P1 Prophage Research Articles

Bacteriophage P1-mediated generalized transduction in Escherichia coli: Structure of abortively transduced DNA

DNA was extracted from [ 3H]thymidine-labeled Escherichia coli infected with 32P-5-bromouracil-labeled transducing particles, and analyzed in equilibrium CsCl gradients. About 75% of the 32P-labeled transducing DNA equilibrated in the heavy region of the gradient. This nonchromosomally associated DNA, which represented abortively transduced DNA, remained fully heavy up to 5 hr after infection. Sedimentation of abortively transduced DNA in neutral and in alkaline sucrose gradients showed that both strands remained intact up to 3 hr after infection. When infected recipient bacteria were lysed by a nonionic detergent procedure, about 60% of the abortively transduced DNA could be recovered in a form which sedimented 1.7 to 1.8 times faster than linear P1 phage DNA in neutral sucrose gradients. Sedimentation of abortively transduced DNA and P1 prophage DNA in neutral sucrose gradients showed faster and slower sedimenting forms of abortively transduced DNA which corresponded to supercoiled and relaxed circular prophage DNA, respectively. Agarose gel electrophoresis of abortively transduced DNA showed bands which comigrated with circular prophage DNA. Treatment of abortively transduced DNA with sodium lauryl sulfate, with heat (70°), or with Pronase converted the DNA to a form which sedimented with linear P1 phage DNA in neutral sucrose gradients, and which migrated with linear P1 transducing particle DNA in agarose gels. The data suggest that abortively transduced DNA exists as a circular DNA-protein complex.

A new gene of bacteriophage P22 which regulates synthesis of antirepressor

Two new mutants of bacteriophage P22 are described which define a new regulatory gene, arc (for anti repressor control). The properties of the arc mutants and of 31 phenotypic revertants indicate that the arc gene codes for a trans-acting protein whose primary role is to depress synthesis of P22 antirepressor protein during the lytic cycle of infection. Failure to regulate antirepressor production apparently leads secondarily to a lethal defect (i.e. failure to produce progeny phage). Although under certain conditions the arc function can be expressed by P22 prophages and can act as a weak barrier to superinfecting homologous phage, the arc product is neither necessary nor sufficient for maintenance of the prophage state or superinfection immunity in lysogens. Instead, as shown previously by others (Levine et al., 1975; Botstein et al., 1975), the prophage mnt gene product is responsible for repressing antirepressor synthesis, both by the prophage and by superinfecting phage.

A trans-acting factor mediates inversion of a specific DNA segment in flagellar phase variation of Salmonella.

Inversions of specific DNA segments have been shown to be involved in regulating gene expression in several systems, and flagellar phase variation in Salmonella is one example. Strains of diphasic Salmonella species, such as S. typhimurium, possess two non-allelic structural genes, H1 and H2, for flagellin, the component protein of flagellar filaments. One or the other is expressed in a bacterial clone. Lederberg and Iino1 reported that the switch from the expression of one gene to that of the other was controlled by a genetic element linked to the H2 gene and that the state of H2 determined the flagellar phase of the bacterium. Simon et al.2–4 have shown that the expression of H2 is controlled by a recombination which inverts a region of DNA containing an element necessary for the transcription of H2. We report here that prophages P1 and Mu produce a trans-acting cytoplasmic factor which mediates the inversion of a specific DNA segment in flagellar phase variation of Salmonella.

Rex-dependent exclusion of lambdoid phages I. Prophage requirements for exclusion

Certain λ lysogens are able to inhibit growth of superinfecting φ80 and λ-P22 hybrid phages with the OPQ region from P22. Superinfection exclusion requires expression of the prophage rex gene and is most evident in λ lysogens in which the prophage carries a mutation (either is or am) in the cI gene. These lysogens also inhibit growth of leaky rII − mutants of T4 which are able to grow in λ + lysogens. Rex-dependent exclusion of lambdoid phages is not strictly a superinfection exclusion system, as thermal induction of a λ rex +clts85 (OP)-P22 prophage results in “self-exclusion,” that is, in low phage production. These observations are explained by proposing that Rex activity of a λ cits lysogen (and other lysogens which produce a modified repressor) is greater than in lysogens carrying a prophage with a wild-type cI gene. The effects of thermal induction and mitomycin C induction on Rex activity are described, and explanations for increased Rex activity in lysogens carrying a prophage with an altered repressor are discussed.

Genetic interaction between the nip1 mutation and genes affecting integration and excision in phage P2.

The excision of prophage P2 is controlled by two genes, int and cox. (The cox gene discussed in this report is defined by the cox class II mutants, defined by Six and Lindqvist, 1978). The combined activity of these two genes is rather inefficient, however, since only about 1% of the lysogens carrying an int+cox+ prophage actually produce phage when derepressed. The efficiency of phage production (and presumably excision) can be increased 100-fold by an additional mutation called nip1 (Calendar et al., 1972), which is dominant and is located in or near the int gene.

Immunity and repression in bacteriophages P1 and P7.

The temperate phage P1 (Bertani 1951) is a large complex phage whose double stranded DNA has a molecular weight of 66 × 106 (Yun and Vapnek 1977). The DNA in the phage particles is terminally redundant and cyclically permuted (Ikeda and Tomizawa 1968). P1 is best known for its ability to perform generalized transduction (Lennox 1955; Ikeda and Tomizawa 1965) and has been used extensively as a tool in Escherichia coli genetics. Because of its broad host range, P1 also serves as a transducing phage for other bacterial species (Goldberg et al. 1974; Kaiser and Dworkin 1975). Phage P1 is the prototype of a class of temperate phages whose prophage is not integrated into the host chromosome (Ikeda and Tomizawa 1968), and thus the strategies it uses to maintain lysogeny may differ from those used by integrative prophages. The P1 prophage is a plasmid (of molecular weight 61 × 106: Yun and Vapnek 1977) and is maintained at about one copy per cell nucleoid (Ikeda and Tomizawa 1968). So, in addition to regulation of transcription, the P1 prophage must also regulate its replication. P1 has all of the characteristics of both a temperate phage and a plasmid. Like other temperate phages, P1 prophage is repressed for production of lytic phage functions and a P1 lysogen exhibits immunity to superinfection by other P1 phage. Like other plasmids, the P1 prophage exhibits incompatibility with related plasmids, i. e., it cannot coexist in the same cell with another member of the same incompatibility group (Hedges et al. 1975). The mechanism for this phenomenon is not yet understood.

Prophage substitution and prophage loss from superinfected Escherichia coli recA(P1) lysogens

It is shown that the plasmid prophage P1 can be displaced by a superinfecting P1 phage in Escherichia coli recA(P1) lysogens. Six widely separated phage markers were used to distinguish between residual recombination and total substitution. It is further shown that superinfection of recA lysogens can lead to loss of both phage (curing). These two phenomena, previously reported in Rec+ strains, are thus independent of host recombination and may result from perturbations of some function involved in plasmid maintenance.

Integration of the plasmid prophages P1 and P7 into the chromosome of Escherichia coli

The prophages of the related temperate bacteriophages P1 and P7, which normally exist as plasmids, suppress Escherichia coli dnaA (ts) mutants by integrating into the host chromosome. The locations of the sites on the prophage used for integrative recombination were identified by restriction nuclease analysis and DNA-DNA hybridization techniques. The integration of P1 and P7 often involves a specific site on the host DNA and a specific site on the phage DNA; the latter is probably the end of the phage genetic map. When this site is utilized, the host Rec + function is not required. In Rec + strains, P1 and P7 may also recombine with homologous regions on the host chromosome; at least one of these regions is an IS1 element. In some integration events, prophage deletions are observed which are often associated with inverted repeat structures on the phage DNA. Thus, P1 and P7 may employ one of several different mechanisms for integration.

Studies of a plasmid coding for tetracycline resistance and hydrogen sulfide production incompatible with the prophage P1

The plasmid pIP231, determining tetracycline resistance and hydrogen sulfide production is shown to belong to incompatibility group Y and to code for a restriction and modification system. Unlike the IncY plasmids, P7 and P15B, plasmid pIP231 shows only little genetic and physical homology with P1 prophage.

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

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 c1 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 degrees. 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.

A dnaB-Andlog DNA-replication Protein of Phage P1

Phage P1 codes for a dnaB-analog (ban) protein (D'Ari et al. 1975; Ogawa 1975). During P1 infection of an Escherichia coli dnaBts mutant at 41°C, synthesis of ban protein enables the phage to grow (Lanka and Schuster 1970), and bacterial DNA synthesis recovers transiently (Beyersmann and Schuster 1971). Synthesis of ban protein is repressed in the wild-type P1 prophage and expressed constitutively in P1 bac mutants. P1 bac lysogens of E. coli dnaBts mutants are therefore able to grow at temperatures nonpermissive for the nonlysogens (D'Ari et al. 1975; Ogawa 1975). This suppressing action of prophage P1 bac was simulated in an in vitro system for DNA replication. Conversion of ϕX174 single-stranded (SS) DNA to its duplex form is dependent on (among other proteins) the dnaB protein (Schekman et al. 1972; Wright et al. 1973). With crude enzyme fractions from P1 bac lysogens containing a temperature-sensitive (ts) dnaB protein, the...

Mutations of bacteriophage P2 which prevent activation of P2 late genes by satellite phage P4

Satellite phage P4 multiplies only in the presence of its helper P2 and requires each morphogenesis function of P2 for phage maturation. The helper phage P2 can be present either as prophage or as a co-infecting phage. We have isolated mutant P2 lysogens unable to support P4 growth. These carry mutations lying in the region of P2 early genes. Measurement of P2 lysozyme synthesis indicates that in these mutants, P2 late genes are activated inefficiently during P4 infection. Complementation tests show that the P4 stimulation function, which is absent in the defective lysogens, can be provided in trans by another prophage. This function cannot be assigned to either of the two known early genes of P2 which are required for late gene transcription during P2 vegetative growth; P4 multiplies normally when both P2 early genes A and B are inactivated by amber mutations. A dominant mutation of P2 has been isolated which makes repressed P2 incapable of supporting P4 multiplication. The mutation prevents derepression of the P2 prophage and may alter the P2 repressor. We propose that two different pathways exist for activation of P2 late genes: (1) a pathway dependent on P2 replication and (2) a pathway activated by satellite P4. One component of the P4-dependent pathway is a diffusible P2 early gene function under the control of P2 repressor. This function acts to stimulate the P2 late gene transcription in the P4-dependent pathway.

Control of gene activation of P2 prophage by satellite phage P4

In the preceding paper we report that satellite phage P4 can derepress P2 lysogens ( Six and Lindqvist, 1978). We now show that derepression leads to the expression of the early as well as the late gene regions of the P2 prophage. In the absence of transactivation, P2 prophage late genes will be expressed only if the early P2 gene A function is available. This was demonstrated with P4 mutants defective in gene α. Such mutants are proficient in derepression but were found to be transactivation deficient, hence restricted to derepression as a means of turning on P2 prophage late genes. Thus two modes of P4-induced late gene expression can be distinguished: transactivation and derepression. Derepression precedes transactivation by about 30 min, but the extra time needed for transactivation appears not to be due to a requirement for early P2 gene products.

Chromosome replication in an Escherichia coli dnaA mutant integratively suppressed by prophage P2

Escherichia coli CRT4624-P2sig5 is a dnaA mutant in which integration of the prophage P2sig5 has occurred at the attP2II site (min 85). This strain was integratively suppressed, and when cells were shifted to 42 degrees C replication was initiated at a site in or near the P2 prophage. Initially, this replication occurred primarily in the direction that corresponds to the clockwise direction on the genetic map. Replication also occurred in the counterclockwise direction, but the initiation of replication in this direction occurred approximately 40 min later than the initiation of replication in the other direction. Because of this delay, the replication forks that traveled in the clockwise direction were the first to arrive in the region of the replication terminus. These replication forks ceased replication near the aroD locus (min 37), and it is proposed that the replication terminus is between the aroD and rac loci (min 31). A model is proposed for the cycle of chromosome replication in this strain at 42 degrees C.

Mutual derepression in the P2–P4 bacteriophage system

P4 lysogens are derepressed by infection with P2 as is evident from the induction of P4 phage production by P2 infection of P4 lysogens. Mutations interfering with P2 DNA synthesis greatly increase the P4 yield, but DNA replication by the infecting phage does not preclude P4 induction. Of four P2 mutations known to block spontaneous phage production by P2 lysogens, three ( cox-2, cox-3, cox-4) also prevent derepression of P4 lysogens by P2. These studies also revealed that, under certain conditions, P4 can complement P2 mutants deficient in gene B function which is required for P2 DNA replication. P2 lysogens are derepressed by infection with P4. This derepression leads to a P2 gene A-dependent P2 prophage replication and, in P2 lysogens mixedly infected with P2 and P4, also to the replication of the DNA of the coinfecting P2. If the derepressing P4 is deficient in gene a function, then derepression of the P2 lysogen will lead to the production of 10 or more P2 phages per cell, provided that P2 prophage excision is enhanced by the P2 nip mutation or is bypassed by infecting the P2 lysogen with P2 as well as with P4. Gene α proficient P4 interferes with the P2 phage production by P2 lysogens; possible causes for this interference are considered, including P4 DNA replication which is known to require gene a function.

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.

Genetic and physical characterization of P1 dlw prophage and its derivatives

P1 dlw, a defective derivative of coliphage P1, carries the chromosomal lac region. Marker rescue for 10 different P1 alleles distributed along the known P1 genetic map indicates that the P1 dlw prophage probably carries the entire P1 genome in addition to the chromosomal lac region. Results of genetic crosses are compatible with insertion of the lac region between the 36 and vir loci, suggesting 34–36-lac-35 linkages. The molecular weight of the P1 dluw prophage, as determined by electron microscopical analysis, is 103.2 ± 5.1 × 10 6. The excision of lac from P1 dlw produces two P1-immune lac − segregant types. Type I carries all 10 markers tested with a distinct defect between genes 1 and 34. The molecular weight of this defective prophage is 63.7 ± 2.4 × 10 6, larger by 2.8 × 10 6 than P1 prophage. The second lac − segregant type (Type II) is deleted for genes 34, 36, and 35. The molecular weight of the defective Type II prophage is 51.1 ± 1.5 × 10 6. The two segregation patterns as well as their frequency of occurrence are independent of the RecA pathway. Marker rescue for P1 genes in the vicinity of inserted bacterial DNA appears to depend on the bacterial RecA pathway. pIH1972, a lac + plasmid derived from P1 dlw, has a molecular weight of 23.6 ± 1.0 × 10 6 and no rescuable PI markers but is incompatible with P1 prophage. It is assumed that the 48.4 × 10 6 chromosomal IS3-bounded lac segment or a major part of this segment is integrated in P1 to produce P1 dlw, which is 42.3 × 10 6 larger than the P1 prophage. The frequency and the pattern of the recA-independent segregation of lac suggest that the chromosomal insertion may carry IS-like elements.

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.

Escherichia coli dnaB mutant defective in DNA initiation: isolation and properties of the dnaB protein.

Extracts of the DNA initiation-defective mutant Escherichia coli dnaB252 are inactive in a dnaB complementation assay but yield a ribonucleoside triphosphatase activity of native molecular weight of about 270,000 (60,000-dalton polypeptide as subunit) that can be inactivated by antibody to dnaB. On the other hand, extracts of a dnaB252(P1 bac) lysogen, in which the dnaB mutation is suppressed in vivo by the constitutive expression of the P1 dnaB analog (ban protein), are active in dnaB complementation and the activity is also sensitive to dnaB antibody. Upon further purification two proteins (with polypeptide molecular weights of 60,000 and 56,000, respectively) are found associated with each other (native molecular weight about 270,000). The larger and the smaller protein are tentatively identified as the dnaB and P1 ban protein. It is suggested that suppression of the dnaB mutation by prophage P1 bac is accomplished by a stabilization of dnaB252 by P1 ban subunit molecules in a heteromultimer.

Ant-mediated transactivation of early genes in Salmonella prophage P22 by superinfecting virulent P22 mutants.

The virulent mutants P22 virB vy and P22 vy mutants, both insensitive to mnt-repressor, transactivate the early genes of a P22 prophage. The transactivation of early P22 prophage genes depends strictly on the expression of gene ant ("antirepressor"-protein) by the superinfecting P22 mutant and therefore occurs by derepression.