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PubMed2006

Effect of systemic herbicides on N2-fixing and phosphate solubilizing microorganisms in relation to availability of nitrogen and phosphorus in paddy soils of West Bengal.

A field experiment has been conducted with four systemic herbicides viz., butachlor [N-(butoxymethyl)-2-chloro-2',6'-diethyl-acetanilide], fluchloralin [N-(2-chloroethyl)-(2,6-dinitro-N-propyl-4-trifluoromethyl) aniline], oxadiazon [5-terbutyl-3-(2,4-dichloro-5-isopro poxyphenyl)-1,3,4-oxadiazol-2-one] and oxyfluorfen [2-chloro-1-(3-ethoxy-4-nitrophenyl)-4-(trifluoromethyl) benzene] at their recommended field rates (2.0, 1.5, 0.4 and 0.12kga.i.ha(-1), respectively) to investigate their effects on growth and activities of aerobic non-symbiotic N(2)-fixing bacteria and phosphate solubilizing microorganisms in relation to availability of nitrogen and phosphorus in the rhizosphere soils as well as yield of the rice crop (Oryza sativa L cv. IR-36). Application of herbicides, in general, highly stimulated the population and activities of the target microorganisms, which resulted in a greater amount of atmospheric nitrogen fixation and phosphate solubilization in the rhizosphere soils of the test crop. The greater microbial activities subsequently augmented the mineralization and availability of nitrogen and phosphorus in the soil solution, which in turn increased the yield of the crop. Among the herbicides, oxyfluorfen was most stimulative followed by fluchloralin and oxadiazon in augmenting the microbial activities in soil. Butachlor also accentuated the mineralization and availability of nitrogen due to higher incitement of non-symbiotic N(2)-fixing bacteria in paddy soil. The grain and straw yields of the crop were also significantly increased due to the application of oxyfluorfen (20.2% and 21%) followed by fluchloralin (13.1% and 15.4%) and butachlor (9.1% and 10.2%), respectively.

Acetanilides↗

PubMed2004

Mutagenesis of the cysteine residues in the transcription factor NtcA from Anabaena PCC 7120 and its effects on DNA binding in vitro.

NtcA is a transcription factor found in a wide variety of cyanobacteria. It is a key component in the control of the nitrogen metabolism, and regulates genes involved in ammonia assimilation, heterocyst differentiation and nitrogen fixation. NtcA expression is subject to nitrogen control, but there is also evidence that the binding of NtcA to DNA can be regulated by a redox mechanism involving the two cysteine residues in the NtcA protein from Anabaena PCC 7120. In order to investigate this further, the two cysteine residues in NtcA were mutated into alanine to give four variants of the protein: wild-type NtcA, the point-mutated variants Cys157Ala and Cys164Ala, as well as the double mutant Cys157Ala/Cys164Ala. The binding of a DNA probe containing a palindromic NtcA-binding motif was investigated by gel mobility shift analysis under non-reducing and reducing conditions. The experiments show that the DNA binding in vitro is stronger in the presence of the reducing agent DTT than in its absence. However, this effect is not due to breaking of a disulfide bond between the cysteine residues, since the double mutant containing no cysteines was also affected by DTT. A molecular model of a monomer of NtcA, based on the homologous cAMP receptor protein structure, was created in order to locate the positions of the cysteine residues. The NtcA model suggested that the positions of the sulfur atoms are not compatible with formation of a bond between them.

Anabaena↗

PubMed2003

Phylogenetic diversity of nitrogenase (nifH) genes in deep-sea and hydrothermal vent environments of the Juan de Fuca Ridge.

The subseafloor microbial habitat associated with typical unsedimented mid-ocean-ridge hydrothermal vent ecosystems may be limited by the availability of fixed nitrogen, inferred by the low ammonium and nitrate concentrations measured in diffuse hydrothermal fluid. Dissolved N2 gas, the largest reservoir of nitrogen in the ocean, is abundant in deep-sea and hydrothermal vent fluid. In order to test the hypothesis that biological nitrogen fixation plays an important role in nitrogen cycling in the subseafloor associated with unsedimented hydrothermal vents, degenerate PCR primers were designed to amplify the nitrogenase iron protein gene nifH from hydrothermal vent fluid. A total of 120 nifH sequences were obtained from four samples: a nitrogen-poor diffuse vent named marker 33 on Axial Volcano, sampled twice over a period of 1 year as its temperature decreased; a nitrogen-rich diffuse vent near Puffer on Endeavour Segment; and deep seawater with no detectable hydrothermal plume signal. Subseafloor nifH genes from marker 33 and Puffer are related to anaerobic clostridia and sulfate reducers. Other nifH genes unique to the vent samples include proteobacteria and divergent Archaea. All of the nifH genes from the deep-seawater sample are most closely related to the thermophilic, anaerobic archaeon Methanococcus thermolithotrophicus (77 to 83% amino acid similarity). These results provide the first genetic evidence of potential nitrogen fixers in hydrothermal vent environments and indicate that at least two sources contribute to the diverse assemblage of nifH genes detected in hydrothermal vent fluid: nifH genes from an anaerobic, hot subseafloor and nifH genes from cold, oxygenated deep seawater.

Archaea↗

PubMed1985

Identification of facultatively heterotrophic, N2-fixing cyanobacteria able to receive plasmid vectors from Escherichia coli by conjugation.

Plasmid vectors transferable by conjugation from Escherichia coli to obligately photoautotrophic strains of Anabaena spp. are also transferred to and maintained in heterotrophic, filamentous cyanobacteria of the genus Nostoc. These organisms can be used for the genetic analysis of oxygenic photosynthesis, chromatic adaptation, nitrogen fixation, and heterocyst development.

Conjugation, Genetic↗

PubMed2006

High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry.

BACKGROUND: Secondary-ion mass spectrometry (SIMS) is an important tool for investigating isotopic composition in the chemical and materials sciences, but its use in biology has been limited by technical considerations. Multi-isotope imaging mass spectrometry (MIMS), which combines a new generation of SIMS instrument with sophisticated ion optics, labeling with stable isotopes, and quantitative image-analysis software, was developed to study biological materials. RESULTS: The new instrument allows the production of mass images of high lateral resolution (down to 33 nm), as well as the counting or imaging of several isotopes simultaneously. As MIMS can distinguish between ions of very similar mass, such as 12C15N- and 13C14N-, it enables the precise and reproducible measurement of isotope ratios, and thus of the levels of enrichment in specific isotopic labels, within volumes of less than a cubic micrometer. The sensitivity of MIMS is at least 1,000 times that of 14C autoradiography. The depth resolution can be smaller than 1 nm because only a few atomic layers are needed to create an atomic mass image. We illustrate the use of MIMS to image unlabeled mammalian cultured cells and tissue sections; to analyze fatty-acid transport in adipocyte lipid droplets using 13C-oleic acid; to examine nitrogen fixation in bacteria using 15N gaseous nitrogen; to measure levels of protein renewal in the cochlea and in post-ischemic kidney cells using 15N-leucine; to study DNA and RNA co-distribution and uridine incorporation in the nucleolus using 15N-uridine and 81Br of bromodeoxyuridine or 14C-thymidine; to reveal domains in cultured endothelial cells using the native isotopes 12C, 16O, 14N and 31P; and to track a few 15N-labeled donor spleen cells in the lymph nodes of the host mouse. CONCLUSION: MIMS makes it possible for the first time to both image and quantify molecules labeled with stable or radioactive isotopes within subcellular compartments.

Adipocytes↗

PubMed1980

Mapping and characterization of the hisD-unlinked nif mutants in Klebsiella pneumoniae.

Two hisD-unlinked genes NifC5 and NifC7 are mapped in the chromosome of K. pneumoniae. The sequence of NifC5 and NifC7 is suggested as NifC5--gltB--NifC7--argG. The P1 infected E. coli lysate can transduce the mutant C-7 to be Nif+ transductant, yet fails to transduce the hisD-linked nif mutants to be Nif+ ones. This indicates that the gene encoding C-7 is not the structural gene of nitrogen fixation and is present in E. coli. It is actually a gene specifying the glutamate synthetase. SDS electrophoresis shows the marked low content of nitrogen reductase and immunoelectrophoretic test reveals the reduced amount of both nitrogenase and nitrogen reductase in the mutant cells.

Chromosome Mapping↗

PubMed2026

Mikania micrantha invasion restructures rhizosphere nitrogen cycling through enzyme activation, microbial recruitment, and allelopathic regulation.

BACKGROUND: Plant invasions profoundly influence terrestrial ecosystems by reshaping nutrient cycling processes. However, the mechanisms through which invasive plants such as Mikania micrantha modulate soil nitrogen (N) cycling and microbial communities remain insufficiently explored. Moreover, comparative studies with indigenous congener are scarce, limiting insights into whether such effects reflect species-specific strategies or genus-wide traits. This study investigates how M. micrantha modulates nitrogen metabolic pathways and rhizosphere microecology using combined metagenomic and metabolomic analyses. RESULTS: Integrated analyses revealed that M. micrantha established a distinctive "high total nitrogen-low mineral nitrogen" profile in the rhizosphere soil. Metagenomic profiling showed consistent enrichment of key ammonium assimilation enzymes, including glutamine synthetase and glutamate dehydrogenase, promoting enhanced incorporation of NH₄⁺ into organic nitrogen pools. In contrast, genes encoding nitrate reductase and nitrate transporters were significantly lower in relative abundance, limiting nitrate assimilation. Mikania micrantha also selectively enriched nitrogen-fixing microbes (notably rhizobia genera) and plant growth-promoting rhizobacteria (PGPR), thereby enhancing biological nitrogen fixation capacity. Metabolomic analysis further identified several allelopathic compounds in invaded soils at higher relative abundance, particularly epicatechin, which exhibited inhibitory effects on nitrifying bacteria. Compared with the congener Mikania cordata, which exerted weaker impacts on soil nitrogen cycling and microbial assembly, M. micrantha deployed a more comprehensive strategy integrating biochemical, microbial, and metabolic regulation. CONCLUSIONS: These findings demonstrate that under greenhouse-controlled conditions, M. micrantha reconfigures rhizosphere nitrogen cycling through a multi-dimensional strategy that couples biochemical regulation, microbial recruitment, and metabolite-mediated interference, thereby suggesting a potential mechanism that may contribute to its ecological advantage in natural settings. Video Abstract.

Rhizosphere↗

PubMed2002

Characterization of an autotrophic sulfide-oxidizing marine Arcobacter sp. that produces filamentous sulfur.

A coastal marine sulfide-oxidizing autotrophic bacterium produces hydrophilic filamentous sulfur as a novel metabolic end product. Phylogenetic analysis placed the organism in the genus Arcobacter in the epsilon subdivision of the Proteobacteria. This motile vibrioid organism can be considered difficult to grow, preferring to grow under microaerophilic conditions in flowing systems in which a sulfide-oxygen gradient has been established. Purified cell cultures were maintained by using this approach. Essentially all 4',6-diamidino-2-phenylindole dihydrochloride-stained cells in a flowing reactor system hybridized with Arcobacter-specific probes as well as with a probe specific for the sequence obtained from reactor-grown cells. The proposed provisional name for the coastal isolate is "Candidatus Arcobacter sulfidicus." For cells cultured in a flowing reactor system, the sulfide optimum was higher than and the CO(2) fixation activity was as high as or higher than those reported for other sulfur oxidizers, such as Thiomicrospira spp. Cells associated with filamentous sulfur material demonstrated nitrogen fixation capability. No ribulose 1,5-bisphosphate carboxylase/oxygenase could be detected on the basis of radioisotopic activity or by Western blotting techniques, suggesting an alternative pathway of CO(2) fixation. The process of microbial filamentous sulfur formation has been documented in a number of marine environments where both sulfide and oxygen are available. Filamentous sulfur formation by "Candidatus Arcobacter sulfidicus" or similar strains may be an ecologically important process, contributing significantly to primary production in such environments.

Arcobacter↗

PubMed1986

Proline inhibits N2-fixation in Anabaena 7120.

The uptake and metabolism of proline by Anabaena 7120 is demonstrated. Uptake is linear over the first hour and it appears that the rate of proline oxidation is sufficient to account for growth is suggested by the finding that exogenous proline represses heterocyst differentiation and inhibits nitrogen fixation in Anabaena 7120, without affecting growth rate.

Cyanobacteria↗

PubMed1993

Homology of the NifS family of proteins to a new class of pyridoxal phosphate-dependent enzymes.

Iterative profile sequence analysis reveals a remote homology of peroxisomal serine-pyruvate aminotransferases from mammals to the small subunit of soluble hydrogenases from cyanobacteria, an isopenicillin N epimerase, the NifS gene products from bacteria and yeast, and the phosphoserine aminotransferase family. All members of this new class whose function is known are pyridoxal phosphate-dependent enzymes, yet they have distinct catalytic activities. Upon alignment, a lysine around position 200 remains invariant and is predicted to be the pyridoxal phosphate-binding residue. Based on the detected homology, it is predicted that NifS has also a pyridoxal phosphate-dependent serine (or related) aminotransferase function associated with nitrogen economy and/or protection during nitrogen fixation.

Amino Acid Sequence↗

PubMed1988

Transformation of Azotobacter vinelandii OP with a broad host range plasmid containing a cloned chromosomal nif-DNA marker.

The non-nitrogen-fixing (Nif-) strain UW10 of Azotobacter vinelandii OP (UW) was naturally induced to competence and transformed with broad host range plasmid pKT210 containing the cloned wild-type nif-10 locus from A. vinelandii UW (Nif+); this marker was unable to complement the nif-10 mutation in trans, but could through recombination with the chromosome. The most frequent type of transformation event observed was recombination between the homologous regions of the plasmid and chromosome (producing Nif+ transformants) with loss of the plasmid vector. At a substantially lower frequency, transformants expressing the plasmid-encoded antibiotic resistance determinants were isolated which were phenotypically Nif-. Agarose gel electrophoresis showed that these transformants contained a plasmid migrating with the same mobility as the original donor plasmid. During culture these transformants acquired a Nif+ phenotype without the loss of the plasmid, as judged by the use of a hybridization probe specific for the cloned nif-DNA fragment. These data indicate that plasmids carrying sequences homologous to chromosomal sequences could be maintained in recombination-proficient A. vinelandii UW. The introduction of plasmids containing sequences homologous to chromosomal sequences was facilitated by prelinearization of the plasmid using a restriction endonuclease generating cohesive ends. Because the site of linearization could be chosen outside the region of shared homology, it was unlikely that the route of plasmid establishment occurred via a homology-facilitated transformation mechanism. The data also indicated that A. vinelandii UW could harbor broad host range cloning vectors based on plasmid RSF1010 without significant impairment of its nitrogen-fixation ability.

Azotobacter↗

PubMed1983

Regulation of nitrogen metabolism genes by nifA gene product in Klebsiella pneumoniae.

The Klebsiella pneumoniae nifA gene product, which is known to activate expression of the nitrogen fixation (nif) structural genes, is shown here also to be able to substitute for the product of the gene glnG (ntrC) in the regulation of other nitrogen metabolism genes. An evolutionary relationship between the nifA and glnG genes is suggested.

Biological Evolution↗

PubMed2026

Azotobacter vinelandii gene fitness following carbon shift from sucrose to acetate, succinate and glycerol.

Nitrogen-fixing microbes are a primary contributor of this important nutrient to the global nitrogen cycle. Biological nitrogen fixation (BNF) through the enzyme nitrogenase requires extensive energy that in whole cells is generally studied during the oxidation of carbohydrates such as sugars. The nitrogen-fixing bacterium Azotobacter vinelandii is a model diazotroph for the study of aerobic BNF. Much is known about metabolism in A. vinelandii when cultured on a simple medium where energy is provided primarily in the form of sucrose or glucose. Outside of the laboratory, this soil bacterium grows on metabolites primarily derived from plant root exudates or from the degradation of dead plant matter. In this work, we expand on previous studies looking at genes that are essential to BNF in A. vinelandii when grown on sucrose medium using transposon sequencing (Tn-seq). We applied Tn-seq to determine the genes essential to growth when the medium was shifted to acetate, succinate or glycerol as the primary carbon and energy source to fuel both growth and BNF. A global overview of the genes of central metabolism and those directing substrates toward central metabolism, along with a selection of unexpected genes that were essential for specific growth substrates, is provided.

Azotobacter vinelandii↗

PubMed2001

Role of a ferredoxin gene cotranscribed with the nifHDK operon in N(2) fixation and nitrogenase "switch-off" of Azoarcus sp. strain BH72.

The endophytic diazotroph Azoarcus sp. strain BH72 is capable of infecting rice roots and of expressing the nitrogenase (nif) genes there. In order to study the genetic background for nitrogen fixation in strain BH72, the structural genes of nitrogenase (nifHDK) were cloned and sequenced. The sequence analysis revealed an unusual gene organization: downstream of nifHDK, a ferredoxin gene (fdxN; 59% amino acid sequence identity to R. capsulatus FdxN) and open reading frames showing 52 and 36% amino acid sequence identity to nifY of Pseudomonas stutzeri A15 and ORF1 of Azotobacter vinelandii were located. Northern blot analysis, reverse transcriptase PCR and primer extension analysis revealed that these six genes are located on one transcript transcribed from a sigma(54)-type promoter. Shorter transcripts sequentially missing genes of the 3' part of the full-length mRNA were more abundantly detected. Mutational analyses suggested that FdxN is an important but not the essential electron donor for dinitrogenase reductase. An in-frame deletion of fdxN resulted in reduced growth rates (59% +/- 9%) and nitrogenase activities (81%) in nitrogen-fixing pure cultures in comparison to the wild type. Nitrogenase activity was fully complemented in an fdxN mutant which carried a nifH promoter-driven fdxN gene in trans. Also, in coculture with the ascomycete Acremonium alternatum, where strain BH72 develops intracytoplasmic membrane stacks, the nitrogenase activity in the fdxN deletion mutant was decreased to 56% of the wild-type level. Surprisingly, the fdxN deletion also had an effect on the rapid "switch-off" of nitrogenase activity in response to ammonium. Wild-type strain BH72 and the deletion mutant complemented with fdxN in trans showed a rapid reversible inactivation of acetylene reduction, while the deletion mutant did not cease to reduce acetylene. In concordance with the hypothesis that changes in the redox state of NifH or electron flux towards nitrogenase may be involved in the mechanism of physiological nitrogenase switch-off, our results suggest that the ferredoxin may be a component involved in this process.

Azoarcus↗

PubMed1990

A Photobacterium-like bacterium able to fix nitrogen.

A Photobacterium-like bacterium isolated from the roots of eelgrass (Zostera marina) was shown to fix nitrogen under anaerobic conditions. Nitrogen fixation by Photobacterium spp. has not been reported previous to this.

Culture Media↗

PubMedDate not supplied

Bacteriology and nutrition of environmental strains of Klebsiella pneumoniae involved in wood and bark decay.

Strains of Klebsiella pneumoniae were isolated from screening procedures for wood- and bark-decaying bacteria. Their bacteriological and biochemical properties, their sensitivity to antibiotics and assimilation of wood components were analysed. Most were pectinolytic, degraded xylan and different phenolic compounds. Unusual associations of capsular polysaccharides and biotypes were observed. The nitrogen fixation ability of these strains was also tested.

Klebsiella pneumoniae↗

PubMed1990

Expression of glutamine synthetase genes in roots and nodules of Phaseolus vulgaris following changes in the ammonium supply and infection with various Rhizobium mutants.

In this paper we have examined whether the four glutamine synthetase (gln) genes, expressed in roots and nodules of Phaseolus vulgaris are substrate-inducible by ammonium. Manipulation of the ammonium pool in roots, through addition and removal of exogenous ammonium, did not elicit any changes in the abundances of the four mRNAs thus suggesting that the gln genes in roots of this legume are neither substrate-inducible by ammonium nor derepressed during nitrogen starvation. In nodules the effect of the ammonium supply on expression of the gln genes has been examined by growing nodules under argon/oxygen atmospheres, or with a number of Fix- Rhizobium mutants, and following addition of exogenous ammonium. The results of these experiments suggest that the expression of the gln-gamma gene, which is strongly induced during nodule development, is primarily under a developmental control. However nitrogen fixation appears to have a quantitative effect on expression of gln-gamma as the abundance of this mRNA is about 2 to 4-fold higher under nitrogen-fixing conditions. This effect could not be mimicked by addition of exogenous ammonium and moreover is not specific to the gln-gamma gene as mRNA from a leghaemoglobin gene was similarly affected. Taken together these results have failed to find an effect of ammonium on specifically inducing the expression of glutamine synthetase genes in roots and nodules of P. vulgaris.

Ammonia↗

PubMed1988

Interaction of nod and exo Rhizobium meliloti in alfalfa nodulation.

Among the genes of Rhizobium meliloti SU47 that affect nitrogen-fixing symbiosis with alfalfa are nod genes, in which mutations block nodule induction, and exo genes, in which mutations allow nodule formation but block rhizobial exopolysaccharide production as well as nodule invasion and nitrogen fixation. To investigate whether an exo+ bacterium can "help" (that is, reverse the symbiotic defect of) an exo mutant in trans, we have coinoculated alfalfa with pairs of rhizobia of different genotypes. Coinoculant genotypes were chosen so that the exo+ helper strain was nif while the exo "indicator" strain was nif+, and thus any fixation observed was carried out by the exo coinoculant. We find that a nod exo+ coinoculant can help an exo mutant both to invade nodules and to fix nitrogen. However, a nod+ exo+ coinoculant cannot help an exo mutant: Few exo bacteria are recovered from nodules, some bacteroids differentiate into bizarre aberrant forms, and the nodules fail to fix nitrogen. In a triple coinoculation, the effect of nod+ helper supersedes that of nod helper. Implications of these results for interaction of nod and exo gene products are discussed.

Genes, Bacterial↗