Trehalose metabolism in Escherichia coli is complicated by the fact that cells grown at high osmolarity synthesize internal trehalose as an osmoprotectant, independent of the carbon source, although trehalose can serve as a carbon source at both high and low osmolarity. The elucidation of the pathway of trehalose metabolism was facilitated by the isolation of mutants defective in the genes encoding transport proteins and degradative enzymes. The analysis of the phenotypes of these mutants and of the reactions catalyzed by the enzymes in vitro allowed the formulation of the degradative pathway at low osmolarity. Thus, trehalose utilization begins with phosphotransferase (IITreHIlI c)-mediated uptake delivering trehalose-6-phosphate to the cytoplasm. It continues with hydrolysis to trehalose and proceeds by splitting trehalose, releasing one glucose residue with the simultaneous transfer of the other to a polysaccharide acceptor. The enzyme catalyzing this reaction was named amylotrehalase. Amylotrehalase and EfiTre were induced by trehalose in the medium but not at high osmolarity. treC and treB encoding these two enzymes mapped at 96.5 min on the E. coli linkage map but were not located in the same operon. Use of a mutation in trehalose-6-phosphate phosphatase allowed demonstration of the phosphoenolpyruvate-and HTre-dependent in vitro phosphorylation of trehalose. The phenotype of this mutant indicated that trehalose-6-phosphate is the effective in vivo inducer of the system.The synthesis of internal trehalose in Escherichia coli in response to high osmolarity has been studied in detail on a genetic and biochemical level (12, 30), yet little is known about trehalose transport and metabolism. Early reports have described E. coli mutants that were partially defective in the utilization of trehalose. The mutations mapped at 26 min on the linkage map (3; for Salmonella typhimurium, see reference 29). Marechal (20) later reported the existence of a specific enzyme II of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) by demonstrating the PEP-dependent phosphorylation of trehalose. He also claimed, on the basis of biochemical studies, the existence of an enzyme able to hydrolyze trehalose-6-phosphate to glucose-6-phosphate and glucose (20). Different results were obtained by Postma et al. (23), who reported that trehalose is transported in S. typhimurium via the mannose-PTS without phosphorylation. Both studies reported the existence of a trehalose-inducible trehalase in crude extracts.A periplasmic trehalase was subsequently discovered and purified from E. coli, and mutants, termed treA, were isolated that lacked this enzyme. treA was mapped at 26 min (5), and the treA gene was cloned, sequenced, and found to be the only gene in the operon (14). Periplasmic trehalase synthesis is not induced by trehalose but rather by growth in the presence of 250 mM NaCl (5). Apparently, the function of the periplasmic trehalase is to ensure the utilization of trehalose under conditions of high osmolarity...

The treA gene of Escherichia coli K12 codes for a periplasmic trehalase that is induced by growth at high osmolarity. The position of treA within a cloned chromosomal DNA fragment was identified by subcloning of restriction fragments and analysis of the gene product in minicells. The nucleotide sequence of the treA coding region as well as its upstream control region was determined. The treA gene consists of 1695 bp encoding 565 amino acids. The amino-terminus of the mature trehalase was found to begin with the amino acid Glu at position 31 of the open reading frame. The first 30 amino acids resemble a typical signal sequence, consistent with trehalase being a secreted periplasmic enzyme. Two previously isolated phoA fusions to the osmA gene were transferred by homologous recombination on to a treA-containing plasmid and found to be within treA. Analysis of the hybrid genes and their gene products aided the localization of treA and the determination of its direction of transcription within the cloned chromosomal segment. The treA-phoA fusions encoded hybrid proteins which could be found in the periplasm. We found that at high osmolarity the normal pathway for the uptake and utilization of trehalose is blocked. Therefore, the function of the periplasmic trehalase is to provide the cell with the ability to utilize trehalose at high osmolarity by splitting it into glucose molecules that can subsequently be taken up by the phosphotransferase-mediated uptake system.

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The reconstitution of active transport by the Ca2+-induced import of exogenous binding protein was studied in detail in whole cells of a malE deletion mutant lacking the periplasmic maltose-binding protein. A linear increase in reconstitution efficiency was observed by increasing the Ca2+concentration in the reconstitution mixture up to 400 mM. A sharp pH optimum around pH 7.5 was measured for reconstitution. Reconstitution efficiency was highest at 0°C and decreased sharply with increasing temperature. The time necessary for optimal reconstitution at 0°C and 250 mM Ca was about 1 min. The competence for reconstitution was highest in exponentially growing cultures with cell densities up to 1 x 109/ml and declined when the cells entered the stationary-growth phase. The apparent Km for maltose uptake was the same as that of wild-type cells (1 to 2 puM). Vmax at saturating maltose-binding protein concentration was 125 pmol per min per 7.5 x 107 cells (30% of the wild-type activity). The concentration of maltose-binding protein required for half-maximal reconstitution was about 1 mM. The reconstitution procedure appears to be generally applicable. Thus, galactose transport in Escherichia coli could also be reconstituted by its respective binding protein. Maltose transport in E. coli was restored by maltose-binding protein isolated from Salmonella typhimurium. Finally, in S. typhimurium, histidine transport was reconstituted by the addition of shock fluid containing histidinebinding protein to a hisJ deletion mutant lacking histidine-binding protein. The method is fast and general enough to be used as a screening procedure to distinguish between transport mutants in which only the binding protein is affected and those in which additional transport components are affected.

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