Evidence for involvement of proteins HU and RpoS in transcription of the osmoresponsive proU operon in Escherichia coli

Abstract

Transcription of the proU operon of Escherichia coli is induced several hundred-fold upon growth at elevated osmolarity, but the underlying mechanisms are incompletely understood. Three cis elements appear to act additively to mediate proU osmoresponsivity: (i) sequences around a promoter, P1, which is situated 250 bp upstream of the first structural gene proV; (ii) sequences around another (σ ⁷⁰-dependent) promoter, P2, which is situated 60 bp upstream of proV; and (iii) a negative regulatory element present within the proV coding region. These three cis elements are designated, respectively, P1R, P2R, and NRE. trans-acting mutants with partially derepressed proU expression have been obtained earlier, and a vast majority of the mutations affect the gene encoding the nucleoid protein HNS. In this study we employed a selection for trans-acting mutants with reduced proU⁺ expression, and we obtained a derivative that had suffered mutations in two separate loci designated dpeA and dpeB. The dpeB mutation caused a marked reduction in promoter P1 expression and was allelic to rpoS, the structural gene for the stationary-phase-specific sigma factor of RNA polymerase. Expression from P1 was markedly induced, in an RpoS-dependent manner, in stationary-phase cultures. In contrast to the behavior of the isolated P1 promoter, transcription from a construct carrying the entire proU cis-regulatory region (P1R plus P2R plus NRE) was not significantly affected by either growth phase or RpoS. The dpeA locus was allelic to hupB, which along with hupA encodes the nucleoid protein HU. hupA hupB double mutants exhibited a pronounced reduction in proU osmotic inducibility. HU appears to affect proU regulation through the P2R mechanism, whereas the effect of HNS is mediated through the NRE.