Physical mapping of coliphage λatt2

The transducing phage lines of the type λatt², which arise by abnormal excision of an integrated prophage, carry bacterial DNA from both the left and right of the prophage including the two prophage attachment sites, attL and attR. The excision sites, X_L and X_R, have been mapped with respect to the attachment sites by DNA heteroduplex analysis of a particular λatt². The X_L-attL segment, containing no known E. coli genes, is about 1300 base-pairs long (2.78 ± 0.13 %λ units). The att_R-X_R segment, including the bio operons and the uvrB locus, is about 7800 base-pairs long (16.8 ± 0.5 %λ units). X_L and X_R appear to be favored sites for the generation of λatt².     

Studies of the stimulation by helper of λ site-specific recombination in lytic crosses

In a lytic phage cross λimm434 helper can enhance site-specific recombination between λN^? phages while λimm21 helper cannot. An analysis of the reciprocal products of the phage crosses shows that formation of each reciprocal product is stimulated by λimm434. The deficiency of λimm21 has been observed for recombination involving several att pairs: attL x attR, attP x attR, attL x attL, and attP x attP. No deficiency was observed for attP x attB.     

A factor in the b2 region affecting site-specific recombinations in lambda

Under certain conditions site-specific recombination of N^? lambda is enhanced with λimm434 helper but not with λimm21 helper. The stimulation has been previously observed in prophage excision and phage-phage recombination involving several att pair combinations. The defect with λimm21 is observed only when the b2 region of the helper is intact. Three mutants have been isolated that mimic the effect of a b2 deletion in λimm21 helpers. The mutants are not large deletions and have a normal att. The alteration has been mapped to the 3.5% of lambda immediately to the left of att. Additionally, it has been found that an int^c mutation on the prophage overcomes the deficiency of λimm21 helper.     

LambdaattB-attP, a lambda derivative containing both sites involved in integrative recombination

A phage genome has been isolated which contains both attB and attP, the bacterial and phage sites which are recombined in the process of formation of stable lysogens. The derivative was created by an illegitimate recombination during mixed vegetative growth of two phage strains which contained attB and attP separately. The attachment sites on the double site derivative can undergo site-specific recombination to to yield a recombinant deleted for about 15% of the parental DNA. This recombination is dependent on the product of the int gene and demonstrates a great preference for recombination between two sites located on the same molecule. The int gene product used for recombination of λattB-attP may be provided either from the int gene residing either on λattB-attP or on another phage genome. In both cases, ochre suppression of amber mutations of the int gene results in extremely depressed levels of integrative recombination.     

New att mutants of phage λ

Phage λ inserts its chromosome into that of its host Escherichia coli by recombination at specific attachment sites (atts). New deletion and point mutants affecting att were isolated. The point mutants map as if they are in a region common to the atts and could not be distinguished from att mutants described earlier (Shulman and Gottesman, 1973).Effects of the mutations on the physiology of insertion and excision are presented.     

The isolation of restriction fragments containing the primary and secondary (galT) bacterial att sites of phage lambda.

Transducing derivatives of bacteriophage λ have been used to isolate small DNA restriction fragments which contain the primary bacterial att site (BOB′) for phage insertion and the secondary bacterial att site (ΔOΔ′) at galT. Four hybrid att sites, BOP′, POB, ΔOP′, and POΔ′, have also been isolated in small restriction fragments. Acrylamide-agarose or agarose gel electrophoresis profiles of two analogous isogenic sets of transducing phage were compared with profiles of wild-type λ to identify the att-containing fragments. Each set consisted of phages transducing bacterial DNA originating to the right (e.g., POB′ or POΔ′) or left (e.g., BOP′ or ΔOP′) of the prophage and a recombinant phage, transducing all of the bacterial DNA of both the rightward and leftward tranaducing lines (e.g., a regenerated BOB′ or ΔOΔ′). The att-containing fragment of each phage was identified by virtue of the fact that it was the only fragment which was unique to the gel profile of that phage. For the mutants transducing the primary bacterial att site, the restriction enzymes HindII + III and MboII were used sequentially to obtain fragments containing BOB′, POB′, and BOP′ of approximately 560, 580, and 340 base pairs in length, respectively. For the mutants transducing the secondary att site at galT, restriction enzymes HindIII, BamHI, EcoRI, and Hinfl were used to identify fragments containing ΔOΔ′, POΔ′, and ΔOP′ of approximately 755, 475, and 580 base pairs in length, respectively. These fragments can be isolated in sufficient quantity for biochemical studies on att site interactions and in sufficient purity for sequence analysis.     

Formation of lambda lysogens by IS2 recombination: gal operon--lambda pR promoter fusions.

When Escherichia coli carrying an IS2 element in the gal operon are infected with lambda which also carry an IS2 element, some lysogens are formed by recombination between the two insertion elements. The resulting prophage are abnormally permuted. Whereas normal prophages are bracketed by two attachment sites, the abnormal prophages carry an internal phage attachment site. Int-promoted recombination between the internal attachment site and the bacterial attachment site results in an inversion of the portion of the prophage and bacterial genome between these two sites. In addition, the expression of the gal operon in these lysogens is under the control of the prophage P_R promoter and may be used to study the factors that influence this promoter.     

Focus assay and defectiveness of avian myeloblastosis virus.

A lysogen for P1d91tet, a defective prophage produced by recombination between P1 and an R factor, is not immune to superinfection by phage P1. However, marker rescue experiments demonstrate that the wild type form of the P1 repressor gene (c1⁺), whose action is necessary for the maintenance of a P1 prophage, is present in d91tet. Furthermore, the c1⁺ gene is active in a P1d91tet lysogen, since a single lysogen for a recombinant P1d91tet plasmid into which a c1ts mutation has been introduced is thermo-inducible. It is therefore concluded that superinfection immunity of a prophage is controlled, at least in part, by a different part of the P1 genome from that needed for prophage repression (gene c1).Marker rescue experiments using the d91tet lysogen allow us to determine the relative order of the genes near the “right-hand” end of the vegetative P1 map, including gene c1.     

A mutant of Escherichia coli that prevents growth of phage lambda and is bypassed by lambda mutants in a nonessential region of the genome

A mutant strain of E. coli (rap^?) has been isolated which blocks the growth of phages λ, 434, and ?80. Phage 424 and various non-lambdoid phages are not affected. λ mutants that can bypass the rap block are of two general types. First, a presumed point mutant mapping between h and att provides partial bypass of the block. Second, all deletions and substitutions that eliminate or substitute one or both recognition regions of the attachment site restore a high level of growth. However, a point mutation in the crossover region of the attachment site does not bypass the block, and the rap mutation does not affect Int-promoted λ recombination. While the mechanism by which rap blocks phage development is unknown, the block implies a new and potentially interesting form of phage-host interaction.     

Specialized transduction of pro genes by coliphage P1: Structure of a partly diploid P1-pro prophage

Bacteria carrying P1-pro hybrid prophages have been isolated as rare (10—⁹Pro⁺ per pfu) Pro⁺ transductants of recA proA Escherichia coli. The proA gene is inserted into the P1 prophage since the transductants become Pro^? when the resident P1 is lost. Furthermore, when induced, these recA transductants produce HFT pro lysates which contain defective pro-transducing particles as well as plaque-forming particles which do not transduce pro. The number of PFUs obtained is about one-tenth the number obtained from normal lysogens. The density of the transducing particles was not distinguishable from that of the PFUs. The Pro⁺ transductants obtained by use of these HFT pro lysates (at low m.o.i.) carry only a small segment of the P1 genome, including cam and gene 2, and are not P1-immune. The number of Pro⁺ transductants increases about 20-fold when the recipient contains a helper P1 either as superinfecting phage or as prophage, The Pro⁺ transductants that arise in the presence of helper P1 are P1-pro lysogens formed by recombination between the DNA of the helper P1 and of the transducing particles. Like the original P1-pro lysogens, they produce lysates which contain plaque-forming and pro transducing particles. The circular P1-pro prophage is inferred to be oversized (at least as large in size as a P1 headful) and to have duplicate cam-gene 2 regions, one copy at each junction of P1 and Pro DNA. The DNA packaged after induction of P1-pro lysogens is not terminally redundant. However, after injection into recipient bacteria, recombination can occur between the redundant cam-gene 2 regions giving rise to either circular pro-cam-gene 2 transducing DNA or to circular P1 plaque-forming DNA. Evidence for this partly diploid prophage is the construction of P1-pro prophages which are heterozygous for gene 2.     

Cooperative interactions between bacteriophage P2 integrase and its accessory factors IHF and Cox

Bacteriophage P2 integrase (Int) mediates site-specific recombination leading to integration or excision of the phage genome in or out of the bacterial chromosome. Int belongs to the large family of tyrosine recombinases that have two different DNA recognition motifs binding to the arm and core sites, respectively, which are located within the phage attachment sites (attP). In addition to the P2 integrase, the accessory proteins Escherichia coli IHF and P2 Cox are needed for recombination. IHF is a structural protein needed for integration and excision by bending the DNA. As opposed to lambda, only one IHF site is found in P2 attP. P2 Cox controls the direction of recombination by inhibiting integration but being required for excision. In this work, the effects of accessory proteins on the capacity of Int to bind to its DNA recognition sequences are analyzed using electromobility shifts. P2 Int binds with low affinity to the arm site, and this binding is greatly enhanced by IHF. The arm binding domain of Int is located at the N-terminus. P2 Int binds with high affinity to the core site, and this binding is also enhanced by IHF. The fact that the cooperative binding of Int and IHF is strongly reduced by lengthening the distance between the IHF and core binding sites indicates that the distance between these sites may be important for cooperative binding. The Int and Cox proteins also bind cooperatively to attP.     

Architectural flexibility in lambda site-specific recombination: three alternate conformations channel the attL site into three distinct pathways

Background :  In the phage lambda life cycle, the Integrase (Int) protein carries out recombination between two different sets of DNA substrates: attP and attB in integration, attL and attR in excision. In each case, the partners are very different in structure from each other and the recombination reaction between them is effectively irreversible. For comparison, we have studied the recombination mediated by Int between two identical attL sites. Both in vitro and in vivo , recombination between two attL sites can be mediated inefficiently by Int alone. But, while IHF can stimulate recombination 5–10-fold in vivo (to the level of excision and integration), this stimulation is not observed under standard conditions in vitro
Results :  We find that IHF can stimulate the in vitro recombination between two attL s that are modified to be defective in one of the high affinity binding sites for Int, P'1. With such substrates, the efficiency of IHF-stimulated recombination is comparable to that seen in vivo . The requirements for this reaction distinguish it from other lambda recombination pathways, as does the performance of several mutant Int proteins. Recombination of attL sites on intracellular plasmids suggests that this pathway is effective in vivo , but that some unknown factor or condition permits it to operate on wild-type as well as mutated attL sites.
Conclusions :  The recombination pathway described in this work apparently uses a unique attL architecture, one which requires bending by IHF and is inhibited by Int bound at the P'1 site. In addition to demonstrating the architectural flexibity of the lambda system, this pathway should be a valuable resource for separating the basic requirements of strand exchange chemistry from the features which impart directionality.     

Characterization of P1argF derivatives from Escherichia coli K12 transduction. III. P1Cm13argF derivatives

In a system where generalized transduction cannot occur, transduction of argF with P1Cm13 as a vector occurred with a frequency of 9.2 × 10^?8. This frequency of specialized transduction was 30 times lower than we have observed for other PI vectors. Genetic analysis indicated that though the transductants harbored P1Cm13argF prophage, most (six out of seven) prophage were defective in some way. Physical analysis confirmed that two of the prophage had large deletions of P1 genes located near or at an IS1 element. From these results we concluded that the insertion of argF into P1Cm13 is often accompanied by deletions in the prophage genome. The additional DNA in P1Cm13 consists of 2 IS1 elements and the chloramphenicol resistance determinant. The deletions in the transductant prophage could be a result of enhanced IS1 × IS1 recombination due to the additional IS1 elements in the P1Cm13 genome or the orientation of these IS1 elements.     

Bipartite control of immunity conferred by the related heteroimmune plasmid prophages,P1 and P7 (formerly φAmp)

Coliphages P1 and P7 (formerly known as φAmp) exhibit a number of similarities including sensitivity to a repressor of lytic functions specified by the c1 gene of either prophage. The ability of bacteria harboring one of these plasmid prophages to allow the growth of the other phage is shown to depend upon the product of a viral gene ant that is not closely linked to c1 and that antagonizes repression. The ant gene of P1 and P7 prophage is normally repressed by an immunity-specific repressor, but ant is expressed constitutively in members of a class of virulent mutants. In one such vir mutant of P7, a nonsense ant mutation was obtained. The ant mutation has been separated by recombination from the closely linked cis-dominant vir mutation. The properties of this mutant, among other P1 and P7 mutants affecting immunity, conform to the bipartite scheme of immunity control established in Salmonella phage P22 and independently concluded by J. R. Scott and her collaborators to apply to P1 and P7. The essentially superfluous ant circuit may possibly permit an enlarged scope for parasitism.     

Characterization of bacteriophage D6

Bacteriophage D6, described by Mise and Suzuki [J. Virol.6, 253–255 (1970)], is a temperate coliphage capable of generalized transduction. D6 resembles phage P1 morphologically and is slightly sensitive to anti-P1 serum. We have found that like P1, the D6 prophage is a plasmid, approximately 90 kb in size. The D6 prophage is compatible with both P1 and the closely related heteroimmune prophage P7, both of which are in incompatibility group Y. D6 DNA is restricted by the P1 and P7 prophages, but in the absence of restriction, D6 is able to grow on P1 and P7 lysogens. Neither P1 nor P7 is able to grow on a D6 lysogen. This is not due to DNA restriction or to the elimination of phage receptor sites on the host's surface. Therefore a nonreciprocal immunity relationship appears to exist between P1–P7 and D6. Mutants of P1 and P7 which are not subject to repression by the P1–P7 c 1⁺ gene product are able to grow on a D6 lysogen, implying that D6 has a repressor activity like that of P1c 1. There are two regions of strong hybridization between P1 and D6 DNA; one includes the invertible C loop of Pl. Attempts to demonstrate complementation or recombination between P1 or P7 and D6 were not successful, even for markers in the regions of homology.