Early events in the replication of Mu prophage DNA.

To determine whether the early replication of Mu prophage DNA proceeds beyond the termini of the prophage into hose DNA, the amounts of both Mu DNA and the prophage-adjacent host DNA sequences were measured using a DNA-DNA annealing assay after induction of the Mu vegetative cycle. Whereas Mu-specific DNA synthesis began 6 to 8 min after induction, no amplification of the adjacent DNA sequences was observed. These data suggest that early Mu-induced DNA synthesis is constrained within the boundaries of the Mu prophage. Since prophage Mu DNA does not undergo a prophage lambda-like excision from its original site after induction (E. Ljungquist and A. I. Bukhari, Proc. Natl. Acad. Sci. U.S.A. 74:3143--3147, 1977), we propose the existence of a control mechanism which excludes prophage-adjacent sequences from the initial mu prophage replication. The frequencies of the Mu prophage-adjacent DNA sequences, relative to other Escherichia coli genes, were not observed to change after the onset of Mu-specific DNA replication. This suggests that these regions remain associated with the host chromosome and continue to be replicated by the chromosomal replication fork. Therefore, we conclude that both the Mu prophage and adjacent host sequences are maintained in the host chromosome, rather than on an extrachromosomal form containing Mu and host DNA.     

Effect of mutations of Escherichia coli K-12 chromosome on the integration of bacteriophage Mu introduced into cells by infection or conjugation

We present the detailed research on the previously described Escherichia coli K-12 Mud- mutants with impaired development of bacteriophage Mu. The ability of Mu phage DNA to penetrate into mutant cells on infection was shown. If introduced into the cells or combined with mud mutation by recombination, the prophage may be induced, which results in phage Mu lythic development and phage burst from mutant cells. In the course of conjugative transfer into the mutant cells, within a DNA fragment of the lysogenic donor chromosome, MupAp1 prophage is not inherited by recombinants. At the same time, Mu prophage deficient in genes A and B, whose products are required for transposition, is inherited by the mutant with the usual frequency. These data enable us to conclude that the mud mutations disturb the stage of conservative transposition which is connected with the insertion of the Mu prophage into the chromosome, after excision from the linear DNA introduced into the cells via infection or conjugation.     

Events following prophage Mu induction.

Escherichia coli strains lysogenic for a thermoinducible Mu prophage (Mu cts62) undergo rapid lysis about 50 min after heat induction. Induction of Mu cts62 apparently causes damage to the host sequences in which Mu is inserted. The normal expression of A, BU, and X genes of Mu is needed for this specific deleterious effect on the prophage-containing host sequences. Mu deoxyribonucleic acid can be shown to reintegrate extensively at different sites on the host genome during the lytic cycle after prophage induction or after infection of sensitive cells by clear-plaque mutants of Mu. We estimate that approximately 10 copies of Mu deoxyribonucleic acid are inserted per chromosome during vegetative growth. The episome rescue method for detecting vegetative Mu deoxyribonucleic acid insertion, in which an episome is transferred from the lytically infected cells to F- receipient cells, can be applied to study Mu integration without requiring the host cells to survive. It also provides an easy system to isolate Mu insertions in transmissible episomes and plasmids.     

Kinetics of Mu DNA synthesis

Summary Mu specific DNA synthesis starts at 10 min after infection. All essential amber mutants of Mu were tested for the ability to replicate in a non permissive host. Except for the amber mutants A and B, which were already known to be blocked in Mu DNA synthesis (Wijffelman et al., 1974), all the other mutants showed normal Mu DNA replication.Using mitomycin C-treated cells Mu DNA synthesis was found to start at about 20 min after induction. However using the much more sensitive method of DNA-RNA hybridization, it was found that the DNA synthesis starts already at 10 min after induction, and that at 20 min after induction about 7 copies of the Mu DNA are present per cell.     

Deoxyribonucleic Acid Polymerase Activity in a Deoxyribonucleic Acid Polymerase I-Deficient Mutant of Bacillus subtilis Infected with Temperate Bacteriophage SPO2

Increased deoxyribonucleic acid (DNA) polymerase activity is found in soluble extracts from a polymerase I-negative mutant of Bacillus subtilis after infection with temperate phage SPO2, or after induction of SPO2 prophage in lysogenic derivatives of this mutant. No increased enzyme activity is found after SPO2 infection in the presence of chloramphenicol. Infection of the polymerase-negative mutant with the DNA-negative sus mutant SPO2 L244 gives no increased enzyme activity, whereas infection with DNA-negative sus mutant SPO2 J385 gives enzyme activities comparable to those found in wild-type infected cells. These findings suggest that SPO2 determines a DNA polymerase activity essential for synthesis of phage DNA.     

Lethal Transposition of Mud Phages in Rec(-) Strains of Salmonella Typhimurium

Under several circumstances, the frequency with which Mud prophages form lysogens is apparently reduced in rec strains of Salmonella typhimurium. Lysogen formation by a MudI genome (37 kb) injected by a Mu virion is unaffected by a host rec mutation. However when the same MudI phage is injected by a phage P22 virion, lysogeny is reduced in a recA or recB mutant host. A host rec mutation reduces the lysogenization of mini-Mu phages injected by either Mu or P22 virions. When lysogen frequency is reduced by a host rec mutation, the surviving lysogens show an increased probability of carrying a deletion adjacent to the Mud insertion site. We propose that the rec effects seen are due to a failure of conservative Mu transposition. Replicative Mud transposition from a linear fragment causes a break in the host chromosome with a Mu prophage at both broken ends. These breaks are lethal unless repaired; repair can be achieved by Rec functions acting on the repeated Mu sequences or by secondary transposition events. In a normal Mu infection, the initial transposition from the injected fragment is conservative and does not break the chromosome. To account for the conditions under which rec effects are seen, we propose that conservative transposition of Mu depends on a protein that must be injected with the DNA. This protein can be injected by Mu but not by P22 virions. Injection or function of the protein may depend on its association with a particular Mu DNA sequence that is present and properly positioned in Mu capsids containing full-sized Mu or MudI genomes; this sequence may be lacking or abnormally positioned in the mini-Mud phages tested.     

The Escherichia coli protein, Fis: specific binding to the ends of phage Mu DNA and modulation of phage growth

We show, using gel retardation, that crude Escherichia coli cell extracts contain a protein which binds specifically to DNA fragments carrying either end of the phage Mu genome. We have identified this protein as Fis, a factor involved in several site-specific recombinational switches. Furthermore, we show that induction of a Mucts62 prophage in a fis lysogen occurs at a lower temperature than that of a wild-type strain, and that spontaneous induction of Mucts62 is increased in the fis mutant. DNasel footprinting using either crude extracts or purified Fis indicate that binding on the left end of Mu occurs at a site which overlaps a weak transposase binding site. Thus, Fis may modulate Mu growth by influencing the binding of transposase, or other proteins, to the transposase binding site(s), in a way similar to its influence on Xis binding in phage lambda.     

Genetic characterization of Mu-like bacteriophage D108.

Infection of Escherichia coli by bacteriophage D108 was shown to result in the generation of apparently random chromosomal mutations. Approximately 1% of the cells lysogenized by D108, as with Mu, acquired new auxotrophic mutations. D108-induced mutations were nonreverting and were most probably the result of insertion of the D108 genome into regions of genetic function. D108 and Mu shared many similar properties but were heteroimmune and had different host ranges. Lytic infections of Mu lysogens with D108 and D108 lysogens with Mu resulted in 100-fold increases in release of phage with prophage markers over those due to spontaneous induction. Phenotypic mixing was common, with most phage carrying the prophage immunity being packaged in particles with the host range of the superinfecting phage. A fraction of the superinfecting phage genomes were, however, packaged in particles with the prophage-specified host range. Although 10% of the prophage progeny were D108-Mu genetic hybrids, superinfecting phage-induced release of the prophage with reciprocal phenotypic mixing occurred in recA hosts, in which the frequency of D108-Mu genetic hybrids was reduced 100-fold.     

Cellular location of Mu DNA replicas.

To ascertain the form and cellular location of the copies of bacteriophage Mu DNA synthesized during lytic development, DNA from an Escherichia coli lysogen was isolated at intervals after induction of the Mu prophage. Host chromosomes were isolated as intact, folded nucleoids, which could be digested with ribonuclease or heated in the presence of sodium dodecyl sulfate to yield intact, unfolded nucleoid DNA. Almost all of the Mu DNA in induced cells was associated with the nucleoids until shortly before cell lysis, even after unfolding of the nucleoid structure. We suggest that the replicas of Mu DNA are integrated into the host chromosomes, possibly by concerted replication-integration events, and are accumulated there until packaged shortly before cell lysis. Nucleoids also were isolated from induced lambda lysogens and from cells containing plasmid DNA. Most of the plasmid DNA sedimented independently of the unfolded nucleoid DNA, whereas 50% or more of the lambda DNA from induced lysogens cosedimented with unfolded nucleoid DNA. Possible explanations for the association of extrachromosomal DNA with nucleoid DNA are discussed.     

Reversal of Mu gem2ts-induced mutations

Abstract: Mutations induced by the integration of a Mu gem 2ts mutant prophage can revert at frequencies around 1 × 10⁻⁶, more than 10⁴-fold higher than that obtained with Mu wild-type. Several aspects characterize Mu gem 2ts precise excision: (i) the phage transposase is not involved; (ii) the RecA protein is not necessary; and (iii) revertants remain lysogenic with the prophage inserted elsewhere in the host genome. In addition, prophage re-integration seems to be non-randomly distributed, whereas Mu insertion into the host genome is a transposition event without any sequence specificity. In this paper, we describe that the site of re-integration somehow depends on the original site of insertion. Two alternative models are proposed to explain the strong correlation between donor and receptor sites.     

Model for the enchancement of lambde-gal integration into partially induced Mu-1 lysogens.

Temperate phage Mu-1, which is able to integrate at random in its host chromosome, is also able to mediate integration of other circular deoxyribonucleic acid, as a lambda-gal mutant unable to integrate by itself. After mixed infection with lambda-gal and Mucplus, galplus transductants are recovered that have the lambda-gal integrated in any circular permutation, sandwiched between two complete Mu genomes in the same orientation, the whole Mu-lambda-gal-Mu structure being found at any location in the bacterial chromosome. Here we show that such a lambda-gal can integrate in an induced Mu lysogen. In this case the lambda-gal is again in any circular permutation, between two Mu in the same orientation, but it is always located at the site of the original Mu prophage, and the two surrounding Mu have always the same genotype as the original Mu prophage. Active Mu replication functions are not essential for that process to occur. This suggests that bacterial replication may generate two Mu copies that in some way can regenerate a Mu attachment site that recombines with the lambda-gal. A model is presented that accounts for these observations, may be helpful for understanding some complex features of Mu development, and may possibly offer a basis for explaining spontaneous duplications.     

Behavior of bacteriophage Mu DNA upon infecton of Escherichia coli cells.

The question whether the ends of bacteriophage Mu DNA are fused to form a ring in host cells is critical to the understanding of the mechanism of integrative recombination between Mu DNA and host DNA. We have examined the fate of ³²P-labeled Mu DNA, after infection of sensitive and immune (lysogenic) cells, by sedimentation in sucrose gradients, ethidium bromide/CsCl density centrifugation and by electrophoresis of parental Mu DNA and its fragments in agarose gels. We find that the parental Mu DNA cannot be detected as covalently closed circles at any stage during the Mu life cycle. An interesting form of Mu DNA can be seen after superinfection of immune cells. This form sediments about twice as fast as the mature phage DNA marker in neutral sucrose gradients but yields linear molecules upon phenol extraction. Upon infection of sensitive cells, most of the parental DNA associates with a large complex, presumably containing the host chromosome. When Mu-sensitive cells are infected with unlabeled Mu particles and Mu DNA examined at different times after infection by fractionation in 0.3% agarose gels and hybridization with ³²P-labeled Mu DNA, Mu sequences are found to appear with the bulk host DNA as the phage lytic cycle progresses. However, no distinct replicative or integrative intermediate of Mu, that behaves differently from linear Mu DNA and is separate from the host DNA, can be detected.     

Synchronization of bacteriophage Mu DNA replicative transposition: analysis of the first round after induction.

The lytic cycle of bacteriophage Mu includes a large number of coupled DNA replication and integration events, each of which is equivalent in several respects to the process of transposition of genetic elements. To aid us in studying the process of Mu DNA replicative transposition, we developed a technique for synchronizing the first round of replication following induction of a lysogen. Synchronization was achieved by inducing a lysogen in the absence of DNA replication for a time sufficient to develop the potential for Mu DNA replication in all cells in the population; upon release of the inhibition of replication, a synchronized round of Mu DNA replication was observed. Development of the potential for Mu DNA replication in the entire population took approximately 12 min. Protein synthesis was required for development of the potential, but the requirement for protein synthesis was satisfied by approximately 9 min suggesting that other, as yet unspecified, reactions occupied the last 3 min. Replication proceeded predominantly from the left end of the prophage, though a significant amount of initiation from the right end was observed. The usefulness of the technique for studying the mechanism of replicative transposition and the end products of a single round of replication are discussed.     

Regulation of bacteriophage Mu transposition

Bacteriophage Mu is a transposon and a temperate phage which has become a paradigm for the study of the molecular mechanism of transposition. As a prophage, Mu has also been used to study some aspects of the influence of the host cell growth phase on the regulation of transposition. Through the years several host proteins have been identified which play a key role in the replication of the Mu genome by successive rounds of replicative transposition as well as in the maintenance of the repressed prophage state. In this review we have attempted to summarize all these findings with the purpose of emphasizing the benefit the virus and the host cell can gain from those phage-host interactions.     

Evolution of the long range structure of satellite DNAs in the genus Apodemus.

The temperate bacteriophage Mu causes mutations by inserting its DNA randomly into the genes of its host bacterium Escherichia coli. It is shown here that Mu DNA can be precisely excised from the different integration sites and that as a result wild-type function of the gene into which Mu was inserted is restored. The excision of Mu DNA is observable only if the Mu prophage carries mutations at the X locus. Thus, lac⁺ revertants from six strains, containing heat-inducible prophage Mu cts62 at different locations in the Z gene of the lac operon, were readily obtained by first introducing the X mutation into Mu cts62. The lac⁺ revertants produced wild-type β-galactosidase, and no trace of Mu DNA could be detected in them; this indicates that the junction of Mu DNA and host DNA can be specifically recognized. However, the excision of Mu DNA is generally not perfect, because in most cases it does not lead to the wild-type genotype. The function of gene A of Mu appears to be required for excision. Since the lethal functions of Mu are completely blocked in the Mu cts62 X prophage, the X locus probably has a regulatory function. At least one X mutation is caused by an insertion of about 900 base-pairs in Mu DNA. The discovery of the X mutants opens the way for studying the reversible interaction of the host and Mu chromosomes, and for using Mu to manipulate the host genome in various ways.     

Precise excision of bacteriophage Mu DNA

The temperate bacteriophage Mu is a transposable element that can integrate randomly into bacterial DNA, thereby creating mutations. Mutants due to an integrated Mu prophage do not give rise to revertants, as if Mu, unlike other transposable elements, were unable to excise precisely. In the present work, starting with a lacZ::Muc62(Ts) strain unable to form Lac+ colonies, we cloned a lacZ+ gene in vivo on a mini-Mu plasmid, under conditions of prophage induction. In all lac+ plasmids recovered, the wild-type sequence was restored in the region where the Mu prophage had been integrated. The recovery of lacZ+ genes shows that precise excision of Mu does indeed take place; the absence of Lac+ colonies suggests that precise excision events are systematically associated with loss of colony-forming ability.     

The effect of gamma-irradiation on mu DNA transposition and gene expression

Mobile genetic elements are a ubiquitous presence in the genomes of all well-studied organisms. The effect of genomic stress on the status and transposition of these elements has not, as yet, been extensively characterized. We have been using temperate, transposable bacteriophage Mu as a model system to examine the behavior of mobile genetic elements and have previously shown that many DNA-damaging agents did not induce a Mu prophage to enter the lytic cycle of multiple rounds of DNA transposition. To extend these results and to examine the possibility that they were a reflection of damage to the DNA substrate for Mu transposition, we have constructed a mini-Mu plasmid, pMD12, which contains the early region of Mu, flanked by both extremities required for transposition in cis, and the beginning of the transposase gene A fused in frame to the lacZ gene. This A'-lacZ fusion protein maintains beta-galactosidase enzymatic activity under the control of the expression of the Mu transposase A gene and thus, the capacity for Mu transposition can be easily monitored by assaying for beta-galactosidase. By measuring the amount of beta-galactosidase after various doses of gamma-irradiation, we found that doses of up to 75 krad had no effect on the expression of the Mu transposase gene A. This was confirmed by the lack of induction of a Mu prophage in strains containing a chromosomally inserted Mu genome. Although the plaque-forming units per colony-forming unit of strain CSH67, containing a chromosomally inserted lambda prophage, increased approximately 100-fold from 0 to 75 krad, no stimulation of induction of prophage Mu lytic growth was observed. We also found that plasmid pMD12 did not transpose and chromosomally associate upon gamma-irradiation. This supports the assertion that DNA-damaging agents, including gamma-rays, do not induce the transposition of prokaryotic mobile genetic elements.     

Heat Induction of Prophage φ105 in Bacillus subtilis: Replication of the Bacterial and Bacteriophage Genomes

A temperature-inducible mutant of temperate Bacillus bacteriophage phi105 was isolated and used to lysogenize a thymine-requiring strain of Bacillus subtilis 168. Synthesis of phage and bacterial deoxyribonucleic acid (DNA) was studied by sucrose gradient centrifugation and density equilibrium centrifugation of DNA extracted from induced bacteria. The distribution of DNA in the gradients was measured by differential isotope and density labeling of DNA before and after induction and by measuring the biological activity of the DNA in genetic transformation, in rescue of phage markers, and in infectivity assays. At early times after induction, but after at least one round of replication, phage DNA remains associated with high-molecular-weight DNA, whereas, later in the infection, phage DNA is associated with material of decreasing molecular weight. Genetic linkage between phage and bacterial markers can be demonstrated in replicated DNA from induced cells. Prophage induction is shown to affect replication of the bacterial chromosome. The overall rate of replication of prelabeled bacterial DNA is identical in temperature-induced lysogenics and in "mock-induced" wild-type phi105 lysogenics. The rate of replication of the bacterial marker phe-1 (and also of nia-38), located close to the prophage in direction of the terminus of the bacterial chromosome, is increased in induced cells, however, relative to other bacterial markers tested. In temperature-inducible lysogenics, where the prophage also carries a ts mutation which blocks phage DNA synthesis, replication of both phage and bacterial DNA stops after about 50% of the phage DNA has replicated once. The results of these experiments suggest that the prophage is not initially excised in induced cells, but rather it is specifically replicated in situ together with adjacent parts of the bacterial chromosome.