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.     

Effect of the polymerase activity of DNA polymerase I on the development of moderate bacteriophages Mu, lambda red- and lambda red-gam-. II. The effect of the polymerase activity of DNA polymerase I on the development of bacteriophage Mu

The paper reports on the influence of polymerizing activity of DNA-polymerase I on different developmental stages of temperate bacteriophage Mu in Escherichia coli K-12 cells. This activity is shown to be necessary for optimization of phage Mu primary integration into cell chromosomes. The relative frequency of Mu integration into bacterial chromosomes is 5-6 times lower in polA cells than in isogenic polA+ control strains, the phage yield from cells being delayed during the phage infectious development, but not in the course of induction from the prophage state. Data have been obtained that show the process of phage Mu DNA integration into the plasmid pRP1 .2 and the process of Mu transposition from the cell chromosome into the plasmid to be independent of the polymerizing activity of DNA-polymerase I.     

Conditionally transposition-defective derivative of Mu d1(Amp Lac).

A Mu d1 derivative is described which is useful for genetic manipulation of Mu-lac fusion insertions. A double mutant of the specialized transducing phage Mu d1(Amp Lac c62ts) was isolated which is conditionally defective in transposition ability. The Mu d1 derivative, designated Mu d1-8(Tpn[Am] Amp Lac c62ts), carries mutations which virtually eliminate transposition in strains lacking an amber suppressor. In such strains, the Mu d1-8 prophage behaves like a standard transposon. It can be moved from one strain of Salmonella typhimurium to another by the general transducing phage P22 with almost 100% inheritance of the donor insertion mutation. When introduced into a recipient carrying supD, supE, or supF, 89 to 94% of the Ampr transductants were transpositions of the donor Mu d1-8, from the transduced fragment into new sites. The stability of Mu d1-8 in a wild-type, suppressor-free background was sufficient to permit use of the fusion to select constitutive mutations without prior isolation of deletions to stabilize the fusion. Fusion strains could be grown at elevated temperature without induction of the Mu d prophage. The transposition defect of Mu d1-8 was corrected by a plasmid carrying the Mu A and B genes.     

Coupling and rec-independence of the processes of replication and transposition of Pseudomonas aeruginosa phage D3112. The effect of the phage genes controlling the replication of DNA of D3112

D3112 phage was shown to replicate via the process of coupled replication--transposition: the phage DNA is not excised from the chromosome after prophage induction and new phage copies insert into many different sites. The transposition is controlled by two D3112 early genes--A (mapped in the 1.5-3 kbp region) and B (3-4.5 kbp), and requires intact attL site (involvement of the phage right end attR not studied). D3112 is capable to transpose RP4 plasmid into the chromosome; both the D3112 and RP4 transpositions are rec-independent. The product of the early C gene which is not required for D3112 transposition has pleiotropic effect on the development of D3112 and is necessary for the process of D3112 DNA excision from the chromosome, for cell lysis as well as for mature phage production. We suggest that this gene is responsible for positive regulation of D3112 late genes expression, similar to the C gene of Mu phage or Q gene of lambda. Mutations in four D3112 late genes ts25, ts35, ts73 and ts110 do not affect transposition or excision processes. No detectable (less than 0.02 copies per cell) amount of linear or circular D3112 DNA is formed during the replication--transposition. Hence, in the course of replication and transposition processes D3112 genome has its ends permanently bound covalently to the chromosome. The excision of the D3112 DNA takes place at late stages.     

Bacteriophage Mu-1: A tool to transpose and to localize bacterial genes

In a previous publication (Faelen et al., 1975), it was predicted that the temperate phage Mu-1 would mediate transposition of bacterial genes. Here we show that this is indeed the case. By mating either induced F′ strains (which carry a thermoinducible Mu prophage in the bacterial chromosome), or sensitive F′ infected with Mu, with appropriate recipients, we were able to isolate new F′ episomes which carry various lengths of bacterial DNA. The frequency of transposition of a given marker can be as high as 10^?4. The episomes which carry the transposed DNA always carry Mu as well. When this is coupled with the fact that induction or infection with Mu is necessary for transposition to occur, it is probable that both Mu enzymes and Mu DNA are required by the transposition process. Episomes selected for the presence of a given marker were analyzed for the presence of unselected markers. It was found that: (1) only markers linked to the selected marker can be cotransposed with it; (2) when two markers are simultaneously transposed, all markers lying between them on the chromosome are also transposed; (3) the frequency at which an unselected marker is cotransposed is in some way related to the distance between that marker and the selected marker; (4) the transposition process occurs in both Rec⁺ and Rec^? strains. Mu-mediated transposition offers a new way to isolate F′ episomes and to localize and order bacterial genes as far apart as three minutes.     

Packaging Specific Segments of the Salmonella Chromosome with Locked-in Mud-P22 Prophages

Hybrid genetic elements, Mud-P and Mud-Q (collectively, Mud-P22s), have been constructed that carry two-thirds of the temperate Salmonella phage P22 genome sandwiched between the ends of transposon Mu. Insertions of these elements in the Salmonella chromosome generate locked-in P22 prophages that cannot excise. Upon induction (as a consequence of the inactivation of P22 c2 repressor), a locked-in prophage replicates its DNA in situ, resulting in the amplification of neighboring regions of the chromosome and the processive packaging of three contiguous headsful of adjacent DNA in one direction from the P22 packaging site, pac. Phage particles in an induced lysate of a Mud-P22 lysogen contain DNA molecules corresponding to several minutes of chromosomal DNA adjacent to the site of prophage insertion and transduce nearby genetic markers with high efficiencies. Mud-P22 prophages have been introduced into an F' episome by transposition; resident Mud insertions on the Salmonella chromosome may be converted to Mud-P22 insertions by homologous recombination in P22-mediated transductional crosses.     

Chromosomal integration mechanism of infecting mu virion DNA

DNA transposition is central to the propagation of temperate phage Mu. A long-standing problem in Mu biology has been the mechanism by which the linear genome of an infecting phage, which is linked at both ends to DNA acquired from a previous host, integrates into the new host chromosome. If Mu were to use its well-established cointegrate mechanism for integration (single-strand nicks at Mu ends, joined to a staggered double-strand break in the target), the flanking host sequences would remain linked to Mu; target-primed replication of the linear integrant would subsequently break the chromosome. The absence of evidence for chromosome breaks has led to speculation that infecting Mu might use a cut-and-paste mechanism, whereby Mu DNA is cut away from the flanking sequences prior to integration. In this study we have followed the fate of the flanking DNA during the time course of Mu infection. We have found that these sequences are still attached to Mu upon integration and that they disappear soon after. The data rule out a cut-and-paste mechanism and suggest that infecting Mu integrates to generate simple insertions by a variation of its established cointegrate mechanism in which, instead of a "nick, join, and replicate" pathway, it follows a "nick, join, and process" pathway. The results show similarities with human immunodeficiency virus integration and provide a unifying mechanism for development of Mu along either the lysogenic or lytic pathway.     

A novel illegitimate recombination event: precise excision and reintegration with the Mu gem mutant prophage

The bacteriophage Mu is known to insert its DNA more or less randomly within the Escherichia coli chromosome, as do transposable elements, but unlike the latter, precise excision of the prophage, thereby restoring the original sequence, is not observed with wild-type Mu, although it has been reported with certain defective mutants. We show here that the mutant prophage Mu gem2ts can excise precisely from at least three separate loci —malT, Iac and thyA (selected as Mal⁺, Lac⁺ and Thy⁺, respectively). This excision occurs under permissive conditions for phage development, is observed in fully immune (c⁺) lysogens, and is independent of RecA and of Mu transposase. Mu gemts2 excision is invariably accompanied by reintegration of a Mu gem2ts prophage elsewhere in the chromosome, in the case of Mal⁺ revertants, this prophage is systematically located at 94min on the E. coli chromosome. Mu gem2ts excision therefore sheds some light on the long-standing paradox of the lack of precise Mu excisio.     

Genetic analysis of Escherichia coli min81 mutation blocking the development of bacteriophage Mu

Data characterizing mim81 mutation obtained by the method for direct selection of transposition mutations are presented. The development of Mu is shown to be dramatically suppressed in the mutant strain both upon infection and after induction from the lysogenic state. Frequencies of lysogenization and mini-Mu-dependent formation of cointegrates in the mutant strain are comparable with those in the wild-type strain. Mu development prohibition is removed if expression of early Mu gene is provided from the modified Pe promoter. The results obtained make us believe that the mechanism of mim81 mutation action involves reduction of early gene expression to the level that is sufficient for Mu DNA integration into the chromosome during infection and for single replicative events, but insufficient for vegetative development of bacteriophage Mu.     

Isolation and characteristics of mutation pfm affecting the penetration of phage Mu into Escherichia coli K-12 cells

A mutant of Escherichia coli K-12 strain with the destroyed process of establishment of lysogenic state for phage Mu in the course of zygotic induction has been obtained. The mutation revealed, designated pfm (penetration factor for Mu), interferes with adsorption of phage Mu to the surface of E. coli K-12 cells. On the basis of data obtained, there is every reason to believe that the phage Mu DNA connection with the membrane components of the bacterial cell provides optimizing condition for the primary integrative transposition of phage Mu at the stage of Mu DNA introduction into the cell.     

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.     

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.     

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.     

Transduction of bacteriophage Mu by bacteriophage T1.

Phage T1 transduces phage Mu PFU from Mu-lysogenic donor cells to sensitive recipient cells. The efficiency of transduction depends on the chromosomal location of the Mu prophage. T1, therefore, appears to package different regions of the bacterial chromosome with different efficiencies. Although T1 transduces bacterial markers with different efficiencies, there is no direct correlation between the efficiency of transduction of a bacterial marker and the efficiency of transduction of Mu PFU from donor cells with the Mu prophage located in that marker.     

Virulent mutants of temperate phage Mu-1

Summary Virulent mutants of phage Mu have been isolated after mutagenesis. The virulent phenotype results from most probably 2 mutations located in the c-A region of the Mu genome.Vir mutants are trans-dominant; they induce the resident prophage upon infection in broth of any Mu lysogen. They however form plaques only on certain lysogens, that are monolysogenic for a mutant prophage. We further isolated secondary mutations in Mu Vir which suppress the virulent phenotype.