Lysogenic conversion caused by phage phi 80. III. The mapping of the conversion gene and additional characterization of the phenomenon

The cor gene specifies lysogenic conversion caused by the lambdoid phage phi 80. The cor gene product inhibits tonA function in infected and lysogenic cells. The cells harboring pBR322 plasmid with the cloned cor gene of phi 80 became resistant to the phages T1 and phi 80 (TonA phenotype). The cor gene was mapped between 24 and 13 genes on the phi 80 phage genetic map. It is not essential for phage lytic growth. Its presence in lysogens leads up to accumulation of tonA mutants in a cell population.     

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.     

Physiological relationship between the temperate phages lambda and phi80

Summary This work deals with the ability of phage 80 to provide defective mutants of with their missing functions. Functions Involved in Recombination. As shown by others, the Int mechanism of 80 cannot excise prophage . However, 80 efficiently excises recombinants from tandem dilysogens, using its Ter mechanism. Likewise, the nonspecific mechanism Red is interchangeable between 80 and . Maturation of DNA by 80. The Ter recombinants excised by 80 from tandem dilysogens are packaged into a 80 protein coat. This contrasts with the fact, already mentionned by Dove, that 80 is extremely inefficient for packaging phage superinfecting a -lysogen. The latter result is also found when the helper phage is a hybrid with the left arm of (80hy4 or 80hy41 — see Fig. 1). However, the maturation of the superinfecting is much more efficient if the 80hy used as a helper has the att-N region of (like 80hy1). Conversely a with the att-N region of 80 (hy6 — see Fig. 1) is packaged more efficiently by 80 or 80hy4 than by 80hy1. It is suggested that the maturation of chromosome superinfecting an immune cell requires a recombination with the helper phage. Vegetative Functions. Among the replicative functoons O and P, the latter only can be supplied by 80. That N mutants are efficiently helped by 80 does not tell that 80 provides the defective with an active N product; the chromosomes are simply packaged into a 80 coat. This shows that 80 is unable to switch on the late genes of . That neither 80 nor any of the 80hy tested can provide an active N product is shown in a more direct way by their complete failure to help N ^-r₁₄; this phage carries a polar mutation which makes the expression of genes O and P entirely N-dependant. The maturation of a N ^- by 80 contrasts with the fact that mutants affected in late genes (A, F or H) are not efficiently helped by 80. This suggests that the products coded by these genes are not interchangeable between 80 and , and that packaging of DNA into 80 coats is possible but inhibited when late proteins are present in the cell. Activation of the Late Genes. Among the im ₈₀ h ⁺ hybrids tested, only 80hy41 is able to switch on the late genes of a N defective mutant. This hybrid differs from the other hybrids studied here, by the fact that it has the Q-S-R region of (see Fig. 1). The results are consistant with the view that the product of Q gene is sufficient for activating the late genes of a DNA. N would thus control the expression of late genes only indirectly by controlling the expression of gene Q (Couturier & Dambly have independantly reached the same conclusion, 1970). Furthermore the failure of 80 and of the 80hy1 and 80hy4 to activate the late genes of would imply that these phages are unable to provide an Q product active on the chromosome Reciprocally, switches on the late genes of prophage 80hy41, but not of prophages 80hy1 and 80hy4. This suggests that the initiation of late genes expression takes place at a main specific site located in the Q-S-R region of the chromosome. The expression of the late genes would thus be sequential, and proceed through the left arm only when steaky ends cohere. Similar conclusions were reached independantly by Toussaint (1969) and by Herskowitz and Signer (1970).

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Ce travail a été réalisé dans le cadre du contrat d'association Euratom-U. L. B. 007-61-10 ABIB et avec l'aide du Fonds de la Recherche Fondamentale Collective.     

Lysogenic conversion in Klebsiella pneumoniae: system which requires active immunity regulation for expression of the conversion phenomenon.

We have previously described Klebsiella pneumoniae MirM7b, which, although stably lysogenic for the inducible and nondefective phages FR2 and AP3, is not immune to superinfection by these same viruses. MirA12b, a strain which is lysogenic for FR2 and AP3 and immune to superinfection, has been derived from MirM7b. The sensitivity of this strain and that of the nonimmune parent to several bacteriophages have been compared in this work. It has been found that, whereas MirM7b is sensitive to coliphages P1, T3, T7, and phiI, MirA12b is fully resistant to all of them. It is shown that phages FR2 and AP3 convert Klebsiella strains to resistance to coliphage P1 and coliphages T3, T7, and phiI, respectively, and cause loss of surface antigens in lysogenic cells. To determine such a conversion, both FR2 and AP3 require expression of immunity to superinfection. This explains the differences that exist between MirM7b and MirA12b in both phage sensitivity and surface antigens. Hypotheses are presented to explain the peculiar need for an active superinfection repressor to express lysogenic conversion.     

Proteins responsible for lysogenic conversion caused by coliphages N15 and phi80 are highly homologous.

Lysogenic conversion caused by lambdoid bacteriophage phi80 and that caused by coliphage N15 have similar characteristics, suggesting that similarities in their cor genes and Cor proteins are responsible for this effect. Here we present the nucleotide sequence of the N15 cor gene. The N15 cor gene homolog was found in the phi80 cor region, but in the opposite direction of that of the open reading frame to which the phi80 cor gene had previously been assigned (M. Matsumoto, N. Ichikawa, S. Tanaka, T. Morita, and A. Matsushiro, Jpn. J. Genet. 60:475-483, 1985).     

Excision and duplication of su3+-transducing fragments carried by bacteriophage phi 80. I. Novel structure of phi 80sus2psu3+ DNA molecule.

DNA molecules of phi 80sus2psu3+ and phi 80dsu3+ isolated by Andoh and Ozeki (1968) were studied by the electron microscope heteroduplex method. The phi 80sus2psu3+ and phi 80dsu3+ DNA lengths were found to be 108.7 and 103.3% of the phi 80 DNA, respectively. The phi 80sus2psu3+/phi 80 heteroduplex shows an insertion loop of 8.7% of the phi 80 DNA which migrates from 7.7 to 9.7%, as measured relative to the left (0%) and right (100%) termini of the mature phi 80 DNA molecule. The region of loop migration occupies the central region of the phi 80 head gene cluster. The presence of su3+-containing Escherichia coli DNA of 6.7% phi 80 unit flanked by two homologous regions of phage DNA of 2.0% of phi 80 unit gives rise to a movable insertion loop. In phi 80dsu3+, from which phi 80sus2psu3+ was derived, 50.5% of the phi 80 DNA at the left arm was replaced by E. coli DNA containing the su3+ gene, equivalent to about 53.8% phi 80 unit in length. The phi 80sus2psu3+/phi 80dsu3+ heteroduplex appears as a double-stranded molecule that bifurcates into two clearly visible single-stranded regions, rejoins, bifurcates, and rejoins again. The middle double-stranded stretches of 6.7% phi 80 unit correspond to the E. coli DNA inserted in phi 80sus2psu3+. Therefore the transducing fragment carried by phi 80sus2psu3+ originates from the inside region of the transducing fragment of defective phage phi 80dsu3+ by at least two illegitimate recombination events.     

Comparative characteristics of antigenic conversion induced by Shigella flexneri and Escherichia coli 0129 phages

The interaction of S. flexneri converting phages PE5, P90 and fV with E. coli antigenic variant O129, E. coli O129 converting phage VB with the above antigenic variant and with S. flexneri y-variant was studied. Phage PE5 and phage VB were found to induce the conversion of O-antigen in E. coli antigenic variant 0129 and in S. flexneri y-variant with the detection of antigens V and 7,8. Phages P90 and fV induced no conversion of O-antigen. Changes in the antigenic properties of convertants were confirmed by the results obtained in the agglutination test and in the agglutination adsorption test.     

DNA targets for the Escherichia coli K restriction system analysed genetically in recombinants between phages phi80 and lambda

Summary Genetic analyses demonstrate the segregation of three targets for the K restriction system in h ⁸⁰ i hybrid phages. Mutations in each of these three targets have been isolated and shown to confer resistance in cis but not in trans. Two of the three targets (sk-1 and sk-2) have been located on the genome: sk-1 is right of gene R and sk-2 is between genes cIII and N. The third target is in the phi80 genome right of, but close to, att. Phage lacking both sk-1 and sk-2 retains at least 3 targets for the K restriction system.     

Lysogenic conversion of staphylococcal lipase is caused by insertion of the bacteriophage L54a genome into the lipase structural gene.

Staphylococcus aureus PS54 manifests no lipase (geh) activity. This is due to the insertion of bacteriophage L54a DNA into the geh structural gene. The nucleotide sequence of this 2,968-base-pair DNA fragment was determined. Lipase deduced from the nucleotide sequence is a polypeptide of 690 amino acids which extends from nucleotide 706 to 2776.     

Characterization and cloning of gene 5 of Bacillus subtilis phage phi 29

Sequencing of the phi 29 DNA region [open reading frames (ORFs) 12, 11 and 10] between genes 6 and 4 of the mutant ts5(219) showed that a G in the wild-type phage had been changed to an A in the mutant at position 218 of ORF 10 indicating that this ORF corresponds to gene 5. ORF 10 was cloned in plasmid pPLc28 under the control of the PL promoter of phage lambda and, after heat induction of the Escherichia coli cells carrying the recombinant plasmid pGM26, a 12-kDa protein was overproduced, accounting for about 5% of the de novo synthesized protein. Introduction of a nonsense mutation in ORF 10 indicated that the latter codes for the 12-kDa protein. The predicted secondary structure, the hydrophilicity values and the antigenic regions of protein p5 are discussed.     

Analysis of bacteriophage phi X174 gene A protein-mediated termination and reinitiation of phi X DNA synthesis. I. Characterization of the termination and reinitiation reactions

The phi X174 (phi X) gene A protein-mediated termination and reinitiation of single-stranded circular (SS(c] phi X viral DNA synthesis in vitro were directly and independently analyzed. Following incubation together with purified DNA replication enzymes from Escherichia coli, ATP, [alpha-32P]dNTPs, and either the phi X A protein and phi X replicative form I (RF I) DNA, or the purified RF II X A complex, the phi X A protein was detected covalently linked to newly synthesized 32P-labeled DNA. Formation of the phi X A protein-[32P]DNA covalent complex required all the factors necessary for phi X (+) SS(c) DNA synthesis in vitro. Thus, it was a product of the reinitiation reaction and an intermediate of the replication cycle. Identification of this complex provided direct evidence that reinitiation of phi X (+) strand DNA synthesis involved regeneration of the RF II X A complex. Substitution of 2',3'-dideoxyguanosine triphosphate (ddGTP) for dGTP in reaction mixtures resulted in the formation of covalent phi X A protein 32P-oligonucleotide complexes; these complexes were trapped analogues of the regenerated RF II X A complex. They could not act catalytically due to the presence of ddGMP residues at the 3'-termini of the oligonucleotide moieties. Reaction mixtures containing ddGTP also yielded nonradioactive (+) SS(c) DNA products derived from circularization of the displaced (+) strand of the input parental template DNA. The formation of the phi X A protein-32P-oligonucleotide complexes and nonradioactive (+) SS(c) DNA were used to assay both reinitiation and termination reactions, respectively. Both reactions required DNA synthesis from the 3'-hydroxyl primer at nucleotide residue 4305 which was formed by cleavage of phi X RF I DNA by the phi X A protein. Elongation of this primer by 18, but not 11 nucleotides was sufficient to support each reaction. Reinitiation reactions proceeded rapidly and were essentially complete after 90 s. In contrast, when ddGTP was replaced with dGTP in reaction mixtures, DNA synthesis proceeded with linear kinetics for up to 10 min. These results suggested that in the presence of all four dNTPs, active templates supported more than 40 rounds of DNA synthesis.     

A novel DNA polymerase induced by Bacillus subtilis phage phi 29.

A novel DNA polymerase induced by Bacillus subtilis bacteriophage phi 29 has been identified. This polymerase can be separated from host DNA polymerase, by fractionation of extracts prepared from phage infected cells, using phosphocellulose chromatography. The isolated polymerase prefers poly(dA)oligo(dT) as template. The DNA polymerase isolated from the cells infected with a gene 2 temperature sensitive mutant (ts2) showed greater heat-lability than that induced by wild type phi 29. The ts2 DNA polymerase was also thermolabile for its activity in the formation of a covalent complex between phi 29 terminal protein and dAMP, the initiation step of phi 29 DNA replication. These findings indicate that gene 2 is the structural gene for a phi 29 DNA polymerase required for the complex formation step of DNA initiation.     

Identification of related genes in phages phi 80 and P22 whose products are inhibitory for phage growth in Escherichia coli IHF mutants.

Bacteriophage lambda grows in both IHF+ and IHF- host strains, but the lambdoid phage phi 80 and hybrid phage lambda (QSRrha+)80 fail to grow in IHF- host strains. We have identified a gene, rha, in the phi80 region of the lambda(QSRrha+)80 genome whose product, Rha, inhibits phage growth in an IHF- host. A search of the GenBank database identified a homolog of rha, ORF201, a previously identified gene in phage P22, which similarly inhibits phage growth in IHF- hosts. Both rha and ORF201 contain two possible translation start sites and two IHF binding site consensus sequences flanking the translation start sites. Mutations allowing lambda (QSRrha+)80 and P22 to grow in IHF- hosts map in rha and ORF201, respectively. We present evidence suggesting that, in an IHF+ host, lambda(QSRrha+)80 expresses Rha only late in infection but in an IHF- host the phage expresses Rha at low levels early in infection and at levels higher than those in an IHF+ host late in infection. We suspect that the deregulation of rha expression and, by analogy, ORF201 expression, is responsible for the failure of phi80, lambda(QSRrha+)80, and P22 to grow in IHF mutants.