Simoni R D, Shandell A
J Biol Chem. 1975 Dec 25;250(24):9421-7.
Recent genetic analyses of the membrane components involved in energy transduction in Escherichia coli have concentrated on the (Ca2+, Mg2+)-ATPase complex (EC 3.6.1.3). Many mutants have been described with altered biochemical properties and defects in energy-requiring processes such as oxidative phosphorylation, transhydrogenase activity, and active transport of several solutes. This report describes the isolation of a mutant strain of E. coli that is defective in several energy-requiring processes. The strain BG-31 was obtained by "localized mutagenesis" using phage P1c1. The mutation maps at approximately 73.5 min on the E. coli chromosome. Reversion and suppression analyses indicate that the defect is the result of a single amber mutation. This strain is unable to utilize succinate, D-lactate, or malate for growth. Mutant cells are unable to couple the energy derived from the hydrolysis of ATP to the active transport of proline, although coupling of energy derived from electron transport to solute transport appears normal when examined in both cells and isolated membrane vesicles. Isolated membranes of the mutant are unable to couple the energy derived from the hydrolysis of ATP to transhydrogenase activity while they can utilize the energy generated from electron transport to drive transhydrogenase activity. Extracts of strain BG-31 have normal levels of (Ca2+, Mg2+)-ATPase activity. The ATPase portion of the complex, bacterial F1 (BF1), is poorly attached to the membrane portion of the complex. In vitro reconstitution of transhydrogenase activity with stripped membrane fractions and crude preparations of BF1 localize the defect in strain BG-31 to the membrane portion of the complex. Analysis of membranes of the strain BG-31 by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate demonstrate the absence of a single polypeptide of molecular weight about 54,000 and the appearance of a new polypeptide of lower molecular weight, about 25,000. Analysis of a spontaneous revertant of BG-31 shows complete restoration of the parental phenotype including the gel patterns. The characterization of this mutant provides the first demonstration of the consequences of a structural gene mutation on a polypeptide in the membrane portion of the complex and represents the initial stages in what we hope will be the biochemical definition and functional characterization of this important energy-transducing system.
近期对大肠杆菌中参与能量转导的膜成分进行的遗传分析主要集中在（Ca2 +，Mg2 +）-ATP酶复合体（EC 3.6.1.3）上。已经描述了许多具有改变的生化特性以及在诸如氧化磷酸化、转氢酶活性和几种溶质的主动运输等能量需求过程中存在缺陷的突变体。本报告描述了一种在几种能量需求过程中存在缺陷的大肠杆菌突变菌株的分离情况。菌株BG - 31是通过使用噬菌体P1c1进行“局部诱变”获得的。该突变位于大肠杆菌染色体上大约73.5分钟处。回复突变和抑制分析表明，该缺陷是单个琥珀突变的结果。该菌株无法利用琥珀酸、D - 乳酸或苹果酸进行生长。突变细胞无法将ATP水解产生的能量与脯氨酸的主动运输偶联起来，不过当在细胞和分离的膜泡中进行检测时，电子传递产生的能量与溶质运输的偶联看起来是正常的。突变体的分离膜无法将ATP水解产生的能量与转氢酶活性偶联起来，而它们能够利用电子传递产生的能量来驱动转氢酶活性。BG - 31菌株的提取物具有正常水平的（Ca2 +，Mg2 +）-ATP酶活性。该复合体的ATP酶部分，即细菌F1（BF1），与复合体的膜部分结合不紧密。用剥离的膜组分和BF1粗制品进行转氢酶活性的体外重建将BG - 31菌株的缺陷定位到复合体的膜部分。在十二烷基硫酸钠存在下通过丙烯酰胺凝胶电泳对BG - 31菌株的膜进行分析，结果表明缺少一条分子量约为54,000的单一多肽，并且出现了一条分子量较低、约为25,000的新多肽。对BG - 31的自发回复突变体进行分析表明，包括凝胶图谱在内的亲本表型完全恢复。对该突变体的表征首次证明了复合体膜部分中一个结构基因突变对一种多肽的影响，并代表了我们希望对这个重要的能量转导系统进行生化定义和功能表征的初始阶段。
