Rhodobacter capsulatus nifA mutants mediating nif gene expression in the presence of ammonium

Paschen, A

doi:10.1016/s0378-1097(01)00230-0

Cited by 12 publications

(25 citation statements)

References 24 publications

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“…In contrast to other diazotrophic organisms, R. capsulatus contains two NifA proteins, NifA1 and NifA2, which differ only in their 19 N-terminal amino acid residues (26,54). These NifA proteins can substitute for each other to activate transcription of all the other nif genes under nitrogen-limiting conditions (47).…”

Section: Resultsmentioning

confidence: 99%

Yeast Two-Hybrid Studies on Interaction of Proteins Involved in Regulation of Nitrogen Fixation in the Phototrophic BacteriumRhodobacter capsulatus

et al. 2003

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Rhodobacter capsulatus contains two PII-like proteins, GlnB and GlnK, which play central roles in controlling the synthesis and activity of nitrogenase in response to ammonium availability. Here we used the yeast two-hybrid system to probe interactions between these PII-like proteins and proteins known to be involved in regulating nitrogen fixation. Analysis of defined protein pairs demonstrated the following interactions: GlnBNtrB, GlnB-NifA1, GlnB-NifA2, GlnB-DraT, GlnK-NifA1, GlnK-NifA2, and GlnK-DraT. These results corroborate earlier genetic data and in addition show that PII-dependent ammonium regulation of nitrogen fixation in R. capsulatus does not require additional proteins, like NifL in Klebsiella pneumoniae. In addition, we found interactions for the protein pairs GlnB-GlnB, GlnB-GlnK, NifA1-NifA1, NifA2-NifA2, and NifA1-NifA2, suggesting that fine tuning of the nitrogen fixation process in R. capsulatus may involve the formation of GlnBGlnK heterotrimers as well as NifA1-NifA2 heterodimers. In order to identify new proteins that interact with GlnB and GlnK, we constructed an R. capsulatus genomic library for use in yeast two-hybrid studies. Screening of this library identified the ATP-dependent helicase PcrA as a new putative protein that interacts with GlnB and the Ras-like protein Era as a new protein that interacts with GlnK.

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Section: Resultsmentioning

confidence: 99%

Yeast Two-Hybrid Studies on Interaction of Proteins Involved in Regulation of Nitrogen Fixation in the Phototrophic BacteriumRhodobacter capsulatus

et al. 2003

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“…1a). For this purpose, cells were cultured under nitrogenasederepressing (2N) or -repressing (+N) conditions prior to protein extraction and Western analysis using a NifA1-specific antiserum raised against a synthetic oligopeptide corresponding to the N-terminal 17 amino acid residues (Paschen et al, 2001). In the wild-type strain, NifA1 accumulated only in N-limited cells, which is consistent with NtrC-mediated transcriptional control of the nifA1 gene (Fig.…”

Section: Ammonium-dependent Regulation Of Nifa Expressionmentioning

confidence: 99%

“…Previous studies demonstrated that constitutive (NtrCindependent) expression of nifA1 leads to a high-level accumulation of the NifA1 protein in both the absence and the presence of ammonium, but NifA1-mediated nifH transcription was still inhibited by ammonium (Paschen et al, 2001). To further analyse the roles of GlnB and GlnK in this post-translational ammonium control of NifA activity, we examined the same protein extracts which were prepared for the NifA1 detection (Fig.…”

Section: Post-translational Ammonium Control Of Nifa Activitymentioning

confidence: 99%

“…In addition to the NtrC-dependent activation, anfA transcription is repressed by MopA and MopB in the presence of molybdenum (Kutsche et al, 1996). At a second regulatory level, the activity of NifA1 and NifA2 is inhibited in response to ammonium (Hübner et al, 1993;Paschen et al, 2001). The characterization of R. capsulatus NifA1 mutants which are able to activate nif gene transcription in the presence of ammonium revealed that the N-terminal domain of NifA is involved in this post-translational control mechanism (Paschen et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

“…At a second regulatory level, the activity of NifA1 and NifA2 is inhibited in response to ammonium (Hübner et al, 1993;Paschen et al, 2001). The characterization of R. capsulatus NifA1 mutants which are able to activate nif gene transcription in the presence of ammonium revealed that the N-terminal domain of NifA is involved in this post-translational control mechanism (Paschen et al, 2001). Finally, at a third level of control, the activity of both nitrogenases is regulated in response to ammonium and darkness via reversible ADP-ribosylation of the dinitrogenase reductases NifH and AnfH mediated by DraT (dinitrogenase reductase ADP-ribosyltransferase) and DraG (dinitrogenase-reductase-activating glycohydrolase) and by a DraT/DraG-independent switch-off mechanism Pierrard et al, 1993;Yakunin & Hallenbeck, 1998).…”

Section: Introductionmentioning

confidence: 99%

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Role of GlnB and GlnK in ammonium control of both nitrogenase systems in the phototrophic bacterium Rhodobacter capsulatus

et al. 2003

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In most bacteria, nitrogen metabolism is tightly regulated and P II proteins play a pivotal role in the regulatory processes. Rhodobacter capsulatus possesses two genes (glnB and glnK ) encoding P II -like proteins. The glnB gene forms part of a glnB-glnA operon and the glnK gene is located immediately upstream of amtB, encoding a (methyl-) ammonium transporter. Expression of glnK is activated by NtrC under nitrogen-limiting conditions. The synthesis and activity of the molybdenum and iron nitrogenases of R. capsulatus are regulated by ammonium on at least three levels, including the transcriptional activation of nifA1, nifA2 and anfA by NtrC, the regulation of NifA and AnfA activity by two different NtrC-independent mechanisms, and the post-translational control of the activity of both nitrogenases by reversible ADP-ribosylation of NifH and AnfH as well as by ADP-ribosylation independent switch-off. Mutational analysis revealed that both P II -like proteins are involved in the ammonium regulation of the two nitrogenase systems. A mutation in glnB results in the constitutive expression of nifA and anfA. In addition, the post-translational ammonium inhibition of NifA activity is completely abolished in a glnB-glnK double mutant. However, AnfA activity was still suppressed by ammonium in the glnB-glnK double mutant. Furthermore, the P II -like proteins are involved in ammonium control of nitrogenase activity via ADP-ribosylation and the switch-off response. Remarkably, in the glnB-glnK double mutant, all three levels of the ammonium regulation of the molybdenum (but not of the alternative) nitrogenase are completely circumvented, resulting in the synthesis of active molybdenum nitrogenase even in the presence of high concentrations of ammonium.

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Derepressive effect of NH on hydrogen production by deleting the glnA1 gene in Rhodobacter sphaeroides

Liu

et al. 2010

Biotech & Bioengineering

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Purple non-sulfur (PNS) bacteria produce hydrogen by photofermentation of organic acids in wastewater. However, NH(4)(+) in wastewater may inhibit hydrogen synthesis by repressing the expression and activity of nitrogenase, the enzyme catalyzing hydrogen production in PNS bacteria. In this study, the Rhodobacter sphaeroides 6016 glnA gene encoding glutamine synthetase (GS) was knocked out by homologous recombination, and the effects on hydrogen production and nitrogenase activity were examined. Using 3 mM glutamine as the nitrogen source, hydrogen production (1,245-1,588 mL hydrogen/L culture) and nitrogenase activity were detected in the mutant in the presence of relatively high NH(4)(+) concentrations (15-40 mM), whereas neither was detected in the wild-type strain under the same conditions. Further analysis indicated that high NH(4)(+) concentrations greatly inhibited the expression of nifA and nitrogenase gene in the wild-type strain but not in the glnA1(-) mutant. These observations suggest that GS is essential to NH(4)(+) repression of nitrogenase and that deletion of glnA1 results in the complete derepression of nitrogenase by preventing NH(4)(+) assimilation in vivo, thus relieving the inhibition of nifA and nitrogenase gene expression. Knocking out glnA1 therefore provides an efficient approach to removing the inhibitory effects of ammonium ions in R. sphaeroides and possibly in other hydrogen-producing PNS bacteria.

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Rhodobacter capsulatus nifA mutants mediating nif gene expression in the presence of ammonium

Cited by 12 publications

References 24 publications

Yeast Two-Hybrid Studies on Interaction of Proteins Involved in Regulation of Nitrogen Fixation in the Phototrophic BacteriumRhodobacter capsulatus

Yeast Two-Hybrid Studies on Interaction of Proteins Involved in Regulation of Nitrogen Fixation in the Phototrophic BacteriumRhodobacter capsulatus

Role of GlnB and GlnK in ammonium control of both nitrogenase systems in the phototrophic bacterium Rhodobacter capsulatus

Derepressive effect of NH on hydrogen production by deleting the glnA1 gene in Rhodobacter sphaeroides

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