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PubMed2004

Functional difference between Sinorhizobium meliloti NifA and Enterobacter cloacae NifA.

The nifA gene is an important regulatory gene and its product, NifA protein, regulates the expression of many nif genes involved in the nitrogen fixation process. We introduced multiple copies of the constitutively expressed Sinorhizobium meliloti (Sm) or Enterobacter cloacae (Ec) nifA gene into both the nifA mutant strain SmY and the wild-type strain Sm1021. Root nodules produced by SmY containing a constitutively expressed Sm nifA gene were capable of fixing nitrogen, while nodules produced by SmY containing the Ec nifA gene remained unable to fix nitrogen, as is the case for SmY itself. However, transfer of an additional Sm nifA gene into Sm1021 improved the nitrogen-fixing efficiency of root nodules to a greater extent than that observed upon transfer of the Ec nifA gene into Sm1021. Comparative analysis of amino acid sequences between Sm NifA and Ec NifA showed that the N-terminal domain was the least similar, but this domain is indispensable for complementation of the Fix- phenotype of SmY by Sm NifA. We conclude that more than one domain is involved in determining functional differences between Sm NifA and Ec NifA.

Amino Acid Sequence↗

PubMed1975

Ammonia assimilation by rhizobium cultures and bacteroids.

The enzymes involved in the assimilation of ammonia by free-living cultures of Rhizobium spp. are glutamine synthetase (EC. 6.o.I.2), glutamate synthase (L-glutamine:2-oxoglutarate amino transferase) and glutamate dehydrogenase (ED I.4.I.4). Under conditions of ammonia or nitrate limitation in a chemostat the assimilation of ammonia by cultures of R. leguminosarum, R. trifolii and R. japonicum proceeded via glutamine synthetase and glutamate synthase. Under glucose limitation and with an excess of inorganic nitrogen, ammonia was assimilated via glutamate dehydrogenase, neither glutamine synthetase nor glutamate synthase activities being detected in extracts. The coenzyme specificity of glutamate synthase varied according to species, being linked to NADP for the fast-growing R. leguminosarum, R. melitoti, R. phaseoli and R. trifolii but to NAD for the slow-growing R. japonicum and R. lupini. Glutamine synthetase, glutamate synthase and glutamate dehydrogenase activities were assayed in sonicated bacteroid preparations and in the nodule supernatants of Glycine max, Vicia faba, Pisum sativum, Lupinus luteus, Medicago sativa, Phaseolus coccineus and P. vulgaris nodules. All bacteroid preparations, except those from M. sativa and P. coccineus, contained glutamate synthase but substantial activities were found only in Glycine max and Lupinus luteus. The glutamine synthetase activities of bacteroids were low, although high activities were found in all the nodule supernatants. Glutamate dehydrogenase activity was present in all bacteroid samples examined. There was no evidence for the operation of the glutamine synthetase/glutamate synthase system in ammonia assimilation in root nodules, suggesting that ammonia produced by nitrogen fixation in the bacteroid is assimilated by enzymes of the plant system.

Acetylene↗

PubMedDate not supplied

[Peculiarities of structure-function organization of microbial groups of soil contaminated by cement dust].

Long-term contamination of the forest soil by the cement dust leads to disturbances of stability of natural microbial groups, changes their quantitative and qualitative composition, results in substitution of some microbial associations by other ones. The intensive contamination of the soil by the effluents of cement integrated works, inhibits the intensity of nitrogen fixation processes and decay of the forest litter, development of micromycetes, results in the decrease of the length of mycelium and its biomass.

Bacteria↗

PubMed2006

Cloning, sequencing, and characterization of the Azospirillum brasilense fhuE gene.

The fhuE gene of Escherichia coli encodes the FhuE protein, which is a receptor protein in the coprogen-mediated siderophore iron-transport system. A fhuE gene homologue from Azospirillum brasilense, a nitrogen-fixing soil bacterium that lives in association with the roots of cereal grasses, was cloned, sequenced, and characterized. The A. brasilense fhuE encodes a protein of 802 amino acids with a predicted molecular weight of approximately 87 kDa. The deduced amino-acid sequence showed a high level of homology to the sequences of all the known fhuE gene products. The fhuE mutant was sensitive to iron starvation and defective in coprogen-mediated iron uptake. The mutant failed to express one membrane protein of approximately 78 kDa that was induced by iron starvation in the wild type. Complementation studies showed that the A. brasilense fhuE gene, when present on a low-copy number plasmid, could restore the functions of the mutant. Mutation in fhuE gene did not affect nitrogen fixation.

Amino Acid Sequence↗

PubMedDate not supplied

Characterization of a nodule-enhanced glutamine synthetase from alfalfa: nucleotide sequence, in situ localization, and transcript analysis.

We have characterized two glutamine synthetase (GS) cDNA clones (pGS13 and pGS100) representing mRNA from root nodules of alfalfa. pGS13 is a full-length version of a previously isolated partial cDNA from an alfalfa nodule cDNA library, while pGS100 was previously isolated from an alfalfa suspension culture cDNA library. Using the 3' untranslated region of the two cDNAs as gene-specific probes, we have shown that the GS genes represented by pGS100 and pGS13 are expressed in all organs tested, although at varying levels. pGS13, however, represents the nodule-enhanced GS gene class. Genomic Southern blot analysis using gene-specific probes shows multiple hybridizing bands, in each case suggesting multiple genes and/or alleles for each class of cytoplasmic GS genes. In situ hybridization of alfalfa nodule sections with gene-specific antisense RNA probes has shown that the nodule-enhanced GS genes are induced in the invasion zone and that their expression is limited to the symbiotic zone, while the GS genes represented by pGS100 are induced in the early symbiotic zone and are expressed throughout the symbiotic and senescent zones. Transcripts for both sets of GS genes are localized in the infected cells and based on the spatial expression pattern it would appear that the two gene classes are induced independently of the onset of nitrogen fixation.

Amino Acid Sequence↗

PubMed2005

Regulation of CO on heterocyst differentiation and nitrate uptake in the cyanobacterium Anabaena sp. PCC 7120.

AIMS: The aim of the present investigation was to study the effects of different inorganic carbon and nitrogen sources on nitrate uptake and heterocyst differentiation in the culture of cyanobacterium Anabaena sp. PCC 7120. METHODS AND RESULTS: Anabaena was cultivated in media BG11 containing combined nitrogen and supplementary NaHCO3 or CO2. Cell growth, heterocyst differentiation, nitrate reductase (NR, EC 1.7.7.2), glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and NO uptake were analysed. The cells cultivated in BG11(0) medium with aeration were taken as reference. Experimental results showed that the differentiation frequency of heterocysts when the cells were cultivated with elevated CO2 was higher than that of the cells grown with air or bicarbonate. Heterocysts appeared unexpectedly when CO2 was introduced into the medium containing nitrate. However, no heterocysts emerged when CO2 was added to medium containing NH or urea, or when NaHCO3 was supplied to the medium with nitrate. Both nitrate uptake rate and nitrate reduction enzyme activity were depressed by the supplement of CO2 to the culture. The activity of G6PDH was enhanced with the increase in heterocyst differentiation frequency. CONCLUSION: CO2 might compete with NO for energy and electrons in the uptake process and CO2 appears favoured. This led to a high intracellular C/N ratio and a relative N limitation. So the process of heterocyst differentiation was activated to supplement nitrogen uptake. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided an attractive possibility to form more heterocysts by rapid growth of Anabaena cells cultivated in the medium containing nitrate in order to increase nitrogen fixation and hydrogen production.

Anabaena↗

PubMed1979

Phosphoglucose isomerase mutant of Rhizobium meliloti.

A mutant strain of complex phenotype was selected in Rhizobium meliloti after nitrosoguanidine mutagenesis. It failed to grow on mannitol, sorbitol, fructose, mannose, ribose, arabitol, or xylose, but grew on glucose, maltose, gluconate, L-arabinose, and many other carbohydrates. Assay showed the enzyme lesion to be in phosphoglucose isomerase (pgi), and revertants, which were of normal growth phenotype, contained the enzyme again. Nonpermissive substrates such as fructose and xylose prevented growth on permissive ones such as L-arabinose, and in such situations there was high accumulation of fructose 6-phosphate. The mutant strain had about 20% as much exopolysaccharide as the parent. Nitrogen fixation by whole plants was low and delayed when the mutant strain was the inoculant.

Carbohydrate Metabolism↗

PubMed1989

Symbiotic properties of rhizobia containing a flavonoid-independent hybrid nodD product.

A hybrid nodD gene consisting of 75% of the nodD1 gene of Rhizobium meliloti at the 5' end and 27% of the nodD gene of Rhizobium trifolii at the 3' end activates the six tested inducible nod promoters of Rhizobium leguminosarum, R. trifolii, or R. meliloti to maximal levels, even in the absence of flavonoids. In strains containing such a constitutive activating nodD gene, transcription of nod genes started at the same site as in flavonoid-induced strains containing a wild-type nodD gene. In contrast to heterologous wild-type nodD products, the constitutive activating nodD gene does not cause a limitation of the host range. Furthermore, R. leguminosarum, R. trifolii, and R. meliloti strains containing the constitutive activating nodD gene induce (pseudo) nodules on tropical leguminous plants. Comparison of the symbiotic properties of rhizobia containing the constitutive nodD hybrid gene with those of rhizobia containing various wild-type nodD genes indicates that the activation of the nodD product by flavonoids is of crucial importance during the process of infection thread formation and, surprisingly, also during nitrogen fixation.

Bacterial Proteins↗

PubMed1996

Cloning, characterization, and regulation of nifF from Rhodobacter capsulatus.

The Rhodobacter capsulatus nifF gene and upstream sequence were cloned by using a probe based on the N-terminal sequence of NifF. nifF was found to not be contained in the previously described nif regions I, II, and III. Comparison of the deduced amino acid sequence showed that it is highly similar to NifF from Azotobacter vinelandii and NifF from Klebsiella pneumoniae. Analysis of translational fusions demonstrated that the regulation of transcription was the same as previously reported at the protein level. Insertional mutagen esis showed that NifF contributes significantly to nitrogenase activity under normal nitrogen-fixing conditions and that it is absolutely required for nitrogen fixation under iron limitation.

Amino Acid Sequence↗

PubMed2001

Role for draTG and rnf genes in reduction of 2,4-dinitrophenol by Rhodobacter capsulatus.

The phototrophic bacterium Rhodobacter capsulatus is able to reduce 2,4-dinitrophenol (DNP) to 2-amino-4-nitrophenol enzymatically and thus can grow in the presence of this uncoupler. DNP reduction was switched off by glutamine or ammonium, but this short-term regulation did not take place in a draTG deletion mutant. Nevertheless, the target of DraTG does not seem to be the nitrophenol reductase itself since the ammonium shock did not inactivate the enzyme. In addition to this short-term regulation, ammonium or glutamine repressed the DNP reduction system. Mutants of R. capsulatus affected in ntrC or rpoN exhibited a 10-fold decrease in nitroreductase activity in vitro but almost no DNP activity in vivo. In addition, mutants affected in rnfA or rnfC, which are also under NtrC control and encode components involved in electron transfer to nitrogenase, were unable to metabolize DNP. These results indicate that NtrC regulates dinitrophenol reduction in R. capsulatus, either directly or indirectly, by controlling expression of the Rnf proteins. Therefore, the Rnf complex seems to supply electrons for both nitrogen fixation and DNP reduction.

2,4-Dinitrophenol↗

PubMed2004

The Sinorhizobium meliloti fur gene regulates, with dependence on Mn(II), transcription of the sitABCD operon, encoding a metal-type transporter.

Sinorhizobium meliloti is an alpha-proteobacterium able to induce nitrogen-fixing nodules on roots of specific legumes. In order to propagate in the soil and for successful symbiotic interaction the bacterium needs to sequester metals like iron and manganese from its environment. The metal uptake has to be in turn tightly regulated to avoid toxic effects. In this report we describe the characterization of a chromosomal region of S. meliloti encoding the sitABCD operon and the putative regulatory fur gene. It is generally assumed that the sitABCD operon encodes a metal-type transporter and that the fur gene is involved in iron ion uptake regulation. A constructed S. meliloti sitA deletion mutant was found to be growth dependent on Mn(II) and to a lesser degree on Fe(II). The sitA promoter was strongly repressed by Mn(II), with dependence on Fur, and moderately by Fe(II). Applying a genome-wide S. meliloti microarray it was shown that in the fur deletion mutant 23 genes were up-regulated and 10 genes were down-regulated when compared to the wild-type strain. Among the up-regulated genes only the sitABCD operon could be associated with metal uptake. On the other hand, the complete rhbABCDEF operon, which is involved in siderophore synthesis, was identified among the down-regulated genes. Thus, in S. meliloti Fur is not a global repressor of iron uptake. Under symbiotic conditions the sitA promoter was strongly expressed and the S. meliloti sitA mutant exhibited an attenuated nitrogen fixation activity resulting in a decreased fresh weight of the host plant Medicago sativa.

Bacterial Proteins↗

PubMed2003

[Creation and genetic study of a collection of symbiotic mutants of the pea (Pisum sativum L.)].

A collection of symbiotic pea mutants consisting of 34 lines was created. The morphobiological and symbiotic description of different types of pea mutants is presented. The mode of inheritance of symbiotic characters is analyzed. For the first time in pea, hypernodulation mutations with dominant inheritance were isolated. Differences in the structure of bacteroid tissues of root nodules were revealed between mutants with effective and ineffective nodules. Using reciprocal root/stem and stem/root grafts, the plant organ producing nodulation-controlling factors was established. New hypernodulation-controlling genes--nod4 and nod6--were identified. For the first time in pea, using pure lines derived from cultivars with contrasting nodulation, the new gene Nod5--nod5 was found. The dominant allele of this gene controls abundant nodulation, high nitrogen fixation, and nitrate resistance. Pea cultivars carrying the Nod5 gene that can be used in selection for improved symbiotic properties were identified.

Alleles↗

PubMedDate not supplied

Characterization of two Azospirillum brasilense Sp7 plasmid genes homologous to Rhizobium meliloti nodPQ.

Bacteria belonging to the Azospirillum genus are nitrogen fixers that colonize the roots of grasses, but do not cause the formation of differentiated structures. Sequences from total DNA of several Azospirillum strains are homologous to restriction fragments containing Rhizobium meliloti nodulation genes. A 10-kilobase (kb) EcoRI fragment from A. brasilense Sp7, sharing homology with a 6.8-kb EcoRI fragment carrying nodGEFH and part of nodP of R. meliloti 41, was cloned in pUC18 to yield pAB502. The nucleotide sequence of a 3.5-kb EcoRI-SmaI fragment of the pAB502 insert revealed 60% homology with R. meliloti nodP and nodQ genes. The nodP gene product shares no homology to any known protein sequence. The Azospirillum nodQ gene product shares homology with a family of initiation and elongation factors as does the R. meliloti nodQ gene product. Since the nodQ gene overlaps the nodP gene, the two genes might be cotranscribed. Azospirillum contains large plasmids, and the nodPQ genes were found on the 90-MDa plasmid (p90). A translational nodP-lacZ fusion was constructed in the broad host range plasmid pGD926. No beta-galactosidase activity was detected in Escherichia coli, but the fusion was functional in Azospirillum and constitutively expressed. Deletions and mutations of nodPQ did not modify growth, nitrogen fixation, or interaction with wheat seedlings.

Amino Acid Sequence↗

PubMed2004

The fixABCX genes in Rhodospirillum rubrum encode a putative membrane complex participating in electron transfer to nitrogenase.

In our efforts to identify the components participating in electron transport to nitrogenase in Rhodospirillum rubrum, we used mini-Tn5 mutagenesis followed by metronidazole selection. One of the mutants isolated, SNT-1, exhibited a decreased growth rate and about 25% of the in vivo nitrogenase activity compared to the wild-type values. The in vitro nitrogenase activity was essentially wild type, indicating that the mutation affects electron transport to nitrogenase. Sequencing showed that the Tn5 insertion is located in a region with a high level of similarity to fixC, and extended sequencing revealed additional putative fix genes, in the order fixABCX. Complementation of SNT-1 with the whole fix gene cluster in trans restored wild-type nitrogenase activity and growth. Using Western blotting, we demonstrated that expression of fixA and fixB occurs only under conditions under which nitrogenase also is expressed. SNT-1 was further shown to produce larger amounts of both ribulose 1,5-bisphosphate carboxylase/oxygenase and polyhydroxy alkanoates than the wild type, indicating that the redox status is affected in this mutant. Using Western blotting, we found that FixA and FixB are soluble proteins, whereas FixC most likely is a transmembrane protein. We propose that the fixABCX genes encode a membrane protein complex that plays a central role in electron transfer to nitrogenase in R. rubrum. Furthermore, we suggest that FixC is the link between nitrogen fixation and the proton motive force generated in the photosynthetic reactions.

Bacterial Proteins↗

PubMed1988

[Augmentation of nitrogen efficiency and hyperphagia associated with refeeding after prolonged fast in the domestic goose].

The domestic goose is hyperphagic after a fast resulting in a 40% decrease in body mass (a decrease similar to that during breeding anorexia). Food intake is maximum on the 8th day of refeeding. As it is then an average of 2.5 times higher than before the fast, food intake may thus reach the value during forced-feeding for "foie gras". Since nitrogen assimilation rate also increases 2.5 times, nitrogen fixation is increased 6.8 times, suggesting a high level of protein synthesis.

Animals↗

PubMed2006

Nitrogen transformations in stratified aquatic microbial ecosystems.

New analytical methods such as advanced molecular techniques and microsensors have resulted in new insights about how nitrogen transformations in stratified microbial systems such as sediments and biofilms are regulated at a microm-mm scale. A large and ever-expanding knowledge base about nitrogen fixation, nitrification, denitrification, and dissimilatory reduction of nitrate to ammonium, and about the microorganisms performing the processes, has been produced by use of these techniques. During the last decade the discovery of anammmox bacteria and migrating, nitrate accumulating bacteria performing dissimilatory reduction of nitrate to ammonium have given new dimensions to the understanding of nitrogen cycling in nature, and the occurrence of these organisms and processes in stratified microbial communities will be described in detail.

Aerobiosis↗

PubMed2004

Rhizosheath of sinai desert plants is a potential repository for associative diazotrophs.

Among 42 plant species representing the flora of north Sinai, two possessed sand grain sheath encasing the roots. They are Panicum turgidum Forssk. and Stipagrostis scoparia (Trin.and Rupr.) deWinter. Rhizosheaths, compared to surrounding free sand, accommodated higher population density of microorganisms including associative diazotrophs. Isolates secured belonged to the species of Bacillus circulans, Paenib. macerans (Bacillus macerans), Enterobacter agglomerans, Agrobacterium radiobacter and Chryseomonas luteola (Pseudomonos luteola). The rhizosheath potentiality in re-vegetating sand dunes and arid lands, through nitrogen fixation, plant-water relationship and root continuity for nutrient uptake, are discussed.

Desert Climate↗

PubMed2004

Discovery of symbiotic nitrogen-fixing cyanobacteria in corals.

Colonies of the Caribbean coral Montastraea cavernosa exhibit a solar-stimulated orange-red fluorescence that is spectrally similar to a variety of fluorescent proteins expressed by corals. The source of this fluorescence is phycoerythrin in unicellular, nonheterocystis, symbiotic cyanobacteria within the host cells of the coral. The cyanobacteria coexist with the symbiotic dinoflagellates (zooxanthellae) of the coral and express the nitrogen-fixing enzyme nitrogenase. The presence of this prokaryotic symbiont in a nitrogen-limited zooxanthellate coral suggests that nitrogen fixation may be an important source of this limiting element for the symbiotic association.

Animals↗