The Transcriptional Regulation Mechanism Of RPFA Gene In Mycobacterium Smegmatis MC~2155

TB now ranks alongside HIV as a leading cause of death worldwide.Tuberculosis is mainly caused by Mycobacterium tuberculosis.Mycobacterium smegmatis mc2155 is widely used as a model organism in mycobacterial research works because of its rapid growth rate and failure to cause diseases.RpfA is one of the resuscitation promoting factors and a peptidoglycan hydrolase.The resuscitation promoting factor is related to the change of the cell from dormancy to the recovery.It can also stimulate the growth of viable cells.The transcription of rpfA in M.tuberculosis is activated by the cAMP receptor protein CRP?Rv3676?,and the rpfA in Streptomyces coelicolor is also reported to be regulated by the CRP and the ydaO riboswitch.In this study,the transcription initiation site?TSS?of rpfA transcripts was identified by the 5-RACE experiments.The co-transcription of the upstream gene moaD2 and rpfA was identified by RT-PCR.Then the binding site of CRP?MSMEG₆189?upstream of the promoter region of rpfA was determined by the site directed mutagenesis,promoter activity assay and electrophoretic mobility shift assay?EMSA?.By means of the P-gal activity assay,quantitative Real-Time PCR and other experiments,it was confirmed that CRP could activate the transcription of rpfA and the co-transcription of rpfA with the upstream gene moaD2.Sequence analysis predicted that there is a ydaO like riboswitch at the 5'-UTR of the rpfA.The promoter activity assays in M.smegmatis and E.coli confimed that this region was regulated by c-di-AMP.When the concentration of c-di-AMP was low,the expression of the downstream gene increased.In addition,it was found that the transcription of rpfA was activated by the transcription regulator CarD.The transcription of rpfA was up-regulated in carD over expression strain,but this was not dependent on CRP.EMSA indicated that CarD activate the transcription of rpfA through binding to its promoter region.In conclusion,this study confirmed the complex regulation mechanism of rpfA in M.smegmatis.Those results provide the foundation for studying long distance regulation mechanism of CRP,and increase the possibility for finding new drug targets.