The effect of CheY and fumarate on switching frequency and rotational bias of the bacterial flagellar motor was analyzed by computer-aided tracking of tethered Escherichia coli. Plots of cells overexpressing CheY in a gutted background showed a bell-shaped correlation curve of switching frequency and bias centering at about 50% clockwise rotation. Gutted cells (i.e., withcheA to cheZ deleted) with a low CheY level but a high cytoplasmic fumarate concentration displayed the same correlation of switching frequency and bias as cells overexpressing CheY at the wild-type fumarate level. Hence, a high fumarate level can phenotypically mimic CheY overexpression by simultaneously changing the switching frequency and the bias. A linear correlation of cytoplasmic fumarate concentration and clockwise rotation bias was found and predicts exclusively counterclockwise rotation without switching when fumarate is absent. This suggests that (i) fumarate is essential for clockwise rotation in vivo and (ii) any metabolically induced fluctuation of its cytoplasmic concentration will result in a transient change in bias and switching probability. A high fumarate level resulted in a dose-response curve linking bias and cytoplasmic CheY concentration that was offset but with a slope similar to that for a low fumarate level. It is concluded that fumarate and CheY act additively presumably at different reaction steps in the conformational transition of the switch complex from counterclockwise to clockwise motor rotation.
展开▼
机译：通过计算机辅助追踪大肠杆菌分析了CheY和富马酸酯对细菌鞭毛运动的开关频率和旋转偏向的影响。在去掉内脏的背景中过表达CheY的细胞图显示了开关频率和偏心的钟形相关曲线，其偏心点约为顺时针旋转50％。 CheY水平低但胞质富马酸盐浓度高的肠壁细胞（即删除了 cheA 至 cheZ ）显示的开关频率和偏差的相关性与过表达CheY的细胞相同野生型富马酸酯水平。因此，高富马酸酯水平可以通过同时改变开关频率和偏置来在表型上模拟CheY过表达。发现胞质富马酸盐浓度与顺时针旋转偏差呈线性关系，并且仅在缺乏富马酸盐时预测逆时针旋转而无需切换。这表明（i）富马酸盐对于体内顺时针旋转至关重要，并且（ii）任何代谢诱导的其胞质浓度波动都会导致偏差和转换概率的瞬时变化。高富马酸酯水平导致剂量-反应曲线将偏倚和细胞质CheY浓度联系起来，但相互抵消，但斜率类似于低富马酸酯水平。结论是，在开关复合体从逆时针旋转到顺时针旋转的构象转变中，富马酸酯和CheY可能在不同的反应步骤上加性起作用。
展开▼
