ObjectiveTo establish a way for screeningMycobacteriummutants through adding the screening markers into pJV53.MethodsThe sucrose counter selection geneSacBand mutant hygromycin-resistant genehygSwere inserted into pJV53; The recovery of the hygromycin-resistance indicated the successful homologous recombination inMycobacterium smegmatis(Ms), which could serve as mutant screening marker; The sucrose counter selection could be used to screen the plasmid-free mutants.ResultsThe recombinant plasmid pJV53-SacB-hygSwere successfully constructed. The rifampin-resistantrpoBD516Y andrpoBH526Q mutants andMSMEG_4487 G188A mutant were efficiently screened out. All mutants had shed the plasmid successfully.ConclusionpJV53-SacB-hygScan 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 generpoBofMycobacterium tuberculosiscontribute to its rifampin-resistance.
