title: MtrA, an essential response regulator of the MtrAB two-component system, regulates the transcription of resuscitation-promoting factor B of Mycobacterium tuberculosis.
abstract: The resuscitation-promoting factors 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 identified the transcriptional and translational start points of rpfB, and suggested from rpf promoter-driven GFP expression and in vitro transcription assays that its transcription possibly occurs in a SigB-dependent manner. We further demonstrated 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 EMSAs showing diminished association of RNA polymerase (RNAP) with the rpfB promoter in the presence of MtrA. In vitro transcription assays confirmed that MtrA inhibits RNAP-driven rpfB transcription.
authors.affiliation: 1Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Kolkata 700009, India.
authors.forename: Arun Kumar
authors.initials: AK
authors.lastname: Sharma
authors.affiliation: 1Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Kolkata 700009, India.
authors.forename: Ayan
authors.initials: A
authors.lastname: Chatterjee
authors.affiliation: 1Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Kolkata 700009, India.
authors.forename: Shamba
authors.initials: S
authors.lastname: Gupta
authors.affiliation: 1Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Kolkata 700009, India.
authors.forename: Rajdeep
authors.initials: R
authors.lastname: Banerjee
authors.affiliation: 2Department of Biochemistry, Bose Institute, P-1/12 CIT Scheme VII M, Kolkata 700054, India.
authors.forename: Sukhendu
authors.initials: S
authors.lastname: Mandal
authors.affiliation: 1Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Kolkata 700009, India.
authors.forename: Jayanta
authors.initials: J
authors.lastname: Mukhopadhyay
authors.affiliation: 1Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Kolkata 700009, India.
authors.forename: Joyoti
authors.initials: J
authors.lastname: Basu
authors.affiliation: 1Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Kolkata 700009, India.
authors.forename: Manikuntala
authors.initials: M
authors.lastname: Kundu
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial; D006867:Hydrolases / Q000096:biosynthesis*; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics*; D010442:Peptide Chain Initiation, Translational; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; C100938:MtrA protein, Bacteria; D004247:DNA; D006867:Hydrolases