IS-Integrase of B.Pertussis
B. pertussis IS-Integrase: Characterization of DNA-Binding Sites and Site-Specific Recombination in E.coli
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
3 Approved without Funding
Gamalei Institute of Epidemiology and Microbiology, Russia, Moscow
- State Research Center for Applied Microbiology, Russia, Moscow reg., Obolensk
- Institut Pasteur, France, Paris\nUniversity of Wisconsin-Madison / Department of Biochemistry, USA, WI, Madison\nInstitut Pasteur / Department de Biochimie et Genetique Moleculaire, France, Paris
Project summaryOver last decades, a focus of numerous investigations has been placed on mobile genetic elements (MGE), enzymes responsible for site-specific recombination, and a role MGE may play in persity and evolution of bacteria. Information on new MGE not only contributes much to understanding of mechanisms of variability of the genome in host’s microorganism, but also extends the knowledge about mechanisms and enzymes involved in site-specific bacterial recombination.
In recent years, our research has been focused on mobile genetic elements of B.pertussis, which have been found to enhance structural re-arrangements in recombinant plasmids and chromosome, fusion of plasmid replicons, integration of replicons of the donor into E.coli chromosome, and transposition of MGE, both within and between genomes.
The objective of the project is to isolate enzyme (integrase) responsible for site-specific recombination of these elements of B.pertussis, and to detect its MGE DNA-target binding sites.
The following tasks will be pursued:
1. Express ORF1 of the sequence RSBP3 in E.coli followed by isolation and characterization of the produced protein (p36 in the text).
2. Assess topoisomerase activity in the protein and determine its superhelical DNA-binding sites.
3. Study site-specific recombination leading to formation of intra-plasmid cointegrates.
The proposed research is basic. The expected results are:
– a producer of integrase is IS B.pertussis; new enzyme responsible for site-specific recombination in bacteria will be isolated and characterized;
– extra-data on molecular mechanisms underlying action of enzymes of transposition in bacteria will be obtained;
– conclusions on mechanisms of site-specific recombination in B.pertussis, a human causative agent, and, probable, on a role these processes may play in variability of this pathogen will be put forward.
Staff who has long experience in genetics, molecular biology and biochemistry will be involved in the project.
The Project meets goals of the ISTC, providing scientists and technicians engaged earlier in counter-biological defense opportunities to integrate into the international scientific community. Moreover, results from the project would promote solving global problems – prevention and treatment of infectious diseases.
Project duration is 2 years. Its key point is to express B.pertussis ORF1 IS-sequence in E.coli, to isolate and purified the end product. It is thought to take a year to solve this task, while the other will stick the technical schedule. Foreign collaborators are expected to promote information exchange, assist participants in attending international meetings and arranging international symposia and workshops.