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.

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).

[Characteristics of the bacteriophage N15 lysogenic conversion gene and identification of its product]. / Kharakteristika gena lizogennoi konversii bakteriofaga N15 i identifikatsiia ego produkta.

The plasmids containing EcoRV fragments of N15 phage DNA and inhibiting the adsorption of T1, phi 80 and N15 phages were selected and characterized. The N15 lysogenic conversion gene (cor) was mapped in the SalI-PstI fragment of 700 bp in length which is localized near SalI site with the coordinates 40.1 kb on the N15 plasmid prophage DNA physical map. The cor gene was recloned on a multicopy vector in the both possible orientations and its expression was shown to occur most probably under control of its own promoter in the direction from SalI to the ClaI site of the SalI-PstI fragment. The molecular weight of the Cor protein (7-9 kD) was determined by the analysis in the system of mini-cells. Initiation of transcription to wards the cor gene from the external promoter led to the bacteriostatic effect.

[Isolation, characterization and mapping of the N15 phasmid insertion mutations]. / Poluchenie, kharakteristika i kartirovanie insertsionnykh mutatsii fazmidy N15.

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).

[Construction of linear plasmid vectors for cloning in Escherichia coli cells]. / Konstruirovanie lineinykh plazmidnykh vektorov dlia klonirovaniia v kletkakh Escherichia coli.

Prophage of the temperate coliphage N15 is a linear plasmid with covalently closed ends. The central part of plasmid N15 genome responsible for vegetative phage growth was replaced by DNA fragments containing genes for selective markers which have unique restriction sites. As a result a family of linear plasmid vectors was constructed. Their size is about 20 kb and their capacity is comparable with that of cosmid vectors.

[Structure of ends of linear plasmid N15]. / Struktura kontsov lineinoi plazmidy 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.

[Structure of a region in the genome of bacteriophage N15, necessary for formation of hairpins at ends of the linear plasmid prophage]. / Struktura oblasti v genome bakteriofaga N15, neobkhodimaia dlia obrazovaniia shpilek na kontsakh lineinogo plazmidnogo profaga.

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.

[Lysogenic conversion caused by phage phi 80. III. The mapping of the conversion gene and additional characterization of the phenomenon]. / Lizogennaia konversiia, vyzyvaemaia fagom phi 80. Soobshchenie III. Utochnenie lokalizatsii gena konversii i dopolnitel'naia kharakteristika iavleniia.

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.

[Physical mapping of the immunity region of phage phi 80]. / Fizicheskoe kartirovanie oblasti immuniteta faga phi 80.

Mapping of the sites of cleavage of five restriction endonucleases (BglI, BglII, EcoRV, KpnI and PvuII) in the immunity region of bacteriophage phi 80 DNA is described. The order of the 27 restriction sites was established. This helped to localize the phi 80 cI gene within the 640 bp fragment of the immunity region. Recombinant plasmids carrying phi 80 "kil" function were constructed. The function is suppressed by the cI-coded repressor. The deletion AB43 which is present in the phi 80 vir mutant is located within the phi 80 DNA fragment carrying the cI gene.

[The role of genes of the system of mismatched base correction in genetic recombination of Escherichia coli]. / Ob uchastii genov sistemy korrektsii nesparennykh osnovanii v geneticheskoi rekombinantsii E. coli.

A genetic system was elaborated to study intramolecular recombination of bacteriophages lambda and phi 80. Practically, the ideal complementation of nucleotide sequences in recombining DNA molecules is required to obtain recombinants resulting from RecA-dependent recombination in Escherichia coli cells. a hypothesis is proposed to which the correction of mismatched bases hinders recombinant formation during recombination of fully homologous DNAs. The increased yields of hybrid molecules during interaction of the same DNA in the cells with deficient genes for correction support the hypothesis as well as independent demonstration of mutation in a gene for correction according to the effect of the increased yield of recombinants. A series of Escherichia coli cells mutants with increased formation of recombinant clones has been obtained.

[Mapping of genes 14 and 16 of phage phi 80]. / Kartirovanie genov 14 i 16 faga fi 80.

This study was performed to obtain more detailed information on the early region of phage phi 80. For this purpose, a selective method for isolation of early phage ts mutants was developed. 32 ts mutants of phi 80 obtained and early sus mutants isolated by Sato were characterized by complementation tests and deletion mapping. The results of this study differ from those of Sato in two aspects. Firstly, it was shown that sus mutations 250, 258 and 326 of phi 80 define one gene 16, and that gene 15 previously determined by Sato via sus 326 mutation does not exist. Secondly, we found that the order of the phi 80 early genes 14 and 16 is cI-16-14, contrary to the Sato data (cI-14-16).

Genetics of bacteriophage phi 80--a review.

The genetic maps of bacteriophage lambda and lambdoid phage phi 80 are compared. The gene organization of phi 80 is very similar to that of lambda, as shown by isolation and characterization of many am, ts and c (clear) mutants of the phage. In general, the essential genes located in the same position on the genetic map of the phages lambda and phi 80 fulfill the same functions. These include the gene clusters coding for the head and tail proteins, genes for DNA synthesis, and the genes controlling lysogeny and late gene expression. The specific regulatory features of phi 80 in relation to the N function of lambda are discussed, but they require further clarification. The two phages differ in immunity specificity, host range, conversion property and temperature sensitivity.

[Lysogenic conversion induced by phage phi 80. II. Mapping of the cor locus]. / Lizogennaia konversiia, vyzyvaemaia fagom phi 80. Soobshchenie II. Kartirovanie lokusa cor.

The cor locus determines lysogenic conversion of the lambdoid phi 80 phage. The cor locus was cloned from phi 80, phi 80pt1, phi 80pt0, phi 80pt4A and lambda hy51 DNAs in the pBR322 plasmid. According to the physical and genetic data, the cor locus was located on the genetic map of phi 80 bacteriophage at the 40.3 divided by 43.5% lambda. It is closely linked to the gene 13 of phi 80 phage and has a position similar to that of lom gene of lambda phage.

[Experiments with Bacillus thuringiensis protoplasts. I. Isolation of protoplasts and their reversion to bacillary form]. / Eksperimenty s protoplastami Bacillus thuringiensis. Soobshchenie I. Poluchenie protoplastov i ikh reversiia v batsilliarnuiu formu.

A method for protoplastization of crystal- and spore-forming Bacillus thuringiensis bacterian and consequent cell wall regeneration on a solid hypertonic medium is presented. Up to 50% of the protoplasts prepared were viable and formed colonies under special conditions; at the same time, less than 0,01% of the cells treated with lysozyme were resistant to the osmotic shock; bacterial autolytic system takes part in protoplasts formation. Electron microscopic studies of protoplasts and cells confirm the fact of cell wall removal and support the proposed mechanism of protoplast formation.

[Experiments with Bacillus thuringiensis protoplasts. II. Study of the potential interspecies fusion of bacterial protoplasts: Bacillus thuringiensis and Bacillus megaterium]. / Eksperimenty s protoplastami Bacillus thuringiensis. Soobshchenie II. Issledovanie vozmozhnosti mezhvidovogo sliianiia bakterial'nykh protoplastov: Bacillus thuringiensis i Bacillus megaterium.

In the present study the possibility of obtaining interspecific bacterial hybrids by polyethylene glycol-assisted fusion of protoplasts from Bacillus thuringiensis and Bac. megaterium has been examined. Electron microscopic and genetic data allow to confirm with great probability that cytological fusion takes place. However, genetic analysis revealed that neither of methods applied for protoplast fusion gave stable recombinants. Apparently, it is due to the lack of recombination or the death of recombinants that follows the functioning of the cell correction system. The mechanism of protoplast fusion and its most important steps are also studied in the present work.

[Lysogenic conversion induced by phages phi 80. I. A description of the phenomenon and the cloning of the conversion gene]. / Lizogennaia konversiia, vyzyvaemaia fagom phi 80. Soobshchenie I. Opisanie iavleniia i klonirovanie gena konversii.

Escherichia coli cells lysogenic for coliphage phi 80 stop adsorbing the superinfecting phi 80 phage after having been kept under anaerobic conditions for a long time, which conferred on these cells the TonA phenotype. To determine the location of the gene for lysogenic conversion (cor), BamHI fragments of phi 80 DNA were cloned in pBR322 plasmid. The cells transformed with the recombinant plasmid pDK01 = pBR322 + phi 80 BamHI fragment 1 immediately acquire the TonA phenotype. So, the cor gene(s) is contained in the central phi 80 BamHI fragment (fragment 1) which includes gene 13, the b2 region and the att site.

[Lambda H-lambda T 80 hybrid study of the DNA structural gene region in lambda and phi 80 phages]. / Izuchenie oblasti strukturneykh genov DNK fagov lambda i fi 80 s pomoshch'iu gibridov lambdaH-lambdaT80.

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.

[Restrictase BamHI, HindII, EcoRI and SmaI mapping of hybrid lambda-phi 80 phages with recombination in the structural gene region]. / Kartirovanie restriktazami BamHI, HindII, EcoRI i SmaI gibridnykh fagov lambda-fi 80 s rekombinatsiei v oblasti strukturnykh genov.

Hybrids lambda H lambda T80 with recombination in the region of structural genes have lambda head and phi 80 tail genes. In this paper the molecular structure of 5 independently isolated hybrids was established using restriction endonucleases. It has been shown that all of them have a recombinant head or tail. A deletion of 4,8% lambda was demonstrated in the immunity region of phi 80vir phage. Co-ordinates of restriction sites for BamHI, HindIII, EcoRI and SmaI restriction endonucleases on phi 80 DNA were calculated.