Prasad K, Caplan S R, Eisenbach M
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, 76100, Israel.
J Mol Biol. 1998 Jul 31;280(5):821-8. doi: 10.1006/jmbi.1998.1922.
Switching flagellar rotation from one direction to another is an essential part of bacterial chemotaxis. Fumarate has been shown to possess the capacity to restore to flagella of cytoplasm-free, CheY-containing bacterial envelopes the ability to switch directions and to increase the probability of reversal in intact cells. Neither the target of fumarate action nor the mechanism of function is known. To distinguish between the two potential targets of fumarate, the response regulator CheY and the flagellar switch-motor complex, we compared flagellar rotation between isogenic strains that lacked CheY and had either low or high levels of fumarate. The difference in the fumarate levels was due to a deletion of the genes encoding the enzymes that synthesize and metabolize fumarate; succinate dehydrogenase and fumarase, respectively. The strains were in a gutted background (i.e. a background deleted for the cytoplasmic chemotaxis proteins and some of the receptors), and switching was achieved by carrying out the measurements at 2.5 degreesC, where it has been demonstrated that gutted cells switch spontaneously. The flagellar rotation of the strain with the highest level of fumarate was the most clockwise-biased and had the highest reversal frequency, indicating that fumarate is effective even in the absence of CheY. Fumarate reduced the free energy difference of the counterclockwise-to-clockwise transition and had no appreciable effect on the activation energy of this transition. Similar observations were made at room temperature, provided that intracellular CheY was present. In a wild-type background, both mutants made rings on semi-solid agar typical of normal chemotaxis. Taken together, the results suggest that the target of fumarate is the switch-motor complex, that fumarate acts by increasing the probability of the clockwise state, and that a fumarate level as low as that found in succinate dehydrogenase mutants is sufficient for normal chemotaxis.
将鞭毛旋转从一个方向切换到另一个方向是细菌趋化性的一个重要部分。已表明富马酸盐具有恢复无细胞质、含CheY的细菌包膜鞭毛切换方向的能力，并增加完整细胞中逆转的概率。富马酸盐作用的靶点和功能机制均未知。为了区分富马酸盐的两个潜在靶点，即应答调节因子CheY和鞭毛开关 - 马达复合体，我们比较了缺乏CheY且富马酸盐水平低或高的同基因菌株之间的鞭毛旋转。富马酸盐水平的差异是由于分别缺失了编码合成和代谢富马酸盐的酶（琥珀酸脱氢酶和富马酸酶）的基因。这些菌株处于去除了细胞质趋化性蛋白和一些受体的背景中（即去除背景），并且通过在2.5℃下进行测量来实现切换，已证明在该温度下去除背景的细胞会自发切换。富马酸盐水平最高的菌株的鞭毛旋转最偏向顺时针方向且逆转频率最高，表明即使在没有CheY的情况下富马酸盐也是有效的。富马酸盐降低了逆时针到顺时针转变的自由能差，并且对该转变的活化能没有明显影响。在室温下也观察到了类似的结果，前提是细胞内存在CheY。在野生型背景下，两个突变体在半固体琼脂上形成了典型的正常趋化性的环。综上所述，结果表明富马酸盐的靶点是开关 - 马达复合体，富马酸盐通过增加顺时针状态的概率起作用，并且琥珀酸脱氢酶突变体中发现的低至那样的富马酸盐水平对于正常趋化性就足够了。
