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PubMed2006

Regulation of uptake hydrogenase and effects of hydrogen utilization on gene expression in Rhodopseudomonas palustris.

Rhodopseudomonas palustris is a purple, facultatively phototrophic bacterium that uses hydrogen gas as an electron donor for carbon dioxide fixation during photoautotrophic growth or for ammonia synthesis during nitrogen fixation. It also uses hydrogen as an electron supplement to enable the complete assimilation of oxidized carbon compounds, such as malate, into cell material during photoheterotrophic growth. The R. palustris genome predicts a membrane-bound nickel-iron uptake hydrogenase and several regulatory proteins to control hydrogenase synthesis. There is also a novel sensor kinase gene (RPA0981) directly adjacent to the hydrogenase gene cluster. Here we show that the R. palustris regulatory sensor hydrogenase HupUV acts in conjunction with the sensor kinase-response regulator protein pair HoxJ-HoxA to activate hydrogenase expression in response to hydrogen gas. Transcriptome analysis indicated that the HupUV-HoxJA regulatory system also controls the expression of genes encoding a predicted dicarboxylic acid transport system, a putative formate transporter, and a glutamine synthetase. RPA0981 had a small effect in repressing hydrogenase synthesis. We also determined that the two-component system RegS-RegR repressed expression of the uptake hydrogenase, probably in response to changes in intracellular redox status. Transcriptome analysis indicated that about 30 genes were differentially expressed in R. palustris cells that utilized hydrogen when growing photoheterotrophically on malate under nitrogen-fixing conditions compared to a mutant strain that lacked uptake hydrogenase. From this it appears that the recycling of reductant in the form of hydrogen does not have extensive nonspecific effects on gene expression in R. palustris.

Bacterial Proteins↗
PubMed2002

Nitrogen-fixing phylotypes of Chesapeake Bay and Neuse River estuary sediments.

Sediments often exhibit low rates of nitrogen fixation, despite the presence of elevated concentrations of inorganic nitrogen. The organisms that potentially fix nitrogen in sediments have not previously been identified. Amplification of nifH genes with degenerate primers was used to assess the diversity of diazotrophs in two distinct sediment systems, anoxic muds of Chesapeake Bay and shallow surficial sediments of the Neuse River. Phylogenetic analysis revealed that sequences obtained from mid-Chesapeake Bay, which receive high organic loading and are highly reducing, clustered closely with each other and with known anaerobic microorganisms, suggesting a low abundance of aerobic or facultative diazotrophs in these sediments. Sulfate reduction dominates in the surface, but methanogenesis becomes more important with depth. A thin (<1 cm) oxidized layer is present only in the spring. No archaeal nifH sequences were obtained from Chesapeake Bay. Sequences of nifH amplified from surficial sediments of the Neuse River were distant from Chesapeake Bay sequences and included nif phylotypes related to sequences previously reported from marine mats and the Spartina rhizosphere. Differences in environmental site characteristics appear to select for different types of sediment diazotrophs, which is reflected in the phylogenetic composition of amplified nifH sequences.

Bacteria, Aerobic↗
PubMed2005

Functional genomic analysis of three nitrogenase isozymes in the photosynthetic bacterium Rhodopseudomonas palustris.

The photosynthetic bacterium Rhodopseudomonas palustris is one of just a few prokaryotes described so far that has vnf and anf genes for alternative vanadium cofactor (V) and iron cofactor (Fe) nitrogenases in addition to nif genes for a molybdenum cofactor (Mo) nitrogenase. Transcriptome data indicated that the 32 genes in the nif gene cluster, but not the anf or vnf genes, were induced in wild-type and Mo nitrogenase-expressing strains grown under nitrogen-fixing conditions in Mo-containing medium. Strains that were unable to express a functional Mo nitrogenase due to mutations in Mo nitrogenase structural genes synthesized functional V and Fe nitrogenases and expressed vnf and anf genes in nitrogen-fixing growth media that contained Mo and V at concentrations far in excess of those that repress alternative nitrogenase gene expression in other bacteria. Thus, not only does R. palustris have multiple enzymatic options for nitrogen fixation, but in contrast to reports on other nitrogen-fixing bacteria, the expression of its alternative nitrogenases is not repressed by transition metals. Between 95 and 295 genes that are not directly associated with nitrogenase synthesis and assembly were induced under nitrogen-fixing conditions, depending on which nitrogenase was being used by R. palustris. Genes for nitrogen acquisition were expressed at particularly high levels during alternative nitrogenase-dependent growth. This suggests that alternative nitrogenase-expressing cells are relatively starved for nitrogen and raises the possibility that fixed nitrogen availability may be the primary signal that controls the synthesis of the V and Fe nitrogenases.

Adaptation, Physiological↗
PubMed2026

Isolation and global occurrence of nitrogen-fixing Acidobacteriota in soil environments.

Acidobacteriota, one of the most abundant and ubiquitous bacterial phyla in soils, are well recognized for their role in carbon cycling. In contrast, their roles in soil nitrogen cycling remain largely unexplored, although recent metagenome-assembled genome (MAG) analyses suggest that Acidobacteriota may harbor genes involved in nitrogen cycling. Here, we provide culture-based evidence of diazotrophy within this phylum and demonstrate the widespread occurrence of nitrogen-fixing Acidobacteriota across diverse soil types. From grassland and agricultural soils, we isolated five Acidobacteriota strains representing novel taxonomic lineages, four of which harbor functional nitrogenase (nif) gene clusters. These strains were capable of fixing atmospheric nitrogen in vitro and/or in soil microcosms, as evidenced by acetylene reduction, N2-dependent growth, transcription of nif genes, incorporation of 15N into biomass and soil, and inhibition of nitrogenase activity by ammonium. Furthermore, global-scale meta-analysis of soil metagenomes revealed that nif-harboring Acidobacteriota are widely distributed and locally dominant across soil types. These results demonstrate the nitrogen-fixing capability of Acidobacteriota at the organismal level, complementing MAG-based inferences, and underscore their adaptive capacity in nitrogen-limited environments and their potential contribution to terrestrial nitrogen fixation. We also propose novel taxa within the class Terriglobia of the phylum Acidobacteriota, including diazotrophic strains, comprising one novel family, three novel genera, and four novel species: Koromonadaceae fam. nov., Koromonas soli gen. nov., sp. nov., Koromonas humicola sp. nov., Oryzophilus luti gen. nov., sp. nov., and Humiphilus diazotrophicus gen. nov., sp. nov.

Soil Microbiology↗
PubMed1997

The Rhizobium meliloti PII protein, which controls bacterial nitrogen metabolism, affects alfalfa nodule development.

Symbiotic nitrogen fixation involves the development of specialized organs called nodules within which plant photosynthates are exchanged for combined nitrogen of bacterial origin. To determine the importance of bacterial nitrogen metabolism in symbiosis, we have characterized a key regulator of this metabolism in Rhizobium meliloti, the uridylylatable P(II) protein encoded by glnB. We have constructed both a glnB null mutant and a point mutant making nonuridylylatable P(II). In free-living conditions, P(II) is required for expression of the ntrC-dependent gene glnII and for adenylylation of glutamine synthetase I. P(II) is also required for efficient infection of alfalfa but not for expression of nitrogenase. However alfalfa plants inoculated with either glnB mutant are nitrogen-starved in the absence of added combined nitrogen. We hypothesize that P(II) controls expression or activity of a bacteroid ammonium transporter required for a functional nitrogen-fixing symbiosis. Therefore, the P(II) protein affects both Rhizobium nitrogen metabolism and alfalfa nodule development.

Bacterial Proteins↗
PubMed1998

Physiological role for the GlnK protein of enteric bacteria: relief of NifL inhibition under nitrogen-limiting conditions.

In Klebsiella pneumoniae, NifA-dependent transcription of nitrogen fixation (nif) genes is inhibited by a flavoprotein, NifL, in the presence of molecular oxygen and/or combined nitrogen. We recently demonstrated that the general nitrogen regulator NtrC is required to relieve NifL inhibition under nitrogen (N)-limiting conditions. We provide evidence that the sole basis for the NtrC requirement is its role as an activator of transcription for glnK, which encodes a PII-like allosteric effector. Relief of NifL inhibition is a unique physiological function for GlnK in that the structurally related GlnB protein of enteric bacteria-apparently a paralogue of GlnK-cannot substitute. Unexpectedly, although covalent modification of GlnK by uridylylation normally occurs under N-limiting conditions, several lines of evidence indicate that uridylylation is not required for relief of NifL inhibition. When GlnK was synthesized constitutively from non-NtrC-dependent promoters, it was able to relieve NifL inhibition in the absence of uridylyltransferase, the product of the glnD gene, and under N excess conditions. Moreover, an altered form of GlnK, GlnKY51N, which cannot be uridylylated due to the absence of the requisite tyrosine, was still able to relieve NifL inhibition.

Bacterial Proteins↗
PubMed2004

Gas exchange in the filamentous cyanobacterium Nostoc punctiforme strain ATCC 29133 and Its hydrogenase-deficient mutant strain NHM5.

Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H(2)), which is recaptured by an uptake hydrogenase. Gas exchange in cultures of N. punctiforme ATCC 29133 and its hydrogenase-free mutant strain NHM5 was studied. Exchange of O(2), CO(2), N(2), and H(2) was followed simultaneously with a mass spectrometer in cultures grown under nitrogen-fixing conditions. Isotopic tracing was used to separate evolution and uptake of CO(2) and O(2). The amount of H(2) produced per molecule of N(2) fixed was found to vary with light conditions, high light giving a greater increase in H(2) production than N(2) fixation. The ratio under low light and high light was approximately 1.4 and 6.1 molecules of H(2) produced per molecule of N(2) fixed, respectively. Incubation under high light for a longer time, until the culture was depleted of CO(2), caused a decrease in the nitrogen fixation rate. At the same time, hydrogen production in the hydrogenase-deficient strain was increased from an initial rate of approximately 6 micro mol (mg of chlorophyll a)(-1) h(-1) to 9 micro mol (mg of chlorophyll a)(-1) h(-1) after about 50 min. A light-stimulated hydrogen-deuterium exchange activity stemming from the nitrogenase was observed in the two strains. The present findings are important for understanding this nitrogenase-based system, aiming at photobiological hydrogen production, as we have identified the conditions under which the energy flow through the nitrogenase can be directed towards hydrogen production rather than nitrogen fixation.

Carbon Dioxide↗
PubMed2004

Indole-3-acetic acid biosynthesis is deficient in Gluconacetobacter diazotrophicus strains with mutations in cytochrome c biogenesis genes.

Gluconacetobacter diazotrophicus is an endophyte of sugarcane frequently found in plants grown in agricultural areas where nitrogen fertilizer input is low. Recent results from this laboratory, using mutant strains of G. diazotrophicus unable to fix nitrogen, suggested that there are two beneficial effects of G. diazotrophicus on sugarcane growth: one dependent and one not dependent on nitrogen fixation. A plant growth-promoting substance, such as indole-3-acetic acid (IAA), known to be produced by G. diazotrophicus, could be a nitrogen fixation-independent factor. One strain, MAd10, isolated by screening a library of Tn5 mutants, released only approximately 6% of the amount of IAA excreted by the parent strain in liquid culture. The mutation causing the IAA(-) phenotype was not linked to Tn5. A pLAFR3 cosmid clone that complemented the IAA deficiency was isolated. Sequence analysis of a complementing subclone indicated the presence of genes involved in cytochrome c biogenesis (ccm, for cytochrome c maturation). The G. diazotrophicus ccm operon was sequenced; the individual ccm gene products were 37 to 52% identical to ccm gene products of Escherichia coli and equivalent cyc genes of Bradyrhizobium japonicum. Although several ccm mutant phenotypes have been described in the literature, there are no reports of ccm gene products being involved in IAA production. Spectral analysis, heme-associated peroxidase activities, and respiratory activities of the cell membranes revealed that the ccm genes of G. diazotrophicus are involved in cytochrome c biogenesis.

Bacterial Proteins↗
PubMedDate not supplied

Parasitic origins of nitrogen-mixing Rhizobium-legume symbioses. A review of the evidence.

This paper is divided into two sections. The first part (I) reviews the literature on the legume-Rhizobium association with emphasis on the processes leading to the establishment of the association. In the second part (II) it is proposed that the legume-Rhizobium association was originally necrotrophic , beginning when the free-living, nitrogen-fixing, saprotrophic Rhizobium developed the ability to infect the plant. The pre-infection events, infection processes and nodulation in the colonization of the legumes by the Rhizobium are similar to those of other parasitic associations. Likewise, the host responses to the Rhizobium entry, infection thread synthesis and bacteroid formation are comparable to those of other plants when they encounter phytopathogens . Evolutionary processes acted in the selection of biotrophy , the fine control and regulation of the extracellular enzymes of the necrotrophic Rhizobium converted the association into biotrophy . The nutritional dependence of the Rhizobium on the legume, the requirement of the plant for combined nitrogen and the Rhizobium potential to meet this requirement drove the biotrophic association into mutualism . This became possible when regulation of the nitrogen-fixing system of the Rhizobium was modified and the oxygen carrying protein leghemoglobin was acquired or evolved by the legume to enhance nitrogen fixation.

Biological Evolution↗
PubMedDate not supplied

[Biochemical characterization of noduleless soybean lectins].

The comparative analysis of activity and hydrocarbon specificity of lectins from common soybean and soybean in which roots the nodules do not form has been carried out. It was shown that the root lectins possesses the less activity in comparison with the seed lectins. The both kinds of lectins most specifically link with mannitol. The supposition was made about the role of activity and hydrocarbon specificity of lectins from soybean in the establishment of symbiotic systems of nitrogen fixation.

Carbohydrates↗
PubMed1999

Control of Azospirillum brasilense NifA activity by P(II): effect of replacing Tyr residues of the NifA N-terminal domain on NifA activity.

It was previously reported that the N-terminal domain of Azospirillum brasilense NifA was a negative regulator of the NifA activity and that the P(II) protein prevented this inhibition under nitrogen fixing conditions. Here, we show that a mutation of a single Tyr residue at position 18 of the N-terminal domain of NifA led to an active NifA protein that did not require P(II) for activation under nitrogen fixation conditions.

Amino Acid Sequence↗
PubMed2004

Comparison of 16S rRNA, nifD, recA, gyrB, rpoB and fusA genes within the family Geobacteraceae fam. nov.

The sequences of five conserved genes, in addition to the 16S rRNA gene, were investigated in 30 members of the Geobacteraceae fam. nov. All members of the Geobacteraceae examined contained nifD, suggesting that they are capable of nitrogen fixation, which may explain their ability to compete effectively in nitrogen-poor subsurface environments undergoing remediation for petroleum or metal contamination. The phylogenies predicted from rpoB, gyrB, fusA, recA and nifD were generally in agreement with the phylogeny predicted from 16S rRNA gene sequences. Furthermore, phylogenetic analysis of concatemers constructed from all five protein-coding genes corresponded closely with the 16S rRNA gene-based phylogeny. This study demonstrated that the Geobacteraceae is a phylogenetically coherent family within the delta-subclass of the Proteobacteria that is composed of three distinct phylogenetic clusters: Geobacter, Desulfuromonas and Desulfuromusa. The sequence data provided here will make it possible to discriminate better between physiologically distinct members of the Geobacteraceae, such as Pelobacter propionicus and Geobacter species, in geobacteraceae-dominated microbial communities and greatly expands the potential to identify geobacteraceae sequences in libraries of environmental genomic DNA.

Bacterial Proteins↗
PubMed1964

LEAF-NODULE SYMBIOSIS. I. ENDOPHYTE OF PSYCHOTRIA BACTERIOPHILA.

Centifanto, Ysolina M. (University of Florida, Gainesville), and Warren S. Silver. Leafnodule symbiosis. I. Endophyte of Psychotria bacteriophila. J. Bacteriol. 88:776-781. 1964.-The leaf-nodule endophyte of Psychotria bacteriophila has been repeatedly isolated in pure culture from germinating seedlings and young leaves on a nitrogen-free mineral agar. Its morphological, serological, and cultural characteristics place it within the Klebsiella-Aerobacter group. The endophyte has been provisionally named Klebsiella rubiacearum. Pure cultures fix N(2) under anaerobic conditions with good efficiency (4.54 mug of N fixed/mg of glucose utilized in 3 days). Nitrogen fixation by pure cultures is intimately related to pyruvate and hydrogen metabolism.

Carbohydrate Metabolism↗
PubMed2025

A rhamnose-rich O-antigen of Paraburkholderia phymatum MP20 is required for symbiosis with Mimosa pudica.

Paraburkholderia phymatum, a &#x3b2;-proteobacterium, forms a nitrogen-fixing symbiosis with many species of the large legume genus Mimosa as well as with common bean (Phaseolus vulgaris L.). Paraburkholderia are considered to have evolved nodulation independently from the well-studied &#x3b1;-proteobacteria symbionts of legumes. However, the detailed mechanisms important for &#x3b2;-rhizobia-legume symbiosis have not yet been determined. In this manuscript, we have sequenced the genome of P. phymatum MP20, a strain isolated from Mimosa pudica nodules, and utilized transposon mutagenesis to identify a mutant that showed delayed and ineffective nodulation of M. pudica. Further analysis revealed that the mutant strain produced an altered lipopolysaccharide lacking rhamnose containing O-antigen. Complementation with the wild-type gene restored the symbiosis. Microscopic analysis of the ineffective nodules showed that the mutant strain did not infect the cortical cells but was restricted to the endodermis. The results suggest that the O-antigen of P. phymatum is important for the bacterial infection of cortical cells and for nodule maturation. Further research will unveil the specific involvement of the glycosyltransferase gene in LPS biosynthesis and its impact on successful nodule formation by P. phymatum.IMPORTANCEThe nitrogen-fixing symbiosis between legumes and rhizobia is important for agricultural and environmental sustainability. The mechanisms of the symbiotic interactions are extensively studied using &#x3b1;-rhizobia. In contrast, mechanisms of symbiotic interactions important for &#x3b2;-rhizobia and their Caesalpinioid (mimosoid) legume hosts are not well known. Here, we describe the genome sequence of P. phymatum MP20, a &#x3b2;-rhizobia isolated from the nodules of M. pudica, and isolation and characterization of a transposon mutant defective in symbiosis. We demonstrate that the O-antigen of the LPS is required for nodulation and symbiotic nitrogen fixation. This study broadens our knowledge of symbiotic interactions in &#x3b2;-rhizobia and will lead to a better understanding of the wider rhizobial-legume symbiosis apart from the &#x3b1;-rhizobia.

Symbiosis↗
PubMed1963

SHORT-TERM N2-15 INCORPORATION BY AZOBACTER.

Bulen, W. A. (Charles F. Kettering Research Laboratory, Yellow Springs, Ohio), J. R. LeComte, and H. E. Bales. Short-term N(2) (15)-incorporation by Azotobacter. J. Bacteriol. 85:666-670. 1963.-Short-term N(2) (15)-incorporation measurements were used to determine which of the growth requirements were necessary for nitrogen fixation by Azotobacter agilis (A. vinelandii). Normal cells required neither added iron nor molybdenum, but a marked stimulation by Na(+) and a minor stimulation by Mg(2+) were observed. The Na(+) stimulation was not accompanied by an increase in O(2) uptake. A lag period preceded the response of molybdenum-deficient cells to added Mo. In systems employing 10 and 20% O(2) with 10% N(2) (15) in the gas phase, O(2) appeared to be both required and inhibitory. These observations may be helpful in attempts to fractionate cell-free nitrogen-fixing systems from this aerobe.

Azotobacter↗
PubMed1968

The kinetics of the reduction of isocyanides, acetylenes and the cyanide ion by nitrogenase preparation from Azotobacter chroococcum and the effects of inhibitors.

1. Nitrogen-fixing preparations from Azotobacter chroococcum reduced substrates with the following K(m) values: methyl isocyanide, 1.8x10(-4)m; ethyl isocyanide, 2.5x10(-2)m; cyanide ion, 1.4x10(-3)m; acetylene, 1.2x10(-4)m. 2. Nitrogen, carbon monoxide or hydrogen competitively inhibited isocyanide reduction with the following K(i) values: hydrogen, 1.3x10(-3)m; carbon monoxide, 6.8x10(-6)m; nitrogen, 4.3x10(-4)m. 3. Living nitrogen-fixing bacteria, and isolated clover nodules, formed methane from methyl isocyanide. 4. These results are discussed in relation to other work and possible mechanisms of nitrogen fixation.

Azotobacter↗
PubMed2006

Iron depletion affects nitrogenase activity and expression of nifH and nifA genes in Herbaspirillum seropedicae.

Herbaspirillum seropedicae Z67 is a nitrogen-fixing bacterium able to colonize the rhizosphere and the interior of several plants. As iron is a key element for nitrogen fixation, we examined the response of this microorganism to iron deficiency under nitrogen fixing conditions. We identified a H. seropedicae exbD gene that was induced in response to iron limitation and is involved in iron homeostasis. We found that an exbD mutant grown in iron-chelated medium is unable to fix nitrogen. Moreover, we provide evidence that expression of the nifH and nifA genes is iron dependent in a H. seropedicae genetic background.

Bacterial Proteins↗