Beta-glucose 1-phosphate-interconverting enzymes in maltose- and trehalose-fermenting lactic acid bacteria

Maltose and trehalose catabolic pathways are linked through their common enzyme, beta-phosphoglucomutase, and metabolite, beta-glucose 1-phosphate, in Lactococcus lactis. Maltose is degraded by the concerted action of maltose phosphorylase and beta-phosphoglucomutase, whereas trehalose is assimilated by a novel pathway, including the recently discovered enzyme, trehalose 6-phosphate phosphorylase, and beta-phosphoglucomutase. In the present study, 40 strains of lactic acid bacteria were investigated for utilization of metabolic reactions involving beta-glucose 1-phosphate. All genera of the low G+C content lactic acid bacteria belonging to the clostridial subbranch of Gram-positive bacteria were represented in the study. The strains, which fermented maltose or trehalose, were investigated for beta-phosphoglucomutase, maltose phosphorylase and trehalose 6-phosphate phosphorylase activity, as indications of maltose and trehalose catabolic pathways involving beta-glucose 1-phosphate interconversions. Eighty per cent of all strains fermented maltose and, of these strains, 63% were shown to use a maltose phosphorylase/beta- phosphoglucomutase pathway. One-third of the strains fermenting trehalose were found to harbour trehalose 6-phosphate phosphorylase activity, and these were also shown to possess beta-phosphoglucomutase activity. Mainly L. lactis and Enterococcus faecalis strains were found to harbour the novel trehalose 6-phosphate phosphorylase/beta-phosphoglucomutase pathway. As lower beta-glucose 1-phosphate interconverting enzyme activities were observed in the majority of glucose-cultivated lactic acid bacteria, glucose was suggested to repress the synthesis of these enzymes in most strains. Thus, metabolic reactions involving the beta-anomer of glucose 1-phosphate are frequently found in both maltose- and trehalose-utilizing lactic acid bacteria.     

Trehalose Metabolism by Bacillus popilliae

Trehalose was found to be utilized more readily than glucose for the growth of Bacillus popilliae NRRL B-2309MC. The pathway of degradation of trehalose was elucidated and found to differ from that reported for other organisms. Trehalase and trehalose phosphorylase activities could not be detected. Rather, trehalose was found to undergo phosphoenolpyruvate (PEP)-dependent phosphorylation, and the resulting trehalose 6-phosphate was cleaved by a phosphotrehalase to equimolar amounts of glucose and glucose 6-phosphate. The phosphotrehalase was purified 34-fold and shown to have a pH optimum of 6.5 to 7.0 and a K(m) for trehalose 6-phosphate of 1.8 mM. A mutant missing the phosphotrehalase failed to grow on trehalose but grew normally on other sugars. The mutant accumulated [(14)C]trehalose as [(14)C]trehalose 6-phosphate. Phosphorylation of trehalose by dialyzed extracts was at least 25 times faster with PEP than with adenosine 5'-triphosphate, and the phosphorylation activity was associated primarily with the particulate fraction. These data and the results of studies of [(14)C]trehalose uptake suggest that trehalose is transported into the cell as trehalose 6-phosphate by a PEP:sugar phosphotransferase system. Cell extracts of other strains of B. popilliae were also found to produce [(14)C]sugar phosphate from [(14)C]trehalose and to have phosphotrehalase activity.     

Overproduction of trehalose: heterologous expression of Escherichia coli trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in Corynebacterium glutamicum

Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum. This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C. glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in C. glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C. glutamicum.     

Trehalose as cryoprotectant for preservation of yeast strains

Preservation of genetic banks of yeast strains as well as of any kind of eukaryotic cells during dehydration and subsequent rehydration depends upon the maintenance of the integrity of the cell membrane. Trehalose has been successfully used as a non-toxic cryoprotectant for plant cells (Bhandal et al., 1985), as well as for lobster sarcoplasmic vesicles (Rudolph and Crowe, 1985). The hypothesis underlying these observations is that the disaccharide avoids fusion of membranes by replacing water molecules in the bilayer (Crowe et al., 1984). The viability of yeast strains submitted to different drying techniques is reported in this paper. Mutant strains with defects in the regulation of the trehalose-6-phosphate synthase complex were compared. Yeast strains dried in layers at 37°C for 6 h did not lose their viability, however, they died thereafter at 5°C, unless trehalose was used for resuspending the cells before drying. It should be noted that no trehalose accumulation was seen during drying at 37°C under our experimental conditions. In experiments in which cells were frozen at −120°C, addition of 10% trehalose to the suspending buffer had a significant protective effect. On the other hand, a mutant strain with an extremely high trehalose-6-phosphate synthase activity showed an intrinsic capacity for survival which did not depend upon addition of exogenous trehalose. This raises the question of the location of the internal trehalose pool and whether it could replace the externally added cryoprotectant.     

Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression

Endogenously 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-phaseinduced 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. coli K-12 and its DNA sequence varies among K-12 strains. For trehalose catabolism under osmotic stress E. coli depends on the osmoticcally inducible periplasmic trehalase (TreA). In the absence of osmotic stress, trehalose induces the formation of an enzyme II^Tre (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.     

The control of saccharide synthesis during development of myxamoebae of Dictyostelium discoideum containing differing amounts of glycogen

1. Myxamoebae initially containing 5.59mg of glycogen/10(8) cells accumulate approx. 25% more cell-wall polysaccharide, 100% more mucopolysaccharide, 200% more glucose and 300% more trehalose during their development than do myxamoebae initially containing less than 0.3mg of glycogen/10(8) cells. 2. These observations restrict the number of possible control mechanisms operating to regulate carbohydrate metabolism during development. 3. Cells accumulating a large amount of trehalose (approx. 400mug/10(8) cells) have the same amount and pattern of changes in specific activity of trehalase and trehalose 6-phosphate synthase as do cells accumulating a smaller amount of trehalose (approx. 100mug/10(8) cells). 4. These two populations of cells do, however, differ markedly in the amount of UDP-glucose and glucose 6-phosphate that they contain. 5. It is concluded that this change in the intracellular pools of the metabolic precursors of trehalose accounts for the increased amount of trehalose synthesized by cells derived from myxamoebae containing an increased glycogen content.     

Regulation of trehalose metabolism in Saccharomyces cerevisiae mutants during temperature shifts

Temperature shifts from 23 degrees C to 36 degrees C resulted in trehalose accumulation in Saccharomyces independently of genetic lesions in the cAMP-protein kinase cascade. In parallel, trehalose 6-phosphate synthase activity increased about 3-fold in all strains; the increase could be inhibited by cycloheximide, suggesting that protein synthesis was required. Heat shock treatment after the temperature shift led to a drastic increase in trehalose activity, and deactivation of the biosynthetic enzyme with a consequent drop in trehalose. Up to now no definite correlation between acquisition of thermotolerance and trehalose accumulation has been made.     

Overproduction of Trehalose: Heterologous Expression of Escherichia coli Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase in Corynebacterium glutamicum

Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum. This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C. glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in C. glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C. glutamicum.     

The involvement of hexokinases in trehalose synthesis.

Yeast cells harboring a MAL2-8c gene accumulate trehalose during the transition phase of growth on glucose due to the presence of the ADPG-dependent trehalose 6-phosphate synthase. Under these conditions, glucokinase appeared not to provide G-6-P for trehalose synthesis and the two hexokinases seemed to act synergistically. After incubation in d-xylose, trehalose levels in these cells dropped almost in 90%, confirming the involvement of both hexokinases in the accumulation of this carbohydrate. Nevertheless, G-6-P levels appeared to be similar in all strains. Some explanations for this paradox are discussed. In stationary phase, neither of the three isoenzymes were involved in trehalose synthesis. Possibly, gluconeogenesis provides the substrate for trehalose synthesis at that stage.     

Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli.

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 the flbB-uvrC region. Mutants with an otsA-lacZ or otsB-lacZ operon fusion displayed osmotically inducible beta-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 beta-galactosidase in ots-lacZ fusion mutants.     

Regulation of trehalose metabolism by Adox and AdoMet in Saccharomyces cerevisiae

Effect of a potent methylation inhibitor oxidized adenosine (Adox), and a universal methyl group donor S-adenosyl-L-methionine (AdoMet) on trehalose metabolism was studied in two haploids of S. cerevisiae of mating types MATalpha, met3 (6460 -8D) and MATa, leu2, ura3, his4 (8534 -10A). Trehalose level decreased in presence of Adox in both strains. Both neutral trehalase (NT) and trehalose-6-phosphate (tre-6-p) synthase activities increased in presence of Adox in -8D strain. Decrease in trehalose level in -8D thus could not be explained in the light of increased tre-6-p synthase activity; however, it could be correlated with increased NT activity. In strain -10A, NT activity was reduced in presence of Adox while tre-6-p synthase activity increased. Enzyme activity profiles in -10A thus do not explain the reduced trehalose level on Adox treatment. Effect of AdoMet was not very prominent in either strain, though in -8D a small increase in trehalose level was seen on treatment. Intracellular AdoMet level of untreated cells of -10A was seen to be almost six times higher than that of -8D. Further, AdoMet treatment caused increase in its level compared to untreated cells, suggesting AdoMet uptake. No effect of either compound was seen on acid trehalase (AT) activity in any strain. The results suggest that there was a possible effect of demethylation on trehalose metabolism (particularly in the synthetic direction) in both strains, though effect of methylation was not very prominent, the reason for which is not very clear.     

New insights on trehalose: a multifunctional molecule

Trehalose is a nonreducing disaccharide in which the two glucose units are linked in an alpha,alpha-1,1-glycosidic linkage. This sugar is present in a wide variety of organisms, including bacteria, yeast, fungi, insects, invertebrates, and lower and higher plants, where it may serve as a source of energy and carbon. In yeast and plants, it may also serve as a signaling molecule to direct or control certain metabolic pathways or even to affect growth. In addition, it has been shown that trehalose can protect proteins and cellular membranes from inactivation or denaturation caused by a variety of stress conditions, including desiccation, dehydration, heat, cold, and oxidation. Finally, in mycobacteria and corynebacteria, trehalose is an integral component of various glycolipids that are important cell wall structures. There are now at least three different pathways described for the biosynthesis of trehalose. The best known and most widely distributed pathway involves the transfer of glucose from UDP-glucose (or GDP-glucose in some cases) to glucose 6-phosphate to form trehalose-6-phosphate and UDP. This reaction is catalyzed by the trehalose-P synthase (TPS here, or OtsA in Escherichia coli ). Organisms that use this pathway usually also have a trehalose-P phosphatase (TPP here, or OtsB in E. coli) that converts the trehalose-P to free trehalose. A second pathway that has been reported in a few unusual bacteria involves the intramolecular rearrangement of maltose (glucosyl-alpha1,4-glucopyranoside) to convert the 1,4-linkage to the 1,1-bond of trehalose. This reaction is catalyzed by the enzyme called trehalose synthase and gives rise to free trehalose as the initial product. A third pathway involves several different enzymes, the first of which rearranges the glucose at the reducing end of a glycogen chain to convert the alpha1,4-linkage to an alpha,alpha1,1-bond. A second enzyme then releases the trehalose disaccharide from the reducing end of the glycogen molecule. Finally, in mushrooms there is a trehalose phosphorylase that catalyzes the phosphorolysis of trehalose to produce glucose-1-phosphate and glucose. This reaction is reversible in vitro and could theoretically give rise to trehalose from glucose-1-P and glucose. Another important enzyme in trehalose metabolism is trehalase (T), which may be involved in energy metabolism and also have a regulatory role in controlling the levels of trehalose in cells. This enzyme may be important in lowering trehalose concentrations once the stress is alleviated. Recent studies in yeast indicate that the enzymes involved in trehalose synthesis (TPS, TPP) exist together in a complex that is highly regulated at the activity level as well as at the genetic level.     

Trehalose 6-phosphate synthase from Selaginella lepidophylla: purification and properties

A protein of 440 kDa with trehalose 6-phosphate synthase activity was purified with only one purification step by immobilized metal affinity chromatography, from fully hydrated Selaginella lepidophylla plants. The enzyme was purified 50-fold with a yield of 89% and a specific activity of 7.05 U/mg protein. This complex showed two additional aggregation states of 660 and 230 kDa. The three complexes contained 50, 67, and 115 kDa polypeptides with pI of 4.83, 4.69, and 4.55. The reaction was highly specific for glucose 6-phosphate and UDP-glucose. The optimum pH was 7.0 and the enzyme was stable from pH 5.0 to 10. The enzyme was activated by low concentrations of Ca2+, Mg2+, K+, and Na+ and by fructose 6-phosphate, fructose, and glucose. Proline had an inhibitory effect, while sucrose and trehalose up to 0.4M did not have any effect on the activity. Neither the substrates nor final product had an inhibitory effect.     

Trehalose-6-phosphate synthase/phosphatase complex from bakers' yeast: purification of a proteolytically activated form.

A protein of about 800 kDa with trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) activity was purified from bakers' yeast. This TPS/P complex contained 57, 86 and 93 kDa polypeptides. The 86 and 93 kDa polypeptides both appeared to be derived from a polypeptide of at least 115 kDa in the native enzyme. A TPS-activator (a dimer of 58 kDa subunits) was also purified. It decreased the Michaelis constants for both UDP-glucose (three-fold) and glucose 6-phosphate (G6P) (4.5-fold), and increased TPS activity at 5 mM-UDP-glucose/10 mM-G6P about three-fold. It did not affect TPP activity. The purification of TPS/P included an endogenous proteolytic step that increased TPS activity about three-fold and abolished its requirement for TPS-activator, but did not change TPP activity. This activation was accompanied by a decrease of some 20 kDa in the molecular mass of a cluster of SDS-PAGE bands at about 115 kDa recognized by antiserum to pure TPS/P, but by no change in the 57 kDa band. Phosphate inhibited TPS activity (Ki about 5 mM), but increased TPP activity about six-fold (Ka about 4 mM). Phosphate (6 mM) stimulated the synthesis of trehalose from G6P and UDP-glucose and decreased the accumulation of trehalose 6-phosphate.     

Trehalose 6-phosphate synthase from Dictyostelium discoideum: partial purification and characterization of the enzyme from young sorocarps.

Trehalose 6-phosphate synthase was solubilized from young sorocarps of the cellular slime mold, Dictyostelium discoideum, by a freeze-thaw cycle and was subsequently purified about 160-fold using streptomycin sulfate precipitation, (NH₄)₂SO₄ fractionation, DEAE-cellulose chromatography, heat treatment in the presence of heparin, and molecular sieve chromatography on columns of Bio-Gel A-1.5m. The purified enzyme was maximally active at pH 6.5, showed an absolute specificity for glucose 6-phosphate as glucosyl acceptor and a relative specificity for the glucosyl donor in the order: UDP-glucose, GDP-glucose, and ADP-glucose. Although heparin and chondroitin sulfate activated the synthase, the order of glucosyl donor specificity was not affected. Other activators of trehalose 6-phosphate synthase were KCL, Mg²⁺, and EDTA, while detergents had little effect. Although synthase activity was reduced 60 to 80% upon the omission of Mg²⁺ from the assay mixture, an absolute dependency for Mg²⁺ could not be demonstrated. Evaluation of the apparent K_m values for partially purified synthase preparations demonstrated that for each of the synthase substrates, the Line weaver-Burk plots displayed complex bimodal kinetics. Estimation of the Michaelis constants after extrapolation of the straight line portions of these plots yielded values of (a) 0.2 and 3.2 mm glucose 6-phosphate and (b) 0.5 and 2.2 mm UDP-glucose. Comparison of the latter parameters with the cellular levels of UDP-glucose and glucose 6-phosphate in Dictyostelium suggests that if the observed bimodal kinetics are the consequence of multiple kinetically distinct forms of the synthase, the activation of trehalose synthesis during slime mold culmination could provide a rationale for the presence of these isozymes.     

Engineering trehalose synthesis in Lactococcus lactis for improved stress tolerance

Trehalose accumulation is a common cell defense strategy against a variety of stressful conditions. In particular, our team detected high levels of trehalose in Propionibacterium freudenreichii in response to acid stress, a result that led to the idea that endowing Lactococcus lactis with the capacity to synthesize trehalose could improve the acid tolerance of this organism. To this end, we took advantage of the endogenous genes involved in the trehalose catabolic pathway of L. lactis, i.e., trePP and pgmB, encoding trehalose 6-phosphate phosphorylase and β-phosphoglucomutase, respectively, which enabled the synthesis of trehalose 6-phosphate. Given that L. lactis lacks trehalose 6-phosphate phosphatase, the respective gene, otsB, from the food-grade organism P. freudenreichii was used to provide the required activity. The trehalose yield was approximately 15% in resting cells and in mid-exponential-phase cells grown without pH control. The intracellular concentration of trehalose reached maximal values of approximately 170 mM, but at least 67% of the trehalose produced was found in the growth medium. The viability of mutant and control strains was examined after exposure to heat, cold or acid shock, and freeze-drying. The trehalose-producing strains showed improved tolerance (5- to 10-fold-higher survivability) to acid (pH 3) and cold shock (4°C); there was also a strong improvement in cell survival in response to heat shock (45°C), and no protection was rendered against dehydration. The insight provided by this work may help the design of food-grade strains optimized for the dairy industry as well as for oral drug delivery.     

Proteolytic activation of α,α-trehalose 6-phosphate synthase in Candida utilis

Total trehalose 6-phosphate synthase activity increased in cell-free extracts from Candida utilis following short-term preincubation of the enzyme samples at 37 degrees C. This endogenous activation was prevented by the inhibitors of serine-type proteases, phenylmethylsulfonyl fluoride, antipain or chymostatin, but not by other protease inhibitors such as pepstatin. Fractionation of the cell extracts by Sephadex G-200 gel filtration revealed that the activity of one of the two synthase enzymes present in these cells was enhanced after the activation treatment. These observations indicate the existence of a proteolytically activatable enzyme form in the trehalose 6-phosphate synthase complex of this yeast in addition to the previously characterized enzyme, whose activity appears to be inactivated by reversible phosphorylation.     

Metabolic regulation of the trehalose content of vegetative yeast.

We have investigated the mechanism by which heat shock conditions lead to a reversible accumulation of trehalose in growing yeast. When cells of S. cerevisiae M1 growing exponentially at 30 degrees C were shifted to 45 degrees C for 20 min, or to 39 degrees C for 40 min, the concentration of trehalose increased by about 25-fold; an effect reversed upon lowering the temperature to 30 degrees C. This was compared to the more than 50-fold rise in trehalose levels obtained upon transition from the exponential to the stationary growth phase. Whereas the latter was paralleled by a 12-fold increase in the activity of trehalose-6-phosphate synthase, no significant change in the activities of trehalose-synthesizing and -degrading enzymes was measured under heat shock conditions. Accordingly, cycloheximide did not prevent the heat-induced accumulation of trehalose. However, the concentrations of the substrates for trehalose-6-phosphate synthase, i.e. glucose-6-phosphate and UDP-glucose, were found to rise during heat shock by about 5-10-fold. Since the elevated levels of both sugars are still well below the Km-values determined for trehalose-6-phosphate synthase in vitro, they are likely to contribute to the increase in trehalose under heat shock conditions. A similar increase in the steady-state levels was obtained for other intermediates of the glycolytic pathway between glucose and triosephosphate, including ATP. This suggests that temperature-dependent changes in the kinetic parameters of glycolytic enzymes vary in steady-state levels of intermediates of sugar metabolism, including an increase of those that are required for trehalose synthesis. Trehalose, glucose-6-phosphate, UDP-glucose, and ATP, were all found to increase during the 40 min heat treatment at 39 degrees C. Since this also occurs in a mutant lacking the heat shock-induced protein HSP104 (delta hsp104), this protein cannot be involved in the accumulation of trehalose under these heat shock conditions. However, mutant delta hsp104, in contrast to the parental wild-type, was sensitive towards a 20 min incubation at 50 degrees C. Since this mutant also accumulated normal levels of trehalose, we conclude that HSP104 function, and not towards a 20 min incubation at 50 degrees C. Since this mutant also accumulated normal levels of trehalose, we conclude that HSP104 function, and not the accumulation of trehalose, protects S. cerevisiae from the damage caused by a 50 degrees C treatment.     

Improvement of drought tolerance and grain yield in common bean by overexpressing trehalose-6-phosphate synthase in rhizobia

Improving stress tolerance and yield in crops are major goals for agriculture. Here, we show a new strategy to increase drought tolerance and yield in legumes by overexpressing trehalose-6-phosphate synthase in the symbiotic bacterium Rhizobium etli. Phaseolus vulgaris (common beans) plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene had more nodules with increased nitrogenase activity and higher biomass compared with plants inoculated with wild-type R. etli. In contrast, plants inoculated with an R. etli mutant in trehalose-6-phosphate synthase gene had fewer nodules and less nitrogenase activity and biomass. Three-week-old plants subjected to drought stress fully recovered whereas plants inoculated with a wild-type or mutant strain wilted and died. The yield of bean plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene and grown with constant irrigation increased more than 50%. Macroarray analysis of 7,200 expressed sequence tags from nodules of plants inoculated with the strain overexpressing trehalose-6-phosphate synthase gene revealed upregulation of genes involved in stress tolerance and carbon and nitrogen metabolism, suggesting a signaling mechanism for trehalose. Thus, trehalose metabolism in rhizobia is key for signaling plant growth, yield, and adaptation to abiotic stress, and its manipulation has a major agronomical impact on leguminous plants.     

Characterization of Borrelia sp. isolated from Ixodes tanuki, I. turdus, and I. columnae in Japan by restriction fragment length polymorphism of rrf (5S)-rrl (23S) intergenic spacer amplicons

Abstract Accumulation of citric acid by Aspergillus niger depends on a high flux through glycolysis. We have investigated the possibility of control of this flux by trehalose 6-phosphate, an inhibitor of hexokinase of Saccharomyces cerevisiae and other eukaryotes (Blasquez et al., FEBS Lett. (1993) 329, 517ndash;54). Hexokinase of A. niger was shown in vitro to be only weakly inhibited by trehalose 6-phosphate (K, 1.5–2 mM). To investigate the in vivo relevance of this inhibition, we used isogenic strains of A. niger , carrying either a disruption or an amplification of the trehalose-6-phosphate synthase A (T6PSA)-encoding gene ( ggsA ) and exhibiting corresponding differences in T6PSA activity. These strains produced citric acid at comparable rates and with similar yields on 1 or 2.5% (w/v) sucrose. At 5–14% (w/v) sucrose, the ggsA disrupted strain initiated citric acid accumulation earlier, whereas the multicopy strain showed the reverse effect. When sucrose was replaced by lactose, which enabled only low rates of catabolism irrespective of its concentration (1–8%), no differences in the initiation or rate of citric acid accumulation were observed between the three strains. The possible mechanisms by which ggsA controls glycolytic flux in A. niger in the presence of high sugar concentrations are discussed.