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PubMed1994

Analysis of the expression from Rhizobium meliloti fix-promoters in other Rhizobium backgrounds.

Using translational fusions to lacZ, we have measured expression from the promoters of Rhizobium meliloti regulatory genes, nifA and fixK, and structural genes, nifH and fixA, in other fast-growing rhizobia whose nitrogen fixation regulation is less known. Neither nifA nor fixK promoters were activated under both free-living microaerobic and symbiotic conditions, except in R. tropici, where clear symbiotic activation of either nifA or fixK expression could be observed. Both nifH and fixA promoters showed strong heterologous activation during symbiosis and weak activation under free-living nitrogen starvation conditions. Only when the nifH promoter was in R. tropici and R. leguminosarum bv. phaseoli, was clear induction observed in the microaerobic free-living state. Deletion analysis of these promoters suggested that a NifA binding site (UAS) was needed for full heterologous activation of nifHp, either in microaerobiosis or symbiosis. In contrast, the UAS region seemed to be unnecessary for fixA activation. However, a region containing a potential integration host factor (IHF) binding site was observed to be needed for complete heterologous symbiotic induction from fixAp. The moderate induction observed in nitrogen-free medium only required the sigma 54 holoenzyme recognition sequence; this may be indicative of the existence of non-specific activation by NtrC-like proteins. Our results suggest possible common and different features in the control mechanisms of the nitrogen fixation gene expression among Rhizobium species.

Base Sequence↗

PubMed1983

Glycine betaine, an osmotic effector in Klebsiella pneumoniae and other members of the Enterobacteriaceae.

Osmoregulation was examined in members of the Enterobacteriaceae. Exogenous glycine betaine at a concentration as low as 1 mM was found to stimulate the growth rate of Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae in media of inhibitory osmotic strength. The stimulation was shown to be independent of any specific solutes, electrolytes, or nonelectrolytes. Therefore, the stimulatory effect of glycine betaine was a consequence of high osmotic potential. This effect was found to be far greater than the proline effect previously observed in S. typhimurium. Whereas nitrogen fixation by K. pneumoniae is completely inhibited under conditions of osmotic stress, nitrogenase activity could be partially restored by the addition of exogenous glycine betaine to the culture medium. Furthermore, glycine betaine in combination with proline, especially proline produced internally at a high level because of regulatory mutations affecting proline biosynthesis, strongly stimulated nitrogen fixation activity during osmotic stress. Glycine betaine was accumulated by the cells, and the amount taken up was correlated with the osmolarity of the medium. These findings are discussed in relation to the possible mechanisms by which glycine betaine might cause enhanced osmotolerance.

Amino Acids↗

PubMed2000

Molybdate-dependent expression of dimethylsulfoxide reductase in Rhodobacter capsulatus.

Expression of the dimethylsulfoxide respiratory (dor) operon of Rhodobacter is regulated by oxygen, light intensity and availability of substrate. Since dimethylsulfoxide reductase contains a pterin molybdenum cofactor, the role of molybdate in the regulation of dor operon expression was investigated. In this report we show that the molybdate-responsive transcriptional regulator, MopB, and molybdate are essential for maximal dimethylsulfoxide reductase activity and expression of a dorA::lacZ transcriptional fusion in Rhodobacter capsulatus. In contrast, mop genes are not required for the expression of the periplasmic nitrate reductase or xanthine dehydrogenase in R. capsulatus under conditions of molybdenum sufficiency. This is the first report demonstrating a clear functional difference between the ModE homologues MopB and MopA in this bacterium. The results suggest that MopA is primarily involved in the regulation of nitrogen fixation gene expression in response to molybdate while MopB has a role in nitrogen fixation and dimethylsulfoxide respiration.

Bacterial Proteins↗

PubMed1993

Characterization of the ntrBC genes of Azospirillum brasilense Sp7: their involvement in the regulation of nitrogenase synthesis and activity.

A 7.1 kb EcoRI fragment from Azospirillum brasilense, that hybridized with a probe carrying the ntrBC genes from Bradyrhizobium japonicum, was cloned. The nucleotide sequence of a 3.8 kb subfragment was established. This led to the identification of two open reading frames, encoding polypeptides of 401 and 481 amino acids, that were similar to NtrB and NtrC, respectively. A broad host range plasmid containing the putative Azospirillum ntrC gene was shown to restore nitrogen fixation under free-living conditions to a ntrC-Tn5 mutant of Azorhizobium caulinodans. Several Tn5 insertion mutants were isolated in the ntrBC coding region in A brasilense. These mutants were prototrophic and Nif+. However, their nitrogenase activity was slightly lower than in the wild type and they were unable to grow on nitrate as sole nitrogen source. Under microaerobiosis and in the absence of ammonia, a nifA-lacZ fusion was expressed in the mutants at about 60% of the level in the wild type. In the presence of ammonia, the fusion was similarly expressed (60% of the maximum) both in the wild type and mutants. Addition of ammonia to a nitrogen-fixing culture of ntrBC mutants did not abolish nitrogenase activity, in contrast with the wild type. It thus appears that in Azospirillum the ntrBC genes are not essential for nitrogen fixation, although NtrC controls nifA expression to some extent. They are, however, required for the switch-off of nitrogenase activity.

Amino Acid Sequence↗

PubMed2001

Azospirillum doebereinerae sp. nov., a nitrogen-fixing bacterium associated with the C4-grass Miscanthus.

A new group of nitrogen-fixing Azospirillum sp. bacteria was isolated from the roots of the C4-gramineous plant Miscanthus. Polyphasic taxonomy was performed, including auxanography using API galleries, physiological tests and 16S rRNA sequence comparison. The ability of the isolates to fix dinitrogen was evaluated by amplification of the nifD gene, immunodetection of the dinitrogenase reductase and acetylene-reduction assay. On the basis of these results, the nitrogen-fixing isolates represent a new species within the genus Azospirillum. Its closest phylogenetic neighbours, as deduced by 16S rDNA-based analysis, are Azospirillum lipoferum, Azospirillum largimobile and Azospirillum brasilense with 96.6, 96.6 and 95.9% sequence similarity, respectively. Two 16S rRNA-targeting oligonucleotide probes were developed which differentiate the new species from the other Azospirillum species by whole-cell fluorescence hybridization. Strains of the new species are curved rods or S-shaped, 1.0-1.5 microm in width and 2,0-3.0 microm in length, Gram-negative and motile with a single polar flagellum. Optimum growth occurs at 30 degrees C and at pH values between 6.0 and 7.0. No growth takes place at 37 degrees C. They have a respiratory type of metabolism, grow well on arabinose, D-fructose, gluconate, glucose, glycerol, malate, mannitol and sorbitol. They differ from A. largimobile and A. lipoferum by their inability to use N-acetylglucosamine and D-ribose, from A. lipoferum by their ability to grow without biotin supplementation and from A. brasilense by their growth with D-mannitol and D-sorbitol as sole carbon sources. Nitrogen fixation occurs in microaerobic nitrogen-limited conditions. For this species, the name Azospirillum doebereinerae sp. nov. is suggested, with strain GSF71T as the type strain (= DSM 13131T; reference strain Ma4 = DSM 13400). Its G+C content is 70.7 mol%.

Azospirillum↗

PubMed2003

Population dynamics of Gluconacetobacter diazotrophicus in sugarcane cultivars and its effect on plant growth.

Different experiments have estimated that the contribution of biological nitrogen fixation (BNF) is largely variable among sugarcane cultivars. Which bacteria are the most important in sugarcane-associated BNF is unknown. However, Gluconacetobacter diazotrophicus has been suggested as a strong candidate responsible for the BNF observed. In the present study, bacteria-free micropropagated plantlets of five sugarcane cultivars were inoculated with three G. diazotrophicus strains belonging to different genotypes. Bacterial colonization was monitored under different nitrogen fertilization levels and at different stages of plant growth. Analysis of the population dynamics of G. diazotrophicus strains in the different sugarcane varieties showed that the bacterial populations decreased drastically in relation to plant age, regardless of the nitrogen fertilization level, bacterial genotype or sugarcane cultivars. However, the persistence of the three strains was significantly longer in some cultivars (e.g., MEX 57-473) than in others (e.g., MY 55-14). In addition, some strains (e.g., PAl 5(T)) persisted for longer periods in higher numbers than other strains (e.g., PAl 3) inside plants of all the cultivars tested. Indeed, the study showed that the inoculation of G. diazotrophicus may be beneficial for sugarcane plant growth, but this response is dependent both on the G. diazotrophicus genotype and the sugarcane variety. The most positive response to inoculation was observed with the combination of strain PAl 5(T) and the variety MEX 57-473. Although the positive effect on sugarcane growth apparently occurred by mechanisms other than nitrogen fixation, the results show the importance of the sugarcane variety for the persistence of the plant-bacteria interaction, and it could explain the different rates of BNF estimated among sugarcane cultivars.

Gluconacetobacter↗

PubMed1981

A general method for site-directed mutagenesis in prokaryotes.

The genetic analysis of genes from prokaryotic species for which experimental genetic systems have not yet been developed is often limited by the difficulty of producing mutations in those genes. We report here a general technique applicable to Gram-negative prokaryotes for site-directed mutagenesis of cloned DNA fragments which we have applied to the study of the symbiotic nitrogen fixation genes of Rhizobium meliloti. In particular, we mutagenized cloned R. meliloti restriction fragments in Escherichia coli with transposon Tn5 and then replaced the wild-type parental DNA sequences with the mutant DNA sequences in the R. meliloti genome. Using this method we show that an R. meliloti DNA restriction fragment, cloned previously on the basis of homology to Klebsiella pneumoniae nif genes, contains gene(s) essential for symbiotic nitrogen fixation. In addition, we use this method to construct a physical genetic map of a subset of the R. meliloti nif genes.

Cells↗

PubMed1992

Interactions of monocrotophos and quinalphos with Anabaena torulosa isolated from rice soil.

The interaction of insecticide combinations of monocrotophos and quinalphos on Anabaena torulosa, by the criteria of absorbance (OD) and packed cell volume (PCV) of the culture, content of chlorophyll a, phycocyanin, carotenoids, total protein, DNA and RNA, heterocyst differentiation and nitrogen fixation, were assessed. In general, monocrotophos and quinalphos, in combination, interacted significantly yielding three different responses viz., additive, antagonistic or synergistic. The nature of the interaction found with OD and PCV was nearly the same for a particular concentration of the insecticides. However, no consistent interaction was observed with respect to carotenoids and phycocyanin. The three types of interaction were noticed for total protein, DNA and RNA. Interestingly, the insecticide combinations at lower concentrations yielded all interaction responses for heterocyst frequency and nitrogenase activity. But, higher concentrations, in combination, resulted in synergism for heterocyst differentiation and nitrogen fixation.

Anabaena↗

PubMedDate not supplied

[Nitrogen fixing activity of pea nodule bacteria during different phases of host plant development].

The activity of nitrogenase in nodules of pea plants and the rate of diurnal nitrogen fixation were studied at different phases of the plant growth using the acetylene and isotope techniques. At the same time, the morphology of nodule bacteria was studied by electron and phase-contrast microscopy. About 90% of the bacteria in nodules were found in the form of bacteroids from the early phase of the plant growth (budding) to ripening when gemmated arthrospores could be observed on some bacteroids. The highest activity of nitrogenase and nitrogen fixation was detected at the reproductive stage of growth. Therefore, in order to obtain more active strains of pea nodule bacteria, they should be isolated from nodules at this stage of plant growth.

Fabaceae↗

PubMed2004

cDNA macroarray analysis of gene expression in ineffective nodules induced on the Lotus japonicus sen1 mutant.

The Lotus japonicus sen1 mutant forms ineffective nodules in which development is arrested at the stage of bacterial differentiation into nitrogen-fixing bacteroids. Here, we used cDNA macroarray systems to compare gene expression in ineffective nodules induced on the sen1 mutant with gene expression in wild-type nodules, in order to identify the host plant genes that are involved in nitrogen fixation. Macroarray analysis coupled with Northern blot analysis revealed that the expression of 18 genes was significantly enhanced in ineffective sen1 nodules, whereas the expression of 30 genes was repressed. Many of the enhanced genes encoded hydrolase enzymes, such as cysteine proteinase and asparaginase, that might function in the early senescence of sen1 nodules. By contrast, the repressed genes encoded nodulins, enzymes that are involved in carbon and nitrogen metabolism, membrane transporters, enzymes involved in phytohormone metabolism and secondary metabolism, and regulatory proteins. These proteins might have a role in the establishment of nitrogen fixation. In addition, we discovered two novel genes that encoded glutamate-rich proteins and were localized in the vascular bundles of the nodules. The expression of these genes was repressed in the ineffective nodules, which had lower levels of nitrogenase activity.

Amino Acid Sequence↗

PubMed2003

The soybean NRAMP homologue, GmDMT1, is a symbiotic divalent metal transporter capable of ferrous iron transport.

Iron is an important nutrient in N2-fixing legume root nodules. Iron supplied to the nodule is used by the plant for the synthesis of leghemoglobin, while in the bacteroid fraction, it is used as an essential cofactor for the bacterial N2-fixing enzyme, nitrogenase, and iron-containing proteins of the electron transport chain. The supply of iron to the bacteroids requires initial transport across the plant-derived peribacteroid membrane, which physically separates bacteroids from the infected plant cell cytosol. In this study, we have identified Glycine max divalent metal transporter 1 (GmDmt1), a soybean homologue of the NRAMP/Dmt1 family of divalent metal ion transporters. GmDmt1 shows enhanced expression in soybean root nodules and is most highly expressed at the onset of nitrogen fixation in developing nodules. Antibodies raised against a partial fragment of GmDmt1 confirmed its presence on the peribacteroid membrane (PBM) of soybean root nodules. GmDmt1 was able to both rescue growth and enhance 55Fe(II) uptake in the ferrous iron transport deficient yeast strain (fet3fet4). The results indicate that GmDmt1 is a nodule-enhanced transporter capable of ferrous iron transport across the PBM of soybean root nodules. Its role in nodule iron homeostasis to support bacterial nitrogen fixation is discussed.

Amino Acid Sequence↗

PubMed1999

High-resolution transcriptional analysis of the symbiotic plasmid of Rhizobium sp. NGR234.

Most of the bacterial genes involved in nodulation of legumes (nod, nol and noe ) as well as nitrogen fixation (nif and fix ) are carried on pNGR234a, the 536 kb symbiotic plasmid (pSym) of the broad-host-range Rhizobium sp. NGR234. Putative transcription regulators comprise 24 of the predicted 416 open reading frames (ORFs) contained on this replicon. Computational analyses identified 19 nod boxes and 16 conserved NifA-sigma54 regulatory sequences, which are thought to co-ordinate the expression of nodulation and nitrogen fixation genes respectively. To analyse transcription of all putative ORFs, the nucleotide sequence of pNGR234a was divided into 441 segments designed to represent all coding and intergenic regions. Each of these segments was amplified by polymerase chain reactions, transferred to filters and probed with radioactively labelled RNA. RNA was extracted from bacterial cultures grown under various experimental conditions, as well as from bacteroids of determinate and indeterminate nodules. Generally, genes involved in the synthesis of Nod factors (e.g. the three hsn loci) were induced rapidly after the addition of flavonoids, whereas others thought to act within the plant (e.g. those encoding the type III secretion system) responded more slowly. Many insertion (IS) and transposon (Tn)-like sequences were expressed strongly under all conditions tested, while a number of loci other than those known to encode nod, noe, nol, nif and fix genes were also transcribed in nodules. Many more diverse transcripts were found in bacteroids of determinate as opposed to indeterminate nodules.

Base Sequence↗

PubMed2004

Nitrogen-fixing symbiosis between photosynthetic bacteria and legumes.

Rhizobia having photosynthetic systems form nitrogen-fixing nodules on the stem and/or root of some species of the legumes Aeschynomene and Lotononis. This review is focused on the recent knowledge about the physiology, genetics and role of the photosystem in these bacteria. Photosynthetic electron transport seems to involve reaction centers, soluble cytochrome c2 and cytochrome bc1. Anaerobically, the electron transport system becomes over-reduced. The photosynthesis genes have been partially characterized; their organization is classical but their regulation is unusual as it is activated by far-red light via a bacteriophytochrome. This original mechanism of regulation seems well adapted to promote photosynthesis during stem symbiosis. Photosynthesis plays a major role in the efficiency of stem nodulation. It is also observed that infrared light stimulates nitrogen fixation in nodules containing photosynthetic bacteroids, suggesting that photosynthesis may additionally provides energy for nitrogen fixation, allowing for more efficient plant growth. Other aspects of these bacteria are discussed, in particular their taxonomic position and nodulation ability, the role of carotenoids and the potential for application of photosynthetic rhizobia in rice culture.

Journal Article↗

PubMed1977

Isolation of an iron-molybdenum cofactor from nitrogenase.

A method for the isolation of an iron-molybdenum cofactor (FeMoCo) from component I of nitrogenase is described. This method is used to isolate FeMoCo from aerobic, anaerobic, facultative, and photosynthetic nitrogen-fixing organisms. The Fe/Mo ratio in the FeMoCo from Azotobacter vinelandii and Clostridium pasteurianum is 8:1. The FeMoCo contains six atoms of acid-labile sulfide per eight Fe atoms. Crystalline component I from A. vinelandii contains 2 Mo, 33 Fe, and 27 acid-labile sulfide atoms per molecular weight of 250,000. The specific activity of FeMoCo is 425 nmol of C(2)H(4) formed/min per nmol of Mo. There is better than 98% reconstitution between FeMoCo and inactive component I in A. vinelandii mutant strain UW45. The FeMoCo yield from component I is about 90%. FeMoCo from nitrogenase component I of C. pasteurianum, Klebsiella pneumoniae, Bacillus polymyxa, and Rhodospirillum rubrum activates inactive component I in an extract from mutant strain UW45 and follows saturation kinetics. The FeMoCo in various nitrogen-fixing organisms seems to be very similar. Wild-type A. vinelandii derepressed for nitrogenase synthesis in tungsten-containing medium and K. pneumoniae mutant strain UN109 are also activated in vitro by FeMoCo. The FeMoCo is very sensitive to oxygen, but is stable even at room temperature as long as it is kept anaerobic and in N-methylformamide, the solvent used for its isolation. FeMoCo is unstable in an aqueous environment, even though it is kept strictly anaerobic. Knowledge of the structure of this cofactor should be useful for understanding the role of molybdenum at the active site of nitrogenase, role of ligands close to molybdenum in electron and proton transfer, and the catalytic mechanism of nitrogen fixation. The FeMoCo might be used as a model for synthesizing catalysts for chemical nitrogen fixation.

Aerobiosis↗

PubMedDate not supplied

Modeling culture profiles of the heterocystous N2-fixing cyanobacterium Anabaena flos-aquae.

Heterocyst differentiation is a unique feature of nitrogen-fixing cyanobacteria, potentially important for photobiological hydrogen production. Despite the significant advances in genetic investigation on heterocyst differentiation, there were no quantitative culture-level models that describe the effects of cellular activities and cultivation conditions on the heterocyst differentiation. Such a model was developed in this study, incorporating photosynthetic growth of vegetative cells, heterocyst differentiation, self-shading effect on light penetration, and nitrogen fixation. The model parameters were determined by fitting experimental results from the growth of the heterocystous cyanobacterium Anabaena flos-aquae CCAP 1403/13f in media without and with different nitrate concentrations and under continuous illumination of white light at different light intensities (2, 5, 10, 17, 20 and 50 microE m-2 s-1). The model describes the experimental profiles well and gives reasonable predictions even for the transition of growth from that on external N source to that via nitrogen fixation, responding to the change in external N concentrations. The significance and implications of the best-fit values of the model parameters are discussed.

Bioreactors↗

PubMed1980

[Glucose metabolism in isolated bacteroids of lupine nodules].

The ability of the bacteroids Rhizobium lupini isolated from the nodules at the stage of active nitrogen fixation to consume [1-14C]- and [2-14C] glucose was studied. 5 min after glucose addition to the cell suspension the label is rapidly incorporated into the organic acids, amino acids and sugars, which is indicative of glucose penetration inside the bacteroids and of a high rate of its catabolism. The rapid incorporation of the label into all Krebs cycle metabolites and the activities of citrate (si)-synthase, isocitrate dehydrogenase, succinate dehydrogenase and malate dehydrogenase, which are positively correlated with the rate of the nitrogen fixation process in the course of plant vegetation provide evidence for the occurrence of the tricarboxylic acid cycle in the bacteroids.

Carbon Radioisotopes↗

PubMedDate not supplied

Activation of extra copies of genes coding for nitrogenase in Rhodopseudomonas capsulata.

Biological nitrogen fixation requires the nitrogenase enzyme complex, ATP, and a strong reductant. Klebsiella pneumoniae contains 15 linked nitrogen fixation (nif) genes, three of which, nifH, nifD and nifK have been sufficiently conserved in evolution that cloned K. pneumoniae nifHDK DNA will hybridize to DNA sequences from every nitrogen-fixing bacterium examined to date, including the purple, non-sulphur bacterium Rhodopseudomonas capsulata, in which one complete nifHDK operon has been mapped. Using cloned K. pneumoniae nifHDK DNA we report here that R. capsulata contains multiple copies of the genes for nitrogenase components. Two regions containing sequences homologous to all three nif structural genes have been identified, and mutations in one region produced a Nif- phenotype. Nif+ pseudorevertants were derived from these mutants, some of which retained the original mutation suggesting that some of the extra nif gene sequences can be functionally activated.

Chromosome Deletion↗

PubMed2001

Nitrogenase activity in cyanobacteria measured by the acetylene reduction assay: a comparison between batch incubation and on-line monitoring.

A new on-line method for measuring acetylene reduction is described. It consists of a gas-flow cell connected to an electronic gas-mixing system and an automatic sample loop in the gas chromatograph. Alternatively, ethylene can be determined by using laser-based trace gas detection. The laser-based trace gas detection technique achieves a detection limit that is three orders of magnitude better than gas chromatography. We have applied the on-line method to the measurement of nitrogen fixation in a culture of the heterocystous cyanobacterium Nodularia spumigena and compared it with conventional batch-type incubations. Incubation of N. spumigena in the gas-flow cell resulted in very short response times with a steady-state flux of ethylene obtained within 2 min. Nitrogenase was shown to respond immediately to changes in light and oxygen. Monitoring of nitrogenase activity could be continued for several hours without having a negative impact on nitrogen fixation rates in N. spumigena. This was not the case in batch incubations, in which changes in nitrogenase activities were recorded during incubations, probably as a result of varying oxygen concentrations. It was therefore concluded that the on-line method is superior to batch incubations when rates of nitrogenase activity are to be measured. The method is suitable for natural samples (water or sediment).

Acetylene↗