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| 2. |
- Ahlgren-Berg, Alexandra, et al.
(författare)
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A comparative analysis of the bifunctional Cox proteins of two heteroimmune P2-like phages with different host integration sites
- 2009
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Ingår i: Virology. - : Elsevier BV. - 0042-6822 .- 1096-0341. ; 385:2, s. 303-12
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Ahlgren-Berg, Alexandra, et al.
(författare)
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A comparison of the DNA binding and bending capacities and the oligomeric states of the immunity repressors of heteroimmune coliphages P2 and W Phi
- 2007
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 35:10, s. 3167-3180
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Ahlgren Berg, Alexandra, 1972-
(författare)
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Developmental switches in a family of temperate phages
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- P2 is the prototype phage of the non-lambdoid P2 family of temperate phages. A developmental switch determines whether a temperate phage will grow lytically or form lysogeny after infection. P2 related phages have two face-to-face located promoters controlling the lysogenic and the lytic operon respectively, and two repressors. The immunity C repressor of P2 is the first gene of the lysogenic operon and it represses the lytic promoter. The Cox protein, the first gene of the lytic operon, is multifunctional. It represses the lysogenic promoter, acts as a directionality factor in site-specific recombination and activates the PLL promoter of satellite phage P4. This thesis focuses on comparisons between the developmental switches of P2 and the two heteroimmune family members, P2 Hy dis and WΦ. A characterization of the developmental switch region of P2 Hy dis identifies a directly repeated sequence which is important for C repression. P2 Hy dis Cox can substitute for P2 Cox in repression of the P2 lysogenic promoter, excision of a P2 prophage and activation of P4 PLL. The P4 ε protein can derepress the developmental switch of P2 Hy dis. Functional characterizations of the C repressors and Cox proteins of P2 and WΦ show that both C repressors induce bending of their respective DNA targets. WΦ C, like P2 C, has a strong dimerization activity in vivo, but there are no indications of higher oligomeric forms. Despite the high degree of identity in the C-terminus, required for dimerization in P2 C, they seem to be unable to form heterodimers. The two Cox proteins are predicted to have identical secondary structures containing a helix-turn-helix motif believed to be involved in DNA binding. It is, however, not possible to change the DNA specificity of P2 Cox to that of WΦ Cox by swapping the presumed recognition helix. P2 Cox recognizes a sequence repeated at least six times in the different targets, while WΦ Cox seems to recognize a single direct repeat. In contrast to P2 Cox, WΦ Cox binds with a stronger affinity to the switch region than to the attachment site (attP). The Cox proteins induce a strong bend in their DNA targets, strengthening the hypothesis that they have a structural role at site-specific recombination. Both proteins show a capacity to oligomerize, but P2 Cox has a higher tendency to form oligomers than WΦ Cox. The P2 integrase mediates site-specific recombination leading to integration or excision of the P2 genome in or out of the host chromosome. P2 Cox controls the direction by inhibiting integration and promoting excision. In this work it is shown that Cox and Int bind cooperatively to attP.
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| 7. |
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| 8. |
- Cardoso-Palacios, Carlos, et al.
(författare)
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A structure-function analysis of P2 integrase
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 9. |
- Cardoso Palacios, Carlos, 1970-
(författare)
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Site-specific recombination in P2-like coliphages
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The scope of these studies has been to investigate the site-specific recombination systems of P2-like coliphages, both in an evolutionary perspective by a comparative analysis of related phages as well as in a functional perspective. Surveys of P2-like phages in Escherichia coli isolated from nature reveal the existence of seven discrete immunity classes and three integration sites, one of them previously unknown. Phylogenetic analysis of the repressor proteins and other analyses show that homologous recombination plays a role in the appearance of new immunities. Other studies of P2-like prophages from sequenced genomes from public databases show that the P2-like phages grow in different γ-proteobacteria. Based on the type of immunity and site-specific recombination system they can be roughly subdivided in two distinct subgroups and some new host integration sites could be identified. Some of the host attachment sites have a high identity to the sequences in the human genome, making them interesting as potential tools for targeted gene insertions into unmodified human cells. The functional studies have been focused on the identification of the determinants for site specificity, which is important for the use of the enzyme for targeted gene insertions into unmodified genomes. Two approaches have been used. In one, we have performed a structure-function analysis of P2 Int that has identified several presumptive residues involved in specific binding to the core sequence, all of them located in the same alpha-helix. This knowledge could be a base for an in vitro evolution of the integrase to enable it to accept new DNA targets with a high affinity. With respect to the excisionases from P2-like coliphages integrating in different sites, we found that they share some common features when they bind and bend to their DNA targets, but there are also significant differences, especially those related to the number of binding sites and the distribution of these and the IHF binding sites in the attP regions. In the other approach we have started to characterize the site-specific recombination system of another P2-like phage, ΦD145, that has a host target with a high identity to a site in the human genome. This looks promising since the human sequence can be used in vivo in E. coli with a rather high efficiency.
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| 10. |
- Eriksson, Harald, 1977-
(författare)
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Bacterial viruses targeting multi-resistant Klebsiella pneumoniae and Escherichia coli
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The global increase in antibiotic resistance levels in bacteria is a growing concern to our society and highlights the need for alternative strategies to combat bacterial infections. Bacterial viruses (phages) are the natural predators of bacteria and are as diverse as their hosts, but our understanding of them is limited. The current levels of knowledge regarding the role that phage play in the control of bacterial populations are poor, despite the use of phage therapy as a clinical therapy in Eastern Europe.The aim of this doctoral thesis is to increase knowledge of the diversity and characteristics of bacterial viruses and to assess their potential as therapeutic agents towards multi-resistant bacteria.Paper I is the product of de novo sequencing of newly isolated phages that infect and kill multi-resistant Klebsiella pneumoniae. Based on similarities in gene arrangement, lysis cassette type and conserved RNA polymerase, the creation of a new phage genus within Autographivirinae is proposed.Paper II describes the genomic and proteomic analysis of a phage of the rare C3 morphotype, a Podoviridae phage with an elongated head that uses multi-resistant Escherichia coli as its host.Paper III describes the study of a pre-made phage cocktail against 125 clinical K. pneumoniae isolates. The phage cocktail inhibited the growth of 99 (79 %) of the bacterial isolates tested. This study also demonstrates the need for common methodologies in the scientific community to determine how to assess phages that infect multiple serotypes to avoid false positive results.Paper IV studies the effects of phage predation on bacterial virulence: phages were first allowed to prey on a clinical K. pneumoniae isolate, followed by the isolation of phage-resistant bacteria. The phage resistant bacteria were then assessed for their growth rate, biofilm production in vitro. The virulence of the phage resistant bacteria was then assessed in Galleria mellonella. In the single phage treatments, two out of four phages showed an increased virulence in the in G. mellonella, which was also linked to an increased growth rate of the phage resistant bacteria. In multi-phage treatments however, three out of five phage cocktails decreased the bacterial virulence in G. mellonella compared to an untreated control.
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