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...

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Main Authors: Schmitz, R A, He, L, Kustu, S
Formato: Artigo
Idioma:en
Publicado em: 1996
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Research Article
Acesso em linha:https://ncbi.nlm.nih.gov/pmc/articles/PMC178239/
https://ncbi.nlm.nih.gov/pubmed/8755900
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spelling pubmed-1782392003-10-28 Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae. Schmitz, R A He, L Kustu, S J Bacteriol Research Article 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. 1996-08 /pmc/articles/PMC178239/ /pubmed/8755900 Text en
institution US National Library of Medicine
collection PubMed Central
language en
format Article
topic Research Article
spellingShingle Research Article
Schmitz, R A
He, L
Kustu, S
Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae.
description 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.
author Schmitz, R A
He, L
Kustu, S
author_facet Schmitz, R A
He, L
Kustu, S
author_sort Schmitz, R A
title Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae.
title_short Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae.
title_full Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae.
title_fullStr Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae.
title_full_unstemmed Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae.
title_sort iron is required to relieve inhibitory effects on nifl on transcriptional activation by nifa in klebsiella pneumoniae.
publishDate 1996
url https://ncbi.nlm.nih.gov/pmc/articles/PMC178239/
https://ncbi.nlm.nih.gov/pubmed/8755900
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