title: The multiple stress responsive transcriptional regulator Rv3334 of Mycobacterium tuberculosis is an autorepressor and a positive regulator of kstR.
abstract: Rv3334 protein of Mycobacterium tuberculosis belongs to the MerR family of transcriptional regulators and is upregulated during hypoxia and other stress conditions. Employing GFP reporter constructs, mobility shift assays and ChIP assays, we demonstrate that Rv3334 binds to its own promoter and acts as an autorepressor. We were able to locate a 22 bp palindrome in its promoter that we show to be the cognate binding sequence of Rv3334. Using chase experiments, we could conclusively prove the requirement of this palindrome for Rv3334 binding. Recombinant Rv3334 readily formed homodimers in vitro, which could be necessary for its transcriptional regulatory role in vivo. Although the DNA-binding activity of the protein was abrogated by the presence of certain divalent metal cations, the homodimer formation remained unaffected. In silico predictions and subsequent assays using GFP reporter constructs and mobility shift assays revealed that the expression of ketosteroid regulator gene (kstR), involved in lipid catabolism, is positively regulated by Rv3334. ChIP assays with aerobically grown M. tuberculosis as well as dormant bacteria unambiguously prove that Rv3334 specifically upregulates expression of kstR during dormancy. Our study throws light on the possible role of Rv3334 as a master regulator of lipid catabolism during hypoxia-induced dormancy.
authors.affiliation: Mycobacterium Research Group, Tropical Disease Biology Division, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India.
authors.forename: Roshna Lawrence
authors.initials: RL
authors.lastname: Gomez
authors.affiliation: Mycobacterium Research Group, Tropical Disease Biology Division, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India.
authors.forename: Leny
authors.initials: L
authors.lastname: Jose
authors.affiliation: Mycobacterium Research Group, Tropical Disease Biology Division, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India.
authors.forename: Ranjit
authors.initials: R
authors.lastname: Ramachandran
authors.affiliation: Mycobacterium Research Group, Tropical Disease Biology Division, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India.
authors.forename: Sajith
authors.initials: S
authors.lastname: Raghunandanan
authors.affiliation: Mycobacterium Research Group, Tropical Disease Biology Division, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India.
authors.forename: Balaji
authors.initials: B
authors.lastname: Muralikrishnan
authors.affiliation: Viral Disease Biology Division, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India.
authors.forename: John Bernet
authors.initials: JB
authors.lastname: Johnson
authors.affiliation: Bioinformatics Facility, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India.
authors.forename: Krishnankutty Chandrika
authors.initials: KC
authors.lastname: Sivakumar
authors.affiliation: Mycobacterium Research Group, Tropical Disease Biology Division, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India.
authors.forename: Sathish
authors.initials: S
authors.lastname: Mundayoor
authors.affiliation: Mycobacterium Research Group, Tropical Disease Biology Division, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India.
authors.forename: Ramakrishnan Ajay
authors.initials: RA
authors.lastname: Kumar
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D055029:Inverted Repeat Sequences; D019216:Metals, Heavy / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D055503:Protein Multimerization; D012097:Repressor Proteins / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D001426:Bacterial Proteins; D019216:Metals, Heavy; D012097:Repressor Proteins; D004247:DNA
keywords: Mycobacterium tuberculosis; autoregulation; hypoxia; metal binding; transcriptional regulator