IRON IS REQUIRED TO RELIEVE INHIBITORY EFFECTS OF NIFL ON TRANSCRIPTIONAL ACTIVATION BY NIFA IN KLEBSIELLA-PNEUMONIAE

In Klebsiella pneumoniae, products of the nitrogen fixation nifLA oper on regulate transcription of the other nif operons, NifA activates tra nscription by sigma(54)-holoenzyme. In vivo, NifL antagonizes the acti on of NifA under aerobic conditions or in the presence of combined nit rogen, Hn contrast to a previous report, we show that depletion of iro n (Fe) from the growth medium with the chelating agent o-phenanthrolin e (20 mu M) mimics aerobiosis or combined nitrogen in giving rise to i nhibition of NifA activity even under anaerobic, nitrogen-limiting con ditions, Adding back Fe in only twofold molar excess over phenanthroli ne restores NifA activity, whereas adding other metals fails to do so, By using strains that lack NifL, we showed that NifA activity itself does not require Fe and is not directly affected by phenanthroline, He nce, Pe is required to relieve the inhibition of NifA activity by NifL in vivo, Despite the Fe requirement in vivo, we have found no evidenc e that NifL, contains Fe or an iron-sulfur (Fe-S) cluster, Determinati on of the molecular mass of an inhibitory form of NifL overproduced un der aerobic conditions indicated that it was not posttranslationally m odified, When NifL was synthesized in vitro, it inhibited transcriptio nal activation by NifA even when it was synthesized under anaerobic co nditions in the presence of a high Fe concentration or of superoxide d ismutase, which is known to protect some Fe-S clusters, Moreover, over production of superoxide dismutase in vivo did not relieve NifL inhibi tion under aerobic conditions, and attempts to relieve NifL inhibition in vitro by reconstituting Fe-S clusters with the NifS enzyme (Azotob acter vinelandii) were unsuccessful, Since we obtained no evidence tha t Fe acts directly on NifL or NifA, we postulate that an additional Fe -containing protein, not yet identified, may be required to relieve Ni fL inhibition under anaerobic, nitrogen-limiting conditions.