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.     

A new pleiotropic bacteriophage P1 mutation, bof, affecting c1 repression activity, the expression of plasmid incompatibility and the expression of certain constitutive prophage genes.

Summary In bacteriophage P1 an amber mutation in a new gene, bof, has been isolated. The bof-1 phage mutant exhibits a pleiotropic phenotype; bof product is non-essential, and acts as a positive modulator. In P1 bac-1 mutants, in which a dnaB analog product, ban, is expressed constitutively, the bof product activates ban expression both in the prophage state and in lytic growth: P1 bof bac prophages have a reduced ban activity and in lytic growth P1 bof bac phages show a lower ban activity than P1 wild type. This effect on ban activity is observed specifically in P1 bac-1 mutants; it is not mediated by the cl repressor of the lytic functions (repressor of the ban operon) since this effect occurs even if the phage carries a heat sensitive c1 repressor. Thus we concluded that the bac mutation put the ban operon under an abnormal, unknown control, modulated by the bof product. P1 bof lysogens show an increased immunity to superinfecting P1 phage and are affected in their inducibility properties; in the presence of the altered c1-100 repressor, bof product is required for maintenance of lysogeny, as shown by the induction of P1 c1-100 bof-1 lysogens at 30°. P1 bof superinfecting phage can be established together with a resident P1 bof prophage in a recA host, unlike P1 wild type which cannot form double lysogens. P1 bof double lysogens are unstable and segregate one or the other prophage. P1 Cm bof and P1 Km bof lysogens show higher levels of antibiotic resistance than the corresponding bof ⁺ lysogens. The bof gene has been mapped, in an interval defined by P1 prophage deletion end points, far from both ban and c1. All bof phenotypes are reversed by single mutations.     

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.     

Synthesis of P1 ban protein in minicells infected by P1 mutants

Summary Phage P1 encodes a dnaB analog (ban) protein. Synthesis of ban protein has been studied in minicells infected by P1 mutants and has been identified as a polypeptide of 56,000 molecular weight by immunoprecipitation using antibody directed against E. coli dnaB protein. The amount of ban protein synthesized by P1 mutants increases in the order: P1 wild type, P1bac, P1crr, and P1bac crr. The relative amount of ban protein identified in P1bac- and P1bac crr-infected minicells is approximately the same as that previously found in dnaBsdban heteromultimers isolated from the corresponding P1 lysogens.     

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.     

A dnaB analog function specified by bacteriophage P7 and its comparison to the similar function specified by bacteriophage P1

Summary Evidence is presented that bacteriophage P7 specifies an analog of the E. coli DNA replication protein, dnaB. As in the related bacteriophage P1 (D'Ari et al., 1975; Ogawa, 1975), in lysogens of P7, the production of the analog protein is repressed and constitutive mutants could be isolated. Such constitutive of several dnaB(ts) mutations and also rescue a strain carrying a dnaB amber mutation. While neither P7 nor the mutant P1bacban (defective in the structural gene ban) could suppress dnaB(ts) mutations efficiently, recombinants between these two phages could do so, indicating the presence of a functional dnaB analog gene (called sdb) on P7. In a dnaB amber strain suppressed by the presence of the constitutive mutant P7csb, bacteriophage failed to replicate which is a further similarity between P7 and P1. P7csb mutants or P7-P1bacban recombinants were found to be less thermoresistant than P1bac1 suggesting that the P7-specified dnaB analog protein or its production is relatively less tolerant of temperatures above 37°C.     

DNA synthesis in an Escherichia coli dna B dnaC mutant.

Summary An Escherichia coli K12 dnaB dnaC mutant was constructed by P1 transduction of the dnaC allele into a dnaB recipient strain dnaB dnaC transductants were discriminated from dnaB mutants by their inability to grow at 40° C after lysogenization with phage P1bac. The dnaB dnaC mutant character was verified by 1. P1 transduction, and 2. by in vitro complementation with dnaB and dnaC wild type protein fractions.DNA synthesis was studied in strains containing dnaB, dnaC, or dnaB dnaC alleles in an otherwise uniform genetic background with the dnaB character either unsuppressed or suppressed by P1bac prophage. Degradation at 42° C of [³H]-thymidine pulselabeled DNA in dnaB and dnaB dnaC mutants is suppressed by P1bac. However, unlike the dnaC mutant, the P1bac lysogen of the dnaB dnaC mutant exhibits an abrupt cessation of DNA synthesis and less residual cell divisions at 42° C indicating an inhibition of DNA chain elongation rather than a defect in DNA initiation. It is suggested that denaturation of the dnaB protein affects the dnaC function.     

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.     

Isolation and structure of phage lambda head-mutant DNA

High molecular weight DNA accumulates in bacteria in which λ is multiplying but cannot complete the formation of new phage particles due to a defect in head assembly. Accumulated λ DNA has been isolated from induced mitomycin C-treated lysogens by means of a shift in buoyant density labels from heavy to light and fractionation by density-gradient sedimentation for completely light DNA. Head formation was blocked in these lysogens by amber mutations in genes D or E, which specify the two major head proteins. The purified DNA is at least 80% λ by DNA-DNA hybridization and some preparations are close to 100% λ by this test.     

Regulation of the ban gene containing operon of prophage P1

Summary A physical map of the ban gene of P1 and sites relevant to its regulation has been deduced from cloning of the appropriate regions of P1 wild-type and of P1 ban regulatory mutants. The cloning required the presence of P1 repressor in the cell confirming the existence of a repressible ban operon (Austin et al. 1978). Evidence for additional member(s) of that operon is presented. Of particular interest for understanding the regulation of ban are the relative positions of a binding site for the P1 repressor and of the regulatory mutations bac and crr that render ban expression constitutive. The results reveal a repressible operon-like structure of about 4 kb within the P1 EcoRI-3 fragment that comprises a c1 repressor binding site/bac additional gene(s) — crr/ban in the clockwise direction of the circular map of P1.     

Bacteriophage P2-lambda interference. III. Essential role of an early step in the initiation of lambda replication.

Induction of bacteriophage λ in the presence of a P2 prophage results in inactivation of cellular transfer RNA, inhibition of amino acid and uridine incorporation in the host, as well as inhibition of phage replication. A red gam double mutation allows λ to escape from interference, and a mutation in gene O or P abolishes the effects on the host.It is shown here that phage and plasmid DNA extracted from cells undergoing P2-λ interference are still active in a transfection assay. Mutations in bacterial gene dna B or in phage site ori suppress the inhibition of amino acid incorporation, whereas genes dnaE and dna G have no such effect. Derepression of bacterial exonuclease VIII totally suppresses the interference, and mutations in genes recA and lexA, which control the SOS functions, suppress it partially if the λ phage is red⁺. Our results suggest that P2-λ interference is due to the action of old at an early step of the initiation of λ replication.     

Initiation of recA+-dependent recombination in Escherichia coli (lambda). II. Specificity in the induction of recombination and strand cutting in undamaged covalent circular bacteriophage 186 and lambda DNA molecules in phage-infected cells.

Covalent circular λ DNA molecules produced in Escherichia coli (λ) host cells by infection with labeled λ bacteriophages are cut following superinfection with λ phages damaged by exposure to psoralen and 360 nm light. This cutting of undamaged covalent circular molecules is referred to as “cutting in trans”, and could be a step in damage-induced recombination (Ross &; Howard-Flanders, 1977). Similar experiments performed with the temperate phage 186, which is not homologous with phage λ, showed cutting in trans and damage-induced recombination to occur in homoimmune crosses with phage 186 also. Double lysogens carrying both λ and 186 prophages were used in a test for specificity in cutting in trans and in damage-induced recombination. The double lysogens were infected with ³H-labeled 186 and ³²P-labeled λ phages. When these doubly infected lysogens containing covalent circular phage DNA molecules of both types were superinfected with psoralen-damaged 186 phages and incubated, the covalent circular 186 DNA was cut, while λ DNA remained intact. Similarly, superinfection with damaged λ phages caused λ, but not 186, DNA to be cut. Evidently, cutting in trans was specific to the covalent circular DNA homologous to the DNA of the damaged phages. Homoimmune phage-prophage genetic crosses were performed in the double lysogenic host infected with genetically marked λ and 186 phages. Damage-induced recombination was observed in this system only between the damaged phage DNA and the homologous prophage, none being detected between other homolog pairs present in the same cell. This result makes it unlikely that the damaged phage DNA induces a general state of enhanced strand cutting and genetic recombination affecting all homolog pairs present in the host cell. The simplest interpretation of the specificity in cutting and in recombination is as follows. When they have been incised, the damaged phage DNA molecules are able to pair directly with their undamaged covalent circular homologs. The latter molecules are cut in a recA ⁺ -dependent reaction by a recombination endonuclease that cuts the intact member of the paired homologs.     

Suppression of the lexC (ssbA) mutation of Escherichia coli by a mutant of bacteriophage P1

Summary A new mutant of bacteriophage P1 designated lxc that suppresses the phenotype of lexC and ssbA mutants of Escherichia coli was isolated and characterized. The properties of lexC mutants suppressed by the lxc mutation include temperature sensitive growth at 42° C, sensitivity to ultraviolet light and alkylating agents, and a nonmutagenic response following exposure to ultraviolet irradiation. A bac mutant of bacteriophage P1 that suppresses the temperature sensitivity of dnaB mutants does not affect the phenotype of lexC or ssbA mutants. Neither the lxc or bac mutations affect the ultraviolet light sensitivity of strains with the mutations uvrA155, lexA102, or recA56.     

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.     

A novel assay for illegitimate recombination in Escherichia coli: Stimulation of λbio transducing phage formation by ultra-violet light and its independence from RecA function

We developed a novel assay system for illegitimate recombination, in which the frequency of the formation of λ Spi⁻ phages formed during prophage induction was measured with an E. coli P2 lysogen as the indicator bacteria. Since almost all of the λ Spi⁻ phages thus detected contain attR, they have essentially the same structures as λbio transducing phages, indicating that this assay system enables us to detect specialized transducing phages that produce heterogenote transductants, thus ignoring the occurrences of docL and docR particles which carry only one cohesive end. The following results on the formation of specialized transducing phages have been obtained by this assay system to date. (1) Irradiation with UV light greatly enhanced the formation of λ Spi⁻ phages. (2) Treatments with other DNA-damaging agents also enhanced the formation of λ Spi⁻ phages. (3) Illegitimate recombination during prophage induction does not require the RecA function, indicating that enhancement of λ Spi⁻ phage formation is not controlled by the SOS regulatory system. (4) Preliminary results suggested that DNA gyrase is involved in the formation of λ Spi⁻ phage during prophage induction. Since the above results were consistent with most of the previous observations on the illegitimate recombination in other systems, the Spi⁻ assay system can provide important clues to the mechanism of illegitimate recombination.     

Ribosomal assembly defectivity in a set of amber mutants of Escherichia coli.

Five amber mutations affecting essential genes of $\text{Escherichia coli}$ have been isolated. The procedure relies on P1-mediated localized mutagenesis(1) and on the use of a recipient strain carrying a strong but instable suppressor gene and a particular thermoinducible λ prophage which kills suppressor hosts at 42°C (2). All five mutations map close to the $\text{spcA}$ gene, in a region which codes essentially for ribosomal proteins. Strains harboring the mutations were studied biochemically; all five exhibit defective ribosomal assembly upon loss of suppression.     

int-h: An int mutation of phage lambda that enhances site-specific recombination

The mutation int-h3 maps in the int gene of coliphage λ and results in the synthesis of an integrase with enhanced activity, which is manifested by an ability to support λ site-specific recombination relatively efficiently under conditions where the wild-type integrase functions inefficiently. The level of site-specific recombination seen in the presence of the int⁺ integrase in himA^? hosts is greatly reduced, as measured by lysogen formation, intramolecular site-specific integration and excision, and excision of a cryptic λ prophage. In contrast, the int-h3 integrase shows relatively high levels of activities under these conditions. Int-h3 is also more active in other host mutants (himB and hip) that reduce λ site-specific recombination. In the absence of the normal attB site, the frequency of lysogen formation (at secondary sites) by λ int⁺ is reduced 200 fold. Although λ int-h3 will integrate preferentially at the attB site if it is present, the mutant phage forms lysogens at a high frequency in attB-deleted hosts. λ int-h3 requires himA function for integration at secondary sites. The fact that the int-h3 integrase uses the same att sites as well as the same host functions as the int⁺ integrase suggests that the mutation results in a quantitative rather than a qualitative change in integrase activity; that is, the int-h3 integrase is more active. The mutant integrase supports site-specific recombination with att sites that carry the att24 mutation. We propose that the int-h3 integrase is endowed with an enhanced ability to recognize att sequences, including some that are not effectively recognized by wild-type integrase.     

Suppression of a thermosensitive dnaA mutation of Escherichia coli by bacteriophage P1 and P7.

Like other plasmids, the P1 and P7 prophages suppress E. coli dnaA(Ts) mutations by integrating into the host chromosome. This conclusion is supported by three lines of evidence: (1) Alkaline sucrose gradients reveal the absence of plasmid DNA in suppressed lysogens; (2) the prophage is linked to host chromosomal markers in conjugation; and (3) auxotrophs whose defect is linked to the prophage are found among suppressed colonies. No phage or bacterial mutation is required for suppression. Integrative suppression by P1 and P7, unlike suppression by F, does not require the host recA⁺ function. Among suppressed P7 lysogens are some that do not produce phage; these contain defective prophages. The genetic extent of the deletions contained by these defective prophages delineates the prophage regions which are not necessary for suppression of dnaA(Ts). The possible mechanisms of integration and deletion formation are discussed.     

UV-mutagenesis at a cloned target sequence: converted suppressor mutation is insensitive to mutation frequency decline regardless of the gene orientation

Premutational lesions produced by ultraviolet radiation in the Gln2 tRNA genes of E. coli B/r show differing sensitivities to a mutation avoidance phenomenon known as mutation frequency decline (MFD). A mutation event that changes the wild-type gene to an amber (UAG) suppressor is normally sensitive to MFD. Mutation of this amber suppressor to an ochre (UAA) suppressor is not sensitive to MFD. These two mutation events occur in the same anticodon region of the DNA. The dissimilarity of MFD sensitivity between these two mutations may result because the respective premutational photoproducts for the two are located in opposite strands of duplex DNA. To examine the effect of strand position of the premutational lesions on MFD, recombinant lambda phage were constructed that contained the amber suppressor as a mutation target in the two possible orientations. Comparison of MFD in bacterial lysogens containing either of the two types of recombinant prophage indicated that reversing the orientation of the target sequence relative to adjacent bacterial DNA had no effect on MFD. Since rotational inversion of the target sequence did not alter the sensitivity to MFD of mutation occurring at the cloned target gene, the antimutation process inherent to MFD can not be attributed to an asymmetrical interaction between the template strands and the DNA-replication complex.