Cloning and disruption of a putative NaH-antiporter gene of Enterococcus hirae.
Cloning and disruption of a putative NaH-antiporter gene of Enterococcus hirae.
复制标题
DOI：
10.1016/s0021-9258(18)42779-2
复制
发表时间：
1992-03
期刊：
影响因子：
--
通讯作者：
M. Waser;Denise Hess-BienzS;K. Davies;Marc Soliozg
中科院分区：
文献类型：
--
作者：
M. Waser;Denise Hess-BienzS;K. Davies;Marc Soliozg
当在富钠环境中生长时，野生型肝炎肠球菌通过两种机制挤出钠，即atp驱动的钠挤出和NaH-antiport。突变体7683不能在富含钠的培养基上生长。这是由于两种突变，一种使atp驱动的钠转运失活，另一种使钠-反港失活。7683在大肠杆菌-大肠杆菌中通过电穿孔转化了一个来自E. hirae的基因库。Hirae穿梭矢量。选择在富钠培养基上恢复生长能力的转化子，并对转化质粒进行分析。在7683中发现了一个能够恢复nah -anti - port活性的基因。这个基因被命名为napA。它编码一种由383个氨基酸组成的极度疏水蛋白。该蛋白的亲水分析表明它可能形成12个跨膜螺旋。在一个突变体中，只有nah -反转运蛋白，napA基因被同源重组破坏。所得到的菌株在富钠培养基中不能生长，从这些细胞中分离出的囊泡表现出钠质子反港活性的缺陷。我们得出结论，napA基因编码一个nah -反转运蛋白。NapA蛋白与EMBL遗传数据库中的任何蛋白都没有明显的同源性。
When growing in a sodium-rich environment, wild-type Enterococcus hirae extrudes sodium by two mechanisms, ATP-driven sodium extrusion, and NaH-antiport. Mutant 7683 is unable to grow on sodium-rich media. This is due to two mutations, one inactivating ATP-driven sodium transport and a second rendering NaH-antiport inoperative. 7683 was transformed by electroporation with a gene bank, derived from E. hirae, in an Escherichia coli-E. hirae shuttle vector. Transformants which had regained the ability to grow on sodium-rich media were selected for and the transforming plasmids analyzed. A gene able to restore NaH-antiport activity in 7683 was identified. This gene was named napA. It codes for an extremely hydrophobic protein of 383 amino acids. Hydropathy analysis of this protein indicates that it probably forms 12 transmembraneous helices. In a mutant, possessing only the NaH-antiporter, the napA gene was disrupted by homologous recombination. The resultant strain failed to grow in sodium-rich media, and vesicles isolated from these cells exhibited a defect in sodium proton antiport activity. We conclude that the napA gene codes for a NaH-antiporter. The NapA protein does not exhibit significant homology to any protein in the EMBL genetic data bank.