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A. D. Elbein, Department of Biochemistry and Molecular Biology, UAMS, 4301 West Markham Street, Slot 516, Little Rock, AR 72205, USA Fax: +1 501 686 8169 Tel: +1 501 686 5176 E-mail: email@example.com
Trehalose (α,α-1,1-glucosyl-glucose) is essential for the growth of mycobacteria, and these organisms have three different pathways that can produce trehalose. One pathway involves the enzyme described in the present study, trehalose synthase (TreS), which interconverts trehalose and maltose. We show that TreS from Mycobacterium smegmatis, as well as recombinant TreS produced in Escherichia coli, has amylase activity in addition to the maltose ↔ trehalose interconverting activity (referred to as MTase). Both activities were present in the enzyme purified to apparent homogeneity from extracts of Mycobacterium smegmatis, and also in the recombinant enzyme produced in E. coli from either the M. smegmatis or the Mycobacterium tuberculosis gene. Furthermore, when either purified or recombinant TreS was chromatographed on a Sephacryl S-200 column, both MTase and amylase activities were present in the same fractions across the peak, and the ratio of these two activities remained constant in these fractions. In addition, crystals of TreS also contained both amylase and MTase activities. TreS produced both radioactive maltose and radioactive trehalose when incubated with [3H]glycogen, and also converted maltooligosaccharides, such as maltoheptaose, to both maltose and trehalose. The amylase activity was stimulated by addition of Ca2+, but this cation inhibited the MTase activity. In addition, MTase activity, but not amylase activity, was strongly inhibited, and in a competitive manner, by validoxylamine. On the other hand, amylase, but not MTase activity, was inhibited by the known transition-state amylase inhibitor, acarbose, suggesting the possibility of two different active sites. Our data suggest that TreS represents another pathway for the production of trehalose from glycogen, involving maltose as an intermediate. In addition, the wild-type organism or mutants blocked in other trehalose biosynthetic pathways, but still having active TreS, accumulate 10- to 20-fold more glycogen when grown in high concentrations (≥ 2% or more) of trehalose, but not in glucose or other sugars. Furthermore, trehalose mutants that are missing TreS do not accumulate glycogen in high concentrations of trehalose or other sugars. These data indicate that trehalose and TreS are both involved in the production of glycogen, and that the metabolism of trehalose and glycogen is interconnected.
Trehalose is a nonreducing disaccharide of d-glucose in which the two glucoses are linked in an α,α-1,1-glycosidic linkage [1,2]. Trehalose can play a number of different roles in biological systems, including serving as a reservoir of glucose for energy and/or carbon ; functioning as a stabilizer or protectant of proteins and membranes during times of stress ; acting as a regulatory molecule in the control of glucose metabolism ; serving as a transcriptional regulator ; and playing a structural and functional role as a component of various cell wall glycolipids in mycobacteria and related organisms .
In Mycobacterium smegmatis and related organisms, there are at least three different pathways that can give rise to trehalose [1,8]. The best known and most widespread pathway in many biological systems is referred to as the TPS/TPP or OtsA/OtsB pathway, which involves two enzymes. The first enzyme, trehalose phosphate synthase (TPS or OtsA), transfers glucose from UDP-glucose to glucose 6-phosphate to form trehalose phosphate and UDP . The second enzyme is a highly specific phosphatase, trehalose-phosphate phosphatase (TPP or OtsB), that removes the phosphate to produce free trehalose plus inorganic phosphate . A second pathway of more limited scope in biological systems also involves two enzymes that convert glycogen to trehalose . The first enzyme of this pathway is maltooligosyl trehalose synthase (TreY), which changes the α1-4 linkage at the reducing end of bacterial glycogen to the α,α,1,1-linkage of trehalose. The second enzyme, maltooligosyl trehalose trehalohydrolase (TreZ), cleaves the α1,4-glycosidic linkage to which the newly-formed trehalose is attached, producing free trehalose and leaving a glycogen chain minus two glucoses . The third pathway involves a single enzyme, trehalose synthase (TreS), which catalyzes the interconversion of maltose and trehalose [13,14]. Although TreS can produce trehalose from maltose, it has been postulated that its real role, at least in corynebacteria, is to control intracellular levels of trehalose by converting excess trehalose to maltose, which can then be converted by α-glucosidases to glucose [15,16]. By contrast, mycobacteria have a potent trehalase , whereas corynebacteria do not. Therefore, the TreS of mycobacteria may have a different and more significant role in the synthesis of trehalose from maltose. However, until now, it has not been clear where mycobacteria could obtain the maltose to transform into trehalose because M. smegmatis grows very poorly on maltose.
Our preliminary experiments suggested that TreS was somehow involved in glycogen synthesis and degradation. Thus, it was important to determine how the presence of TreS affects the levels of glycogen and trehalose in cells. Accordingly, mutants of M. smegmatis that were missing TreS or one of the other trehalose biosynthetic pathways were prepared (for designation of mutants, see Table 1) and the levels of glycogen and trehalose were compared in these cells. In addition, either recombinant TreS made in Escherichia coli, or TreS purified from the wild-type M. smegmatis, was assayed to determine its substrate specificity, and its sensitivity to various inhibitors of trehalose or glycogen metabolism. These studies demonstrated that TreS contains amylase activity, in addition to its maltose ↔ trehalose interconverting activity (referred to as MTase). These experiments also show that all of the M. smegmatis stains that contain TreS accumulate large amounts of glycogen when grown in high concentrations of trehalose, but mutants missing TreS activity do not accumulate glycogen, regardless of the amount of trehalose in the media. The results obtained indicate that TreS plays an key role in the utilization of trehalose for the production of glycogen. We hypothesize that TreS acts as a sensor or regulator of trehalose levels in these cells by catalyzing the conversion of glycogen to trehalose when cytoplasmic trehalose levels are low, but this enzyme also can expedite or promote the conversion of trehalose to glycogen when cytoplasmic trehalose levels become too high.
Table 1. Enzymatic profiles of various mycobacterial trehalose biosynthetic mutants.
Enzyme(s) missing (trehalose biosynthesis)
Trehalose biosynthetic pathways (active)
All (i.e. TPS/TPP; TreS TreY/TreZ)
TPS, TPP, TreY
TPS/TPP, TreS, TreY
Purification and demonstration of two activities
TreS was initially purified to near homogeneity from extracts of M. smegmatis as previously described . The final preparation showed one major band on SDS gels with a molecular mass of approximately 68 kDa. This activity of TreS, referred to here as MTase, catalyzed the conversion of trehalose to maltose as measured by the reducing sugar method, or by the formation of maltose on the Dionex carbohydrate analyzer . MTase also catalyzed the reverse reaction (i.e. the conversion of maltose to trehalose). Studies on the substrate specificity of TreS showed that the purified enzyme could also produce maltose from either glycogen or maltooligosaccharides (amylase activity). This second activity was of considerable interest because it suggested that at least one function of TreS could be to convert glycogen to trehalose by a series of reactions: glycogen → maltose ↔ trehalose. Trehalose has been shown to be essential for the growth of mycobacteria [18,19]; therefore, TreS could have an important function under certain conditions, such as when cytoplasmic trehalose levels are low, where this enzyme could provide the essential trehalose from glycogen.
The TreS gene from both M. smegmatis and M. tuberculosis was cloned and expressed in E. coli with a (His)6 tag at the amino terminus, and active enzyme was produced in good yield. The expressed proteins were applied to a Ni column and the 100 mm imidazole eluate of the column containing the purified TreS was concentrated on the Amicon filtration apparatus (Millipore, Billerica, MA, USA) several times to remove imidazole. Both recombinant TreS preparations made from either the M. tuberculosis or the M. smegmatis gene, as well as TreS purified directly from extracts of M. smegmatis, undergo a self-induced or autocatalytic proteolysis upon long-term storage on ice, during which time the 68 kDa protein is slowly converted to a 58 kDa protein. This transformation is shown in Fig. 1. In this experiment, recombinant M. smegmatis TreS, purified on the Ni column, was kept on ice for 43 days and, at various times, samples were removed and subjected to SDS/PAGE and also assayed for MTase and amylase activities. The MTase activity increased as the protein was degraded and was approximately two-fold higher in the 58 kDa protein as in the 68 kDa MTase. On the other hand, the amylase activity remained constant during this change, but it was present in all of the intermediate proteins, as well as in the 58 kDa protein. The 58 kDa band was eluted from the gel and subjected to tryptic digestion and Q-TOF MS to identify the peptides. These data indicated that the 58 kDa protein was identical to the 68 kDa TreS, except for the loss of approximately 10 kDa of peptide from the carboxy terminus. Thus, these data indicate that the MTase activity is increased by the loss of the carboxy-terminal region of the protein, but the amylase activity remains at the same level in the various intermediate forms of the enzyme.
Additional evidence that both MTase and amylase activities reside in the same protein is demonstrated by the experiment shown in Fig. 2. In this case, recombinant M. smegmatis TreS was purified on a Ni column and, after removal of imidazole, the protein was allowed to remain in an ice bath for several weeks until most of the protein had been converted to the 58 kDa form. This protein preparation was then applied to a Sephacryl S-200 column (GE Healthcare, Uppsala, Sweden), and fractions from the column were collected. Starting at tube number 41, every 10 tubes were pooled to give seven fractions as follows: A = 41–50; B = 51––60; C = 61–70; D = 71–80; E = 81–90; F = 91–100; G = 101–110; and H = 111–120. An aliquot of each pooled fraction was subjected to SDS/PAGE (Fig. 2) and MTase activity and amylase activity were also assayed in each of these fractions. Figure 2 shows that the 58 kDa protein was clearly evident on SDS/PAGE gels in fractions C to G, but was present in highest amounts in fractions D and E. In addition, both MTase and amylase activities were present in fractions B to H but, more importantly, the ratio of MTase to amylase remained fairly constant in fractions C to F (Fig. 2, bottom). These data strongly suggest that these two activities reside in the same protein. As a control for these experiments, we prepared a cell-free extract of the untransfected vector and put it through the same purification procedure. In this case, we did not find any amylase activity in the imidazole elutions of the Ni column.
Finally, as further proof that amylase and MTase activities reside in the same protein, we demonstrated the presence of both activities in crystals of TreS. These crystals had both MTase activity for converting trehalose to maltose and amylase activity that converted either glycogen or maltoheptaose to maltose (Table 2). The amylase activity was better with maltoheptaose as a substrate than with glycogen. A second set of crystals was also isolated and tested in the same way and showed both activities, although at slightly different levels.
Table 2. Enzymatic activities of MTase and amylase in crystals of TreS. ND, not determined.
Time of incubation (min)
Amylase activity on [amount of maltose (μg)]:
MTase activity [maltose produced (μg)]
Demonstration of amylase activity
As described in the Experimental procedures, the Dionex analyzer readily separates trehalose, maltose and glucose from each other and quantifies the amount of each sugar using an amperometric detection system. Figure 3A shows that the amount of maltose produced from glycogen by the recombinant TreS was linear with time of incubation for up to 24 h, and was also proportional to the amount of enzyme added (Fig. 3B), for up to at least 3 μg of protein. These data also indicate that the amylase activity was quite stable at 37 °C in the presence of glycogen because the rate of production of maltose remained linear for at least 24 h of incubation. In these experiments, very little trehalose was detected at early times, probably because the Km of MTase for maltose is approximately 10 mm  and, therefore, even at 6 h of incubation, the amount of maltose produced is far below the Km. However, the production of trehalose from glycogen could be demonstrated using radioactive glycogen as the substrate, as described below.
The production of maltose from glycogen, as well as the production of trehalose, could be demonstrated using the Dionex carbohydrate analyzer (Fig. 4). [3H]glycogen was incubated either with the purified TreS (lower profile), or with a commercial preparation of pancreatic amylase to serve as a control (upper profile). After incubation for 6 h, the reaction mixtures were passed through a column of Biogel P-4, and those column fractions representing the monosaccharide to tetrasaccharide elution region of the column were pooled, concentrated, and the radioactive sugars were identified on the Dionex HPLC by analyzing an aliquot of each fraction for its radioactive content. The upper profile shows that the pancreatic amylase generated a large peak of [3H]maltose and a smaller peak of [3H]glucose, but no radioactive trehalose was produced by this enzyme. By contrast, incubation with the TreS generated a large peak of radioactive maltose as well as a substantial peak of radioactive trehalose and a small peak of [3H]glucose. The radioactive peak corresponding to trehalose was completely susceptible to digestion by a specific recombinant trehalase produced in E. coli, and this digestion resulted in the production of radioactive glucose as the only product (data not shown). That maltose is the initial product produced from glycogen was previously demonstrated by the experiment shown in Fig. 3A when the time course fractions were analyzed on the Dionex and essentially no trehalose was observed at the early time points, but was clearly evident at later times of incubation. Thus, TreS not only has MTase activity, but also it has amylase activity that produces the initial maltose.
Properties of the M. smegmatis amylase activity
As indicated in Fig. 3, the production of maltose from glycogen by TreS increased in a linear fashion with increasing time of incubation and with increasing amounts of protein. The pH requirement for the conversion of glycogen to maltose was determined and the pH optimum was found to be in the range 6.0–6.2 (data not shown). Interestingly, the pH optimum for the MTase activity (conversion of trehalose to maltose) of TreS was previously determined to be 7.0 .
TreS can also use maltooligosaccharides as substrates to produce maltose and then trehalose. A comparison of the activity of TreS on glycogen and on maltoheptaose is presented in Table 3. Maltoheptaose was a good substrate for the production of maltose and the rate of maltose formation increased with increasing amounts of substrate. In the presence of higher amounts of maltoheptaose and with longer incubations, trehalose was also identified in these incubations. The production of maltose was measured by determining the area of the maltose peak on the Dionex analyzer. It is not possible to directly compare the effectiveness of maltoheptaose to glycogen because the commercial glycogen is a mixture of glucose polymers of different molecular masses, and probably different degrees of branching. However, this experiment demonstrates that TreS can use various maltooligosaccharides, as well as glycogen, to produce maltose and trehalose.
Table 3. Comparison of maltoheptaose and glycogen as substrates for TreS.
Amount of substrate (μg added to incubation)
Maltose (μg) produced from:
The amylase activity of TreS was stimulated by the addition of Ca2+ (Fig. 5). Although amylase activity was not completely dependent on the presence of this divalent cation, the activity was stimulated by as much as four-fold in the presence of 14 mm Ca2+. Other metal ions such as Mg2+, Mn2+, and Co2+ were also somewhat effective but Hg2+, Cu2+, Ni2+ and Zn2+ were inhibitory. The requirement for Ca2+ for activity, and its ability to stabilize this group of glucosylhydrolases, has been demonstrated for a number of α-amylases [20–22]. Whereas the amylase activity of TreS is stimulated by calcium ions, the MTase is inhibited by divalent cations. This effect is also shown in Fig. 5. These data show that MTase not only does not require Ca2+, but also that this activity is strongly inhibited by calcium concentrations of 5 mm or higher. Other cations were also inhibitory to MTase activity.
Selective inhibition of MTase and amylase activities
Two inhibitors have been identified that selectively inhibit either the amylase activity or the MTase activity, suggesting the possibility of two different active sites in the TreS. Validoxylamine is a known inhibitor of trehalases  and has a structure that mimics a number of known glycosidase inhibitors . Figure 6A shows that validoxylamine is a potent inhibitor of the MTase activity of TreS, with a Ki of approximately 25 nm. However, validoxylamine had no effect on the amylase activity, even at concentrations of 2 μm. Figure 6B shows a Lineweaver–Burk plot of the effect of trehalose concentration on MTase activity at two different concentrations of validoxylamine. These data clearly demonstrate that validoxylamine is a competitive inhibitor of MTase with respect to trehalose. Although the amylase activity of TreS is not inhibited by validoxylamine, it is inhibited by the known amylase inhibitor, acarbose [24–27]. These experiments with acarbose (Fig. 7) demonstrate that the amylase activity is strongly inhibited by acarbose, with 50% inhibition occurring at a concentration of approximately 5 μg·mL−1. On the other hand, MTase activity was not susceptible to inhibition by acarbose, even at concentrations of 100 μg·mL−1. Other studies on the crystal structure of pancreatic amylase demonstrated that acarbose binds at the active site of this enzyme [26,27]. Additional studies with acarbose have suggested that it acts as a transition state inhibitor with amylase-like enzymes, also indicating that it binds at the active sites of these enzymes.
Additional support for the hypothesis that TreS has two different binding sites is provided by the finding that adding glycogen to incubations of MTase with trehalose does not inhibit the conversion of trehalose to maltose. In this experiment, increasing amounts of glycogen were added to incubation mixtures containing buffer, trehalose and TreS (Table 4). Following incubation for 15 min, reactions were stopped by heating, and the amount of maltose was determined by the reducing sugar method. A series of control incubations were also run that contained the same amounts of glycogen, buffer and enzyme, but trehalose was omitted from these incubations. Glycogen had no effect on the MTase activity because the amount of reducing sugar did not change in these incubations containing trehalose, regardless of the amount of glycogen added (Table 4). The product from several of these incubations was also examined on the Dionex analyzer, and trehalose and maltose were the only oligosaccharides detected. On the other hand, no reducing sugar or maltose was detected in the control incubations of glycogen and enzyme, but without trehalose. It should be noted that incubations were only 15 min in length; therefore, the amylase activity on the added glycogen was not sufficient to produce detectable amounts of maltose. Thus, the MTase site of TreS appears to be distinct from the amylase site.
Table 4. Effect of glycogen on MTase activity (trehalose → maltose). Incubations with trehalose as substrate were as described in the text. The amount of maltose formed was determined by the reducing sugar test.
Amount of glycogen added to incubations (μg)
Reducing sugar (A620)
Importance of TreS in homeostasis of mycobacteria
Although the exact function of TreS is not known, and mutants lacking TreS can still grow if trehalose is added to the medium, this enzyme does appear to play a key role in the interactions between glycogen and trehalose. Thus, under some circumstances, such as low levels of cytoplasmic trehalose, it is likely that the cells would degrade glycogen to maltose, and this maltose would then be converted to trehalose to raise trehalose levels. Interestingly, as shown in the present study, high levels of trehalose in the cytoplasm also appear to cause/or stimulate the accumulation of glycogen in these cells. This effect is shown by the data presented in Table 5, where the levels of cytoplasmic glycogen are compared in wild-type M. smegmatis, or in various trehalose mutants (for identification of mutants, see Table 1) grown in a mineral salts medium with low (0.1%) or high (2% or 4%) amounts of trehalose. Those flasks containing 0.1% trehalose also had 1.9% glucose. With 2% or higher concentrations of trehalose in the media, cells containing TreS (wild-type and mutants #47 and #74; Table 1) had 10 to 30-fold more glycogen than cells grown in low trehalose, or in cells lacking TreS (mutants #80 and #91) (Table 5). Furthermore, Table 6 shows that this increase in glycogen levels only occurred when trehalose was in the media at concentrations of 25 mm (∼ 1%) or higher, but did not occur in the presence of high levels of glucose, or other sugars such as sucrose or lactose (not shown). The level of cytoplasmic trehalose in wild-type M. smegmatis was not significantly altered by high (100 mm) concentrations of trehalose or glucose in the media (Table 6), suggesting that the level of intracellular trehalose is carefully regulated. There is some evidence from other systems that high intracellular concentrations of trehalose may be toxic to cells.
Table 5. Effect of exogenous trehalose on accumulation of glycogen. In all cases, the sugar content of the media was 2% or higher (i.e. 0.1% trehalose + 1.9% glucose, etc.).
Amount of trehalose in media (%/weight)
Amount of glycogen in cells (nmol glycogen as glucose·mg−1 dry cells) in the following wild-type or mutant:
0.1 (+1.9 glucose)
Presence of TreS in cells
Table 6. Effect of trehalose concentration in the media on levels of glycogen and trehalose in cells of M. smegmatis. All experiments were performed with the wild-type organism (i.e. B11).
Sugar in the media (glucose or trehalose) (mm)
Amount of trehalose in cytoplasm (μg·mg−1 cells)
Amount of glycogen in (nmol as glucose/mg cells)
TreS is a 68 kDa protein that is present in a number of bacteria, including mycobacteria, corynebacteria, nocardia and streptomyces, as well as arthrobacter, sulfolobus and rhizobium [8,11–13]. TreS has been purified to near homogeneity from M. smegmatis, and the gene for this protein was cloned and expressed in E. coli . The expressed protein had a subunit molecular mass of 68 kDa on SDS gels, but active enzyme eluted as a 390 kDa protein upon gel filtration, suggesting that active TreS is a hexamer of six identical subunits. TreS catalyzes the reversible interconversion of trehalose and maltose. The reaction kinetics favor the conversion of maltose to trehalose, with a Km for maltose of approximately 10 mm, whereas the Km for trehalose is approximately 90 mm.
In Corynebacterium glutamicum, TreS has been proposed to function as a substitute for a trehalase to control intracellular levels of trehalose because no ORF homologous to known trehalase genes have been identified, nor has any trehalase activity been demonstrated in this organism . However, M. smegmatis does have a highly specific and active trehalase , in addition to the TreS described above . Another report on the TreS of C. glutamicum suggests that this enzyme is only involved in trehalose biosynthesis when these organisms are growing on maltose. Thus, a critical question with regard to the production of trehalose by TreS remains. What is the possible source of maltose that TreS could use as a substrate to produce trehalose?
Exogenous maltose is not a likely source of maltose for M. smegmatis because this organism grows very poorly on maltose. However, the results obtained in the present study indicate that endogenous maltose can be produced from glycogen by the amylase activity of TreS, and that this maltose is readily converted to trehalose by the MTase activity of TreS.
The present study provides evidence indicating that both activities reside in the same protein. First, TreS, purified from M. smegmatis as well as recombinant TreS produced in E. coli, had both MTase activity and amylase activity. Second, the 68 kDa TreS undergoes auto-proteolysis to give a 58 kDa protein, which also contains both MTase and amylase activity. Third, the 58 kDa protein was subjected to gel filtration and fractions were collected. Six fractions across the protein peak had variable amounts of MTase activity with the highest activity corresponding to fractions showing the most 58 kDa protein by SDS/PAGE. Importantly, the ratio of MTase/amylase, but not the absolute activity, remained fairly constant in fractions having different amounts of the 58 kDa protein. Finally, crystals of TreS were obtained, and these isolated crystals have both amylase activity and MTase activity.
These results strongly indicate that the MTase activity and the amylase activity are in the same protein, and suggest that this multifunctional protein has the capacity to convert glycogen to trehalose.
The partial amino acid sequence of TreS from M. smegmatis allowed us to locate the ORF for this protein and a blastp search indicated that it had approximately 83% identity to a gene (Rv 0126) for a hypothetical α-amylase in the M. tuberculosis genome . It also has 72% identity to a putative TreS from Streptomyces avermitilis, 69% identity in C. glutamicum, and 61% identity to the putative TreS from Pseudomonas sp. Because there are no reports on the isolation or characterization of these TreS proteins, it is not known whether they also have amylase activity, but it will be interesting to determine whether the TreS of corynebacteria also shares this activity. It will also be important to determine ways to test this amylase activity for function in vivo to establish whether it can really act in collaboration with the TreS activity to convert glycogen glucoses into cytoplasmic trehalose.
We propose that TreS has two distinct active sites: one catalyzing the interconversion of maltose and trehalose (referred to here as MTase activity) and the other catalyzing the breakdown of glycogen to maltose (amylase activity).
The present study provides evidence supporting the existence of the two sites. First, the amylase site is activated by Ca2+ whereas the MTase activity is inhibited by Ca2+ and other cations. Second, we have identified two inhibitors each of which competitively inhibits one activity and not the other. Thus, validoxylamine competitively inhibits MTase but not amylase, whereas acarbose competitively inhibits amylase but not MTase. Third, glycogen, which is a substrate for the amylase activity of TreS, has no effect on the MTase activity of TreS. That is, incubations of MTase with trehalose produce the same amount of maltose, even in the presence of high amounts of glycogen.
These data suggest that these two activities reside in different sites on the protein. However, it will require site-directed mutagenesis studies, or deletions of various parts of the protein, to conclusively prove that there are indeed two sites. Once we have identified active site amino acids for each catalytic activity, it will be possible to perform site-directed mutagenesis to modify one activity and not the other. We have been able to obtain small-sized crystals of TreS but they do not have sufficiently high resolution for structural analysis. Attempts to improve the resolution of these crystals is in progress.
Our hypothesis on the function of TreS is that it serves as a sensor and/or controller of the cellular trehalose levels in mycobacteria and perhaps other organisms. The present studies show that TreS can mediate the formation of trehalose from glycogen. In addition, growth studies with the wild-type M. smegmatis show that, when this organism is grown in a mineral salts medium with high concentrations (1–4%) of trehalose as the major carbon source, these cells contain 10- to 30-fold higher amounts of glycogen than cells grown in the same concentration of glucose or other sugars. Furthermore, additional studies with a number of trehalose mutants that are missing one, two or all three of the trehalose biosynthetic pathways (Table 1) demonstrate that any of the mutants still containing TreS (including the mutant that only has TreS) show this accumulation of glycogen in the presence of high trehalose, but any mutants that are missing TreS do not accumulate glycogen at any level of trehalose, or any other sugar. Thus, TreS not only is involved in the production of trehalose from glycogen, but also appears to play an essential role in the formation, and/or accumulation, of glycogen. This accumulation somehow involves the utilization of trehalose as the carbon source, but the mechanism of this conversion is not known. We propose that when high levels of trehalose are produced in the cell, perhaps as a result of exposure to stress, TreS may function to convert this trehalose to maltose and then to glycogen when the stress is removed. Removal of trehalose is probably essential because high levels of trehalose may be toxic. On the other hand, if trehalose falls to a dangerously low level, TreS may function to convert glycogen to maltose and then to trehalose. Ongoing studies are attempting to determine how trehalose is involved in the formation of glycogen, and how TreS functions as a sensor or regulator of trehalose and/or glycogen levels in these cells.
Bacterial strains and culture conditions
M. smegmatis was obtained from the American Type Culture Collection (ATCC 14468). It was maintained on slants of Trypticase Soy Agar and was grown at 37 °C in 2 L Erlenmeyer flasks containing 1 L of Trypticase Soy Broth (Becton Dickinson, Franklin Lakes, NJ, USA). The E. coli strains DH5α and HMS-F were used for cloning and expression studies, respectively. HMS-F is a derivative of the expression strain HMS174(DE-3) (Novagen, Madison, WI, USA). HMS174(DE-3) contains a chromosomal isopropyl thio-β-d-galactoside-inducible T7 RNA pol gene. HMS-F contains an additional copy of the lac repressor lacIq on an F episome, which was transferred from the E. coli cloning strain XL-1 (Stratagene, La Jolla, CA, USA). This addition essentially represses expression from the T7 promoter on the E. coli expression vector pET15b (Novagen) in the absence of isopropyl thio-β-d-galactoside. HMS-F was routinely cultured in the presence of tetracycline (10 μg·mL−1) to maintain carriage of the F episome. E. coli strains were cultured in LB-broth and on LB-agar supplemented with 100 μg·mL−1 ampicillin, 20 μg·mL−1 kanamycin or 10 μg·mL−1 tetracycline, individually or in combination, where applicable.
Preparation of mutant strains of M. smegmatis missing various trehalose synthetic pathways
Mutants were prepared by allele replacement mutagenesis. Target genes were PCR amplified using gene specific primers from M. smegmatis genomic DNA. The cloned target gene was mutagenized by generating an internal deletion in the target ORF. The deletion was confirmed by sequencing the mutagenized allele. The PCR product was ligated into the plasmid pMAR1, a mycobacterial suicide vector constructed by introducing a unique PacI restriction site and a wild-type allele of M. smegmatis rspL  into the E. coli cloning vector pSP72 (Promega, Madison, WI, USA). A PacI-ended selection cassette, containing a positive selector hyg (hygromycin resistant), the reporter gene lacZ and the negative selector sacB (each driven by separate mycobacterial promotors), was inserted into the pMAR1 PacI site . The resulting plasmid was then transformed into the wild-type M. smegmatis . Duplication insertions, resulting from homologous recombination between the plasmid-borne mutant allele and the chromosomal wild-type target gene, were recovered on medium containing hygromycin and X-Gal. These transformants were also streptomycin-sensitive, as a result of acquisition of the plasmid-borne rspLwt gene. Wild-type rspL-mediated streptomycin sensitivity (Strs) is dominant over mutant rspL-mediated streptomycin resistance . The presence of both wild-type and mutant alleles were confirmed by PCR. Resolution of the duplication and loss of one of the target gene alleles by homologous recombination was selected for by growing the culture in nonselective medium and plating on medium containing streptomycin and X-Gal. Loss of the integrated pMAR1 plasmid restored streptomycin resistance (Strr). Resultant StrrLac– colonies were screened by PCR for the carriage of the wilt-type or mutant allele. As this strategy generates unmarked deletion mutants that are fully amenable to further rounds of mutagenesis, multiple pathways were mutagenized in the same M. smegmatis strain . The mutants used are shown in Table 1 and comprise: mutant #47 missing TPP and the TPS/TPP pathway, but having functional TreS and TreY/TreZ pathways; mutant #74 missing TPS, TPP and TreY and the TPS/TPP and TreY/TreZ pathways, but having a functional TreS pathway; mutant #91 missing TreS and the TreS pathway but having functional TPS/TPP and TreY/TreZ pathways; mutant #80 missing TPS, TPP, TreS and TreY and having no trehalose biosynthetic pathways. mutant #80 absolutely requires trehalose in the media for growth.
Materials and reagents
Trehalose, maltose, isomaltose, malto-oligosaccharides and other sugars were purchased from Sigma Chemical Co. (St Louis, MO, USA). DEAE-cellulose and various other chromatographic resins for protein purification, molecular markers for gel filtration and buffers were also obtained from Sigma. Bio-Rad protein reagent and DE-52 were from Bio-Rad Laboratories Inc. (Hercules, CA, USA). Trypticase soy broth was from Becton Dickinson, and LB broth was from Fisher Scientific Co. (Pittsburgh, PA, USA).
Radioactive glycogen was made by growing M. smegmatis in high-specific activity [3H]glucose in a mineral salts medium for 48 h. The glycogen was isolated and purified as previously described . One hundred to five hundred μCi of [U-3H]-glucose was added to Trypticase Soy Broth that did not contain any unlabeled glucose. The flasks containing this radioactivity were inoculated with a small innoculum of a growing culture of M. smegmatis and the cultures were grown for 2 days at 37 °C on a recriprocal shaker. At the end of this time, cells were harvested by centrifugation, washed with NaCl/Pi and sonicated in water. The cell debris was removed by high-speed centrifugation, and the supernatant (cytosolic) fraction was cooled and cold trichloroacetic acid was added with stirring to a final concentration of 5% to precipitate the protein. The precipitated protein was removed by centrifugation and discarded, and the supernatant liquid was placed in a large separatory funnel and extracted four times with large volumes of ethyl ether to remove the trichloroacetic acid. The aqueous fraction from these extractions was concentrated to a smaller volume and three volumes of ice cold methanol was added with stirring to precipitate the glycogen. The precipitate was isolated by centrifugation, dissolved in water and subjected to descending paper chromatography on 3MM paper (Whatman, Clifton, NJ, USA) in n-butanol/pyridine/water (4 : 3 : 4, v/v/v). As glycogen is a large molecule, it remains at the origin, but monosaccharides and oligosaccharides migrate down the paper and away from the origin. The papers were dried and the glycogen at the origins was eluted with water and passed through a column of Biogel P-4 (Bio-Rad Laboratories Inc.). A large symmetrical peak of radioactive glycogen emerged from the P-4 column at the void volume of the column. This fraction was used as the glycogen substrate. It reacted with iodine and this complex gave a spectrum in the same visible range as that produced by authentic glycogen.
Purification of TreS from M. smegmatis
TreS was purified from cell free extracts of M. smegmatis as previously described . This included ammonium sulfate fractionation, gel filtration, chromatography on columns of DEAE-cellulose, hydroxyapatite, aminohexyl-agarose and then phenyl-sepharose. At the final stage of purification, the enzyme preparation showed one major protein band of 68 kDa on SDS gels, which had both MTase activity and amylase activity (see Results).
Crystals of TreS were obtained using the crystallization kits, and these crystals were tested for the presence of both MTase activity and amylase activity. Although these crystals were small and not of high enough resolution for structural studies, they were isolated by centrifugation in a microfuge tube, washed several times with the same fresh crystallization fluid, and then dissolved in the assay buffer. Both MTase activity and amylase activity were measured in these dissolved crystals using the assay methods described below. The results of those assays are presented in Table 2.
Assay of TreS activities
The MTase activity of TreS was measured by determining the formation of reducing sugar resulting from the formation of maltose, when the enzyme was incubated with trehalose. Assays were performed in a final volume of 100 μL containing 40 mm potassium phosphate buffer (pH 6.8), various amounts of trehalose (usually 50–100 mm) and an appropriate amount of enzyme. After incubation at 37 °C for various time periods, the mixture was heated in a boiling water bath for several minutes to stop the reaction, and the amount of maltose produced was determined by the reducing sugar method . The production of maltose could also be determined by subjecting the heated reaction mixture to HPLC on the Dionex carbohydrate analyzer (Dionex, Sunnyvale, CA, USA). In addition, the activity could also be assayed in the opposite direction by measuring the formation of trehalose when TreS (MTase) was incubated with maltose. This was best perfomred using the Dionex carbohydrate analyzer that readily separates trehalose from maltose, glucose and other sugars (Fig. 4). As shown in the present study, TreS can also convert glycogen to maltose and trehalose. For assay of these reactions, incubations contained 0.5 mg of glycogen in 100 μL of 40 mm potassium phosphate buffer (pH 6.0) or sodium acetate buffer (pH 6.0), 10 mm CaCl2, and various amounts of enzyme. After incubation as described above, the reaction mixtures were subjected to HPLC on the Dionex carbohydrate analyzer and the amounts of maltose and trehalose produced from glycogen were measured.
Separation and identification of sugars
Sugars were separated and identified using high-performance anion-exchange chromatography on the Dionex carbohydrate analyzer. Eluents were distilled water (E1) and 400 nm NaOH (E2). Appropriate aliquots (0–3 nmol) from each sample were injected into a CarboPac PA-1 column equilibrated with a mixture of E1 and E2 (E1/E2 = 98/2). The elution and resolution of the carbohydrate mixtures was performed as follows: T0 – T20 min = 2% E2 (v/v); T20 min – T30 min = gradient 2% E2 to 100% E2 (v/v); T30 min – T∞ = 100% E2 (v/v). Each constituent was detected by pulse amperometry as recommended by the manufacturer (Dionex, technical note, 20 March 1989) at a range setting of 300 K. In some cases, an aliquot of the elution fraction was subjected to liquid scintillation counting to determine the radioactive content of each peak. These aliquots were mixed with scintillation fluid and counted in a Beckman scintillation counter (Beckman Coulter Inc., Fullerton, CA, USA).
Protein was measured with the Bio-Rad protein reagent using BSA as the standard. Sugars were analyzed using the Dionex carbohydrate analyzer to separate maltose, trehalose and other sugars. Reducing sugars were measured and quantified by the copper colorimetric method of Nelson . SDS/PAGE was performed according to Laemmli in 10% polyacrylamide gel using 0.1% SDS . The gels were stained with 0.5% Coomassie blue in 10% acetic acid.
We thank Dr Alan Tackett (Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences) for sequencing the 58 kDa TreS and comparing this sequence to that of the 68 kDa TreS. We also thank Drs Reha Celikel and Kottayil Varughese (Department of Physiology and Biophysics, University of Arkansas for Mediucal Sciences) for obtaining the crystals of TreS.