SITE-SPECIFIC RECOMBINATION OF THE MYCOBACTERIUM TUBERCULOSIS PROPHAGE-LIKE ELEMENT PHIRV1
The site-specific recombination systems of bacteriophages and other mobile elements fall into two categories that are named for the integrase protein that catalyzes integration and excision of the phage genome. These integrases utilize either a tyrosine or a serine residue to carry out the nucleophilic attack of the DNA. In many cases the directionality of the reaction, that is whether the enzyme catalyzes integration or excision, is determined by an additional phage encoded protein referred to as RDF, or recombination directionality factor.
Two prophage-like elements, phiRv1 and phiRv2, were found through sequencing Mycobacterium tuberculosis strain H37Rv. These are absent from the vaccine bacillus M. bovis BCG, and are often found in virulent strains of M. bovis and M. tuberculosis. Through the work presented here, one of these elements, phiRv1, was found to encode an active recombination system, with a serine integrase and RDF. The phiRv1 element is found within a degenerate repeated element, REP13E12, which is present in seven non-identical copies in M. tuberculosis and M. bovis BCG. In vivo studies have revealed that four of the seven 13E12 elements can serve as integration sites (attBs) for a plasmid carrying a reconstructed attP and integrase, and that multiple integrations can occur. The fast growing saprophyte, M. smegmatis, also supports integration, although inefficiently. In M. smegmatis, the phiRv1 plasmid integrates into at least two 13E12 repeats that are quite different from those found in M. bovis BCG and M. tuberculosis. Inefficiency is overcome by providing M. smegmatis with an attB site from BCG. These integrated plasmids are stable, and excision occurs in the presence of the phiRv1 RDF encoded by Rv1584c. In vitro assays were developed for both integration and excision. All the substrate site requirements are relatively small. Integration occurs slowly but efficiently in the presence of excess attB or on an intramolecular attP-attB plasmid. In the presence of RDF integration is inhibited and excision is stimulated. The RDF binds to a specific sequence in both attB and attL, although the way in which it functions may be through protein-protein interactions with integrase.
Advisor:Graham F. Hatfull; Karen Arndt; JoAnne Flynn; Craig Peebles; Roger Hendrix
School:University of Pittsburgh
School Location:USA - Pennsylvania
Source Type:Master's Thesis
Date of Publication:06/24/2004