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Characterization of the mycobacteriophage L5 attachment site, attP
C E Peña1, M H Lee, M L Pedulla
1Department of Biological Sciences, University of Pittsburgh, PA 15260, USA.
Abstract:
Lysogenization of mycobacteriophage L5 involves integration of the phage genome into the Mycobacterium smegmatis chromosome. Integration occurs by a site-specific recombination event between a phage attachment site, attP, and a bacterial attachment site, attB, which is catalyzed by the phage-encoded integrase protein. DNase I footprinting reveals that L5 integrase binds to two types of sites within attP which span an unexpectedly large region of 413 bp: seven arm-type sites (P1 to P7) each of which correspond to a consensus sequence 5'-TGCaaCtcYy, and core-type sites at the points of strand exchange. Mutational analyses indicate that not all of the arm-type sites are required for integration, and that the P3 site and the rightmost pair of sites (P6 and P7) are dispensable for integration. We show that a 252 bp segment of attP DNA is sufficient for efficient integrative recombination and that int can be provided in trans for simple and efficient transformation of the mycobacteria.
Insights
Mycobacteriophage L5 integrates into Mycobacterium smegmatis via site-specific recombination. Researchers identified key DNA binding sites for the L5 integrase, simplifying phage integration and transformation.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Lysogenization is a critical step in the life cycle of many bacteriophages, involving the integration of the phage genome into the host chromosome.
- Mycobacteriophage L5 integration into Mycobacterium smegmatis is mediated by a specific recombination event between phage (attP) and bacterial (attB) attachment sites, catalyzed by the L5 integrase protein.
Purpose of the Study:
- To investigate the DNA binding characteristics of the L5 integrase within the attP site.
- To determine the essential regions of attP required for efficient integrative recombination.
- To explore the potential for simplified mycobacterial transformation using the L5 integrase system.
Main Methods:
- DNase I footprinting was employed to map the binding sites of L5 integrase on the attP DNA.
- Site-directed mutagenesis was used to analyze the functional significance of different regions within attP.
- In vitro recombination assays were performed to assess the efficiency of integration with varying attP segments.
Main Results:
- L5 integrase binds to seven arm-type sites and core-type sites within the 413 bp attP region.
- Mutational analysis revealed that not all arm-type sites are essential, with P3 and the P6/P7 pair being dispensable.
- A minimal 252 bp segment of attP was found sufficient for efficient integrative recombination.
- Providing the integrase (int) in trans enabled simple and efficient transformation of Mycobacterium smegmatis.
Conclusions:
- The L5 integrase exhibits specific binding patterns within attP, with some sites being more critical than others for integration.
- A reduced attP fragment is sufficient for efficient recombination, offering a streamlined approach.
- The ability to provide integrase in trans facilitates straightforward and effective transformation of mycobacteria, with potential applications in genetic engineering.