Effects of H-NS and potassium glutamate on σ^S- and σ⁷⁰-directed transcription in vitro from osmotically regulated P1 and P2 promoters of proU in Escherichia coli

Abstract

We have used supercoiled DNA templates in this study to demonstrate that transcription in vitro from the P1 and P2 promoters of the osmoresponsive proU operon of Escherichia coli is preferentially mediated by the σ^s and σ⁷⁰-bearing RNA polymerase holoenzymes, respectively. Addition of potassium glutamate resulted in the activation of transcription from both P1 and P2 and also led to a pronounced enhancement of σ^s selectivity at the P1 promoter. Transcription from P2, and to a lesser extent from P1, was inhibited by the nucleoid protein H-NS but only in the absence of potassium glutamate. This study validates the existence of dual promoters with dual specificities for proU transcription. Our results also support the proposals that potassium, which is known to accumulate in cells grown at high osmolarity, is at least partially responsible for effecting the in vivo induction of proU transcription and that it does so through two mechanisms, directly by the activation of RNA polymerase and indirectly by the relief of repression imposed by H-NS.