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N15: The linear phage–plasmid
Plasmid 65 (2011) 102–109
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Review
N15: The linear phage–plasmid Nikolai V. Ravin ⇑ Centre ‘‘Bioengineering’’, Russian Academy of Sciences, Prosp. 60-let Oktiabria, bld. 7-1, Moscow 117312, Russia
a r t i c l e
i n f o
Article history: Received 9 November 2010 Accepted 17 December 2010 Available online 23 December 2010 Communicated by Dhruba K. Chattoraj Keywords: Linear plasmid Bacteriophage N15 Covalently closed telomere DNA replication Partition
a b s t r a c t 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, uKO2 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 UHAP-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. Ó 2010 Elsevier Inc. All rights reserved.
Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.
History of isolation and study . . . . . . . . . . . . . . . . . . . . Organization of the genome . . . . . . . . . . . . . . . . . . . . . Lysogeny control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prophage partitioning system . . . . . . . . . . . . . . . . . . . . Generation of covalently closed telomeres . . . . . . . . . . Replication mechanism . . . . . . . . . . . . . . . . . . . . . . . . . Biotech applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . N15 – related marine phages UHAP-1, VP882, VP58.5 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.......... .......... .......... .......... .......... .......... .......... and VHML . .......... .......... ..........
⇑ Fax: +7 4991350571 E-mail addresses: [email protected], [email protected] 0147-619X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2010.12.004
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1. History of isolation and study Bacteriophage N15 was isolated by Victor Ravin in 1964 and was initially studied in Russia (Golub and Ravin, 1967; Ravin, 1968, 1971; Ravin and Golub, 1967; Ravin and Shulga, 1970). N15 belongs to the lambdoid phage family that was suggested on the basis of cross-hybridization of their DNAs (Ravin and Shulga, 1970) and is similar to k with respect to the genome size, burst size, morphology of phage particles, latent period and frequency of lysogenization (Ravin, 1971). As was shown by Victor Ravin and coworkers in 1967–70, an unusual feature of phage N15 is that its prophage replicates extrachromosomally (Ravin, 1968, 1972; Ravin and Shulga, 1970). The next important step in investigation the N15 biology was performed by Valentin Rybchin and colleagues who showed (Svarchevsky and Rybchin, 1984b; Malinin et al., 1992b) that the N15 prophage is a linear plasmid with covalently closed ends (telomeres). It was the first example of linear DNA with covalently closed ends in prokaryotes; several years later the same structure was found for plasmids of Spirochete genus Borrelia (Barbour and Garon, 1987). The mature N15 phage DNA, like the k DNA, has 12 bp – single stranded cohesive ends, named cosL and cosR. The gene order in plasmid DNA is a circular permutation of
Fig. 1. (A) Mechanism of conversion of phage DNA into linear plasmid. cosL, cosR, single stranded cohesive ends; cosRL, cos site after annealing and ligation of cohesive ends; telRL, uncut target site of protelomerase; telL and telR, left and right hairpin ends of the prophage created by protelomerase. (B) Sequences of telRL site and hairpin ends of the prophage. The positions of the cleavage sits are marked by a filled triangle.
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that in the virion phage DNA, and the telomere-forming site telRL in phage DNA is a 56 bp inverted repeat. The above data suggest the following mechanism of conversion of phage DNA to the prophage plasmid (Malinin et al., 1992a). After infection of an E. coli cell, the phage DNA becomes circularised via its cohesive termini. Then a special phage-encoded enzyme, protelomerase (prokaryotic telomerase) cuts telRL sequence and joins the phosphodiester bonds making covalently closed ends, telL and telR (Fig. 1). 2. Organization of the genome The nucleotide sequence of the N15 genome has been determined in 1998 (Ravin et al., 2000). The genome contains 46,363 base pairs, of which about one half is similar to bacteriophage k sequence (Fig. 2). This is the left arm of the phage genome (Fig. 2) that contains the structural genes for the proteins required for virion head and tail assembly. From N15 genes 1 through gene 21 there is a one to one correlation with the phage k genes A through J. There is as much as 90% identity between the amino acid sequences of N15 and the k head gene products. Some parts of this region of N15 are more closely related to other lambdoid phages and starting from gene 17 (the k tail assembly gene M analogue) to gene 25, except for gene 24, N15 matches lambdoid phages HK97 and HK022 better than k. The above observation that N15 carries a k-like head and tail protein genes correlates well with the observation that N15 virion morphology is similar to that of lambdoid phages (Ravin, 1971). The N15 gene 24 is the homolog and functional analogue of the cor gene of phage u80 (Vostrov et al., 1996) and is responsible for unability of N15 lysogenes to adsorb bacteriophages N15, T1 and /80 (Ravin and Golub, 1967). To the right of the block of morphogenic genes, there is gene 26, a homolog of the E. coli umuD gene, which is involved in error-prone DNA damage repair. Promoter of this gene is overlapped by potential LexA binding site suggesting expression of N15 umuD upon DNA damage. The next two genes in the left half of the genome are homologs of the sopA and sopB genes of the F plasmid and determines the segregation stability of the N15 prophage (see below). The division between the left and right arms of the N15 genome is determined by the site (telRL) at which phage DNA is cut by protelomerase to make the linear plasmid prophage at infection (Fig. 1). Contrary to the left arm, only 10 of the 35 N15 right arm genes have homologs in lambdoid phages. Among them are genes 38, 39 and 40 homologues to cB, cro and Q respectively; they are responsible for the control of lysogeny (see below). Genes 53, 54, 55 and 55.1 are supposed to encode lysis function and also have homologues in the lambdoid phage family (Ravin et al., 2000). Two operons located in the right arm are specific to N15 and reflect its unusual lifestyle: the protelomerase gene (29) located rightward of telRL, and the replication region comprising gene 37(repA) encoding multifunctional replication protein and ancillary genes 33–36. Genes of replication region are supposed to be cotranscribed from the promoter controlled by the CB repressor. Detail description of all N15 genes and discussion about their possible functions could be found in (Ravin et al., 2000).
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Fig. 2. (A) Map of N15 virion chromosome. The N15 linear virion chromosome is shown with a scale in kbp. Rectangles immediately above and below the scale represent predicted genes that are transcribed rightward and leftward, respectively; their colours indicate similarity to known genes in the following way: genes that have been found on lambdoid phages (grey); genes that have been found on plasmids and non-lambdoid phages (black); no database match (white). The N15 gene names are given within or near the rectangles and alternate descriptive names are indicated above or below. Asterisks (⁄) mark the position of the centromere sites involved in plasmid partition. (B) Mosaic relationship between the whole genomes of phages k, N15, uKO2 and PY54. Grey areas between the genomes indicate main regions of homology. The ends of each phage’s circularly permuted prophage is marked by a black vertical line.)
The nucleotide sequences of the genomes of two other phage of N15 family, uKO2 and pY54, were also determined (Casjens et al., 2004; Hertwig et al., 2003b). Overall genome structures of three genomes are similar, although genetic mosaicism is also evident, – the virion proteins genes of PY54 and uKO2 are very similar and both rather different from N15 structural genes set (Fig. 2). On the contrary, the regions of the uKO2 genome outside the late operon are largely similar to, but are mosaically related to, those of N15 (Fig. 2). Phage PY54 right arm contains N15-like protelomerase, replication and primary immunity regions; other N15 related ORFs are not clustered, but are scattered over the whole PY54 right arm and there are a number of unrelated ORFs lying in between. In addition, the order and orientation of the N15 related genes in PY54 are not the same as in N15. The similar overall genome organizations of phages N15, PY54, uKO2, and k suggest that it is legitimate to include the former three within the lambdoid phage group, but in a subgroup that has a different strategy of lysogeny. 3. Lysogeny control The extrachomosomal location of the N15 prophage apparently requires controlled expression of not only the repressor function but also the genes responsible for
prophage maintenance. One could expect that at least the replication, prophage partition and protelomerase gene clusters will be expressed, but, in fact, analysis of N15 transcription patterns showed that about a half of the N15 genes are transcribed in the lysogen (Ravin et al., 2000). This situation differs from that of k and suggests the possibility of more complex regulatory mechanisms. At least three distinct loci are involved in the control of lysogeny (Fig. 3). Prophage superinfection immunity is encoded at immB, which was found to be structurally and functionally similar to the lambdoid phages immunity regions (Lobocka et al., 1996). immB contains three genes (Fig. 3). Gene 38 (cB) encodes the repressor protein, homologous to k CI. Clear plaque mutants, mapping at immB, were found in the cB gene supporting its role as a primary repressor. Genes 39 and 40 shows homology to lambda genes cro and Q, respectively. The cB gene is flanked by a complex array of divergent operator–promoter sites. The two operators leftward of the cB overlap the predicted promoter of the N15 repA gene, implying that binding of CB at these operators represses and regulates transcription of repA. This supposition is further supported by an observation that the N15 based miniplasmids lacking the cB gene have a higher copy number than similar plasmids with an intact cB (Ravin and Ravin, 1994). The three operators rightward from cB overlap the predicted promoter of the cB itself and the predicted
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Fig. 3. Three lysogeny control regions. Protein-encoding genes are shown by grey boxes, cA, which encodes an RNA, by open box across the main line, bent arrows indicate promoters (P), positions of transpiration terminators (T) are shown by solid triangles. CB binding sites (O) are shown by black rectangles, LexA binding site at P471 – by black circle. Genes and sites shown above (below) the main line apply to transcription from left to right (from right to left). The transcripts of the immA region are indicated by arrows. The CA RNA is represented by a closed bar.
promoters of the ‘‘late’’ operon containing cro and Q. It was proposed (Lobocka et al., 1996) that CB, by binding to these operators, represses both its own transcription and transcription of cro, Q and late genes. The secondary immunity region, immA, is located between the protelomerase gene and replication operon (Fig. 3). Ravin et al. (1999) have characterized this region and found that it contains antirepressor operon. Three open reading frames at the immA encode an inhibitor of cell division (icd), an antirepressor protein (antA) and a gene that may play an ancillary role in antirepression (antB). The operon may be transcribed from two promoters: the upstream promoter Pa could be repressed by the CB repressor, whereas the weaker downstream promoter Pb is constitutive. Full repression of the antirepressor operon is achieved by premature transcription termination elicited by a small RNA (CA RNA) produced by processing of the leader transcript of the operon – the mechanism similar to the one used in the anti-immunity system of phage P1 and the lysogeny control region of phage P4 (Citron and Schuster, 1992; Deho et al., 1992). The CA RNA thus acts as a secondary repressor and clear plaque mutants mapped at immA were found within the cA sequence. The antirepressor functions encoded at the immA are involved in the lysis-lysogeny decision of N15 early upon infection. Analysis of transcription patterns of the immA locus showed that the structural genes (icd, antA and antB) of the ant operon can only be expressed very soon after infection from the two promoters, before the CA RNA is produced by processing of the leader region of the transcript, and then be rapidly turned off. In the lysogen, the CB repressor turns off promoter Pa, while the second promoter, Pb, allows production of the immunity factor, CA RNA. The third region involved in the control of lysogeny, was characterized in (Mardanov and Ravin, 2007). It contains gene encoding antirepressor protein, AntC (Fig. 3). Expres-
sion of this gene (antC) from a plasmid is sufficient to prevent lysogenization by an infecting phage and to induce lytic development in N15 lysogens. Expression of antC counteracts the repression of promoters controlled by the primary N15 repressor, CB, resulting in activation of replication of N15-based minplasmids and expression of late gene clusters. Using a bacterial two-hybrid system it was shown that AntC binds to CB in vivo. The antC seems to be involved in the switch from lysogeny to lytic development in the process of prophage induction. Phage N15 mutants in antC can infect E. coli cells, as well as establish stable lysogens, but are deficient in prophage induction. The antC expression is controlled by one of the major components of SOS system, the LexA protein, which binding site overlaps the antC promoter. Exposure of the host cell to DNA damaging agents that challenge cell survival results in RecA-dependent autocleavage of LexA, derepression of antC promoter, synthesis of this antirepressor protein and, finally, activation of lytic development. In this way, the cellular SOS response controls N15 prophage induction (Mardanov and Ravin, 2007). Analysis of the nucleotide sequences of linear phage– plasmids uKO2 and pY54 revealed that both phages encode AntC-like antirepressors and that these genes may be controlled by LexA. In phage uKO2 the LexA binding site directly overlaps gene 47 promoter while in phage PY54 the LexA operator controls expression of a two-gene operon comprising gene 56 which encodes a DinI-like protein (Hertwig et al., 2003b) and the antirepressor gene 57.
4. Prophage partitioning system The N15 plasmid prophage is maintained at three–five copies per bacterial chromosome and is very stable – its rate of spontaneous loss is less than 104 per generation (Svarchevsky and Rybchin, 1984a). This is much less then would be expected in the case of random distribution of
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plasmid copies between the daughter cells, and implies the existence of special stabilisation machinery. Two principal mechanisms ensuring stable inheritance of bacterial plasmids have been described: active partition of plasmid copies to daughter cells prior to division and addiction systems responsible for post-segregational killing of plasmidless cells. It is unlikely that the addiction system is active in N15 since prophage-free cells are easily accumulated at the non-permissive temperature in the N15 lysogens carrying ‘‘early’’ temperature-sensitive mutations (Ravin, 1968). However, report of Dziewit et al. (2007) suggest that phage N15 operon consisting of genes 49 and 48 constitute toxin– antitoxin module functional in E. coli. The N15 partitioning system was characterized (Ravin and Lane, 1998; Grigoriev and Lobocka, 2001). The regions near the right end of the prophage show remarkable similarity to the sop locus, which governs partition of F plasmid copies to daughter cells. Ravin and Lane (1998) demonstrated that the sop locus of N15 in fact determines stability of the prophage since sop proteins, encoded at this locus, can stabilise the partition-defective N15 derivatives. PY54 and uKO2 also contain the N15-like sop loci close to the right ends of their prophages. The structural and functional organization of the N15 sop is similar to that of other partition loci including F sop and P1 par. These loci consist of a two-gene operon and an adjacent cis-acting site (Hiraga, 1992). The first gene (gene 28 = sopA) encodes a protein which binds to the promoter of the partition operon to repress transcription (the operon is thus negatively autoregulated) and also acts directly in the partition process itself, while the product of the second gene (gene 27 = sopB) binds to the cis acting centromere site (C) to form a partition complex, and acts as a corepressor of operon expression. The N15 and F partition functions appeared to be partly interchangeable: N15 SopA and SopB can partly stabilise partition-defective mini F and repress the F sop promoter and vice versa (Ravin and Lane, 1998). However, the N15 partition system, although a functional analogue of F sop system, differs from it in several important respects. The centromere site of N15 is not composed of a cluster of multiple inverted repeats (IR sites) adjacent to the sop operon, as in the case of F, P1 and other circular plasmids (except RK2), but is represented by four inverted repeats located in different regions of the N15 genome. Each of these sites binds SopB and acts as a centromere (Ravin and Lane, 1998; Grigoriev and Lobocka, 2001). Like N15, genomes of PY54 and uKO2 also contain multiple centromere IR sites, respectively, 10 and 4. It was shown (Dorokhov et al., 2010) that a single site is sufficient to stabilise a circular, but not a linear N15-based plasmid. Stabilisation of a linear plasmid increased in proportion to the number of IR sites and the distance between them (Dorokhov et al., 2010), however, the molecular mechanisms of this phenomenon remain to be determined. Transcription of the F sop operon is driven from one autoregulated promoter while transcription of N15 sop is driven by two major promoters (Dorokhov et al., 2003). The first is similar in sequence and function to the F sop promoter; it is repressed by Sop proteins. The second stronger promoter is insensitive to regulation by Sop pro-
teins but is tightly repressed by protelomerase, the N15 enzyme that completes prophage replication by generating hairpin telomeres. These establish a regulatory link between the partition system and other processes of N15 maintenance. N15 centromere sites are located in the regions of N15 genome that are supposed to be essential for replication and control of gene expression. One site, IR1, is located within the coding sequence of the replication gene repA; the second, IR2, is located downstream of gene Q encoding antiterminator, the other two, IR3 and IR4, are located close to the late promoters. This suggests that the N15 partition functions may be involved in the regulation of gene expression and replication. Particularly, IR3 is located immediately upstream of the main promoter P52 of the late operon, controlled by Q-dependent antitermination. The Sop-mediated interaction between IR2 and IR3 is important for ‘‘delivery’’ of Q to its target site qut located at P52 and activation of late transcription (Ravin et al., 2008). Phages uKO2 and PY54 both carry centromeres near their presumed Q genes and late promoters. Although we do not know whether the three prophages represent a succession or convergent evolution, they provide the raw material for addressing such intriguing questions as which function of sop system came first and was co-opted to the other, partition or regulation? 5. Generation of covalently closed telomeres The N15 protelomerase was first hypothesized by Valentin Rybchin as an enzyme responsible for the formation of a linear hairpin prophage molecule from the circularised phage DNA (Fig. 1). In this model N15 protelomerase is a functional analogue of lambdoid phage integrases. The protelomerase gene, telN, was identified upon sequencing of the N15 genome, its predicted product, TelN, has limited sequence homology with the tyrosine recombinases and type IB topoisomerases, as well as with the ResT telomere resolvase of Borrelia burgdorferi (GenBank AF064539 annotation; Kobryn and Chaconas, 2002; Ravin et al., 2000; Rybchin and Svarchevsky, 1999). Later the protelomerase genes were identified in the genomes of phages uKO2 and PY54 (Casjens et al., 2004; Hertwig et al., 2003a). The cleavage-joining activity of TelN and protelomerase TelK of uKO2 was analysed in vitro (Deneke et al., 2000, 2002; Huang et al., 2004), and a structure of the TelK in complex with the target site has been solved (Aihara et al., 2007). Protelomerases and tyrosine recombinases have similar catalytic mechanisms for DNA cleavage and ligation, generating a 30 -phosphotyrosine DNA intermediate that enables the covalent rejoining of cleaved DNA strands without the use of a high-energy cofactor. Two protelomerase molecules bind as dimer to a doublestranded DNA and generate a pair of transient staggered cleavages six base-pairs apart the axis of symmetry of the palindromic target site (Huang et al., 2004), as shown in Fig. 1. Protelomerase molecules form a pair of proteinlinked DNA intermediates at each 30 end of the cleaved openings leaving a 50 -OH. Then the partners of the two initial openings are exchanged, and the transient breaks are resealed to generate hairpin ends.
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Ravin and colleagues analysed the protelomerase activity in vivo and demonstrated that this enzyme is required for processing of replicative intermediates. The telN gene and the telRL site constitute an independent functional unit: cloning of this module in circular mini-F and miniP1 plasmids resulted in their linearization and further maintenance as linear plasmids with hairpin telomeres (Ravin et al., 2001). Functional independence of the protelomerase unit was used to develop linear derivatives of commonly used BAC vectors (Ooi et al., 2008). 6. Replication mechanism All cells with linear chromosomes must utilize special mechanisms to replicate the extreme termini of their DNA molecules, since DNA polymerases alone are unable to perform this function (Watson, 1972). Most eukaryotes have open-ended DNAs and employ special ‘‘telomerase’’ enzymes for this purpose, but there are other solutions that ensure complete replication of linear DNA: protein priming, recombination and covalently closed terminal hairpins. In order to identify the minimal set of genes able to drive replication of the N15 prophage, a set of miniplasmids consisting of different fragments of N15 DNA and a antibiotic resistance gene has been constructed (Ravin et al., 2003). The shortest circular miniplasmid contained only gene 37 (repA) that is thus necessary and sufficient to drive replication of circular miniplasmid. The shortest constructed linear plasmid consists of repA and a protelomerase module (telN gene and telRL site). The RepA is a multifunctional protein combining primase, helicase and origin-binding activities (Mardanov and Ravin, 2006) thus resembling phage P4 a replication protein (Ziegelin et al., 1993). The replication initiation site (ori) is located within the repA, replication initiated at this site proceeds bidirectionally (Ravin et al., 2003). Various models involving processing of replicative intermediates by an end-resolving enzyme have been pro-
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posed for replication of linear DNAs with covalently closed telomeres (see for review Casjens, 1999). One of the crucial points allowing discriminating between different models of replication is the structure of replicative intermediate processed by the end-resolving enzyme. Ravin et al. (2001) constructed the N15 mutant carrying deletion in the protelomerase gene and cloned the telN gene in the expression vector under the control of a regulatable promoter. The mutant may be maintained as a linear plasmid if the telN is expressed from the vector plasmid present in the same cell; repression of the telN results in accumulation of unprocessed replicative intermediates which were found to be circular head-to-head dimer molecules (Ravin et al., 2001). These data suggest the following model of N15 plasmid replication (Fig. 4; Ravin et al., 2003). Replication is initiated from an internal ori site, located within repA, follows the h mode and proceeds bidirectionally. After duplication of telL, protelomerase cuts this site creating hairpin ends, and thus a Y-shaped structure is formed. After the replication of the right telomere and subsequent cutting, two linear molecules are produced (Fig 4, pathway A1). Alternatively, full replication of the molecule with the formation of a full head-to-head circular dimer may precede ends resolution (see Fig 4, pathway A2). The intermediates of replication predicted by this model were actually observed in electron microscopy analysis (Ravin et al., 2003). The above model could be extended to explain the mechanism of N15 lytic replication. It is very likely, in view of the strong similarity of N15 morphogenetic genes to those of phage k, that late steps of N15 lytic replication and encapsidation follow the lambda model. Mardanov and Ravin (2009) analysed the structures of phage N15 DNA in course of lytic development upon infection and found that upon circularised infecting phage DNA is not used to initiate lambda-like lytic replication but is converted into a linear plasmid as shown in Fig. 1. Replication of N15 DNA then follows the plasmid mode and only at late steps the circular unit-length molecules that could start
Fig. 4. Model of N15 plasmid prophage and lytic replication. A – Replication of N15 plasmid prophage. A1 – protelomerase cuts before completion of replication: Y-like structure. A2 – replication is finished before protelomerase cutting: circular head-to-head dimer. B – lytic replication initiated in the lysogen. Note that circular head-to-head dimer is supposed to be processed by protelomerase into two circular monomers (only one is shown). The known or suggested participation of the N15 genes TelN (T) and RepA (R) at the individual steps is shown.
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lambda-like late replication and appeared (Fig. 4, pathway B). Consistently, the protelomerase is required for phage N15 lytic development. The circular monomer results from TelN-mediated resolution of a circular dimer into two circular molecules rather then being generated directly from a linear plasmid in reverse ‘‘telomere fusion’’ reaction (Mardanov and Ravin, 2009). The switch to circular plasmid formation in course of lytic replication of N15 may result from depletion of protelomerase or modification of the protelomerase and/or its target site by some phage-encoded factor at late steps of lytic growth. Phage–plasmids uKO2 and pY54 seem to follow the same mode of replication as N15. As already mentioned above, both phages contain N15-like protelomerase genes. Replication regions of N15, uKO2 and PY54 has similar organization and contains homologues repA genes. Like N15, the repA gene of PY54 was shown to function as a minimal circular replicon in E. coli (Ziegelin et al., 2005) and contain the ori site. 7. Biotech applications One particular property of N15 prompted its biotechnological exploitation, – linearity of the plasmid prophage, presumably resulting in the absence of supercoiling. It is well known that plasmid supercoiling can induce cruciforms and other secondary structures, favouring deletions or rearrangements. N15 based linear miniplasmids have been used as cloning vectors (Ravin and Ravin, 1994), which appeared to be particularly suitable for cloning DNA sequences with inverted repeats (Ravin and Ravin, 1999). More sophisticated N15-based linear cloning system, the ‘pJAZZ’ series of transcription-free, linear cloning vectors, can stably maintain templates that are difficult or impossible to clone in circular vectors, including AT-rich inserts of up to 30 kb and short tandem repeats of up to 2 kb (Godiska et al., 2010). N15 was also used to develop E. coli host/vector system allowing a combination of two principles of regulation of protein synthesis: use of an inducible promoter and regulation of the copy number of vector (Mardanov et al., 2007). The pN15E vectors are low copy number plasmids based on the N15 replicon, comprising the repA replicase gene and cB repressor gene. Regulation of pN15E copy number is achieved through arabinose-inducible expression of phage N15 antirepressor protein, AntA, whose gene was integrated into the chromosome of the host strain under control of the regulatable promoter. The low copy number of these vectors ensures very low basal level of expression allowing cloning genes encoding toxic products, while full induction of the inducible promoter and elevation of the copy number of the vector allow high level expression of the target protein. 8. N15 – related marine phages UHAP-1, VP882, VP58.5 and VHML Besides N15, uKO2 and PY54, genes encoding homologs of N15 protelomerase are present in genomes of four recently described marine phages, – the Vibrio harveyi phage
VHML (Oakey et al., 2002), the Vibrio parahaemolyticus phages VP882 (Lan et al., 2009) and VP58.5 (Zabala et al., 2009), and the Halomonas aquamarina phage UHAP-1 (Mobberley et al., 2008). The later three phages were shown to lysogenize their hosts as a linear plasmids with covalently closed telomeres, while VHML was described as integrative. The overall genomic organization of the functional modules was similar across these phages and N15, with packaging, structural, and phage metabolism genes present. The capsid-associated proteins are similar to those from lambda-like siphoviruses, while tail proteins are similar to that from P2-like temperate myoviruses (Mobberley et al., 2008). Homologs of another protein specific for N15-like viruses, RepA replicase, are encoded at UHAP-1, VP882, VP58.5 and VHML genomes. Like in N15, protelomerase and counteroriented repA-like gene are located between the structural gene cluster and k-type lysogeny control region.
9. Conclusions N15-like group of viruses is unique among bacteriophages in genetic organization. The similar overall genome organization, presence of genes associated with linear plasmid lifestyle, – protelomerase and repA-like replicase, in N15, uKO2, PY54, UHAP-1, VP882, VP58.5 and VHML suggest that these phages may belong to a group diverged from a common ancestor. Such ancestor of N15-like phages must have arisen either through the accumulation of new genetic modules from plasmid and bacterial sources by lambdoid progenitor or by an unknown plasmid acquiring a lambdoid set of ‘‘virion’’ genes. Subsequent evolution followed the Botstein’s modular theory (Botstein, 1980), where evolution occurs via the exchange of groups of functional genes between different phages and plasmids. Therefore, N15 and its relatives provide a very interesting model system for the study of phage and plasmid evolution and interactions between phages, plasmids and bacterial hosts.
Acknowledgments The author’s work was supported by the Program ‘‘Molecular and Cell Biology’’ of RAS, and the grant 1004-01204 from Russian Foundation for Basic Research.
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Mobberley, J.M., Authement, R.N., Segall, A.M., Paul, J.H., 2008. The temperate marine phage PhiHAP-1 of Halomonas aquamarina possesses a linear plasmid-like prophage genome. J. Virol. 82, 6618– 6630. Oakey, H.J., Cullen, B.R., Owens, L., 2002. The complete nucleotide sequence of the Vibrio harveyi bacteriophage VHML. J. Appl. Microbiol. 93, 1089–1098. Ooi, Y.S., Warburton, P.E., Ravin, N.V., Narayanan, K., 2008. Recombineering linear DNA that replicate stably in E. coli. Plasmid 59 (1), 63–71. Ravin, N.V., Kuprianov, V.V., Gilcrease, E.B., Casjens, S.R., 2003. Bidirectional replication from an internal ori site of the linear N15 plasmid prophage. Nucleic Acids Res. 31, 6552–6560. Ravin, N., Lane, D., 1999. Partition of the linear plasmid, N15: functional interactions with the sop locus of the F plasmid. J. Bacteriol. 181, 6898–6906. Ravin, N.V., Ravin, V.K., 1994. An ultrahigh-copy plasmid based on the mini-replicone of the temperate phage N15. Mol. Gen. Mikrobiol. Virusol. 1, 37–39. Ravin, N.V., Ravin, V.K., 1999. Use of a linear multicopy vector based on the mini-replicon of temperate coliphage N15 for cloning DNA with abnormal secondary structures. Nucleic Acids Res. 27, e13. Ravin, N.V., Rech, J., Lane, D., 2008. Extended function of plasmid partition genes: Sop system of linear phage–plasmid N15 facilitates late gene expression. J. Bacteriol. 190, 3538–3545. Ravin, N.V., Strakhova, T.S., Kuprianov, V.V., 2001. The protelomerase of the phage–plasmid N15 is responsible for its maintenance in linear form. J. Mol. Biol. 312, 899–906. Ravin, N.V., Svarchevsky, A.N., Deho, G., 1999. The antiimmunity system of phage–plasmid N15: identification of the antirepressor gene and its control by a small processed RNA. Mol. Microbiol. 34, 980–994. Ravin, V.K., 1968. The functioning of the genes of temperate bacteriophage in lysogenic cells. Genetika 4, 119–124. Ravin, V.K., 1971. Lysogeny. Nauka Press, Moscow. pp. 106. Ravin, V., 1972. Lack of prophage N15 in Escherichia coli K12 chromosome. Genetika 8, 189–191. Ravin, V., Golub, E., 1967. A study of phage conversion in Escherichia coli. I. The aquisition of resistance to bacteriophage T1 as a result of lysogenization. Genetika 4, 113–121. Ravin, V., Ravin, N., Casjens, S., Ford, M., Hatfull, G., Hendrix, R., 2000. Genomic sequence and analysis of the atypical bacteriophage N15. J. Mol. Biol. 299, 53–73. Ravin, V.K., Shulga, M.G., 1970. The evidence of extrachromosomal location of prophage N15. Virology 40, 800–807. Rybchin, V.N., Svarchevsky, A.N., 1999. The plasmid prophage N15, a linear DNA with covalently closed ends. Mol. Microbiol. 33, 895–903. Svarchevsky, A.N., Rybchin, V.N., 1984a. Characteristics of plasmid properties of bacteriophage N15. Mol. Gen. Mikrobiol. Virusol. 10, 34–39. Svarchevsky, A.N., Rybchin, V.N., 1984b. Physical mapping of plasmid N15 DNA. Mol. Gen. Mikrobiol. Virusol. 10, 16–22. Vostrov, A., Vostrukhina, O., Svarchevsky, A., Rybchin, V., 1996. Proteins responsible for lysogenic conversion caused by coliphages N15 and phi80 are highly homologous. J. Bacteriol. 178, 1484–1486. Watson, J., 1972. Origin of concatemeric T7 DNA. Nature New Biol. 239, 197–201. Zabala, B., Hammerl, J.A., Espejo, R.T., Hertwig, S., 2009. 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. J. Virol. 83, 9313–9320. Ziegelin, G., Scherzinger, E., Lurz, R., Lanka, E., 1993. Phage P4 a protein is multifunctional with origin recognition, helicase and primase activities. EMBO J. 12, 3703–3708. Ziegelin, G., Tegtmeyer, N., Lurz, R., Hertwig, S., Hammerl, J., Appel, B., Lanka, E., 2005. The repA gene of the linear Yersinia enterocolitica prophage PY54 functions as a circular minimal replicon in Escherichia coli. J. Bacteriol. 187, 3445–3454.
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Plasmid journal homepage: www.elsevier.com/locate/yplas
Review
N15: The linear phage–plasmid Nikolai V. Ravin ⇑ Centre ‘‘Bioengineering’’, Russian Academy of Sciences, Prosp. 60-let Oktiabria, bld. 7-1, Moscow 117312, Russia
a r t i c l e
i n f o
Article history: Received 9 November 2010 Accepted 17 December 2010 Available online 23 December 2010 Communicated by Dhruba K. Chattoraj Keywords: Linear plasmid Bacteriophage N15 Covalently closed telomere DNA replication Partition
a b s t r a c t 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, uKO2 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 UHAP-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. Ó 2010 Elsevier Inc. All rights reserved.
Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.
History of isolation and study . . . . . . . . . . . . . . . . . . . . Organization of the genome . . . . . . . . . . . . . . . . . . . . . Lysogeny control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prophage partitioning system . . . . . . . . . . . . . . . . . . . . Generation of covalently closed telomeres . . . . . . . . . . Replication mechanism . . . . . . . . . . . . . . . . . . . . . . . . . Biotech applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . N15 – related marine phages UHAP-1, VP882, VP58.5 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.......... .......... .......... .......... .......... .......... .......... and VHML . .......... .......... ..........
⇑ Fax: +7 4991350571 E-mail addresses: [email protected], [email protected] 0147-619X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2010.12.004
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1. History of isolation and study Bacteriophage N15 was isolated by Victor Ravin in 1964 and was initially studied in Russia (Golub and Ravin, 1967; Ravin, 1968, 1971; Ravin and Golub, 1967; Ravin and Shulga, 1970). N15 belongs to the lambdoid phage family that was suggested on the basis of cross-hybridization of their DNAs (Ravin and Shulga, 1970) and is similar to k with respect to the genome size, burst size, morphology of phage particles, latent period and frequency of lysogenization (Ravin, 1971). As was shown by Victor Ravin and coworkers in 1967–70, an unusual feature of phage N15 is that its prophage replicates extrachromosomally (Ravin, 1968, 1972; Ravin and Shulga, 1970). The next important step in investigation the N15 biology was performed by Valentin Rybchin and colleagues who showed (Svarchevsky and Rybchin, 1984b; Malinin et al., 1992b) that the N15 prophage is a linear plasmid with covalently closed ends (telomeres). It was the first example of linear DNA with covalently closed ends in prokaryotes; several years later the same structure was found for plasmids of Spirochete genus Borrelia (Barbour and Garon, 1987). The mature N15 phage DNA, like the k DNA, has 12 bp – single stranded cohesive ends, named cosL and cosR. The gene order in plasmid DNA is a circular permutation of
Fig. 1. (A) Mechanism of conversion of phage DNA into linear plasmid. cosL, cosR, single stranded cohesive ends; cosRL, cos site after annealing and ligation of cohesive ends; telRL, uncut target site of protelomerase; telL and telR, left and right hairpin ends of the prophage created by protelomerase. (B) Sequences of telRL site and hairpin ends of the prophage. The positions of the cleavage sits are marked by a filled triangle.
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that in the virion phage DNA, and the telomere-forming site telRL in phage DNA is a 56 bp inverted repeat. The above data suggest the following mechanism of conversion of phage DNA to the prophage plasmid (Malinin et al., 1992a). After infection of an E. coli cell, the phage DNA becomes circularised via its cohesive termini. Then a special phage-encoded enzyme, protelomerase (prokaryotic telomerase) cuts telRL sequence and joins the phosphodiester bonds making covalently closed ends, telL and telR (Fig. 1). 2. Organization of the genome The nucleotide sequence of the N15 genome has been determined in 1998 (Ravin et al., 2000). The genome contains 46,363 base pairs, of which about one half is similar to bacteriophage k sequence (Fig. 2). This is the left arm of the phage genome (Fig. 2) that contains the structural genes for the proteins required for virion head and tail assembly. From N15 genes 1 through gene 21 there is a one to one correlation with the phage k genes A through J. There is as much as 90% identity between the amino acid sequences of N15 and the k head gene products. Some parts of this region of N15 are more closely related to other lambdoid phages and starting from gene 17 (the k tail assembly gene M analogue) to gene 25, except for gene 24, N15 matches lambdoid phages HK97 and HK022 better than k. The above observation that N15 carries a k-like head and tail protein genes correlates well with the observation that N15 virion morphology is similar to that of lambdoid phages (Ravin, 1971). The N15 gene 24 is the homolog and functional analogue of the cor gene of phage u80 (Vostrov et al., 1996) and is responsible for unability of N15 lysogenes to adsorb bacteriophages N15, T1 and /80 (Ravin and Golub, 1967). To the right of the block of morphogenic genes, there is gene 26, a homolog of the E. coli umuD gene, which is involved in error-prone DNA damage repair. Promoter of this gene is overlapped by potential LexA binding site suggesting expression of N15 umuD upon DNA damage. The next two genes in the left half of the genome are homologs of the sopA and sopB genes of the F plasmid and determines the segregation stability of the N15 prophage (see below). The division between the left and right arms of the N15 genome is determined by the site (telRL) at which phage DNA is cut by protelomerase to make the linear plasmid prophage at infection (Fig. 1). Contrary to the left arm, only 10 of the 35 N15 right arm genes have homologs in lambdoid phages. Among them are genes 38, 39 and 40 homologues to cB, cro and Q respectively; they are responsible for the control of lysogeny (see below). Genes 53, 54, 55 and 55.1 are supposed to encode lysis function and also have homologues in the lambdoid phage family (Ravin et al., 2000). Two operons located in the right arm are specific to N15 and reflect its unusual lifestyle: the protelomerase gene (29) located rightward of telRL, and the replication region comprising gene 37(repA) encoding multifunctional replication protein and ancillary genes 33–36. Genes of replication region are supposed to be cotranscribed from the promoter controlled by the CB repressor. Detail description of all N15 genes and discussion about their possible functions could be found in (Ravin et al., 2000).
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Fig. 2. (A) Map of N15 virion chromosome. The N15 linear virion chromosome is shown with a scale in kbp. Rectangles immediately above and below the scale represent predicted genes that are transcribed rightward and leftward, respectively; their colours indicate similarity to known genes in the following way: genes that have been found on lambdoid phages (grey); genes that have been found on plasmids and non-lambdoid phages (black); no database match (white). The N15 gene names are given within or near the rectangles and alternate descriptive names are indicated above or below. Asterisks (⁄) mark the position of the centromere sites involved in plasmid partition. (B) Mosaic relationship between the whole genomes of phages k, N15, uKO2 and PY54. Grey areas between the genomes indicate main regions of homology. The ends of each phage’s circularly permuted prophage is marked by a black vertical line.)
The nucleotide sequences of the genomes of two other phage of N15 family, uKO2 and pY54, were also determined (Casjens et al., 2004; Hertwig et al., 2003b). Overall genome structures of three genomes are similar, although genetic mosaicism is also evident, – the virion proteins genes of PY54 and uKO2 are very similar and both rather different from N15 structural genes set (Fig. 2). On the contrary, the regions of the uKO2 genome outside the late operon are largely similar to, but are mosaically related to, those of N15 (Fig. 2). Phage PY54 right arm contains N15-like protelomerase, replication and primary immunity regions; other N15 related ORFs are not clustered, but are scattered over the whole PY54 right arm and there are a number of unrelated ORFs lying in between. In addition, the order and orientation of the N15 related genes in PY54 are not the same as in N15. The similar overall genome organizations of phages N15, PY54, uKO2, and k suggest that it is legitimate to include the former three within the lambdoid phage group, but in a subgroup that has a different strategy of lysogeny. 3. Lysogeny control The extrachomosomal location of the N15 prophage apparently requires controlled expression of not only the repressor function but also the genes responsible for
prophage maintenance. One could expect that at least the replication, prophage partition and protelomerase gene clusters will be expressed, but, in fact, analysis of N15 transcription patterns showed that about a half of the N15 genes are transcribed in the lysogen (Ravin et al., 2000). This situation differs from that of k and suggests the possibility of more complex regulatory mechanisms. At least three distinct loci are involved in the control of lysogeny (Fig. 3). Prophage superinfection immunity is encoded at immB, which was found to be structurally and functionally similar to the lambdoid phages immunity regions (Lobocka et al., 1996). immB contains three genes (Fig. 3). Gene 38 (cB) encodes the repressor protein, homologous to k CI. Clear plaque mutants, mapping at immB, were found in the cB gene supporting its role as a primary repressor. Genes 39 and 40 shows homology to lambda genes cro and Q, respectively. The cB gene is flanked by a complex array of divergent operator–promoter sites. The two operators leftward of the cB overlap the predicted promoter of the N15 repA gene, implying that binding of CB at these operators represses and regulates transcription of repA. This supposition is further supported by an observation that the N15 based miniplasmids lacking the cB gene have a higher copy number than similar plasmids with an intact cB (Ravin and Ravin, 1994). The three operators rightward from cB overlap the predicted promoter of the cB itself and the predicted
N.V. Ravin / Plasmid 65 (2011) 102–109
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Fig. 3. Three lysogeny control regions. Protein-encoding genes are shown by grey boxes, cA, which encodes an RNA, by open box across the main line, bent arrows indicate promoters (P), positions of transpiration terminators (T) are shown by solid triangles. CB binding sites (O) are shown by black rectangles, LexA binding site at P471 – by black circle. Genes and sites shown above (below) the main line apply to transcription from left to right (from right to left). The transcripts of the immA region are indicated by arrows. The CA RNA is represented by a closed bar.
promoters of the ‘‘late’’ operon containing cro and Q. It was proposed (Lobocka et al., 1996) that CB, by binding to these operators, represses both its own transcription and transcription of cro, Q and late genes. The secondary immunity region, immA, is located between the protelomerase gene and replication operon (Fig. 3). Ravin et al. (1999) have characterized this region and found that it contains antirepressor operon. Three open reading frames at the immA encode an inhibitor of cell division (icd), an antirepressor protein (antA) and a gene that may play an ancillary role in antirepression (antB). The operon may be transcribed from two promoters: the upstream promoter Pa could be repressed by the CB repressor, whereas the weaker downstream promoter Pb is constitutive. Full repression of the antirepressor operon is achieved by premature transcription termination elicited by a small RNA (CA RNA) produced by processing of the leader transcript of the operon – the mechanism similar to the one used in the anti-immunity system of phage P1 and the lysogeny control region of phage P4 (Citron and Schuster, 1992; Deho et al., 1992). The CA RNA thus acts as a secondary repressor and clear plaque mutants mapped at immA were found within the cA sequence. The antirepressor functions encoded at the immA are involved in the lysis-lysogeny decision of N15 early upon infection. Analysis of transcription patterns of the immA locus showed that the structural genes (icd, antA and antB) of the ant operon can only be expressed very soon after infection from the two promoters, before the CA RNA is produced by processing of the leader region of the transcript, and then be rapidly turned off. In the lysogen, the CB repressor turns off promoter Pa, while the second promoter, Pb, allows production of the immunity factor, CA RNA. The third region involved in the control of lysogeny, was characterized in (Mardanov and Ravin, 2007). It contains gene encoding antirepressor protein, AntC (Fig. 3). Expres-
sion of this gene (antC) from a plasmid is sufficient to prevent lysogenization by an infecting phage and to induce lytic development in N15 lysogens. Expression of antC counteracts the repression of promoters controlled by the primary N15 repressor, CB, resulting in activation of replication of N15-based minplasmids and expression of late gene clusters. Using a bacterial two-hybrid system it was shown that AntC binds to CB in vivo. The antC seems to be involved in the switch from lysogeny to lytic development in the process of prophage induction. Phage N15 mutants in antC can infect E. coli cells, as well as establish stable lysogens, but are deficient in prophage induction. The antC expression is controlled by one of the major components of SOS system, the LexA protein, which binding site overlaps the antC promoter. Exposure of the host cell to DNA damaging agents that challenge cell survival results in RecA-dependent autocleavage of LexA, derepression of antC promoter, synthesis of this antirepressor protein and, finally, activation of lytic development. In this way, the cellular SOS response controls N15 prophage induction (Mardanov and Ravin, 2007). Analysis of the nucleotide sequences of linear phage– plasmids uKO2 and pY54 revealed that both phages encode AntC-like antirepressors and that these genes may be controlled by LexA. In phage uKO2 the LexA binding site directly overlaps gene 47 promoter while in phage PY54 the LexA operator controls expression of a two-gene operon comprising gene 56 which encodes a DinI-like protein (Hertwig et al., 2003b) and the antirepressor gene 57.
4. Prophage partitioning system The N15 plasmid prophage is maintained at three–five copies per bacterial chromosome and is very stable – its rate of spontaneous loss is less than 104 per generation (Svarchevsky and Rybchin, 1984a). This is much less then would be expected in the case of random distribution of
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plasmid copies between the daughter cells, and implies the existence of special stabilisation machinery. Two principal mechanisms ensuring stable inheritance of bacterial plasmids have been described: active partition of plasmid copies to daughter cells prior to division and addiction systems responsible for post-segregational killing of plasmidless cells. It is unlikely that the addiction system is active in N15 since prophage-free cells are easily accumulated at the non-permissive temperature in the N15 lysogens carrying ‘‘early’’ temperature-sensitive mutations (Ravin, 1968). However, report of Dziewit et al. (2007) suggest that phage N15 operon consisting of genes 49 and 48 constitute toxin– antitoxin module functional in E. coli. The N15 partitioning system was characterized (Ravin and Lane, 1998; Grigoriev and Lobocka, 2001). The regions near the right end of the prophage show remarkable similarity to the sop locus, which governs partition of F plasmid copies to daughter cells. Ravin and Lane (1998) demonstrated that the sop locus of N15 in fact determines stability of the prophage since sop proteins, encoded at this locus, can stabilise the partition-defective N15 derivatives. PY54 and uKO2 also contain the N15-like sop loci close to the right ends of their prophages. The structural and functional organization of the N15 sop is similar to that of other partition loci including F sop and P1 par. These loci consist of a two-gene operon and an adjacent cis-acting site (Hiraga, 1992). The first gene (gene 28 = sopA) encodes a protein which binds to the promoter of the partition operon to repress transcription (the operon is thus negatively autoregulated) and also acts directly in the partition process itself, while the product of the second gene (gene 27 = sopB) binds to the cis acting centromere site (C) to form a partition complex, and acts as a corepressor of operon expression. The N15 and F partition functions appeared to be partly interchangeable: N15 SopA and SopB can partly stabilise partition-defective mini F and repress the F sop promoter and vice versa (Ravin and Lane, 1998). However, the N15 partition system, although a functional analogue of F sop system, differs from it in several important respects. The centromere site of N15 is not composed of a cluster of multiple inverted repeats (IR sites) adjacent to the sop operon, as in the case of F, P1 and other circular plasmids (except RK2), but is represented by four inverted repeats located in different regions of the N15 genome. Each of these sites binds SopB and acts as a centromere (Ravin and Lane, 1998; Grigoriev and Lobocka, 2001). Like N15, genomes of PY54 and uKO2 also contain multiple centromere IR sites, respectively, 10 and 4. It was shown (Dorokhov et al., 2010) that a single site is sufficient to stabilise a circular, but not a linear N15-based plasmid. Stabilisation of a linear plasmid increased in proportion to the number of IR sites and the distance between them (Dorokhov et al., 2010), however, the molecular mechanisms of this phenomenon remain to be determined. Transcription of the F sop operon is driven from one autoregulated promoter while transcription of N15 sop is driven by two major promoters (Dorokhov et al., 2003). The first is similar in sequence and function to the F sop promoter; it is repressed by Sop proteins. The second stronger promoter is insensitive to regulation by Sop pro-
teins but is tightly repressed by protelomerase, the N15 enzyme that completes prophage replication by generating hairpin telomeres. These establish a regulatory link between the partition system and other processes of N15 maintenance. N15 centromere sites are located in the regions of N15 genome that are supposed to be essential for replication and control of gene expression. One site, IR1, is located within the coding sequence of the replication gene repA; the second, IR2, is located downstream of gene Q encoding antiterminator, the other two, IR3 and IR4, are located close to the late promoters. This suggests that the N15 partition functions may be involved in the regulation of gene expression and replication. Particularly, IR3 is located immediately upstream of the main promoter P52 of the late operon, controlled by Q-dependent antitermination. The Sop-mediated interaction between IR2 and IR3 is important for ‘‘delivery’’ of Q to its target site qut located at P52 and activation of late transcription (Ravin et al., 2008). Phages uKO2 and PY54 both carry centromeres near their presumed Q genes and late promoters. Although we do not know whether the three prophages represent a succession or convergent evolution, they provide the raw material for addressing such intriguing questions as which function of sop system came first and was co-opted to the other, partition or regulation? 5. Generation of covalently closed telomeres The N15 protelomerase was first hypothesized by Valentin Rybchin as an enzyme responsible for the formation of a linear hairpin prophage molecule from the circularised phage DNA (Fig. 1). In this model N15 protelomerase is a functional analogue of lambdoid phage integrases. The protelomerase gene, telN, was identified upon sequencing of the N15 genome, its predicted product, TelN, has limited sequence homology with the tyrosine recombinases and type IB topoisomerases, as well as with the ResT telomere resolvase of Borrelia burgdorferi (GenBank AF064539 annotation; Kobryn and Chaconas, 2002; Ravin et al., 2000; Rybchin and Svarchevsky, 1999). Later the protelomerase genes were identified in the genomes of phages uKO2 and PY54 (Casjens et al., 2004; Hertwig et al., 2003a). The cleavage-joining activity of TelN and protelomerase TelK of uKO2 was analysed in vitro (Deneke et al., 2000, 2002; Huang et al., 2004), and a structure of the TelK in complex with the target site has been solved (Aihara et al., 2007). Protelomerases and tyrosine recombinases have similar catalytic mechanisms for DNA cleavage and ligation, generating a 30 -phosphotyrosine DNA intermediate that enables the covalent rejoining of cleaved DNA strands without the use of a high-energy cofactor. Two protelomerase molecules bind as dimer to a doublestranded DNA and generate a pair of transient staggered cleavages six base-pairs apart the axis of symmetry of the palindromic target site (Huang et al., 2004), as shown in Fig. 1. Protelomerase molecules form a pair of proteinlinked DNA intermediates at each 30 end of the cleaved openings leaving a 50 -OH. Then the partners of the two initial openings are exchanged, and the transient breaks are resealed to generate hairpin ends.
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Ravin and colleagues analysed the protelomerase activity in vivo and demonstrated that this enzyme is required for processing of replicative intermediates. The telN gene and the telRL site constitute an independent functional unit: cloning of this module in circular mini-F and miniP1 plasmids resulted in their linearization and further maintenance as linear plasmids with hairpin telomeres (Ravin et al., 2001). Functional independence of the protelomerase unit was used to develop linear derivatives of commonly used BAC vectors (Ooi et al., 2008). 6. Replication mechanism All cells with linear chromosomes must utilize special mechanisms to replicate the extreme termini of their DNA molecules, since DNA polymerases alone are unable to perform this function (Watson, 1972). Most eukaryotes have open-ended DNAs and employ special ‘‘telomerase’’ enzymes for this purpose, but there are other solutions that ensure complete replication of linear DNA: protein priming, recombination and covalently closed terminal hairpins. In order to identify the minimal set of genes able to drive replication of the N15 prophage, a set of miniplasmids consisting of different fragments of N15 DNA and a antibiotic resistance gene has been constructed (Ravin et al., 2003). The shortest circular miniplasmid contained only gene 37 (repA) that is thus necessary and sufficient to drive replication of circular miniplasmid. The shortest constructed linear plasmid consists of repA and a protelomerase module (telN gene and telRL site). The RepA is a multifunctional protein combining primase, helicase and origin-binding activities (Mardanov and Ravin, 2006) thus resembling phage P4 a replication protein (Ziegelin et al., 1993). The replication initiation site (ori) is located within the repA, replication initiated at this site proceeds bidirectionally (Ravin et al., 2003). Various models involving processing of replicative intermediates by an end-resolving enzyme have been pro-
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posed for replication of linear DNAs with covalently closed telomeres (see for review Casjens, 1999). One of the crucial points allowing discriminating between different models of replication is the structure of replicative intermediate processed by the end-resolving enzyme. Ravin et al. (2001) constructed the N15 mutant carrying deletion in the protelomerase gene and cloned the telN gene in the expression vector under the control of a regulatable promoter. The mutant may be maintained as a linear plasmid if the telN is expressed from the vector plasmid present in the same cell; repression of the telN results in accumulation of unprocessed replicative intermediates which were found to be circular head-to-head dimer molecules (Ravin et al., 2001). These data suggest the following model of N15 plasmid replication (Fig. 4; Ravin et al., 2003). Replication is initiated from an internal ori site, located within repA, follows the h mode and proceeds bidirectionally. After duplication of telL, protelomerase cuts this site creating hairpin ends, and thus a Y-shaped structure is formed. After the replication of the right telomere and subsequent cutting, two linear molecules are produced (Fig 4, pathway A1). Alternatively, full replication of the molecule with the formation of a full head-to-head circular dimer may precede ends resolution (see Fig 4, pathway A2). The intermediates of replication predicted by this model were actually observed in electron microscopy analysis (Ravin et al., 2003). The above model could be extended to explain the mechanism of N15 lytic replication. It is very likely, in view of the strong similarity of N15 morphogenetic genes to those of phage k, that late steps of N15 lytic replication and encapsidation follow the lambda model. Mardanov and Ravin (2009) analysed the structures of phage N15 DNA in course of lytic development upon infection and found that upon circularised infecting phage DNA is not used to initiate lambda-like lytic replication but is converted into a linear plasmid as shown in Fig. 1. Replication of N15 DNA then follows the plasmid mode and only at late steps the circular unit-length molecules that could start
Fig. 4. Model of N15 plasmid prophage and lytic replication. A – Replication of N15 plasmid prophage. A1 – protelomerase cuts before completion of replication: Y-like structure. A2 – replication is finished before protelomerase cutting: circular head-to-head dimer. B – lytic replication initiated in the lysogen. Note that circular head-to-head dimer is supposed to be processed by protelomerase into two circular monomers (only one is shown). The known or suggested participation of the N15 genes TelN (T) and RepA (R) at the individual steps is shown.
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lambda-like late replication and appeared (Fig. 4, pathway B). Consistently, the protelomerase is required for phage N15 lytic development. The circular monomer results from TelN-mediated resolution of a circular dimer into two circular molecules rather then being generated directly from a linear plasmid in reverse ‘‘telomere fusion’’ reaction (Mardanov and Ravin, 2009). The switch to circular plasmid formation in course of lytic replication of N15 may result from depletion of protelomerase or modification of the protelomerase and/or its target site by some phage-encoded factor at late steps of lytic growth. Phage–plasmids uKO2 and pY54 seem to follow the same mode of replication as N15. As already mentioned above, both phages contain N15-like protelomerase genes. Replication regions of N15, uKO2 and PY54 has similar organization and contains homologues repA genes. Like N15, the repA gene of PY54 was shown to function as a minimal circular replicon in E. coli (Ziegelin et al., 2005) and contain the ori site. 7. Biotech applications One particular property of N15 prompted its biotechnological exploitation, – linearity of the plasmid prophage, presumably resulting in the absence of supercoiling. It is well known that plasmid supercoiling can induce cruciforms and other secondary structures, favouring deletions or rearrangements. N15 based linear miniplasmids have been used as cloning vectors (Ravin and Ravin, 1994), which appeared to be particularly suitable for cloning DNA sequences with inverted repeats (Ravin and Ravin, 1999). More sophisticated N15-based linear cloning system, the ‘pJAZZ’ series of transcription-free, linear cloning vectors, can stably maintain templates that are difficult or impossible to clone in circular vectors, including AT-rich inserts of up to 30 kb and short tandem repeats of up to 2 kb (Godiska et al., 2010). N15 was also used to develop E. coli host/vector system allowing a combination of two principles of regulation of protein synthesis: use of an inducible promoter and regulation of the copy number of vector (Mardanov et al., 2007). The pN15E vectors are low copy number plasmids based on the N15 replicon, comprising the repA replicase gene and cB repressor gene. Regulation of pN15E copy number is achieved through arabinose-inducible expression of phage N15 antirepressor protein, AntA, whose gene was integrated into the chromosome of the host strain under control of the regulatable promoter. The low copy number of these vectors ensures very low basal level of expression allowing cloning genes encoding toxic products, while full induction of the inducible promoter and elevation of the copy number of the vector allow high level expression of the target protein. 8. N15 – related marine phages UHAP-1, VP882, VP58.5 and VHML Besides N15, uKO2 and PY54, genes encoding homologs of N15 protelomerase are present in genomes of four recently described marine phages, – the Vibrio harveyi phage
VHML (Oakey et al., 2002), the Vibrio parahaemolyticus phages VP882 (Lan et al., 2009) and VP58.5 (Zabala et al., 2009), and the Halomonas aquamarina phage UHAP-1 (Mobberley et al., 2008). The later three phages were shown to lysogenize their hosts as a linear plasmids with covalently closed telomeres, while VHML was described as integrative. The overall genomic organization of the functional modules was similar across these phages and N15, with packaging, structural, and phage metabolism genes present. The capsid-associated proteins are similar to those from lambda-like siphoviruses, while tail proteins are similar to that from P2-like temperate myoviruses (Mobberley et al., 2008). Homologs of another protein specific for N15-like viruses, RepA replicase, are encoded at UHAP-1, VP882, VP58.5 and VHML genomes. Like in N15, protelomerase and counteroriented repA-like gene are located between the structural gene cluster and k-type lysogeny control region.
9. Conclusions N15-like group of viruses is unique among bacteriophages in genetic organization. The similar overall genome organization, presence of genes associated with linear plasmid lifestyle, – protelomerase and repA-like replicase, in N15, uKO2, PY54, UHAP-1, VP882, VP58.5 and VHML suggest that these phages may belong to a group diverged from a common ancestor. Such ancestor of N15-like phages must have arisen either through the accumulation of new genetic modules from plasmid and bacterial sources by lambdoid progenitor or by an unknown plasmid acquiring a lambdoid set of ‘‘virion’’ genes. Subsequent evolution followed the Botstein’s modular theory (Botstein, 1980), where evolution occurs via the exchange of groups of functional genes between different phages and plasmids. Therefore, N15 and its relatives provide a very interesting model system for the study of phage and plasmid evolution and interactions between phages, plasmids and bacterial hosts.
Acknowledgments The author’s work was supported by the Program ‘‘Molecular and Cell Biology’’ of RAS, and the grant 1004-01204 from Russian Foundation for Basic Research.
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