YongHao Guo, YanFen Xue, Jun Liu, QuanHui Wang, YanHe Ma
{"title":"Characterization and function analysis of a Halo-alkaline-adaptable Trk K+ uptake system in Alkalimonas amylolytica strain N10.","authors":"YongHao Guo, YanFen Xue, Jun Liu, QuanHui Wang, YanHe Ma","doi":"10.1007/s11427-009-0132-2","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":49127,"journal":{"name":"Science in China. Series C, Life Sciences / Chinese Academy of Sciences","volume":"52 10","pages":"949-57"},"PeriodicalIF":0.0000,"publicationDate":"2009-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s11427-009-0132-2","citationCount":"6","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Science in China. Series C, Life Sciences / Chinese Academy of Sciences","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1007/s11427-009-0132-2","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2009/11/13 0:00:00","PubModel":"Epub","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 6
Abstract
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.