We have studied the transport of trehalose and maltose in the thernophilic bacterium Thermus thermophilus HB27, which grows optimally in the range of 70 to 75°C. The K m values at 70°C were 109 nM for trehalose and 114 nM for maltose; also, a high K m (424 nM) was found for the uptake of sucrose. Competition studies showed that a single transporter recognizes trehalose, maltose, and sucrose, while D-galactose, D-fucose, L-rhamnose, L-arabinose, and D-mannose were not competitive inhibitors. In the recently published genome of T. thermophilus HB27, two gene clusters designated malEFG1 (TTC1627 to -1629) and malEFG2 (TTC1288 to -1286) and two monocistronic genes designated malK1 (TTC0211) and malK2 (TTC0611) are annotated as trehalose/ maltose and maltose/maltodextrin transport systems, respectively. To find out whether any of these systems is responsible for the transport of trehalose, the malE1 and malE2 genes, lacking the sequence encoding the signal peptides, were expressed in Escherichia coli. The binding activity of pure recombinant proteins was analyzed by equilibrium dialysis. MalE1 was able to bind maltose, trehalose, and sucrose but not glucose or maltotetraose (K d values of 103, 67, and 401 nM, respectively). Mutants with disruptions in either malF1 or malK1 were unable to grow on maltose, trehalose, sucrose, or palatinose, whereas mutants with disruption in malK2 or malF2 showed no growth defect on any of these sugars. Therefore, malEFG1 encodes the binding protein and the two transmembrane subunits of the trehalose/maltose/sucrose/palatinose ABC transporter, and malK1 encodes the ATP-binding subunit of this transporter. Despite the presence of an efficient transporter for trehalose, this compound was not used by HB27 for osmoprotection. MalE1 and MalE2 exhibited extremely high thermal stability: melting temperatures of 90°C for MalE1 and 105°C for MalE2 in the presence of 2.3 M guanidinium chloride. The latter protein did not bind any of the sugars examined and is not implicated in a maltose/maltodextrin transport system. This work demonstrates that malEFG1 and malK1 constitute the high-affinity ABC transport system of T. thermophilus HB27 for trehalose, maltose, sucrose, and palatinose.The superfamily of ATP-binding cassette (ABC) transport systems comprises a large diversity of primary pumps that use ATP hydrolysis to translocate substrates across biological membranes. Prokaryotic ABC importers typically consist of an extracytoplasmic or membrane-anchored binding protein that provides the recognition site for the substrate, a translocation complex formed by two membrane components, and two copies of an ATP-hydrolyzing protein. In the archetypal maltose/ maltodextrin transporter of Escherichia coli, MalE designates the substrate binding protein, MalF and MalG are the two membrane components of the translocation complex, and two MalK subunits form the ATP-hydrolyzing complex. The encoding genes form a cluster on the chromosome of E. coli where malE/malF/malG constitutes an operon with an orientation ...
2-O-␣-Mannosyl-D-glycerate (MGs)has been recognized as an osmolyte in hyperthermophilic but not mesophilic prokaryotes. We report that MG is taken up and utilized as sole carbon source by Escherichia coli K12, strain MC4100. Uptake is mediated by the P-enolpyruvatedependent phosphotransferase system with the MGinducible HrsA (now called MngA) protein as its specific EIIABC complex. The apparent K m of MG uptake in induced cells was 10 M, and the V max was 0.65 nmol/min/ 10 9 cells. Inverted membrane vesicles harboring plasmid-encoded MngA phosphorylated MG in a P-enolpyruvate-dependent manner. A deletion mutant in mngA was devoid of MG transport but is complemented by a plasmid harboring mngA. Uptake of MG in MC4100 also caused induction of a regulon specifying the uptake and the metabolism of galactarate and glucarate controlled by the CdaR activator. The ybgG gene (now called mngB) the gene immediately downstream of mngA encodes a protein with ␣-mannosidase activity. farR, the gene upstream of mngA (now called mngR) had previously been characterized as a fatty acyl-responsive regulator; however, deletion of mngR resulted in the up-regulation of only two genes, mngA and mngB. The mngR deletion caused constitutive MG transport that became MG-inducible after transformation with plasmid expressed mngR. Thus, MngR is the regulator (repressor) of the MG transport/metabolism system. Thus, the mngR mngA mngB gene cluster encodes an MG utilizing system.
Trehalases play a central role in the metabolism of trehalose and can be found in a wide variety of organisms. A periplasmic trehalase (alpha,alpha-trehalose glucohydrolase, EC 3.2.1.28) from the thermophilic bacterium Rhodothermus marinus was purified and the respective encoding gene was identified, cloned and overexpressed in Escherichia coli. The recombinant trehalase is a monomeric protein with a molecular mass of 59 kDa. Maximum activity was observed at 88 degrees C and pH 6.5. The recombinant trehalase exhibited a K(m) of 0.16 mM and a V(max) of 81 micromol of trehalose (min)(-1) (mg of protein)(-1) at the optimal temperature for growth of R. marinus (65 degrees C) and pH 6.5. The enzyme was highly specific for trehalose and was inhibited by glucose with a K(i) of 7 mM. This is the most thermostable trehalase ever characterized. Moreover, this is the first report on the identification and characterization of a trehalase from a thermophilic bacterium.
We report the construction of an Escherichia coli mutant that harbors two compatible plasmids and that is able to synthesize labeled 2-O-␣-D-mannosyl-D-glycerate from externally added labeled mannose without the loss of specific isotopic enrichment. The strain carries a deletion in the manA gene, encoding phosphomannose isomerase. This deletion prevents the formation of fructose-6-phosphate from mannose-6-phosphate after the uptake of mannose from the medium by mannose-specific enzyme II of the phosphotransferase system (PtsM). The strain also has a deletion of the cps gene cluster that prevents the synthesis of colanic acid, a mannosecontaining polymer. Plasmid-encoded phosphomannomutase (cpsG) and mannose-1-phosphate guanylyltransferase ( 2-O-␣-D-Mannosyl-D-glycerate (MG) is one of the most widespread compatible solutes of thermophilic or hyperthermophilic bacteria and archaea (21). This compound has otherwise been encountered only in the red algae of the order Ceramiales (4). The highly preferential distribution of MG among organisms requiring high temperatures for growth led to the hypothesis that it could play a role in the thermoprotection of cell components in vivo. This speculation still lacks experimental proof, but at least in vitro, MG has been shown to be highly efficient in the protection of enzymes against thermal inactivation (3,19).In order to study the metabolic fate of MG and the mechanisms underlying its stabilizing effect on proteins, it is desirable for this compound to be available in a labeled form with a high level of specific isotopic enrichment ( 14 C, 13 C); at present, such a compound is not commercially available.The biosynthesis of MG has been characterized in detail for the thermophilic bacterium Rhodothermus marinus (12) and for the hyperthermophilic archaeon Pyrococcus horikoshii (7). The known pathways involve the transfer of ␣-mannosyl residues from GDP-mannose to either D-glycerate, forming MG, or D-3-phosphoglycerate, followed by hydrolysis of mannosyl-3-phosphoglycerate to MG (7, 12). Most organisms in which the synthesis of MG has been observed are not accessible for easy genetic manipulation (21). Thus, the conversion of a simple radioactive sugar for the formation of MG is plagued by its fast metabolism after entering the producing organism. This situation is due to the common separation of catabolic and anabolic pathways seen for sugar-type compatible solutes. The strict separation of trehalose metabolism and its internal synthesis is a typical example of this phenomenon (10). Therefore, we thought of using a genetically altered Escherichia coli strain to synthesize MG from external mannose. E. coli does produce GDP-mannose, mainly for the synthesis of colanic acid via GDP-fucose (2, 9). The cpsB gene, encoding the enzyme for GDP-mannose synthesis (mannose-1-phosphate guanylyltransferase), and the cpsG gene, encoding phosphomannomutase, form an operon that is part of the cps gene cluster necessary for the synthesis of colanic acid (25).By constructing an E. coli strain ...
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