Modeling the dynamic regulation of nitrogen fixation in the cyanobacterium Trichodesmium sp

A physiological, unbalanced model is presented that explicitly describes growth of the marine cyanobacterium Trichodesmium sp. at the expense of N(2) (diazotrophy). The model involves the dynamics of intracellular reserves of carbon and nitrogen and allows the uncoupling of the metabolism of these elements. The results show the transient dynamics of N(2) fixation when combined nitrogen (NO(3)(-), NH(4)(+)) is available and the increased rate of N(2) fixation when combined nitrogen is insufficient to cover the demand. The daily N(2) fixation pattern that emerges from the model agrees with measurements of rates of nitrogenase activity in laboratory cultures of Trichodesmium sp. Model simulations explored the influence of irradiance levels and the length of the light period on fixation activity and cellular carbon and nitrogen stoichiometry. Changes in the cellular C/N ratio resulted from allocations of carbon to different cell compartments as demanded by the growth of the organism. The model shows that carbon availability is a simple and efficient mechanism to regulate the balance of carbon and nitrogen fixed (C/N ratio) in filaments of cells. The lowest C/N ratios were obtained when the light regime closely matched nitrogenase dynamics.     

Environmental forcing of nitrogen fixation in the eastern tropical and sub-tropical North Atlantic Ocean

During the winter of 2006 we measured nifH gene abundances, dinitrogen (N(2)) fixation rates and carbon fixation rates in the eastern tropical and sub-tropical North Atlantic Ocean. The dominant diazotrophic phylotypes were filamentous cyanobacteria, which may include Trichodesmium and Katagnymene, with up to 10(6) L(-1)nifH gene copies, unicellular group A cyanobacteria with up to 10(5) L(-1)nifH gene copies and gamma A proteobacteria with up to 10(4) L(-1)nifH gene copies. N(2) fixation rates were low and ranged between 0.032-1.28 nmol N L(-1) d(-1) with a mean of 0.30 ± 0.29 nmol N L(-1) d(-1) (1σ, n = 65). CO(2)-fixation rates, representing primary production, appeared to be nitrogen limited as suggested by low dissolved inorganic nitrogen to phosphate ratios (DIN:DIP) of about 2 ± 3.2 in surface waters. Nevertheless, N(2) fixation rates contributed only 0.55 ± 0.87% (range 0.03-5.24%) of the N required for primary production. Boosted regression trees analysis (BRT) showed that the distribution of the gamma A proteobacteria and filamentous cyanobacteria nifH genes was mainly predicted by the distribution of Prochlorococcus, Synechococcus, picoeukaryotes and heterotrophic bacteria. In addition, BRT indicated that multiple a-biotic environmental variables including nutrients DIN, dissolved organic nitrogen (DON) and DIP, trace metals like dissolved aluminum (DAl), as a proxy of dust inputs, dissolved iron (DFe) and Fe-binding ligands as well as oxygen and temperature influenced N(2) fixation rates and the distribution of the dominant diazotrophic phylotypes. Our results suggest that lower predicted oxygen concentrations and higher temperatures due to climate warming may increase N(2) fixation rates. However, the balance between a decreased supply of DIP and DFe from deep waters as a result of more pronounced stratification and an enhanced supply of these nutrients with a predicted increase in deposition of Saharan dust may ultimately determine the consequences of climate warming for N(2) fixation in the North Atlantic.     

Comparative proteomic profiles of the marine cyanobacterium Trichodesmium erythraeum IMS101 under different nitrogen regimes

Trichodesmium is a marine filamentous diazotrophic cyanobacterium and an important contributor of "new" nitrogen in the oligotrophic surface waters of the tropical and sub-tropical oceans. It is unique in that it exclusively fixes N(2) at daytime, although it belongs to the non-heterocystous filamentous segment of the cyanobacterial radiation. Here we present the first quantitative proteomic analysis of Trichodesmium erythraeum IMS101 when grown under different nitrogen regimes using 2-DE/MALDI-TOF-MS. Addition of combined nitrogen (NO3-) prevented development of the morphological characteristics of the N(2)-fixing cell type (diazocytes), inhibited expression of the nitrogenase enzyme subunits and consequently N(2) fixation activity. The diazotrophic regime (N(2) versus NO3- cultures) elicited the differential expression of more than 100 proteins, which represented 13.5% of the separated proteins. Besides proteins directly related to N(2) fixation, proteins involved in the synthesis of reducing equivalents and the generation of a micro-oxic environment were strongly up-regulated, as was in particular Dps, a protein related to iron acquisition and potentially other vital cellular processes. In contrast, proteins involved in the S-adenosylmethionine (SAM) cycle, synthesis of amino acids and production of carbon skeletons for storage and synthesis of amino acids were suppressed. The data are discussed in the context of Trichodesmium's unusual N(2)-fixing physiology.     

Uptake of dissolved organic nitrogen by size-fractionated plankton along a salinity gradient from the North Sea to the Baltic Sea

The Baltic Sea is known for its ecological problems due to eutrophication caused by high nutrient input via nitrogen fixation and rivers, which deliver up to 70% of nitrogen in the form of dissolved organic nitrogen (DON) compounds. We therefore measured organic nitrogen uptake rates using self produced ¹⁵N labeled allochthonous (derived from Brassica napus and Phragmites sp.) and autochthonous (derived from Skeletonema costatum) DON at twelve stations along a salinity gradient (34 to 2) from the North Sea to the Baltic Sea in August/September 2009. Both labeled DON sources were exploited by the size fractions 0.2–1.6 μm (bacteria size fraction) and >1.6 μm (phytoplankton size fraction). Higher DON uptake rates were measured in the Baltic Sea compared to the North Sea, with rates of up to 1213 nmol N l^?1 h^?1. The autochthonous DON was the dominant nitrogen form used by the phytoplankton size fraction, whereas the heterotrophic bacteria size fraction preferred the allochthonous DON. We detected a moderate shift from >1.6 μm plankton dominated DON uptake in the North Sea and central Baltic Sea towards a 0.2–1.6 μm dominated DON uptake in the Bothnian Bay and a weak positive relationship between DON concentrations and uptake. These findings indicate that DON is an important component of plankton nutrition and can fuel primary production. It may therefore also contribute substantially to eutrophication in the Baltic Sea especially when inorganic nitrogen sources are depleted.     

Microplanktonic regeneration of ammonium and dissolved organic nitrogen in the upwelling area of the NW of Spain: relationships with dissolved organic carbon production and phytoplankton size-structure

Ammonium regeneration and dissolved organic nitrogen (DON) releasewere studied experimentally in the euphotic zone of shelf andoceanic waters of NW Spain in relation to coastal upwellingdynamics and the size-structure of phytoplankton communities.Incubations of plankton labelled with [¹⁵N]ammonium were madeduring four cruises, two of which also included size-fractionateddeterminations of chlorophyll a and primary production, andexperimental determinations of production rates of dissolvedorganic carbon (DOC) using ¹⁴C. Inorganic nitrogen concentrationswere mainly related to nitrate enrichment by upwelling pulses,while ammonium concentrations were generally low in all situations.Ammonium did not accumulate in the study area, suggesting adaily time scale coupling between regeneration and uptake. Incontrast, DON largely exceeded inorganic nitrogen in all situationsand generally increased from spring to autumn. Ammonium regenerationwas positively correlated with DON release and both rates showedthe largest variation in summer, with minimum values duringactive upwelling and maximum values when upwelling relaxed.Comparison of DON stocks and rates in different shelf areassuggests that DON release near the coast during summer was morepersistent in the water than DON release in off-shelf and oceanicareas. The carbon:nitrogen ratio of DOC and DON release rateswas highly variable, revealing a large excess of DOC comparedwith DON. This excess can be attributed to either an underestimateof total DON release (as only release from ammonium was measured)or to an enhanced production of carbon-rich organic substancesby diatoms in coastal areas. By considering a broad range oftrophic situations, this study reveals a fundamental differencebetween short term release of DOC and DON by plankton. Physiologicalprocesses (such as carbohydrate exudation by diatoms) seem tobe the cause of large DOC excess, whereas trophic processes(such as grazing) are more likely the cause of enhanced DONrelease.     

Immunochemical localization of nitrogenase in marine trichodesmium aggregates: relationship to n(2) fixation potential

Colonial aggregation among nonheterocystous filaments of the planktonic marine cyanobacterium Trichodesmium is known to enhance N(2) fixation, mediated by the O(2)-sensitive enzyme complex nitrogenase. Expression of nitrogenase appears linked to the formation of O(2)-depleted microzones within aggregated bacterium-associated colonies. While this implies a mechanism by which nonheterocystous N(2) fixation can take place in an oxygenated water column, both the location and regulation of the N(2)-fixing apparatus remain unknown. We used an antinitrogenase polyclonal antibody together with postsection immunocolloidal gold staining and transmission electron microscopy to show that (i) virtually all Trichodesmium cells within a colony possessed nitrogenase, (ii) nitrogenase showed no clear intracellular localization, and (iii) certain associated bacteria contained nitrogenase. Our findings emphasize the critical role coloniality plays in regulating nitrogenase expression in nature. We interpret the potential for a large share of Trichodesmium cells to fix N(2) as an opportunistic response to the dynamic nature of the sea state; during quiescent conditions, aggregation and consequent expression of nitrogenase can proceed rapidly.     

Heterogeneous dissolved organic nitrogen supply over a coral reef: first evidence from nitrogen stable isotope ratios

Dissolved organic nitrogen (DON) potentially plays a major role in sustaining the high productivity and biological diversity of coral reefs. However, data are scarce regarding sources and sinks of DON. This study, for the first time, determined the ¹⁵N isotopic composition of total dissolved nitrogen (δ¹⁵N_TDN), reflecting the isotopic signature of DON, in the water column over a coral reef. The uniformity in δ¹⁵N_TDN during high tide (3.2 ± 0.3 ‰) indicated that the DON was mainly derived from offshore waters. In contrast, higher spatial heterogeneity of δ¹⁵N_TDN (3.1 ± 0.9 ‰) and DON concentrations during low tide indicated the existence of local DON sources patchily distributed over the reef. Low δ¹⁵N_TDN values located mid-reef were indicative of DON release from organisms that obtained their N via N₂ fixation, whereas high δ¹⁵N_TDN appeared to reflect localized release of DON by organisms exposed to dissolved inorganic nitrogen with elevated ¹⁵N, such as from terrestrial and offshore inputs. Collectively, the results highlight the importance of spatial patterns in DON release from reef communities in the N cycling of coral reefs.     

A molecular assessment of the iron stress response in the two phylogenetic clades of Trichodesmium

Trichodesmium spp. play key roles in global carbon and nitrogen budgets and thus defining what controls their productivity is important for understanding climate change. While iron availability has been shown to be an important chemical factor for controlling both growth and nitrogen fixation rates in Trichodesmium , all culture experiments to date have focused solely on representatives from one clade of Trichodesmium . Genomic sequence analysis determined that the Trichodesmium erythraeum (IMS101) genome contains many of the archetypical genes involved in the prokaryotic iron stress response. Focusing on three of these genes, isiB , idiA and feoB , we found that all three showed an iron stress response in axenic T. erythraeum (IMS101), and their sequences were well conserved across four species in our Trichodesmium culture collection [consisting of two T. erythraeum strains (IMS101 and GBRTRLI101), two Trichodesmium tenue strains (Z-1 and H9-4), Trichodesmium thiebautii and Trichodesmium spiralis ]. With clade-specific quantitative PCR (qPCR) primers for one of these genes, isiB , we found that high isiB expression at low Fe levels corresponded to specific reductions in N₂ fixation rates in both major phylogenetic clades of Trichodesmium (the T. erythraeum clade and T. tenue clade). With regard to the two clades, the most significant difference determined was temperature optima, while more subtle differences in growth, N₂ fixation rate and gene expression responses to Fe stress were also observed. However the apparent conservation of the Fe stress response in the Trichodesmium genus suggests that it is an important adaptation for their niche in the oligotrophic ocean.     

Rates of NH4+ uptake, intracellular transformation and dissolved organic nitrogen release in two clones of marine Synechococcus spp

Evidence suggests that phytoplankton can release significant amounts of dissolved organic carbon (DOC), particularly when they are nitrogen (N) deficient. To investigate this phenomenon from the perspective of dissolved organic N (DON), ¹⁵N tracer techniques were used to trace the flow of N through two clones of Synechococcus, WH7803 and WH8018, which were grown in batch culture under N-sufficient and N-deficient conditions. At various stages during the experiment, a subsample of the culture was removed and the response to a pulse of ¹⁵N-labeled NH4⁺ was measured with respect to rates of NH4⁺ uptake, intracellular NH4⁺ transformation, and release of total and low-molecular-weight (LMW; <10 000 Da) DON. In contrast to what is commonly observed for DOC, high rates of DON release were not observed during N starvation in either clone. The highest rates of DON release, both absolute and as a percentage of gross N uptake, occurred during N-sufficient growth, and release rates decreased by a factor of 4-7 when NH4⁺ was depleted in the medium.      

The nitrogen physiology of the marine N2-fixing cyanobacteria Trichodesmium spp

Trichodesmium spp. have proved to be enigmatic organisms, and their ecology and physiology are unusual among diazotrophs. Recent research shows that they can simultaneously fix N2 and take up combined nitrogen. The co-occurrence of these two processes is thought to be incompatible, but they could be obligatory in Trichodesmium spp. if only a small fraction of cells within a colony or along a filament are capable of N2 fixation. Combined nitrogen is released from cells during periods of active growth and N2 fixation, and concomitantly taken up by Trichodesmium spp. or cells living in association with colonies. Although the nitrogenase of Trichodesmium spp. is affected by high concentrations of combined nitrogen, it might be relatively less sensitive to low concentrations of combined nitrogen typical of the oligotrophic ocean and culture conditions. Nitrogenase activity and synthesis exhibits an endogenous rhythm in Trichodesmium spp. cultures, which is affected by the addition of nitrogen.     

Net and gross incorporation of nitrogen by marine copepods fed on N-labelled diatoms: Methodology and trophic studies

The stable isotope of nitrogen (¹⁵N) and an appropriate three-compartment model were used in two 24-h lasting feeding experiments to trace the flow of N through the copepod Acartia discaudata and Calanus helgolandicus fed on ¹⁵N-labelled Skeletonema costatum and Thalassiosira weissflogii, respectively. Details of the labelling technique and principles of the computation of N transport rates are given. At the end of a single 24-h feeding period only about one third of the total amount of N ingested by A. discaudata was incorporated into the copepod's body N; we refer to this rate as net incorporation. Most of the N ingested was lost as ammonium (48% of total N ingested), followed by losses in the form of eggs + fecal pellets (13%) and dissolved organic N (DON, 9%). The sum of net incorporation and the latter losses is defined as gross incorporation. Net incorporation by C. helgolandicus and N losses did not vary over time during a 24 h lasting time-series feeding experiment. On average, 79% of total N ingested was actually incorporated by the copepod whereas mean N losses as ammonium, eggs + fecal pellets represented only 12 and 9%, respectively. After a 24-h feeding period only 2% of N ingested was lost as DON. Inspection of individual DON pathways showed that both A. discaudata and C. helgolandicus highly contributed to total DON production via direct excretion (79 and 64%, respectively). The remaining DON appearing in the DON pool was derived from phytoplankton via direct release and/or indirect release (copepod ‘sloppy feeding’).     

Phenotypic and genotypic characterization of multiple strains of the diazotrophic cyanobacterium, Crocosphaera watsonii, isolated from the open ocean

Diazotrophic cyanobacteria have long been recognized as important sources of reduced nitrogen (N) and therefore are important ecosystem components. Until recently, species of the filamentous cyanobacterium Trichodesmium were thought to be the primary sources of fixed N to the open ocean euphotic zone. It is now recognized that unicellular cyanobacteria are also important contributors, with members of the oligotrophic genus Crocosphaera being the only cultured examples. Herein we genetically and phenotypically characterize 10 strains isolated from the tropical Atlantic and North Pacific Oceans, and show that although all of the strains are highly similar at the genetic level, with the internal transcribed sequence (ITS) region sequence varying by ∼2 bp on average, there are many unexpected phenotypic differences between the isolates (e.g. cell size, temperature optima and range, extracellular material excretion and variability in rates of nitrogen fixation). However based on the observed sequence similarity, we propose that all of these isolates are members of the genus Crocosphaera (type strain Crocosphaera watsonii WH8501), and that the phenotypic diversity we see may reflect ecologically important variation relevant for modelling N₂ fixation in the oligotrophic ocean.     

Patterns in the Chemical Fractionation of Organic Nitrogen in Rocky Mountain Streams

The intraannual dynamics of particulate organic nitrogen (PON) and two fractions of dissolved organic nitrogen (DON) were investigated in two Rocky Mountain streams draining watersheds with low rates of N deposition. Organic nitrogen accounted for over 60% of the total annual nitrogen export and consisted mostly of DON. Nitrate peaked during winter months and declined considerably during the growing season (less than 10 µg/L) suggesting the importance of biotic uptake. Concentrations of PON, total DON, and two DON fractions (humic and non-humic) peaked during spring runoff and were positively related to discharge, indicating hydrologic influence. Total DON and its two fractions showed significant inverse relationships to nitrate, indicating that DON and nitrate followed different intraannual patterns. Despite its seasonal fluctuations in concentration, PON showed a consistent carbon–nitrogen (C:N) ratio suggesting that it was relatively uniform in composition. Fractionation studies indicated that DON was primarily of non-humic origin, whereas dissolved organic carbon (DOC) was mainly derived from humic sources. The two DON fractions differed from each other in seasonal patterns of concentration and C:N ratio. The proportion of humic DON increased during snowmelt, and there were diverging seasonal patterns in the C:N ratio of the two fractions implying variations in bioavailability. Although organic nitrogen is commonly treated as a single pool in ecological studies, our results indicated that DON consists of fractions that undergo large intraannual changes in proportions and chemical composition. Treatment of DON as a single pool may be misleading from the viewpoint of understanding ecosystem processes directly related to changes in its sources and biological reactivity.     

IRON STIMULATION OF PHOTOSYNTHESIS AND NITROGEN FIXATION IN ANABAENA 7120 AND TRICHODESMIUM (CYANOPHYCEAE)1

Iron availability may limit carbon and nitrogen fixation in the oceans. The freshwater cyanobacterium, Anabaena, was used as a laboratory model for the biochemical and physiological effects of iron. Increased iron nutrition, in the range of 10^?8 M to 10^?6 M resulted in increases of approximately four fold in carbon and nitrogen fixation rates. Chlorophyll concentration increased, and the relative amount of in vivo fluorescence was reduced with more iron. Natural samples of Trichodesmium, collected off Barbados and incubated with increased iron for two days, showed similar effects. Trichodesmium responded to iron additions indicating that it may be Fe limited in its natural environment. These responses to iron are consistent with the biochemical roles of iron in photosynthesis and nitrogen fixation. The results are discussed in the geochemical context of the sporadic total iron input to tropical oceans and possible implications to spatial and temporal patterns of productivity.     

Nutrient and mineralogical control on dissolved organic C, N and P fluxes and stoichiometry in Hawaiian soils

We measured DOM fluxes from the O horizon of Hawaiiansoils that varied in nutrient availability and mineralcontent to examine what regulates the flux ofdissolved organic carbon (DOC), nitrogen (DON) andphosphorus (DOP) from the surface layer of tropicalsoils. We examined DOM fluxes in a laboratory study from N, P and N+Pfertilized and unfertilized sites on soils that rangedin age from 300 to 4 million years old. The fluxesof DOC and DON were generally related to the % Cand % N content of the soils across the sites. Ingeneral, CO₂ and DOC fluxes were not correlatedsuggesting that physical desorption, dissolution andsorption reactions primarily control DOM release fromthese surface horizons. The one exception to thispattern was at the oldest site where there was asignificant relationship between DOC and CO₂flux. The oldest site also contained the lowestmineral and allophane content of the three sites andthe DOC-respiration correlation indicates arelationship between microbial activity and DOC fluxat this site. N Fertilization increased DON fluxes by50% and decreased DOC:DON ratios in the youngest,most N poor site. In the older, more N rich sites, Nfertilization neither increased DON fluxes nordecreased DOM C:N ratios. Similarly, short termchanges in N availability in laboratory-based soil Nand P fertilization experiments did not affect the DOMC:N ratios of leachate. DOM C:N ratios were similar tosoil organic matter C:N ratios, and changes in DOM C:Nratios with fertilization appeared to have beenmediated through long term effects on SOM C:N ratiosrather than through changes in microbial demand for Cand N. There was no relationship between DON andinorganic N flux during these incubations suggestingthat the organic and inorganic components of N fluxfrom soils are regulated by different factors and thatDON fluxes are not coupled to immediate microbialdemand for N. In contrast to the behavior of DON, thenet flux of dissolved organic phosphorus (DOP) and DOMC:P ratios responded to both long-term P fertilizationand natural variation in reactive P availability. There was lower DOP flux and higher DOM C:P ratiosfrom soils characterized by low P availability andhigh DOP flux and narrow DOM C:P ratios in sites withhigh P availability. DOP fluxes were also closelycorrelated with dissolved inorganic P fluxes. PFertilization increased DOP fluxes by 73% in theyoungest site, 31% in the P rich intermediate agesite and 444% in the old, P poor site indicating thatDOP fluxes closely track P availability in soils.     

Simultaneous measurement of denitrification and nitrogen fixation using isotope pairing with membrane inlet mass spectrometry analysis

A method for estimating denitrification and nitrogen fixation simultaneously in coastal sediments was developed. An isotope-pairing technique was applied to dissolved gas measurements with a membrane inlet mass spectrometer (MIMS). The relative fluxes of three N(2) gas species ((28)N(2), (29)N(2), and (30)N(2)) were monitored during incubation experiments after the addition of (15)NO(3)(-). Formulas were developed to estimate the production (denitrification) and consumption (N(2) fixation) of N(2) gas from the fluxes of the different isotopic forms of N(2). Proportions of the three isotopic forms produced from (15)NO(3)(-) and (14)NO(3)(-) agreed with expectations in a sediment slurry incubation experiment designed to optimize conditions for denitrification. Nitrogen fixation rates from an algal mat measured with intact sediment cores ranged from 32 to 390 microg-atoms of N m(-2) h(-1). They were enhanced by light and organic matter enrichment. In this environment of high nitrogen fixation, low N(2) production rates due to denitrification could be separated from high N(2) consumption rates due to nitrogen fixation. Denitrification and nitrogen fixation rates were estimated in April 2000 on sediments from a Texas sea grass bed (Laguna Madre). Denitrification rates (average, 20 microg-atoms of N m(-2) h(-1)) were lower than nitrogen fixation rates (average, 60 microg-atoms of N m(-2) h(-1)). The developed method benefits from simple and accurate dissolved-gas measurement by the MIMS system. By adding the N(2) isotope capability, it was possible to do isotope-pairing experiments with the MIMS system.     

Glutamine synthetase and nitrogen cycling in colonies of the marine diazotrophic cyanobacteria Trichodesmium spp.

We examined freshly collected samples of the colonial planktonic cyanobacterium Trichodesmium thiebautii to determine the pathways of recently fixed N within and among trichomes. High concentrations of glutamate and glutamine were found in colonies. Glutamate and glutamine uptake rates and concentrations in cells were low in the early morning and increased in the late morning to reach maxima near midday; then uptake and concentration again fell to low values. This pattern followed that previously observed for T. thiebautii nitrogenase activity. Our results suggest that recently fixed nitrogen is incorporated into glutamine in the N2-fixing trichomes and may be passed as glutamate to non-N2-fixing trichomes. The high transport rates and concentrations of glutamate may explain the previously observed absence of appreciable uptake of NH4+, NO3-, or urea by Trichodesmium spp. Immunolocalization, Western blots (immunoblots), and enzymatic assays indicated that glutamine synthetase (GS) was present in all cells during both day and night. GS appeared to be primarily contained in cells of T. thiebautii rather than in associated bacteria or cyanobacteria. Double immunolabeling showed that cells with nitrogenase (Fe protein) contained levels of the GS protein that were twofold higher than those in cells with little or no nitrogenase. GS activity and the uptake of glutamine and glutamate dramatically decreased in the presence of the GS inhibitor methionine sulfoximine. Since no glutamate dehydrogenase activity was detected in this species, GS appears to be the primary enzyme responsible for NH3 incorporation.     

Glutamine synthetase and nitrogen cycling in colonies of the marine diazotrophic cyanobacteria Trichodesmium spp.

We examined freshly collected samples of the colonial planktonic cyanobacterium Trichodesmium thiebautii to determine the pathways of recently fixed N within and among trichomes. High concentrations of glutamate and glutamine were found in colonies. Glutamate and glutamine uptake rates and concentrations in cells were low in the early morning and increased in the late morning to reach maxima near midday; then uptake and concentration again fell to low values. This pattern followed that previously observed for T. thiebautii nitrogenase activity. Our results suggest that recently fixed nitrogen is incorporated into glutamine in the N2-fixing trichomes and may be passed as glutamate to non-N2-fixing trichomes. The high transport rates and concentrations of glutamate may explain the previously observed absence of appreciable uptake of NH4+, NO3-, or urea by Trichodesmium spp. Immunolocalization, Western blots (immunoblots), and enzymatic assays indicated that glutamine synthetase (GS) was present in all cells during both day and night. GS appeared to be primarily contained in cells of T. thiebautii rather than in associated bacteria or cyanobacteria. Double immunolabeling showed that cells with nitrogenase (Fe protein) contained levels of the GS protein that were twofold higher than those in cells with little or no nitrogenase. GS activity and the uptake of glutamine and glutamate dramatically decreased in the presence of the GS inhibitor methionine sulfoximine. Since no glutamate dehydrogenase activity was detected in this species, GS appears to be the primary enzyme responsible for NH3 incorporation.