Changing the direction of flagellar rotation in bacteria by modulating theratio between the rotational states of the switch protein FliM

One of the major questions in bacterial chemotaxis is how the switch, which controls the direction of flagellar rotation, functions. It is well establ ished that binding of the signaling molecule CheY to the switch protein FUM shifts the rotation from the default direction, counterclockwise, to clock wise. How this shift is done is still a mystery. Our aim in this study was to determine the correlation between the fraction of FliM molecules in the clockwise state (i.e. occupied by CheY) and the probability of clockwise ro tation. For this purpose we gradually expressed, from a plasmid, a clockwis e FliM mutant protein in cells that express, from the chromosome, wild-type FliM but no chemotaxis proteins. We verified that plasmid-borne FliM excha nges chromosomal FIM in the switch. Surprisingly, a substantial clockwise p robability was not obtained before the large majority of the FEM molecules in the switch were clockwise molecules. Thereafter, the rise in clockwise p robability was very steep. These results suggest that an increase in the cl ockwise probability requires a high level of FliM occupancy by CheY similar to P. They further suggest that the steep increase in clockwise rotation u pon increasing CheY levels, reported in several studies, is due, at least i n part, to cooperativity of post-binding interactions within the switch. We also carried out the inverse experiment, in which wild-type FliM was gradu ally expressed in a background of a clockwise fliM mutant. In this case, th e level of the clockwise mutant protein, required for establishing a certai n clockwise probability, was lower than in the original experiment. If our system (in which the ratio between the rotational states of FliM in the swi tch is established by slow exchange) and the native system (in which the ra tio is established by fast changes in FliM occupancy) are comparable, the r esults suggest that hysteresis is involved in the switch function. Such a s ituation might reflect a damping mechanism, which prevents a situation in w hich fluctuations in the phosphorylation level of CheY throw the switch fro m one direction of rotation to the other. (C) 2001 Academic Press.