OXYGEN REGULATION OF NIFA TRANSCRIPTION INVITRO

In Rhizobium meliloti, transcription of the key nitrogen-fixation regu latory genes nifA and fixK is induced in response to microaerobiosis t hrough the action of the FixL and FixJ proteins. These two proteins ar e sensor and regulator homologues, respectively, of a large family of bacterial two-component systems involved in sensing and responding to environmental changes. A soluble, truncated form of the membrane prote in FixL, FixL, has been shown to be a hemoprotein that phosphorylates and dephosphorylates FixJ in response to oxygen tension. Here we use an in vitro transcription system to prove that FixJ is a transcription al activator of both nifA and fixK and that phosphorylation of FixJ ma rkedly increases its activity. Phosphorylation was achieved either by preincubating FixJ with FixL and ATP or by exposing FixJ to the inorg anic phospho donor ammonium hydrogen phosphoramidate. Both FixJ and Fi xJ-phosphate formed heparin-resistant complexes under the assay condit ions used. Lastly, we were able to show that anaerobiosis, in the pres ence of FixL and ATP, greatly stimulates FixJ activity at the nifA pr omoter with either Escherichia coli or R. meliloti RNA polymerase. Thi s use of atmospheric oxygen to control nifA transcription in vitro rep resents a reconstitution of a bacterial two-component signal transduct ion system in its entirety, from effector to ultimate target, by the u se of purified components.