Regulation of Phosphatase Activity in Bacterial Chemotaxis Yuval Blat 1 , Blake Gillespie 2 , Anat Bren 1 , Frederick W. Dahlquist 2 * and Michael Eisenbach 1 * 1 Department of Biological Chemistry, The Weizmann Institute of Science 76100 Rehovot, Israel 2 Institute of Molecular Biology University of Oregon, Eugene Oregon 97403, USA Bacterial chemotaxis is the most studied model system for signaling by the widely spread family of two-component regulatory systems. It is con- trolled by changes in the phosphorylation level of the chemotactic response regulator, CheY, mediated by a histidine kinase (CheA) and a speci®c phosphatase (CheZ). While it is known that CheA activity is regulated, via the receptors, by chemotactic stimuli, the input that may regulate CheY dephosphorylation by CheZ has not been found. We measured, by using stopped-¯ow ¯uorometry, the kinetics of CheZ- mediated dephosphorylation of CheY. The onset of dephosphorylation was delayed by 50 ms after mixing phosphorylated CheY (CheY  P) with CheZ, and a distinct overshoot was observed in the approach to the new steady state of CheY  P. The delay and overshoot were not observed in a hyperactive mutant CheZ protein (CheZ54RC) that does not support chemotaxis in vivo and appears to be constitutively active. CheZ activity was cooperative with respect to CheY  P, with a Hill-coef- ®cient of 2.5. The observed delayed modulation of CheZ activity and its cooperativity suggest that the phosphatase activity is regulated at the level of CheY  P-CheZ interaction. This novel kind of interplay between a response regulator and its phosphatase may be involved in signal tun- ing and in adaptation to chemotactic signals. # 1998 Academic Press Keywords: bacterial chemotaxis; phosphorylation; phosphatase; CheY; CheZ *Corresponding authors Introduction Most prokaryotic responses as well as some archeal and eukaryotic responses are mediated by signal transduction proteins belonging to the family of two-component regulatory systems (Appleby et al., 1996). Of these, bacterial chemo- taxis is the best understood. The chemotaxis- speci®c response regulator, CheY, is activated by phosphorylation mediated by CheA, a speci®c receptor-bound histidine kinase (Stock & Surette, 1996). Phosphorylated CheY (CheY  P) can be dephosphorylated spontaneously or, in an enhanced manner, by a speci®c phosphatase, CheZ (Eisenbach, 1996; Hess et al., 1988; Wylie et al., 1988), yielding non-active CheY (Barak & Eisenbach, 1992; Welch et al., 1993). (The term phosphatase is used here in the broader sense and it does not connote the mechanism of CheZ action.) While CheY phosphorylation is known to be regulated by chemotactic stimuli (Borkovich et al., 1989; Ninfa et al., 1991), no such regulation of CheZ-mediated dephosphorylation is known. Recently, we found that the ability of CheZ to dephosphorylate CheY  P is correlated with its ability to undergo oligomerization upon binding to CheY  P, and that the oligomerization is depen- dent on the level of CheY  P (Blat & Eisenbach, 1996b,c). This suggested that CheY  P depho- sphorylation might be indeed regulated and that mutual interactions between CheZ and CheY  P are important for this regulation (Blat & Eisenbach, 1996b). Here, we addressed the question of depho- sphorylation regulation by measuring the kinetics Present address: Y. Blat, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA. Abbreviations used: CheY, chemotaxis response regulator; CheY  P, phosphorylated CheY; CheA, speci®c receptor-bound histidine kinase; CheZ, speci®c phosphatase. E-mail addresses of the corresponding authors: BMEISEN@WEIZMANN.WEIZMANN.AC.IL; fwd@nmr.uoregon.edu Article No. mb982224 J. Mol. Biol. (1998) 284, 1191±1199 0022 ± 2836/98/491191±09 $30.00/0 # 1998 Academic Press