PHOSPHORYLATION OF THE RHIZOBIUM-MELILOTI FIXJ PROTEIN INDUCES ITS BINDING TO A COMPOUND REGULATORY REGION AT THE FIXK PROMOTER

The FixJ protein is a member of the regulator class of two-component s ystems involved in the transcriptional activation of nitrogen fixation genes in Rhizobium meliloti. Phosphorylation of FixJ was previously d emonstrated to dramatically enhance its transcriptional activity at th e nifA and fixK promoters. Here we show that the isolated carboxyl-ter minal domain of FixJ, FixJC, binds the fixK promoter, whereas binding of the full-length FixJ protein requires its phosphorylation. By analy zing the DNase I and Exonuclease III protection patterns of the wild-t ype and a mutant fixK promoter, we have identified two overlapping bin ding regions for both phosphorylated FixJ and FixJC. A higher affinity region is located between positions -69 and -44 relative to the trans cription start site, and a lower affinity region, between positions -5 7 and -31, overlaps the -35 region of the promoter.