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Leprosy, an important infectious disease in humans caused by  Mycobacterium leprae  ( Mle ), remains endemic in many countries. Notably, the pathogen cannot be cultured  in vitro , except in mouse footpads  in vivo . The molecular basis of these characteristics and the mechanisms remain unknown. Consequently, analysis of  Mle  growth and survival is urgently needed to develop novel therapies against leprosy, including rapid, simple, and specific methods to detect infection. Here, we demonstrated the functional role and contribution of  Mle- DNA gyrase, which regulates DNA topology, DNA replication, and chromosome segregation to promote bacterial growth and survival, in  Mle  growth and survival  in vitro  and  in vivo . The optimum temperature for  Mle -DNA gyrase activity was 30 °C. When the DNA  gyrB-gyrA  genes in  Mycobacterium smegmatis  were replaced with the  Mle  gyrase genes by allelic exchange, the recombinants could not grow at 37 °C. Moreover, using radiorespirometry analysis for viability of  Mle  bacilli, we found that  Mle  growth was more vigorous at 25–30 °C than at 37 °C, but was inhibited above 40 °C. These results propose that DNA gyrase is a crucial factor for  Mle  growth and survival and its sensitivity to temperature may be exploited in heat-based treatment of leprosy.

DNA gyrase could be a crucial regulatory factor for growth and survival of Mycobacterium leprae