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.     

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 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.     

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.     

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.     

Structure of a region in the genome of bacteriophage N15, necessary for formation of hairpins at ends of the linear plasmid prophage]

A fragment containing telRL site of bacteriophage N15 has been cloned in the vector plasmid pUC19. The nucleotide sequence of a small region from EcoRV-PstI fragment has been defined by Maxam-Gilbert technique. The analysis of the obtained sequence has shown the telRL site to be a nonideal palindrome (the size of 56 nucleotide ops) in which two nucleotide pairs differ in the positions 12 and 14 on both sides of the palindrome centre. The DNA region with alteration of purines and pyrimidines (GC) surrounded by AT-rich regions: 5'-ATTATACGCGCGTATAAT-3'--in the symmetry centre of palindrome is characteristic of the telRL site structure. This characteristic of the region may play a key role in recognition of the site by the specific enzyme at formation of linear prophage-plasmid during lysogenization.     

Microscale method for rapid isolation of covalently closed circular plasmid DNA from group N streptococci

A method for rapid purification of plasmid DNA from lactic streptococci, utilizing microliter quantities of reagents, was developed by combination of a short lysozyme-mutanolysin cell wall digestion with a modification of the Escherichia coli plasmid isolation procedure of McMaster et al. (Anal. Biochem. 109:47-54, 1980). The preparations obtained were highly enriched for covalently closed circular DNA, and the method was applicable to plasmids of at least 40 megadaltons. Centrifugation in CsCl-ethidium bromide density gradients was not required.     

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.     

Microscale method for rapid isolation of covalently closed circular plasmid DNA from group N streptococci.

A method for rapid purification of plasmid DNA from lactic streptococci, utilizing microliter quantities of reagents, was developed by combination of a short lysozyme-mutanolysin cell wall digestion with a modification of the Escherichia coli plasmid isolation procedure of McMaster et al. (Anal. Biochem. 109:47-54, 1980). The preparations obtained were highly enriched for covalently closed circular DNA, and the method was applicable to plasmids of at least 40 megadaltons. Centrifugation in CsCl-ethidium bromide density gradients was not required.     

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.     

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 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.     

Genomic replacement in Escherichia coli K-12 using covalently closed circular plasmid DNA

A number of gene replacements at different loci were constructed using covalently closed circular (ccc) plasmid DNA in the recB21 recC22 sbcB15 sbcC201 mutant of Escherichia coli (JC7623). Selected constructs representing deletions and insertion mutations formed from double-crossover events involving the ccc plasmid molecules and the genome were confirmed by Southern blots, and the frequency of double-crossover events was evaluated. It is reported that such mutants may be constructed without linearizing plasmid DNA, as described previously.     

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.     

An improved method for rapid purification of covalently closed circular plasmid DNA over a wide size range

An improved method has been developed for the large-scale purification of covalently closed circular (CCC) plasmid DNA molecules of sizes ranging from 4·3 to 73 kb. This protocol uses an alkaline-lysis procedure followed by acid-phenol extraction but with several modifications to previously reported methods. The principal modification is the replacement of NaCl by MgCl₂ in the extraction buffer to improve yield and to remove chromosomal and other non-CCC plasmid DNA. Plasmid DNA can be purified in less than 1 h and used successfully in restriction enzyme analysis and cloning experiments.     

A simple method for the preparation of the covalently closed circular form of plasmid DNA

We describe a simple, rapid, inexpensive method for isolation of covalently closed circular plasmid DNA. The method involves the electrophoresis of crude DNA preparations in an agarose gel, electrotransfer onto a dialysis membrane and elution of the highly purified circular covalently closed plasmid DNA. Native and recombinant plasmid DNA have been purified by this method and shown to be suitable for restriction enzyme digestion and transformation of bacteria. The yield of this rapid purification procedure makes it a good alternative method to standard centrifugation in cesium chloride ethidium bromide gradients.     

Analysis of linear plasmids isolated from Streptomyces: association of protein with the ends of the plasmid DNA

Linear DNA plasmids, designated pSLA1 and pSLA2, which were isolated from two strains of Streptomyces sp. producing lankacidin group antibiotics, were analyzed by using restriction endonucleases. Cleavage patterns of these plasmids were very similar, indicating that they are closely related. Cleavage maps of pSLA2 were constructed with BamHI, SalI, BglII, and EcoRI. A protein was associated with the restriction fragments of both ends of pSLA2 and this was not removed by sodium dodecyl sulfate-phenol treatment. pSLA2 was senstive to a 3′-exonuclease, exonuclease III, but was resistant to the 5′-exonuclease, λ-exonuclease. These results suggest that the protein is associated with the 5′ termini of pSLA2. The same terminal structure was also found on pSLA1. The approximate copy number of the plasmid was estimated by a brief method using agarose gel electrophoresis.     

Yeast episome: oligomycin resistance associated with a small covalently closed non-mitochondrial circular DNA

We have isolated a single step spontaneous mutant of S. cerevisiae resistant simultaneously to oligomycin, venturicidin, chloramphenicol, cycloheximide and triethyltin. This multiple drug resistance results from the interaction of two genetic factors showing both chromosomal location and episomal characteristics. One factor (π) confers oligomycin resistance, the other (τ) confers the other resistances. π can be lost spontaneously while τ can be completely eliminated with ethidium bromide. All π⁺ strains, whether grande or petite, τ⁺ or τ^?, carry a covalently closed circular DNA while π^? strains are devoid of it. We hypothesise that this circular DNA may play an informational role in the biogenesis and/or function of membranes.