It has been shown previously that Escherichia coli accumulates endogenously synthesized trehalose under osmotic stress. We report here that E. coli contained an osmotically regulated trehalose-phosphate synthase which utilized UDP-glucose and glucose 6-phosphate as substrates. In the wild type, the synthase was induced by growth in glucose-mineral medium of elevated osmotic strength and the synthase itself was strongly stimulated by K+ and other monovalent cations. A laboratory strain which expressed the synthase at a high constitutive level was found. GalU mutants, defective in synthesis of UDP-glucose, did not accumulate trehalose. Two genes governing the synthase were identified and named otsA and otsB (osmoregulatory trehalose synthesis). They mapped near 42 min in theflbB-uvrC region. Mutants with an otsA-lacZ or otsB-lacZ operon fusion displayed osmotically inducible I8-galactosidase activity; i.e., the activity was increased fivefold by growth in medium of elevated osmotic strength. Mutants unable to synthesize trehalose (galU, otsA, and otsB) were osmotically sensitive in glucose-mineral medium. But an osmotically tolerant phenotype was restored in the presence of glycine betaine, which also partially repressed the synthesis of synthase in the wild type and of 0-galactosidase in ots-lacZ fusion mutants.

The sequence was determined of 6493 nucleotides encompassing the bet genes of Escherichia coli which encode the osmoregulatory choline-glycine betaine pathway. Four open reading frames were identified: betA encoding choline dehydrogenase, a flavoprotein of 61.9kDa; betB encoding betaine aldehyde dehydrogenase (52.8kDa); betT encoding a proton-motive-force-driven, high-affinity transport system for choline (75.8kDa); and betl, capable of encoding a protein of 21.8kDa, implicated as a repressor involved in choline regulation of the bet genes. Identification of the genes was supported by subcloning, physical mapping of lambda placMu53 insertions, amino acid sequence similarity, or N-terminal amino acid sequencing. The bet genes are tightly spaced, with betT located upstream of, and transcribed divergently to, the tandemly linked betIBA genes.

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.

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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