IN-VITRO ACTIVITY OF NIFL, A SIGNAL-TRANSDUCTION PROTEIN FOR BIOLOGICAL NITROGEN-FIXATION

In the free-living diazotroph Klebsiella pneumoniae, the NifA protein is required for transcription of all nif (nitrogen fixation) operons e xcept the regulatory nifLA operon itself. NifA activates transcription of nif operons by the alternative holoenzyme form of RNA polymerase, sigma(54) holoenzyme. In vivo, NifL is known to antagonize the action of NifA in the presence of molecular oxygen or combined nitrogen. We n ow demonstrate inhibition by NifL in vitro in both a coupled transcrip tion-translation system and a purified transcription system. Crude cel l extracts containing NifL inhibit NifA activity in the coupled system , as does NifL that has been solubilized with urea and allowed to refo ld. Inhibition is specific to NifA in that it does not affect activati on by NtrC, a transcriptional activator homologous to NifA, or transcr iption by sigma(70) holoenzyme. Renatured NifL also inhibits transcrip tional activation by a maltose-binding protein fusion to NifA in a pur ified transcription system, indicating that no protein factor other th an NifL is required. Since inhibition in the purified system persists anaerobically, our NifL, preparation does not sense molecular oxygen d irectly.