Microbiology MtrA, an essential response regulator of the MtrAB two component system regulates the transcription of resuscitation promoting factor B (RpfB) of Mycobacterium tuberculosis --Manuscript Draft-- Manuscript Number: MIC-D-14-00188R2 Full Title: MtrA, an essential response regulator of the MtrAB two component system regulates the transcription of resuscitation promoting factor B (RpfB) of Mycobacterium tuberculosis Short Title: MtrA of Mycobacterium tuberculosis regulates rpfB Article Type: Standard Section/Category: Regulation Corresponding Author: Joyoti Basu, Ph.D. Bose Institute Kolkata, West Bengal, INDIA First Author: Arun Kumar Sharma, M.Sc. Order of Authors: Arun Kumar Sharma, M.Sc. Ayan Chatterjee, M.Sc. Shamba Gupta, M.Sc. Rajdeep Banerjee, Ph.D. Sukhendu Mandal Jayanta Mukhopadhyay, Ph.D. Joyoti Basu Manikuntala Kundu Abstract: The resuscitation promoting factors (Rpfs) of Mycobacterium tuberculosis are hydrolytic enzymes which are required for resuscitation of dormant cells. RpfB, a peptidoglycan remodelling enzyme similar to the lytic transglycosylase of Escherichia coli, is required for reactivation of M. tuberculosis from chronic infection in vivo, underscoring the need to understand its transcriptional regulation. Here we identify the transcriptional and translational start points of rpfB, and suggest from rpf promoter driven GFP expression and in vitro transcription assays that its transcription possibly occurs in a SigB-dependent manner. We further demonstrate that rpfB transcription is regulated by MtrA, the response regulator of the essential two-component system, MtrAB. Association of MtrA with the rpfB promoter region in vivo, was confirmed by chromatin immunoprecipitation analysis. Electrophoretic mobility shift assays (EMSAs) revealed a loose direct repeat sequence associated with MtrA binding. Binding of MtrA was enhanced upon phosphorylation. MtrA could be pulled down from lysates of M. tuberculosis using a biotinylated DNA fragment encompassing the MtrA binding site on the rpfB promoter, confirming that MtrA binds to the rpfB promoter. Enhanced GFP fluorescence driven by the rpfB promoter, upon deletion of the MtrA binding site, and repression of rpfB expression upon overexpression of MtrA, suggested that MtrA functions as a repressor of rpfB transcription. This was corroborated by EMSA showing diminished association of RNA polymerase with the rpfB promoter in the presence of MtrA. In vitro transcription assay confirmed that MtrA inhibits RNAP-driven rpfB transcription. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Microbiology Papers in Press. Published April 1, 2015 as doi:10.1099/mic.0.000087