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 N₂ (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 N₂ fixation when combined nitrogen (NO₃⁻, NH₄⁺) is available and the increased rate of N₂ fixation when combined nitrogen is insufficient to cover the demand. The daily N₂ 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.     

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.     

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.     

Iron-Stimulated N(2) Fixation and Growth in Natural and Cultured Populations of the Planktonic Marine Cyanobacteria Trichodesmium spp

In light of recent proposals that iron (Fe) availability may play an important role in controlling oceanic primary production and nutrient flux, its regulatory impact on N(2) fixation and production dynamics was investigated in the widespread and biogeochemically important diazotrophic, planktonic cyanobacteria Trichodesmium spp. Fe additions, as FeCl(3) and EDTA-chelated FeCl(3), enhanced N(2) fixation (nitrogenase activity), photosynthesis (CO(2) fixation), and growth (chlorophyll a production) in both naturally occurring and cultured (on unenriched oligotrophic seawater) Trichodesmium populations. Maximum enhancement of these processes occurred under FeEDTA-amended conditions. On occasions, EDTA alone led to enhancement. No evidence for previously proposed molybdenum or phosphorus limitation was found. Our findings geographically extend support for Fe limitation of N(2) fixation and primary production to tropical and subtropical oligotrophic ocean waters often characterized by Trichodesmium blooms.     

Arrangement of nitrogenase structural genes in an aerobic filamentous nonheterocystous cyanobacterium.

Members of the marine filamentous, nonheterocystous cyanobacterial genus Trichodesmium not only are capable of fixing nitrogen aerobically in the light but when grown under a light-dark cycle will fix nitrogen only during the light phase. In this study, we constructed a restriction map of the structural nitrogen fixation genes (nifHDK) in Trichodesmium sp. strain NIBB 1067. We found that the organization of the nif genes in Trichodesmium sp. strain NIBB 1067 is contiguous, as found in other nonheterocystous cyanobacteria and in heterocysts. Furthermore, the nif gene arrangement was identical when the cultures were grown with combined nitrogen or under nitrogen-fixing conditions. Therefore, no gene rearrangements occur, such as those that occur during the development of heterocysts in heterocystous species.     

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.     

Release of Dissolved Organic Nitrogen by Marine Diazotrophic Cyanobacteria, Trichodesmium spp

Trichodesmium sp. is a filamentous, colonial cyanobacterium which contributes substantially to the input of nitrogen in tropical and subtropical oceanic waters through nitrogen fixation (N(2) fixation). We applied a N tracer technique to assess the rate of release of dissolved organic nitrogen (DON) from this cyanobacterium and compared those rates with rates of N(2) fixation determined for the same assemblages at the same times of day. Rates of release of DON showed considerable variation within replicate experiments and were variable depending on time of day and duration of time course experiments. On average, rates of DON release were ca. 50% the rates of N(2) fixation. We also fractionated the DON released by using ultrafiltration and found that 60 to 80% of the total organic release was of the size class <10,000 Da. The release of these organic compounds by Trichodesmium spp. is likely a significant source of new nitrogen for the associated bacteria or the non-nitrogen-fixing filaments of the Trichodesmium colonies.     

Incorporation of Different N Sources and Light Response Curves of Nitrogenase and Photosynthesis by Cyanobacterial Blooms from Rice Fields

In this work, we estimate the contributions of the different sources of N incorporated by two N₂-fixing cyanobacterial blooms (Anabaena sp. and Microchaete sp.) in the rice fields of Valencia (Spain) during the crop cycles of 1999 and 2000, and evaluate the response of nitrogenase and C assimilation activities to changing irradiances. Our results show that, far from the generally assumed idea that the largest part of the N incorporated by N₂-fixing cyanobacterial blooms in rice fields comes from N₂ fixation, both cyanobacterial blooms incorporated about three times more N from dissolved combined compounds than from N₂ fixation (only about 33–41% of the N incorporated came from N₂ fixation). Our results on the photodependence of C and N₂ fixation indicate that in both cyanobacterial blooms, N₂ fixation showed a steeper initial slope (α) and was saturated with less irradiance than C fixation, suggesting that N₂ fixation was more efficient than photosynthesis under conditions of light limitation. At saturating light, N₂ fixation and C fixation differed depending on the bloom and on the environmental conditions created by rice plant growth. Carbon assimilation but not nitrogenase activity appeared photoinhibited in the Anabaena but not in the Microchaete bloom in August 1999, when the plants were tall and the canopy was important, and there was no limitation of dissolved inorganic carbon. The opposite was found in the Microchaete bloom of June 2000, when plants were small and produced little shade, and dissolved inorganic carbon was very low.     

Control by light of whole-cell nitrogenase activity and of nitrogenase and bacteriochlorophyll a formation in Rhodobacter capsulatus strain B10S

Control of nitrogenase and bacteriochlorophyll a (BChl) by light was studied under steady-state conditions with continuous cultures of Rhodobacter capsulatus B10S supplied with malate and growth-limiting amounts of ammonium. Consumption of malate and, correspondingly, the C/N ratio at which malate and ammonium were consumed increased when illumination was increased from 3 to approximately 20 klx and became constant at higher illuminations of up to 40 klx. Essentially the same kinetics were observed with respect to nitrogenase activity of cells, contents of nitrogenase polypeptides, and nifH promoter activity. Substrate consumption was half-maximal at 8 klx and was independent of the presence of nitrogenase. Therefore, it is concluded that light controls the C/N ratio (a quantitative measure of the nitrogen status of cells), which in turn is involved in the control of nitrogenase at the level of nif promoter activity. Post-translational regulation of nitrogenase activity by ADP-ribosylation was not observed under steady-state conditions, but it took place when illumination was suddenly decreased to the range where malate consumption and, consequently, the C/N ratio decreased. Irrespective of the presence or absence of nitrogenase, specific BChl contents of the cultures were constant above 20 klx, and they increased at lower illuminations. These results do not confirm a recently proposed link between nitrogen fixation and photosynthesis as represented by BChl. Received: 29 October 1998 / Accepted: 30 December 1998     

Azolla filiculoides Nitrogenase Activity Decrease Induced by Inoculation with Chlamydomonas sp

Experiments were conducted to determine the influence of Chlamydomonas sp. on nitrogen fixation (C(2)H(2) --> C(2)H(4)) in Azolla filiculoides and on the nitrogen fixation and growth of free-living Anabaena azollae 2B organisms. Inoculation of azolla medium with Chlamydomonas sp. was associated with decreased nitrogenase activity in A. filiculoides and with increases in the density of a fungal population identified as Acremonium sp. Subsequent inoculation of azolla medium with this fungus was also accompanied by a significant decrease in nitrogenase activity of A. filiculoides. However, the extent of depression of nitrogenase activity was significantly higher when azolla medium was inoculated with Chlamydomonas sp. than when it was inoculated with Acremonium sp. Inoculation of nitrogen-free Stanier medium with either Acremonium sp. or Chlamydomonas sp. did not adversely affect the growth or nitrogenase activity of free-living A. azollae. Decreased nitrogenase activity in A. filiculoides is apparently related to the adverse influence of the green alga and the fungus on the macrosymbiont. The mechanisms that might be involved are discussed.     

Molecular analysis of the phosphorus starvation response in Trichodesmium spp.

The marine diazotroph Trichodesmium is a major contributor to primary production and nitrogen fixation in the tropical and subtropical oceans. These regions are often characterized by low phosphorus (P) concentrations, and P starvation of Trichodesmium could limit growth, and potentially constrain nitrogen fixation. To better understand how this genus responds to P starvation we examined four genes involved in P acquisition: two copies of a high-affinity phosphate binding protein ( pstS and sphX ) and two putative alkaline phosphatases ( phoA and phoX ). Sequence analysis of these genes among cultured species of Trichodesmium ( T. tenue, T. erythraeum, T. thiebautii and T. spiralis ) showed that they all are present and conserved within the genus. In T. erythraeum IMS101, the expression of sphX , phoA and phoX were sensitive to P supply whereas pstS was not. The induction of alkaline phosphatase activity corresponded with phoA and phoX expression, but enzyme activity persisted after the expression of these genes returned to basal levels. Additionally, nifH (nitrogenase reductase; involved in nitrogen fixation) expression was downregulated under P starvation conditions. These data highlight molecular level responses to low P and lay a foundation for better understanding the dynamics of Trichodesmium P physiology in low-P environments.     

In vivo energetics and control of nitrogen fixation: changes in the adenylate energy charge and adenosine 5'-diphosphate/adenosine 5'-triphosphate ratio of cells during growth on dinitrogen versus growth on ammonia.

The effects of the intracellular energy balance and adenylate pool composition on N2 fixation were examined by determining changes in the energy charge (EC) and the ADP/ATP (D/T) ratio of cells in chemostat and batch cultures of Clostridium pasteurianum, Klebsiella pneumoniae, and Azotobacter vinelandii. When cells of C. pasteurianum, K. pneumoniae, and A. vinelandii in sucrose-limited chemostats were examined, in all cases the EC increased greater than or equal to 15% when the nitrogen source was switched from N2 to NH3 and decreased greater than or equal to 15% when the nitrogen source was switched from NH3 to N2. The D/T ratio of the same cultures decreased greater than or equal to 70% when they were switched from N2 to NH3. In such cultures the adenylate pools remained constant when the cells were grown on either NH3 or N2. In nitrogen (NH3)-limited cultures, the adenylate pool was two- to threefold higher than the adenylate pool in sucrose-limited cultures, and the nitrogenase content of such cells was two- to threefold greater than the nitrogenase content of sucrose-limited N2-fixing cells. The EC and D/T ratio of cells from batch cultures of C. pasteurianum growing on NH3 in the presence of N2 were 0.82 and 0.83, respectively, but when the NH3 was consumed and the cells were switched to a nitrogen-fixing metabolism, the EC and D/T ratio changed to 0.70 and 0.90, respectively. Conversely, when NH3 was added to N2-fixing cultures the EC and D/T ratio changed within 1.5 h the EC and D/T ratio of NH3-grown cells. The nitrogen content of N2-fixing cells to which NH3 was added decreased at a rate greater could be accounted for by cell growth in the absence of further synthesis. This decay of nitrogenase activity (with a half-life about 1.2 to 1.4 h) suggests that some type of inactivation of nitrogenase occurs during repression. The nitrogenase of whole cells was estimated to be operating at about 32% of its theoretical maximum activity during steady-state N2-fixing conditions. Similarities in the data from chemostat and batch cultures of both aerobic and anaerobic N2-fixing organisms suggest that low EC and high D/T ratio are normal manifestations of an N2-fixing physiology.     

N(2) Fixation Associated with Decaying Leaves of the Red Mangrove (Rhizophora mangle)

N(2) (C(2)H(2)) fixation was associated with decaying leaves of Rhizophora mangle. The process was predominantly anaerobic, with about two-thirds of the nitrogenase activity being light dependent. Average N(2) fixation rates in the light were 11 mug of N per g (dry weight) per h for leaves that had decayed for 2 to 3 weeks. This nitrogen input is probably significant in the estuarine, detrital food chains linked to R. mangle.     

Environmental factors affectingin vitro nitrogenase activity of cyanobacteria isolated from rice-fields

The nitrogen-fixing capacity of four cyanobacterial strains was tested in relation to heterotrophic ability, tolerance to combined nitrogen and adaptive capacity to changes in light intensity and pH. Strains (Anabaena variabilis UAM 202;Calothrix marchica UAM 214;Nodularia spumigena UAM 204,Nostoc punctiforme UAM 205) were isolated from the rice-fields of Valencia (Spain).C. marchica, was the only strain able to grow and to fix dinitrogen under heterotrophic conditions, with fructose and glucose. Fructose was the best substrate supporting growth and dinitrogen fixation in mixotrophy (growth in the light under conditions where CO₂ and organic carbon are assimilated simultaneously), photoheterotrophy (growth in the light on an organic compound in the absence of net CO₂ fixation) and heterotrophy (growth on an organic compound in the dark). Ammonium repressed nitrogenase more than nitrate. Full repression was observed only at concentrations of combined nitrogen higher than those usually found in rice-fields. Carbohydrates had a protective effect on nitrogenase against ammonium inhibition inC. marchica. All four strains showed increased nitrogenase activity when the light intensity was increased during assays. Variations of pH normally occurring in rice fields led to no important changes in nitrogenase activity inC. marchica. This fact, together with its potential for heterotrophic growth and tolerance to combined nitrogen, make this the most suitable of the four strains for inoculation experiments in rice fields.     

Low temperature delays timing and enhances the cost of nitrogen fixation in the unicellular cyanobacterium Cyanothece

Marine nitrogen-fixing cyanobacteria are largely confined to the tropical and subtropical ocean. It has been argued that their global biogeographical distribution reflects the physiologically feasible temperature range at which they can perform nitrogen fixation. In this study we refine this line of argumentation for the globally important group of unicellular diazotrophic cyanobacteria, and pose the following two hypotheses: (i) nitrogen fixation is limited by nitrogenase activity at low temperature and by oxygen diffusion at high temperature, which is manifested by a shift from strong to weak temperature dependence of nitrogenase activity, and (ii) high respiration rates are required to maintain very low levels of oxygen for nitrogenase, which results in enhanced respiratory cost per molecule of fixed nitrogen at low temperature. We tested these hypotheses in laboratory experiments with the unicellular cyanobacterium Cyanothece sp. {"type":"entrez-nucleotide","attrs":{"text":"BG043511","term_id":"12489990","term_text":"BG043511"}}BG043511. In line with the first hypothesis, the specific growth rate increased strongly with temperature from 18 to 30 °C, but leveled off at higher temperature under nitrogen-fixing conditions. As predicted by the second hypothesis, the respiratory cost of nitrogen fixation and also the cellular C:N ratio rose sharply at temperatures below 21 °C. In addition, we found that low temperature caused a strong delay in the onset of the nocturnal nitrogenase activity, which shortened the remaining nighttime available for nitrogen fixation. Together, these results point at a lower temperature limit for unicellular nitrogen-fixing cyanobacteria, which offers an explanation for their (sub)tropical distribution and suggests expansion of their biogeographical range by global warming.     

Isolation and characterization of the unicellular diazotrophic cyanobacterium Group C TW3 from the tropical western Pacific Ocean

A unicellular diazotrophic cyanobacterium strain of Group C, designated TW3, was isolated from the oligotrophic Kuroshio Current of the western Pacific Ocean. To our knowledge, this represents the first successful laboratory culture of a Group C unicellular diazotroph from oceanic water. TW3 cells are green rods, 2.5-3.0 μm in width and 4.0-6.0 μm in length. Phylogenetic analyses of both 16S rRNA and nifH gene fragments indicated that the TW3 sequences were over 98% identical to those of the previously isolated Cyanothece sp. ATCC51142 and Gloeocapsa sp., suggesting that TW3 is a member of the Group C unicellular diazotrophs. In addition, both TW3 and Cyanothece sp. ATCC51142 share morphological characteristics; both strains are sheathless and rod-shaped, display binary fission in a single plane, and possess dispersed thylakoids. TW3 grows aerobically in nitrogen-deficient artificial seawater, and exhibited the highest observed growth rate of 0.035 h(-1) when cultured at 30°C and 140 μmol m(-2) s(-1) of light intensity. The nitrogen fixation rate, when grown optimally using a 12 h/12 h light-dark cycle, was 7.31 × 10(-15) mol N cell(-1) day(-1) . Immunocytochemical staining using Trichodesmium sp. NIBB1067 nitrogenase antiserum revealed the existence of diazotrophic cells sharing morphological characteristics of TW3 in the Kuroshio water from which TW3 was isolated.     

Nitrogenase. VI. Acetylene reduction assay: Dependence of nitrogen fixation estimates on component ratio and acetylene concentration.

Acetylene reduction, an assay for nitrogenase activity (nitrogen:(acceptor) oxidoreductase, EC 1.7.99.2), Is dependent on the ratio of the two protein components of nitrogenase as well as on C2H2 concentration. As the component I : component II ratio (based on activity) is increased, the C2H2 reduction : N2 fixation ratio decreases to a minimum of 3.4 and then increases. The minimum is found at a ratio near 1 : 1. At a component I : component II ratio of 20 : 1, the C2H2 reduction : N2 fixation ratio is 5.3. Acetylene exhibits substrate inhibition in assays for nitrogenase activity. Both the apparent Km and Ki for acetylene vary as a function of the relative concentrations of components I and II present in the assay. When the more labile component II is limiting in the assay and "saturating" levels of C2H2 (above 0.1 atm) are used, N2-fixation capacity may be greatly under-estimated.     

Light-dark (12:12) cycle of carbon and nitrogen metabolism in Crocosphaera watsonii WH8501: relation to the cell cycle

This study provides with original data sets on the physiology of the unicellular diazotrophic cyanobacterium Crocosphaera watsonii WH8501, maintained in continuous culture in conditions of obligate diazotrophy. Cultures were exposed to a 12:12 light-dark regime, representative of what they experience in nature and where growth is expected to be balanced. Nitrogen and carbon metabolism were monitored at high frequency and their dynamics was compared with the cell cycle. Results reveal a daily cycle in the physiological and biochemical parameters, tightly constrained by the timely decoupled processes of N(2) fixation and carbon acquisition. The cell division rate increased concomitantly to carbon accumulation and peaked 6 h into the light. The carbon content reached a maximum at the end of the light phase. N(2) fixation occurred mostly during the dark period and peaked between 9 and 10 h into the night, while DNA synthesis, reflected by DNA fluorescence, increased until the end of the night. Consequently, cells in G1- and S-phases present a marked decrease in their C:N ratio. Nitrogen acquisition through N(2) fixation exceeded 1.3- to 3-fold the nitrogen requirements for growth, suggesting that important amounts of nitrogen are excreted even under conditions supposed to favour balanced, carbon and nitrogen acquisitions.