Regulation of nitrogen fixation and assimilation genes in the free-living versus symbiotic state

deBruijn, Frans J.; Hilgert, Uwe; Stigter, John; Schneider, Maria Victoria; Meyer, H. E.; Klosse, Ulrike; Pawlowski, Katharina

doi:10.1007/978-1-4684-6432-0_4

Cited by 18 publications

(4 citation statements)

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“…We have been studying the structure and regulation of early and late nodulin genes from the tropical leguminous shrub Sesbania rostrata (see de Bruijn, 1989;de Bruijn et a/., 199Oa). S. rostratais nodulated by its symbiotic partner Azorhizobium caulinodans (Dreyfus et a/., 1988) not only on the roots, but also on the stem (see de Bruijn, 1989).…”

Section: Introductionmentioning

confidence: 99%

The early nodulin gene SrEnod2 from Sesbania rostrata is inducible by cytokinin

1992

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Summary The structure and expression of the early nodulin gene Enod2 from the stem‐nodulated tropical legume Sesbania rostrata (SrEnod2) was examined. Genomic clones carrying the single SrEnod2locus were isolated and the DMA sequence of the gene was determined. The SrEnod2 gene was found to lack introns and to encode a protein consisting primarily of a 55‐fold repeat of short proline‐rich oligopeptides. A putative signal sequence, which may be responsible for targeting of the Enod2 protein to the cell wall, was found to precede this repeat. The temporal expression of the SrEnod2 gene was found to be different in S. rostrata stem versus root nodules induced by Azorhizobium caulinodans ORS571. SrEnod2 gene expression was shown to be induced specifically and rapidly by physiologically significant concentrations of exogenously supplied cytokinins. The SrEnod2gene was also found to be highly expressed in S. rostrata crown gall tumors induced by wild‐type Agrobacterium tumefaciens strains, but not in tumors induced by an A. tumefaciens strain carrying a mutation in the cytokinin biosynthesis gene 4. Implications of these observations with regard to cytokinin‐induced plant gene expression and a possible role for cytokinin as a symbiotic signal are discussed.

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Section: Introductionmentioning

confidence: 99%

The early nodulin gene SrEnod2 from Sesbania rostrata is inducible by cytokinin

1992

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“…In previous works, it has been suggested that glnA was required only for the establishment of functional bacteroids. This was based on the fact that GS activity was not detected in mature bacteroids, while Gln Ϫ strains displayed a Fix Ϫ phenotype (10,14). In the present work, whether GS is required for symbiotic nitrogen fixation in addition to P II cannot be elucidated.…”

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Characterization of Azorhizobium caulinodans glnB and glnA genes: involvement of the P(II) protein in symbiotic nitrogen fixation

et al. 1997

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The nucleotide sequence and transcriptional organization of Azorhizobium caulinodans ORS571 glnA, the structural gene for glutamine synthetase (GS), and glnB, the structural gene for the P II protein, have been determined. glnB and glnA are organized as a single operon transcribed from the same start site, under conditions of both nitrogen limitation and nitrogen excess. This start site may be used by two different promoters since the expression of a glnB-lacZ fusion was high in the presence of ammonia and enhanced under conditions of nitrogen limitation in the wild-type strain. The increase was not observed in rpoN or ntrC mutants. In addition, this fusion was overexpressed under both growth conditions, in the glnB mutant strain, suggesting that P II negatively regulates its own expression. A DNA motif, similar to a 54 -dependent promoter consensus, was found in the 5 nontranscribed region. Thus, the glnBA operon seems to be transcribed from a 54 -dependent promoter that operates under conditions of nitrogen limitation and from another uncharacterized promoter in the presence of ammonia. Both glnB and glnBA mutant strains derepress their nitrogenase in the free-living state, but only the glnBA mutant, auxotrophic for glutamine, does not utilize molecular nitrogen for growth. The level of GS adenylylation is not affected in the glnB mutant as compared to that in the wild type. Under symbiotic conditions, the glnB and glnBA mutant strains induced Fix ؊ nodules on Sesbania rostrata roots. P II is the first example in A. caulinodans of a protein required for symbiotic nitrogen fixation but dispensable in bacteria growing in the free-living state.Azorhizobium caulinodans ORS571, isolated from stem nodules of its host plant, the tropical legume Sesbania rostrata, fixes nitrogen both during symbiosis and in the free-living state (15). In the free-living state, this strain assimilates fixed ammonium for growth, via the glutamine synthetase (GS)/glutamate synthetase pathway (14). During symbiosis, ammonium produced by nitrogen fixation is exported from the bacteroid to the vegetal cell, where it is assimilated by the plant GS. As in enteric bacteria, only one GS (GSI) has been characterized in A. caulinodans (14), whereas two forms (GSI and GSII) have been identified in most rhizobia (9). In enteric bacteria and rhizobia, GS activity is modulated by reversible adenylylation in response to changes in the intracellular glutamine/2-ketoglutarate ratio, reflecting the level of cellular nitrogen (20). In Escherichia coli, under conditions of nitrogen limitation, the P II protein, encoded by glnB, is uridylylated by the glnD gene product. Under conditions of nitrogen excess, P II is deuridylylated and activates an adenylyltransferase (28). This enzyme transfers an AMP group to a tyrosine residue in each of the 12 subunits of the GS; the fully adenylylated form of the enzyme is less active (33). The structural gene for GS (glnA) is part of the glnALG operon, where glnL and glnG (also designated ntrB and ntrC) are the structura...

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“…DNA gyrase activity has been found to be important for the anaerobic transcription of the nifLA promoter. Our results suggest that anaerobic regulation of the nifLA operon is mediated through the control of the promoter region-binding trans-acting factor at the transcriptional level, while DNA supercoiling functions in providing a topological requirement for the activation of transcription.In the nitrogen-fixing (nif) enteric bacteria Klebsiella pneumoniae and Enterobacter cloacae, the nif genes are regulated by the activity of the ntrC, ntrA, nifA, and nifL genes at the transcriptional level (2,3,4,24). nifL and nifA constitute an operon which is regulated by the product of ntrC or autoregulated by the product of nifA, NifA (5, 9).…”

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“…In the nitrogen-fixing (nif) enteric bacteria Klebsiella pneumoniae and Enterobacter cloacae, the nif genes are regulated by the activity of the ntrC, ntrA, nifA, and nifL genes at the transcriptional level (2,3,4,24). nifL and nifA constitute an operon which is regulated by the product of ntrC or autoregulated by the product of nifA, NifA (5, 9).…”

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A Promoter Region Binding Protein and DNA Gyrase Regulate Anaerobic Transcription of nifLA in Enterobacter cloacae

Zhu

Shen

et al. 2000

J Bacteriol

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Our work provides evidence that a sequence characteristic of FNR binding sites, when interacted with by a trans-acting factor, activates anaerobic transcription of the nifLA operon in Enterobacter cloacae. DNA gyrase activity has been found to be important for the anaerobic transcription of the nifLA promoter. Our results suggest that anaerobic regulation of the nifLA operon is mediated through the control of the promoter region-binding trans-acting factor at the transcriptional level, while DNA supercoiling functions in providing a topological requirement for the activation of transcription.In the nitrogen-fixing (nif) enteric bacteria Klebsiella pneumoniae and Enterobacter cloacae, the nif genes are regulated by the activity of the ntrC, ntrA, nifA, and nifL genes at the transcriptional level (2,3,4,24). nifL and nifA constitute an operon which is regulated by the product of ntrC or autoregulated by the product of nifA, NifA (5, 9). NifA acts as a positive regulator in conjunction with NtrA to activate nif genes (19), while the product of nifL, NifL, acts as a repressor of nif genes under oxygen or in an excess of fixed nitrogen (13).Our previous investigations showed that the nifLA promoter is highly sensitive to oxygen and that NifA is inactivated by NifL under oxygen (5,15,23). Accordingly, we hypothesized that nif regulation by oxygen is mediated at two different levels. First, at the transcriptional level, the oxygen sensitivity of the nifLA promoter ensures that the nifLA promoter and, hence, all other nif promoters are repressed by oxygen; second, at the posttranslational level, NifL interacts with NifA in the presence of oxygen. As the result of a shortage of active NifA, the expression of other nif genes is blocked.Direct interaction of NifL and NifA has been demonstrated by the two-hybrid system test (16, 23). As to the question of aerial regulation of the nifLA promoter, most investigators have claimed that neither FNR nor the oxrC gene product is involved in the expression of nifLA in response to oxygen (14). Since the activity of the K. pneumoniae nifLA promoter can be prevented by inhibition of DNA gyrase activity, they thus held that aerobic-anaerobic regulation of the nifLA promoter is mainly mediated through the level of DNA supercoiling (6,8). Our present work demonstrated that an upstream sequence of the nifLA promoter characteristic of the FNR binding sequence, when bound by the trans-acting factor from the bacterial cells, activates anaerobic expression of the nifLA operon. We also proved that DNA gyrase is truly important for anaerobic transcription of the nifLA promoter. A mechanism for aerobic regulation of the nifLA promoter is proposed. MATERIALS AND METHODSStrains and plasmids. The bacterial strains and plasmids used in this work are listed in Table 1. DNA manipulations.Preparation of plasmids DNA, endonuclease digestion, ligation, and transformation were carried out essentially as described by Sambrook et al. (21).Construction of nifLA promoter mutants. Initially, the E. cloacae nif...

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Regulation of nitrogen fixation and assimilation genes in the free-living versus symbiotic state

Cited by 18 publications

References 73 publications

The early nodulin gene SrEnod2 from Sesbania rostrata is inducible by cytokinin

The early nodulin gene SrEnod2 from Sesbania rostrata is inducible by cytokinin

Characterization of Azorhizobium caulinodans glnB and glnA genes: involvement of the P(II) protein in symbiotic nitrogen fixation

A Promoter Region Binding Protein and DNA Gyrase Regulate Anaerobic Transcription of nifLA in Enterobacter cloacae

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