Tuberculosis is the leading cause of death from a single infectious agent, i.e. Mycobacterium tuberculosis. Development of genetic tools for understanding mycobacterial biology has been advanced through the study of their viruses, the mycobacteriophages. Several well-characterized mycobacteriophages form lysogens in which the phage DNA is integrated into the host chromosome and these integration systems have proven useful both for constructing site-specific integration-proficient vectors but also for elucidating the mechanism and control of site-specific recombination. Several newly characterized mycobacteriophages integrate and excise their DNA using unusual serine-integrases, which contain a catalytic motif similar to those in transposon resolvases and DNA-invertases. A similar protein is involved in the mobility of the prophage-like element, phiRvl, in M. tuberculosis. However, little is known about how this class of enzymes catalyzes integrative recombination between their attP and attB sites, or how this same protein catalyzes recombination between attL and attR for excision. Since attP and attB are different, an interesting biochemical and structural problem arises as to how these serine-integrases choose the correct site pairs for productive recombination. Defined in vitro reactions have been established for both phiRvl and phage Bxbl integration and are simple, requiring just the correct DNA partners, purified integrase and a simple buffer. There is no requirement for either DNA supercoiling, additional proteins or high-energy cofactors. An in vitro assay has also been established for phiRvl excision, which requires a phiRvl-encoded recombination directionality factor (RDF). The mechanism and control of these reactions will be determined through analysis of the requirements at the attachment sites, the nature of the interaction between the integrase and RDF proteins and DNA, and mutational dissection of the integrase proteins. These studies will provide important insights into how phage integration and excision operate and how they can be harnessed as genetical tools for understanding M. tuberculosis.
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