Pinto Crystal T, Nano Francis E
Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V6P 4W8, Canada.
Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V6P 4W8, Canada
FEMS Microbiol Lett. 2015 Sep;362(18):fnv152. doi: 10.1093/femsle/fnv152. Epub 2015 Sep 3.
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.
编码来自北极细菌嗜冷栖假交替单胞菌的NAD依赖性DNA连接酶的ligA基因的合成版本，被用于替换耻垢分枝杆菌染色体中的同源基因。所得重组菌株在允许温度下的生长情况与亲本菌株相同，但在37°C以上无法生长。温度敏感表型是稳定的，该菌株在可检测水平上未能产生耐温形式。杂交菌株的反复传代并未导致嗜冷ligA基因发生变化。鉴于分枝杆菌物种间DNA连接酶的高度同一性，这些结果表明，使用此处描述的方法可以产生稳定的结核分枝杆菌温度敏感菌株。
