The yeast two-hybrid system detects interactions between Bacillus subtilis sigmaB regulators

Abstract

B , the general stress response factor of Bacillus subtilis, is regulated by the products of seven genes (rsbR, S, T, U, V, W, and X) with which it is cotranscribed. Biochemical techniques previously revealed physical associations among RsbW, RsbV, and B but failed to detect interactions of RsbR, S, T, U, or X with each other or RsbV, RsbW, or B . Using the yeast two-hybrid system, we have now obtained evidence for such interactions. The yeast reporter system was activated when RsbS was paired with either RsbR or RsbT, RsbR was paired with RsbT, and RsbV was paired with either RsbU or RsbW. In addition, RsbW 2 and RsbR 2 dimer formation was detected. RsbX failed to show interactions with itself or any of the other sigB operon products.

Mutational Analysis of RsbT, an Activator of theBacillus subtilisStress Response Transcription Factor, σB

“…These plasmids were transformed, according to established protocols (Clontech) into yeast strain Y190 harboring a resident plasmid that encoded the GAL4 activation domain fused to either RsbU, RsbR, RsbS, RsbT, or Obg (Table 1). Yeast strains carrying both plasmids were selected on the basis of plasmid-encoded prototrophic markers and screened for GAL4-dependent histidine prototrophy and ␤-galactosidase activity (34).…”

“…Transformation of naturally competent B. subtilis cells was carried out as described by Yasbin et al (38). Yeast ␤-galactosidase assays were performed as done previously (34). Sequencing of rsbT mutant alleles and plasmids was performed by the Center for Advanced DNA Technologies at the University of Texas Health Science Center.…”

“…In the yeast dihybrid assay, RsbT binds to itself, RsbR, RsbS, and RsbU, as well as to the GTP-binding protein Obg, with sufficient affinity to activate Gal4-dependent transcription (27,34). In vitro, RsbT can form a high-molecular-weight complex with RsbR and RsbS, phosphorylate either of these proteins with ATP as a P0 4 donor and activate the RsbU-dependent dephosphorylation of RsbV-P (12,16,37).…”

“…Previously, the yeast dihybrid system demonstrated that RsbT is able to interact with itself, RsbR, RsbS, RsbU, and Obg, a ribosome-binding protein needed for stress activation of B (27,34). In an attempt to further characterize the six mutant RsbTs that accumulate but fail to activate B , we used the yeast dihybrid system to examine the binding properties of the RsbT variants.…”

“…The recipient yeast strains lacked an intact GAL4 gene but carried both a gene needed for histidine prototrophy (GAL1 UAS -HIS3 TATA -HIS3) and an E. coli lacZ gene (GAL1 UAS -GAL1 TATA -lacZ) expressible from GAL4-activated promoters. Plasmid-containing yeast were selected on the basis of Trp and Leu prototrophy, (markers carried on each of the plasmids) and screened for GAL4-dependent histidine prototrophy and ␤-galactosidase activity (34). The Gal4 activation data are summarized in Table 4.…”