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.

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...

Osmotically stressed Escherichia coli cells synthesize the osmoprotectant glycine betaline by oxidation of cholibe through glycine betaine aldehyde (choline -* glycine betalne aldehyde glycine betaine;Landfald and A. R. Str0m, J. Ilacteriol. 165:849-855, 1986. Mutants blocked at the level of choline dehydrogenase were isolated by selection of strains which did not grow at elevated osmotic strength in the presence of choline but grew when supplemented with glycine betaine. A gene governing the choline dehydrogenase activity was named betA. Mapping by P1 transduction, F' complementation, and deletion mutagenesis showed the betA gene to be located at 7.5 min in the argF-codB region of the chromosome. Mutants carrying deletions of this region also lacked glycine betaine aidehyde dehydrogeniase activity and high-affinity uptake activity for choline; these deletions did not influence the activities of glycine betalne uptake or low-affinity choline uptake, both of which were osmotically regulated.Glycine betaine is accumulated under osmotic stress in widely different-organisms such as chemoheterotrophic bacteria (16, 22, 26-28, 33. 35), halotolerant photosynthetic bacteria (12, 29, 34), marine animals, and halophytic plants (36,37). Whereas the biological activity of glycine betaine in osmoregulation cannot -be easily demonstrated for higher organisms, this is possible for Esckerichia coli and other enteric bacteria. Addition of relatively low concentrations (e.g., 1 mM) of glycine betaine or its precursors choline or glycine betaine aldehyde to medium of inhibitory osmotic strength stimulates the growth of these bacteria (11,22,(26)(27)(28)35). In the companion paper (22) we show that the choline-glycine betaine pathway of E. coli contains two enzymes: a membrane-bound, 02-dependent choline dehydrogenase and a soluble, NAD-dependent glycine betaine aldehyde dehydrogenase. The pathway is osmotically regulated, i.e., appreciable dehydrogenase activities are synthesized only in cells grown -under osmotic stress. Thus, glycine betaine synthesis ard accumulation in E. coli represent an intriguing system for studies of the molecular mechanism of osmotic tolerance (27). In this communication we describe the selection and characterization of mutants blocked in the choline-glycine betaine pathway. Genes governing several critical activities including choline and glycine betaine aldehyde oxidation and choline uptake were mapped -on -the chromosome of E. coli. MATERIALS AND METHODSBacterial and bacteriophage strains. The strains used are listed in Table 1. The betA mutations-generated in K10 were transferred to CSH7 by P1 transduction and conjugation.

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It has been reported previously that Escherichia coli K-12 carries an amber mutation that prevents osmotic stress-dependent accumulation of trehalose (M. L. Rod, K. Y. Alam, P. R. Cunningham, and D. P. Clark, J. Bacteriol. 170:3601-3610, 1988). We report that E. coli K-12 and W1485 (sup0) accumulated trehalose but that they required a higher osmotic strength in the growth medium than that required by their sup+ derivatives. Furthermore, the sup+ derivatives displayed both strongly increased trehalose-6-phosphate synthase activity and expression of otsA-lacZ and otsB-lacZ operon fusions compared with their parental strains. It is suggested that the amber mutation in question may be in a gene system encoding a transcriptional activator of the ots genes which govern the synthase. The much-used sup0 strain MC4100 behaved like the sup+ derivatives of W1485 with respect to trehalose synthesis. treA mutants with a defective periplasmic trehalase accumulated trehalose extracellularly under osmotic stress. The amount of trehalose excreted correlated with their synthase activity. Strains with an intact trehalase did not display extracellular trehalose accumulation. Thus, stressed E. coli cells regulate the cytoplasmic level of trehalose by a futile cycle involving overproduction, excretion, and degradation to glucose, which is reutilized.

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