Formation of a functionally active sodium-translocating hybrid F1F0 ATPase in Escherichia coli by homologous recombination.
A deletion mutant of Escherichia coli lacking the genes for ATPase subunits a, c, b, delta and part of the alpha subunit was transformed with a plasmid containing the corresponding genes of the sodium-translocating ATPase of Propionigenium modestum. The respective DNA fragment of P. modestum was integrated into the genome of the E. coli deletion mutant by site-specific homologous recombination. The sites of this recombination event were identified by cloning and DNA sequencing. As a consequence of the recombination event, a functionally active hybrid ATPase was obtained by in vivo complementation. The biochemical characterization of this hybrid ATPase revealed high sensitivity to dicyclohexylcarbodiimide as well as strong activation by the addition of sodium ions. After reconstitution into liposomes, the hybrid ATPase catalysed the transport of Na+ upon ATP addition. In the absence of Na+, the ATPase hybrid was able to pump protons, as was shown by the ATP-dependent fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine.
Kaim
G
G
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Switzerland.
Dimroth
P
P
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001693:Biological Transport, Active; D027682:Cation Transport Proteins; D002876:Chromosomes, Bacterial / Q000378:metabolism; D003001:Cloning, Molecular; D004024:Dicyclohexylcarbodiimide / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011510:Proteolipids / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D012964:Sodium / Q000378:metabolism*; D014169:Transformation, Bacterial
D027682:Cation Transport Proteins; D011510:Proteolipids; C020485:proteoliposomes; D004024:Dicyclohexylcarbodiimide; D012964:Sodium; D000251:Adenosine Triphosphatases; C021838:sodium-translocating ATPase; D006180:Proton-Translocating ATPases