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In this paper we have studied the localisation of expression of the two functional cytosolic glutamine synthetase (GS) genes, MtGSa and MtGSb, in root nodules of the model legume Medicago truncatula. We have used a combination of different techniques, including immunocytochemistry, in situ hybridisation and promoter beta-glucuronidase (GUS) fusions in transgenic plants, to provide the means of correlating gene expression with protein localisation. These studies revealed that transcriptional regulation (mRNA synthesis) plays an important part in controlling GS protein levels in nodules of M. truncatula. The major locations of cytosolic GS mRNA and protein are the central tissue, the parenchyma and the pericycle of the vascular bundles. These findings indicate that in nodules, GS might be involved in other physiological processes in addition to the primary assimilation of ammonia released by the bacterial nitrogenase. The two genes show different but overlapping patterns of expression with MtGSa being the major gene expressed in the infected cells of the nodule. Promoter fragments of 2.6 kb and 3.1 kb of MtGSa and MtGSb, respectively, have been sequenced and primer extension revealed that the MtGSb promoter is expressed in nodules from an additional start site that is not used in roots. Generally these fragments in the homologous transgenic system were sufficient to drive GUS expression in almost all the tissues and cell types where GS proteins and transcripts are located except that the MtGSa promoter fragment did not express GUS highly in the nodule infected cells. These results indicate that the cis-acting regulatory elements responsible for infected-cell expression are missing from the MtGSa promoter fragment.
Plant Mol Biol 2000 Mar
PMID:Cellular expression and regulation of the Medicago truncatula cytosolic glutamine synthetase genes in root nodules. 1080 46

The pairs of nitrogen fixation genes nifDK and nifEN encode for the alpha and beta subunits of nitrogenase and for the two subunits of the NifNE protein complex, involved in the biosynthesis of the FeMo cofactor, respectively. Comparative analysis of the amino acid sequences of the four NifD, NifK, NifE, and NifN in several archaeal and bacterial diazotrophs showed extensive sequence similarity between them, suggesting that their encoding genes constitute a novel paralogous gene family. We propose a two-step model to reconstruct the possible evolutionary history of the four genes. Accordingly, an ancestor gene gave rise, by an in-tandem paralogous duplication event followed by divergence, to an ancestral bicistronic operon; the latter, in turn, underwent a paralogous operon duplication event followed by evolutionary divergence leading to the ancestors of the present-day nifDK and nifEN operons. Both these paralogous duplication events very likely predated the appearance of the last universal common ancestor. The possible role of the ancestral gene and operon in nitrogen fixation is also discussed.
J Mol Evol 2000 Jul
PMID:Molecular evolution of nitrogen fixation: the evolutionary history of the nifD, nifK, nifE, and nifN genes. 1090 67

The molybdopterin cofactor (MoCF) is required for the activity of a variety of oxidoreductases. The xanthine oxidase class of molybdoenzymes requires the MoCF to have a terminal, cyanolysable sulphur ligand. In the sulphite oxidase/nitrate reductase class, an oxygen is present in the same position. Mutations in both the ma-l gene of Drosophila melanogaster and the hxB gene of Aspergillus nidulans result in loss of activities of all molybdoenzymes that necessitate a cyanolysable sulphur in the active centre. The ma-l and hxB genes encode highly similar proteins containing domains common to pyridoxal phosphate-dependent cysteine transulphurases, including the cofactor binding site and a conserved cysteine, which is the putative sulphur donor. Key similarities were found with NifS, the enzyme involved in the generation of the iron-sulphur centres in nitrogenase. These similarities suggest an analogous mechanism for the generation of the terminal molybdenum-bound sulphur ligand. We have identified putative homologues of these genes in a variety of organisms, including humans. The human homologue is located in chromosome 18.q12.
Mol Microbiol 2000 Oct
PMID:Comparison of the sequences of the Aspergillus nidulans hxB and Drosophila melanogaster ma-l genes with nifS from Azotobacter vinelandii suggests a mechanism for the insertion of the terminal sulphur atom in the molybdopterin cofactor. 1102 94

In nitrogen-poor soils, rhizobia elicit nodule formation on legume roots, within which they differentiate into bacteroids that fix atmospheric nitrogen. Protection against reactive oxygen species (ROS) was anticipated to play an important role in Rhizobium-legume symbiosis because nitrogenase is extremely oxygen sensitive. We deleted the sodA gene encoding the sole cytoplasmic superoxide dismutase (SOD) of Sinorhizobium meliloti. The resulting mutant, deficient in superoxide dismutase, grew almost normally and was only moderately sensitive to oxidative stress when free living. In contrast, its symbiotic properties in alfalfa were drastically affected. Nitrogen-fixing ability was severely impaired. More strikingly, most SOD-deficient bacteria did not reach the differentiation stage of nitrogen-fixing bacteroids. The SOD-deficient mutant nodulated poorly and displayed abnormal infection. After release into plant cells, a large number of bacteria failed to differentiate into bacteroids and rapidly underwent senescence. Thus, bacterial SOD plays a key protective role in the symbiotic process.
Mol Microbiol 2000 Nov
PMID:Critical protective role of bacterial superoxide dismutase in rhizobium-legume symbiosis. 1111 10

To gain insight into the molecular processes occurring in root nodule metabolism after stress, we used a mRNA differential display (DDRT-PCR) approach to identify cDNAs corresponding to genes whose expression is enhanced in nodules of decapitated Medicago truncatula plants. Two full-length cDNAs of plant origin were isolated (MTD1 and MTD2). Sequence analysis revealed that MTD1 is identical to an EST clone (accession number AW559774) expressed in roots of M. truncatula upon infection with Phytophthora medicaginis, while MTD2 is highly homologous to an Arabidopsis thaliana gene (accession number AL133292) coding for a RNA binding-like protein. The two mRNAs started to accumulate in root nodules at 4 h after plant decapitation and reached even higher transcript levels at 24 h from the imposition of the treatment. MTD1 and MTD2 mRNAs were mainly induced in nodules, with very little induction in roots. The abundance of the two transcripts did not change in response to other perturbations known to decrease nitrogenase activity, such as nitrate and Ar/O2 treatments. Our results suggest that MTD1 and MTD2 represent transcripts that accumulate locally in nodules and may be involved in changes in nodule metabolism in response to decapitation.
Plant Mol Biol 2000 Nov
PMID:Increased abundance of MTD1 and MTD2 mRNAs in nodules of decapitated Medicago truncatula. 1119 23

Biological nitrogen fixation involves the reduction of atmospheric N2 to ammonia by the bacterial enzyme nitrogenase. In legume-rhizobium symbioses, the nitrogenase-producing bacteria (bacteroids) are contained in the infected cells of root nodules within which they are enclosed by a plant membrane to form a structure known as the symbiosome. The plant provides reduced carbon to the bacteroids in exchange for fixed nitrogen, which is exported to the rest of the plant. This exchange is controlled by plant-synthesised transport proteins on the symbiosome membranes. This review summarises our current understanding of these transport processes, focusing on ammonia and amino acid transport.
Cell Mol Life Sci 2001 Jan
PMID:Ammonia and amino acid transport across symbiotic membranes in nitrogen-fixing legume nodules. 1122 17

Acidaminococcus fermentans degrades glutamate via the hydroxyglutarate pathway, which involves the syn-elimination of water from (R)-2-hydroxyglutaryl-CoA in a key reaction of the pathway. This anaerobic process is catalyzed by 2-hydroxyglutaryl-CoA dehydratase, an enzyme with two components (A and D) that reversibly associate during reaction cycles. Component A (CompA), a homodimeric protein of 2x27 kDa, contains a single, bridging [4Fe-4S] cluster and uses the hydrolysis of ATP to deliver an electron to the dehydratase component (CompD), where the electron is used catalytically. The structure of the extremely oxygen-sensitive CompA protein was solved by X-ray crystallography to 3 A resolution. The protein was found to be a member of the actin fold family, revealing a similar architecture and nucleotide-binding site. The key differences between CompA and other members of the actin fold family are: (i) the presence of a cluster binding segment, the "cluster helix"; (ii) the [4Fe-4S] cluster; and (iii) the location of the homodimer interface, which involves the bridging cluster. Possible reaction mechanisms are discussed in light of the close structural similarity to members of the actin-fold family and the functional similarity to the nitrogenase Fe- protein.
J Mol Biol 2001 Mar 16
PMID:Crystal structure of the Acidaminococcus fermentans 2-hydroxyglutaryl-CoA dehydratase component A. 1124 21

Ferredoxins are small iron sulfur proteins necessary for electron donation. FdxH1 and FdxH2 are associated with two different nif gene clusters where they transfer electrons for the reduction of nitrogenase complex. FdxH1 was observed to be stable towards oxygen, whereas, FdxH2 was relatively unstable. We had identified the amino acid involved in oxygen sensitivity of ferredoxin protein using protein modeling. The exchange of valine to leucine at position 77 was critical for ferredoxin proteins in relation to its oxygen sensitivity. This exchange leads to a longer side chain, which inhibits the accessibility of oxygen to the iron sulfur cluster. Site directed mutagenesis and in vitro experiments confirms that valine indeed is involved in the oxygen sensitivity. The exchange of leucine to valine in FdxH1 makes it oxygen unstable. Thus, from the above results we can conclude that the position of leucine at position 77 is critical for oxygen sensitivity of ferredoxin and protein modeling can be used to identify specific amino acids in other oxygen-sensitive proteins.
Mol Cell Biochem 2001 Jan
PMID:Valine 77 of heterocystous ferredoxin FdxH2 in Anabaena variabilis strain ATCC 29413 is critical for its oxygen sensitivity. 1126 58

Nitrogenase is the complex metalloenzyme responsible for biological dinitrogen reduction. This reaction represents the single largest contributor to the reductive portion of the global nitrogen cycle. Recent developments in understanding the mechanism of the Mo-based nitrogenase are reviewed. Topics include how nucleotide binding and hydrolysis are coupled to electron transfer and substrate reduction, how electrons are accumulated and transferred within the MoFe-protein, and how substrates bind and are reduced at the active site metal cluster.
Annu Rev Plant Physiol Plant Mol Biol 2001 Jun
PMID:MECHANISTIC FEATURES OF THE MO-CONTAINING NITROGENASE. 1133 99

In specific plant organs, namely the root nodules of alfalfa, fixed nitrogen (ammonia) produced by the symbiotic partner Sinorhizobium meliloti supports the growth of the host plant in nitrogen-depleted environment. Here, we report that a derivative of S. meliloti carrying a mutation in the chromosomal ntrR gene induced nodules with enhanced nitrogen fixation capacity, resulting in an increased dry weight and nitrogen content of alfalfa. The efficient nitrogen fixation is a result of the higher expression level of the nifH gene, encoding one of the subunits of the nitrogenase enzyme, and nifA, the transcriptional regulator of the nif operon. The ntrR gene, controlled negatively by its own product and positively by the symbiotic regulator syrM, is expressed in the same zone of nodules as the nif genes. As a result of the nitrogen-tolerant phenotype of the strain, the beneficial effect of the mutation on efficiency is not abolished in the presence of the exogenous nitrogen source. The ntrR mutant is highly competitive in nodule occupancy compared with the wild-type strain. Sequence analysis of the mutant region revealed a new cluster of genes, termed the "ntrPR operon," which is highly homologous to a group of vap-related genes of various pathogenic bacteria that are presumably implicated in bacterium-host interactions. On the basis of its favorable properties, the strain is a good candidate for future agricultural utilization.
Mol Plant Microbe Interact 2001 Jul
PMID:Mutation in the ntrR gene, a member of the vap gene family, increases the symbiotic efficiency of Sinorhizobium meliloti. 1143 62


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