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 replication. These studies strongly suggest thatgrpD55 is an allele ofdnaB and is localized near 92 min on theE. coli linkage map.     

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.     

Suppression and enhancement of temperature sensitivity of dnaB mutations of Escherichia coli K12 by conjugative plasmids.

A majority of wild-type, conjugative antibiotic-resistance plasmids from a standard collection had the ability to suppress or to enhance the temperature sensitivity of dnaB mutants of Escherichia coli K12. This ability appears to be widely dispersed among all plasmid groups. The mode of suppression does not involve the insertion of the plasmid into the chromosome. Plasmid-induced suppression or enhancement is not as a rule mutation specific and can extend to several mutations within the dnaB region. The patterns of suppression and enhancement suggest a direct or indirect interaction between a plasmid-specified product and the dnaB protein.     

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.     

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.     

Genetic location ofrra mutation and its role in alleviation of rgl restriction

Restriction of glucosyl-free HMC-DNA mediated by RglB is alleviated inrecBC sbcA strains ofEscherichia coli K12. Mutation in the unlinkedrra gene reverses thisrecBC sbcA-mediated alleviation. The map position ofrra is 90.16 min on the standard map, and therra ⁺ gene product counteracts Rgl restriction. The activation of therra gene is controlled by thesbcA gene, and this regulation does not seem to require the involvement of other gene functions.     

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.     

A new mutation inEscherichia coli K12,isfA,which is responsible for inhibition of SOS functions

A new mutation inEscherichia coli K12,isfA, is described, which causes inhibition of SOS functions. The mutation, discovered in a ΔpolA ⁺ mutant, is responsible for inhibition of several phenomena related to the SOS response inpolA ⁺ strains: UV- and methyl methanesulfonate-induced mutagenesis, resumption of DNA replication in UV-irradiated cells, cell filamentation, prophage induction and increase in UV sensitivity. TheisfA mutation also significantly reduces UV-induced expression of β-galactosidase fromrecA::lacZ andumuC′::lacZ fusions. The results suggest that theisfA gene product may affect RecA^* coprotease activity and may be involved in the regulation of the termination of the SOS response after completion of DNA repair. TheisfA mutation was localized at 85 min on theE. coli chromosome, and preliminary experiments suggest that it may be dominant to the wild-type allele.     

Analysis of theilv-linked genes that determine the morphology ofEscherichia coli Cells

The genetic location ofwee relative tocya was measured by cotransduction with a Tn5 insertion inilv. These experiments locatedwee at 84.8 min in the standardEscherichia coli map. Mutations incya andwee give rise to morphological changes, coccal morphology incya and short rods inwee, suggesting that both may be involved in the pathways of cell elongation. Addition of cAMP to the cultures reverted thecya but not thewee phenotype. Cells ofE. coli in the absence of thewee gene product were, contrary to what has been described forcya cells, as sensitive to mecillinam as in its presence. These results suggested that the action of Wee on elongation is exerted at a level different from that of adenyl cyclase.     

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.     

Investigation of a phage resistantSerratia entomophila strain (BC4B), establishment of generalised transduction and construction ofS. entomophila RecA mutants

ArecA clone was isolated from a cosmid library ofSerratia entomophila constructed in theEscherichia coli strain HB101. Subcloning and transposon mutagenesis were used to identify a 1.36 kb fragment containing therecA gene. A clonedrecA mutation, generated by transposon mutagenesis and the replacement of a portion of therecA gene with an antibiotic resistance cassette, was introduced into the chromosome via a marker exchange technique. TherecA strains created were deficient in DNA repair, homologous recombination and both the spontaneous and UV induction of prophages.S. entomophila recA strains showed continued pathogenicity towards the New Zealand grass grub,Costelytra zealandica. Simple procedures for further construction ofS. entomophila recA strains have been demonstrated.     

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.     

Emergence of a mutagenic ochre suppressor mutation under lactose selection in appm mutant ofEscherichia coli harbouring the F′lacZU118 episome

WhenEscherichia coli harbouring theppm (earlier calledadi) mutation and the F′lacZU118 episome is subjected to lactose selection in the presence of suboptimal concentrations of glycerol, Lac⁺ colonies emerge after 5–6 days. They are shown to harbour an ochre suppressor mutation at 15.15 min. Inactivation ofrecA results in approximately four-fold reduction in the response. In theppm — ochre suppressor double mutant background the leakiness of thelacZ allele carried by F′ CC105 is enhanced, suggesting misreading of a valine codon (GUG) as glutamic acid codon (GAG). This is accompanied by reversion of thelacZ mutation tolacZ ⁺ (GTG → GAG). In LB medium both the leakiness and reversion are inhibited by streptomycin. Inactivation ofrecA did not affect leakiness but abolished reversion. These data are discussed in relation to the importance of allele leakiness and restricted growth in stationary-phase (adaptive) mutagenesis.     

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.