Construction And Application Of Homologous Recombination System Of Mycobacterium

Genus Mycobacterium, including the pathogenic species tuberculosis and leprae, remains a huge threat to human health. Both scientific study and medical control of mycobacteria require efficient molecular tools for genetic manipulation, especially directed mutagenesis and gene deletion. However, due to the slow growth of the Mycobacterium, the low recombinase activity, genetic manipulation techniques, including gene knockout technology development has been lagging behind other model organisms. In this study, we developed an efficient, versatile and improved deletion system based on the Xer/dif knockout system. We modified pJV53 plasmid by introducing a gfp gene for rapid screening of recombinant. We also created dif-flanked Zeo- and Hyg-resistance cassettes containing multiple restriction enzyme sites for homology arms inserted. We tested this system by using it to sequentially delete four pairs of toxin-antitoxin (TA) genes in Mycobacterium smegmatis. This system provides a new genetic tool for the study of functional genomics of Mycobacterium.We further systematically analyzed the TA genes of Mycobacterium tuberculosis in M. smegmatis and identified six new functional TA genes. In addition, we found 12 TA genes increase the plasmid stability, indicating these genes might be involved in chromosome stability of M. tuberculosis.