In vitro reconstitution of osmoregulated expression of proU of Escherichia coli.

Ramírez, Rosalía; Prince, William S.; Bremer, Erhard; Villarejo, Merna

doi:10.1073/pnas.86.4.1153

Cited by 52 publications

(45 citation statements)

References 32 publications

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“…(i) Increase in the osmolarity of the growth medium leads to an increase in supercoiling of DNA, which in turn affects gene expression (18)(19)(20); (ii) growth in high-osmolarity medium is associated with intracellular accumulation of potassium glutamate, which was shown to directly control proU expression (24,41,42). These two mechanisms might be complementary to each other rather than exclusive.…”

Section: Discussionmentioning

confidence: 99%

Osmotic repression of anaerobic metabolic systems in Escherichia coli

Gouesbet

Abaibou

et al. 1993

J Bacteriol

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The influence of the osmolarity of the growth medium on anaerobic fermentation and nitrate respiratory pathways was analyzed. The levels of several enzymes, including formate dehydrogenase, hydrogenase, and nitrate reductase, plus a nickel uptake system were examined, as was the expression of the corresponding structural and regulatory genes. While some functions appear to be only moderately affected by an increase in osmolarity, others were found to vary considerably. An increase in the osmolarity of the medium inhibits both fermentation and anaerobic respiratory pathways, though in a more dramatic fashion for the former. fnr expression is affected by osmolarity, but the repression of anaerobic gene expression was shown to be independent of FNR regulatory protein, at least for hyd-17 andfdhF. This repression could be mediated by the intracellular concentration of potassium and is reversed by glycine betaine.

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Section: Discussionmentioning

confidence: 99%

Osmotic repression of anaerobic metabolic systems in Escherichia coli

Gouesbet

Abaibou

et al. 1993

J Bacteriol

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“…According to one, increase in osmolarity of the growth medium leads to increased supercoiling of DNA within the cells, and this in turn leads to proU induction (17)(18)(19)(20)30). According to the other, growth in high-osmolarity media is associated with intracellular accumulation of potassium glutamate, which then directly activates proU expression (24, [35][36][37][38]. However, neither mechanism by itself has so far been shown capable of accounting quantitatively for the several-hundredfold magnitude of proU induction observed in vivo, and evidence presented in this report for the existence of multiple controls on proU transcription suggests that the two themes discussed above might be complementary to one another rather than mutually exclusive.…”

Section: Discussionmentioning

confidence: 99%

Multiple mechanisms contribute to osmotic inducibility of proU operon expression in Escherichia coli: demonstration of two osmoresponsive promoters and of a negative regulatory element within the first structural gene

1991

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Transcription of the proU operon in Escherichia coli is induced several hundredfold upon growth of cells in media of elevated osmolarity. A low-copy-number promoter-cloning plasmid vector, with lacZ as the reporter gene, was used for assaying the osmoresponsive promoter activity of each of various lengths of proU DNA, generated by cloning of discrete restriction fragments and by an exonuclease III-mediated deletion approach. The results indicate that expression of proU in E. coli is directed from two promoters, one (P2) characterized earlier by other workers with the start site of transcription 60 nucleotides upstream of the initiation codon of the first structural gene (proV), and the other (P1) situated 250 nucleotides upstream of proV. Furthermore, a region of DNA within proV was shown to be involved in negative regulation of proU transcription; phage Mu dII1681-generated lac fusions in the early region ofproV also exhibited partial derepression ofproU regulation, in comparison with fusions further downstream in the operon. Sequences around promoter P1, sequences around P2, and the promoter-downstream negative regulatory element, respectively, conferred approximately 5-, 8-, and 25-fold osmoresponsivity on proU expression. Within the region genetically defined to encode the negative regulatory element, there is a 116-nucleotide stretch that is absolutely conserved between the proU operons of E. coli and Salmonella typhimurium and has the capability of exhibiting alternative secondary structure. Insertion of this region of DNA into each of two different plasmid vectors was associated with a marked reduction in the mean topological linking number in plasmid molecules isolated from cultures grown in high-osmolarity medium. We propose that this region of DNA undergoes reversible transition to an underwound DNA conformation under high-osmolarity growth conditions and that this transition mediates its regulatory effect on proU expression.The growth rate of Escherichia coli and Salmonella typhimurium in high-osmolarity media is promoted by the addition of small concentrations of L-proline or glycine betaine to the culture medium. The osmoprotective effect of both of these compounds is presumed to be consequent upon their intracellular accumulation under conditions of water stress and is in part dependent on the presence of a functional ProU transporter in these cells, encoded by genes of the proU locus (for a review, see reference 6).Complementation studies using a number of proU mutants, in combination with nucleotide sequence analysis of the locus, have shown that proU is an operon composed of three structural genes, proV, proW, and proX (7, 13). The product of proX is a periplasmic protein which has been purified and shown to be a glycine betaine-binding protein in vitro; furthermore, the deduced amino acid sequences of the products of proV and proW each show similarities to components of other well-characterized transport systems such as those for histidine, maltose, or arabinose, thus permitting the inference t...

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“…First, in vivo activation ofproU transcription requires intracellular K+ accumulation (44). Second, added potassium glutamate enhanced expression of proU in in vitro coupled transcription-translation S-30 systems (18,37). Finally, potassium glutamate specifically stimulatedproU transcription in an in vitro assay with purified components (36), suggesting that potassium glutamate acts directly on the transcription complex.…”

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confidence: 99%

osmY, a new hyperosmotically inducible gene, encodes a periplasmic protein in Escherichia coli

1992

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A new osmotically inducible gene in Escherichia coli, osmY, was induced 8- to 10-fold by hyperosmotic stress and 2- to 3-fold by growth in complex medium. The osmY gene product is a periplasmic protein which migrates with an apparent molecular mass of 22 kDa on sodium dodecyl sulfate-polyacrylamide gels. A genetic fusion to osmY was mapped to 99.3 min on the E. coli chromosome. The gene was cloned and sequenced, and an open reading frame was identified. The open reading frame encoded a precursor protein with a calculated molecular weight of 21,090 and a mature protein of 18,150 following signal peptide cleavage. Sequencing of the periplasmic OsmY protein confirmed the open reading frame and defined the signal peptide cleavage site as Ala-Glu. A mutation caused by the osmY::TnphoA genetic fusion resulted in slightly increased sensitivity to hyperosmotic stress.

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In vitro reconstitution of osmoregulated expression of proU of Escherichia coli.

Cited by 52 publications

References 32 publications

Osmotic repression of anaerobic metabolic systems in Escherichia coli

Osmotic repression of anaerobic metabolic systems in Escherichia coli

Multiple mechanisms contribute to osmotic inducibility of proU operon expression in Escherichia coli: demonstration of two osmoresponsive promoters and of a negative regulatory element within the first structural gene

osmY, a new hyperosmotically inducible gene, encodes a periplasmic protein in Escherichia coli

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