N15: the linear phage-plasmid

The lambdoid phage N15 of Escherichia coli is very unusual among temperate phages in that its prophage is not integrated into chromosome but is a linear plasmid molecule with covalently closed ends. Upon infection the phage DNA circularises via cohesive ends, then phage-encoded enzyme, protelomerase, cuts at an inverted repeat site and forms hairpin ends (telomeres) of the linear plasmid prophage. Replication of the N15 prophage is initiated at an internally located ori site and proceeds bidirectionally resulting in formation of duplicated telomeres. Then the N15 protelomerase cuts duplicated telomeres generating two linear plasmid molecules with hairpin telomeres. Stable inheritance of the plasmid prophage is ensured by partitioning operon similar to the F factor sop operon. Unlike F sop, the N15 centromere consists of four inverted repeats dispersed in the genome. The multiplicity and dispersion of centromeres are required for efficient partitioning of a linear plasmid. The centromeres are located in N15 genome regions involved in phage replication and control of lysogeny, and binding of partition proteins at these sites regulates these processes. Two N15-related lambdoid Siphoviridae phages, φKO2 in Klebsiella oxytoca and pY54 in Yersinia enterocolitica, also lysogenize their hosts as linear plasmids, as well as Myoviridae marine phages VP882 and VP58.5 in Vibrio parahaemolyticus and ΦHAP-1 in Halomonas aquamarina. The genomes of all these phages contain similar protelomerase genes, lysogeny modules and replication genes, as well as plasmid-partitioning genes, suggesting that these phages may belong to a group diverged from a common ancestor.     

Bidirectional replication from an internal ori site of the linear N15 plasmid prophage

The prophage of coliphage N15 is not integrated into the chromosome but exists as a linear plasmid molecule with covalently closed hairpin ends (telomeres). Upon infection the injected phage DNA circularizes via its cohesive ends. Then, a phage-encoded enzyme, protelomerase, cuts the circle and forms the hairpin telomeres. N15 protelomerase acts as a telomere-resolving enzyme during prophage DNA replication. We characterized the N15 replicon and found that replication of circular N15 miniplasmids requires only the repA gene, which encodes a multidomain protein homologous to replication proteins of bacterial plasmids replicated by a theta-mechanism. Replication of a linear N15 miniplasmid also requires the protelomerase gene and telomere regions. N15 prophage replication is initiated at an internal ori site located within repA and proceeds bidirectionally. Electron microscopy data suggest that after duplication of the left telomere, protelomerase cuts this site generating Y-shaped molecules. Full replication of the molecule and subsequent resolution of the right telomere then results in two linear plasmid molecules. N15 prophage replication thus appears to follow a mechanism that is distinct from that employed by eukaryotic replicons with this type of telomere and suggests the possibility of evolutionarily independent appearances of prokaryotic and eukaryotic replicons with covalently closed telomeres.     

Mechanisms of replication and telomere resolution of the linear plasmid prophage N15

The prophage of coliphage N15 is not integrated into the bacterial chromosome but exists as a linear plasmid molecule with covalently closed ends. Upon infection of an Escherichia coli cell, the phage DNA circularizes via cohensive ends. A phage-encoded enzyme, protelomerase, then cuts at another site, telRL, and forms hairpin ends (telomeres). Purified protelomerase alone processes circular and linear plasmid DNA containing the target site telRL to produce linear double-stranded DNA with covalently closed ends in vitro. N15 protelomerase is necessary for replication of the linear prophage through its action as a telomere-resolving enzyme. Replication of circular N15-based miniplasmids requires the only gene repA that encodes multidomain protein homologous to replication proteins of bacterial plasmids replicated by theta-mechanism, particularly, phage P4 alpha-replication protein. Replication of the N15 prophage is initiated at an internal ori site located within repA. Bidirectional replication results in formation of the circular head-to-head, tail-to-tail dimer molecule. Then the N15 protelomerase cuts both duplicated telomeres generating two linear plasmid molecules with covalently closed ends. The N15 prophage replication thus appears to follow the mechanism distinct from that employed by poxviruses and could serve as a model for other prokaryotic replicons with hairpin ends, and particularly, for linear plasmids and chromosomes of Borrelia burgdorferi.     

The plasmid prophage N15: a linear DNA with covalently closed ends

Coliphage N15 is a temperate bacteriophage whose prophage is a linear plasmid molecule with covalently closed ends (telomeres). The N15 prophage provided the first example of such DNA in prokaryotes and, up to now, it is the only known example of a linear plasmid in Escherichia coli. The linear N15 mature phage DNA has single-stranded cohesive ends. The phage and plasmid prophage DNAs are circularly permuted. The nucleotide structure of the telomere-forming site tel RL in phage DNA corresponds to the structures of the terminal hairpin loops. It suggests a unique mechanism for conversion of the circular phage DNA to the linear plasmid form, which is performed by the prokaryotic telomerase (protelomerase). The results of a comparison of the protelomerase with integrases lead us to suggest that these proteins may have evolved from a common ancestor. The mechanism of plasmid N15 replication is unknown. We propose that the protelomerase participates in linear plasmid replication, acting as a resolvase of replicative intermediates that are tail-to-tail linear dimers. The sequence analysis of the N15 DNA showed that it represents an evolutionary 'link' between plasmids F, P1, P4 and lambdoid bacteriophages.     

The protelomerase of the phage-plasmid N15 is responsible for its maintenance in linear form

The prophage of coliphage N15 is not integrated into the bacterial chromosome but exists as a linear plasmid molecule with covalently closed ends. Upon infection of an Escherichia coli cell, the phage DNA circularises via cohesive ends. A phage-encoded enzyme, protelomerase, then cuts at another site, telRL, and forms hairpin ends (telomeres). We demonstrate that this enzyme acts in vivo on specific substrates, and show that it is necessary for replication of the linear prophage. We show that protelomerase is an end-resolving enzyme responsible for processing of replicative intermediates. Removal of protelomerase activity resulted in accumulation of replicative intermediates that were found to be circular head-to-head dimers. N15 protelomerase and its target site constitute a functional unit acting on other replicons independently of other phage genes; a mini-F or mini-P1 plasmid carrying this unit replicates as a linear plasmid with covalently closed ends. Our results suggest the following model of N15 prophage DNA replication. Replication is initiated at an internal ori site located close to the left end of plasmid DNA and proceeds bidirectionally. After replication of the left telomere, protelomerase cuts this sequence and forms two hairpin loops telL. After duplication of the right telomere (telR) the same enzyme resolves this sequence producing two linear plasmids. Alternatively, full replication of the linear prophage to form a circular head-to-head dimer may precede protelomerase-mediated formation of hairpin ends.     

Expression regulation of the protelomerase gene of the bacteriophage N15

The N15 bacteriophage, when in the lysogenic state, does not integrate into the chromosome; in fact, it exists as a linear plasmid with the covalently closed ends. Upon infection, the phage DNA circularizes via its cohesive ends, after which a specific enzyme, the N15 protelomerase, cuts the circular molecule thus generating a linear plasmid with the covalently closed telomeres. Protelomerase generates, as the replication of plasmid prophage proceeds, the hairpin telomeres in replicated molecules. We identified the promoter of the protelomerase gene and demonstrated that it could be repressed presumably due to its binding with 3 tosL sites overlapping the promoter. We also found the transformation efficiency of E. coli cells of linear DNA with hairpin telomeres to be approximately 100-fold lower versus the circular DNA of the same size. At the same time, presence of the N15 prophage or of the protelomerase-expressing vector enhances, in a strain being transformed, the efficiency of its transformation by linear DNA up to a level ensured in transformation by circular plasmids. We believe that protelomerase, while binding with the hairpin telomeres, protects the latter from degradation by cellular nucleases.     

Cloning and sequence analysis of linear plasmid telomeres of the bacterium Borrelia burgdorferi

Borrelia burgdorferi, the Lyme disease agent, has double-stranded linear plasmids with covalently closed ends. DNA at the ends, or telomeres, of two linear plasmids of B. burgdorferi strain B31 was examined. Telomeric sequences from both ends of a 16 kb linear plasmid and from one end of a 49 kb linear plasmid were cloned and sequenced. An 18 bp AT-rich inverted repeat was found at each end of the 16 kb linear plasmid. The sequences of the two ends of this plasmid were different beyond these short inverted terminal repeats. The cloned end of the 49 kb linear plasmid had sequence identity with one end of the 16 kb linear plasmid. The end sequence common to both plasmids contained a series of phased, short direct repeats and a 52 bp palindrome adjacent to a highly AT-rich region. These findings indicate that Borrelia linear plasmid telomeres have structural features different from those of other known replicons.     

The pKO2 linear plasmid prophage of Klebsiella oxytoca

Temperate bacteriophages with plasmid prophages are uncommon in nature, and of these only phages N15 and PY54 are known to have a linear plasmid prophage with closed hairpin telomeres. We report here the complete nucleotide sequence of the 51,601-bp Klebsiella oxytoca linear plasmid pKO2, and we demonstrate experimentally that it is also a prophage. We call this bacteriophage phiKO2. An analysis of the 64 predicted phiKO2 genes indicate that it is a fairly close relative of phage N15; they share a mosaic relationship that is typical of different members of double-stranded DNA tailed-phage groups. Although the head, tail shaft, and lysis genes are not recognizably homologous between these phages, other genes such as the plasmid partitioning, replicase, prophage repressor, and protelomerase genes (and their putative targets) are so similar that we predict that they must have nearly identical DNA binding specificities. The phiKO2 virion is unusual in that its phage lambda-like tails have an exceptionally long (3,433 amino acids) central tip tail fiber protein. The phiKO2 genome also carries putative homologues of bacterial dinI and umuD genes, both of which are involved in the host SOS response. We show that these divergently transcribed genes are regulated by LexA protein binding to a single target site that overlaps both promoters.     

Interplay between the temperate phages PY54 and N15, linear plasmid prophages with covalently closed ends

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.     

The temperate marine phage PhiHAP-1 of Halomonas aquamarina possesses a linear plasmid-like prophage genome

A myovirus-like temperate phage, PhiHAP-1, was induced with mitomycin C from a Halomonas aquamarina strain isolated from surface waters in the Gulf of Mexico. The induced cultures produced significantly more virus-like particles (VLPs) (3.73 x 10(10) VLP ml(-1)) than control cultures (3.83 x 10(7) VLP ml(-1)) when observed with epifluorescence microscopy. The induced phage was sequenced by using linker-amplified shotgun libraries and contained a genome 39,245 nucleotides in length with a G+C content of 59%. The PhiHAP-1 genome contained 46 putative open reading frames (ORFs), with 76% sharing significant similarity (E value of <10(-3)) at the protein level with other sequences in GenBank. Putative functional gene assignments included small and large terminase subunits, capsid and tail genes, an N6-DNA adenine methyltransferase, and lysogeny-related genes. Although no integrase was found, the PhiHAP-1 genome contained ORFs similar to protelomerase and parA genes found in linear plasmid-like phages with telomeric ends. Southern probing and PCR analysis of host genomic, plasmid, and PhiHAP-1 DNA indicated a lack of integration of the prophage with the host chromosome and a difference in genome arrangement between the prophage and virion forms. The linear plasmid prophage form of PhiHAP-1 begins with the protelomerase gene, presumably due to the activity of the protelomerase, while the induced phage particle has a circularly permuted genome that begins with the terminase genes. The PhiHAP-1 genome shares synteny and gene similarity with coliphage N15 and vibriophages VP882 and VHML, suggesting an evolutionary heritage from an N15-like linear plasmid prophage ancestor.     

The Linear Plasmid Prophage Vp58.5 of Vibrio parahaemolyticus Is Closely Related to the Integrating Phage VHML and Constitutes a New Incompatibility Group of Telomere Phages

Vibrio parahaemolyticus O3:K6 pandemic strains recovered in Chile frequently possess a 42-kb plasmid which is the prophage of a myovirus. We studied the prototype phage VP58.5 and show that it does not integrate into the host cell chromosome but replicates as a linear plasmid (Vp58.5) with covalently closed ends (telomeres). The Vp58.5 replicon coexists with other plasmid prophages (N15, PY54, and ΦKO2) in the same cell and thus belongs to a new incompatibility group of telomere phages. We determined the complete nucleotide sequence (42,612 nucleotides) of the VP58.5 phage DNA and compared it with that of the plasmid prophage. The two molecules share the same nucleotide sequence but are 35% circularly permuted to each other. In contrast to the hairpin ends of the plasmid, VP58.5 phage DNA contains 5′-protruding ends. The VP58.5 sequence is 92% identical to the sequence of phage VHML, which was reported to integrate into the host chromosome. However, the gene order and termini of the phage DNAs are different. The VHML genome exhibits the same gene order as does the Vp58.5 plasmid. VHML phage DNA has been reported to contain terminal inverted repeats. This repetitive sequence is similar to the telomere resolution site (telRL) of VP58.5 which, after processing by the phage protelomerase, forms the hairpin ends of the Vp58.5 prophage. It is discussed why these closely related phages may be so different in terms of their genome ends and their lifestyle.Most temperate bacteriophages integrate into the host chromosome during lysogeny. However, there are some phages (telomere phages) whose prophages are linear plasmids with covalently closed ends. Members of this group of phages are the siphoviruses N15, PY54, and ΦKO2 isolated from Escherichia coli, Yersinia enterocolitica, and Klebsiella oxytoca, respectively, and the recently described myoviruses ΦHAP-1 of Halomonas aquamarina and VP882 of Vibrio parahaemolyticus (6, 20, 23, 26, 37). Despite their different origins (enterobacteria versus marine bacterium) and morphologies, all known telomere phages share similar genome organizations and some protein similarities. The linear DNA of each phage is a circular permutation of the respective linear plasmid prophage. For the generation of the terminal hairpins of the linear plasmid, the protelomerase (Tel) is essential (8). This enzyme has cleaving/joining activity; its target is a large palindromic DNA sequence called the telomere resolution site (telRL) located upstream of tel on the phage genome. After cleaving telRL by staggered cuts, the resulting self-complementary single-stranded DNA overhangs fold back and are rejoined by the protelomerase (9). Besides tel, all telomere phages possess the gene repA, encoding a multifunctional replication protein. repA of N15 and PY54 was shown to harbor the prophage replication origin and to function as a circular minimal replicon (35, 42). Compatibility studies demonstrated that the N15 and ΦKO2 plasmids belong to the same incompatibility group, whereas the PY54 plasmid is able to coexist with these two prophages in doubly lysogenic E. coli and Y. enterocolitica hosts (19).There are some reports on the presence of tel and repA in prophages (VP882, VHML, and Vp58.5) of marine Vibrio strains (28, 41). V. parahaemolyticus phage VP882 is a close relative of the Halomonas phage ΦHAP-1 (26). VHML was isolated from a toxin-producing Vibrio harveyi strain, pathogenic for some crustaceans and fish (30). Similarly to ΦHAP-1 and VP882, VHML has a myovirus-like morphology. The phage contains genes for products similar to Tel and RepA, suggesting that its prophage is a linear plasmid with terminal hairpins. However, it was surmised that VHML integrates into the Vibrio chromosome (28, 29). Phage VP58.5 was isolated from a V. parahaemolyticus strain belonging to the serovar O3:K6 pandemic clonal complex (41). During the last several years, this clone has been associated with many seafood-borne diarrhea outbreaks in Southeast Asia and South America, particularly Chile (5, 12, 13, 15). Up to 33% of the Chilean isolates harbored a 42-kb plasmid which was shown to be the prophage of a myovirus inducible by mitomycin C. VP58.5 is the prototype of these phages.In this work we demonstrate that VP58.5 is closely related to the V. harveyi phage VHML but that its prophage is a linear plasmid with covalently closed ends. The Vp58.5 prophage belongs to a new incompatibility group of telomere phages.     

Linear chromosome-generating system of Agrobacterium tumefaciens C58: protelomerase generates and protects hairpin ends

Agrobacterium tumefaciens C58, the pathogenic bacteria that causes crown gall disease in plants, harbors one circular and one linear chromosome and two circular plasmids. The telomeres of its unusual linear chromosome are covalently closed hairpins. The circular and linear chromosomes co-segregate and are stably maintained in the organism. We have determined the sequence of the two ends of the linear chromosome thus completing the previously published genome sequence of A. tumefaciens C58. We found that the telomeres carry nearly identical 25-bp sequences at the hairpin ends that are related by dyad symmetry. We further showed that its Atu2523 gene encodes a protelomerase (resolvase) and that the purified enzyme can generate the linear chromosomal closed hairpin ends in a sequence-specific manner. Agrobacterium protelomerase, whose presence is apparently limited to biovar 1 strains, acts via a cleavage-and-religation mechanism by making a pair of transient staggered nicks invariably at 6-bp spacing as the reaction intermediate. The enzyme can be significantly shortened at both the N and C termini and still maintain its enzymatic activity. Although the full-length enzyme can uniquely bind to its product telomeres, the N-terminal truncations cannot. The target site can also be shortened from the native 50-bp inverted repeat to 26 bp; thus, the Agrobacterium hairpin-generating system represents the most compact activity of all hairpin linear chromosome- and plasmid-generating systems to date. The biochemical analyses of the protelomerase reactions further revealed that the tip of the hairpin telomere may be unusually polymorphically capable of accommodating any nucleotide.     

Isolation, characterization and mapping of the N15 phasmid insertion mutations]

Prophage of N15 temperate bacteriophage is stably maintained in Escherichia coli lysogens as a 46.33 kb linear plasmid. Using different transposons we obtained 18 insertion mutants of the N15 plasmid prophage. They were analysed for plaque formation ability, stability of the plasmid state and lysogenic conversion. Restriction mapping of the insertions allowed us to localize on the map the regions necessary for lytic growth and to map the lysogenic conversion gene. A recombinant phage encoding two antibiotic resistance genes was obtained. The phage contains an additional 4.77 kb DNA fragment (over 10% of the N15 genome).     

An interlocked dimer of the protelomerase TelK distorts DNA structure for the formation of hairpin telomeres

The termini of linear chromosomes are protected by specialized DNA structures known as telomeres that also facilitate the complete replication of DNA ends. The simplest type of telomere is a covalently closed DNA hairpin structure found in linear chromosomes of prokaryotes and viruses. Bidirectional replication of a chromosome with hairpin telomeres produces a catenated circular dimer that is subsequently resolved into unit-length chromosomes by a dedicated DNA cleavage-rejoining enzyme known as a hairpin telomere resolvase (protelomerase). Here we report a crystal structure of the protelomerase TelK from Klebsiella oxytoca phage varphiKO2, in complex with the palindromic target DNA. The structure shows the TelK dimer destabilizes base pairing interactions to promote the refolding of cleaved DNA ends into two hairpin ends. We propose that the hairpinning reaction is made effectively irreversible by a unique protein-induced distortion of the DNA substrate that prevents religation of the cleaved DNA substrate.     

Construction and characteristics of mini-Mu phages

The paper reports on the principles of construction, physical characterization and results of preliminary genetic investigation of hybrid plasmids containing Mu DNA sequences or deletion derivatives of phage Mu, the so-called mini-Mu phages. The mini-Mu were obtained by joining both phage ends within one plasmid in a regular orientation. A collection obtained by in vitro manipulations included 14 recombinant plasmids containing different DNA fragments of the Mu genome. Seven plasmids have both ends of phage Mu, three plasmids containing regularly oriented ends, i.e. mini-phages of different size: the mini-Mu5 (11 kb) within pRM8 plasmid, the mini-Mu4 Ap (18 kb) within pRM6 and the mini-mini-Mu (4.4 kb) within pRM5. The collection comprises mini-Mu phages with the gene kil inactivated after treatment with hydroxylamine. Biological properties of the hybrid plasmids have been preliminary studied.     

Infectious circular DNA of human adenovirus type 5: regeneration of viral DNA termini from molecules lacking terminal sequences.

A series of plasmids containing the entire human adenovirus genome with viral DNA termini joined 'head to tail' has been isolated. Several plasmids were able to generate infectious virus following transfection of human cells in spite of having small deletions and rearrangements at the junctions of termini. One plasmid has lost 2 bp of DNA from one end of the viral genome and 11 bp from the other end yet produced viruses with complete wild-type sequences at both ends of the genome. We propose a model for replication of viral DNA off circular templates in which regeneration of terminal information involves translocation of primer and polymerase during initiation of DNA replication. The model suggests a novel mechanism for extension of the 5' ends of linear DNA molecules which could be applicable to chromosomal telomeres.     

Sop proteins can cause transcriptional silencing of genes located close to the centromere sites of linear plasmid N15

Stable inheritance of bacterial chromosomes and low copy number plasmids is ensured by accurate partitioning of replicated molecules between the daughter cells at division. Partitioning of the prophage of the temperate bacteriophage N15, which exists as a linear plasmid molecule with covalently closed ends, depends on the sop locus, comprising genes sopA and sopB, as well as four centromere sites in different regions of the N15 genome essential for replication and the control of lysogeny. We found that binding of SopB to the centromere could silence centromere-proximal promoters, presumably due to subsequent polymerization of SopB along the DNA. Close to the IR4 centromere site we identified a promoter, P59, which was able to drive the expression of phage late genes encoding structural proteins of virion. We found that, following binding to IR4, the N15 Sop proteins could induce repression of this promoter. The repression depended on SopB and was enhanced in the presence of SopA. Sop-dependent silencing of centromere-proximal promoters may control gene expression in phage N15, particularly preventing undesired expression of late genes in the N15 prophage. Thus, the phage N15 sop system not only ensures plasmid partitioning but is also involved in the genetic network controlling prophage replication and the maintenance of lysogeny.