Structure of ends of linear plasmid N15]

The complete structure of the pins on the ends of the linear plasmid N15 (telomers) has been defined for procaryotic organisms for the first time. The ends of the plasmid DNA contain the short nonideal inverted repeats (the size of 28 nucleotide bps) differing in only two nucleotide pairs.     

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.     

Characterization of the primary immunity region of the Escherichia coli linear plasmid prophage N15.

N15 is the only bacteriophage of Escherichia coli known to lysogenize as a linear plasmid. Clear-plaque mutations lie in at least two regions of the 46-kb genome. We have cloned, sequenced, and characterized the primary immunity region, immB. This region contains a gene, cB, whose product shows homology to lambdoid phage repressors. The cB3 mutation confers thermoinducibility on N15 lysogens, consistent with CB being the primary repressor of N15. Downstream of cB lies the locus of N15 plasmid replication. Upstream of cB lies an operon predicted to encode two products: one homologous to the late repressor of P22 (Cro), the other homologous to the late antiterminator of phi 82 (Q). The Q-like protein is essential for phage development. We show that CB protein regulates the expression of genes that flank the cB gene by binding to DNA at symmetric 16-bp sites. Three sites are clustered upstream of cB and overlap a predicted promoter of the cro and Q-like genes as well as two predicted promoters of cB itself. Two sites downstream of cB overlap a predicted promoter of a plasmid replication gene, repA, consistent with the higher copy number of the mutant, N15cB3. The leader region of repA contains terminators in both orientations and a putative promoter. The organization of these regulatory elements suggests that N15 plasmid replication is controlled not only by CB but also by an antisense RNA and by a balance between termination and antitermination.     

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.     

PY54, a linear plasmid prophage of Yersinia enterocolitica with covalently closed ends

PY54 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 tel RL). 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.     

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.     

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.     

Structure of the ends of the coliphage N4 genome

The coliphage N4 genome, a linear and double-stranded DNA of approximately 72,000 bases in length, has unique (non-permuted) direct terminal repeats of 390 to 440 base-pairs in length with 3' extensions. The very terminal sequences were determined by the Maxam-Gilbert method after 5' or 3' labeling, while sequences of internal fragments were determined by the dideoxy chain terminator method after cloning them onto M13 phage DNA. The left end of the N4 genome is relatively precise at its 5' terminus, while microheterogeneity of length exists at the 3'-terminal extensions. The predominant species had a 5 or 6 base 3' protruding sequence, 3' CATAA or 3' CATAAA. On the other hand, the right end is variable; there are at least six discrete ends differing from each other by approximately ten base-pairs and giving rise to the variability of the length of the terminal repeats. Each of the six discrete ends has a microheterogeneity of length, especially at the 3' termini. These properties of the terminal redundancy are discussed in conjunction with the mechanism whereby N4 DNA is replicated and processed.     

Independence of bacteriophage N15 lytic and linear plasmid replication from the heat shock proteins DnaJ, DnaK, and GrpE.

The chromosome of the temperate bacteriophage N15 replicates as a linear plasmid with covalently closed ends (or hairpins) when it forms a lysogen. I found that, in contrast to the cases for lambda and the low-copy-number plasmids F and P1, both phage and plasmid replication of N15 are independent of the heat shock proteins DnaJ, DnaK, and GrpE.     

Partition of the linear plasmid N15: interactions of N15 partition functions with the sop locus of the F plasmid

A locus close to one end of the linear N15 prophage closely resembles the sop operon which governs partition of the F plasmid; the promoter region contains similar operator sites, and the two putative gene products have extensive amino acid identity with the SopA and -B proteins of F. Our aim was to ascertain whether the N15 sop homologue functions in partition, to identify the centromere site, and to examine possible interchangeability of function with the F Sop system. When expressed at a moderate level, N15 SopA and -B proteins partly stabilize mini-F which lacks its own sop operon but retains the sopC centromere. The stabilization does not depend on increased copy number. Likewise, an N15 mutant with most of its sop operon deleted is partly stabilized by F Sop proteins and fully stabilized by its own. Four inverted repeat sequences similar to those of sopC were located in N15. They are distant from the sop operon and from each other. Two of these were shown to stabilize a mini-F sop deletion mutant when N15 Sop proteins were provided. Provision of the SopA homologue to plasmids with a sopA deletion resulted in further destabilization of the plasmid. The N15 Sop proteins exert effective, but incomplete, repression at the F sop promoter. We conclude that the N15 sop locus determines stable inheritance of the prophage by using dispersed centromere sites. The SopB-centromere and SopA-operator interactions show partial functional overlap between N15 and F. SopA of each plasmid appears to interact with SopB of the other, but in a way that is detrimental to plasmid maintenance.     

The SXT conjugative element and linear prophage N15 encode toxin-antitoxin-stabilizing systems homologous to the tad-ata module of the Paracoccus aminophilus plasmid pAMI2

A group of proteic toxin-antitoxin (TA) cassettes whose representatives are widely distributed among bacterial genomes has been identified. These cassettes occur in chromosomes, plasmids, bacteriophages, and noncomposite transposons, as well as in the SXT conjugative element of Vibrio cholerae. The following four homologous loci were subjected to detailed comparative studies: (i) tad-ata from plasmid pAMI2 of Paracoccus aminophilus (the prototype of this group), (ii) gp49-gp48 from the linear bacteriophage N15 of Escherichia coli, (iii) s045-s044 from SXT, and (iv) Z3230-Z3231 from the genomic island of enterohemorrhagic Escherichia coli O157:H7 strain EDL933. Functional analysis revealed that all but one of these loci (Z3230-Z3231) are able to stabilize heterologous replicons, although the host ranges varied. The TA cassettes analyzed have the following common features: (i) the toxins are encoded by the first gene of each operon; (ii) the antitoxins contain a predicted helix-turn-helix motif of the XRE family; and (iii) the cassettes have two promoters that are different strengths, one which is located upstream of the toxin gene and one which is located upstream of the antitoxin gene. All four toxins tested are functional in E. coli; overexpression of the toxins (in the absence of antitoxin) results in a bacteriostatic effect manifested by elongation of bacterial cells and growth arrest. The toxins have various effects on cell viability, which suggests that they may recognize different intracellular targets. Preliminary data suggest that different cellular proteases are involved in degradation of antitoxins encoded by the loci analyzed.     

The Bacillus thuringiensis linear double-stranded DNA phage Bam35, which is highly similar to the Bacillus cereus linear plasmid pBClin15, has a prophage state

Bam35, a 15-kbp double-stranded DNA phage, infects Bacillus thuringiensis. Recently, sequencing of the related Bacillus cereus revealed a 15.1-kbp linear plasmid, pBClin15. We show that pBClin15 closely resembles Bam35 and demonstrate conversion of Bam35 to a prophage. This state is common, as several B. thuringiensis strains release Bam35-related viruses.     

Evidence for circular permutation of the prophage genome of Bacillus subtilis bacteriophage phi 105.

Analysis of DNA extracted from Bacillus subtilis lysogenic for bacteriophage phi 105 was performed by restriction endonuclease digestion and Southern hybridization using mature phi 105 DNA as a probe. The data revealed that the phi 105 prophage is circularly permuted. Digests using the enzymes EcoRI, SmaI, PstI, and HindIII localized the bacteriophage attachment site (att) to a region 63.4 to 65.7% from the left end of the mature bacteriophage genome. The phi 105 att site-containing SmaI C, PstI J, and HindIII L fragments were not present in digests of phi 105 prophage DNA. phi 105-homologous "junction" fragments were visualized by probing digests of prophage DNA with the purified PstI J fragment isolated from the mature bacteriophage genome. The excision of the phi 105 prophage was detected by observing the appearance of the mature PstI J fragment and the concomitant disappearance of a junction fragment during the course of prophage induction.     

The genome localization of the plasmid pAP27 fin N system]

Plasmid transfer genetic regulatory system fin N of F-like plasmid pAP27 have been localized on BamHI-restriction fragment f3 (length 8.7 kb). Plasmid pUC19 Fin-activity have been cleared up and characterized from the point of view of its specificity of action on some plasmid transfer functions.     

Viral R plasmid Rphi6P: properties of the penicillinase plasmid prophage and the supercoiled, circular encapsidated genome.

Properties of the viral R plasmid Rphi6P are described. As a temperate bacteriophage, it plaques on the facultative phototroph Rhodopseudomonas sphaeroides. Under aerobic conditions the phage had a latent period of 180 min, a burst time of 200 min, and a burst size of 15 to 20 particles per infective center. The encapsidated viral genome occurred as a supercoiled, circular DNA duplex with a mean contour length of 16.5 +/- 10 micron. Percent guanine plus cytosine, as calculated from thermal denaturation profiles, was 63.5. Mitomycin C-induced loss of the prophage suggested an extrachromosomal location in the host cell. Use of this curing agent enabled the isolation of a plasmid-free strain of R. sphaeroides. Biophysical analysis of the plasmid-free strain lysogenized with Rphi6P confirmed that the prophage occurred as a plasmid in the host cell.     

Cloning of both ends and the thermo-inducible genes A and B of bacteriophage Mu on a multicopy plasmid

Employing the recombinant DNA technique, two hybrid plasmids were constructed carrying both ends of bacteriophage Mu DNA in two different orientations. The expression of the early Mu genes located on these plasmids is thermo-inducible.