Characterization and function analysis of a Halo-alkaline-adaptable Trk K+ uptake system in Alkalimonas amylolytica strain N10.
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.
Guo
YongHao
Y
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
Xue
YanFen
Y
Liu
Jun
J
Wang
QuanHui
Q
Ma
YanHe
Y
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D000222:Adaptation, Physiological; D000468:Alkalies; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001692:Biological Transport / Q000187:drug effects; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D020563:Gammaproteobacteria / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009997:Osmotic Pressure; D011188:Potassium / Q000378:metabolism* / Q000493:pharmacokinetics; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D012965:Sodium Chloride / Q000494:pharmacology
D018528:ATP-Binding Cassette Transporters; D000468:Alkalies; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D015221:Potassium Channels; C549116:TrkA protein, E coli; C443122:TrkH protein, E coli; C068653:TrkG protein, E coli; D012965:Sodium Chloride; D011188:Potassium