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A Genetic Switch in Bacteriophages within the Peduovirinae Subfamily: Structure, Function and Evolution
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The temperate bacteriophages in the Peduovirinae subfamily can either grow lytically or integrate into their bacterial host and form lysogeny. Which one of the two life cycles the phage will enter after infection is controlled by a transcriptional switch. The switch also controls the induction of genes necessary for an integrated phage, a prophage, to excise out of the host genome and propagate lytically. In its most simple form, the transcriptional switch consists of two proteins repressing each other’s promoters, which are oriented face to face in close proximity.

The Peduovirinae phages contain two types of transcriptional switches. They were studied with phylogenetic methods to determine their evolution and distribution. Bioinformatic analyses showed that there were several new E. coli integration sites and new inferred immunity classes among the Peduovirinae phages. The two switch types fell into two distinct groups, with no overlap in any of the proteins, but these groups were not defined by host barriers. But in vivo distribution did show a host preference.

The P2 C protein was crystallized and its 3D structure determined. It forms a symmetrical dimer in vitro, with an unstructured C-terminal end. The DNA binding domain was determined to lie in alpha helix three and narrowed down to three residues. The C terminal end of the protein is suggested to be part of tetramerization, but a nine amino acid truncation does not affect activity in vitro.

In an attempt to discover the mechanism between the switch from lysogeny to lysis in phage P2 the interactions between the two switch proteins and the proteins of its host E. coli was analyzed. Eight E. coli proteins interacted with protein C or Cox, but no interaction between the two switch proteins was detected. Two E. coli proteins showed a distinct effect on the expression of C, and several affected the level of phage lysis. The mechanisms behind these effects are still unclear.

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University , 2012. , 88 p.
National Category
Genetics
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-74031ISBN: 978-91-7447-467-1 (print)OAI: oai:DiVA.org:su-74031DiVA: diva2:506167
Public defence
2012-03-30, Magnelisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defence the following paper was unpublished and had a status as follows: Paper nr 3: ManuscriptAvailable from: 2012-03-08 Created: 2012-02-27 Last updated: 2012-03-01Bibliographically approved
List of papers
1. 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
2. Crystal structure of the P2 C-repressor: a binder of nonpalindromic direct DNA repeats
Open this publication in new window or tab >>Crystal structure of the P2 C-repressor: a binder of nonpalindromic direct DNA repeats
Show others...
2010 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 21, 7778-7790 p.Article in journal (Refereed) Published
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.

Keyword
DNA-binding protein, direct repeats, P2 C repressor
National Category
Structural Biology
Research subject
Structural Biology; Biochemistry
Identifiers
urn:nbn:se:su:diva-42003 (URN)10.1093/nar/gkq626 (DOI)000284952000042 ()
Funder
The Wenner-Gren FoundationSwedish Foundation for Strategic Research Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2010-08-13 Created: 2010-08-13 Last updated: 2017-12-12Bibliographically approved
3. Interactions between the regulatory repressors of phage P2 and host proteins, a puzzling story
Open this publication in new window or tab >>Interactions between the regulatory repressors of phage P2 and host proteins, a puzzling story
Show others...
(English)Manuscript (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.

Keyword
bacteriophages, P2, E. coli, transcriptional switch, protein-protein interactions, KEIO, yeast-two-hybrid
National Category
Genetics
Identifiers
urn:nbn:se:su:diva-74030 (URN)
Available from: 2012-02-28 Created: 2012-02-27 Last updated: 2012-02-28Bibliographically approved

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