The nifA gene product from Rhizobium leguminosarum biovar trifolii lacks the N‐terminal domain found in other NifA proteins

Iismaa, Siiri E.; Watson, John M.

doi:10.1111/j.1365-2958.1989.tb00244.x

Cited by 31 publications

(22 citation statements)

References 66 publications

(17 reference statements)

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“…3). Like other N 2 -fixing bacteria, such as A. brasilense, R. capsulatus, Rhizobium meliloti, Rhizobium leguminosarum, and Bradyrhizobium japonicum (14,21,40,44,50,59), NifA from R. rubrum also has an interdomain linker between the central domain and the C terminus. Four cysteines were located in this linker, which is thought to be involved in O 2 sensing (14) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Mutagenesis and Functional Characterization of the glnB , glnA , and nifA Genes from the Photosynthetic Bacterium Rhodospirillum rubrum

et al. 2000

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Nitrogen fixation is tightly regulated in Rhodospirillum rubrum at two different levels: transcriptional regulation of nif expression and posttranslational regulation of dinitrogenase reductase by reversible ADPribosylation catalyzed by the DRAT-DRAG (dinitrogenase reductase ADP-ribosyltransferase-dinitrogenase reductase-activating glycohydrolase) system. We report here the characterization of glnB, glnA, and nifA mutants and studies of their relationship to the regulation of nitrogen fixation. Two mutants which affect glnB (structural gene for P II ) were constructed. While P II -Y51F showed a lower nitrogenase activity than that of wild type, a P II deletion mutant showed very little nif expression. This effect of P II on nif expression is apparently the result of a requirement of P II for NifA activation, whose activity is regulated by NH 4 ؉ in R. rubrum. The modification of glutamine synthetase (GS) in these glnB mutants appears to be similar to that seen in wild type, suggesting that a paralog of P II might exist in R. rubrum and regulate the modification of GS. P II also appears to be involved in the regulation of DRAT activity, since an altered response to NH 4 ؉ was found in a mutant expressing P II -Y51F. The adenylylation of GS plays no significant role in nif expression or the ADP-ribosylation of dinitrogenase reductase, since a mutant expressing GS-Y398F showed normal nitrogenase activity and normal modification of dinitrogenase reductase in response to NH 4 ؉ and darkness treatments.Biological nitrogen fixation is catalyzed by the nitrogenase complex, which consists of two proteins: dinitrogenase (also referred to as MoFe protein) and dinitrogenase reductase (also referred to as Fe protein) (7). Dinitrogenase is an ␣ 2 ␤ 2 tetramer of the nifDK gene products and contains the active site (FeMo-co) for reduction of N 2 , C 2 H 2 , and other substrates. Dinitrogenase reductase is an ␣ 2 dimer of the nifH gene product and is the unique electron donor to dinitrogenase. This biological process is very energy demanding and is thus tightly controlled.In all studied nitrogen-fixing bacteria, nitrogen fixation is regulated at the transcriptional level. Transcriptional regulation has been best studied in Klebsiella pneumoniae, a freeliving nitrogen-fixing bacterium, and it involves the general nitrogen regulation (ntr) system (47). This ntr regulatory system has also been intensively studied in Escherichia coli (52), and it has been successfully reconstituted in vitro (25-28). This regulatory system involves the products of at least five genes: ntrA, ntrB, ntrC, glnB, and glnD. glnD encodes a bifunctional, uridylyltransferase-uridylyl-removing enzyme (UTase-UR) that is believed to be the sensor of the intracellular concentration of glutamine in the cell. UTase-UR reversibly controls the activity of the P II protein (the gene product of glnB) by uridylylation or deuridylylation (46, 52). P II was recently found to be responsible for the sensing of ␣-ketoglutarate (␣-KG) in E. coli (32), and its roles are descr...

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Section: Resultsmentioning

confidence: 99%

Mutagenesis and Functional Characterization of the glnB , glnA , and nifA Genes from the Photosynthetic Bacterium Rhodospirillum rubrum

et al. 2000

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“…Thus, nifA R. meliloti, B. japonicum, and R. leguminosarum mutants show no N2-fixing activity (26,58,59). The nifA-independent activation of nif genes in freeliving R. meliloti has already been described (57).…”

Section: Resultsmentioning

confidence: 99%

Symbiotic nitrogen fixation by a nifA deletion mutant of Rhizobium meliloti: the role of an unusual ntrC allele

et al. 1993

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In the N2-fixing alfalfa symbiont Rhizobium meliloti, the three r,4 (NTRA)-dependent positively acting regulatory proteins NIFA, NTRC, and DCTD are required for activation of promoters involved in N2 fixation (pnifHDKE and pfixABCX), nitrogen assimilation (pglnII), and C4-dicarboxylate transport (pdct4), respectively. Here, we describe an allele of ntrC which results in the constitutive activation of the above NTRC-, NIFA-, and DCTD-regulated promoters. The expression and activation of wild-type NTRC occur in response to nitrogen availability, whereas in cells carrying the ntrC283 allele, the NTRC283 protein appears constitutively active and is constitutively expressed. The ntrC283 allele was shown to carry a single mutation resulting in the replacement of an Asp by a Tyr residue in the helix-turn-helix motif of ntrC283. Introduction of the ntrC283 allele into a nifA deletion mutant restores the N2-fixation ability to 70 to 80%o of the wild-type level. Thus, the nifA gene is dispensable for symbiotic N2 fixation.Nitrogen-fixing root nodules develop through an intimate interaction between bacteria (rhizobia) and plant cells. In one of the early steps in this interaction, flavonoids produced by the plant induce the expression of a set of nodulation (nod) genes in the bacteria (31,40,43). In Rhizobium meliloti and Rhizobium leguminosarum, which induce root nodules on alfalfa and pea plants, respectively, the nod gene products function in the synthesis of a lipooligosaccharide signal molecule which triggers root nodule formation (62). The bacteria enter the root via tube-like structures, or infection threads, and are released into the plant cortical cells via an endocytosis-like process in which they are surrounded by the host plasma membrane. Both the bacteria and plant cells then undergo many biochemical changes, one of which is the expression of the plant globin genes whose products are involved in oxygen transport in the nodule. In the bacteria, the nitrogenase (nif) and other genes required for N2 fixation are induced. Expression of many nif genes and associated genes in nodules is regulated positively by the nifA gene product. Thus, nifA null mutants show no symbiotic N2-fixation activity. Expression of nifA is, in turn, regulated in response to oxygen concentration by a two-component environmental sensor-regulator pair, fixL and fixi, and by fixK (4,11,12). Recent data have shown that, within a nodule, the expression of the nifgenes in the bacteria and the globin genes in the plant cells occurs synchronously within a few layers and thus shows a high level of control (10, 69).The N2-fixing bacteria within nodules (called bacteroids) reduce N2 gas to ammonia which is then supplied to the plant. Because the bacteroids are within the plant cells, they are totally dependent on the plant for a source of energyreduced carbon, which is necessary for the high-energyrequiring N2-fixation reaction. The supply of a carbon source thus potentially represents a key point in control of N2 fixation and bacterial proliferation...

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“…The rationale for choosing this gene was as follows. (i) It codes for a transcriptional regulator protein, which probably means that it is transcribed at a low level comparable to that of the nod genes, (ii) the size of the transcript is of the same order as those of the nod genes, and (iii) the gene is probably transcribed only in bacteroids (25,39).…”

Section: Methodsmentioning

confidence: 99%

Suppression of nodulation gene expression in bacteroids of Rhizobium leguminosarum biovar viciae

et al. 1991

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The expression of nod genes of Rhizobium leguminosarum bv. viciae in nodules of Pisum sativum was investigated at both the translational and transcriptional levels. By using immunoblots, it was found that the levels of NodA, NodI, NodE, and NodO proteins were reduced at least 14-fold in bacteroids compared with cultured cells, whereas NodD protein was reduced only 3-fold. Northern (RNA) blot hybridization, RNase protection assays, and in situ RNA hybridization together showed that, except for the nodD transcript, none of the other nod gene transcripts were present in bacteroids. The amount of nodD transcript in bacteroids was reduced only two-to threefold compared with that in cultured cells. Identical results were found with a Rhizobium strain harboring multicopies of nodD and with a strain containing a NodD protein (NodD604) which is activated independently of flavonoids. Furthermore, it was found that mature pea nodules contain inhibitors of induced nod gene transcription but that NodD604 was insensitive to these compounds. In situ RNA hybridization on sections from P. sativum and Vicia hirsuta nodules showed that transcription of inducible nod genes is switched off before the bacteria differentiate into bacteroids. This is unlikely to be due to limiting amounts of NodD, the absence of inducing compounds, or the presence of anti-inducers. The observed switch off of transcription during the development of symbiosis is a general phenomenon and is apparently caused by a yet unknown negative regulation mechanism.Bacteria of the genus Rhizobium are able to establish a symbiosis with leguminous plants, resulting in formation of root nodules in which the bacteria, in an altered form designated as bacteroids, reduce atmospheric nitrogen to ammonia. Successful nodulation is a host-specific process in the sense that Pisum and Vicia species are host plants for Rhizobium leguminosarum biovar (bv.) viciae, alfalfa is a host for R. meliloti, and Trifolium sp. is a host for R. leguminosarum bv. trifolii.Bacterial nod (for nodulation) genes localized on a Sym (for symbiosis) plasmid code for proteins involved in early steps in nodulation. The nodD gene is the only constitutively transcribed nod gene in free-living cells. In R. leguminosarum bv. viciae and R. leguminosarum bv. trifolii, nodD is present as a single copy whereas in R. meliloti four allelic forms, designated nodDi, nodD2, nodD3, and syrM, have been identified. The NodD protein binds specifically to nod boxes (18,19,22,28), conserved DNA sequences in the upstream untranslated region of other nod genes (11,40,46,49), and induces transcription of the other nod genes, provided that NodD protein is activated by an inducer of plant origin. These inducers have been identified as flavones and flavanones (17,34,37,65), while isoflavones and coumarins act as anti-inducers for these species (13,17 early steps of nodulation, as reflected by the Nod-phenotype of nodABC mutants and the strongly reduced nodulation of nodFEL mutants. The products of these genes function in root...

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The nifA gene product from Rhizobium leguminosarum biovar trifolii lacks the N‐terminal domain found in other NifA proteins

Cited by 31 publications

References 66 publications

Mutagenesis and Functional Characterization of the glnB , glnA , and nifA Genes from the Photosynthetic Bacterium Rhodospirillum rubrum

Mutagenesis and Functional Characterization of the glnB , glnA , and nifA Genes from the Photosynthetic Bacterium Rhodospirillum rubrum

Symbiotic nitrogen fixation by a nifA deletion mutant of Rhizobium meliloti: the role of an unusual ntrC allele

Suppression of nodulation gene expression in bacteroids of Rhizobium leguminosarum biovar viciae

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