Role of conserved cysteine residues in Herbaspirillum seropedicae NifA activity

Oliveira, Marco Aurélio Schüler de; Baura, Valter A.; Aquino, Bruno Pinheiro; Huergo, Luciano F.; Kadowaki, Marco Antonio Seiki; Chubatsu, Leda S.; Souza, Emanuel M.; Dixon, Ray; Pedrosa, Fábio O.; Wassem, Roseli; Monteiro, Rose A.

doi:10.1016/j.resmic.2009.06.002

Cited by 13 publications

(13 citation statements)

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“…To examine this further, we assayed nifB::lacZ activity in cultures grown in the absence of fixed nitrogen under defined initial oxygen concentrations of oxygen in the gas phase (Figure 1 B). As anticipated, nifB expression was not detected under either 8% or 20.8% oxygen in both wild-type and the fnr triple deletion mutant, presumably because H. seropedicae NifA is inactivated at high oxygen concentrations [ 8 , 19 ]. However, the activity of the nifB::lacZ promoter fusion was markedly higher in the triple fnr deletion strain (MB231) compared with the parental strain, when cultures were incubated under an initial oxygen concentration of 4% or 6% in the gas phase (Figure 1 B).…”

Section: Resultsmentioning

confidence: 94%

“…As the H. seropedicae NifA protein is sensitive to oxygen, being inactivated and degraded upon exposure to O 2 [ 8 , 19 ], we hypothesized that NifA might be protected in its active form in fnr deletion strains if these strains exhibit a higher oxygen consumption rate. To further test this hypothesis we measured oxygen depletion during the growth of bacterial cultures in the same growth conditions as described for the assay of the nifA::lacZ fusions.…”

Section: Resultsmentioning

confidence: 99%

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Enhanced oxygen consumption in Herbaspirillum seropedicae fnr mutants leads to increased NifA mediated transcriptional activation

et al. 2015

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BackgroundOrthologous proteins of the Crp/Fnr family have been previously implicated in controlling expression and/or activity of the NifA transcriptional activator in some diazotrophs. This study aimed to address the role of three Fnr-like proteins from H. seropedicae SmR1 in controlling NifA activity and consequent NifA-mediated transcription activation.ResultsThe activity of NifA-dependent transcriptional fusions (nifA::lacZ and nifB::lacZ) was analysed in a series of H. seropedicae fnr deletion mutant backgrounds. We found that combined deletions in both the fnr1 and fnr3 genes lead to higher expression of both the nifA and nifB genes and also an increased level of nifH transcripts. Expression profiles of nifB under different oxygen concentrations, together with oxygen consumption measurements suggest that the triple fnr mutant has higher respiratory activity when compared to the wild type, which we believe to be responsible for greater stability of the oxygen sensitive NifA protein. This conclusion was further substantiated by measuring the levels of NifA protein and its activity in fnr deletion strains in comparison with the wild-type.ConclusionsFnr proteins are indirectly involved in controlling the activity of NifA in H. seropedicae, probably as a consequence of their influence on respiratory activity in relation to oxygen availability. Additionally we can suggest that there is some redundancy in the physiological function of the three Fnr paralogs in this organism, since altered respiration and effects on NifA activity are only observed in deletion strains lacking both fnr1 and fnr3.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0432-6) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Enhanced oxygen consumption in Herbaspirillum seropedicae fnr mutants leads to increased NifA mediated transcriptional activation

et al. 2015

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“…Models for the regulation of NifA activity of A. brasilense and H. seropedicae in response to the levels of ammonium and oxygen have been proposed (6,7,18,19). According to these models, the N-terminal domain folds over the catalytic central domain inhibiting transcription activation under high ammonium conditions.…”

Section: Resultsmentioning

confidence: 99%

“…Monteiro et al (7), using an N-truncated form of H. seropedicae NifA, also showed that an aerobically purified NifA retained DNA-binding activity. Moreover, Oliveira et al (18) produced mutants for each of the four cysteine residues of the putative Fe-S cluster binding motif in H. seropedicae NifA, and observed that the mutant proteins were still able to bind DNA although the ability to activate transcription was completely abolished. Oxidation of the Fe-S cluster may trigger conformational changes affecting nucleotide binding and/or hydrolysis necessary for transcriptional activation, as proposed (18,20).…”

Section: Resultsmentioning

confidence: 99%

“…Moreover, Oliveira et al (18) produced mutants for each of the four cysteine residues of the putative Fe-S cluster binding motif in H. seropedicae NifA, and observed that the mutant proteins were still able to bind DNA although the ability to activate transcription was completely abolished. Oxidation of the Fe-S cluster may trigger conformational changes affecting nucleotide binding and/or hydrolysis necessary for transcriptional activation, as proposed (18,20). Therefore, under aerobic conditions, the oxidized Fe-S cluster would lead to a transcriptionally inactive form of NifA possibly compromising the catalytic activity of the central domain.…”

Section: Resultsmentioning

confidence: 99%

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Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense

Nishikawa

Araújo

Kadowaki

et al. 2012

Braz J Med Biol Res

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Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ54 factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH4Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.

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Regulation of Nitrogen Fixation in Photosynthetic Purple Nonsulfur Bacteria

Masepohl

2017

Modern Topics in the Phototrophic Prokaryotes

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Role of conserved cysteine residues in Herbaspirillum seropedicae NifA activity

Cited by 13 publications

References 28 publications

Enhanced oxygen consumption in Herbaspirillum seropedicae fnr mutants leads to increased NifA mediated transcriptional activation

Enhanced oxygen consumption in Herbaspirillum seropedicae fnr mutants leads to increased NifA mediated transcriptional activation

Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense

Regulation of Nitrogen Fixation in Photosynthetic Purple Nonsulfur Bacteria

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