No abstract

SummaryPY54 is a temperate phage isolated from Yersinia enterocolitica . Lysogenic Yersinia strains harbour the PY54 prophage as a plasmid (pY54). The plasmid has the same size (46 kb) as the PY54 genome isolated from phage particles. By electron microscopy, restriction analysis and DNA sequencing, it was demonstrated that the phage and the plasmid DNAs are linear, circularly permuted molecules. Unusually for phages of Gram-negative bacteria, the phage genome has 3 ¢ ¢ ¢ ¢ -protruding ends. The linear plasmid pY54 has covalently closed ends forming telomere-like hairpins. The equivalent DNA sequence of the phage genome is a 42 bp perfect palindrome. Downstream from the palindrome, an open reading frame (ORF) was identified that revealed strong DNA homology to the telN gene of Escherichia coli phage N15 encoding a protelomerase. Similar to PY54, the N15 prophage is a linear plasmid with telomeres. The N15 protelomerase has cleaving/joining activity generating the telomeres by processing a 56 bp palindrome (telomere resolution site telRL ). To study the activity of the PY54 protein, the telN -like gene was cloned and expressed in E. coli . A 77 kDa protein was obtained and partially purified. The protein was found to process recombinant plasmids containing the 42 bp palindrome. Telomere resolution of plasmids under in vivo conditions was also investigated in Yersinia infected with PY54. Processing required a plasmid containing the palindrome as well as adjacent DNA sequences from the phage including an additional inverted repeat. Regions on the phage genome important for plasmid maintenance were defined by the construction of linear and circular miniplasmid derivatives of pY54, of which the smallest miniplasmid comprises a 4.5 kb DNA fragment of the plasmid prophage.

Yersinia strains frequently harbor plasmids, of which the virulence plasmid pYV, indigenous in pathogenic strains, has been thoroughly characterized during the last decades. Yet, it has been unknown whether the nonconjugative pYV can be transferred by helper plasmids naturally occurring in this genus. We have isolated the conjugative plasmids pYE854 (95.5 kb) and pYE966 (70 kb) from a nonpathogenic and a pathogenic Yersinia enterocolitica strain, respectively, and demonstrate that both plasmids are able to mobilize pYV. The complete sequence of pYE854 has been determined. The transfer proteins and oriT of the plasmid reveal similarities to the F factor. However, the pYE854 replicon does not belong to the IncF group and is more closely related to a plasmid of gram-positive bacteria. Plasmid pYE966 is very similar to pYE854 but lacks two DNA regions of the larger plasmid that are dispensable for conjugation.

The objective of this study was to determine whether the temperate Yersinia enterocolitica phage PY54 may interact with the related Escherichia coli phage N15 during both the lysogenic and the lytic cycle in the same cell. The PY54 and N15 prophages are linear plasmids which have been shown to be compatible and stably replicating in E. coli and Yersinia. In E. coli, the PY54 prophage does not restrict N15 propagation. In contrast, N15 reduces by use of its cor gene the susceptibility of Yersinia strains to PY54. Doubly lysogenic E. coli strains release PY54 virions, some of which apparently contain the N15 genome. Further experiments with replicative miniplasmid derivatives of PY54, N15, and the related Klebsiella oxytoca phage KO2 demonstrated that the KO2 and N15 plasmid prophages belong to the same incompatibility group.Escherichia coli phage N15 (16), PY54 isolated from Yersinia enterocolitica (10), and the Klebsiella oxytoca phage KO2 (5) are the only known temperate phages whose prophages are linear plasmids with terminal hairpins (telomeres). Virions of the three phages contain a linear genome with cohesive ends. Each phage and its respective plasmid prophage are circularly permuted molecules with sizes of 46.3 kb (N15 and PY54) or 51.6 kb (KO2). Sequence analyses revealed similar genome organizations of the three phages (5). The left arm of the genomes carries mainly genes for virion assembly and plasmid partitioning (sop and spy). The right arm contains the protelomerase gene (tel) essential for the generation of the telomeres, a large gene encoding a multifunctional replication protein (RepA), immunity and anti-immunity genes (e.g., cB, cro, and antA), and genes contributing to host cell lysis. Overall the left arm of PY54 is more closely related to KO2 than to N15, whereas N15 and KO2 show the strongest similarities in the right arm. As the genomes of all three phages are mosaically related to and due to homologies to lambda-like phages, it has been suggested to include the telomere phages as a subgroup of the lambdoid phage family (5). Members of this family can interact with each other or with unrelated phages. Phage P22, for example, encodes an antirepressor (Ant) which is capable of inducing resident lambdoid phages by neutralizing the repressor already present in the cell (3). On the other hand, lambda rex genes prevent the growth of rII mutants of phage T4 (2), whereas the HK022 transcription termination factor Nun prematurely terminates early transcripts (13). Besides these interactions, some lambdoid phages can easily recombine with each other, producing functional hybrids (7). Campbell and Botstein (4) defined lambdoid phages as being capable of productive genetic recombination with . Horizontal gene exchange is therefore suggested to be a major component of evolution for these viruses (8).In this work, we studied possible interactions between the telomere phages during their life cycles. The experiments were carried out to answer the question whether the detected DNA similarities between the...

Simian virus 40 (SV40) is capable of inducing cellular DNA synthesis in permissive and nonpermissive cells. Utilizing flow cytometry, we analyzed the DNA content changes in two diploid human cell strains and two monkey cell lines. The osteogenesis imperfecta (OI) human skin fibroblasts were induced into DNA synthesis, and within one to two cell generations, a polyploid cell population was produced. With WI-38 phase II cells, a similar pattern of increased cycling of cells into DNA synthesis was observed; however, the majority (approximately 60%) of the cells were blocked in the G2 + M phase of the cell cycle. At later time intervals, an increase in the G1 population was demonstrated. The two monkey cell lines responded to SV40 virus with an accumulation of cells in the G2 + M phase of the cell cycle. Thus, two diploid human cell strains exhibited different cell cycle kinetics early after infection with SV40 virus. The one strain (WI-38) behaved similarly to the two monkey cell lines studied. The other strain (OI) responded similarly to nonpermissive (transforming) cells infected with SV40 virus.