Recently developed techniques for introducing and expressing genes in mycobacteria have opened the way for molecular genetic manipulation as a means to obtain further understanding of these species (Jacobs et al., 1991). Usually, virulence factors are genetically characterized following Koch's molecular postulates (Falkow, 1988): (i) cloning of the gene of interest; (ii) effects on the virulence of specific inactivation of the gene; and (iii) restoration of the pathogenicity by complementing with the wild-type allele. However, the genetic analysis of mycobacteria has been hampered by a lack of efficient tools for generating defined mutants by homologous recombination (Jacobs, 1992).
The `reverse genetics` approach to understanding gene function is to specifically disrupt the gene of interest by exploiting the homologous recombination properties of the cell to replace the functional allele with an inactivated copy (Ruvkun and Ausubel, 1981). Usually, the gene is inactivated by the insertion of an antibiotic-resistance marker, such that the recombination event is easily detected on a selective medium. It was demonstrated that this methodology is applicable to Mycobacterium smegmatis using the pyrF gene (Husson et al., 1990). However, performing allelic exchange has been relatively cumbersome due to the rarity of double-cross-over events, requiring an extensive screening to isolate a gene-exchange mutant. This method has proven particularly inefficient in slow-growing mycobacteria where homologous recombination is less frequent than illegitimate recombination, i.e., recombination at a site other than the selected gene by an unknown mechanism. Several workers have been unable to detect any gene replacement (Aldovini et al., 1993; Kalpana et al., 1991). However, several recent studies have succeeded in identifying allelic exchange, albeit at low frequency, in slow-growing mycobacteria (Marklund et al., 1995; Norman et al., 1995; Reyrat et al., 1995; Balasubramanian et al., 1996). Clearly, allelic exchange would greatly benefit from a system allowing positive selection of mutants resulting from gene replacement.
High levels of illegitimate recombination and low frequency of recombination have also been described in gene-replacement experiments in eukaryotic cells and some bacteria (Cai and Wolk, 1990; Desomer et al., 1991). These problems can be overcome by employing a double-selection strategy (Stibitz, 1994). The vector used for mutagenesis should bear an antibiotic marker for the primary selection of transformants, and a second marker with a conditionally dominant lethal effect to counter-select clones which have lost the vector DNA, eliminating the need for extensive screening. No counter-selectable marker was available for mycobacteria until recently, when the rpsL gene was shown to exhibit a dominant lethal effect in M. smegmatis. It was used to demonstrate that double selection is possible in this bacterium (Sander et al., 1995).
There has existed a need to design a general method for gene exchange mutagenesis, which would overcome the problems arising from high levels of illegitimate recombination and low frequency of homologous recombination. A possible strategy, based on the use of much longer stretches of linear homologous DNA (20 kb or more) was recently proposed (Balasubramanian et al., 1996). In this way, it was shown that the frequency of homologous recombination for the M. tuberculosis leuD gene could be increased. The proportion of allelic exchange mutants rose from an undetectable level to approximately 6% of the transformants (Balasubramanian et al., 1996). However, a possible limitation to this methodology is the fact that manipulation of cosmids is relatively difficult due to their extensive length.
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