Correlation between phosphorylation of the chemotaxis protein CheY and its activity at the flagellar motor

Abstract

Phosphorylation of the chemotaxis protein CheY by its kinase CheA appears to play a central role in the process of signal transduction in bacterial chemotaxis. It is presumed that the role is activation of CheY which results in clockwise (CW) flagellar rotation. The aim of this study was to determine whether this activity of CheY indeed depends on the protein being phosphorylated. Since the phosphorylation of CheY can be detected only in vitro, we studied the ability of CheY to cause CW rotation in an in vitro system, consisting of cytoplasm-free envelopes of Salmonella typhimurium or Escherichia coli having functional flagella. Envelopes containing just buffer rotated only counterclockwise. Inclusion of CheY caused 14% of the rotating envelopes to go CW. This fraction of CW-rotating envelopes was not altered when the phosphate potential in the envelopes was lowered by inclusion of ADP together with CheY in them, indicating that CheY has a certain degree of activity even without being phosphorylated. Attempts to increase the activity of CheY in the envelopes by phosphorylation were not successful. However, when CheY was inserted into partially-lysed cells (semienvelopes) under phosphorylating conditions, the number of CW-rotating cells increased 3-fold. This corresponds to more than a 100-fold increase in the activity of a single CheY molecule upon phosphorylation. It is concluded that nonphosphorylated CheY can interact with the flagellar switch and cause CW rotation, but that this activity is increased by at least 2 orders of magnitude by phosphorylation. This increase in activity requires additional cytoplasmic constituents, the identity of which is not yet known.

Uncoupled Phosphorylation and Activation in Bacterial Chemotaxis

“…Thus it is possible that FliM binding and promotion of clockwise flagellar rotation may be separable events. It has been proposed elsewhere (10,34,55) that binding of CheY-P to the flagellar motor is a necessary, but not sufficient, event for signal transduction. The average values of 1 and 2 are 208°and 84°, respectively, for the apo forms (q), excluding D13K, and 177°and 33°, respectively, for the cation bound forms (f).…”

“…First, the hyper-signaling activity of CheY D13K is observed in a strain containing a multicopy plasmid (25) but is substantially reduced when the mutant protein is expressed from a single copy chromosomal gene (26). Second, the in vivo activity of wild-type CheY is increased by simple overexpression (52), and there is evidence that nonphosphorylated wild-type CheY possesses a low level of clockwise-generating activity (10). Finally, in the case of …”

“…The best characterized response regulator is the bacterial chemotaxis protein CheY, which has been the subject of extensive genetic, biochemical, and biophysical analyses. Phosphorylated CheY (CheY-P) 1 causes flagellar rotation to change from the default counterclockwise direction to clockwise (7)(8)(9)(10). The coordination of changes in swimming behavior with the temporal changes in chemical concentrations experienced by cells swimming in chemical gradients results in chemotaxis (11,12).…”

Intragenic suppressors of a mutation in the aspartate chemoreceptor gene that abolishes binding of the receptor to methyltransferase

“…The unliganded (or repellent-bound) receptor stimulates the activity of CheA, which auto- D. Shiomi, M. Homma and I. Kawagishi phosphorylates itself and then transfers the phosphate group to the response regulators CheY and CheB (Hess et al, 1988 ;Stock et al, 1988 ;Wylie et al, 1988). Phospho-CheY binds to FliM, which is a component of the flagellar motor, and induces the clockwise rotation of the motor, which causes tumbling (Barak & Eisenbach, 1992 ;Welch et al, 1993). Dephosphorylation of phospho-CheY is facilitated by CheZ.…”

“…When attractant binds to a chemoreceptor, it inhibits the activity of CheA (Borkovich et al, 1989). Consequently, the concentration of phospho-CheY decreases and the flagella rotate counterclockwise, which results in smooth swimming (Barak & Eisenbach, 1992 ;Welch et al, 1993). The inhibition of the CheA kinase also decreases the concentration of phospho-CheB, the active form of the methylesterase, which reduces demethylation activity and hence increases methylation of the receptors.…”