The High-Affinity Maltose/Trehalose ABC Transporter in the Extremely Thermophilic Bacterium Thermus thermophilus HB27 Also Recognizes Sucrose and Palatinose

Abstract

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 ...

Living with Genome Instability: the Adaptation of Phytoplasmas to Diverse Environments of Their Insect and Plant Hosts

“…In contrast, phytoplasmas possess ABC transporters for the import of maltose. The maltose-binding protein (MalE) ( Table 4) may have affinity to maltose, trehalose, sucrose, and palatinose (88). Affinity of MalE to trehalose is likely, as trehalose is a major sugar in the insect hemolymph.…”

Genomic analysis of Chthonomonas calidirosea, the first sequenced isolate of the phylum Armatimonadetes

“…Many other microbial species (including most Archaea) lack the PTS system (Koning et al, 2002;Silva et al, 2005), but the prevalence of diauxic versus non-diauxic growth as a physiological trait within the bacterial and archaeal domains is currently not well described. Archaeal and bacterial species without PTS transporters employ alternative carbohydrate transporter systems, including ABC transporters, and secondary transporters such as the major facilitator superfamily and major intrinsic protein (Koning et al, 2002;Silva et al, 2005;Joshua et al, 2011). These alternative transport systems are thought to be less involved in the regulation of metabolism and transcription than PTS (Saier, 2001).…”

Role of Periplasmic Trehalase in Uptake of Trehalose by the Thermophilic Bacterium Rhodothermus marinus

“…In fact, the occurrence of PTS in thermophiles and hyperthermophiles is extremely rare; the only exception is the thermophilic bacterium Bacillus stearothermophilus, which possesses PTSs for mannitol and cellobiose (8,12). On the other hand, all thermophilic and hyperthermophilic organisms examined thus far transport trehalose via ABC transport systems (5,9,11,18,24,25). The strong reduction in trehalose transport induced by arsenate in R. marinus is consistent with this type of system.…”

“…The capacity of the trehalose transporter in R. marinus is 1 order of magnitude lower than that of trehalose transporters in other thermophilic and hyperthermophilic prokaryotes (18,25). As the genomes of thermophiles and hyperthermophiles available in databanks lack homologs of trehalase genes, it appears that the strategy used by R. marinus to take up trehalose is unique at least among organisms from hot environments.…”

“…In mesophilic bacteria, like Escherichia coli, the uptake of trehalose is typically mediated by the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) (2). In contrast, the PTS has not been found in extreme thermophiles or hyperthermophiles, and the ATP-binding cassette (ABC) transport system is used for trehalose uptake in the few cases examined thus far (5,9,18,24,25). The high affinity of the ABC transporters seems to be crucial for utilization of the scarce carbon sources typical of the hostile environments where thermophiles and hyperthermophiles are found.…”