Integration of the related prophages lambda, phi 80 and their hybrid lambda att80 into the secondary chromosomal att sites of wild-type Escherichia coli

The family of lambdoid phages displays a varying specificity of integration into the host chromosome. The lambda phage DNA failed to get inserted at the secondary attachment site(s) of the gal operon (frequency less than 2.6 X 10(-8)) in the presence of the primary (normal) one. By contrast, phi 80 and the lambda att80 hybrid integrated into wild-type Escherichia coli at least, at two secondary att sites of the btuB locus, the latter phage being also capable of integration in the vicinity of purE and purC (frequency 2 X 10(-3) to 10(-4)). Integration of phi 80 and lambda att80 into btuB occurred with about the same frequency as in cells deleted for normal insertion site (0.7 divided by 4.0 X 10(-6)). An analysis of the secondary lysogens with the prophage in btuB showed them to be polylysogens; the additional prophage(s) was found in the primary att site. We also failed to observe integration of phi 80 and lambda att80 with formation of secondary monolysogens into other foci (frequency less than 0.0035, if multiplicity of infection was 10(-3) or 10). It is presumed that phi 80 and lambda att80 prophages get only integrated at secondary att sites in case the primary site is occupied.     

Mechanism of non-tandem integration of prophage phi 80 into the chromosome of the wild-type Escherichia coli

We studied the ability of lambda, phi 80 and their hybrid lambda att80 to lysogenize homoimmune monolysogens and examined the prophage locations on the chromosome of the resulting polylysogens. We observed an effective integration of phi 80 and lambda att80, in contrast to lambda, into the host chromosome, exclusively, at the attachment sites that were not occupied by the resident prophage (nontandem). Besides, the lambda att80 (int+) prophage was observed to ensure effective nontandem integration of a homoimmune int mutant DNA. Hence, we inferred that the expression of the int gene in the phi 80 prophage is constitutive, cI-independent and results in nontandem integration of the homoimmune prophage. The validity of this inference has been supported experimentally: (i) the only lysogen that was found to contain a phi 80 tandem was highly unstable (spontaneous segregation of monolysogens occurred 6-7 times more frequently than with the lambda tandem); (ii) an int inactivating mutation stabilized the phi 80 tandem; as a result, the int mutant has the frequency of tandem integration as high as that of lambda, while no nontandem integration was observed. A hypothesis is proposed which accounts for the instability of the phi 80 tandems and explains the relation between this phenomenon and the prophage ability to integrate into secondary attachment sites in the presence of the primary (normal) one.     

Integration of bacteriophages lambda and phi 80 in wild-type Escherichia coli at secondary attachment sites. II. Genetic structure and mechanism of polylysogen formation for lambda, phi 80 and the lambda att80 hybrid

Summary The frequency of occurrence and the genetic structure of polylysogens were studied for phages , 80 and att80. In the case of , frequency of polylysogenization is high (0.20 to 0.41) with a tandem integration of prophages at the primary att site (att). With 80 and att80, this frequency is about 10 times lower, and usually one prophage becomes integrated at the primary att site (att80-I) while another (sometimes two others) integrates at one of the secondary sites. At least four secondary att80 sites have been found in wild-type Escherichia coli , two of which (near the his and tolC loci) are preferred. The frequency of secondary integration of 80 and att80 does not differ significantly in the wild-type host and in that deleted for the primary att site (0.041 and 0.045, respectively, among surviving cells at an MOI of 10).Homoimmune superinfection has revealed a constitutive cI-independent expression of the 80 int gene in the prophage state. The only 80 tandem detected proved to be unstable. With the 80int ^- mutant, we observed stabilization of 80 tandems; as a consequence, their frequency of occurrence during coinfection with 80int ⁺ was up to the level and no nontandem insertions were found. A model is proposed for the 80 and att80 nontandem integration.Abbreviations TP transducing phage(s) - PFU plaque-forming units - PC pink clear-resistant colonies on EMBO plates - MOI multiplicity of infection - O origin of Hfr transfer

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New transducing phage with RNA polymerase beta- and beta'-subunit genes derived from a hybrid phage lambda att80: isolation, genetic analysis and physical mapping]

A hybrid lambda att 80 phage with the genetic structure lambda (A-J) phi 80 (att-int-xis) imm lambda..cI857s7 is shown to be a convenient vector for creating transducing phages. On the one hand, the restriction analysis indicates that it has 3 restriction sites for EcoRI in comparison with 5 and 9 sites for parental phages lambda and phi 80 respectively. On the other hand, its buoyant density is less than that of phage lambda and under centrifugation it is easier separated from the phage transducing particles. When lambda att 80 prophage was excluded from the bfe locus of Escherichia coli, transducing phages with genes of two RNA polymerase beta-subunits (rpoB and rpoC) were isolated. To identify the latter, a convenient genetic test was worked out. A physical map of lambda att 80 drifd 35 transducing phage, carrying rpoB and rpoC genes has been constructed using endonucleases EcoRI and HindIII. A comparison of this map and the corresponding maps of transducing phages lambda drifd 18 and lambda drifd 47, studied earlier, led to the discovery of two integration sites of phage lambda within the locus bfe spaced apart by about 1800 nucleotide pairs. At all the sites both phages (lambda and lambda att 80) have integrated in the locus bfe in the counter clockwise order.     

Integration of bacteriophages lambda and phi 80 in wild-type Escherichia coli at secondary attachment sites. I. Formation of secondary lysogens

Summary The family of lambdoid phages displays a varying specificity of integration into the host chromosome. The phage DNA failed to get inserted at the secondary site(s) of the gal operon (frequency <2.6x10^-8) in the presence of the primary (normal) att site. By contrast, 80 and the att80 hybrid (x80) became integrated into wild-type Escherichia coli at at least two secondary att sites of the btuB locus, and the latter near purE and purC as well (frequency 2x10^-3-10^-4). The integration of 80 and att80 into btuB occurred with about the same frequency as in cells in which the normal insertion site had been deleted (0.7-4.0x10^-6). An analysis of the secondary lysogens with the prophage in btuB showed them to be polylysogens; the additional prophage(s) was found at the primary att site. We also failed to observe the integration into other loci of 80 and att80 with the formation of secondary monolysogens (frequency <0.0035 at MOI-10^-3 or 10). It is presumed that these prophages become integrated at secondary att sites only if the primary site is occupied.Abbreviations MOI multiplicity of infection (PFU/cell) - PFU plaque-forming unit - TP transducing phage - P1/HfrH P1vir multiplied on HfrH - Rif-R rifampin-resistance - Int int protein     

Genetics of bacteriophage phi 80--a review

The genetic maps of bacteriophage lambda and lambdoid phage phi 80 are compared. The gene organization of phi 80 is very similar to that of lambda, as shown by isolation and characterization of many am, ts and c (clear) mutants of the phage. In general, the essential genes located in the same position on the genetic map of the phages lambda and phi 80 fulfill the same functions. These include the gene clusters coding for the head and tail proteins, genes for DNA synthesis, and the genes controlling lysogeny and late gene expression. The specific regulatory features of phi 80 in relation to the N function of lambda are discussed, but they require further clarification. The two phages differ in immunity specificity, host range, conversion property and temperature sensitivity.     

The phi 80 and P22 attachment sites. Primary structure and interaction with Escherichia coli integration host factor

Although the lambdoid bacteriophage phi 80 and P22 possess site-specific recombination systems analogous to bacteriophage lambda, they have different attachment (att) site specificities. We have identified and determined the nucleotide sequences of the att sites of phi 80 and P22 and have examined the interaction of these sites with purified Escherichia coli integration host factor (IHF). The sizes of the homologous core regions of the att sites vary greatly: P22 has a 46-base pair core, while phi 80 and lambda have 17- and 15-base pair cores, respectively. The core sequences of the three phage show no significant homology, although dispersed regions of homology in arm sequences indicate that the three phage att sites are related. All three att sites have a high A + T composition, and restriction fragments carrying these sites migrate anomalously upon polyacrylamide gel electrophoresis. IHF binds to a site to the left of the common core in the phi 80 and P22 phage att sites (attP) and to a site to the right of the core in P22 attP and attB (the bacterial att site). In the lambda system, IHF interacts with three regions on attP (designated H1, H2, and H') and none on attB (Craig N., and Nash, H.A. (1984) Cell 39, 707-716). Alignment of the IHF sites of all three phage results in a consensus sequence for IHF binding, Pyr-AANNNNTTGATAT. Among the three phage, the number of IHF sites differs; however, the location and orientation of the binding sites in relation to the respective core regions are well conserved. An IHF site analogous to lambda H2 is present in both phi 80 and P22 attP, while a site analogous to lambda H' is present in P22 attP. This conservation suggests that IHF plays a very similar role in the site-specific recombination pathways of all three phage, and that the flanking arm sequences are necessary for phi 80 and P22 attP function, as is the case for lambda attP function. These structural similarities presumably reflect a conservation of the mechanism of site-specific recombination for the three phage.     

Transposition of the kanamycin resistance determinant (Tn601) from plasmid 5T to the genome of bacteriophage lambda and the expression of this gene after prophage induction

Tn601, determinging kanamycin resistance of Escherichia coli, has been transposed into the bacteriophage lambda genome from R6 plasmid. After curing lambda gtc1857 (Tn601) lysogenes on the kanamycin containing medium, the clones with stable and unstable integrations of the Tn6-1 into the chromosome were obtained. After the lysogenization of these clones with the phage lambda att80c1857S7, the phages lambda att80c1857S7 (Tn601) were obtained. These phages contained the Tn601 from the sites of stable or unstable integrations. The frequency of the Tn601 transposition from the sites of unstable integration was 10(-7), that was two order of magnitude higher than the frequency of the Tn601 transpostion from the site of stable integration. Temperature induction of the lambda att80c1857 (Tn601) prophage resulted in 10--15 times increase of the yeild of aminoglycoside-3'-phosphotransferase I, the enzyme coded by the aphA gene of the Tn601.     

Specialized transduction with lambda plac5: dependence on recA and on configuration of lac and att lambda.

The construction of lambda plac5 transducing phages carrying various lacZ alleles is described. Genetically disabled (N- N- P-) lambda plac transducing the phages were used to study the dependence of specialized transduction on host RecA function and on the location of the lacZ gene in the recipient strain. In the absence of site-specific recombination at att lambda, transduction was completely dependent on host RecA function. Regardless of the configuration of att lambda, lambda plac transducing phages recombined at a 20- to 50-fold higher frequency with F42 lac than with a lac gene located in the cellular chromosome. Deletion mutants of lacZ in the recipient strain were used to show that the probability of lac recombination resulting from lambda plac infection is apparently proportional to the amount of homology between the parental lacZ genes.     

Specificity of Tn9 insertion into the genome of bacteriophage lambda att80 depending on its preceding location

It was shown that the site of previous integration (the donor site) of Tn9 affects the specificity of its next integration into the target molecule--phage lambda att80 DNA. The transposon integration sites were mapped by restriction and heteroduplex analysis following Tn9 transposition from chromosomal sites of Escherichia coli K-12 differing in location and Tn9 stability. When transposed from chromosomal galT::IS1 gene, Tn9 inserted into the site with coordinates 44,5 +/- 2 kb of lambda att80; when transposed from chromosomal attTn9A site, the transposon inserted into the sites with coordinates 31 +/- 0,7 kb or 33,3 +/- 0,5 kb. In the course of transposition of Tn9 from chromosomal attTn9N site the transposon inserted into the lambda att80 site with coordinates 26,5 +/- 5 kb. In the latter case, the increase of Tn9 single-stranded loop and the appearance of two new HindIII cleavage sites were observed in heteroduplex experiments. The data were interpreted as indicating structural rearrangements of Tn9 or linked sequences in the course of transposition.     

Lambda H-lambda T 80 hybrid study of the DNA structural gene region in lambda and phi 80 phages

Hybrids lambda H lambda T80 are formed due to recombination of the phage lambda att80 and phi 80 prophage partially deleted in the region of structural genes. Genetic structure of 22 independently isolated lambda H lambda T80 hybrids was determined by the restriction method and it was shown that recombination took place in the genes A, C, D and H. The frequencies of hybrid formation diminish from 1.10(-3) to 4.10(-5) for this gene order, which suggests that the polar divergence of nucleotide sequencies in the region of structural genes exists. It was found that formation of hybrids with recombination in the region of "weak" homology (gene H) was possible only when the region of "strong" homology was present in the deleted phi 80 prophage to initiate recombination.     

Analysis of insertions of the determinant of drug resistance to kanamycin and chloramphenicol into bacteriophage lambda att80

The insertion sites of elements Tn9 and Tn601 which determine chloramphenicol and kanamycin resistance have been detected restriction analysis. The functioning of transposons i.e. their stability or instability, has been found to influence the specificity of their insertions into the genome of lambda att80 bacteriophage. During transposition from stable integration sites both transposons are inserted into the regions of the lambda att80 bacteriophage genome, definite for each transposon. However, during transposition from the site of unstable integration both determinants of drug resistance are inserted into different regions of the phage genome.     

Restriction alleviation by bacteriophages lambda and lambda reverse.

Deletion analysis indicated that the phage lambda restriction alleviation gene(s) ral resides between the cIII and N genes. The Ral+ phenotype was expressed only when lambda ral+ carried a modification such that it was resistant to restriction by the host specificity system. Under these conditions, Ral function protected superinfecting unmodified phages from restriction by EcoK or EcoB but not from restriction by EcoP1. Ral-protected phage DNA was not concomitantly K and B modified, but rather received only the modification specified by the system of the restricting host. Possible mechanisms for Ral action are discussed. Of the other lambdoid phages tested, the hybrid phage lambda rev had Ral activity, whereas phi 80vir and one lambda-P22 hybrid did not. The restriction alleviation activity of lambda rev called Lar, may be the same as the activity expressed in sbcA- strains of Escherichia coli, but it was functionally separable from exonuclease VIII activity (the product of the recE gene), which is also expressed in sbcA- strains.     

Molecular structure of hybrid phage phi80hy43

The phage hybrid phi80hy43 derived from a vegetative cross phi80cIhlambda x lambdacIc17 was constructed for discrimination phi80mono- and polylysogens. Molecular structure of this hybrid was established using heteroduplex analysis and restriction endonuclease EcoRI. It is found that the hybrid phi80hy43 represents a phage phi80 containing a foreign piece of DNA between genes cI and 0. The length of this piece of DNA comprises 0.7%, corresponding to the length of cy-cII region of th phage lambda. So it is believed that the hybrid phi80hy43 was formed due to insertion of the lambdacy region with the mutation c17 into phi80hlambda phage genome.     

Incorporation of phi80 phage into unusual chromosome sites and isolation of a specialized transducing bacteriophage]

The mutant strain KS713 of Escherichia coli K-12 deleted for the normal insertion site and secondary preferable one was obtained. The insertion frequency of phage phi80 into the double deletion strain is reduced about 30-fold with respect to integration into the strain H47 with deletion of the primary phi80 attachment site and about 500-fold relative to integration into wild type Escherichia coli. Analysis of the rare abnormal lysogens of KS 713 strain indicates that there are secondary sites on the chromosome, which are utilized for prophage attachment if insertion at preferable secondary att80-II site is eliminated too. The insertion of phi80 phage into the bfe locus was obtained by the appropriate selection technique. Induced prophage excision from the bfe site was rather efficient and lysates contained phi80 phage particles that could specificically transduce the argH+ gene. Upon transduction into a recipient strain carrying recA, heterogenotes harbouring both the wild-type and the mutant argH genes were isolated. These heterogenotes were used for producing high-frequency transducing lysates.     

Sequence of a secondary phage lambda attachment site located between the pentitol operons of Klebsiella aerogenes.

We have determined the nucleotide sequence of a secondary phage lambda attachment site (att) located between the structural genes of the ribitol and D-arabitol catabolic operons of Klebsiella aerogenes. The core region of this secondary attachment site (sequence: GGTTTTTTCGATTAT) shows considerable homology with the 15-base-pair core region common to both the phage att and the primary bacterial att of Escherichia coli K12 (sequence: GCTTTTTTACTAA); however, there is no such clear homology between the sequences flanking the cores of the primary att and this secondary att. Integration of phage lambda into the K. aerogenes secondary att occurred by recombination between the core region of the phage att and an oligo(T.A) stretch located within the K. aerogenes secondary att.     

Formation of lambda transducing phage in vitro: involvement of DNA gyrase

We found that transducing phages carrying the gal or bio regions of the Escherichia coli genome were formed during in vitro packaging of endogenous lambda DNA. Structural analysis of the transducing phage genomes indicated that they were formed by abnormal excision of lambda prophage. Formation of transducing phages was stimulated by oxolinic acid, an inhibitor of DNA gyrase, implying that DNA gyrase participates in the abnormal excision of lambda prophage. When pBR322 DNA was added to the reaction mixture, transducing phages into which pBR322 had been inserted were produced at a high frequency. This reaction was also stimulated by oxolinic acid. Sequence analyses revealed that pBR322 is inserted into the sites of abnormal excision of the prophage. These results show that transducing phages can be formed by DNA gyrase-dependent illegitimate recombination in an in vitro system and that secondary recombination takes place frequently at the site where the first recombination occurs.     

Phi X174 E complements lambda S and R dysfunction for host cell lysis.

Hybrid lambda phages which have the E lysis gene of the bacteriophage phi X174 in cis to defective nonsense and deletion alleles of the normal lambda lysis genes S and R have been constructed and shown to be fully competent for plaque-forming ability, which demonstrates that the single-gene, lysozyme-independent lysis system of phi X174 and related phages can serve the lytic function for large complex phages. These hybrid phages are unable to form plaques on a slyD host. Moreover, plaque morphology indicates that in E-mediated lysis the soluble lambda R endolysin can participate in lysis, indicating that the protein E-mediated lesions are not completely sealed off from the periplasm.     

Effects of recB21, recF143, and uvrD152 on recombination in lambda bacteriophage-prophage and Hfr by F- crosses.

The effects of the mutation pairs recB21 recF143 and recB21 uvrD152 on the frequency of genetic recombination were investigated in lambda phage-prophage crosses under homoimmune conditions. To prevent recombinants from being formed by the phage red system, these experiments were performed with phages and prophages carrying red and gam mutations. Both spontaneous and damage-induced recombination was measured, the phages being either undamaged or treated with trimethylpsoralen and 360-nm light to cross-link the phage DNA. Control and damaged phages were allowed to infect lysogenic host cells under conditions in which phage gene expression was repressed and phage DNA replication was blocked by lambda immunity. Although the double mutations recB21 recF143 and recB21 uvrD152 reduced recombination in Hfr by F- crosses to 0.3 to 0.02% of the wild-type controls, the presence of these pairs of mutations in the host lysogens had relatively little effect on the results of the phage-prophage crosses. In the latter system, recB21 recF143 reduced spontaneous and damaged-induced recombination by less than threefold whereas recB21 uvrD152 increased it to three times the wild-type level, the increase being attributable to the uvrD mutation. Evidently, the gene products of recB,C uvrD, and recF wee not needed for lambda phage-prophage recombination under repressed conditions.