Control of transmembrane ion fluxes to select halorhodopsin-deficient and other energy-transduction mutants of Halobacterium halobium.
Control of transmembrane ion fluxes to select halorhodopsin-deficient and other energy-transduction mutants of Halobacterium halobium.
复制标题
控制跨膜离子通量以选择盐杆菌的盐视紫红质缺陷和其他能量转导突变体。
DOI：
10.1073/pnas.79.14.4308
复制
发表时间：
1982
影响因子：
11.1
通讯作者：
Spudich,JL
中科院分区：
文献类型：
--
作者：
Spudich,EN;Spudich,JL
我们描述了一种突变体的选择方法，这种突变体改变了盐杆菌跨膜离子通量的产生和调节。该方法是基于通过光、离子载体和外部pH值的组合来实验控制离子通量，从而在细胞通过膜的质子循环中产生不平衡。稳态质子循环通过引入一个带有质子团的小的向内质子泄漏而增加。然后将细胞照射以激发盐视紫红质，使膜超极化并驱使质子进入细胞。因此，野生型细胞遭受细胞质酸化，这导致运动能力的急剧丧失，并抑制了它们的生长。这些特性可以用来选择逃避细胞质酸化的突变体，因为它们要么缺乏盐视紫红质功能，要么在光照下有更大的喷射质子的能力。在一个流行的选择，这种方法，97%的单个细胞在离子通量特性明显改变。盐菌紫红质改变的细胞被选择，特别是盐菌紫红质产生膜电位的缺陷和细菌紫红质增加的细胞质子喷射。我们描述了一种缺盐紫红质菌株Flx37的特性。
We describe a selection method for mutants altered in the generation and regulation of transmembrane ion flux in Halobacterium halobium. The method is based on experimental control of ion fluxes by a combination of light, ionophore, and external pH to generate an imbalance in the cells' proton circulation through their membranes. The steady-state proton circulation is increased by the introduction of a small inward proton leak with a protonophore. The cells are then illuminated to excite halorhodopsin, which hyperpolarizes the membrane and drives protons into the cells. As a result, wild-type cells suffer cytoplasmic acidification, which causes a dramatic loss of motility and suppresses their growth. These properties can be used to select for mutants that escape cytoplasmic acidification because either they lack halorhodopsin function or they have a greater capacity to eject protons during the illumination. In a popular selected by this method, 97% of the individual cells were demonstrably altered in ion flux properties. Cells were selected with alterations in the halobacterial rhodopsin, specifically with deficiencies in membrane potential generation by halorhodopsin and with increased cellular proton ejection by bacteriorhodopsin. We describe properties of one of the halorhodopsin-deficient strains, Flx37.