Inorganic nitrogen regulation of glutamate uptake in the cyanobacterium Nostoc muscorum

In Nostoc muscorum (Anabaena ATCC 27893) glutamate was not metabolised as a fixed nitrogen source, rather it functioned as an inhibitor of growth. The latter effect was nitrogen source specific and occurred in N₂-fixing cultures but not in cultures assimilating nitrate or ammonium. NO₃^--grown cultures lacked heterocysts and nitrogenase activity and showed a nearly 50% reduction in glutamate uptake rates, as well as in the final extent of glutamate taken up, compared to N₂-fixing or nitrogen-limited control cultures. NH₄⁺-grown cultures showed a similar response, except that the reduction in glutamate uptake rates and the final exten of glutamate taken up was over 80%. The present results suggest a relation between nitrate/ammounium nitrogen-dependent inhibition of glutamate uptake, probably via repression of the glutamate transport system, and glutamate toxicity.     

Properties of the glyceraldehyde-3-P dehydrogenase in heterocysts and vegetative cells of cyanobacteria

Abstract Glyceraldehyde-3-P dehydrogenase (GAPDH) in heterocysts and vegetative cells of 3 N₂-fixing cyanobacteria was found to utilize both NAD⁺ and NADP⁺. The enzyme activity was enhanced by thiols (glutathione, reduced lipoic acid and dithiothreitol). GAPDH of the 3 cyanobacterial species was not activated by thioredoxin. Heterocysts have now been shown to possess all the enzymes of glycolysis and the tricarboxylic acid cycle to convert glyceraldehyde-3-phosphate (GAP) to oxoglutarate and glutamate. The GAPDH reaction is a major source for the generation of NADH, which is oxidized by a thylakoid-bound NADH:plastoquinone oxidoreductase in heterocysts.     

Aerobic nitrogen fixation is confined to a subset of cells in the non-heterocystous cyanobacterium Symploca PCC 8002

Symploca PCC 8002 Kützing is a filamentous cyanobacterium that lacks the specialized cells, known as heterocysts, that protect nitrogenase from O₂ in most aerobic N₂-fixing cyanobacteria. Nevertheless, Symploca is able to carry out N₂ fixation in the light under aerobic conditions. When cultures were grown under light/dark cycles, nitrogenase activity commenced and increased in the light phase and declined towards zero in the dark. Immunolocalization of dinitrogenase reductase in sectioned Symploca trichomes showed that the enzyme was present only in 9% of the cells. These cells lacked any obvious mechanical protection against atmospheric O₂ and their ultrastructural characteristics were similar to those of cells that did not contain any dinitrogenase reductase. The nitrogenase-containing cells possessed carboxysomes that were rich in ribulose-1,5-bisphosphate carboxylase/oxygenase and phycoerythrin, a light harvesting pigment of PS II. This indicates that these cells had a capacity for both N₂ fixation and photosynthesis. The significance of the localization pattern for dinitrogenase reductase is discussed in the context of N₂ fixation in Symploca PCC 8002.     

Nitrogen fixation by Baltic cyanobacteria is adapted to the prevailing photon flux density

N₂ fixation, measured as acetylene reduction, was studied in laboratory cultures and in natural assemblages (both as a mixed population and as individually picked colonies) of the heterocystous cyanobacteria Aphanizomenon sp. and Nodularia spp. from the Baltic Sea. During a diurnal cycle of alternating light and darkness, these organisms reduced acetylene predominantly during the period of illumination, although considerable activity was also observed during the dark period. In both laboratory cultures and natural populations N₂ fixation was saturated below a photon flux density of 600 μm⁻² s⁻¹. In cyanobacterial blooms in the Baltic Sea, nitrogenase activity was mostly confined to the surface layers. Samples collected from greater depths did not possess the same capacity for acetylene reduction as samples from the surface itself, even when incubated at the photon flux density prevailing in surface waters. This suggests that, with respect to N₂ fixation, Baltic cyanobacteria are adapted to the intensity of illumination that they are currently experiencing.     

Photosynthetic capacity in relation to nitrogen content and its partitioning in lichens with different photobionts

We tested the hypothesis that lichen species with a photosynthetic CO₂-concentrating mechanism (CCM) use nitrogen more efficiently in photosynthesis than species without this mechanism. Total ribulose bisphosphate carboxylase-oxygenase (Rubisco; EC 4.1.1.39) and chitin (the nitrogenous component of fungal cell walls), were quantified and related to photosynthetic capacity in eight lichens. The species represented three modes of CO₂ acquisition and two modes of nitrogen acquisition, and included one cyanobacterial ( Nostoc ) lichen with a CCM and N₂ fixation, four green algal ( Trebouxia ) lichens with a CCM but without N₂ fixation and three lichens with green algal primary photobionts ( Coccomyxa or Dictyochloropsis ) lacking a CCM. The latter have N₂-fixing Nostoc in cephalodia. When related to thallus dry weight, total thallus nitrogen varied 20-fold, chitin 40-fold, Chl a 5-fold and Rubisco 4-fold among the species. Total nitrogen was lowest in three of the four Trebouxia lichens and highest in the bipartite cyanobacterial lichen. Lichens with the lowest nitrogen invested a larger proportion of this into photosynthetic components, while the species with high nitrogen made relatively more chitin. As a result, the potential photosynthetic nitrogen use efficiency was negatively correlated to total thallus nitrogen for this range of species. The cyanobacterial lichen had a higher photosynthetic capacity in relation to both Chl a and Rubisco compared with the green algal lichens. For the range of green algal lichens both Chl a and Rubisco contents were linearly related to photosynthetic capacity, so the data did not support the hypothesis of an enhanced photosynthetic nitrogen use efficiency in green-algal lichens with a CCM.     

New type of N2-fixing unicellular cyanobacterium (blue-green alga)

Abstract A new N₂-fixing unicellular cyanobacterium identified as a Synechococcus sp. was isolated and purified as an axenic culture. It fixed N₂ aerobically either under continuous illumination or in alternating light-dark cycle. The N₂-fixing properties of the new isolate and Gloeocapsa are discussed.     

Nitrogen use efficiency. 3. Nitrogen fixation: genes and costs

The nitrogen use efficiencies (NUE) of N₂ fixation, primary NH ₄⁺ assimilation and NO ₃⁻ assimilation are compared. The photon and water costs of the various biochemical and transport processes involved in plant growth, N-assimilation, pH regulation and osmolarity generation, per unit N assimilated are respectively likely to be around 5 and 7% greater for N₂ fixation than for a combination of NH ₄⁺ and root and shoot NO ₃⁻ assimilation as occurs with most crops. Studies on plant and rhizobial genes involved in nodulation and N₂ fixation may lead to more rapid nodulation, production of more stress-tolerant N₂ fixing systems and transfer of the hydrogenase system to rhizobium/legume symbioses which currently do not have this ability. The activity of an uptake hydrogenase is predicted to decrease the photon cost of diazotrophic plant growth by about 1%.     

Sustainable hydrogen production in the Cyanobacterium Nostoc sp. ARM 411 grown in fructose- and magnesium sulphate-enriched culture

We show that high levels of both carbon sources (sugars) and cations (notably magnesium) in the growth medium enhance the frequency of heterocyst formation by three-fold over the control in Nostoc sp. Such an alteration in the frequency and in enzyme activity (hydrogenase and nitrogenase), induced a proportional increase in hydrogen evolution capacity under a variety of conditions. Nitrogenase and hydrogenase activity also showed an increase in the enriched cultures. Our results therefore suggest that enriching the Nostoc culture with some selected carbon source or a low cost inorganic source like magnesium would result in a high yield of heterocysts in heterocystous cyanobacteria which would be helpful in the production of sustainable bio-fuels.     

Distribution of sym-homosperimidine in eubacteria, cyanobacteria, algae and ferns

Abstract Polyamines were analyzed in 12 of N₂-fixing aerobic eubacteria and other eubacteria, cyanobacteria, algae and ferns. sym -Homospermidine (homospermidine) was found to be widely distributed as a major polyamine in various N₂-fixing eubacteria which belong to Azospirillum, Agromonas, Beijerinckia, Bradyrhizobium, Rhizobium and Xathnbacter . 3 species of Azotobater contained spermidine but not homospermidine, though they are N₂-fixing eubactera. Homospermidine is also distributed in some eubacteria, i.e., the photosynthetic Rhodopseudomanas rutila and the sulfur-oxidizing Thiobacillus denitrificans , a cyanobacterium, Synechococcus sp., and in the cyanobacterium-symbiotic ferns, Azolla imbircatta and Azolla japonica .     

Consortial N2 fixation: a strategy for meeting nitrogen requirements of marine and terrestrial cyanobacterial mats

Abstract: Four microbial mat-forming, non-axenic, strains of the non-heterocystous, filamentous, cyanobacterial genus Microcoleus were maintained in culture and examined for the ability to fix atmospheric nitrogen (N₂). Each was tested for nitrogenase activity using the acetylene reduction assay (ARA) and for the presence of the dinitrogenase reductase gene ( nifH ), an essential gene for N₂ fixation, using the polymerase chain reaction (PCR). The Microcoleus spp. cultures were incapable of growth without an exogenous nitrogen source and never exhibited nitrogenase activity. Attempts to amplify a 360-bp segment of the nifH gene using DNA purified from the cyanobacterial cultures did not produce any cyanobacteria-specific nifH sequences. However, several non-cyanobacterial homologous nifH sequences were obtained. Phylogenetic analysis showed these sequences to be most similar to sequences from heterotrophic bacteria isolated from a marine microbial mat in Tomales Bay (California, USA), and bulk DNA extracted from a cryptobiotic soil crust in Moab (Utah, USA). Microcoleus spp. dominated the biomass of both systems. Cyanobacteria-specific 16S rDNA sequences obtained from the cultured cyanobacterial strains demonstrate that the lack of cyanobacteria-specific nifH sequences was not due to inefficiency of extracting Microcoleus DNA. Hence, both the growth and genetic data indicate that, contrary to earlier reports, Microcoleus spp. appear incapable of fixing N₂ because they lack at least one of the requisite genes for this process. Furthermore, our study suggests epiphytic N₂-fixing bacteria form a diazotrophic consortium with these Microcoleus spp. and are likely key sources of fixed N₂ generated within soil crusts and marine microbial mats.     

Hydrogenases and Hydrogen Metabolism of Cyanobacteria

Cyanobacteria may possess several enzymes that are directly involved in dihydrogen metabolism: nitrogenase(s) catalyzing the production of hydrogen concomitantly with the reduction of dinitrogen to ammonia, an uptake hydrogenase (encoded by hupSL) catalyzing the consumption of hydrogen produced by the nitrogenase, and a bidirectional hydrogenase (encoded by hoxFUYH) which has the capacity to both take up and produce hydrogen. This review summarizes our knowledge about cyanobacterial hydrogenases, focusing on recent progress since the first molecular information was published in 1995. It presents the molecular knowledge about cyanobacterial hupSL and hoxFUYH, their corresponding gene products, and their accessory genes before finishing with an applied aspect—the use of cyanobacteria in a biological, renewable production of the future energy carrier molecular hydrogen. In addition to scientific publications, information from three cyanobacterial genomes, the unicellular Synechocystis strain PCC 6803 and the filamentous heterocystous Anabaena strain PCC 7120 and Nostoc punctiforme (PCC 73102/ATCC 29133) is included.     

Hydrogenases and hydrogen metabolism of cyanobacteria.

Cyanobacteria may possess several enzymes that are directly involved in dihydrogen metabolism: nitrogenase(s) catalyzing the production of hydrogen concomitantly with the reduction of dinitrogen to ammonia, an uptake hydrogenase (encoded by hupSL) catalyzing the consumption of hydrogen produced by the nitrogenase, and a bidirectional hydrogenase (encoded by hoxFUYH) which has the capacity to both take up and produce hydrogen. This review summarizes our knowledge about cyanobacterial hydrogenases, focusing on recent progress since the first molecular information was published in 1995. It presents the molecular knowledge about cyanobacterial hupSL and hoxFUYH, their corresponding gene products, and their accessory genes before finishing with an applied aspect--the use of cyanobacteria in a biological, renewable production of the future energy carrier molecular hydrogen. In addition to scientific publications, information from three cyanobacterial genomes, the unicellular Synechocystis strain PCC 6803 and the filamentous heterocystous Anabaena strain PCC 7120 and Nostoc punctiforme (PCC 73102/ATCC 29133) is included.     

Effects of mycorrhizal colonization on biomass production and nitrogen fixation of black locust (Robinia pseudoacacia) seedlings grown under elevated atmospheric carbon dioxide

Interactive effects of elevated atmospheric CO₂ and arbuscular mycorrhizal (AM) fungi on biomass production and N₂ fixation were investigated using black locust ( Robinia pseudoacacia ). Seedlings were grown in growth chambers maintained at either 350 μmol mol⁻¹ or 710 μmol mol⁻¹ CO₂. Seedlings were inoculated with Rhizobium spp. and were grown with or without AM fungi. The ¹⁵N isotope dilution method was used to determine N source partitioning between N₂ fixation and inorganic fertilizer uptake. Elevated atmospheric CO₂ significantly increased the percentage of fine roots that were colonized by AM fungi. Mycorrhizal seedlings grown under elevated CO₂ had the greatest overall plant biomass production, nodulation, N and P content, and root N absorption. Additionally, elevated CO₂ levels enhanced nodule and root mass production, as well as N₂ fixation rates, of non- mycorrhizal seedlings. However, the relative response of biomass production to CO₂ enrichment was greater in non-mycorrhizal seedlings than in mycorrhizal seedlings. This study provides strong evidence that arbuscular mycorrhizal fungi play an important role in the extent to which plant nutrition of symbiotic N₂-fixing tree species is affected by enriched atmospheric CO₂.     

Microbiological, molecular biological and stable isotopic evidence for nitrogen fixation in the open waters of Lake Michigan

We have used a combination of microbiological, molecular biological and stable isotope methods to relate specific microbial populations to elemental cycling at an offshore site in Lake Michigan. Several lines of evidence suggest that atmospheric N₂ may be a significant source of nitrogen to the lake. Particulate organic nitrogen (PON) at ≈ 10–15 m depth in July and October had a δ¹⁵N of 0.5–1.5‰. These values closely reflect the ¹⁵N composition of atmospheric N₂, suggesting biological nitrogen fixation. Historical data show a developing late-summer N:P minimum at ≈ 15 m; low abundance of inorganic nitrogen relative to phosphorus favours species able to acquire atmospheric nitrogen. Microscopic examination of October water samples revealed abundant heterocystous cyanobacteria, including Nodularia sp. Potentially nitrogen-fixing Anabaena spp. have been found in Lake Michigan before but, to our knowledge, this is the first report of Nodularia . Finally, we have amplified both cyanobacterial and non-cyanobacterial nifH sequences (encoding the nitrogenase iron protein) from lakewater samples, evidence for the presence of bacteria capable of nitrogen fixation. The surface waters of Lake Michigan are considered to be phosphate limited in the stratified season and, under these conditions, energetically expensive nitrogen fixation is expected to be uncompetitive with assimilation of combined nitrogen. Our results suggest that, from both microbiological and biogeochemical perspectives, this may be an oversimplification.     

Photosynthetic oxygen evolution within Sesbania rostrata stem nodules

The tropical wetland legume, Sesbania rostrata Brem. forms N₂-fixing nodules along its stem and on its roots after infection by Azorhizobium caulinodans . The N₂-fixing tissue is surrounded by a cortex of uninfected cells which, in the stem nodules (but not the root nodules), contain chloroplasts. The photosynthetic competence of these chloroplasts was assessed through a novel technique involving image analysis of chlorophyll a fluorescence. Calculation of the quantum efficiency of photosystem II (PS II) photochemistry from these images indicated that most of the chloroplasts with potential for non-cyclic photosynthetic electron transport were concentrated within the mid- and inner-cortex, close to the edge of the N₂-fixing tissue. PS II activity in the cortical cells was confirmed in vivo using O₂-specific microelectrodes which showed that the concentration of O₂ (pO₂) in the outer cortex could rise from less than 1% up to 23.4% upon increased irradiance of the nodule, but that the pO₂ of the inner cortex and infected tissue remained less than 0.0025%. Nitrogenase activity of stem nodules, as measured using a flow-through acetylene reduction assay (no H₂ evolution was evident), showed a reversible increase of 28% upon exposure of the nodules to supplemental light. This increase resembled that obtained with stem nodules upon their exposure to an external pO₂ of 40%.     

Isolation and identification of a N2-fixing zoogloea-forming bacterium from kallar grass histoplane

Semi-solid medium was used to isolate an aerobic, N₂-fixing (C₂H₂-reducing), H₂-utilizing bacterium from the roots of kallar grass ( Leptochloa fusca ). The organism was identified by morphological, cultural and biochemical characteristics. The N₂-fixing, zoogloeal floc-forming isolate described here is a new species.     

Oxygen and the regulation of nitrogen fixation in legume nodules

In N₂-fixing legume nodules, O₂ is required in large amounts for aerobic respiration, yet nitrogenase, the bacterial enzyme that fixes N₂, is O₂ labile. A high rate of O₂ consumptition and a cortical barrier to gas diffusion work together to maintain a low, non-inhibitory O₂ concentration in the central, infected zone of the nodule. At this low O₂ concentration, cytosolic leghemoglobin is required to facilitate the diffusion of O₂ through the infected cell to the bacteria. The resistance of the cortical diffusion barrier is variable and is used by legume nodules to regulate the O₂ concentration in the infected cells such that it limits aerobic respiration and N₂ fixation at all times. The resistance of the diffusion barrier and therefore the degree of O₂ limitation seems to be regulated in response to changes in the O₂ concentration of the central infected zone, the supply of phloem sap to the nodule, and the rate of N assimilation into the end products of fixation.