The high-affinity maltose/trehalose ABC transporter in the extremely thermophilic bacterium Thermus thermophilus HB27 also recognizes sucrose and palatinose.
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 degrees C. The K(m) values at 70 degrees 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 degrees C for MalE1 and 105 degrees 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.
Silva
Zélia
Z
Centro de Neurociências e Biologia Celular, Departamento de Zoologia, Universidade de Coimbra, Coimbra, Portugal.
Sampaio
Maria-Manuel
MM
Henne
Anke
A
Böhm
Alex
A
Gutzat
Ruben
R
Boos
Winfried
W
da Costa
Milton S
MS
Santos
Helena
H
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D005798:Genes, Bacterial; D007534:Isomaltose / Q000031:analogs & derivatives* / Q000378:metabolism*; D007700:Kinetics; D008320:Maltose / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008689:Methacrylates; D005810:Multigene Family; D016254:Mutagenesis, Insertional; D009154:Mutation; D021382:Protein Sorting Signals / Q000235:genetics; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism*; D016963:Thermus thermophilus / Q000235:genetics / Q000378:metabolism*; D014199:Trehalose / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; C068021:MalK protein, Bacteria; D008565:Membrane Proteins; D008689:Methacrylates; D021382:Protein Sorting Signals; D021122:Protein Subunits; D011994:Recombinant Proteins; C483029:Temphase; D013395:Sucrose; D007534:Isomaltose; D008320:Maltose; D014199:Trehalose; C008189:isomaltulose