Optimizing Mycobacterium Recombineering System (pJV53) to Promote the Screening of the Mycobacterium Mutants

  Objective   To establish a way for screening Mycobacterium mutants through adding the screening markers into pJV53.

  Methods   The sucrose counter selection gene SacB and mutant hygromycin-resistant gene hyg^S were inserted into pJV53; The recovery of the hygromycin-resistance indicated the successful homologous recombination in Mycobacterium smegmatis (Ms), which could serve as mutant screening marker; The sucrose counter selection could be used to screen the plasmid-free mutants.

  Results   The recombinant plasmid pJV53-SacB-hyg^S were successfully constructed. The rifampin-resistant rpoB D516Y and rpoB H526Q mutants and MSMEG_4487 G188A mutant were efficiently screened out. All mutants had shed the plasmid successfully.

  Conclusion   pJV53-SacB-hyg^S can efficiently contribute to construct and screen the mutants and to get the mutants shedding the plasmid self, which has high value of extensive application; the D516Y and H526Q mutations in gene rpoB of Mycobacterium tuberculosis contribute to its rifampin-resistance.