SummaryColiphage N15 is a temperate bacteriophage whose prophage is a linear plasmid molecule with covalently closed ends (telomeres). The N15 prophage provided the ®rst 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.
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 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 betweem termination and antitermination.N15 is unique among known bacteriophages of Escherichia coli in that it lysogenizes cells as a linear plasmid (40). Despite this unusual mode of lysogenization, numerous properties of N15 resemble those of lambdoid phages, which, as prophages, are normally integrated into a bacterial chromosome. N15 is similar to with respect to length of the genome (46.3 kb, N15; 48.5 kb, ), morphology of phage particles and plaques, burst size, and lysogenization frequency (27). N15 phage DNA, like phage DNA, is a double-stranded molecule with singlestranded, cohesive ends (40). In both cases, phage and prophage DNAs are circularly permuted with respect to each other. Kinship of N15 and is suggested on the basis of cross-hybridization of their DNAs (29). Cells lysogenized with N15 are unable to adsorb bacteriophages N15, T1, and 80 (28). In this respect, they resemble lysogens of another lambdoid phage, 80 (15-17). This lysogenic conversion depends upon cor (Fig. 1) (20), which is homologous to the 80 gene with a similar function (44).The N15 prophage is maintained in cells as a low-copynumber plasmid and is stable (29). Its DNA is a doublestranded linear molecule with covalently closed ends (40). The ends have a telomeric structure similar to those found in the DNAs of poxviruses, African swine fever virus, mitochondria of cells of the yeast genus Pichia, and linear plasmids found in spirochetes of the genus Borrelia (11,12,18). Each telomere of N15 consists of a palindromic terminal hairpin loop and a 28-bp inverted repeat (19).Ravin and Shulga (29) isolated temperature-sensitive early muta...
Bacteriophage 80 is a member of the lambda phage family (16), and bacteriophage N15 is a temperate phage of Escherichia coli isolated by Ravin in 1964 (13). It was shown that N15 prophage exists in a plasmid state (15). Later, we found that N15 plasmid prophage is a double-stranded linear DNA molecule with covalently closed ends (19).Lysogenization by some temperate bacteriophages changes the host cell phenotype. The characteristics of this phenomenon, named lysogenic conversion, are similar for phages 80 and N15. Neither 80 lysogens (4) nor N15 lysogens (14) adsorb superinfecting phages 80, N15, and T1. We previously described in detail this phenomenon for phage 80 (7) and subsequently cloned and mapped the gene of conversion, which we named cor (5, 6). The phenotype induced by this gene was designated Cor ϩ . The nucleotide sequence of the 80 cor gene region was determined by Matsumoto et al. (9). The N15 cor gene was also cloned and mapped. By using a minicell system, N15 Cor protein was shown to have a molecular mass of about 8 kDa (8).Further localization, sequencing, and analysis of the N15 cor gene. Elsewhere, it was shown that E. coli cells acquired the Cor phenotype after being transformed with plasmid pNC304 or pNC3044, which contain the N15 plasmid DNA PstI restriction fragment (coordinates 39.43 to 41.68 kb) and SalI-PstI restriction fragment (coordinates 40.98 to 41.68 kb) (8), respectively.A series of deletions was introduced into pNC304, producing plasmids pNC3041, pNC3042, and pNC3043 (Fig. 1). Transformation with plasmid pNC3041 or pNC3042, but not with plasmid pNC3043, permitted host E. coli HB101 cells to grow on plates covered with 80 vir phage (10 8 PFU/cm 2 ), i.e., caused the Cor ϩ phenotype. The results suggested that the cor gene resides between the ClaI and PvuII restriction sites.We determined the nucleotide sequence of the N15 plasmid DNA SalI-PstI restriction fragment. The data obtained are summarized in Fig. 2.An open reading frame (ORF) was found in the ClaI-PstI direction, starting at nucleotide 196 and terminating at nucleotide 432 (ORF78; Fig. 2). This ORF encodes a protein with a molecular mass of 8.6 kDa. Putative Ϫ35 and Ϫ10 promoter elements were found upstream of this ORF. The sequences of both elements as well as the putative Shine-Dalgarno sequence are very close to the consensus sequence. Both the reading direction of ORF78 and the size of the protein it encodes are consistent with the results reported earlier for the cor gene and its product (8). No other extensive ORF was found in either direction of the ClaI-PstI restriction fragment. Therefore, we assigned the N15 cor gene to ORF78.Computer-assisted analysis based on the Argos and Rao method (12) revealed that the 21 N-terminal amino acids of the deduced N15 Cor protein are capable of forming a transmembrane helix. This suggests that the protein could be integrated into the membrane, where it may interact with common receptors for phages N15 and 80.Comparison of the N15 and 80 cor gene and protein sequences. Initiall...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.