Gene Replacement in Mycobacterium smegmatis Using a Dominant Negative Selectable Marker

Abstract

The process of homologous recombination IS essential to all living organisms. It is important for the generation of genetic diversity, the maintenance of genomic integrity, and the proper segregation of chromosomes. At least 25 gene products (RecA, helicases, nucleases, ligases, single-strand binding protein, etc.) are involved in the process of homologous recombination. Homologous recombination can occur at any site in the chromosome between homologous, but not necessarily identical, DNA duplexes. The first step in homologous recombination involves breaking of the DNA double helices (single- or double-stranded break), followed by pairing of homologous strands, which results in heteroduplex formation, branch migration, and finally resolution of the intermediate by cleavage and rejoining (1). Homologous recombination has to be distinguished first from the process of site-specific recombination, in which the two parental strands pair at sites of very limited homology (e.g. site-specific integration of phage λ or mycobacteriophage L5 [2), and second from illegitimate recombination. Illegitimate recombination, which uses neither extensive sequence homology nor specific sites, is a process that in most bacteria occurs at a low frequency. Illegitimate recombination however, seems to be the predominant type of integration of foreign DNA m Mycobacterium tuberculosis complex (e.g., Mycobacterium tuberculosis or Mycobacterium bovis BCG [3). It has been hypothesized that this phenomenon might be owing to the unusual structure of the recA gene in M tuberculosis, which is interrupted by an intern (4).