Fumarate Modulates Bacterial Flagellar Rotation by Lowering the Free Energy Difference between the Clockwise and Counterclockwise States of the Motor Krishna Prasad, S. Roy Caplan and Michael Eisenbach* Department of Biological Chemistry, The Weizmann Institute of Science 76100 Rehovot, Israel Switching ¯agellar rotation from one direction to another is an essential part of bacterial chemotaxis. Fumarate has been shown to possess the capacity to restore to ¯agella 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 poten- tial targets of fumarate, the response regulator CheY and the ¯agellar switch-motor complex, we compared ¯agellar 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 car- rying out the measurements at 2.5  C, where it has been demonstrated that gutted cells switch spontaneously. The ¯agellar 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 suf®cient for normal chemotaxis. # 1998 Academic Press Keywords: chemotaxis; motility; ¯agellar motor; fumarate; switch *Corresponding author Introduction Bacteria approach attractants and avoid repel- lents by modulating the direction of rotation of their ¯agella (Larsen et al., 1974). Therefore, the question of how the chemotaxis process is regu- lated in bacteria reduces to how the direction of ¯agellar rotation is regulated. In bacteria such as Escherichia coli, the default direction of rotation is counterclockwise (CCW), i.e. the ¯agellar motor always rotates CCW in the temperature range 20 to 37  C, unless it receives a signal to do otherwise (for a review, see Eisenbach, 1996). The signal is the chemotaxis protein CheY. CheY acts on a com- plex of three proteins, termed a ``switch'', which is located at the base of the ¯agellar motor and deter- mines its direction of rotation (for a review, see Macnab, 1995). CheY, when phosphorylated, binds to the switch protein FliM (Welch et al., 1993) and, as a result, the motor rotates clockwise (CW; Barak & Eisenbach (1992a); for reviews, see Barak & Eisenbach, 1996; Eisenbach, 1996). Very little is known about the molecular mechanism of switch- E-mail address of the corresponding author: bmeisen@weizmann.weizmann.ac.il Abbreviations used: CW, clockwise; CCW, counterclockwise. Article No. mb981922 J. Mol. Biol. (1998) 280, 821±828 0022 ± 2836/98/300821±08 $30.00/0 # 1998 Academic Press