Jiang P, Cronan J E
Department of Microbiology, University of Illinois at Urbana, Champaign 61801.
J Bacteriol. 1994 May;176(10):2814-21. doi: 10.1128/jb.176.10.2814-2821.1994.
The effects of inhibition of Escherichia coli phospholipid synthesis on the accumulation of intermediates of the fatty acid synthetic pathway have been previously investigated with conflicting results. We report construction of an E. coli strain that allows valid [14C]acetate labeling of fatty acids under these conditions. In this strain, acetate is a specific precursor of fatty acid synthesis and the intracellular acetate pools are not altered by blockage of phospholipid synthesis. By use of this strain, we show that significant pools of fatty acid synthetic intermediates and free fatty acids accumulate during inhibition of phospholipid synthesis and that the rate of synthesis of these intermediates is 10 to 20% of the rate at which fatty acids are synthesized during normal growth. Free fatty acids of abnormal chain length (e.g., cis-13-eicosenoic acid) were found to accumulate in glycerol-starved cultures. Analysis of extracts of [35S]methionine-labeled cells showed that glycerol starvation resulted in the accumulation of several long-chain acyl-acyl carrier protein (ACP) species, with the major species being ACP acylated with cis-13-eicosenoic acid. Upon the restoration of phospholipid biosynthesis, the abnormally long-chain acyl-ACPs decreased, consistent with transfer of the acyl groups to phospholipid. The introduction of multicopy plasmids that greatly overproduced either E. coli thioesterase I or E. coli thioesterase II fully relieved the inhibition of fatty acid synthesis seen upon glycerol starvation, whereas overexpression of ACP had no effect. Thioesterase I overproduction also resulted in disappearance of the long-chain acyl-ACP species. The release of inhibition by thiosterase overproduction, together with the correlation between the inhibition of fatty acid synthesis and the presence of abnormally long-chain acyl-ACPs, suggests with that these acyl-ACP species may act as feedback inhibitors of a key fatty acid synthetic enzyme(s).
先前已对抑制大肠杆菌磷脂合成对脂肪酸合成途径中间产物积累的影响进行了研究，但结果相互矛盾。我们报告构建了一种大肠杆菌菌株，该菌株能在这些条件下实现对脂肪酸有效的[14C]乙酸盐标记。在该菌株中，乙酸盐是脂肪酸合成的特定前体，并且磷脂合成的阻断不会改变细胞内乙酸盐库。通过使用该菌株，我们表明在磷脂合成受到抑制期间，大量的脂肪酸合成中间产物和游离脂肪酸会积累，并且这些中间产物的合成速率是正常生长期间脂肪酸合成速率的10%至20%。发现在甘油饥饿的培养物中积累了链长异常的游离脂肪酸（例如顺式-13-二十碳烯酸）。对[35S]甲硫氨酸标记细胞提取物的分析表明，甘油饥饿导致几种长链酰基-酰基载体蛋白（ACP）种类的积累，主要种类是被顺式-13-二十碳烯酸酰化的ACP。恢复磷脂生物合成后，异常长链的酰基-ACP减少，这与酰基转移到磷脂上一致。引入大量过量表达大肠杆菌硫酯酶I或大肠杆菌硫酯酶II的多拷贝质粒可完全缓解甘油饥饿时所见的脂肪酸合成抑制，而过量表达ACP则没有效果。硫酯酶I的过量表达还导致长链酰基-ACP种类消失。硫酯酶过量表达导致抑制解除，以及脂肪酸合成抑制与异常长链酰基-ACP的存在之间的相关性，表明这些酰基-ACP种类可能作为关键脂肪酸合成酶的反馈抑制剂。
