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PubMedDate not supplied

[Study of nucleotide sequences of nifH genes in methanotrophic bacteria].

Using a previously developed primer system, nifH gene fragments 450 nucleotides long were amplified, cloned, and sequenced for representatives of nitrogen-fixing methanotrophic bacteria of the genera Methylococcus, Methylocystis and Methylosinus. Fragments of nifH genes were also detected and sequenced in representatives of the genera Methylomonas and Methylobacter, which were not considered diazotrophs until recently. Phylogenetic analysis revealed remoteness of nifH genes sequences of methanotroph types I and II. At the same time, close relationship was found between nifH of type I methanotrophs and representatives of gamma-proteobacteria and between nifH genes of type II methanotrophs and representatives of alpha-proteobacteria. The results obtained in this study are in good accordance with the data of phylogenetic analysis based on 16S rRNA sequence comparison with the only exception of Methylococcus capsulatus strains, whose nifH genes proved to be closely related to nifH genes of Methylocystis and Methylosinus representatives. Our findings extend the database of primary sequences of nifH genes and allow the contribution of methanotrophs to the process of nitrogen fixation to be estimated.

Base Sequence↗

PubMedDate not supplied

[Production of soy bean inoculants. Behavior of supports based on peat from Tierra del Fuego sterilized by vapor and ethylene oxide].

The survival of Rhizobium japonicum was studied in neutralized and sterilized peats from Ushuaia and Rio Grande. The carriers were sterilized by ethylene oxide and by autoclaving. Similar counts for Rhizobium (5 x 10(8) cel/g) were obtained in peat-cultures sterilized by both methods, after eight months. A good nodulation and nitrogen fixation capacity was observed with inoculated soybean plants. Using the strain Rhizobium japonicum E-45, no appreciable difference in symbiotic effectiveness was found between the inoculants prepared with the two peats.

Argentina↗

PubMed2007

Inactivation of the nodH gene in Sinorhizobium sp. BR816 enhances symbiosis with Phaseolus vulgaris L.

Sulfate modification on Rhizobium Nod factor signaling molecules is not a prerequisite for successful symbiosis with the common bean (Phaseolus vulgaris L.). However, many bean-nodulating rhizobia, including the broad host strain Sinorhizobium sp. BR816, produce sulfated Nod factors. Here, we show that the nodH gene, encoding a sulfotransferase, is responsible for the transfer of sulfate to the Nod factor backbone in Sinorhizobium sp. BR816, as was shown for other rhizobia. Interestingly, inactivation of nodH enables inoculated bean plants to fix significantly more nitrogen under different experimental setups. Our studies show that nodH in the wild-type strain is still expressed during the later stages of symbiosis. This is the first report on enhanced nitrogen fixation by blocking Nod factor sulfation.

Bacterial Proteins↗

PubMed2005

Sinorhizobium meliloti dctA mutants with partial ability to transport dicarboxylic acids.

Sinorhizobium meliloti dctA encodes a transport protein needed for a successful nitrogen-fixing symbiosis between the bacteria and alfalfa. Using the toxicity of the DctA substrate fluoroorotic acid as a selective agent in an iterated selection procedure, four independent S. meliloti dctA mutants were isolated that retained some ability to transport dicarboxylates. Two mutations were located in a region called motif B located in a predicted transmembrane helix of the protein that has been shown in other members of the glutamate transporter family to be involved in cation binding. A G114D mutation was located in the third transmembrane helix, which had not previously been directly implicated in transport. Multiple sequence alignment of more than 60 members of the glutamate transporter family revealed a glycine at this position in nearly all members of the family. The fourth mutant was able to transport succinate at almost wild-type levels but was impaired in malate and fumarate transport. It contains two mutations: one in a periplasmic domain and the other predicted to be in the cytoplasm. Separation of the mutations showed that each contributed to the altered substrate preference. dctA deletion mutants that contain the mutant dctA alleles on a plasmid can proceed further in symbiotic development than null mutants of dctA, but none of the plasmids could support symbiotic nitrogen fixation, although they can transport dicarboxylates, some at relatively high levels.

Amino Acid Sequence↗

PubMedDate not supplied

[Macromolecular subunits on the surfaces of the cell wall of Thiocapsa roseopersicina].

A layer of fungiform macromolecular subunits was found on the surface of the cell wall of Thiocapsa roseopersicina, a purple sulphur bacterium, strain BBS. The cap of a particle has a diameter of 40 to 60 A; the stalk is 80 to 100 A long and 20 to 30 A thick. Under the conditions of nitrogen fixation and a low content of vitamin B12 (0.1 mcg/litre) in the cultural broth, a second layer of similar particles is formed over the first layer.

Cell Membrane↗

PubMed1991

Synchronous expression of leghaemoglobin genes in Medicago truncatula during nitrogen-fixing root nodule development and response to exogenously supplied nitrate.

Two leghaemoglobin genes from the diploid, autogamous Medicago truncatula (Mtlb1 and Mtlb2) have been cloned and their nucleotide sequences determined. The deduced amino acid sequences encoded by these two genes differ significantly (18%), confirming that they belong to different sub-groups of Medicago leghaemoglobin genes. RNAse protection experiments have been used to show that both genes are transcriptionally active, and are expressed specifically in the nitrogen-fixing root nodule of M. truncatula. Whilst Mtlb1 mRNA is present at approximatively 3-fold higher steady-state levels than Mtlb2 mRNA, the transcription of both genes is triggered concomitantly during nodule development (5 days after inoculation with Rhizobium meliloti), and the ratio of the steady-state levels of the two mRNA species remains constant throughout nodule maturation. When the growth medium of nodulated M. truncatula is supplemented with 5 mM KNO3 over a period of 2-3 days there is a progressive drop in specific nitrogen fixation activity to only 20-25% of the original level. This is accompanied with a parallel and synchronous reduction in the quantities of mRNA corresponding to both Mtlb1 and Mtlb2. By contrast, the expression of the nodule parenchyma-specific gene ENOD2 is not significantly modified following nitrate treatment, clearly demonstrating differences in tissue-specific gene regulation in response to combined nitrogen.

Amino Acid Sequence↗

PubMed2001

Ammonia and amino acid transport across symbiotic membranes in nitrogen-fixing legume nodules.

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.

Amino Acids↗

PubMedDate not supplied

Mineral nutrition of plants: a short history of plant physiology.

The development of the knowledge on the mineral nutrition of plants begins between the 17th and 18th centuries when some European naturalists gave the first experimental evidences of what had been empirically known for about two millennia. The works of Hales and Ingenhousz were of absolute importance in relation to the transport of water and solutes, and assimilation of "fixed air" (carbon dioxide), respectively. The early chemistry introduced by Lavoisier benefited the first physiologists Senebier and De Saussure to reject the "theory of humus", which imposed the soil as the unique source of carbon. During the first half of the 19th century, Sprengel and Liebig investigated on the problems related to some indispensable mineral salts, while Boussingault and Ville attempted to prove the nitrogen fixation from air without giving any convincing evidence. Liebig was the pioneer of the agricultural chemistry: he epitomised the knowledge of that period by imposing the so-called "law of the minima", already acknowledged by Sprengel, and patronised the use of mineral fertilisers in Europe by devising several formulas of mineral manure. He, however, did not recognise the needs of external supplies of nitrogen salts for the crops, in open dispute with the English school of Lawes and Gilbert, who were instead convinced assertors of such needs. At the end of the 19th century Hellriegel showed that leguminous plants presenting peculiar nodules on their roots could really fix the gaseous nitrogen. From these nodules Beijerinck and Prazmowski isolated for the first time some bacteria which were recognised as the real agents fixing nitrogen. This discovery was of fundamental importance for plant nutrition, only second to the discovery of photosynthesis. Another basic contribution came from early research of Sachs on plants grown on aqueous solutions: these techniques allowed to impose the concept of "essential elements", which was fixed as a principle by Arnon and Stout in 1939. This principle benefited further research concerning the effects of states of deficiency on plant growth and development through investigation on the anatomical, histologic and biochemical nutritional disorders of plants.

Biology↗

PubMed2003

Nitrogenase activity of Herbaspirillum seropedicae grown under low iron levels requires the products of nifXorf1 genes.

Herbaspirillum seropedicae strains mutated in the nifX or orf1 genes showed 90% or 50% reduction in nitrogenase activity under low levels of iron or molybdenum respectively. Mutations in nifX or orf1 genes did not affect nif gene expression since a nifH::lacZ fusion was fully active in both mutants. nifX and the contiguous gene orf1 are essential for maximum nitrogen fixation under iron limitation and are probably involved in synthesis of nitrogenase iron or iron-molybdenum clusters.

Bacterial Proteins↗

PubMed2006

[Screening of mixed crop disease-resistant bacterial inoculants from N2-fixing and P-solubilzing bacterial isolates].

In this paper, a total of 22 bacterial isolates capable of fixing nitrogen and/or solubilizing phosphorus were inoculated, and mixed incubated in 15 liquid media. The isolates were obtained from soil with selective media, and not purified. After grown for 48 hours, the mixed cultures were continuously inoculated to the same media, and then incubated under the same conditions. The procedures were repeated for 12 times, and 15 stable bacterial mixtures (SBM) were obtained. Each SBM was purified, which contained two or three bacterial species. Agar plate bacteriostasis test showed that 6 mixed cultures of the SBM exhibited significant inhibition effects to one or more plant pathogens of Verticillium dahliae, Rhizoctonia solani, and Pseudomonas syringae. It was found that C7 and C15 were the two SBM with most obvious antibiological effect. SMB C15 had the ability of nitrogen fixation, and C4, C5 C7, C9, C14 and C15 showed the ability of phosphorus solubilization. These results suggested a possible approach to develop stable, synergistic, and multi-functional antibiological agents.

Crops, Agricultural↗

PubMed1995

The C-terminal domain of NifL is sufficient to inhibit NifA activity.

In Klebsiella pneumoniae, transcription of all nif (nitrogen fixation) operons except the regulatory nifLA operon itself is regulated by the proteins NifA and NifL. NifA, an enhancer-binding protein, activates transcription by RNA polymerase containing the alternative sigma factor sigma 54. The central catalytic domain of NifA is sufficient for transcriptional activation, which can occur from solution. In vivo, NifL antagonizes the action of NifA in the presence of molecular oxygen or combined nitrogen. Inhibition has also been shown in vitro, but it was not responsive to environmental signals. Assuming a two-domain structure of NifL, we localized inhibition by NifL to its carboxy (C)-terminal domain, which is more soluble than the intact protein. The first line of evidence for this is that internal deletions of NifL containing an intact C-terminal domain were able to inhibit transcriptional activation by NifA in a coupled transcription-translation system. The second line of evidence is that the isolated C-terminal domain of NifL (assayed as a fusion to the soluble maltose-binding protein [MBP]) was sufficient to inhibit transcriptional activation by the central domain of NifA in a purified transcription system. The final line of evidence is that an MBP fusion to the C-terminal domain of NifL inhibited transcriptional activation by NifA in vivo. On the basis of these data, we postulate that the inhibitory function of NifL lies in its C-terminal domain and hence infer that this domain is responsible for interaction with NifA. Gel filtration experiments with MBP-NifL fusion derivatives lacking portions of the N- or C-terminal domain of the protein revealed that the C-terminal domain is the most soluble part of NifL. Up to 50% of two MBP-NifL truncations containing only the C-terminal domain appeared to be in a defined dimeric state.

Bacterial Proteins↗

PubMed1998

Poly-beta-hydroxybutyrate turnover in Azorhizobium caulinodans is required for growth and affects nifA expression.

Azorhizobium caulinodans is able to fix nitrogen in the free-living state and in symbiosis with the tropical legume Sesbania rostrata. The bacteria accumulate poly-beta-hydroxybutyrate (PHB) under both conditions. The structural gene for PHB synthase, phbC, was inactivated by insertion of an interposon. The mutant strains obtained were devoid of PHB, impaired in their growth properties, totally devoid of nitrogenase activity ex planta (Nif-), and affected in nucleotide pools and induced Fix- nodules devoid of bacteria. The Nif- phenotype was the consequence of the lack of nifA transcription. Nitrogenase activity was partially restored to a phbC mutant by constitutive expression of the nifA gene. However, this constitutive nifA expression had no effect on the nucleotide content or on growth of the phbC mutant. It is suggested that PHB is required for maintaining the reducing power of the cell and therefore the bacterial growth. These observations also suggest a new control of nifA expression to adapt nitrogen fixation to the availability of carbon and reducing equivalents.

Acyltransferases↗

PubMedDate not supplied

Identification and characterization of a Rhizobium leguminosarum bv. phaseoli gene that is important for nodulation competitiveness and shows structural homology to a Rhizobium fredii host-inducible gene.

DNA sequence analysis of a 1.4-kb SalI-HindIII segment located approximately 2 kb upstream of the Rhizobium leguminosarum bv. phaseoli syrM gene revealed the presence of an open reading frame (ORF3) encoding a putative 295-amino acid polypeptide with a molecular mass of 33,401 Da. ORF3 is homologous to a R. fredii host-inducible gene. The proteins encoded by R. l. bv. phaseoli ORF3 and by the R. fredii host-inducible gene share 37% sequence identity. In contrast to the R. fredii host-inducible gene, expression of ORF3 is not induced in the presence of Phaseolus vulgaris root exudates or by specific flavonoids, able to induce nodulation genes in R. l. bv. phaseoli. A R. l. bv. phaseoli ORF3 mutant was constructed by site-directed deletion/replacement mutagenesis. This mutant strain is not affected in symbiotic nitrogen fixation but exhibits a delay in nodulation on Phaseolus vulgaris. Moreover, this mutant was shown to be defective in competition for nodulation.

Amino Acid Sequence↗

PubMed2002

Nodule ultrastructure and initial growth of Anadenanthera peregrina (L.) Speg. var. falcata (Benth.) altschul plants infected with rhizobia.

The anatomy and ultrastructure of root nodules of Anadenanthera peregrina var. falcata (Leguminosae-Mimosoideae) were analysed, as was plant growth. To ensure that nodules developed, seedlings were inoculated with a mixture of six strains of rhizobia. Nodules were produced that differed in appearance-and probably also effectiveness-but their structure was similar and they showed characteristics typical of indeterminate nodules, such as persistent meristematic tissue and a gradient of cells at different stages of development. Many starch grains were present in inner cortex cells and interstitial cells of infected tissue. Infected cells were densely packed with bacteroids, which contained many poly-beta-hydroxybutyrate granules. The high incidence of these granules, together with high levels of starch accumulation in interstitial cells, suggested low N2-fixation efficiency of the rhizobia isolates used for inoculation. In the symbiosomes of early-senescent infected cells, reticulum-like structures, small vesicles and a fibrillar material were observed; these may be related to bacteroid degradation. In the cytoplasm of late-senescent infected cells, many vesicles and membrane-like structures were observed, probably associated with membrane degradation of bacteroids and peribacteroids. The total biomass of plants inoculated with rhizobia was low and their xylopodia and shoots had low levels of N compared with noninoculated plants fertilized with ammonium nitrate. However, inoculated plants did not show N-deficiency symptoms and grew better than non-inoculated plants without N fertilization. These growth results, together with ultrastructural observations of nodules, suggest that nitrogen fixation of rhizobia isolates associated with Anadenanthera peregrina var. falcata roots is poor.

Biomass↗

PubMed1978

The effect of ammonium nitrate on the synthesis of nitrogenase and the concentration of leghemoglobin in pea root nodules induced by Rhizobium leguminosarum.

The effects of NH4NO3 on the development of root nodules of Pisum sativum after infection with Rhizobium leguminosarum (strain PRE) and on the nitrogenase activity of the bacteroids in the nodule tissue were studied. The addition of NH4NO3 decreased the nitrogenase activity measured on intact nodules. This reduction of nitrogen fixation did not result from a reduced number of bacteroids or a decreased amount of bacteroid proteins per gram of nodule. The synthesis of nitrogenase, measured as the relative amount of incorporation of [35S]sulfate into the components I and II of nitrogenase was similarly not affected. The addition of NH4NO3 decreased the amount of leghemoglobin in the nodules and there was a quantitative correlation between the leghemoglobin content and the nitrogen-fixing capacity of the nodules. The conclusion is that the decrease of nitrogen-fixing capacity is caused by a decrease of the leghemoglobin content of the root nodules and not by repression of the nitrogenase synthesis.

Enzyme Precursors↗

PubMed2003

The Sinorhizobium meliloti glycine betaine biosynthetic genes (betlCBA) are induced by choline and highly expressed in bacteroids.

The symbiotic soil bacterium Sinorhizobium meliloti has the capacity to synthesize the osmoprotectant glycine betaine from choline-O-sulfate and choline. This pathway is encoded by the betICBA locus, which comprises a regulatory gene, betI, and three structural genes, betC (choline sulfatase), betB (betaine aldehyde dehydrogenase), and betA (choline dehydrogenase). Here, we report that betICBA genes constitute a single operon, despite the existence of intergenic regions containing mosaic elements between betI and betC, and betB and betA. The regulation of the bet operon was investigated by using transcriptional lacZ (beta-galactosidase) fusions and has revealed a strong induction by choline at concentrations as low as 25 microM and to a lesser extent by choline-O-sulfate and acetylcholine but not by osmotic stress or oxygen. BetI is a repressor of the bet transcription in the absence of choline, and a nucleotide sequence of dyad symmetry upstream of betI was identified as a putative betI box. Measurements of intracellular pools of choline, well correlated with beta-galactosidase activities, strongly suggested that BetI senses the endogenous choline pool that modulates the intensity of BetI repression. In contrast to Escherichia coli, BetI did not repress choline transport. During symbiosis with Medicago sativa, S. meliloti bet gene expression was observed within the infection threads, in young and in mature nodules. The existence of free choline in nodule cytosol, peribacteroid space, and bacteroids was demonstrated, and the data suggest that bet regulation in planta is mediated by BetI repression, as in free-living cells. Neither Nod nor Fix phenotypes were significantly impaired in a betI::omega mutant, indicating that glycine betaine biosynthesis from choline is not crucial for nodulation and nitrogen fixation.

Alcohol Oxidoreductases↗

PubMed1996

Aspartate transport by the Dct system in Rhizobium leguminosarum negatively affects nitrogen-regulated operons.

Amino acid uptake by the general amino acid permease (Aap) of Rhizobium leguminosarum strain 3841 was severely reduced by the presence of aspartate in the growth medium when glucose was the carbon source. The reduction in transport by the Aap appeared to be caused by inhibition of uptake and not by transcriptional repression. However, as measured with lacZ fusions, the Ntr-regulated gene glnII was repressed by aspartate. The negative regulatory effect on both the Aap and glnII was prevented by mutation of any component of the dicarboxylate transport (Dct) system or by the inclusion of a C4- dicarboxylate in the growth medium, including the non-metabolizable analogue 2-methylsuccinate. As measured by total uptake and with a dctA-lacZ fusion, aspartate was an efficient inducer of the Dct system, but slightly less so than succinate alone or succinate and aspartate together. Thus, aspartate does not cause overexpression of DctA leading to improper regulation of other operons. Transport measurements revealed that the Dct system has an apparent Km for succinate of 5 microM and an apparent Ki for aspartate inhibition of succinate uptake of 5 mM. These data imply that the Dct-mediated accumulation of aspartate causes an unregulated build-up of aspartate or a metabolic product of it in the cell. This accumulation of aspartate is prevented either by mutation of the dct system or by the presence of a higher affinity substrate that will reduce access of aspartate to the carrier protein. Elevation or disruption of the intracellular aspartate pool is predicted to disrupt N-regulated operons and nitrogen fixation.

Amino Acid Transport Systems↗

PubMed2003

Identification of a third sulfate activation system in Sinorhizobium sp. strain BR816: the CysDN sulfate activation complex.

Sinorhizobium sp. strain BR816 possesses two nodPQ copies, providing activated sulfate (3'-phosphoadenosine-5'-phosphosulfate [PAPS]) needed for the biosynthesis of sulfated Nod factors. It was previously shown that the Nod factors synthesized by a nodPQ double mutant are not structurally different from those of the wild-type strain. In this study, we describe the characterization of a third sulfate activation locus. Two open reading frames were fully characterized and displayed the highest similarity with the Sinorhizobium meliloti housekeeping ATP sulfurylase subunits, encoded by the cysDN genes. The growth characteristics as well as the levels of Nod factor sulfation of a cysD mutant (FAJ1600) and a nodP1 nodQ2 cysD triple mutant (FAJ1604) were determined. FAJ1600 shows a prolonged lag phase only with inorganic sulfate as the sole sulfur source, compared to the wild-type parent. On the other hand, FAJ1604 requires cysteine for growth and produces sulfate-free Nod factors. Apigenin-induced nod gene expression for Nod factor synthesis does not influence the growth characteristics of any of the strains studied in the presence of different sulfur sources. In this way, it could be demonstrated that the "household" CysDN sulfate activation complex of Sinorhizobium sp. strain BR816 can additionally ensure Nod factor sulfation, whereas the symbiotic PAPS pool, generated by the nodPQ sulfate activation loci, can be engaged for sulfation of amino acids. Finally, our results show that rhizobial growth defects are likely the reason for a decreased nitrogen fixation capacity of bean plants inoculated with cysD mutant strains, which can be restored by adding methionine to the plant nutrient solution.

Bacterial Proteins↗