Stable, temperature-sensitive recombinant strain of Mycobacterium smegmatis generated through the substitution of a psychrophilic ligA gene.

A synthetic version of the ligA gene encoding the NAD-dependent DNA ligase from the Arctic bacterium Pseudoalteromonas haloplanktis was substituted for its homolog in the chromosome of Mycobacterium smegmatis. The resulting recombinant strain grew identically to the parent strain at permissive temperatures but failed to grow above 37°C. The temperature-sensitive phenotype was stable, and the strain failed to generate temperature-resistant forms at a detectable level. Repeated passage of the hybrid strain resulted in no changes in the psychrophilic ligA gene. Given the high identity of the DNA ligases among mycobacterial species, these results suggest that a stable temperature-sensitive strain of M. tuberculosis could be generated using the approach described here.