Characterization and function analysis of a Halo-alkaline-adaptable Trk K+ uptake system in Alkalimonas amylolytica strain N10.

YongHao Guo, YanFen Xue, Jun Liu, QuanHui Wang, YanHe Ma
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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.

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溶淀粉碱单胞菌N10耐碱性Trk - K+吸收系统的特性及功能分析
通过对吸收钾(K(+))缺陷的大肠杆菌突变体的功能互补,克隆了嗜碱性解淀粉碱单胞菌N10吸收钾(+)所需的两个基因。这两个基因Aa-trkA (1337 bp)和Aa-trkH (1452 bp)在解淀粉拟南蝽N10染色体上相邻,转录方向相反。互补实验表明,解淀粉芽孢杆菌菌株N10的Aa-TrkA和Aa-TrkH分别恢复了大肠杆菌DeltatrkA和DeltatrkG DeltatrkH菌株在低K(+)浓度下的生长能力。此外,Aa-TrkAH支持大肠杆菌DeltasapD菌株的生长,这表明atp结合蛋白TrkE对于解淀粉A.菌株N10的Trk系统是不可缺少的。在不同pH水平下检测了净K(+)吸收量,临界NaCl浓度表明Aa-TrkAH是一种碱适应性和部分光晕适应性的K(+)转运体。大肠杆菌DeltatrkA菌株的非均相K(+)转运实验表明,Aa-TrkAH的最佳碱性pH接近8.5或更高。Aa-TrkH的定点突变表明,Phe103和Ser229在K(+)的选择和转运中起着关键作用。分子伴侣groES-groEL和tig促进Aa-TrkH和Aa-TrkA在体外过表达。
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