Activation of RpoS-dependent proP P2 transcription by the Fis protein in vitro

The proP gene, encoding a transporter of the osmoprotecting compounds proline and glycine betaine, is expressed from two promoters. Transcription of the P2 promoter occurs at a transient period in late exponential phase and is dependent upon Fis and the RpoS (σ 38 ) sigma factor. Here we characterize Fis-mediated activation of the P2 promoter in vitro . We find that this promoter displays unusually high specificity for σ 38 . Fis strongly activates P2 when bound to site I centered at −41 within the promoter region. There is a complex relationship involving DNA supercoiling and potassium glutamate concentration on Fis activation, but most efficient transcription occurs under high salt conditions when the superhelical density is above −0.03. The major stimulatory effect of DNA supercoiling occurs between superhelical densities of 0 to −0.02 suggesting that, while supercoiling is mechanistically important, it may not be a physiologically relevant controlling factor. However, the stimulation of transcription by high potassium glutamate concentrations may contribute to the osmotic inducibility of the P2 promoter. We show that Fis and Eσ 38 bind cooperatively on supercoiled DNA to form a stable complex at P2 that involves promoter melting. Fis also binds to a second site within the proP regulatory region. While binding to this site appears to play no role in Fis activation of the P2 promoter, it functions as a repressor of transcription initiating from the P1 promoter by either σ 70 or σ 38 .