Phage N15 Research Articles

Phage N15-Based Vectors for Gene Cloning and Expression in Bacteria and Mammalian Cells.

Bacteriophage N15 is the first virus known to deliver linear prophage into Escherichia coli. During its lysogenic cycle, N15 protelomerase (TelN) resolves its telomerase occupancy site (tos) into hairpin telomeres. This protects the N15 prophage from bacterial exonuclease degradation, enabling it to stably replicate as a linear plasmid in E. coli. Interestingly, purely proteinaceous TelN can retain phage DNA linearization and hairpin formation without involving host- or phage-derived intermediates or cofactors in the heterologous environment. This unique feature has led to the advent of synthetic linear DNA vector systems derived from the TelN-tos module for the genetic engineering of bacterial and mammalian cells. This review will focus on the development and advantages of N15-based novel cloning and expression vectors in the bacterial and mammalian environments. To date, N15 is the most widely exploited molecular tool for the development of linear vector systems, especially the production of therapeutically useful miniDNA vectors without a bacterial backbone. Compared to typical circular plasmids, linear N15-based plasmids display remarkable cloning fidelity in propagating unstable repetitive DNA sequences and large genomic fragments. Additionally, TelN-linearized vectors with the relevant origin of replication can replicate extrachromosomally and retain transgenes functionality in bacterial and mammalian cells without compromising host cell viability. Currently, this DNA linearization system has shown robust results in the development of gene delivery vehicles, DNA vaccines and engineering mammalian cells against infectious diseases or cancers, highlighting its multifaceted importance in genetic studies and gene medicine.

The coevolution of large and small terminases of bacteriophages is a result of purifying selection leading to phenotypic stabilization

Genome packaging in many dsDNA phages requires a series of precisely coordinated actions of two phage-coded proteins, namely, large terminase (TerL) and small terminase (TerS) with DNA and ATP, and with each other. Despite the strict functional conservation, TerL and TerS homologs exhibit large sequence variations. We investigated the sequence variability across eight phage types and observed a coevolutionary framework wherein the genealogy of TerL homologs mirrored that of the corresponding TerS homologs. Furthermore, a high purifying selection observed (dN/dS«1) indicated strong structural constraints on both TerL and TerS, and identify coevolving residues in TerL and TerS of phage T4 and lambda. Using the highly coevolving (correlation coefficient of 0.99) TerL and TerS of phage N4, we show that their biochemical features are similar to the phylogenetically divergent phage λ terminases. We also demonstrate using the Surface Plasma Resonance (SPR) technique that phage N4 TerL transiently interacts with TerS.

High-throughput mapping of the phage resistance landscape in E. coli.

Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage-specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and high-throughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phage-mediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.

From Orphan Phage to a Proposed New Family-the Diversity of N4-Like Viruses.

Escherichia phage N4 was isolated in 1966 in Italy and has remained a genomic orphan for a long time. It encodes an extremely large virion-associated RNA polymerase unique for bacterial viruses that became characteristic for this group. In recent years, due to new and relatively inexpensive sequencing techniques the number of publicly available phage genome sequences expanded rapidly. This revealed new members of the N4-like phage group, from 33 members in 2015 to 115 N4-like viruses in 2020. Using new technologies and methods for classification, the Bacterial and Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) has moved the classification and taxonomy of bacterial viruses from mere morphological approaches to genomic and proteomic methods. The analysis of 115 N4-like genomes resulted in a huge reassessment of this group and the proposal of a new family “Schitoviridae”, including eight subfamilies and numerous new genera.

A Self-Replicating Linear DNA.

TelN and tos are a unique DNA linearization unit isolated from bacteriophage N15. While being transferable, the TelN cleaving-rejoining activities remained stable to function on tos in both bacterial and mammalian environments. However, TelN contribution in linear plasmid replication in mammalian cells remains unknown. Herein, we investigated the association of TelN in linear tos-containing DNA (tos-DNA) replication in mammalian cells. Additionally, the mammalian origin of replication (ori) that is well-known to initiate the replication event of plasmid vectors was also studied. In doing so, we identified that both TelN and mammalian initiation sites were essential for the replication of linear tos-DNA, determined by using methylation sensitive DpnI/MboI digestion and polymerase chain reaction (PCR) amplification approaches. Furthermore, we engineered the linear tos-DNA to be able to retain in mammalian cells using S/MAR technology. The resulting S/MAR containing tos-DNA was robust for at least 15 days, with (1) continuous tos-DNA replication, (2) correct splicing of gene transcripts, and (3) stable exogenous gene expression that was statistically comparable to the endogenous gene expression level. Understanding the activities of TelN and tos in mammalian cells can potentially provide insights for adapting this simple DNA linearization unit in developing novel genetic engineering tools, especially to the eukaryotic telomere/telomerase study.

Diversity of Sinorhizobium (Ensifer) meliloti Bacteriophages in the Rhizosphere of Medicago marina: Myoviruses, Filamentous and N4-Like Podovirus.

Using different Sinorhizobium meliloti strains as hosts, we isolated eight new virulent phages from the rhizosphere of the coastal legume Medicago marina. Half of the isolated phages showed a very narrow host range while the other half exhibited a wider host range within the strains tested. Electron microscopy studies showed that phages M_ort18, M_sf1.2, and M_sf3.33 belonged to the Myoviridae family with feature long, contractile tails and icosaedral head. Phages I_sf3.21 and I_sf3.10T appeared to have filamentous shape and produced turbid plaques, which is a characteristic of phages from the Inoviridae family. Phage P_ort11 is a member of the Podoviridae, with an icosahedral head and a short tail and was selected for further characterization and genome sequencing. P_ort11 contained linear, double-stranded DNA with a length of 75239 bp and 103 putative open reading frames. BLASTP analysis revealed strong similarities to Escherichia phage N4 and other N4-like phages. This is the first report of filamentous and N4-like phages that infect S. meliloti.

Complete Genome Sequence of Klebsiella pneumoniae Podophage Pylas.

Carbapenemase-producing Klebsiella pneumoniae is an important opportunistic pathogen due to its drug resistance. This study reports on the isolation and characterization of a podophage, named Pylas, infecting this bacterium. The complete genome of phage Pylas is described, and it is distantly related to the well-studied phage N4.

Complete Genome Sequence of Stenotrophomonas Phage Pokken.

Stenotrophomonas maltophilia is a Gram-negative bacterium associated with multidrug-resistant nosocomial infections, a problem for immunocompromised patients and those with cystic fibrosis. Here, we present the new S. maltophilia-infecting podophage Pokken. Its 76,239-bp genome, with 92 protein-coding genes and 5 tRNA genes predicted, is similar to that of phage N4.

Phage N15 protelomerase resolves its tos recognition site into hairpin telomeres within mammalian cells

Phage N15 protelomerase (TelN) cleaves double-stranded circular DNA containing a telomerase-occupancy-site (tos) and rejoins the resulting linear-ends to form closed-hairpin-telomeres in Escherichia coli (E. coli). Continued TelN expression is essential to support resolution of the linear structure. In mammalian cells, no enzyme with TelN-like activities has been found. In this work, we show that phage TelN, expressed transiently and stably in human and mouse cells, recapitulates its native activities in these exogenous environments. We found TelN to accurately resolve tos-DNA in vitro and in vivo within human and mouse cells into linear DNA-containing terminal telomeres that are resistant to RecBCD degradation, a hallmark of protelomerase processing. In stable cells, TelN activity was detectable for at least 60 days, which suggests the possibility of limited silencing of its expression. Correspondingly, linear plasmid containing a 100 kb human β-globin gene expressed for at least 120 h in non-β-globin-expressing mouse cells with TelN presence. Our results demonstrate TelN is able to cut and heal DNA as hairpin-telomeres within mammalian cells, providing a tool for creating novel structures by DNA resolution in these hosts. The TelN protelomerase may be useful for exploring novel technologies for genome interrogation and chromosome engineering.

Extreme divergence between one-to-one orthologs: the structure of N15 Cro bound to operator DNA and its relationship to the λ Cro complex

The gene cro promotes lytic growth of phages through binding of Cro protein dimers to regulatory DNA sites. Most Cro proteins are one-to-one orthologs, yet their sequence, structure and binding site sequences are quite divergent across lambdoid phages. We report the cocrystal structure of bacteriophage N15 Cro with a symmetric consensus site. We contrast this complex with an orthologous structure from phage λ, which has a dissimilar binding site sequence and a Cro protein that is highly divergent in sequence, dimerization interface and protein fold. The N15 Cro complex has less DNA bending and smaller DNA-induced changes in protein structure. N15 Cro makes fewer direct contacts and hydrogen bonds to bases, relying mostly on water-mediated and Van der Waals contacts to recognize the sequence. The recognition helices of N15 Cro and λ Cro make mostly nonhomologous and nonanalogous contacts. Interface alignment scores show that half-site binding geometries of N15 Cro and λ Cro are less similar to each other than to distantly related CI repressors. Despite this divergence, the Cro family shows several code-like protein–DNA sequence covariations. In some cases, orthologous genes can achieve a similar biological function using very different specific molecular interactions.

Minimalism and functionality: Structural lessons from the heterodimeric N4 bacteriophage RNA polymerase II

Genomes of phages, mitochondria, and chloroplasts are transcribed by a diverse group of transcriptional machineries with structurally related single-subunit RNA polymerases (RNAPs). Our understanding of transcription mechanisms of these enzymes is predominantly based on biochemical and structural studies of three most-studied members, transcription factor-independent phage T7 RNAP, transcription factor-dependent phage N4 virion-encapsidated RNAP, and transcription factor-dependent mitochondrial RNAPs (mtRNAP). Although these RNAPs employ completely different mechanisms for promoter recognition and transcription termination, these enzymes are relatively large and formed by single polypeptides. Historically being a model enzyme for studying the mechanisms of transcription by T7-like RNAPs, however, T7 RNAP represents only a small group of RNAPs in this family. The vast majority of T7-like RNAPs are transcription factor-dependent, and several of them are heterodimeric enzymes. Here, we report X-ray crystal structures of transcription complexes of the smallest and heterodimeric form of T7-like RNAP, bacteriophage N4 RNAPII, providing insights into the structural organization of a minimum RNAP in this family. We analyze structural and functional aspects of heterodimeric architecture of N4 RNAPII concerning the mechanisms of transcription initiation and transition to processive RNA elongation. Interestingly, N4 RNAPII maintains the same conformation in promoter-bound and elongation transcription complexes, revealing a novel transcription mechanism for single-subunit RNAPs. This work establishes a structural basis for studying mechanistic aspects of transcription by factor-dependent minimum RNAP.

Novel N4-Like Bacteriophages of Pectobacterium atrosepticum.

Pectobacterium atrosepticum is an economically important phytopathogen that is responsible for potato blackleg and soft rot, and for which current control strategies are limited. In this study, stem samples of potato crops exhibiting blackleg were taken from three farms in Co. Cork, Ireland, and they were found to be infected with P. atrosepticum. Three closely related bacteriophages (phages) that are specific to this phytopathogen were isolated and characterized, namely vB_PatP_CB1, vB_PatP_CB3, and vB_PatP_CB4 (abbreviated as CB1, CB3, and CB4). Both CB1 and CB3 were determined to infect 12 strains and CB4 10 strains of the 19 strains of P. atrosepticum tested. Morphology, latent periods, burst sizes, and their stability at various temperatures and pHs were also examined. Genome sequencing of the three phages revealed that they shared a minimum nucleotide identity of 93% with each other. Their genomes exhibited an Enquartavirinae genome organization, possessing several conserved proteins that were associated with phages of this group, like the type species Escherichia virus N4. Tandem electrospray ionization-mass spectrometry (ESI-MS/MS) allowed for the identification of ten structural proteins that form the virion of CB1, six that are conserved in phage N4. Biocontrol experiments demonstrated that the phages suppress soft rot formation upon co-inoculation with P. atrosepticum on whole tubers. The results of this study indicate that CB1 related phages could be good candidates for phage-based control.

Characterization of Tail Sheath Protein of N4-Like Phage phiAxp-3.

Achromobacter phage phiAxp-3, an N4-like bacteriophage, specifically recognize Achromobacter xylosoxidans lipopolysaccharide (LPS) as its receptor. PhiAxp-3 tail sheath protein (TSP, ORF69) shares 54% amino acid sequence identity with the TSP of phage N4 (gp65); the latter functions as a receptor binding protein and interacts with the outer membrane receptor NfrA of its host bacterium. Thus, we hypothesized that ORF69 is the receptor-binding protein of phiAxp-3. In the present study, a series of ORF69 truncation variants was constructed to identify the part(s) of this protein essential for binding to A. xylosoxidans LPS. Phage adsorption and enzyme-linked immunosorbent assay showed that amino acids 795–1195 of the TSP, i.e., ORF69(795–1195), are sufficient and essential for receptor and binding. The optimum temperature and pH for the functions of ORF69 and ORF69(795–1195) are 4/25°C and 7, respectively. In vitro cytotoxicity assays showed that ORF69 and ORF69(795–1195) were respectively toxic and non-toxic to a human immortalized normal hepatocyte cell line (LO2; doses: 0.375–12 μg). The potential of this non-toxic truncated version of phiASP-3 TSP for clinical applications is discussed.

Binding Specificities of the Telomere Phage ϕKO2 Prophage Repressor CB and Lytic Repressor Cro.

Temperate bacteriophages possess a genetic switch which regulates the lytic and lysogenic cycle. The genomes of the temperate telomere phages N15, PY54, and ϕKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor (cI or cB), the lytic repressor (cro) and a putative antiterminator (q). The roles of these products are thought to be similar to those of the lambda proteins CI (CI prophage repressor), Cro (Cro repressor), and Q (antiterminator Q), respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ϕKO2 are reminiscent of lambda-like phages. We determined binding sites of the ϕKO2 prophage repressor CB and lytic repressor Cro on the ϕKO2 genome in detail by electrophoretic mobility shift assay (EMSA) studies. Unexpectedly, ϕKO2 CB and Cro revealed different binding specificities. CB was bound to three OR operators in the intergenic region between cB and cro, two OL operators between cB and the replication gene repA and even to operators of N15. Cro bound exclusively to the 16 bp operator site OR3 upstream of the ϕKO2 prophage repressor gene. The ϕKO2 genes cB and cro are regulated by several strong promoters overlapping with the OR operators. The data suggest that Cro represses cB transcription but not its own synthesis, as already reported for PY54 Cro. Thus, not only PY54, but also phage ϕKO2 possesses a genetic switch that diverges significantly from the switch of lambda-like phages.

The diverse genetic switch of enterobacterial and marine telomere phages.

Temperate bacteriophages possess a genetic switch which regulates the lytic and lysogenic cycle. The genomes of the enterobacterial telomere phages N15, PY54 and ϕKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator, similar to CI, Cro and Q of lambda, respectively. Moreover, N15 and ϕKO2 contain 3 related operator (OR) sites between cI and cro, while only one site (OR3) has been detected in PY54. Marine telomere phages possess a putative cI gene but not a cro-like gene. Instead, a gene is located at the position of cro, whose product shows some similarity to the PY54 ORF42 product, the function of which is unknown. We have determined the transcription start sites of the predicted repressor genes of N15, PY54, ϕKO2 and of the marine telomere phage VP58.5. The influence of the genes on phage propagation was analyzed in E. coli, Y. enterocolitica and V.parahaemolyticus. We show that the repressors and antiterminators of N15, ϕKO2 and PY54 exerted their predicted activities. However, while the proteins of both N15 and ϕKO2 affected lysis and lysogeny by N15, they did not affect PY54 propagation. On the other hand, the respective PY54 proteins exclusively influenced the propagation of this phage. The immB region of VP58.5 contains 2 genes that revealed prophage repressor activity, while a lytic repressor gene could not be identified. The results indicate an unexpected diversity of the growth regulation mechanisms in these temperate phages.

DNA Packaging Specificity of Bacteriophage N15 with an Excursion into the Genetics of a Cohesive End Mismatch.

During DNA replication by the λ-like bacteriophages, immature concatemeric DNA is produced by rolling circle replication. The concatemers are processed into mature chromosomes with cohesive ends, and packaged into prohead shells, during virion assembly. Cohesive ends are generated by the viral enzyme terminase, which introduces staggered nicks at cos, an approx. 200 bp-long sequence containing subsites cosQ, cosN and cosB. Interactions of cos subsites of immature concatemeric DNA with terminase orchestrate DNA processing and packaging. To initiate DNA packaging, terminase interacts with cosB and nicks cosN. The cohesive ends of N15 DNA differ from those of λ at 2/12 positions. Genetic experiments show that phages with chromosomes containing mismatched cohesive ends are functional. In at least some infections, the cohesive end mismatch persists through cyclization and replication, so that progeny phages of both allelic types are produced in the infected cell. N15 possesses an asymmetric packaging specificity: N15 DNA is not packaged by phages λ or 21, but surprisingly, N15-specific terminase packages λ DNA. Implications for genetic interactions among λ-like bacteriophages are discussed.

Taxonomic reassessment of N4-like viruses using comparative genomics and proteomics suggests a new subfamily - "Enquartavirinae".

The GenBank database currently contains sequence data for 33 N4-like viruses, with only one, Escherichia phage N4, being formally recognized by the ICTV. The genus N4likevirus is uniquely characterized by that fact that its members possess an extremely large, virion-associated RNA polymerase. Using a variety of proteomic, genomic and phylogenetic tools, we have demonstrated that the N4-like phages are not monophyletic and that N4 is actually a genomic orphan. We propose to create four new genera: "G7cvirus" (consisting of phages G7C, IME11, KBNP21, vB_EcoP_PhAPEC5, vB_EcoP_PhAPEC7, Bp4, EC1-UPM and pSb-1), "Lit1virus" (LIT1, PA26 and vB_PaeP_C2-10_Ab09), "Sp58virus" (SP058 and SP076), and "Dss3virus" (DSS3φ2 and EE36φ1). We propose that coliphage N4, the members of "G7cvirus", Erwinia phage Ea9-2, and Achromobacter phage JWAlpha should be considered members of the same subfamily, which we tentatively call the "Enquartavirinae".

The Molecular Switch of Telomere Phages: High Binding Specificity of the PY54 Cro Lytic Repressor to a Single Operator Site.

Temperate bacteriophages possess a molecular switch, which regulates the lytic and lysogenic growth. The genomes of the temperate telomere phages N15, PY54 and ϕKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator. The roles of these products are thought to be similar to those of the lambda proteins CI, Cro and Q, respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ϕKO2 are also reminiscent of lambda-like phages. By contrast, in silico analyses revealed the presence of only one operator (OR3) in PY54. The purified PY54 Cro protein was used for EMSA studies demonstrating that it exclusively binds to a 16-bp palindromic site (OR3) upstream of the prophage repressor gene. The OR3 operator sequences of PY54 and ϕKO2/N15 only differ by their peripheral base pairs, which are responsible for Cro specificity. PY54 cI and cro transcription is regulated by highly active promoters initiating the synthesis of a homogenious species of leaderless mRNA. The location of the PY54 Cro binding site and of the identified promoters suggests that the lytic repressor suppresses cI transcription but not its own synthesis. The results indicate an unexpected diversity of the growth regulation mechanisms in lambda-related phages.

Novel DNA packaging recognition in the unusual bacteriophage N15

Phage lambda׳s cosB packaging recognition site is tripartite, consisting of 3 TerS binding sites, called R sequences. TerS binding to the critical R3 site positions the TerL endonuclease for nicking cosN to generate cohesive ends. The N15 cos (cosN15) is closely related to cosλ, but whereas the cosBN15 subsite has R3, it lacks the R2 and R1 sites and the IHF binding site of cosBλ. A bioinformatic study of N15-like phages indicates that cosBN15 also has an accessory, remote rR2 site, which is proposed to increase packaging efficiency, like R2 and R1 of lambda. N15 plus five prophages all have the rR2 sequence, which is located in the TerS-encoding 1 gene, approximately 200bp distal to R3.An additional set of four highly related prophages, exemplified by Monarch, has R3 sequence, but also has R2 and R1 sequences characteristic of cosB–λ. The DNA binding domain of TerS-N15 is a dimer.

Replication and Maintenance of Linear Phage-Plasmid N15.

The lambdoid phage N15 of Escherichia coli is very unusual among temperate phages in that its prophage is not integrated into the chromosome but is a linear plasmid molecule with covalently closed ends (telomeres). Upon infection, the phage DNA circularizes via cohesive ends, and then a special phage enzyme of the tyrosine recombinase family, protelomerase, cuts at another site and joins the ends, forming hairpin telomeres of the linear plasmid prophage. Replication of the N15 prophage is initiated at an internally located ori site and proceeds bidirectionally, resulting in the formation of duplicated telomeres. The N15 protelomerase cuts them, generating two linear plasmid molecules with hairpin telomeres. Stable inheritance of the plasmid prophage is ensured by a partitioning operon similar to the F factor sop operon. Unlike the F centromere, 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 the N15 genome regions involved in phage replication and control of lytic development, and binding of partition proteins at these sites regulates these processes. The family of N15-like linear phage-plasmids includes lambdoid phages ɸKO2 and pY54, as well as Myoviridae phages ΦHAP-1, VHML, VP882, Vp58.5, and vB_VpaM_MAR of marine gamma-proteobacteria. The genomes of these phages contain similar protelomerase genes, lysogeny control modules, and replication genes, suggesting that these phages may belong to a group diverged from a common ancestor.