MTB H37Ra strain was selected as the original strain, and the Ra1362 knock out strain (△Ra1362) was constructed. Change in biofilm formation was compared in vitro; gene expression differences between the wild type (WT) strain and △Ra1362 were detected by transcriptional microarray and real-time fluorescent quantitative PCR experiments. Also, rapid anaerobic dormancy model (RADM) was established in vitro to compare the formation of clones, expression of dormancy-related genes and result of live-cell staining.

△ Ra1362 strian had formed a thicker biofilm in the sputum tube on the 26th day of culture, and the formation speed was 5 days faster than that of WT strain. The results of transcriptional microarray and real-time fluorescent quantitative PCR showed that the expression levels of 19 dormancy-related genes were downregulated in △Ra1362, which could be recovered in Ra1362 complementary strain or after adding exogenous c-di-GMP. In the RADM, it was found that the oxygen consumption in △Ra1362 was 12 h faster than that in WT after the lag phase, and the strians were in a dormant state in which normal growth could not be restored.

Ra1362 can modulate the biofilm development and dormancy of MTB H37Ra in vitro, which is based on the gene expression of DosR regulons under hypoxic condition by sensing hypoxia or oxidation-reduction pressure through controling c-di-GMP synthesis.