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  • 1. Ahlgren-Berg, Alexandra
    et al.
    Cardoso-Palacios, Carlos
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Eriksson, Jesper M.
    Mandali, Sridhar
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sehlén, Wilhelmina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sylwan, Lina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    A comparative analysis of the bifunctional Cox proteins of two heteroimmune P2-like phages with different host integration sites2009In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 385, no 2, p. 303-12Article in journal (Refereed)
    Abstract [en]

    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.

  • 2.
    Ahlgren-Berg, Alexandra
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Henriksson-Peltola, Petri
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sehlén, Wilhelmina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    A comparison of the DNA binding and bending capacities and the oligomeric states of the immunity repressors of heteroimmune coliphages P2 and W Phi2007In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 35, no 10, p. 3167-3180Article in journal (Refereed)
    Abstract [en]

    Bacteriophages P2 and W Phi 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 convergent 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 W Phi are compared. An in vivo genetic analysis shows that W Phi C, like P2C, 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. P2C is unable to bind to one half-site as opposed to W Phi, but both half-sites are required for repression of W Phi Pe. A reduction from three to two helical turns between the centers of the half-sites in W Phi 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.

  • 3.
    Cardoso-Palacios, Carlos
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sylwan, Lina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Mandali, Sridhar
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Frumerie, Clara
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    A structure-function analysis of P2 integraseManuscript (preprint) (Other academic)
    Abstract [en]

    Bacteriophage P2 integrase catalyzes site-specific recombination between the phage DNA and the host chromosome thereby promoting integration or excision of the phage genome. P2 integrase belongs to the large tyrosine family of integrases that shows little sequence identity besides some conserved boxes and patches in the catalytic domain. However, the overall structure of the tyrosine family of integrases seems to be similar. Phage integrases have the potential as tools for site-specific gene insertions into eukaryotic genomes provided that target sequences are available. To elucidate the possibility of evolving the P2 integrase to accept new targets, we have in this work initiated a structure-function analysis of the P2 integrase using two approaches based on a comparison of the predicted secondary structure of P2 integrase with that determined for the lambda integrase. First, we have made hybrids between P2 integrase and the related WΦ integrase that has a different host DNA target, to locate the region promoting specificity between the integrases. This, however, has not been possible, the N-terminal domains can be exchanged without losing biological activity and this will not affect the specificity. All other hybrids made were biological inactive. Next we have made an alanine scanning of the alpha helices believed to be involved in specific interactions with the target, and four amino acids have been identified as candidates for sequence-specific interactions with the core.

  • 4.
    Frumerie, Clara
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sylwan, Lina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Ahlgren-Berg, Alexandra
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Cooperative interactions between bacteriophage P2 integrase and its accessory factors IHF and Cox2005In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 332, no 1, p. 284-294Article in journal (Refereed)
    Abstract [en]

    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.

  • 5.
    Frumerie, Clara
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sylwan, Lina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Yu, Anna
    Department of Clinical Chemistry, Danderyd Hospital and Karolinska Institute, Stockholm, Sweden.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Bacteriophage P2 integrase: another possible tool for site-specific recombination in eukaryotic cells2008In: Journal of Applied Microbiology, ISSN 1364-5072, E-ISSN 1365-2672, Vol. 105, no 1, p. 290-299Article in journal (Refereed)
    Abstract [en]

    AIMS: To investigate if the site-specific tyrosine integrase (Int) from phage P2 has features that would make it interesting for use of gene transfer into eukaryotic cells. These include the possibility of promoting recombination with a nonphage sequence, abolishing the requirement for the bacterial DNA-binding and -bending protein integration host factor (IHF), and localization to the nucleus of eukaryotic cells. METHODS AND RESULTS: We show that the Int protein catalyzes site-specific recombination using a human sequence in Escherichia coli and in vitro although not as efficiently as with the wild-type bacterial sequence, and that insertion of high mobility group recognition boxes in the phage attachment site substrate abolish the requirement of IHF and allows efficient recombination in vitro in a eukaryotic cell extract. Furthermore, we show by fluorescence that the Int protein contains a functional intrinsic nuclear localization signal, localizing it to the nucleus in both HeLa and 293 cells. CONCLUSIONS: We conclude that P2 Int may be a potential tool for site-specific integration of genes into the human chromosome. SIGNIFICANCE AND IMPACT OF THE STUDY: The study implies the possibility of using multiple prokaryotic Int proteins with different specific integration sites in human cells for future gene therapy programmes.

  • 6. Haeuser, Roman
    et al.
    Blasche, Sonja
    Dokland, Terje
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    von Brunn, Albrecht
    Salas, Margarita
    Casjens, Sherwood
    Molineux, Ian
    Uetz, Peter
    Bacteriophage Protein-Protein Interactions2012In: Advances in virus research, vol 83: Bacteriophages, pt B, ELSEVIER ACADEMIC PRESS , 2012, p. 219-298Chapter in book (Refereed)
    Abstract [en]

    Bacteriophages T7, lambda, P22, and P2/P4 (from Escherichia coli), as well as phi 29 (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 lambda and T7. For example, the approximate to 55 proteins encoded by the T7 genome are connected by approximate to 43 interactions with another approximate to 15 between the phage and its host. The chapter compiles published interactions for the well-studied phages lambda (33 intra-phage/22 phage-host), P22 (38/9), P2/P4 (14/3), and phi 29 (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 lambda 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.

  • 7.
    Henriksson-Peltola, Petri
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sehlén, Wilhelmina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Determination of the DNA-binding kinetics of three related but heteroimmune bacteriophage repressors using EMSA and SPR analysis.2007In: Nucleic Acids Res, ISSN 1362-4962, Vol. 35, no 10, p. 3181-91Article in journal (Refereed)
  • 8.
    Khan Mirzaei, Mohammadali
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Eriksson, Harald
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kasuga, Kie
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nilsson, Anders S.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Genomic, Proteomic, Morphological, and Phylogenetic Analyses of vB_EcoP_SU10, a Podoviridae Phage with C3 Morphology2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 12, article id e116294Article in journal (Refereed)
    Abstract [en]

    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.

  • 9.
    Mandali, Sridhar
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Cardoso-Palacios, Carlos
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sylwan, Lina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Characterization of the site-specific recombination system of phage ΦD145, and its capacity to promote recombination in human cells2010In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 408, no 1, p. 64-70Article in journal (Refereed)
    Abstract [en]

    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.

  • 10.
    Mandali, Sridhar
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Phosphorylation affects the biological activity of the integrase of the P2-likephage ΦD145Manuscript (preprint) (Other academic)
  • 11.
    Massad, Tariq
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Papadopoulos, Evangelos
    Beth Israel Deaconess Med. Center Harvard Institute of Medicin.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Damberg, Peter
    Department of Neurobiology, Care Sciences and Society, Karolinska Institutet .
    NMR Structure Note: The C Repressor of the P2 BacteriophageManuscript (preprint) (Other academic)
  • 12.
    Massad, Tariq
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Papadopoulos, Evangelos
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Henriksson-Peltola, Petri
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Damberg, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Assignment of 1H, 13C, and 15N chemical shift resonances of P2 C-repressor protein2008In: Biomolecular NMR Assignments, ISSN 1874-270X, Vol. 2, no 2, p. 215-217Article in journal (Refereed)
  • 13.
    Massad, Tariq
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Skaar, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, Hanna
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Damberg, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Henriksson-Peltola, Petri
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Crystal structure of the P2 C-repressor: a binder of nonpalindromic direct DNA repeats2010In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 21, p. 7778-7790Article in journal (Refereed)
    Abstract [en]

    As opposed to the vast majority of prokaryoticrepressors, the immunity repressor of temperateEscherichia coli phage P2 (C) recognizes nonpalindromicdirect repeats of DNA rather thaninverted repeats. We have determined the crystalstructure of P2 C at 1.8A ° . This constitutes the firststructure solved from the family of C proteins fromP2-like bacteriophages. The structure reveals thatthe P2 C protein forms a symmetric dimer orientedto bind the major groove of two consecutive turns ofthe DNA. Surprisingly, P2 C has great similarities tobinders of palindromic sequences. Nevertheless, thetwo identical DNA-binding helixes of the symmetricP2 C dimer have to bind different DNA sequences.Helix 3 is identified as the DNA-recognition motif inP2 C by alanine scanning and the importance for theindividual residues in DNA recognition is defined.A truncation mutant shows that the disorderedC-terminus is dispensable for repressor function.The short distance between the DNA-bindinghelices together with a possible interaction betweentwo P2 C dimers are proposed to be responsible forextensive bending of the DNA. The structure providesinsight into the mechanisms behind the mutants ofP2 C causing dimer disruption, temperature sensitivityand insensitivity to the P4 antirepressor.

  • 14.
    Nilsson, Anders S
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Evolution of P2-like phages and their impact on bacterial evolution.2007In: Res Microbiol, ISSN 0923-2508, Vol. 158, no 4, p. 311-7Article in journal (Other academic)
  • 15.
    Nilsson, Anders S
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    The P2-like bacteriophages2006In: The Bacteriophages, Oxford University Press , 2006Chapter in book (Other academic)
  • 16.
    Nilsson, Hanna
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Cardoso-Palacios, Carlos
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Nilsson, Anders
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Phylogenetic structure and evolution of regulatory genes and integrases of P2-like phages2011In: Bacteriophage, ISSN 2159-7073, Vol. 1, no 4, p. 207-218Article in journal (Refereed)
    Abstract [en]

    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 to 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.

  • 17.
    Nilsson, Hanna
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Odegrip, Richard
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Franca-Koh, Jonathan
    J. Craig venter institute.
    Nilsson, Anders
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Interactions between the regulatory repressors of phage P2 and host proteins, a puzzling storyManuscript (preprint) (Other academic)
    Abstract [en]

    Bacteriophage P2 belongs to a group of P2-like phages that have been classified as non-inducible. This is based on the fact that they are not induced by UV light, and upon inactivation of the repressor the bacteria will die but no progeny phage is produced. When the prophage is derepressed it is replicated in situ, but unable to excise due to a lack of integrase. The transcriptional switch of phage P2 contains two repressors, the immunity repressor C and the Cox repressor. The C gene is transcribed from the Pc promoter that also controls the integrase gene, and the C repressor controls the early Pe promoter. The cox gen is transcribed from the Pe promoter and the Cox repressor controls the Pc promoter, making the two promoters mutually exclusive. Thus, the integrase cannot be expressed at the same time as Cox and both proteins are required for phage excision. To try to resolve this paradox, a two-hybrid screen has been performed to find possible host proteins that interact with C or Cox that could control the transcriptional switch.

    Eight E. coli proteins showed interactions with C and three with Cox, out of which all also interacted with C. One of the candidate genes is known to be a "sticky" protein, and was not analysed further. Using a plasmid containing the transcriptional switch, we found that deletions of two of the candidate genes encoding proteins interacting with C or Cox gave a reduced percentage of plasmids choosing the lysogenic pathway; the E. coli yeeD and yqjG genes. YeeD interacts with C as well as Cox, and it is a conserved 8 kD hypothetical proteins with a SirA motif, and YqjG is a predicted glutathione S-transferase. More studies are required to clarify their involvement of these genes in regulating the transcriptional switch.

  • 18.
    Skaar, Karin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Claesson, Magnus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Odegrip, Richard
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Magnus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Crystal structure of the bacteriophage P2 integrase catalytic domain2015In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 589, no 23, p. 3556-3563Article in journal (Refereed)
    Abstract [en]

    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 several non-functional P2 integrase mutants. The DNA binding area is characterized by a large positively charged patch, harbouring key residues. The structure reveals potential for large dimer flexibility, likely essential for rearrangement of DNA strands upon integration and excision.

  • 19.
    Sylwan, Lina
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Frumerie, Clara
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Identification of bases required for P2 integrase core binding and recombination2010In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 404, p. 240-245Article in journal (Refereed)
    Abstract [en]

    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.

  • 20.
    Sylwan, Lina
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Frumerie, Clara
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Identification of bases required for P2 Integrase core-binding and recombinationManuscript (preprint) (Other academic)
    Abstract [en]

    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 share 27 nucleotides with the core of attP (COC´). In the present study we determine the minimal attB site using an in vivo recombination assay and 10 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 hypotetical overlap region is essential for efficient recombination in vivo.

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