title: The mycobacterial antibiotic resistance determinant WhiB7 acts as a transcriptional activator by binding the primary sigma factor SigA (RpoV).
abstract: Tuberculosis therapeutic options are limited by the high intrinsic antibiotic resistance of Mycobacterium tuberculosis. The putative transcriptional regulator WhiB7 is crucial for the activation of systems that provide resistance to diverse antibiotic classes. Here, we used in vitro run-off, two-hybrid assays, as well as mutagenic, complementation and protein pull-down experiments, to characterize WhiB7 as an auto-regulatory, redox-sensitive transcriptional activator in Mycobacterium smegmatis. We provide the first direct biochemical proof that a WhiB protein promotes transcription and also demonstrate that this activity is sensitive to oxidation (diamide). Its partner protein for transcriptional activation was identified as SigA, the primary sigma factor subunit of RNA polymerase. Residues required for the interaction mapped to region 4 of SigA (including R515H) or adjacent domains of WhiB7 (including E63D). WhiB7's ability to provide a specific spectrum of antibiotic-resistance was dependent on these residues as well as its C-terminal AT-hook module that binds to an AT-rich motif immediately upstream of the -35 hexamer recognized by SigA. These experimentally established constrains, combined with protein structure predictions, were used to generate a working model of the WhiB7-SigA-promoter complex. Inhibitors preventing WhiB7 interactions could allow the use of previously ineffective antibiotics for treatment of mycobacterial diseases.
authors.affiliation: Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada V6T 1Z3, Centre for Tuberculosis Research, University of British Columbia, Vancouver, Canada V6T 1Z3 and Vancouver Prostate Centre, University of British Columbia, Vancouver, Canada V6T 1Z3.
authors.forename: Ján
authors.initials: J
authors.lastname: Burian
authors.forename: Grace
authors.initials: G
authors.lastname: Yim
authors.forename: Michael
authors.initials: M
authors.lastname: Hsing
authors.forename: Peter
authors.initials: P
authors.lastname: Axerio-Cilies
authors.forename: Artem
authors.initials: A
authors.lastname: Cherkasov
authors.forename: George B
authors.initials: GB
authors.lastname: Spiegelman
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Thompson
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000187:drug effects / Q000235:genetics*; D059372:Nucleotide Motifs; D011401:Promoter Regions, Genetic; D012808:Sigma Factor / Q000737:chemistry / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D012808:Sigma Factor; D015534:Trans-Activators; D004247:DNA