Characterization and function analysis of a Halo-alkaline-adaptable Trk K+ uptake system in Alkalimonas amylolytica...

By functional complementation of Escherichia coli mutants defective in potassium (K(+)) uptake, two genes that are required for K(+) uptake in halo-alkaliphilic Alkalimonas amylolytica strain N10 were cloned. These two genes, Aa-trkA (1337 bp) and Aa-trkH (1452 bp), were adjacent on the A. amylolytica N10 chromosome and transcribed in opposite directions. Complementation experiments revealed that Aa-TrkA and Aa-TrkH from A. amylolytica strain N10 restored the ability to grow at low K(+) concentration in E. coli DeltatrkA and DeltatrkG DeltatrkH strains, respectively. In addition, Aa-TrkAH supported the growth of an E. coli DeltasapD strain, indicating that the ATP-binding protein TrkE was dispensable for the Trk system of A. amylolytica strain N10. The net K(+) uptake was detected at different pH levels and the critical NaCl concentration indicated that Aa-TrkAH is an alkaline-adaptable and partially halo-adaptable K(+) transporter. Kinetics determined by heterogeneous K(+) transport experiments with an E. coli DeltatrkA strain revealed that Aa-TrkAH has an alkaline pH optimum close to 8.5 or higher. Site-directed mutagenesis of Aa-TrkH showed that Phe103 and Ser229 play certain key roles in K(+) selection and transportation. The molecular chaperones groES-groEL and tig promoted Aa-TrkH and Aa-TrkA overexpression in vitro.