Regulation of Switching Frequency and Bias of the Bacterial Flagellar Motor by CheY and Fumarate

Abstract

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.

The bacterial flagellar switch complex is getting more complex

“…In contrast, the switching frequency was higher than that in the wild type (line 4), possibly reflecting futile attempts to switch to clockwise rotation. These results are consistent with the clockwise‐promoting effect of fumarate in the intact cells of E. coli (Montrone et al , 1998; Prasad et al , 1998) and with the possibility, investigated below, that FRD is the protein that transmits the fumarate effect to the switch.…”

“…(It should be mentioned, however, that although removal of FRD did not have a significant effect on cellular ATP levels, its absence might have other, unexplored effects on cellular metabolism.) The possible contribution of an elevated fumarate level was ruled out earlier by showing that, in cells deleted of the genes encoding fumarase, elevated cellular fumarate has no effect on the motility level (Montrone et al , 1998; Prasad et al , 1998).…”

“…Next, we measured the effect of fumarate on flagellar rotation using tethered, cytoplasm‐free envelopes containing His‐tagged CheY. In intact bacteria, fumarate mainly increases the fraction of time spent in clockwise rotation (Montrone et al , 1998; Prasad et al , 1998). In envelopes, fumarate enables switching: tethered envelopes require this dicarboxylate to switch from one direction of rotation to the other (Barak and Eisenbach, 1992; Barak et al , 1996).…”

“…In cytoplasm‐free but CheY‐containing cell envelopes, it enabled the flagella to switch their direction of rotation (Barak and Eisenbach, 1992; Barak et al , 1996). By employing mutants having exceptionally low or high intracellular levels of fumarate due to the absence of one of the enzymes that act on fumarate, that is, succinate dehydrogenase (SDH) or fumarase, Prasad et al (1998) and Montrone et al (1998) found that, in intact E. coli cells, fumarate increases both the fraction of time spent in clockwise rotation and switching frequency. These effects were due, in part, to reduction of the standard free energy difference between the clockwise and counterclockwise states of the switch (Prasad et al , 1998).…”

Chemotactic‐like response of Escherichia coli cells lacking the known chemotaxis machinery but containing overexpressed CheY

“…(b) CheY may not be involved, and the regulation of the direction of flagellar rotation may be carried out by fumarate. Fumarate was shown to decrease the CCW bias of flagellar rotation by direct interaction with the switchmotor complex (Montrone et al, 1998;Prasad et al, 1998). Furthermore, the repellents indole and benzoate were shown to inhibit reversibly the enzyme fumarase and consequently to increase the fumarate level in the cell and decrease the CCW bias (Montrone et al, 1996).…”