Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae.

In Klebsiella pneumoniae, products of the nitrogen fixation nifLA operon regulate transcription of the other nif operons. NifA activates transcription by sigma54-holoenzyme. In vivo, NifL antagonizes the action of NifA under aerobic conditions or in the presence of combined nitrogen. In contrast to a previous report, we show that depletion of iron (Fe) from the growth medium with the chelating agent o-phenanthroline (20 microM) mimics aerobiosis or combined nitrogen in giving rise to inhibition of NifA activity even under anaerobic, nitrogen-limiting conditions. Adding back Fe in only twofold molar excess over phenanthroline 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. Hence, Fe is required to relieve the inhibition of NifA activity by NifL in vivo. Despite the Fe requirement in vivo, we have found no evidence that NifL contains Fe or an iron-sulfur (Fe-S) cluster. Determination of the molecular mass of an inhibitory form of NifL overproduced under aerobic conditions indicated that it was not posttranslationally modified. When NifL was synthesized in vitro, it inhibited transcriptional activation by NifA even when it was synthesized under anaerobic conditions in the presence of a high Fe concentration or of superoxide dismutase, which is known to protect some Fe-S clusters. Moreover, overproduction of superoxide dismutase in vivo did not relieve NifL, inhibition under aerobic conditions, and attempts to relieve NifL inhibition in vitro by reconstituting Fe-S clusters with the NifS enzyme (Azotobacter vinelandii) were unsuccessful. Since we obtained no evidence that Fe acts directly on NifL or NifA, we postulate that an additional Fe-containing protein, not yet identified, may be required to relieve NifL inhibition under anaerobic, nitrogen-limiting conditions.