RESUMEN
Bacteriophage P2 is a temperate phage capable of integrating its DNA into the host genome by site-specific recombination upon lysogenization. Integration and excision of the phage genome requires P2 integrase, which performs recognition, cleavage and joining of DNA during these processes. This work presents the high-resolution crystal structure of the catalytic domain of P2 integrase, and analysis of the structure-function relationship of several previously identified non-functional P2 integrase mutants. The DNA binding area is characterized by a large positively charged patch, harboring key residues. The structure reveals potential for large dimer flexibility, likely essential for rearrangement of DNA strands upon integration and excision of the phage DNA.
Asunto(s)
Bacteriófago P2/enzimología , Dominio Catalítico , Integrasas/química , Integrasas/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Integrasas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Relación Estructura-ActividadRESUMEN
The Cox protein from bacteriophage P2 is a small multifunctional DNA-binding protein. It is involved in site-specific recombination leading to P2 prophage excision and functions as a transcriptional repressor of the P2 Pc promoter. Furthermore, it transcriptionally activates the unrelated, defective prophage P4 that depends on phage P2 late gene products for lytic growth. In this article, we have investigated the structural determinants to understand how P2 Cox performs these different functions. We have solved the structure of P2 Cox to 2.4 Å resolution. Interestingly, P2 Cox crystallized in a continuous oligomeric spiral with its DNA-binding helix and wing positioned outwards. The extended C-terminal part of P2 Cox is largely responsible for the oligomerization in the structure. The spacing between the repeating DNA-binding elements along the helical P2 Cox filament is consistent with DNA binding along the filament. Functional analyses of alanine mutants in P2 Cox argue for the importance of key residues for protein function. We here present the first structure from the Cox protein family and, together with previous biochemical observations, propose that P2 Cox achieves its various functions by specific binding of DNA while wrapping the DNA around its helical oligomer.
Asunto(s)
Proteínas de Unión al ADN/química , ADN/química , Proteínas Virales/química , Secuencia de Aminoácidos , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Multimerización de Proteína , Alineación de Secuencia , Proteínas Virales/metabolismoRESUMEN
A recently isolated phage, vB_EcoP_SU10 (SU10), with the unusual elongated C3 morphotype, can infect a wide range of Escherichia coli strains. We have sequenced the genome of this phage and characterized it further by mass spectrometry based proteomics, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and ultra-thin section electron microscopy. The genome size is 77,327 base pairs and its genes, and genome architecture, show high similarity to the phiEco32 phage genes and genome. The TEM images reveal that SU10 have a quite long tail for being a Podoviridae phage, and that the tail also changes conformation upon infection. The ultra-thin section electron microscopy images of phages at the stage of replication within the host cell show that the phages form a honeycomb-like structure under packaging of genomes and assembly of mature capsids. This implies a tight link between the replication and cutting of the concatemeric genome, genome packaging, and capsid assembly. We have also performed a phylogenetic analysis of the structural genes common between Podoviridae phages of the C1 and C3 morphotypes. The result shows that the structural genes have coevolved, and that they form two distinct groups linked to their morphotypes. The structural genes of C1 and C3 phages appear to have diverged around 280 million years ago applying a molecular clock calibrated according to the presumed split between the Escherichia - Salmonella genera.
Asunto(s)
Genoma Viral , Filogenia , Podoviridae/genética , Proteómica , Mapeo Cromosómico , ADN Viral/química , Genómica , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Podoviridae/ultraestructura , Análisis de Secuencia de ADN , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Bacteriophages T7, λ, P22, and P2/P4 (from Escherichia coli), as well as Ï29 (from Bacillus subtilis), are among the best-studied bacterial viruses. This chapter summarizes published protein interaction data of intraviral protein interactions, as well as known phage-host protein interactions of these phages retrieved from the literature. We also review the published results of comprehensive protein interaction analyses of Pneumococcus phages Dp-1 and Cp-1, as well as coliphages λ and T7. For example, the ≈55 proteins encoded by the T7 genome are connected by ≈43 interactions with another ≈15 between the phage and its host. The chapter compiles published interactions for the well-studied phages λ (33 intra-phage/22 phage-host), P22 (38/9), P2/P4 (14/3), and Ï29 (20/2). We discuss whether different interaction patterns reflect different phage lifestyles or whether they may be artifacts of sampling. Phages that infect the same host can interact with different host target proteins, as exemplified by E. coli phage λ and T7. Despite decades of intensive investigation, only a fraction of these phage interactomes are known. Technical limitations and a lack of depth in many studies explain the gaps in our knowledge. Strategies to complete current interactome maps are described. Although limited space precludes detailed overviews of phage molecular biology, this compilation will allow future studies to put interaction data into the context of phage biology.
Asunto(s)
Fagos de Bacillus/fisiología , Colifagos/fisiología , Mapeo de Interacción de Proteínas , Fagos de Streptococcus/fisiología , Proteínas Virales/metabolismo , Bacillus subtilis/virología , Escherichia coli/virología , Interacciones Huésped-Parásitos , Unión Proteica , Streptococcus pneumoniae/virologíaRESUMEN
The phylogenetic relationships and structural similarities of the proteins encoded within the regulatory region (containing the integrase gene and the lytic-lysogenic transcriptional switch genes) of P2-like phages were analyzed, and compared with the phylogenetic relationship of P2-like phages inferred from four structural genes. P2-like phages are thought to be one of the most genetically homogenous phage groups but the regulatory region nevertheless varies extensively between different phage genomes. The analyses showed that there are many types of regulatory regions, but two types can be clearly distinguished; regions similar either to the phage P2 or to the phage 186 regulatory regions. These regions were also found to be most frequent among the sequenced P2-like phage or prophage genomes, and common in phages using Escherichia coli as a host. Both the phylogenetic and the structural analyses showed that these two regions are related. The integrases as well as the cox/apl genes show a common monophyletic origin but the immunity repressor genes, the type P2 C gene and the type 186 cI gene, are likely of different origin. There was no indication of recombination between the P2-186 types of regulatory genes but the comparison of the phylogenies of the regulatory region with the phylogeny based on four structural genes revealed recombinational events between the regulatory region and the structural genes. Less common regulatory regions were phylogenetically heterogeneous and typically contained a fusion of genes from distantly related or unknown phages and P2-like genes.
RESUMEN
Phage integrases have the potential of becoming tools for safe site-specific integration of genes into unmodified human genomes. The P2-like phages have been found to have different bacterial host integration sites and consequently they have related integrases with different sequence specificities. In this work the site-specific recombination system of the P2-like phage ΦD145 is characterized. The minimal attB site is determined to 22 nt with 18 nt identity to the core region of attP. A non-coding sequence on the human chromosome 13 is shown to be a rather good substrate for recombination in vivo in bacteria as well as in a plasmid system in HeLa cells when HMG protein recognition sequences are inserted between the left arm-binding site and the core in the complex phage attachment site attP. Thus ΦD145 integrase that belongs to the tyrosine family shows potential as a tool for site-specific integration into the human genome.
Asunto(s)
Bacteriófagos/enzimología , Integrasas/metabolismo , Recombinación Genética , Sitios de Ligazón Microbiológica , Secuencia de Bases , Escherichia coli/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Plásmidos , Homología de SecuenciaRESUMEN
Temperate coliphage P2 integrates its genome into the host chromosome upon lysogenization via a site-specific recombination event mediated by an integrase belonging to the complex family of tyrosine recombinases. The host integration site attB (BOB') is localized in the end of the cyaR gene and shares 27 nucleotides with the core of attP (COC'). In the present study we determine the minimal attB site using an in vivo recombination assay. Ten nt on the left side (B) are found to be nonessential for recombination. We show that the integrase has higher affinity for the right side (B') compared to B and that artificial B'OB' and an attP site with a matching core (C'OC') are efficient substrates for recombination in vitro. We have analyzed single nucleotides in attB and find that sequence homology within a non-centrally located quadruplet in the hypothetical overlap region is essential for efficient recombination in vivo.
Asunto(s)
Bacteriófago P2/enzimología , Bacteriófago P2/genética , Regulación Viral de la Expresión Génica/fisiología , Integrasas/metabolismo , Unión Proteica , Recombinación Genética , Sitios de Ligazón Microbiológica/genética , Secuencia de Bases , Escherichia coli/clasificación , Escherichia coli/virología , Regulación Enzimológica de la Expresión Génica , Integrasas/genética , Mutación Puntual , Alineación de SecuenciaRESUMEN
As opposed to the vast majority of prokaryotic repressors, the immunity repressor of temperate Escherichia coli phage P2 (C) recognizes non-palindromic direct repeats of DNA rather than inverted repeats. We have determined the crystal structure of P2 C at 1.8 Å. This constitutes the first structure solved from the family of C proteins from P2-like bacteriophages. The structure reveals that the P2 C protein forms a symmetric dimer oriented to bind the major groove of two consecutive turns of the DNA. Surprisingly, P2 C has great similarities to binders of palindromic sequences. Nevertheless, the two identical DNA-binding helixes of the symmetric P2 C dimer have to bind different DNA sequences. Helix 3 is identified as the DNA-recognition motif in P2 C by alanine scanning and the importance for the individual residues in DNA recognition is defined. A truncation mutant shows that the disordered C-terminus is dispensable for repressor function. The short distance between the DNA-binding helices together with a possible interaction between two P2 C dimers are proposed to be responsible for extensive bending of the DNA. The structure provides insight into the mechanisms behind the mutants of P2 C causing dimer disruption, temperature sensitivity and insensitivity to the P4 antirepressor.
Asunto(s)
ADN/química , Secuencias Repetitivas de Ácidos Nucleicos , Proteínas Virales/química , Alanina/química , Secuencia de Aminoácidos , Bacteriófago P2/fisiología , Secuencia de Bases , Sitios de Unión , Cristalografía , Dimerización , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Regiones Operadoras Genéticas , Unión Proteica , Eliminación de Secuencia , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The Cox protein of the coliphage P2 is multifunctional; it acts as a transcriptional repressor of the Pc promoter, as a transcriptional activator of the P(LL) promoter of satellite phage P4, and as a directionality factor for site-specific recombination. The Cox proteins constitute a unique group of directionality factors since they couple the developmental switch with the integration or excision of the phage genome. In this work, the DNA binding characteristics of the Cox protein of WPhi, a P2-related phage, are compared with those of P2 Cox. P2 Cox has been shown to recognize a 9 bp sequence, repeated at least 6 times in different targets. In contrast to P2 Cox, WPhi Cox binds with a strong affinity to the early control region that contains an imperfect direct repeat of 12 nucleotides. The removal of one of the repeats has drastic effects on the capacity of WPhi to bind to the Pe-Pc region. Again in contrast to P2 Cox, WPhi Cox has a lower affinity to attP compared to the Pe-Pc region, and a repeat of 9 bp can be found that has 5 bp in common with the repeat in the Pe-Pc region. WPhi Cox, however, is essential for excisive recombination in vitro. WPhi Cox, like P2 Cox, binds cooperatively with integrase to attP. Both Cox proteins induce a strong bend in their DNA targets upon binding.
Asunto(s)
Bacteriófago P2/genética , Bacteriófago P2/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Virales/metabolismo , Integración Viral , Sitios de Ligazón Microbiológica/fisiología , Bacteriófago P2/inmunología , ADN Viral/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Unión Proteica , Proteínas Virales/genéticaRESUMEN
This note presents the (1)H, (13)C, and (15)N resonances assignment of the 22 kDa, dimeric, C-repressor protein from the P2 bacteriophage. The C-repressor controls the genetic switch that determines if the temperate P2 phage should exist in the lytic or lysogenic lifemode.
Asunto(s)
Bacteriófago P2/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Proteínas Represoras/química , Proteínas Virales/química , Secuencia de Aminoácidos , Isótopos de Carbono/química , Datos de Secuencia Molecular , Peso Molecular , Isótopos de Nitrógeno/química , Estructura Terciaria de Proteína , ProtonesRESUMEN
Bacteriophages P2 and WPhi are heteroimmune members of the P2-like family of temperate Escherichia coli phages. Temperate phages can grow lytically or form lysogeny after infection. A transcriptional switch that contains two con-vergent promoters, Pe and Pc, and two repressors regulate what life mode to enter. The immunity repressor C is the first gene of the lysogenic operon, and it blocks the early Pe promoter. In this work, some characteristics of the C proteins of P2 and WPhi are compared. An in vivo genetic analysis shows that WPhi C, like P2 C, has a strong dimerization activity in the absence of its DNA target. Both C proteins recognize two directly repeated sequences, termed half-sites and a strong bending is induced in the respective DNA target upon binding. P2 C is unable to bind to one half-site as opposed to WPhi, but both half-sites are required for repression of WPhi Pe. A reduction from three to two helical turns between the centers of the half-sites in WPhi has no significant effect on the capacity to repress Pe. However, the protein-DNA complexes formed differ, as determined by electrophoretic mobility shift experiments. A difference in spontaneous phage production is observed in isogenic lysogens.
Asunto(s)
Bacteriófago P2/genética , Colifagos/genética , Regiones Operadoras Genéticas , Proteínas Represoras/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Bacteriófago P2/crecimiento & desarrollo , Sitios de Unión , Colifagos/crecimiento & desarrollo , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Dimerización , Regulación Viral de la Expresión Génica , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Proteínas Represoras/química , Proteínas Virales/químicaRESUMEN
The structural genes of P2-like phages are almost identical between different isolates of Escherichia coli, whereas the regulatory genes and host integration sites are more variable. The variation in P2-like phages infecting other gamma-proteobacteria is broader, but their structural genes seem to follow the evolution of their host bacteria. Taken together, this suggests that P2-like phages and their hosts are coevolving.
Asunto(s)
Bacteriófago P2/genética , Evolución Molecular , Gammaproteobacteria/genética , Proteínas Virales/genéticaRESUMEN
Bacteriophages P2, P2 Hy dis and WPhi are very similar but heteroimmune Escherichia coli phages. The structural genes show over 96% identity, but the repressors show between 43 and 63% identities. Furthermore, the operators, which contain two directly repeated sequences, vary in sequence, length, location relative to the promoter and spacing between the direct repeats. We have compared the in vivo effects of the wild type and mutated operators on gene expression with the complexes formed between the repressors and their wild type or mutated operators using electrophoretic mobility shift assay (EMSA), and real-time kinetics of the protein-DNA interactions using surface plasmon resonance (SPR) analysis. Using EMSA, the repressors formed different protein-DNA complexes, and only WPhi was significantly affected by point mutations. However, SPR analysis showed a reduced association rate constant and an increased dissociation rate constant for P2 and WPhi operator mutants. The association rate constants of P2 Hy dis was too fast to be determined. The P2 Hy dis dissociation response curves were shown to be triphasic, while both P2 and WPhi C were biphasic. Thus, the kinetics of complex formation and the nature of the complexes formed differ extensively between these very closely related phages.
Asunto(s)
Colifagos/genética , Regiones Operadoras Genéticas , Proteínas Represoras/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Bacteriófago P2/genética , Sitios de Unión , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Regulación Viral de la Expresión Génica , Genes Reporteros , Cinética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Mutación Puntual , Regiones Promotoras Genéticas , Proteínas Represoras/química , Resonancia por Plasmón de Superficie , Proteínas Virales/químicaRESUMEN
The amount and distribution of variation in the genomic region containing the genes in the lytic-lysogenic genetic switch and the sequence that determines the integration site into the host chromosome were analyzed for 38 P2-like phages from Escherichia coli. The genetic switch consists of two convergent mutually exclusive promoters, Pe and Pc, and two repressors, C and Cox. The immunity repressor C blocks the early Pe promoter, leading to the establishment of lysogeny. The Cox repressor blocks expression of Pc, allowing lytic growth. Phylogenetic analyses showed that the C and Cox proteins were distributed into seven distinct classes. The phylogenetic relationship differed between the two proteins, and we showed that homologous recombination plays a major role in creating alterations in the genetic switch, leading to new immunity classes. Analyses of the host integration site for these phages resulted in the discovery of a previously unknown site, and there were at least four regular integration sites. Interestingly, we found no case where phages of the same immunity class had different host attachment sites. The evolution of immunity and integration sites is complex, since it involves interactions both between the phages themselves and between phages and hosts, and often, both regulatory proteins and target DNA must change.
Asunto(s)
Bacteriófago P2/genética , Bacteriófago P2/inmunología , Cromosomas Bacterianos/genética , Escherichia coli/genética , Escherichia coli/virología , Lisogenia , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Escherichia coli/clasificación , Datos de Secuencia Molecular , Filogenia , Plásmidos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
The P2-like coliphages are highly similar; the structural genes show at least 96% identity. However, at two loci they have genes believed to be horizontally transferred. We show that the genetic content at the second loci, the TO region, contains six completely different sequences with high AT contents and with different open reading frames. The product of one of them exhibits reverse transcriptase activity and blocks infection of phage T5.
Asunto(s)
Bacteriófago P2/genética , Genes Virales , ADN Polimerasa Dirigida por ARN/genética , Bacteriófago P2/enzimología , Secuencia de Bases , ADN Bacteriano , Variación Genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , ADN Polimerasa Dirigida por ARN/metabolismo , Alineación de SecuenciaRESUMEN
Bacteriophage P2 integrase (Int) mediates site-specific recombination leading to integration or excision of the phage genome in or out of the bacterial chromosome. Int belongs to the large family of tyrosine recombinases that have two different DNA recognition motifs binding to the arm and core sites, respectively, which are located within the phage attachment sites (attP). In addition to the P2 integrase, the accessory proteins Escherichia coli IHF and P2 Cox are needed for recombination. IHF is a structural protein needed for integration and excision by bending the DNA. As opposed to lambda, only one IHF site is found in P2 attP. P2 Cox controls the direction of recombination by inhibiting integration but being required for excision. In this work, the effects of accessory proteins on the capacity of Int to bind to its DNA recognition sequences are analyzed using electromobility shifts. P2 Int binds with low affinity to the arm site, and this binding is greatly enhanced by IHF. The arm binding domain of Int is located at the N-terminus. P2 Int binds with high affinity to the core site, and this binding is also enhanced by IHF. The fact that the cooperative binding of Int and IHF is strongly reduced by lengthening the distance between the IHF and core binding sites indicates that the distance between these sites may be important for cooperative binding. The Int and Cox proteins also bind cooperatively to attP.
Asunto(s)
Bacteriófago P2/enzimología , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/virología , Integrasas/metabolismo , Factores de Integración del Huésped/metabolismo , Proteínas Virales/metabolismo , Bacteriófago P2/genética , Bacteriófago P2/metabolismo , Sitios de Unión , Escherichia coli/metabolismo , Integración ViralRESUMEN
Coliphage P2 integrates into the host chromosome upon lysogenization via site-specific recombination mediated by the phage integrase (Int). P2 integrase belongs to the tyrosine family of recombinases. In this work, it is shown that P2 integrase forms dimers but not oligomers in the absence of its DNA target. Furthermore, the C-terminal end of the protein and amino acid (aa) E197 have been found to be involved in dimerization. Amino acid E197 is located in a conserved region of the tyrosine recombinases that has not previously been implicated in dimerization. The dimerization deficient mutants were unaffected in binding to its phage attachment site (attP) substrate, but had a reduced ability to complement an int-defective prophage.
Asunto(s)
Bacteriófago P2/enzimología , ADN/metabolismo , Integrasas/metabolismo , Secuencia de Aminoácidos , Sitios de Ligazón Microbiológica/genética , Sitios de Unión/genética , Dimerización , Electroforesis en Gel de Poliacrilamida , Integrasas/química , Integrasas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Conformación Proteica , Recombinación Genética/genética , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The genome of the tailed temperate coliphage P2 (Myoviridae) contains some genes that probably are horizontally transferred additions to the genome. One of these genes, the Z/fun gene, was recently found intact in the genome of Neisseria meningitidis. We have investigated the presence of P2-like phages, and the genetic variation at the position corresponding to the phage P2 Z/fun locus, in the Escherichia coli reference collection (ECOR). P2-like phages are common in E. coli since they are present in about 30% of the ECOR strains. Hybridizations and PCR amplifications indicate that the overall variation among these phages is small. Amplification of the region corresponding to the phage P2 Z/fun locus in 11 prophages revealed that this is a multivariable locus. Sequencing of the region resulted in 10 completely different sequences but with a similar high AT-content as the Z/fun gene. All sequences contained at least one open reading frame with good transcription and translation signals. All sequences were also surrounded by a highly similar, previously undiscovered, inverted repeat (IR). We also found this IR in genetically unstable regions in pathogenic enterobacteria. This demonstrates that P2-like phages are important factors in the evolution of bacteria, not only because they carry a diversity of lysogenic conversion genes but also because they can act as vectors for single genes. The genes found between the IRs have unknown functions, and only a few clearly similar genes have been found in other bacteria.