranded DNA (ssDNA) related to SOS induction in nalidixilate (Nal)-treated Escherichia coli, using immunofluorescence microscopy accompanied by computer analysis. We found enhancement of both ssDNA concentrations and cells having ssDNA foci that often localized around cellpoles. Analyzing several mutants deficient in DNA repair or replication, we found, after Nal treatment, that recN, recA, uvrD and dnaB failed to increase ssDNA concentration and that recG and particularly ruvA only partially enhanced it. In Nal-treated recB mutant, despite its failure in SOS induction, ssDNA foci positive cells increased with a slight enhancement of its concentration. These observations suggest the existence of a cellular process that sequesters genotoxic ssDNA as inert form, offering a new concept for SOS suppressor genes action.     

Bacteriophage P1 Ban protein is a hexameric DNA helicase that interacts with and substitutes for Escherichia coli DnaB

Since the ban gene of bacteriophage P1 suppresses a number of conditionally lethal dnaB mutations in Escherichia coli, it was assumed that Ban protein is a DNA helicase (DnaB analogue) that can substitute for DnaB in the host replication machinery. We isolated and sequenced the ban gene, purified the product, and analysed the function of Ban protein in vitro and in vivo. Ban hydrolyses ATP, unwinds DNA and forms hexamers in the presence of ATP and magnesium ions. Since all existing conditionally lethal dnaB strains bear DnaB proteins that may interfere with the protein under study, we constructed a dnaB null strain by using a genetic set-up designed to provoke the conditional loss of the entire dnaB gene from E.coli cells. This novel tool was used to show that Ban restores the viability of cells that completely lack DnaB at 30°C, but not at 42°C. Surprisingly, growth was restored by the dnaB252 mutation at a temperature that is restrictive for ban and dnaB252 taken separately. This indicates that Ban and DnaB are able to interact in vivo. Complementary to these results, we demonstrate the formation of DnaB–Ban hetero-oligomers in vitro by ion exchange chromatography. We discuss the interaction of bacterial proteins and their phage-encoded analogues to fulfil functions that are essential to phage and host growth.     

Intragenic and Extragenic Suppressors of Temperature Sensitive Mutations in the Replication Initiation Genes dnaD and dnaB of Bacillus subtilis

Background

The Bacillus subtilis genes dnaD and dnaB are essential for the initiation of DNA replication and are required for loading of the replicative helicase at the chromosomal origin of replication oriC. Wild type DnaD and DnaB interact weakly in vitro and this interaction has not been detected in vivo or in yeast two-hybrid assays.

Methodology/Principal Findings

We isolated second site suppressors of the temperature sensitive phenotypes caused by one dnaD mutation and two different dnaB mutations. Five different intragenic suppressors of the dnaD23ts mutation were identified. One intragenic suppressor was a deletion of two amino acids in DnaD. This deletion caused increased and detectable interaction between the mutant DnaD and wild type DnaB in a yeast two-hybrid assay, similar to the increased interaction caused by a missense mutation in dnaB that is an extragenic suppressor of dnaD23ts. We isolated both intragenic and extragenic suppressors of the two dnaBts alleles. Some of the extragenic suppressors were informational suppressors (missense suppressors) in tRNA genes. These suppressor mutations caused a change in the anticodon of an alanine tRNA so that it would recognize the mutant codon (threonine) in dnaB and likely insert the wild type amino acid (alanine).

Conclusions/Significance

The intragenic suppressors should provide insights into structure-function relationships in DnaD and DnaB, and interactions between DnaD and DnaB. The extragenic suppressors in the tRNA genes have important implications regarding the amount of wild type DnaB needed in the cell. Since missense suppressors are typically inefficient, these findings indicate that production of a small amount of wild type DnaB, in combination with the mutant protein, is sufficient to restore some DnaB function.     

The requirements for conjugal DNA synthesis in the donor strain during flac transfer.

Although neither rifampicin nor spectinomycin had any effect on the frequency of Flac transfer by a sensitive donor, rifampicin but not spectinomycin prevented donor conjugal DNA synthesis as measured in matings between a dnaB donor and a tdk recipient. An untranslated RNA species is therefore probably required for this synthesis, although transfer took place even in its absence. Donor conjugal DNA synthesis was abolished in a dnaE donor, showing that DNA polymerase III is responsible for this process; again, plasmid DNA transfer was not affected.Flac mutants lacking the F pilus gave neither donor conjugal DNA synthesis nor plasmid DNA transfer, probably because they could not receive a “mating signal” to activate the transfer process. The products of traI and traM were also required both for donor conjugal DNA synthesis and for physical transfer of plasmid DNA, probably being involved in the conversion of covalently closed circular plasmid DNA into the open circular form that is the substrate for the independent although normally simultaneous synthesis and transfer steps. In contrast, donor conjugal DNA synthesis took place at a normal rate in both piliated traG and traN mutants, and at a reduced rate in traD mutants, although in no case was there physical transfer of plasmid DNA. These gene products are therefore required for DNA transfer to the recipient, and in addition, the absence of the traD product may hinder DNA synthesis.Based upon these results, a scheme for the processing of DNA during conjugation is presented.     

A dnaB analog ban, specified by bacteriophage P1: genetic and physiological evidence for functional analogy and interactions between the two products.

Summary Bacteriophage P1 has been shown previously to determine a product ban than can substitute in DNA replication for the protein specified by cistron dnaB of Escherichia coli. However, ban product furnished by P1 bac prophage (ban constitutive) substitutes only poorly for DNA replication in the absence of dnaB product in a strain bearing an unsuppressed amber mutation, dnaB266, as shown by the cryosensitivity of the dnaB266 (P1 bac) lysogen and its unability to support growth. An additional mutation (termed crr) in the P1 bac prophage has been obtained which confers cryoresistance to the sup ⁺ dnaB266 (P1 bac crr) lysogen and restores its ability to support growth. ban product produced in P1 bac crr lysogen fulfills all dnaB roles in vivo, especially in the various instances in which ban product expressed in P1 bac lysogens does not. The ban product is expressed constitutively in P1 crr prophage. The crr-1 mutation is tightly linked to the bac-1 and ban-1 mutations and is dominant over crr ⁺. The nature of the crr mutation is discussed: two hypotheses are considered, that of a mutation in the ban gene rendering the ban product more active or that of a site mutation in the ban operon increasing the level of ban expression. Expression of ban product (wild type or altered) leads to interactions with the variously altered dnaB product. Both positive and negative interactions are described. Genetic results presented here suggest that ban and dnaB subunits interact to form hybrid dnaB-like molecules; the average composition of which depends on the relative quantities of ban and dnaB subunits in the cell.     

High Frequency of Genetic Duplications in the dnaB Region of the ESCHERICHIA COLI K12 Chromosome

The region that includes the dnaB locus on the E. coli K12 chromosome was shown to be duplicated at high frequency in cell populations. The duplications were shown to be arranged in tandem and segregated at various frequencies. Segregation was dependent on the recA recombination system, but independent of recB,C. Though most of the data was obtained with dnaB::Tn10 insertion mutants, the duplications were shown to occur in the absence of Tn10.     

Incompatibility between bacteriophage lambda and the sex factor F

Defective lambda plasmids, either λdv or λN, interfere with the replication of the Escherichia coli sex factor F. The incompatibility between these two plasmids is considered due to the P protein of lambda, since non-lambda replicons into which the lambda P gene has been cloned inhibit F replication. The inhibitory effect of the P protein on F replication is reduced in bacteria with the dnaB mutation grpB. This implies that the interference by lambda P protein is mediated, at least in part, through the E. coli dnaB product.     

Calcium enhances Salmonella typhimurium invasion of HeLa cells

Salmonella typhimurium cultured with physiological levels of calcium are significantly enhanced in their ability to penetrate HeLa cells in vitro. Increased infectivity was not observed in magnesium-supplemented media, but was demonstrated in calcium-supplemented minimal defined or calcium-supplemented cation-deficient media. Invasion enhancement was observed for a number of S. typhimurium strains and ranged from 28–390% over calcium-deficient controls. Enhanced HeLa cell infectivity was not dependent on the presence of an autonomous 60-MDa plasmid.