Interactions of the nucleoid-associated DNA-binding protein H-NS with the regulatory region of the osmotically controlled proU operon of Escherichia coli

Abstract

The Escherichia coli hns gene encodes the abundant nucleoid-associated DNA-binding protein H-NS. Mutations in hns alter the expression of many genes with unrelated functions and result in a derepression of the proU operon (proVWX) without abolishing the osmotic control of its transcription. We have investigated the interactions of H-NS with the proU regulatory region by deletion analysis of cis-acting sequences, competitive gel retardation assays, and DNase I footprinting. The negative effect of H-NS on proU transcription was mediated by cis-acting sequences within proV but did not depend on the presence of a curved DNA segment upstream of the proU-35 region previously characterized as a target for H-NS binding in vitro. We detected a 46-base pair high affinity H-NS binding region downstream of the proU promoter at the 5' end of the proV gene and a complex array of additional H-NS binding sites which suggest the presence of an extended H-NS nucleoprotein complex. Most of the H-NS binding sites were highly A+T-rich and carried stretches of 5 or more consecutive A-T base pairs. The implications of our results for the osmotic regulation of proU transcription are discussed.