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

Abstract

Transcription of theproU operon ofEscherichia 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 geneproV; (ii) sequences around another (&0-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, PIR, 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 (PIR 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.The proU locus in Escherichia coli and Salmonella typhimurium encodes an active transport system for glycine betaine and L-proline and has been shown to play an important role in the adaptation of these organisms to growth in media of elevated osmolarity (4). Genetic and molecular characterization of proU has shown that it is composed of three genes (designated, in order, proV, proW, and proX) (5, 10) whose products constitute a binding-protein-dependent porter system that is a member of the large family of traffic ATPases or ABC transporters described for both prokaryotes and eukaryotes (1,16).Transcription ofproU is induced several hundred-fold upon growth in high-osmolarity media (4, 9), making this quantitatively the most prominent example in which a mechanical stimulus controls gene expression. However, the mechanism of proU regulation is as yet poorly understood, and no trans mutant in which osmotic regulation of proU is completely abolished has been obtained.Our studies with E. coli on the cis regulation of proU have shown the presence of two promoters, P1 and P2, with the sequences immediately around each conferring five-and eightfold osmotic inducibility on transcription initiated from the respective promoters (6). The cis elements or mechanisms ...

The bacterial nucleoid: A highly organized and dynamic structure

“…1c), whereas H-NS converts circular DNA substrates into compact, dumbbell-shaped structures . In agreement with an antagonistic function of these proteins, H-NS-mediated transcriptional repression is enhanced in mutants lacking HU, but alleviated when HU is overexpressed [Manna and Gowrishankar, 1994;Delihas and Forst, 2001]. A general connection between HU-induced architectural changes and gene regulation is further supported by numerous reports that link HU to promoter function, although the molecular details are frequently unknown [Wagner, 2000].…”

H‐NS: a modulator of environmentally regulated gene expression

“…The best studied of this class is the proU promoter. In both E. coli and S. typhimurium, proU expression is induced up to 100 times by increases in osmolarity (Lucht et al, 1994;Manna and Gowrishankar, 1994;Fletcher and Csonka, 1995). Full osmoregulation requires sequences located downstream of the promoter (DRE or NRE) that bind H-NS with high affinity (Lucht et al, 1994;Owen-Hughes et al, 1992;Fletcher and Csonka, 1995).…”

A global role for Fis in the transcriptional control of metabolism and type III secretion in Salmonella enterica serovar Typhimurium

“…Here, no effect of the fis mutation on stpA expression was detected at 1 h, although stpA expression was dependent on Fis when wildtype and mutant were compared at 4 h. The hupA and hupB genes encode the subunits of the heterodimeric DNAbinding protein HU (Hillyard et al, 1990;Oberto et al, 1994). In addition to its role in nucleoid organization, HU contributes to the osmotic stress response of the cell and normal regulation of the proU osmotic stress response operon (Manna & Gowrishankar, 1994). Expression of the hupA gene showed a strong requirement for Fis at 1 h and has been described previously as being activated by Fis in E. coli (Claret & Rouvière-Yaniv, 1996) (supplementary data Tables S1 and S2 at http://mic.sgmjournals.org).…”