Assays for CheC, FliY, and CheX as representatives of response regulator phosphatases

Much study of two-component systems deals with the excitation of the histidine kinase, activation of the response regulator, and the ultimate target of the signal. Removal of the message is of great importance to these signaling systems. Many methods have evolved in two-component systems to this end. These include autodephosphorylation of the response regulator, hydrolysis of the phosphoryl group by the kinase, or a dedicated phosphatase protein. It has long been known that CheZ is the phosphatase in the chemotaxis system of Escherichia coli and related bacteria. Most bacteria and archaea, however, do not have a cheZ gene, but instead rely on the CheC, CheX, and FliY family of CheY-P phosphatases. Here, we describe assays to test these chemotactic phosphatases, applicable to many other response regulator phosphatases.