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PubMed1977

Effect of boron and cadmium on nitrogen fixation in soil.

The influence of different doses of boron (100, 500 and 1000 ppm) and cadmium (50, 100, 500, 1000 and 2000 ppm) on the activity of nitrogen fixation in the sandy and alluvial soil has been studied. Almost all doses of boron stimulated this process except 1000 ppm of B added to the sandy soil. All the doses of cadmium also exerted a positive effect on the nitrogen fixation, but only during the first 3 months of the experiment, later (after 12 months) Cd decreased the activity of this process. The most marked effect to the examined elements was pronounced in the sandy soil.

Boron↗
PubMed1976

Nitrogen fixation in the muskeg ecosystem of the James Bay Lowlands, Northern Ontario.

The acetylene-reduction assay was used for in situ and laboratory assessment of biological nitrogen fixation in the acidic, waterlogged, muskeg ecosystem of the southern James Bay area, in the region of Moosonee, Ontario. In situ assays and subsequent laboratory experimentation revealed that nitrogenase activity was predominately a function of the activities of heterocystic blue-green bacteria associated with surface water, with the phyllosphere of mosses, and with at least one lichen, a species of Peltigera. No such in situ activity was detected in the subsurface organic material, even when such material was amended with glucose. However, under laboratory conditions at 20 degrees C, nitrogenase activity was evident in the subsurface layers after an extended lag and was shown to be higher under anaerobic than under aerobic conditions, to have an optimum temperature range extending about a mean of 20 degrees C, and to be stimulated by glucose. This potential for subsurface nitrogen fixation proved to be related to the presence of microorganisms existing in anaerobic microsites within the organic layers and no microorganisms capable of fixation could be detected under aerobic incubation.

Acetylene↗
PubMed1989

In vitro activity of the nitrogen fixation regulatory protein NIFA.

We have detected activity of the nitrogen fixation regulatory protein NIFA of Klebsiella pneumoniae in vitro. To do so we directed synthesis of NIFA in a coupled transcription-translation system and detected its ability to activate expression of a translational fusion between the nifH and lacZ genes. We infer that NIFA stimulates initiation of transcription by sigma 54 holoenzyme from the nifHDK promoter. The activity of NIFA was lost rapidly under both aerobic and anaerobic conditions at 30 degrees C and was lost somewhat less rapidly at 0 degrees C. Loss of activity was not accompanied by degradation of NIFA polypeptide. Loss of activity was approximately exponential and was not affected by NIFA concentration over a 5-fold range. Therefore, NIFA inactivation does not appear to be due to self-association. We found that the factor in crude extracts previously demonstrated to bind to the nifHDK promoter-regulatory region [Beynon, J., Cannon, M., Buchanan-Wollaston, V., and Cannon, F. (1983) Cell 34, 665-671] is the integration host factor, which is known to bend DNA. Since the binding site for integration host factor lies between the upstream binding site for NIFA and the nifHDK promoter, integration host factor may bend the DNA between these two sites to facilitate productive interactions between NIFA and sigma 54 holoenzyme.

Bacterial Proteins↗
PubMed2003

Genomics insights into symbiotic nitrogen fixation.

Following an interaction with rhizobial soil bacteria, legume plants are able to form a novel organ, termed the root nodule. This organ houses the rhizobial microsymbionts, which perform the biological nitrogen fixation process resulting in the incorporation of ammonia into plant organic molecules. Recent advances in genomics have opened exciting new perspectives in this field by providing the complete gene inventory of two rhizobial microsymbionts. The complete genome sequences of Mesorhizobium loti, the symbiont of several Lotus species, and Sinorhizobium meliloti, the symbiont of alfalfa, were determined and annotated in detail. For legume macrosymbionts, expressed sequence tag projects and expression analyses using DNA arrays in conjunction with proteomics approaches have identified numerous genes involved in root nodule formation and nitrogen fixation. The isolation of legume genes by tagging or positional cloning recently allowed the identification of genes that control the very early steps of root nodule organogenesis.

Gene Expression Regulation, Plant↗
PubMed1994

Identification and characterization of a novel Bradyrhizobium japonicum gene involved in host-specific nitrogen fixation.

To understand the genetic mechanism of host specificity in the interaction between rhizobia and their hosts, it is important to identify genes that influence both early and late steps in symbiotic development. This paper focuses on the little-understood genetics of host-specific nitrogen fixation. A deletion mutant of Bradyrhizobium japonicum, strain NAD163, was found to induce effective, nitrogen-fixing nodules on soybean and siratro plants but produced ineffective nodules on cowpea plants. Additional transposon and deletion mutants defined a small region that conferred this phenotype, and this region was sequenced to identify two putative open reading frames (ORFs). Data indicate that only one of these ORFs is detectable in bacteroids. This ORF was termed hsfA, with a predicted protein product of 11 kDa. The transcriptional start site of hsfA was determined and found to coincide with a predicted RpoN-dependent promoter. Microscopic studies of nodules induced by the wild type and hsfA mutants on cowpea and soybean plants indicate that the cowpea mutant nodules are slow to develop. The data indicate that hsfA appears to play a crucial role in bacteroid development on cowpea but does not appear to be essential for nitrogen fixation on the other hosts tested.

Amino Acid Sequence↗
PubMed1978

Nitrogen fixation and hydrogen metabolism in photosynthetic bacteria.

The photosynthetic bacteria are found in a wide range of specialized aquatic environments. These bacteria represent important members of the microbial community since they are capable of carrying out two of the most important processes on earth, namely, photosynthesis and nitrogen fixation, at the expense of solar energy. Since the discovery that these bacteria could fix atmospheric nitrogen, there has been an intensification of studies relating to both the biochemistry and physiology of this process. The practical importance of this field is emphasized by a consideration of the tremendous energy input required for the production of artificial nitrogenous fertilizer. The present communication aims to briefly review the current state of knowledge relating to certain aspects of nitrogen fixation by the photosynthetic bacteria. The topics that will be discussed include a general survey of the nitrogenase system in the various photosynthetic bacteria, the regulation of both nitrogenase biosynthesis and activity, recent advances in the genetics of the nitrogen fixing system, and the hydrogen cycle in these bacteria. In addition, a brief discussion of some of some of the possible practical applications provided by the photosynthetic bacteria will be presented.

Bacteria↗
PubMed1988

Characterization of the fixABC region of Azorhizobium caulinodans ORS571 and identification of a new nitrogen fixation gene.

The fast growing strain, Azorhizobium caulinodans ORS571, isolated from stem nodules of the tropical legume Sesbania rostrata, can grow in the free-living state at the expense of molecular nitrogen. Five point mutants impaired in nitrogen fixation in the free-living state have been complemented by a plasmid containing the cloned fix-ABC region of strain ORS571. Genetic analysis of the mutants showed that one was impaired in fixC, one in fixA and the three others in a new gene, located upstream from fixA and designated nifO. Site-directed Tn5 mutagenesis was performed to obtain Tn5 insertions in fixB and fixC. The four genes are required for nitrogen fixation both in the free-living state and under symbiotic conditions. The nucleotide sequence of nifO was established. The gene is transcribed independently of fixA and does not correspond to fixX, recently identified in Rhizobium meliloti and R. leguminosarum. Biochemical analysis of the five point mutants showed that they synthesized normal amounts of nitrogenase components. It is unlikely that fixA, fixC and nifO are involved in electron transport to nitrogenase. FixC could be required for the formation of a functional nitrogenase component 2.

Amino Acid Sequence↗
PubMed1994

Redundancy of the conserved His residue in Azotobacter vinelandii NifL, a histidine autokinase homologue which regulates transcription of nitrogen fixation genes.

The NifL protein of Azotobacter vinelandii inhibits NifA, the activator of nif (nitrogen fixation) transcription, in response to oxygen and fixed nitrogen. NifL shows strong homology in its C-terminal domain to the histidine autokinase domains of the canonical two-component sensor proteins, including the region around His-304, which corresponds to the residue known to be phosphorylated in other systems. To examine the mechanism of sensory transduction by NifL, mutations encoding 10 substitutions for His-304 were introduced into the A. vinelandii chromosome. Regulation of nif transcription was measured using acetylene reduction and RNA blots. The substitutions His-304-->Arg and His-304-->Pro impaired regulation by both fixed nitrogen and oxygen, but substitution of Ala, Phe, Ile, Lys, Asn, Ser, Thr, Val had no effect. None of the mutants, including His-304-->Arg and His-304-->Pro, excreted ammonium during diazotrophy, a phenotype of nifL deletion mutants, suggesting that the molecular basis of this effect differs from that responsible for the inhibition of nif transcription. The data show conclusively that phosphorylation of His-304 is not essential for any of the known functions of A. vinelandii NifL. Homology to the family of histidine autokinases is therefore inadequate evidence for a mechanism of sensory transduction involving phosphorylation of the conserved histidine residue.

Amino Acid Sequence↗
PubMed1998

The rnf gene products in rhodobacter capsulatus play an essential role in nitrogen fixation during anaerobic DMSO-dependent growth in the dark

The rnf genes in Rhodobacter capsulatus are essential for nitrogen fixation in the light. Because R. capsulatus grows readily on N2 in the dark by anaerobic respiration with dimethylsulfoxide, the diazotrophic capacities of various strains in the dark were examined. No rnf mutants tested grew diazotrophically, and a nonpolar fdxN-null mutant showed decreased diazotrophic growth in the dark, suggesting that the Rnf and FdxN proteins form the primary electron donor pathway to nitrogenase in the dark as well as in the light. Nonphotosynthetic mutants lacking the component of cyclic electron transport grew diazotrophically and the levels of Rnf proteins were similar to those of the wild-type. These results indicate that rnf gene products play an essential role in nitrogen fixation without any functional link to the cyclic electron transport system.

Journal Article↗
PubMed2001

PatS and products of nitrogen fixation control heterocyst pattern.

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 forms a developmental pattern of single heterocysts separated by approximately 10 vegetative cells. Heterocysts differentiate from vegetative cells and are specialized for nitrogen fixation. The patS gene, which encodes a small peptide that inhibits heterocyst differentiation, is expressed in proheterocysts and plays a critical role in establishing the heterocyst pattern. Here we present further analysis of patS expression and heterocyst pattern formation. A patS-gfp reporter strain revealed clusters of patS-expressing cells during the early stage of heterocyst differentiation. PatS signaling is likely to be involved in the resolution of these clusters. Differentiating cells were inhibited by PatS during the time period 6 to 12 h after heterocyst induction, when groups of differentiating cells were being resolved to a single proheterocyst. Increased transcription of patS during development coincided with expression from a new transcription start site. In vegetative cells grown on nitrate, the 5' end of a transcript for patS was localized 314 bases upstream from the first translation initiation codon. After heterocyst induction, a new transcript with a 5' end at -39 bases replaced the vegetative cell transcript. A patS mutant grown for several days under nitrogen-fixing conditions showed partial restoration of the normal heterocyst pattern, presumably because of a gradient of nitrogen compounds supplied by the heterocysts. The patS mutant formed heterocysts when grown in the presence of nitrate but showed no nitrogenase activity and no obvious heterocyst pattern. We conclude that PatS and products of nitrogen fixation are the main signals determining the heterocyst pattern.

Anabaena↗
PubMed2001

Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species.

Nitrogen-fixing bacteria were isolated from the stems of wild and cultivated rice on a modified Rennie medium. Based on 16S ribosomal DNA (rDNA) sequences, the diazotrophic isolates were phylogenetically close to four genera: Herbaspirillum, Ideonella, Enterobacter, and Azospirillum. Phenotypic properties and signature sequences of 16S rDNA indicated that three isolates (B65, B501, and B512) belong to the Herbaspirillum genus. To examine whether Herbaspirillum sp. strain B501 isolated from wild rice, Oryza officinalis, endophytically colonizes rice plants, the gfp gene encoding green fluorescent protein (GFP) was introduced into the bacteria. Observations by fluorescence stereomicroscopy showed that the GFP-tagged bacteria colonized shoots and seeds of aseptically grown seedlings of the original wild rice after inoculation of the seeds. Conversely, for cultivated rice Oryza sativa, no GFP fluorescence was observed for shoots and only weak signals were observed for seeds. Observations by fluorescence and electron microscopy revealed that Herbaspirillum sp. strain B501 colonized mainly intercellular spaces in the leaves of wild rice. Colony counts of surface-sterilized rice seedlings inoculated with the GFP-tagged bacteria indicated significantly more bacterial populations inside the original wild rice than in cultivated rice varieties. Moreover, after bacterial inoculation, in planta nitrogen fixation in young seedlings of wild rice, O. officinalis, was detected by the acetylene reduction and (15)N(2) gas incorporation assays. Therefore, we conclude that Herbaspirillum sp. strain B501 is a diazotrophic endophyte compatible with wild rice, particularly O. officinalis.

Acetylene↗
PubMed1983

Biological nitrogen fixation: primary structure of the Rhizobium trifolii iron protein gene.

Biological nitrogen fixation in the Rhizobium-legume symbiosis is dependent on the induction of a bacterially-encoded enzyme complex, nitrogenase. To examine the organization and expression of the genes encoding the components of nitrogenase in this complex system, these genes have been isolated from the legume symbiont Rhizobium trifolii by molecular cloning. DNA sequence analysis of the entire nifH gene (encoding the Fe-protein component of nitrogenase) and of the amino-terminal 141 codons of the nifD gene (encoding the alpha-subunit of the Mo-Fe protein) indicates that these genes are linked on a single operon in this strain. The Fe-protein amino acid sequence shares considerable homology with the sequence from other organisms, in particular the related organism Rhizobium meliloti (90% homology). The nif structural genes are preceded by a DNA sequence which is repeated at least three times in the Rhizobium trifolii genome.

Amino Acid Sequence↗
PubMed1991

Characterization of a novel Azorhizobium caulinodans ORS571 two-component regulatory system, NtrY/NtrX, involved in nitrogen fixation and metabolism.

Azorhizobium caulinodans ORS571 nifA regulation is partially mediated by the nitrogen regulatory gene ntrC. However, the residual nifA expression in ntrC mutant strains is still modulated by the cellular nitrogen and oxygen status. A second ntrC-homologous region, linked to ntrC, was identified and characterized by site-directed insertion mutagenesis and DNA sequencing. Tn5 insertions in this region cause pleiotropic defects in nitrogen metabolism and affect free-living as well as symbiotic nitrogen fixation. DNA sequencing and complementation studies revealed the existence of a bicistronic operon (ntrYX). NtrY is likely to represent the transmembrane 'sensor' protein element in a two-component regulatory system. NtrX shares a high degree of homology with NtrC proteins of other organisms and probably constitutes the regulator protein element. The regulation of the ntrYX and ntrC loci and the effects of ntrYX, ntrY and ntrX mutations on nifA expression were examined using beta-galactosidase gene fusions. NtrY/NtrX were found to modulate nifA expression and ntrYX transcription was shown to be partially under the control of NtrC.

Amino Acid Sequence↗
PubMed1987

Identification of two classes of Rhizobium phaseoli genes required for melanin synthesis, one of which is required for nitrogen fixation and activates the transcription of the other.

The symbiotic plasmid pRP2JI of Rhizobium phaseoli strain 8002 was shown to contain two separate regions of DNA which are required and sufficient for the synthesis of the pigment melanin. One of these regions containing the class II mel gene(s) was located to other genes involved in nodulation and in nitrogen fixation. Mutations in this region abolished both the ability to synthesize melanin and to fix nitrogen in Phaseolus bean root nodules. Mutations in the other, unlinked region, containing class I mel gene(s), also abolished melanin synthesis but did not affect symbiotic nitrogen fixation. Transcriptional fusions between the class I mel gene and the Escherichia coli lacZ gene were constructed and it was demonstrated that the class II mel gene(s) activated their transcription in free-living culture. Further, strains containing the cloned regulatory class II gene(s) synthesized melanin when growing in minimal medium, in contrast to wild-type strains which became pigmented only in complete medium containing yeast extract and tryptone. It was shown by hybridization experiments that the regulatory mel gene was closely linked to or may correspond to the regulatory nifA gene; a fragment of R. phaseoli DNA which included the class II gene(s) of R. phaseoli hybridized to a previously identified nifA-like gene of R. leguminosarum, the species that nodulates peas.

Bacterial Proteins↗
PubMed1986

Organization and characterization of genes essential for symbiotic nitrogen fixation from Bradyrhizobium japonicum I110.

A total of 96 independent Tn5 insertions within a 39-kilobase-pair (kbp) segment of chromosomal DNA containing the three structural genes for nitrogenase (nifH, nifD, and nifK) from Bradyhizobium japonicum I110 were obtained in Escherichia coli and transferred to the wild-type strain by marker exchange. Individual transconjugants containing a Tn5 insertion were inoculated onto Glycine max cv. Wilkin (soybeans) and analyzed for their effect on symbiotic nitrogen fixation. In addition to the three structural genes, genes essential for nitrogen fixation (fix genes) were located in three separate regions: 9 kbp upstream of the nifDK operon; 1.5 kbp downstream of the nifDK operon; 4.5 kbp upstream of nifH. All of the fix::Tn5 insertion strains formed nodules which contained low or undetectable levels of nitrogenase activity. Bacteroids isolated from these nodules had approximately the same levels of the nifDK and nifH transcripts as those detectable from nodules formed by the wild-type strain. Western blot analysis of bacteroid proteins from nodules formed by the fix::Tn5 mutants or the wild-type strain showed the presence of similar levels of the nitrogenase protein subunits. The region upstream of nifH was characterized further by DNA sequence analysis and was shown to contain the nifB gene. The coding sequence of the nifB gene consisted of 1,494 nucleotides and was preceded by putative promoter (5' GTGG-10 base pairs [bp] TTGCA 3') and upstream activator (5' TGT-4 bp-T-5 bp-ACA 3') sequences.

Bacterial Proteins↗
PubMed1993

Nucleotide sequence and genetic analysis of the region essential for functional expression of the gene for ferredoxin I, fdxN, in Rhodobacter capsulatus: sharing of one upstream activator sequence in opposite directions by two operons related to nitrogen fixation.

Nucleotide sequencing of the region upstream of two ferredoxin genes, fdxC and fdxN, of Rhodobacter capsulatus revealed the existence of one open reading frame (ORF), ORFU1, in the same orientation as these genes and two other ORFs, ORFU2 and ORFU3, in the opposite orientation. Two potential -24/-12 promoters were found in front of ORFU1 and ORFU2, respectively, and there was a putative upstream activator sequence (UAS) or NifA-binding site between them. The ORFs corresponded to no known nif genes. However, analysis of their putative products showed that the product of ORFU1 (M(r) 47,912) and that of ORFU3 (M(r) 19,090) had a flavodoxin-like domain and a 2[4Fe-4S] ferredoxin-like domain, respectively, and that the product of ORFU2 (M(r) 20,424) was a hydrophobic protein with six potential membrane-spanning portions. Results of interposon mutagenesis and complementation experiments indicated that ORFU2 but not ORFU1 is essential for nitrogen fixation and that additional gene(s) essential for nitrogen fixation must be present in the unsequenced region adjacent to ORFU3. Translational fusion analysis involving lacZYA and fdxN or ORFU3 provided evidence that the putative UAS is responsible for regulation of both ORFU1-fdxC-fdxN and ORFU2-ORFU3 operons in opposite orientations, and that the control of the latter is stricter than that of the former.

Amino Acid Sequence↗
PubMed1981

Heterotrophic growth and nitrogen fixation in the filamentous blue-green alga Anabaena sp.

Experiments were conducted to study the effect of five different organic substrates viz. lactose, fructose, mannose, xylose and sodium acetate on the heterotrophic growth characteristics with emphasis on growth, pigment composition, heterocyst frequency and nitrogen fixation of Anabaena sp. under light and dark conditions. Lactose was found to be the best carbon source to support the heterotrophic growth of the alga. Phycoerythrin pigments were synthesized in photoheterotrophic cultures but pigment production was not observed in dark-grown cultures. The alga fixed nitrogen to a limited extent in the dark in pure inorganic medium. But when suitable organic substrates were supplemented, appreciable nitrogen fixation was observed in dark heterotrophic as well as in photoheterotrophic cultures.

Acetates↗