Transcription from the P₃ promoter of the Bacillus subtilis spx gene is induced in response to bisulfide stress

The spx gene of Bacillus subtilis encodes a global regulator that controls transcription initiation in response to oxidative stress by interaction with RNA polymerase (RNAP). It is located in a dicistronic operon with the yjbC gene. The spx gene DNA complements an spx null mutation with respect to disulfide stress resistance, suggesting that spx is transcribed from a promoter located in the intergenic region oiyjbC and spx. Transcription of the yjbC-spx operon has been reported to be driven by four promoters, three (P₁, P ₂, and P_B) residing upstream of yjbC and one (P _M) located in the intergenic region between yjbC and spx. Primer extension analysis uncovered a second intergenic promoter, P₃, from which transcription is elevated in cells treated with the thiol-specific oxidant diamide. P₃ is utilized by the σ^Aform of RNA polymerase in vitro without the involvement of a transcriptional activator. Transcriptional induction from P₃ did not require an Spx-RNAP interaction and was observed in a deletion mutant lacking DNA upstream of position -40 of the P₃ promoter start site. Deletion mutants with endpoints 3′ to the P₃ transcriptional start site (positions +5, +15, and +30) showed near-constitutive transcription at the induced level, indicating the presence of a negative control element downstream of the P ₃ promoter sequence. Point mutations characterized by bgaB fusion expression and primer extension analyses uncovered evidence for a second as-acting site in the P₃ promoter sequence itself. The data indicate that spx transcription is under negative transcriptional control that is reversed when disulfide stress is encountered.