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Xylem colonization of tomato by Azorhizobium caulinodans ORS571.

Tomato seedlings growing aseptically in Murashige and Skoog Medium were inoculated with Azorhizobium caulinodans ORS571 (pXLGD4), carrying the lacZ reporter gene. By microscopic analyses of inoculated tomato roots, it has been demonstrated that the xylem of tomato roots can be colonized by Azorhizobium. We discuss whether this colonization of the xylem of tomato roots by diazotrophic azorhizobia might provide a suitable niche for endophytic nitrogen fixation.

Azorhizobium caulinodans↗

Variability in molybdenum uptake activity in Bradyrhizobium japonicum strains.

Twenty naturally occurring strains of Bradyrhizobium japonicum in 11 serogroups were screened for the ability to take up Mo as bacteroids from soybean root nodules. The strains varied greatly in their ability to take up Mo in a 1-min period. The best strain was USDA 136, which had an Mo uptake activity of almost 3.0 pmol/min per mg of bacteroid (dry weight). In contrast, the poorest strain, USDA 62, had an Mo uptake activity of 0.35 pmol of Mo per min per mg of bacteroid. There were similarities in Mo uptake ability among most of the same serogroup members. The variability in Mo uptake rates between the best (USDA 136 and USDA 122) and poorest (USDA 62 and USDA 140) strains was attributed to their differing affinities for Mo. Double-reciprocal plots of velocity versus substrate indicated a Km for USDA 136 and USDA 122 of 0.045 and 0.054 microM, respectively, whereas strains USDA 62 and USDA 140 both exhibited an apparent Km for MoO42- of about 0.36 microM. The two strains with the higher-affinity Mo binding also accumulated four to five times as much Mo over a 30-min period as the other strains. Soybeans were grown in Mo-deficient and Mo-supplemented conditions after inoculation with the three top-ranking Mo uptake strains and the three poorest Mo uptake strains. Two separate greenhouse studies indicated that Mo supplementation significantly increased the N2 fixation activity of USDA 140 nodules; up to a 35% increase in specific nitrogen fixation activity of nodules due to Mo supplementation was observed. Strain USDA 62 nodule N2 fixation responded positively to Mo supplementation in one of the two experiments. The results indicate that MoO42- transport and, specifically, affinity for Mo by the bacteroid may ultimately affect symbiotic N2 fixation activity. Attempts to reactivate nitrogenase by adding molybdate to bacteroids from plants grown in Mo-deficient conditions were unsuccessful.

Biological Transport↗

Osmoregulatory role of alanine during salt stress in the nitrogen fixing cyanobacterium Anabaena sp. 287.

The freshwater cyanobacterium, Anabaena sp. 287 exhibited enhanced tolerance to NaCl in the presence of ammonium, nitrate, and the amino acids, alanine, valine, proline, lysine, histidine, methionine and aspartic acid. Apart from providing permanent protection to the growth during stress, like NO3- and NH4+, alanine also relieved the initial salt mediated inhibition on enzymes involved in nitrogen fixation, photosynthesis and respiration. The accumulation of sugars was observed only during molecular nitrogen growth in the presence of salt. The cellular total nitrogen content was inversely proportional to the total sugar concentration. All nitrogenous substances which supported growth in the presence of salt curtailed the Na+ influx. Sodium chloride concentration-dependent [3H]D-alanine uptake in this cyanobacterium favours the conclusion that alanine acts as an osmoregulator along with sugars.

Alanine↗

Heavy metal toxicity to microbe-mediated ecologic processes: a review and potential application to regulatory policies.

Microorganisms are sensitive to heavy metal pollution as are other components of the biota. However, most studies on the interactions between microbes and heavy metals have been conducted in synthetic media or in altered (e.g., sterilized) environmental samples and usually have used only single species. Few studies have evaluated the effects of heavy metals on the activities of natural heterogeneous microbial populations, both autotrophic and heterotrophic, in terrestrial and aquatic environments. These latter studies have shown that heavy metals inhibit primary productivity, nitrogen fixation, the mineralization of carbon, nitrogen, sulfur, and phosphorus, litter decomposition, and enzyme synthesis and activity in soils, sediments, and surface waters. The potential adverse effects of heavy metals on such microbe-mediated ecologic processes need to be incorporated into the methodologies used by regulatory agencies, such as the U.S. Environmental Protection Agency, to prepare environmental risk assessments which, in turn, are used to formulate environmental criteria, such as the Water Quality Criteria, and to evaluate the safety to the environment of exposure to "new chemical substances," as mandated by the U.S. Toxic Substances Control Act of 1976. To provide appropriate data that can be assimilated into regulatory policy, it is essential that microbial ecotoxicity tests be standardized, are neither costly nor difficult to train personnel to conduct, and produce data that can be quantitated.

Bacteria↗

Alanine, not ammonia, is excreted from N2-fixing soybean nodule bacteroids.

Symbiotic nitrogen fixation, the process whereby nitrogen-fixing bacteria enter into associations with plants, provides the major source of nitrogen for the biosphere. Nitrogenase, a bacterial enzyme, catalyzes the reduction of atmospheric dinitrogen to ammonium. In rhizobia-leguminous plant symbioses, the current model of nitrogen transfer from the symbiotic form of the bacteria, called a bacteroid, to the plant is that nitrogenase-generated ammonia diffuses across the bacteroid membrane and is assimilated into amino acids outside of the bacteroid. We purified soybean nodule bacteroids by a procedure that removed contaminating plant proteins and found that alanine was the major nitrogen-containing compound excreted. Bacteroids incubated in the presence of 15N2 excreted alanine highly enriched in 15N. The ammonium in these assays neither accumulated significantly nor was enriched in 15N. The results demonstrate that a transport mechanism rather than diffusion functions at this critical step of nitrogen transfer from the bacteroids to the plant host. Alanine may serve only as a transport species, but this would permit physiological separation of the transport of fixed nitrogen from other nitrogen metabolic functions commonly mediated through glutamate.

Journal Article↗

Sinorhizobium medicae genes whose regulation involves the ActS and/or ActR signal transduction proteins.

ActS-ActR proteins belong to a highly conserved family of two-component signal transduction systems involved in global regulation in the alpha-proteobacteria; they were first identified in Sinorhizobium medicae (previously Sinorhizobium meliloti) as essential for acid-tolerance. This paper reports on the identification of genes regulated by ActS and/or ActR in S. medicae. To do this, random gusA fusions were created in S. medicae to follow gene transcription in an actS chromosomal knockout mutant containing plasmid-borne actS. Plasmid borne actS was cured from the mutants and beta-glucuronidase (GUS) activity compared between the different genetic backgrounds. We detected actS-dependent regulation of the genes gst1 (detoxification), hyuA (hydantoin utilization) and fixN2 (microaerobic respiration). We show that ActR is involved in regulating cbbS (CO2 fixation), narB (nitrate assimilation) and required for low pH and microaerobic induction of the nitrogen fixation regulators fixK and nifA. In particular, we demonstrate that the transcriptional activation of fixN2 is regulated by ActR through FixK.

Acids↗

[Biological activity of the soils of the rice irrigation systems in the southern Ukraine].

The biological activity of soils was studied in the irrigation systems of the Southern Ukraina. Aerobic microorganisms were found to be widely distributed in all these soils. The cell number of Azotobacter and the activity of nitrogen fixation differed among various irrigation systems. Active nitrogen-fixing microbial associations were isolated from the soils; some of them were capable of fixation of 19.4 mg N per 1 g of assimilated glucose. The microbiological processes of certain irragation systems were characterized by reductive processes such as the intensive growth of sulphate reducing bacteria and the high activity of denitrification. The activities of protease, catalase and dehydrogenase differed among various soils, and were the lowest at a level of ground waters being 0.8 m. Flooding is one of ecological factors affecting the biological activity of the soils of irrigation systems.

Aerobiosis↗

NrrA, a nitrogen-responsive response regulator facilitates heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120.

The heterocyst is a specialized cell for nitrogen fixation in the filamentous cyanobacteria, and its development is triggered by limitation of combined nitrogen in the medium. During heterocyst development, patterns of gene expression change dramatically. We identified seven genes encoding transcriptional regulators that were upregulated by nitrogen deprivation in Anabaena PCC 7120, using an Anabaena oligonucleotide microarray. Among them, the nrrA gene, which encodes a response regulator of the OmpR family with a DNA-binding domain, has shown the most prominent induction after nitrogen deprivation. Expression of nrrA increased all through the filaments within 3 h of nitrogen deprivation and became higher in proheterocysts than in vegetative cells after 12 h. Sequence analysis of the promoter region of nrrA indicated that the induction of nrrA depended on NtcA, which is the global nitrogen regulator in cyanobacteria. In the nrrA deletion mutant, heterocyst development was delayed and the induction of hetR, which is the master gene in regulation of heterocyst development, was diminished up to 24 h nitrogen deprivation. It is concluded that nrrA facilitates heterocyst development.

Anabaena↗

Fnr Is required for NifL-dependent oxygen control of nif gene expression in Klebsiella pneumoniae.

In Klebsiella pneumoniae, NifA-dependent transcription of nitrogen fixation (nif) genes is inhibited by NifL in response to molecular oxygen and combined nitrogen. We recently showed that K. pneumoniae NifL is a flavoprotein, which apparently senses oxygen through a redox-sensitive, conformational change. We have now studied the oxygen regulation of NifL activity in Escherichia coli and K. pneumoniae strains by monitoring its inhibition of NifA-mediated expression of K. pneumoniae phi (nifH'-'lacZ) fusions in different genetic backgrounds. Strains of both organisms carrying fnr null mutations failed to release NifL inhibition of NifA transcriptional activity under oxygen limitation: nif induction was similar to the induction under aerobic conditions. When the transcriptional regulator Fnr was synthesized from a plasmid, it was able to complement, i.e., to relieve NifL inhibition in the fnr mutant backgrounds. Hence, Fnr appears to be involved, directly or indirectly, in NifL-dependent oxygen regulation of nif gene expression in K. pneumoniae. The data indicate that in the absence of Fnr, NifL apparently does not receive the signal for anaerobiosis. We therefore hypothesize that in the absence of oxygen, Fnr, as the primary oxygen sensor, activates transcription of a gene or genes whose product or products function to relieve NifL inhibition by reducing the flavin adenine dinucleotide cofactor under oxygen-limiting conditions.

Anaerobiosis↗

PATTERNS OF OXIDATIVE ASSIMILATION IN STRAINS OF ACETOBACTER AND AZOTOBACTER.

Tomlinson, Geraldine A. (The University of British Columbia, Vancouver, B.C., Canada), and J. J. R. Campbell. Patterns of oxidative assimilation in strains of Acetobacter and Azotobacter. J. Bacteriol. 86:1165-1172. 1963.-Oxidative assimilation of glucose-U-C(14) was studied with washed-cell suspensions of Acetobacter aceti, A. xylinum, Azotobacter vinelandii, and A. agilis. The suggestion that oxidative assimilation is largely the incorporation of endogenously produced ammonia is tenable. A. aceti did not exhibit oxidative assimilation and it did not incorporate ammonia in the presence of glucose, alpha-ketoglutarate, or pyruvate. A. xylinum, A. vinelandii, and A. agilis incorporated C(14) into the nitrogenous fractions of the cell. The level of assimilation into A. xylinum was low due to the accumulation of extracellular cellulose, and the level of assimilation into the Azotobacter was low presumably because of the requirement of energy for nitrogen fixation. The Azotobacter were characterized by the presence of a high level of radioactivity in the cold trichloroacetic acid-soluble pool. None of the organisms accumulated compounds in the supernatant fluid that might be considered pacemakers in glucose oxidation, and this could be a contributing factor in the low level of assimilation.

Acetobacter↗

The Rhodobacter capsulatus glnB gene is regulated by NtrC at tandem rpoN-independent promoters.

The protein encoded by glnB of Rhodobacter capsulatus is part of a nitrogen-sensing cascade which regulates the expression of nitrogen fixation genes (nif). The expression of glnB was studied by using lacZ fusions, primer extension analysis, and in vitro DNase I footprinting. Our results suggest that glnB is transcribed from two promoters, one of which requires the R. capsulatus ntrC gene but is rpoN independent. Another promoter upstream of glnB is repressed by NtrC; purified R. capsulatus NtrC binds to sites that overlap this distal promoter region.

Bacterial Proteins↗

Symbiotic N2 fixation activity in relation to C economy of Pisum sativum L. as a function of plant phenology.

The relationships between symbiotic nitrogen fixation (SNF) activity and C fluxes were investigated in pea plants (Pisum sativum L. cv. Baccara) using simultaneous 13C and 15N labelling. Analysis of the dynamics of labelled CO2 efflux from the nodulated roots allowed the different components associated with SNF activity to be calculated, together with root and nodule synthetic and maintenance processes. The carbon costs for the synthesis of roots and nodules were similar and decreased with time. Carbon lost by turnover, associated with maintenance processes, decreased with time for nodules while it increased in the roots. Nodule turnover remained higher than root turnover until flowering. The effect of the N source on SNF was investigated using plants supplied with nitrate or plants only fixing N2. SNF per unit nodule biomass (nodule specific activity) was linearly related to the amount of carbon allocated to the nodulated roots regardless of the N source, with regression slopes decreasing across the growth cycle. These regression slopes permitted potential values of SNF specific activity to be defined. SNF activity decreased as the plants aged, presumably because of the combined effects of both increasing C costs of SNF (from 4.0 to 6.7 g C g-1 N) and the limitation of C supply to the nodules. SNF activity competed for C against synthesis and maintenance processes within the nodulated roots. Synthesis was the main limiting factor of SNF, but its importance decreased as the plant aged. At seed-filling, SNF was probably more limited by nodule age than by C supply to the nodulated roots.

Kinetics↗

The genome sequence of an anaerobic aromatic-degrading denitrifying bacterium, strain EbN1.

Recent research on microbial degradation of aromatic and other refractory compounds in anoxic waters and soils has revealed that nitrate-reducing bacteria belonging to the Betaproteobacteria contribute substantially to this process. Here we present the first complete genome of a metabolically versatile representative, strain EbN1, which metabolizes various aromatic compounds, including hydrocarbons. A circular chromosome (4.3 Mb) and two plasmids (0.21 and 0.22 Mb) encode 4603 predicted proteins. Ten anaerobic and four aerobic aromatic degradation pathways were recognized, with the encoding genes mostly forming clusters. The presence of paralogous gene clusters (e.g., for anaerobic phenylacetate oxidation), high sequence similarities to orthologs from other strains (e.g., for anaerobic phenol metabolism) and frequent mobile genetic elements (e.g., more than 200 genes for transposases) suggest high genome plasticity and extensive lateral gene transfer during metabolic evolution of strain EbN1. Metabolic versatility is also reflected by the presence of multiple respiratory complexes. A large number of regulators, including more than 30 two-component and several FNR-type regulators, indicate a finely tuned regulatory network able to respond to the fluctuating availability of organic substrates and electron acceptors in the environment. The absence of genes required for nitrogen fixation and specific interaction with plants separates strain EbN1 ecophysiologically from the closely related nitrogen-fixing plant symbionts of the Azoarcus cluster. Supplementary material on sequence and annotation are provided at the Web page http://www.micro-genomes.mpg.de/ebn1/.

Adaptation, Physiological↗

Key role of bacterial NH(4)(+) metabolism in Rhizobium-plant symbiosis.

Symbiotic nitrogen fixation is carried out in specialized organs, the nodules, whose formation is induced on leguminous host plants by bacteria belonging to the family Rhizobiaceae: Nodule development is a complex multistep process, which requires continued interaction between the two partners and thus the exchange of different signals and metabolites. NH(4)(+) is not only the primary product but also the main regulator of the symbiosis: either as ammonium and after conversion into organic compounds, it regulates most stages of the interaction, from the production of nodule inducers to the growth, function, and maintenance of nodules. This review examines the adaptation of bacterial NH(4)(+) metabolism to the variable environment generated by the plant, which actively controls and restricts bacterial growth by affecting oxygen and nutrient availability, thereby allowing a proficient interaction and at the same time preventing parasitic invasion. We describe the regulatory circuitry responsible for the downregulation of bacterial genes involved in NH(4)(+) assimilation occurring early during nodule invasion. This is a key and necessary step for the differentiation of N(2)-fixing bacteroids (the endocellular symbiotic form of rhizobia) and for the development of efficient nodules.

Bacterial Proteins↗

Epiphytic cyanobacteria on Chara vulgaris are the main contributors to N(2) fixation in rice fields.

The distribution of nitrogenase activity in the rice-soil system and the possible contribution of epiphytic cyanobacteria on rice plants and other macrophytes to this activity were studied in two locations in the rice fields of Valencia, Spain, in two consecutive crop seasons. The largest proportion of photodependent N(2) fixation was associated with the macrophyte Chara vulgaris in both years and at both locations. The nitrogen fixation rate associated with Chara always represented more than 45% of the global nitrogenase activity measured in the rice field. The estimated average N(2) fixation rate associated with Chara was 27.53 kg of N ha(-1) crop(-1). The mean estimated N(2) fixation rates for the other parts of the system for all sampling periods were as follows: soil, 4.07 kg of N ha(-1) crop(-1); submerged parts of rice plants, 3.93 kg of N ha(-1) crop(-1); and roots, 0.28 kg of N ha(-1) crop(-1). Micrographic studies revealed the presence of epiphytic cyanobacteria on the surface of Chara. Three-dimensional reconstructions by confocal scanning laser microscopy revealed no cyanobacterial cells inside the Chara structures. Quantification of epiphytic cyanobacteria by image analysis revealed that cyanobacteria were more abundant in nodes than in internodes (on average, cyanobacteria covered 8.4% +/- 4.4% and 6.2% +/- 5.0% of the surface area in the nodes and internodes, respectively). Epiphytic cyanobacteria were also quantified by using a fluorometer. This made it possible to discriminate which algal groups were the source of chlorophyll a. Chlorophyll a measurements confirmed that cyanobacteria were more abundant in nodes than in internodes (on average, the chlorophyll a concentrations were 17.2 +/- 28.0 and 4.0 +/- 3.8 microg mg [dry weight] of Chara(-1) in the nodes and internodes, respectively). These results indicate that this macrophyte, which is usually considered a weed in the context of rice cultivation, may help maintain soil N fertility in the rice field ecosystem.

Chara↗

A second nitrogenase in vegetative cells of a heterocyst-forming cyanobacterium.

In many filamentous cyanobacteria nitrogen fixation occurs in differentiated cells called heterocysts. Filamentous strains that do not form heterocysts may fix nitrogen in vegetative cells, primarily under anaerobic conditions. We describe here two functional Mo-dependent nitrogenases in a single organism, the cyanobacterium Anabaena variabilis. Using a lacZ reporter with a fluorescent beta-galactoside substrate for in situ localization of gene expression, we have shown that the two clusters of nif genes are expressed independently. One nitrogenase functions only in heterocysts under either aerobic or anaerobic growth conditions, whereas the second nitrogenase functions only under anaerobic conditions in vegetative cells and heterocysts. Differences between the two nif clusters suggest that the nitrogenase that is expressed in heterocysts is developmentally regulated while the other is regulated by environmental factors.

Acetylene↗

GlnD is essential for NifA activation, NtrB/NtrC-regulated gene expression, and posttranslational regulation of nitrogenase activity in the photosynthetic, nitrogen-fixing bacterium Rhodospirillum rubrum.

GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme and is thought to be the primary sensor of nitrogen status in the cell. It plays an important role in nitrogen assimilation and metabolism by reversibly regulating the modification of P(II) proteins, which in turn regulate a variety of other proteins. We report here the characterization of glnD mutants from the photosynthetic, nitrogen-fixing bacterium Rhodospirillum rubrum and the analysis of the roles of GlnD in the regulation of nitrogen fixation. Unlike glnD mutations in Azotobacter vinelandii and some other bacteria, glnD deletion mutations are not lethal in R. rubrum. Such mutants grew well in minimal medium with glutamate as the sole nitrogen source, although they grew slowly with ammonium as the sole nitrogen source (MN medium) and were unable to fix N(2). The slow growth in MN medium is apparently due to low glutamine synthetase activity, because a DeltaglnD strain with an altered glutamine synthetase that cannot be adenylylated can grow well in MN medium. Various mutation and complementation studies were used to show that the critical uridylyltransferase activity of GlnD is localized to the N-terminal region. Mutants with intermediate levels of uridylyltransferase activity are differentially defective in nif gene expression, the posttranslational regulation of nitrogenase, and NtrB/NtrC function, indicating the complexity of the physiological role of GlnD. These results have implications for the interpretation of results obtained with GlnD in many other organisms.

Ammonia↗

[Introduction of the bacteriophage Mu into a nitrogen-fixing strain Klebsiella pneumoniae M 5 a 1].

Mu cts 62 a thermo-inductible mutant of phage Mu was integrated in E. coli in to the broad host range RP4 plasmid. The hybrid plasmid RP4::Mu cts 62 was then transferred by mating to the dinitrogen-fixing strain K. pneumoniae M5al. In Klebsiella Mu cts 62 is still heat inducible and the phage production is similar to that observed in E. coli. Mu Should be a useful tool for the genetic analysis of nitrogen fixation.

Bacteriophages↗