Analysis and DNA sequence of the osmoregulatedtreA gene encoding the periplasmic trehalase ofEscherichia coli K12

Gutierrez, Claude; Ardourel, Maryvonne; Bremer, Erhard; Middendorf, Anke; Boos, Winfried; Ehmann, U

doi:10.1007/bf02464903

Cited by 79 publications

(44 citation statements)

References 31 publications

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“…The operon is induced by high osmolarity, and at least the synthase is activated by potassium (23), which is accumulated in response to osmotic stress (8). Induction of the ots operon is also dependent on RpoS, the alternative sigma factor of stationary-phase cells (26 Under conditions of high osmolarity, trehalose is hydrolyzed to glucose by a periplasmic trehalase, encoded by treA, located at 26 min on the E. coli chromosome (7,24). Glucose is subsequently taken up by the phosphotransferase system (PTS) as glucose 6-phosphate and enters glycolysis.…”

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Trehalose-6-phosphate hydrolase of Escherichia coli

1994

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The disaccharide trehalose acts as an osmoprotectant as well as a carbon source in Escherichia coli. At high osmolarity of the growth medium, the cells synthesize large amounts of trehalose internally as an osmoprotectant. However, they can also degrade trehalose as the sole source of carbon under both high-and low-osmolarity growth conditions. The modes of trehalose utilization are different under the two conditions and have to be well regulated (W. Boos, U. Ehmann, H. Forkl, W. Klein, M. Rimmele, and P. Postma, J. Bacteriol. 172:3450-3461, 1990). At low osmolarity, trehalose is transported via a trehalose-specific enzyme II of the phosphotransferase system, encoded by treB. The trehalose-6-phosphate formed internally is hydrolyzed to glucose and glucose 6-phosphate by the key enzyme of the system, trehalose-6-phosphate hydrolase, encoded by treC. We have cloned treC, contained in an operon with treB as the promoter-proximal gene. We have overproduced and purified the treC gene product and identified it as a protein consisting of a single polypeptide with an apparent molecular weight of 62,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme hydrolyzes trehalose-6-phosphate with a Km of 6 mM and a V.. of at least 5.5iimol of trehalose-6-phosphate hydrolyzed per min per mg of protein. The enzyme also very effectively hydrolyzes p-nitrophenyl-a-D-glucopyranoside, but it does not recognize trehalose, sucrose, maltose, isomaltose, or maltodextrins. treC was sequenced and found to encode a polypeptide with a calculated molecular weight of 63,781. The amino acid sequence deduced from the DNA sequence shows homology (50%6 identity) with those of oligo-1,6-glucosidases (sucrase-isomaltases) of Bacilus spp. but not with those of other disaccharide phosphate hydrolases. This report corrects our previous view on the function of the treC gene product as an amylotrehalase, which was based on the analysis of the metabolic products of trehalose metabolism in whole cells.The disaccharide trehalose serves as an osmoprotectant in many different organisms. Its two glucose molecules are linked 1-1 oa-glycosidically. Thus, the sugar is nonreducing and has the unique quality of maintaining the fluidity of membranes under conditions of dryness and desiccation (16,17). In Escherichia coli, trehalose is synthesized internally as an answer to osmotic stress (23,45,48). Among other, more prominent osmoprotectants such as glycine betaine or proline (11,36), trehalose can contribute up to 20% of the entire capacity for osmotic protection of the cell (21, 32). To synthesize the osmoprotectant trehalose at high osmolarity, UDP-glucose and glucose 6-phosphate are used to form trehalose-6-phosphate, regardless of the carbon source. Trehalose-6-phosphate is subsequently dephosphorylated to give free trehalose. Trehalose-6-phosphate synthase is encoded by otsA (osmotic trehalose synthesis), and trehalose-6-phosphate phosphatase is encoded by otsB. The two genes are localized at 42 min on the chromosome, otsB...

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“…Under conditions of high osmolarity, trehalose is hydrolyzed to glucose by a periplasmic trehalase, encoded by treA, located at 26 min on the E. coli chromosome (7,24). Glucose is subsequently taken up by the phosphotransferase system (PTS) as glucose 6-phosphate and enters glycolysis.…”

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Trehalose-6-phosphate hydrolase of Escherichia coli

1994

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“…Plasmids used were pBAD22, a pBR322-derived plasmid that has the arabinose promoter followed by a linker containing multiple restriction sites (10); pBADtreF has the coding region of wild-type treF in pBAD22 (5); ptre11 has wild-type treA under its own promoter in pBR322 (2).…”

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confidence: 99%

“…Mature TreA has a molecular mass of 58 kDa (1,2). The K m of the purified enzyme is 0.8 mM, the V max is 66 mol of trehalose hydrolyzed/min/mg of protein, and the pH optimum is 5.5 (3).…”

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Determinants of Translocation and Folding of TreF, a Trehalase of Escherichia coli

Uhland¹,

Mondigler²,

Spiess³

et al. 2000

Journal of Biological Chemistry

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One isoform of trehalase, TreF, is present in the cytoplasm and a second, TreA, in the periplasm. To study the questions of why one enzyme is exported efficiently and the other is not and whether these proteins can fold in their nonnative cellular compartment, we fused the signal sequence of periplasmic TreA to cytoplasmic TreF. Even though this TreF construct was exported efficiently to the periplasm, it was not active. It was insoluble and degraded by the periplasmic serine protease DegP. To determine why TreF was misfolded in the periplasm, we isolated and characterized Tre ؉ revertants of periplasmic TreF. To further characterize periplasmic TreF, we used a genetic selection to isolate functional TreA-TreF hybrids, which were analyzed with respect to solubility and function. These data suggested that a domain located between residues 255 and 350 of TreF is sufficient to cause folding problems in the periplasm. In contrast to TreF, periplasmic TreA could fold into the active conformation in its nonnative cellular compartment, the cytoplasm, after removal of its signal sequence.Secretory and cytoplasmic proteins differ not only by the signal sequence but also in their folding properties. It is thought that the export competence of secretory proteins is the result of slow folding prior to export. Cytoplasmic proteins are believed to fold more rapidly and thus are not substrates of the cellular secretion apparatus. For a better understanding of the mechanism of translocation, the folding of cytoplasmic and secretory proteins needs to be characterized. Folding of polypeptides is determined by the primary amino acid sequence but also could be influenced by the particular properties of a cellular compartment. However, little is known about whether and how the cytoplasm and the periplasm specifically influence protein folding, i.e. whether there are other elements besides redox state and the presence/absence of ATP. To study these aspects, we used TreA and TreF, the two trehalases of Escherichia coli, as model proteins.Periplasmic TreA is synthesized as a precursor of 565 amino acids. The signal sequence of 30 amino acids is rather long.Mature TreA has a molecular mass of 58 kDa (1, 2). The K m of the purified enzyme is 0.8 mM, the V max is 66 mol of trehalose hydrolyzed/min/mg of protein, and the pH optimum is 5.5 (3). The expression of treA is independent of the presence of trehalose in the growth medium but is stimulated 10-fold at high osmolarity (1, 2). Also, treA is regulated by RpoS, the stationary phase sigma factor (4).Cytoplasmic TreF has 549 amino acids and a molecular mass of 64 kDa. The K m of the purified enzyme is 1.9 mM, the V max is 54 mol of trehalose hydrolyzed/min/mg of protein, and the pH optimum is 6.0. Like treA,

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