The hyperthermophilic marine archaeon Thermococcus litoralis exhibits high-affinity transport activity for maltose and trehalose at 85؇C. The K m for maltose transport was 22 nM, and that for trehalose was 17 nM. In cells that had been grown on peptone plus yeast extract, the V max for maltose uptake ranged from 3.2 to 7.5 nmol/min/mg of protein in different cell cultures. Cells grown in peptone without yeast extract did not show significant maltose or trehalose uptake. We found that the compound in yeast extract responsible for the induction of the maltose and trehalose transport system was trehalose. [14 C]maltose uptake at 100 nM was not significantly inhibited by glucose, sucrose, or maltotriose at a 100 M concentration but was completely inhibited by trehalose and maltose. The inhibitor constant, K i , of trehalose for inhibiting maltose uptake was 21 nM. In contrast, the ability of maltose to inhibit the uptake of trehalose was not equally strong. With 20 nM [ 14 C]trehalose as the substrate, a 10-fold excess of maltose was necessary to inhibit uptake to 50%. However, full inhibition was observed at 2 M maltose. The detergent-solubilized membranes of trehalose-induced cells contained a high-affinity binding protein for maltose and trehalose, with an M r of 48,000, that exhibited the same substrate specificity as the transport system found in whole cells. We conclude that maltose and trehalose are transported by the same high-affinity membrane-associated system. This represents the first report on sugar transport in any hyperthermophilic archaeon.

The maltose system in Escherichia coli consists of cell envelope-associated proteins and enzymes that catalyze the uptake and utilization of maltose and a,1-4-linked maltodextrins. The presence of these sugars in the growth medium induces the maltose system (exogenous induction), even though only maltotriose has been identified in vitro as an inducer (0. Raibaud and E. Richet, J. Bacteriol., 169:3059-3061, 1987). Induction is dependent on MalT, the positive regulator protein of the system. In the presence of exogenous glucose, the maltose system is normally repressed because of catabolite repression and inducer exclusion brought about by the phosphotransferase-mediated vectorial phosphorylation of glucose. In contrast, the increase of free, unphosphorylated glucose in the cell induces the maltose system. A ptsG ptsM glk mutant which cannot grow on glucose can accumulate ['4CJglucose via galactose permeases. In this strain, internal glucose is polymerized to maltose, maltotriose, and maltodextrins in which only the reducing glucose residue is labeled. This polymerization does not require maltose enzymes, since it still occurs in malT mutants. Formation of maltodextrins from external glucose as well as induction of the maltose system is absent in a mutant lacking phosphoglucomutase, and induction by external glucose could be regained by the addition of glucose-lphosphate entering the cells via a constitutive glucose phosphate transport system. malQ mutants, which lack amylomaltase, are constitutive for the expression of the maltose genes. This constitutive nature is due to the formation of maltose and maltodextrins from the degradation of glycogen.The Escherichia coli maltose system consists of a maltodextrin-specific pore (encoded by lamB) (17, 30) in the outer membrane and a binding-protein-dependent transport system in the cell envelope (encoded by malE malF malG malK) (38), as well as one periplasmic enzyme (encoded by malS) (35) and three cytoplasmic enzymes (encoded by malQ, malP, and malZ) (27,34,41). Expression of all mal genes depends on the positive regulator MalT (33).The transport system (11,12,20,24,40) can recognize and accumulate maltose and linear a,1-4-linked maltodextrins up to a chain length of seven glucose units (15). The major enzymes of the system (see Fig. 1) are the cytoplasmic amylomaltase (MalQ) (42) and maltodextrin phosphorylase (MalP) (34). Amylomaltase recognizes maltotriose and larger maltodextrins (donors), cleaving off the reducing glucose residue and transferring the remaining dextrinyl residue onto the nonreducing end of maltodextrin (acceptors), including maltose and glucose. With maltotriose, the smallest donor substrate, as well as with longer linear maltodextrins, amylomaltase thus produces glucose and longer maltodextrins (26). Maltodextrin phosphorylase subsequently releases glucose-i-phosphate from the nonreducing end of maltodextrins with a minimal chain length of five glucose residues (37). The glucose and glucose-l-phosphate are both transformed into glucose-6-phosphat...

We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeonThermococcus litoralis that is homologous to themalEFG cluster encoding the Escherichia colimaltose transport system. The deduced amino acid sequence of themalE product, the trehalose/maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine. The T. litoralis malE gene was expressed in E. coli under control of an inducible promoter with and without its natural signal sequence. In addition, in one construct the endogenous signal sequence was replaced by the E. coli MalE signal sequence. The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose. The protein bound both sugars at 85°C with aKd of 0.16 μM. Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from T. litoralis membranes as well as the products from all other DNA constructs expressed in E. coli. Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the E. coli MalF protein are missing in the T. litoralis MalF. MalG is homologous throughout the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches.

When sn-glycerol-3-phosphate (G3P) is taken up exclusively by the pho regulon-dependent Ugp transport system, it can be used as the sole source of P i but not as the sole source of carbon. We had previously suggested that the inability of G3P to be used as a carbon source under these conditions is due to trans inhibition of G3P uptake by internal P i derived from the degradation of G3P (P. Brzoska, M. Rimmele, K. Brzostek, and W. Boos, J. Bacteriol. 176:15-20, 1994). Here, we report 31 P nuclear magnetic resonance measurements of intact cells after exposure to G3P as well as to P i , using different mutants defective in pst (high-affinity P i transport), ugp (pho-dependent G3P transport), glpT (glp-dependent G3P transport), and glpD (aerobic G3P dehydrogenase). When G3P was transported by the Ugp system and when metabolism of G3P was allowed (glpD ؉ ), P i accumulated to about 13 to 19 mM. When G3P was taken up by the GlpT system, the preexisting internal P i pool (whether low or high) did not change. Both systems were inversly controlled by internal P i . Whereas the Ugp system was inhibited, the GlpT system was stimulated by elevated internal P i .sn-Glycerol-3-phosphate (G3P) can be taken up in Escherichia coli by several transport systems. When used as a carbon source, G3P is taken up exclusively by the GlpT transport system (1, 11, 13). This system is part of the glp regulon, a number of proteins whose genes are transcribed in response to the presence of glycerol and G3P in the medium and which are geared for the uptake and the metabolism of glycerol, G3P, and glycerol phosphoryl diesters (15). GlpT and all of the other glp-encoded proteins are under the control of GlpR, the repressor of the system (14), with G3P as the effective inducer (16). The GlpT transport system can function in two modes. In the exchange mode, G3P is taken up in exchange with internal P i (1,8). Uptake of G3P without P i exchange is essentially by proton symport. Under these conditions, GlpT-mediated uptake of G3P can serve as the sole source of P i . The GlpT permease is a tightly membrane-bound oligomeric complex of identical polypeptide subunits (13). G3P can also be taken up by the Ugp (uptake of glycerol phosphate) system. The Ugp system is a typical periplasmic binding protein-dependent multicomponent transport system specific for G3P and glyceryl phosphoryl phosphodiesters, the diacylation products of phospholipids (4, 21). The genes ugpB, ugpA, ugpE, ugpC, and ugpQ (18) form an operon located at 75 min on the E. coli chromosome encoding the specific binding protein (ugpB) (2), the two membrane-bound components (ugpA and ugpE), and the ATPbinding-fold-containing subunit (ugpC), which is supposedly the energy module of the system. The ugp operon is under the control of PhoB, the central gene activator of the pho regulon (3).The last transport system recognizing G3P is the Uhp system specific for hexose phosphates (12). Since Uhp is not induced in the absence of glucose-6-phosphate, it is not relevant to the phenomena discusse...

The maltose chemoreceptor in Escherichia coli consists of the periplasmic maltose-binding protein (MBP) and the Tar signal transducer, which is localized in the cytoplasmic membrane. We previously isolated strains containing malE mutations that cause specific defects in the chemotactic function of MBP. Four of these mutations have now been characterized by DNA sequence analysis. Two of them replace threonine at residue 53 of MBP with isoleucine (MBP-TI53), one replaces an aspartate at residue 55 with asparagine (MBP-DN55), and the fourth replaces threonine at residue 345 with isoleucine (MBP-TI345). The chemotactic defects of MBP-TI53 and MBP-DN55, but not of MBP-TI345, are suppressed by mutations in the tar gene. Of the tar mutations, the most effective suppressor (isolated independently three times) replaces Arg-73 of Tar with tryptophan. Two other tar mutations that disrupt the aspartate chemoreceptor function of Tar also suppress the maltose taxis defects associated with MBP-TI53 and MBP-DN55. One of these mutations introduces glutamine at residue 73 of Tar, the other replaces arginine at residue 69 of Tar with cysteine. These results suggest that regions of MBP that include residues 53 to 55 and residue 345 are important for the interaction with Tar. In turn, arginines at residues 69 and 73 of Tar must be involved in the recognition of maltose-bound MBP and/or in the production of the attractant signal generated by Tar in response to maltose-bound MBP.