Functional reconstitution of a maltose ATP-binding cassette transporter from the thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius.
The thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius grows at 60 degrees C and pH 2-3. The organism can utilize maltose and maltodextrins as energy source that are taken up by an ATP-binding cassette (ABC) import system. Genes encoding a maltose binding protein, MalE, and two membrane-integral subunits, MalF and MalG, are clustered on the chromosome but a malK gene translating into a cognate ATPase subunit is lacking. Here we report the cloning of malK from genomic DNA by using the msiK gene of Streptomyces lividans as a probe. Purified MalK exhibited a spontaneous ATPase activity with a Vmax of 0.13 micromol Pi/min/mg and a Km of 330 microM that was optimal at the growth temperature of the organism. Coexpression of malK, malF and malG in Escherichia coli resulted in the formation of a complex that could be coeluted from an affinity matrix after solubilization of membranes with dodecylmaltoside. Proteoliposomes prepared from the MalFGK complex and preformed phospholipid vesicles of A. acidocaldarius displayed a low intrinsic ATPase activity that was stimulated sevenfold by maltose-loaded MalE, thereby indicating coupling of ATP hydrolysis to substrate translocation. These results provide evidence for MalK being the physiological ATPase subunit of the A. acidocaldarius maltose transporter. Moreover, to our knowledge, this is the first report on the functional reconstitution of an ABC transport system from a thermophilic microorganism.
Scheffel
Frank
F
Institut für Biologie, Humboldt Universität zu Berlin, Bakterienphysiologie, Chausseestr. 117, D-10115 Berlin, Germany.
Fleischer
Rebecca
R
Schneider
Erwin
E
D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002244:Carbon / Q000378:metabolism; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D016937:Gram-Positive Endospore-Forming Rods / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism; D011510:Proteolipids / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D018528:ATP-Binding Cassette Transporters; D019843:Archaeal Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; C461639:MalF protein, E coli; C432665:MalG protein, Thermococcus litoralis; C068021:MalK protein, Bacteria; C489398:MalK protein, E coli; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; C020485:proteoliposomes; D002244:Carbon