Osmotic signal transduction to proU is independent of DNA supercoiling in Escherichia coli

Ramírez, Rosalía; Villarejo, Merna

doi:10.1128/jb.173.2.879-885.1991

Cited by 27 publications

(25 citation statements)

References 50 publications

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“…proU transcription is dependent on the housekeeping sigma factor°70 (26), and the osmotic regulation of proU can be explained by a stimulatory effect of potassium glutamate on the transcriptional complex (43), independent of DNA supercoiling (44). Apparently, an intrinsic property of the osmotic stress-dependent proU promoter elicits this potassium glutamate effect without the need of protein factors, at least in vitro (43).…”

Section: Discussionmentioning

confidence: 99%

Molecular cloning and physical mapping of the otsBA genes, which encode the osmoregulatory trehalose pathway of Escherichia coli: evidence that transcription is activated by katF (AppR)

Kaasen¹,

Falkenberg²,

Styrvold³

et al. 1992

J Bacteriol

198

155

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It has been shown previously that the otsA and otsB mutations block osmoregulatory trehalose synthesis in Escherichia coli. We report that the transcription of these osmoregulated ots genes is dependent on KatF (AppR), a putative sigma factor for certain stationary phase-and starvation-induced genes. The transcription of the osmoregulated bet and proU genes was not katF dependent. Our genetic analysis showed that katF carries an amber mutation in E. coli K-12 and many of its derivatives but that katF has reverted to an active form in the much-used strain MC4100. This amber mutation in katF leads to strain variations in trehalose synthesis and other katF-dependent functions of E. coli. We have performed a molecular cloning of the otsBA genes, and we present evidence that they constitute an operon encoding trehalose-6-phosphate phosphatase and trehalose-6-phosphate synthase. A cloning and restriction site analysis, performed by comparing the cloned fragments with the known physical map of the E. coli chromosome, revealed that the otsBA genes are situated on a 2.9-kb HindIII fragment located 8 to 11 kb clockwise of tar (41.6 min).Trehalose, a nonreducing disaccharide of glucose, is a stress metabolite in various organisms (29). Saccharomyces cerevisiae accumulates trehalose when exposed to an elevated temperature of growth (3,24) or to hazardous chemical agents such as ethanol, copper sulfate, or hydrogen peroxide (3). Rhizobia accumulate trehalose when stressed with lowoxygen (e.g., 1%) tension, regardless of the composition of the growth medium (23). Many phototrophic and heterotrophic bacteria, including Escherichia coli, accumulate trehalose in response to osmotic stress (18,31,45,50). Trehalose is shown to preserve the function and integrity of biological membranes exposed to conditions of low water activity (14) and to confer desiccation tolerance to yeasts (24), to spores of Streptomyces sp. (36), and to nematodes (14); frost tolerance to insects (2) and yeasts (22); and osmotic tolerance to E. coli (19). In yeasts, trehalose accumulation during growth in liquid culture coincides with an increased plating efficiency on agar plates of low water activity (34).In E. coli, the osmoregulatory trehalose pathway consists of a trehalose-6-phosphate synthase which converts UDPglucose and glucose-6-phosphate to trehalose-6-phosphate and a phosphatase which dephosphorylates this metabolic intermediate (reference 19 and this study). Two insertion mutations, named otsA and otsB, which block the synthesis of the synthase, have previously been mapped to 42 min, but the trehalose-6-phosphate phosphatase activity of these mutants was not reported (19). However, a point mutation named otsP which causes accumulation of trehalose-6-phosphate in stressed cells, presumably because of a defective phosphatase, was mapped near otsA (27). This mutation appears to be allelic with otsB (reference 27 and this study).Trehalose accumulation in stressed cells of E. coli is regulated at several levels. Experiments with lac fusions have show...

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Section: Discussionmentioning

confidence: 99%

Molecular cloning and physical mapping of the otsBA genes, which encode the osmoregulatory trehalose pathway of Escherichia coli: evidence that transcription is activated by katF (AppR)

Kaasen¹,

Falkenberg²,

Styrvold³

et al. 1992

J Bacteriol

198

155

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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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“…If was previously shown fhaf the in wfro transcription of fhe proU and osmC genes is strongly stimulated by pofassium glufamafe, presumably by a direct action of the potassium salf on fhe RNA polymerase (Prince and Villarejo, 1990;Ramirez and Villarejo, 1991).…”

Section: Trehalose Catabolismmentioning

confidence: 99%

Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression

Strøm

Kaasen

1993

Molecular Microbiology

341

240

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SummaryEndogenously synthesized trehalose is a stress protectant in Escherichia coli. Externally supplied trehalose does not serve as a stress protectant, but it can be utilized as the sole source of carbon and energy. Mutants defective in trehalose synthesis display an impaired osmotic tolerance in minimal growth media without glycine betaine, and an impaired stationary-phase-induced heat tolerance. Mechanisms for stress protection by trehalose are discussed. The genes for trehalose-6-phosphate synthase {otsA) and anabolic trehalose-6-phosphate phosphatase {otsB) constitute an operon. Their expression is induced both by osmotic stress and by growth into the stationary phase and depend on the sigma factor encoded by rpoS {katF). rpoS is amber-mutated in E. coti K-12 and its DNA sequence varies among K-12 strains. For trehalose catabolism under osmotic stress E. coli depends on the osmotically inducible periplasmic trehalase (TreA). In the absence of osmotic stress, trehalose induces the formation of an enzyme W^'^ (TreB) of the group translocation system, a catabolic trehalose-6-phosphate phosphatase (TreE), and an amylotrehalase (TreC) which converts trehalose to free glucose and a glucose polymer.

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Osmotic signal transduction to proU is independent of DNA supercoiling in Escherichia coli

Cited by 27 publications

References 50 publications

Molecular cloning and physical mapping of the otsBA genes, which encode the osmoregulatory trehalose pathway of Escherichia coli: evidence that transcription is activated by katF (AppR)

Molecular cloning and physical mapping of the otsBA genes, which encode the osmoregulatory trehalose pathway of Escherichia coli: evidence that transcription is activated by katF (AppR)

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

Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression

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