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.

WblAch, a Pivotal Activator of Natamycin Biosynthesis and Morphological Differentiation in Streptomyces chattanoogensis L10, Is Positively Regulated by AdpAch

“…In Mycobacterium tuberculosis, WhiB3 acts as a metabolic regulator that binds to the promoters of polyketide biosynthetic genes (13), while WhiB6 acts as an initial phagosomal signal receptor (14). It has also been shown that WhiB7 is a critical protein for generating resistance to antibiotics in M. tuberculosis (15). Recently, whiB3 (16), whiB4 (17), whiB5 (18), and whiB2 (19) were shown to play essential roles in the virulence of M. tuberculosis during progressive infection.…”

WhiB7, an Fe-S-dependent Transcription Factor That Activates Species-specific Repertoires of Drug Resistance Determinants in Actinobacteria

“…Direct biochemical evidence (transcriptional run off experiments) has assigned transcriptional regulatory functions to WhiB proteins, including whiB1 (31,32), a repressor, and whiB7, an activator (33). In vitro transcriptional run-off studies have shown that the M. smegmatis WhiB7 protein is a redox-sensitive transcriptional activator of its own promoter (33).…”

“…Direct biochemical evidence (transcriptional run off experiments) has assigned transcriptional regulatory functions to WhiB proteins, including whiB1 (31,32), a repressor, and whiB7, an activator (33). In vitro transcriptional run-off studies have shown that the M. smegmatis WhiB7 protein is a redox-sensitive transcriptional activator of its own promoter (33). The whiB7 promoter, conserved across mycobacteria and other Actinobacteria, includes an AT-rich motif directly upstream of its Ϫ35 hexamer that is targeted by WhiB7 to promote transcription (9,33).…”

“…In vitro transcriptional run-off studies have shown that the M. smegmatis WhiB7 protein is a redox-sensitive transcriptional activator of its own promoter (33). The whiB7 promoter, conserved across mycobacteria and other Actinobacteria, includes an AT-rich motif directly upstream of its Ϫ35 hexamer that is targeted by WhiB7 to promote transcription (9,33). AT-rich motifs are also found upstream of other promoters in the whiB7 regulon (9) and are necessary for in vitro transcriptional activation (33).…”

“…The whiB7 promoter, conserved across mycobacteria and other Actinobacteria, includes an AT-rich motif directly upstream of its Ϫ35 hexamer that is targeted by WhiB7 to promote transcription (9,33). AT-rich motifs are also found upstream of other promoters in the whiB7 regulon (9) and are necessary for in vitro transcriptional activation (33). WhiB3 or WhiB7 function depends on their direct interactions with the vegetative factor RpoV (33,34), and at least one Streptomyces factor gene encodes an N-terminal extension homologous to Wbl proteins (35).…”