MUTANTS OF THE 2-COMPONENT REGULATORY PROTEIN FIXJ OF RHIZOBIUM-MELILOTI THAT HAVE INCREASED ACTIVITY AT THE NIFA PROMOTER

FixL and FixJ belong to a two-component regulatory system in Rhizobium meliloti that induces the expression of numerous nitrogen-fixation ge nes during symbiosis with alfalfa. FixJ is a positive activator requir ed for transcription of the regulatory genes nifA and fixK, while FixL is an oxygen-binding hemoprotein capable of regulating the phosphoryl ation status of both itself and FixJ, in response to oxygen availabili ty. In this study, we isolated four FixJ mutants that display increase d activity at the nifA promoter (PnifA) in Escherichia coli. All four mutants possess amino acid changes in a domain of FixJ that is conserv ed in other response regulator proteins, and all exhibit increased act ivity at PnifA in R. meliloti that is dependent on the presence of Fix L. One of the mutant proteins, while less efficient at accepting phosp hate from a truncated derivative of FixL (FixL), nevertheless has a p hosphorylated form that is more stable than the phosphorylated form of wild-type (wt) FixJ and is more resistant to the phosphatase activity of FixL. The wt FixJ-phosphate was found to have a half-life of appr oximately 4 h, which makes it an unusually long-lived response regulat or protein. The exceptional stability of wt FixJ-phosphate and the alt ered phosphorylation properties observed for the mutant are discussed in relation to signal transduction in the FixLJ system.