Soupene E, He L, Yan D, Kustu S
Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley, CA 94720-3102, USA.
Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):7030-4. doi: 10.1073/pnas.95.12.7030.
Homologues of the amtB gene of enteric bacteria exist in all three domains of life. Although their products are required for transport of the ammonium analogue methylammonium in washed cells, only in Saccharomyces cerevisiae have they been shown to be necessary for growth at low NH4+ concentrations. We now demonstrate that an amtB strain of Escherichia coli also grows slowly at low NH4+ concentrations in batch culture, but only at pH values below 7. In addition, we find that the growth defect of an S. cerevisiae triple-mutant strain lacking the function of three homologues of the ammonium/methylammonium transport B (AmtB) protein [called methylammonium/ammonium permeases (MEP)] that was observed at pH 6.1 is relieved at pH 7.1. These results provide direct evidence that AmtB participates in acquisition of NH4+/NH3 in bacteria as well as eucarya. Because NH3 is the species limiting at low pH for a given total concentration of NH4+ + NH3, results with both organisms indicate that AmtB/MEP proteins function in acquisition of the uncharged form. We confirmed that accumulation of [14C]methylammonium depends on its conversion to gamma-N-methylglutamine, an energy-requiring reaction catalyzed by glutamine synthetase, and found that at pH 7, constitutive expression of AmtB did not relieve the growth defects of a mutant strain of Salmonella typhimurium that appears to require a high internal concentration of NH4+/NH3. Hence, contrary to previous views, we propose that AmtB/MEP proteins increase the rate of equilibration of the uncharged species, NH3, across the cytoplasmic membrane rather than actively transporting-that is, concentrating-the charged species, NH4+.
肠道细菌的amtB基因同源物存在于生命的所有三个域中。尽管在洗涤过的细胞中，铵类似物甲铵的运输需要它们的产物，但只有在酿酒酵母中，它们才被证明在低NH4+浓度下生长是必需的。我们现在证明，大肠杆菌中的amtB菌株在分批培养中，在低NH4+浓度下也生长缓慢，但仅在pH值低于7时才会如此。此外，我们发现，在pH 6.1时观察到的缺乏铵/甲铵转运B（AmtB）蛋白的三个同源物功能的酿酒酵母三突变体菌株（称为甲铵/铵通透酶（MEP））的生长缺陷，在pH 7.1时得到缓解。这些结果提供了直接证据，表明AmtB参与细菌和真核生物中NH4+/NH3的获取。由于对于给定的NH4+ + NH3总浓度，NH3是低pH下的限制物种，两种生物体的结果都表明AmtB/MEP蛋白在获取不带电荷的形式中起作用。我们证实，[14C]甲铵的积累取决于其转化为γ-N-甲基谷氨酰胺，这是一种由谷氨酰胺合成酶催化的需能反应，并且发现，在pH 7时，AmtB的组成型表达并不能缓解鼠伤寒沙门氏菌突变体菌株的生长缺陷，该菌株似乎需要高内部浓度的NH4+/NH3。因此，与先前的观点相反，我们提出AmtB/MEP蛋白增加了不带电荷的物种NH3跨细胞质膜的平衡速率，而不是主动运输——即浓缩——带电荷的物种NH4+。
