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Site-specific recombination in P2-like coliphages
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Institutionen för genetik, mikrobiologi och toxikologi , 2009. , 41 p.
National Category
Genetics
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-8502ISBN: 978-91-7155-817-6 (print)OAI: oai:DiVA.org:su-8502DiVA: diva2:200378
Public defence
2009-03-06, sal E306, Arrheniuslaboratorierna, Svante Arrhenius väg 14-18, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2009-02-12 Created: 2009-02-03 Last updated: 2012-02-28Bibliographically approved
List of papers
1. A structure-function analysis of P2 integras
Open this publication in new window or tab >>A structure-function analysis of P2 integras
(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-25721 (URN)
Note
Part of urn:nbn:se:su:diva-8502Available from: 2009-02-12 Created: 2009-02-03 Last updated: 2010-05-04Bibliographically approved
2. A comparative analysis of the bifunctional Cox proteins of two heteroimmune P2-like phages with different host integration sites
Open this publication in new window or tab >>A comparative analysis of the bifunctional Cox proteins of two heteroimmune P2-like phages with different host integration sites
Show others...
2009 (English)In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 385, no 2, 303-12 p.Article in journal (Refereed) Published
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.

Keyword
Bacteriophage, Directionality factor, Repressor
Identifiers
urn:nbn:se:su:diva-31971 (URN)10.1016/j.virol.2008.12.002 (DOI)000264252200003 ()19150106 (PubMedID)
Available from: 2009-12-01 Created: 2009-12-01 Last updated: 2010-11-17Bibliographically approved
3. Evolution of immunity and host chromosome integration site of P2-like coliphages
Open this publication in new window or tab >>Evolution of immunity and host chromosome integration site of P2-like coliphages
2006 In: Journal of bacteriology, ISSN 0021-9193, Vol. 188, no 11, 3923-3935 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-25723 (URN)
Note
Part of urn:nbn:se:su:diva-8502Available from: 2009-02-12 Created: 2009-02-03Bibliographically approved
4. Characterization of the site-specific recombination system of phage ΦD145, and its capacity to promote recombination in human cells
Open this publication in new window or tab >>Characterization of the site-specific recombination system of phage ΦD145, and its capacity to promote recombination in human cells
2010 (English)In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 408, no 1, 64-70 p.Article in journal (Refereed) Published
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.

Keyword
Integrase, Site-specific recombination, Bacteriophage
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-25724 (URN)10.1016/j.virol.2010.08.035 (DOI)000283970500007 ()
Available from: 2009-02-12 Created: 2009-02-03 Last updated: 2011-12-07Bibliographically approved
5. Phylogenetic structure and evolution of regulatory genes and integrases of P2-like phages
Open this publication in new window or tab >>Phylogenetic structure and evolution of regulatory genes and integrases of P2-like phages
2011 (English)In: Bacteriophage, ISSN 2159-7073, Vol. 1, no 4, 207-218 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Austin, Texas, USA: Landes Bioscience, 2011
Keyword
Peduovirinae, P2-like bacteriophages, Phylogenetic analysis, Phage integration, Lytic-lysogenic transcriptional switch, Gamma-proteobacteria
National Category
Genetics Microbiology
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-65365 (URN)10.4161/bact.1.4.18470 (DOI)
Projects
The evolution of P2-like bacteriophages
Note
Article has also supplementary material, which is missing in this recordAvailable from: 2011-12-08 Created: 2011-12-08 Last updated: 2012-02-28Bibliographically approved

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