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 (sigma38) sigma factor. Here we characterize Fis-mediated activation of the P2 promoter in vitro. We find that this promoter displays unusually high specificity for sigma38. 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 E sigma38 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 sigma70 or sigma38.