Program in Molecular Microbiology, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, MA, USA.
J Bacteriol. 2013 Apr;195(7):1525-37. doi: 10.1128/JB.01690-12. Epub 2013 Jan 25.
Previously, it was shown that an aconitase (citB) null mutation results in a vast overaccumulation of citrate in the culture fluid of growing Bacillus subtilis cells, a phenotype that causes secondary effects, including the hyperexpression of the citB promoter. B. subtilis aconitase is a bifunctional protein; to determine if either or both activities of aconitase were responsible for this phenotype, two strains producing different mutant forms of aconitase were constructed, one designed to be enzymatically inactive (C450S [citB2]) and the other designed to be defective in RNA binding (R741E [citB7]). The citB2 mutant was a glutamate auxotroph and accumulated citrate, while the citB7 mutant was a glutamate prototroph. Unexpectedly, the citB7 strain also accumulated citrate. Both mutant strains exhibited overexpression of the citB promoter and accumulated high levels of aconitase protein. These strains and the citB null mutant also exhibited increased levels of citrate synthase protein and enzyme activity in cell extracts, and the major citrate synthase (citZ) transcript was present at higher-than-normal levels in the citB null mutant, due at least in part to a >3-fold increase in the stability of the citZ transcript compared to the wild type. Purified B. subtilis aconitase bound to the citZ 5' leader RNA in vitro, but the mutant proteins did not. Together, these data suggest that wild-type aconitase binds to and destabilizes the citZ transcript in order to maintain proper cell homeostasis by preventing the overaccumulation of citrate.
先前的研究表明，在生长的枯草芽孢杆菌细胞的培养液中，乌头酸酶（citB）的缺失突变导致柠檬酸的大量过度积累，这种表型会引起次级效应，包括 citB 启动子的过度表达。枯草芽孢杆菌乌头酸酶是一种双功能蛋白；为了确定乌头酸酶的任一或两种活性是否对此表型负责，构建了两种产生不同突变形式乌头酸酶的菌株，一种设计为无酶活性（C450S [citB2]），另一种设计为 RNA 结合缺陷（R741E [citB7]）。citB2 突变体是谷氨酸营养缺陷型，积累柠檬酸，而 citB7 突变体是谷氨酸原养型。出乎意料的是，citB7 菌株也积累了柠檬酸。两种突变株均表现出 citB 启动子的过度表达和高水平的乌头酸酶蛋白积累。这些菌株和 citB 缺失突变体还在细胞提取物中表现出柠檬酸合酶蛋白和酶活性的增加，并且主要的柠檬酸合酶（citZ）转录物在 citB 缺失突变体中的水平高于正常水平，至少部分原因是 citZ 转录物的稳定性比野生型增加了 3 倍以上。体外纯化的枯草芽孢杆菌乌头酸酶与 citZ 5' 先导 RNA 结合，但突变蛋白没有。这些数据表明，野生型乌头酸酶结合并使 citZ 转录物不稳定，以通过防止柠檬酸的过度积累来维持适当的细胞内稳态。
