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Strains of Thermus thermophilus accumulate primarily trehalose and smaller amounts of mannosylglycerate in response to salt stress in yeast extract-containing media (O. C. Nunes, C. M. Manaia, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 61:2351-2357, 1995). A 2.4-kbp DNA fragment from T. thermophilus strain RQ-I carrying otsA (encoding trehalose-phosphate synthase [TPS]), otsB (encoding trehalose-phosphate phosphatase [TPP]), and a short sequence of the 5' end of treS (trehalose synthase [TreS]) was cloned from a gene library. The sequences of the three genes (including treS) were amplified by PCR and sequenced, revealing that the genes were structurally linked. To understand the role of trehalose during salt stress in T. thermophilus RQ-1, we constructed a mutant, designated RQ-1M6, in which TPS (otsA) and TPP (otsB) genes were disrupted by gene replacement. Mutant RQ-1M6 accumulated trehalose and mannosylglycerate in a medium containing yeast extract and NaCI. However, growth in a defined medium (without yeast extract, known to contain trehalose) containing NaCI led to the accumulation of mannosylglycerate but not trehalose. The deletion of otsA and otsB reduced the ability to grow in defined salt-containing medium, with the maximum salinity being 5% NaCI for RQ-1 and 3% NaCI for RQ-1M6. The lower salt tolerance observed in the mutant was relieved by the addition of trehalose to the growth media. In contrast to trehalose, the addition of glycine betaine, mannosylglycerate, maltose, and glucose to the growth medium did not allow the mutant to grow at higher salinities. The results presented here provide crucial evidence for the importance of the TPS/TPP pathway for the synthesis and accumulation of trehalose and the decisive contribution of this disaccharide to osmotic adaptation in T. thermophilus RQ-1.

The periplasmic cyclodextrin binding protein CymE from Klebsiella oxytoca was characterized and analyzed. Chromatographic techniques were used to analyze the bacterial strains. Alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin was easily bound by CymE. The K. oxytoca cym system efficienty metabolized alpha-cyclodextrin compared to the ability of the E. coli maltose system to take up maltohexaose.

An experiment was conducted to clone and sequence the gene cluster encoding a trehalose/maltose transport system of Thermococcus litoralis. This hyperthermophilic archaeon is homologous to the malEFG cluster that encodes the Escherichia coli maltose transport system. Results reveal a strong homology between the bacterial systems and the archaeal transport system. This indicates the evolutionary conservation of the binding protein-dependent ABC transport systems.

The glk structural gene of Escherichia coli K-12 glucokinase was cloned and sequenced to characterize its biochemical function and kinetics. The purified Escherichia coli glk protein product was characterized as a housekeeping enzyme that was expressed by cells cultured with different carbon sources. Overexpression of the glucokinase gene also effected the mal gene expression which mediated the maltose system in Escherichia coli cells.

A unique cytoplasmic trehalase from Escherichia coli that hydrolyzes the trehalose to glucose is presented. The gene encoding this enzyme, treF, was cloned under ara promoter control. The enzyme (TreF) was purified from extracts of an overexpressing strain and characterized biochemically. The enzyme is monomeric, has broad pH optimum at 6.0 and has no metal dependency. TreF has a molecular weight of 63,703 and is highly homologous to TreA.

The molecular analysis of TreB, the enzyme IITre, the phosphotransferase system (PTS)-dependent transport protein for trehalose, reveals the similarity in protein sequence as well as in the sequence of domains with another PTS enzyme, enzyme IIScr. The disaccharide trehalose, a nonreducing sugar synthesized in Escherichia coli during osmotic stress, maintains the fluidity of the membrane under dry conditions. The genes treB and treC are parts of an operon that is negatively regulated by TreR, which is encoded by a gene found upstream of treB.

The crystal structure of the Escherichia coli trehalose repressor (TreR) in a complex with its inducer trehalose-6-phosphate was determined by the method of multiple isomorphous replacement (MIR) at 2.5 Å resolution, followed by the structure determination of TreR in a complex with its noninducer trehalose at 3.1 Å resolution. The model consists of residues 61 to 315 comprising the effector binding domain, which forms a dimer as in other members of the LacI family. This domain is composed of two similar subdomains each consisting of a central β-sheet sandwiched between α-helices. The effector binding pocket is at the interface of these subdomains. In spite of different physiological functions, the crystal structures of the two complexes of TreR turned out to be virtually identical to each other with the conformation being similar to those of the effector binding domains of the LacI and PurR in complex with their effector molecules. According to the crystal structure, the noninducer trehalose binds to a similar site as the trehalose portion of trehalose-6-phosphate. The binding affinity for the former is lower than for the latter. The noninducer trehalose thus binds competitively to the repressor. Unlike the phosphorylated inducer molecule, it is incapable of blocking the binding of the repressor headpiece to its operator DNA. The ratio of the concentrations of trehalose-6-phosphate and trehalose thus is used to switch between the two alternative metabolic uses of trehalose as an osmoprotectant and as a carbon source. [ABSTRACT FROM AUTHOR]

Reports on an improvement of a procedure using Escherichia coli to synthesize 14C-labeled trehalose from [14C]glucose. Preparative synthesis; Method for producing trehalose from glucose; Advantage of using E. coli strain.