Identification of a gene cluster encoding an arginine ATP-binding-cassette transporter in the genome of the thermophilic Gram-positive bacterium Geobacillus stearothermophilus strain DSMZ 13240.
A single gene cluster encoding components of a putative ATP-binding cassette (ABC) transporter for basic amino acids was identified in the incomplete genome sequence of the thermophilic Gram-positive bacterium Geobacillus stearothermophilus by blast searches. The cluster comprises three genes, and these were amplified from chromosomal DNA of G. stearothermophilus, ligated into plasmid vectors and expressed in Escherichia coli. The purified solute-binding protein (designated ArtJ) was demonstrated to bind L-arginine with high affinity (Kd=0.39+/-0.06 microM). Competition experiments revealed only partial inhibition by excess L-lysine (38 %) and L-ornithine (46 %), while no inhibition was observed with L-histidine or other amino acids tested. The membrane-associated transport complex, composed of a permease (designated ArtM) and an ATPase component (designated ArtP), was solubilized from E. coli membranes by decanoylsucrose and purified by metal-affinity chromatography. The ArtMP complex, when incorporated into liposomes formed from a crude extract of G. stearothermophilus lipids, displayed ATPase activity in the presence of ArtJ only. Addition of L-arginine further stimulated the activity twofold. ATP hydrolysis was optimal at 60 degrees C and sensitive to the specific inhibitor vanadate. Analysis of kinetic parameters revealed a maximal velocity of ATP hydrolysis of 0.71 micromol Pi min(-1) (mg protein)(-1) and a Km(ATP) of 1.59 mM. Together, these results identify the ArtJMP complex as a high-affinity arginine ABC transporter.
Fleischer
Rebecca
R
Humboldt Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, Institut für Biologie, Bakterienphysiologie, Chausseestr. 117, D-10115 Berlin, Germany.
Wengner
Antje
A
Humboldt Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, Institut für Biologie, Bakterienphysiologie, Chausseestr. 117, D-10115 Berlin, Germany.
Scheffel
Frank
F
Humboldt Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, Institut für Biologie, Bakterienphysiologie, Chausseestr. 117, D-10115 Berlin, Germany.
Landmesser
Heidi
H
Humboldt Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, Institut für Biologie, Bakterienphysiologie, Chausseestr. 117, D-10115 Berlin, Germany.
Schneider
Erwin
E
Humboldt Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, Institut für Biologie, Bakterienphysiologie, Chausseestr. 117, D-10115 Berlin, Germany.
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D001120:Arginine / Q000378:metabolism*; D001406:Bacillaceae / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D016680:Genome, Bacterial; D008081:Liposomes / Q000378:metabolism; D005810:Multigene Family; D010957:Plasmids; D013379:Substrate Specificity
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D008081:Liposomes; D001120:Arginine