Coregulation of beta-galactoside uptake and hydrolysis by the hyperthermophilic bacterium Thermotoga neapolitana

Regulation of the beta-galactoside transport system in response to growth substrates in the extremely thermophilic anaerobic bacterium Thermotoga neapolitana was studied with the nonmetabolizable analog methyl-beta-D-thiogalactopyranoside (TMG) as the transport substrate. T. neapolitana cells grown on galactose or lactose accumulated TMG against a concentration gradient in an intracellular free sugar pool that was exchangeable with external galactose or lactose and showed induced levels of beta-galactosidase. Cells grown on glucose, maltose, or galactose plus glucose showed no capacity to accumulate TMG, though these cells carried out active transport of the nonmetabolizable glucose analog 2-deoxy-D-glucose. Glucose neither inhibited TMG uptake nor caused efflux of preaccumulated TMG; rather, glucose promoted TMG uptake by supplying metabolic energy. These data show that beta-D-galactosides are taken up by T. neapolitana via an active transport system that can be induced by galactose or lactose and repressed by glucose but which is not inhibited by glucose. Thus, the phenomenon of catabolite repression is present in T. neapolitana with respect to systems catalyzing both the transport and hydrolysis of beta-D-galactosides, but inducer exclusion and inducer expulsion, mechanisms that regulate permease activity, are not present. Regulation is manifest at the level of synthesis of the beta-galactoside transport system but not in the activity of the system.     

Regulation of beta-galactoside transport and accumulation in heterofermentative lactic acid bacteria.

Galactose-grown cells of the heterofermentative lactic acid bacteria Lactobacillus brevis and Lactobacillus buchneri transported methyl-beta-D-thiogalactopyranoside (TMG) by an active transport mechanism and accumulated intracellular free TMG when provided with an exogenous source of energy, such as arginine. The intracellular concentration of TMG resultant under these conditions was approximately 20-fold higher than that in the medium. In contrast, the provision of energy by metabolism of glucose, gluconate, or glucosamine promoted a rapid but transient uptake of TMG followed by efflux that established a low cellular concentration of the galactoside, i.e., only two- to fourfold higher than that in the medium. Furthermore, the addition of glucose to cells preloaded with TMG in the presence of arginine elicited a rapid efflux of the intracellular galactoside. The extent of cellular TMG displacement and the duration of the transient effect of glucose on TMG transport were related to the initial concentration of glucose in the medium. Exhaustion of glucose from the medium restored uptake and accumulation of TMG, providing arginine was available for ATP generation. The nonmetabolizable sugar 2-deoxyglucose elicited efflux of TMG from preloaded cells of L. buchneri but not from those of L. brevis. Phosphorylation of this glucose analog was catalyzed by cell extracts of L. buchneri but not by those of L. brevis. Iodoacetate, at a concentration that inhibits growth and ATP production from glucose, did not prevent efflux of cellular TMG elicited by glucose. The results suggested that a phosphorylated metabolite(s) at or above the level of glyceraldehyde-3-phosphate was required to evoke displacement of intracellular TMG from the cells. Counterflow experiments suggested that glucose converted the active uptake of TMG in L. brevis to a facilitated diffusion mechanism that allowed equilibrium of TMG between the extra- and intracellular milieux. The means by which glucose metabolites elicited this vectorial regulation is not known, but similarities to the inducer expulsion that has been described for homofermentative Streptococcus and Lactobacillus species suggested the involvement of HPr, a protein that functions as a phosphocarrier protein in the phosphotransferase system, as well as a presumptive regulator of sugar transport. Indeed, complementation assays wit extracts of Staphylococcus aureus ptsH mutant revealed the presence of HPr in L. brevis, although this lactobacillus lacked a functional phaosphoenolpyruvate-dependent phosphortransferase system for glucose, 2-deoxyglucose, or TMG.     

Phosphoenolpyruvate and 2-phosphoglycerate: endogenous energy source(s) for sugar accumulation by starved cells of Streptococcus lactis.

In the absence of an exogenous energy source, galactose-grown cells of Streptococcus lactis ML3 rapidly accumulated thiomethyl-beta-D-galactopyranoside (TMG) and 2-deoxyglucose to intracellular concentrations of 40 to 50 mM. Starved cells maintained the capacity for TMG uptake for many hours, and accumulation of the beta-galactoside was insensitive to proton-conducting ionophores (tetrachlorosalicylanilide and carbonylcyanide-m-chlorophenyl hydrazone) and sulfydryl group reagents including iodoacetate and N-ethylmaleimide. Fluorimetric analysis of glycolytic intermediates in extracts prepared from starved cells revealed (a) high intracellular levels of phosphoenolpyruvate (13 mM; PEP) and 2-phosphoglycerate (approximately 39 mM; 2-PG), but an absence of other metabolites including glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-diphosphate, and triosephosphates. The following criteria showed PEP (and 2-PG) to be the endogenous energy source for TMG accumulation by the phosphotransferase system: the intracellular concentrations of PEP and 2-PG decreased with concomitant uptake of TMG, and a close correlation was observed between maximum accumulation of the beta-galactoside and the total available concentration of the two intermediates; TMG accumulated as an anionic derivative, which after extraction and incubation with alkaline phosphatase (EC 3.1.3.1) formed the original analogue; fluoride inhibition of 2-phospho-D-glycerate hydrolyase (EC 4.2.1.11) prevented the conversion of 2-PG to PEP, and uptake of TMG by the starved cells was reduced by 80%; and the stoichiometric ratio [TMG] accumulated/[PEP] consumed was almost unity (0.93). In cells metabolizing glucose, all intermediates listed in (a) and (b) were found. Upon exhaustion of glucose from the medium, the metabolites in (b) were not longer detectable, while the intracellular concentrations of PEP and 2-PG increased to the levels previously observed in starved cells. The glycolytic intermediates in (b) are all in vitro heterotropic effectors of pyruvate kinase (adenosine 5'-triphosphate:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from S. lactis ML3. It is suggested that the capacity of starved cells to maintain high intracellular concentrations of PEP and 2-PG is a consequence of decreased in vivo activity of this key regulatory enzyme of glycolysis.     

Regulation of the glucose:H+ symporter by metabolite-activated ATP-dependent phosphorylation of HPr in Lactobacillus brevis.

Lactobacillus brevis takes up glucose and the nonmetabolizable glucose analog 2-deoxyglucose (2DG), as well as lactose and the nonmetabolizable lactose analoge thiomethyl beta-galactoside (TMG), via proton symport. Our earlier studies showed that TMG, previously accumulated in L. brevis cells via the lactose:H+ symporter, rapidly effluxes from L. brevis cells or vesicles upon addition of glucose and that glucose inhibits further accumulation of TMG. This regulation was shown to be mediated by a metabolite-activated protein kinase that phosphorylase serine 46 in the HPr protein. We have now analyzed the regulation of 2DG uptake and efflux and compared it with that of TMG. Uptake of 2DG was dependent on an energy source, effectively provided by intravesicular ATP or by extravesicular arginine which provides ATP via an ATP-generating system involving the arginine deiminase pathway. 2DG uptake into these vesicles was not inhibited, and preaccumulated 2DG did not efflux from them upon electroporation of fructose 1,6-diphosphate or gluconate 6-phosphate into the vesicles. Intravesicular but not extravesicular wild-type or H15A mutant HPr of Bacillus subtilis promoted inhibition (53 and 46%, respectively) of the permease in the presence of these metabolites. Counterflow experiments indicated that inhibition of 2DG uptake is due to the partial uncoupling of proton symport from sugar transport. Intravesicular S46A mutant HPr could not promote regulation of glucose permease activity when electroporated into the vesicles with or without the phosphorylated metabolites, but the S46D mutant protein promoted regulation, even in the absence of a metabolite. The Vmax but not the Km values for both TMG and 2DG uptake were affected. Uptake of the natural, metabolizable substrates of the lactose, glucose, mannose, and ribose permeases was inhibited by wild-type HPr in the presence of fructose 1,6-diphosphate or by S46D mutant HPr. These results establish that HPr serine phosphorylation by the ATP-dependent, metabolite-activated HPr kinase regulates glucose and lactose permease activities in L. brevis and suggest that other permeases may also be subject to this mode of regulation.     

Mechanism of inducer expulsion in Streptococcus pyogenes: a two-step process activated by ATP.

The mechanism of methyl-beta-D-thiogalactoside-phosphate (TMG-P) expulsion from Streptococcus pyogenes was studied. The expulsion elicited by glucose was not due to exchange vectorial transphosphorylation between the expelled TMG and the incoming glucose since more beta-galactoside was displaced than glucose taken up, and the stoichiometry between TMG and glucose transport was inconstant. Instead, two distinct and sequential reactions, intracellular dephosphorylation of TMG-P followed by efflux of free TMG, mediated the expulsion. This was shown by temporary accumulation of free TMG effected by competitive inhibition of its efflux and by the aid of arsenate, which arrested dephosphorylation of TMG-P but did not affect efflux of free TMG formed intracellularly before arsenate addition. The competitive inhibition of TMG efflux by its structural analogs suggests that a transport protein facilitates the expulsion. Iodoacetate or fluoride prevented TMG-P dephosphorylation and its expulsion. However, provision of ATP via the arginine deiminase pathway restored these activities in the presence of the glycolytic inhibitors and stimulated expulsion in their absence. Other amino acids tested did not promote this restoration, and canavanine or norvaline severely inhibited it. Arginine without glucose neither elicited the dephosphorylation nor evoked the expulsion of TMG-P. Ionophores or ATPase inhibitors did not prevent the expulsion as elicited by glucose or its restoration by arginine. The results suggest that activation of the dephosphorylation-expulsion mechanism occurs independently of a functional glycolytic pathway, requires ATP provision, and is possibly due to protein phosphorylation controlled by a yet unknown metabolite. The in vivo phosphorylation of a protein (approximate molecular weight - 10,000) under the conditions of expulsion was demonstrated.     

A requirement for ATP for beta-galactoside transport by Bacillus alcalophilus.

Lactose-grown cells of Bacillus alcalophilus actively transported methylthio-beta, D-galactoside (TMG) in a range of pH values from 7.5 to 10.5 with a pH optimum at 8.5. The TMG was accumulated in a chemically unmodified form, and cell extracts failed to catalyze either ATP or P-enolpyruvate-dependent phosphorylation of TMG. At pH 8.5, the lactose-grown cells exhibited a transmembrane proton gradient (deltapH) of 1.38 units, interior acid, and a transmembrane electrical potential (delta psi) of -132 mV. Accordingly, the total protonmotive force at this pH was very low, -51mV. Several lines of evidence indicate that the protonmotive force or delta psi did not directly energize TMG transport but, rather, that ATP was directly required: (a) in cells treated with arsenate so that the delta psi was unaffected and cellular ATP levels were markedly lowered, TMG transport was inhibited in proportion to the reduction of cellular ATP, while electrogenic alpha-aminoisobutyric acid transport was not; (b) when a valinomycin-induced potassium diffusion potential was established in starved cells, alpha-aminoisobutyric acid transport, but not TMG transport, was stimulated; and (c) in a series of experiments in which the delta psi was rapidly abolished by treatment with gramicidin, ATP levels declined slowly and the rate of TMG transport correlated directly with ATP levels rather than with the delta psi. Consumption of cellular ATP concomitant with TMG transport could be demonstrated.     

Effects of colicins E1 and K on transport systems

The effect of colicins E1 and K on active transport of beta-d-galactosides and of alpha-methyl-d-glucoside (alphaMG) by Escherichia coli was studied. These colicins strongly inhibited the accumulation of thio-methyl-galactoside (TMG) by bacteria and caused rapid exit of previously accumulated TMG. The inhibition effect was limited to the accumulation phase of galactoside transport; the rate of hydrolysis of o-nitrophenyl galactoside, which is dependent on transport of the substrate by the lactose-permease system, was only slightly affected. The accumulation of alphaMG was highly resistant to inhibition by these colicins under conditions which caused complete suppression of TMG accumulation. These effects of the colicins on transport resemble qualitatively those of sodium azide. The findings were interpreted by assuming that colicins E1 and K inhibit the energy-dependent steps in the accumulation of TMG but do not affect facilitated diffusion of galactosides mediated by the specific transport mechanism. The continued accumulation of alphaMG was attributed to the fact that this compound is stored by E. coli cells as a phosphorylated compound by a phosphoenolpyruvate-dependent transport system rather than by an adenosine triphosphate-linked accumulation mechanism.     

Substrate selectivity of the melibiose permease (MelY) from Enterobacter cloacae

We have examined the substrate selectivity of the melibiose permease (MelY) from Enterobacter cloacae in comparison with that of the lactose permease (LacY) from Escherichia coli. Both proteins catalyze active transport of lactose or melibiose with comparable affinity and capacity. However, MelY does not transport the analogue methyl-1-thio-β,d-galactopyranoside (TMG), which is a very efficient substrate in LacY. We show that MelY binds TMG and conserves Cys148 (helix V) as a TMG binding residue but fails to transport this ligand. Based on homology modeling, organization of the putative MelY sugar binding site is the same as that in LacY and residues irreplaceable for the symport mechanism are conserved. Moreover, only 15% of the residues where a single-Cys mutant is inactivated by site-directed alkylation differ in MelY. Using site-directed mutagenesis at these positions and engineered cross-homolog chimeras, we show that Val367, at the periplasmic end of transmembrane helix XI, contributes in defining the substrate selectivity profile. Replacement of Val367 with the MelY residue (Ala) leads to impairment of TMG uptake. Exchanging domains N6 and C6 between LacY and MelY also leads to impairment of TMG uptake. TMG uptake activity is restored by the re-introduction of a Val367 in the background of chimera N6(LacY)-C6(MelY). Much less prominent effects are found with the same mutants and chimeras for the transport of lactose or melibiose.     

Photolyase-dimer-DNA complexes and exclusion stimulation in Escherichia coli: Depolarization of the plasma membrane

Using cells that overproduce DNA photolyase, we found that UV irradiation (3 J/m²) efficiently inactivates accumulation of methylthiogalactoside (TMG) when RexAB proteins of phage lambda are present. The effect requires both formation of photolyase-dimer-DNA (PDD) complexes and expression of the RexAB proteins. It is reversed completely by a flash of visible light if given immediately after UV and becomes irreversible after post-UV incubation for about 15 min. Inactivation is significant after only 5 min of post-UV incubation, is accompanied by a loss of previously accumulated TMG, and does not require de novo protein synthesis. Passive transport of O-nitrophenylgalactoside by inactivated cells is typical of energy-depleted membranes. We suggest that PDD complexes mimic a developmental intermediate of phage superinfection and stimulate formation of the RexB membrane channel recently proposed by others to explain classical “exclusion”. This suggestion is supported by additional data showing an inactivation of colony-forming ability by exclusion stimulation and an inability of PDD complexes to inactivate accumulation of TMG if RexB is present in larger relative amounts than RexA (a detail characteristic of exclusion stimulated by phage superinfection).     

Role of Na+ and Li+ in thiomethylgalactoside transport by the melibiose transport system of Escherichia coli.

Thiomethyl-beta-galactoside (TMG) accumulation via the melibiose transport system was studied in lactose transport-negative strains of Escherichia coli. TMG uptake by either intact cells or membrane vesicles was markedly stimulated by Na+ or Li+ between pH 5.5 and 8. The Km for uptake of TMG was approximately 0.2 mM at an external Na+ concentration of 5 mM (pH 7). The alpha-galactosides, melibiose, methyl-alpha-galactoside, and o-nitrophenyl-alpha-galactoside had a high affinity for this system whereas lactose, maltose and glucose had none. Evidence is presented for Li+-TMG or Na+-TMG cotransport.     

Na+-dependent methyl β-thiogalactoside transport in Salmonella typhimurium

We have studied the role of sodium ions in methyl β-thiogalactoside (TMG) transport via the melibiose permease (TMG II) in SalmonellaTMG uptake via TMG Il in anaerobic, starved and metabolically poisoned cells is dependent on an inward-directed Na⁺ gradient.Cells which have been partially depleted of endogenous substrates show H⁺ extrusion upon sodium-stimulated TMG influx.Measurements of the electrochemical H⁺ gradient in cells, starved in different ways for endogenous substrates, suggest that this proton extrusion is probably not linked to the actual translocation mechanism but is the result of metabolism induced by TMG plus Na⁺ uptake.     

Mechanism of the melibiose porter in membrane vesicles of Escherichia coli

The melibiose transport system of Escherichia coli catalyzes sodium--methyl 1-thio-beta-D-galactopyranoside (TMG) symport, and the cation is required not only for respiration-driven active transport but also for binding of substrate to the carrier in the absence of energy and for carrier-mediated TMG efflux. As opposed to the proton--beta-galactoside symport system [Kaczorowski, G. J., & Kaback, H. R. (1979) Biochemistry 18, 3691], efflux and exchange of TMG occur at the same rate, implying that the rates of the two processes are limited by a common step, most likely the translocation of substrate across the membrane. Furthermore, the rate of exchange, as well as efflux, is influenced by imposition of a membrane potential (delta psi; interior negative), suggesting that the ternary complex between sodium, TMG, and the porter may bear a net positive charge. Consistently, energization of the vesicles leads to a large increase in the Vmax for TMG influx, with little or no change in the apparent Km of the process. It is proposed that the sodium gradient (Na+out < Na+in) and the delta psi (interior negative) may affect different steps in the overall mechanism of active TMG accumulation in the following manner: the sodium gradient causes an increased affinity for TMG on the outer surface of the membrane relative to the inside and the delta psi facilitates a reaction involved with the translocation of the positively charged ternary complex to the inner surface of the membrane.     

In Vitro Sugar Transport in Zea mays L. Kernels : II. Characteristics of Sugar Absorption and Metabolism by Isolated Developing Embryos

In vitro sugar transport into developing isolated maize embryos was studied. Embryo fresh and dry weight increased concomitantly with endogenous sucrose concentration and glucose uptake throughout development. However, endogenous glucose and fructose concentration and sucrose uptake remained constant. The uptake kinetics of radiolabeled sucrose, glucose, and fructose showed a biphasic dependence on exogenous substrate concentration. Hexose uptake was four to six times greater than sucrose uptake throughout development. Carbonylcyanide-m-chlorophenylhydrazone and dinitrophenol inhibited sucrose and glucose uptake significantly, but 3-O-methyl glucose uptake was less affected. The uptake of 1 millimolar sucrose was strongly pH dependent while glucose was not. Glucose and fructose were readily converted to sucrose and insoluble products soon after absorption into the embryo. Thus, sucrose accumulated, while glucose pools remained low. Based on the findings of this and other studies a model for sugar transport in the developing maize kernel is presented.     

The regulation of sugar uptake and accumulation in bean pod tissue

The identity, localization and physiological significance of enzymes involved in sugar uptake and accumulation were determined for endocarp tissue of pods of Kentucky Wonder pole beans (Phaseolus vulgaris). An intracellular, alkaline invertase (pH optimum, 8) was assayed in extracted protein, as well as enzymes involved in sucrose synthesis, namely, uridinediphosphate (UDP-glucose pyrophosphorylase and UDP-glucose-fructose transglucosylase). Indirect evidence indicated the presence also of hexokinase, phosphohexoseisomerase and phosphoglucomutase. The data suggested that sucrose synthesis occurred in the cytoplasm, and that both sugar storage and an alkaline invertase occurred in the vacuole. The latter functions to hydrolyze accumulated sucrose. An outer space invertase (pH optimum, 4.0) was detected, but was variable in occurrence. Although its activity at the cell surface enhanced sucrose uptake, sucrose may be taken up unaltered.

Over a wide range of concentrations of exogenous glucose the sucrose/reducing sugar ratio of accumulated sugars remained unchanged at about 20. Synthesis of sucrose appears to be requisite to initial accumulation from glucose or fructose, as free hexoses do not increase at the apparent saturating concentration for uptake. Sucrose accumulation from exogenous hexose represents a steady-state value, in which sucrose is transported across the tonoplast into the vacuole at a rate equivalent to its rate of synthesis. Evidence indicates that this component of the accumulation process involves active transport of sucrose against a concentration gradient. The ratio of sucrose/reducing sugars in the accumulated sugars immediately after a period of uptake was inversely related to the level of inner space invertase. Within 16 hours after a period of accumulation, practically all of the sugar occurs as glucose and fructose.

The absence of competition among hexoses and sucrose indicated that a common carrier was not involved in their uptake. From a series of studies on the kinetics of uptake of glucose and fructose, including competition studies, the effects of inhibitors, radioactive assay of accumulated sugars and the distribution of label in accumulated sucrose it appeared that rate limitation for glucose or fructose uptake resides in the sequence of reactions leading to sucrose synthesis, rather than in a process mediated by a carrier protein.

     

Regulation of methyl-beta-d-thiogalactopyranoside-6-phosphate accumulation in Streptococcus lactis by exclusion and expulsion mechanisms

Starved cells of Streptococcus lactis ML3 (grown previously on galactose, lactose, or maltose) accumulated methyl-beta-D-thiogalactopyranoside (TMG) by the lactose:phosphotransferase system. More than 98% of accumulated sugar was present as a phosphorylated derivative, TMG-6-phosphate (TMG-6P). When a phosphotransferase system sugar (glucose, mannose, 2-deoxyglucose, or lactose) was added to the medium simultaneously with TMG, the beta-galactoside was excluded from the cells. Galactose enhanced the accumulation of TMG-6P. Glucose, mannose, lactose, or maltose plus arginine, was added to a suspension of TMG-6P-loaded cells of S. lactis ML3, elicited rapid expulsion of intracellular solute. The material recovered in the medium was exclusively free TMG. Expulsion of galactoside required both entry and metabolism of an appropriate sugar, and intracellular dephosphorylation of TMG-6P preceded efflux of TMG. The rate of dephosphorylation of TMG-6P by permeabilized cells was increased two-to threefold by adenosine 5'-triphosphate but was strongly inhibited by fluoride. S. lactis ML3 (DGr) was derived from S. lactis ML3 by positive selection for resistance to 2-deoxy-D-glucose and was defective in the enzyme IIMan component of the glucose:phosphotransferase system. Neither glucose nor mannose excluded TMG from cells of S. lactic ML3 (DGr), and these two sugars failed to elicit TMG expulsion from preloaded cells of the mutant strain. Accumulation of TMG-6P by S. lactis ML3 can be regulation by two independent mechanisms whose activities promote exclusion or expulsion of galactoside from the cell.     

Primary structure and characteristics of the melibiose carrier of Klebsiella pneumoniae.

The melB gene coding for the melibiose carrier of Klebsiella pneumoniae was cloned and sequenced. There were two potential translation initiation sites. It was predicted that the melibiose carrier consists of 471 (or 467) amino acid residues. Seventy-eight percent of the 471 amino acids were identical to the Escherichia coli melibiose carrier. Sugar transport characteristics were studied using an E. coli mel- mutant expressing cloned K. pneumoniae melB gene. Accumulation of melibiose via the K. pneumoniae melibiose carrier was not stimulated by adding NaCl or LiCl which stimulates melibiose accumulation via the E. coli melibiose carrier. Lactose was accumulated only in the presence of LiCl. TMG (methyl-1-thio-beta-D-galactopyranoside) was accumulated in the absence of added NaCl or LiCl. The accumulation was stimulated by LiCl but not by NaCl. Rapid H+ uptake was observed when melibiose or TMG was added to cell suspensions. These results suggest that the preferred cation couplings via K. pneumoniae melibiose carrier are H(+)-melibiose, Li(+)-lactose, and H+/Li(+)-TMG. This coupling spectrum is quite different from that of the E. coli melibiose carrier. It is of special interest that the K. pneumoniae melibiose carrier seems to be lacking the ability to recognize Na+ which is a preferred coupling cation of the E. coli melibiose carrier for all known sugar substrates. Further investigation of these two carriers may give us insight into the Na+ recognition site.     

Cation-sugar cotransport in the melibiose transport system of Escherichia coli.

The entry of Na+ or H+ into cells of Escherichia coli via the melibiose transport system was stimulated by the addition of certain galactosides. The principal cell used in these studies (W3133) was a lactose transport negative strain of E. coli possessing an inducible melibiose transport system. Such cells were grown in the presence of melibiose, washed, and incubated in the presence of 25 microM Na+. The addition of thiomethylgalactoside (TMG) resulted in a fall in Na+ concentration in the incubation medium. No TMG-stimulated Na+ movement was observed in uninduced cells. In an alpha-galactosidase negative derivative of W3133 (RA11) a sugar-stimulated Na+ uptake was observed in melibiose-induced cells on the addition of melibiose, thiodigalactoside, methyl-alpha-galactoside, methyl-beta-galactoside, and galactose, but not lactose. It was inferred from these studies that the substrates of the melibiose system enter the cell on the melibiose carrier associated with the simultaneous entry of Na+ when this cation is present in the incubation medium. Extracellular pH was measured in unbuffered suspensions of induced cells in order to study proton movement across the membrane of cells exposed to different galactosides. In the absence of external Na+ or Li+ the addition of melibiose or methyl-alpha-galactoside resulted in marked alkalinization of the external medium (consistent with H+-sugar cotransport). On the other hand TMG, thiodigalactoside, and methyl-beta-galactoside gave no proton movement under these conditions. When Na+ was present, the addition of TMG or melibiose resulted in acidification of the medium. This observation is consistent with the view that the entry of Na+ with TMG or melibiose carries into the cell a positive charge (Na+) which provides the driving force for the diffusion of protons out of the cell. It is concluded that the melibiose carrier recognition of cations differs with different substrates.     

A novel amylolytic enzyme from Thermotoga maritima,resembling cyclodextrinase and alpha-glucosidase,that liberates glucose from the reducing end of the substrates

The gene previously designated as putative cyclodextrinase from Thermotoga maritima (TMG) was cloned and overexpressed in Escherichia coli. The recombinant TMG was partially purified and its enzymatic characteristics on various substrates were examined. The enzyme hydrolyzes various maltodextrins including maltotriose to maltoheptaose and cyclomaltodextrins (CDs) to mainly glucose and maltose. Although TMG could not degrade pullulan, it rapidly hydrolyzes acarbose, a strong amylase and glucosidase inhibitor, to acarviosine and glucose. Also, TMG initially hydrolyzes p-nitrophenyl-alpha-pentaoside to give maltopentaose and p-nitrophenol, implying that the enzyme specifically cleaves a glucose unit from the reducing end of maltooligosaccharides unlike to other glucosidases. Since its enzymatic activity is negligible if alpha-methylglucoside is present in the reducing end, the type of the residue at the reducing end of the substrate is important for the TMG activity. These results support the fact that TMG is a novel exo-acting glucosidase possessing the characteristics of both CD-/pullulan hydrolyzing enzyme and alpha-glucosidase.