Oxygen-Poor Microzones as Potential Sites of Microbial N2 Fixation in Nitrogen-Depleted Aerobic Marine Waters

The nitrogen-deficient coastal waters of North Carolina contain suspended bacteria potentially able to fix N₂. Bioassays aimed at identifying environmental factors controlling the development and proliferation of N₂ fixation showed that dissolved organic carbon (as simple sugars and sugar alcohols) and particulate organic carbon (derived from Spartina alterniflora) additions elicited and enhanced N₂ fixation (nitrogenase activity) in these waters. Nitrogenase activity occurred in samples containing flocculent, mucilage-covered bacterial aggregates. Cyanobacterium-bacterium aggregates also revealed N₂ fixation. In all cases bacterial N₂ fixation occurred in association with surficial microenvironments or microzones. Since nitrogenase is oxygen labile, we hypothesized that the aggregates themselves protected their constituent microbes from O₂. Microelectrode O₂ profiles revealed that aggregates had lower internal O₂ tensions than surrounding waters. Tetrazolium salt (2,3,5-triphenyl-3-tetrazolium chloride) reduction revealed that patchy zones existed both within microbes and extracellularly in the mucilage surrounding microbes where free O₂ was excluded. Triphenyltetrazolium chloride reduction also strongly inhibited nitrogenase activity. These findings suggest that N₂ fixation is mediated by the availability of the appropriate types of reduced microzones. Organic carbon enrichment appears to serve as an energy and structural source for aggregate formation, both of which were required for eliciting N₂ fixation responses of these waters.     

Contemporaneous N(2) Fixation and Oxygenic Photosynthesis in the Nonheterocystous Mat-Forming Cyanobacterium Lyngbya aestuarii

The nonheterocystous filamentous cyanobacterial genus Lyngbya is a widespread and frequently dominant component of marine microbial mats. It is suspected of contributing to relatively high rates of N(2) fixation associated with mats. The ability to contemporaneously conduct O(2)-sensitive N(2) fixation and oxygenic photosynthesis was investigated in Lyngbya aestuarii isolates from a North Carolina intertidal mat. Short-term (<4-h) additions of the photosystem II (O(2) evolution) inhibitor 3(3,4-dichlorophenyl)-1,1-dimethylurea stimulated light-mediated N(2) fixation (nitrogenase activity), indicating potential inhibition of N(2) fixation by O(2) production. However, some degree of light-mediated N(2) fixation in the absence of 3(3,4-dichlorophenyl)-1,1-dimethylurea was observed. Electron microscopic immunocytochemical localization of nitrogenase, coupled to microautoradiographic studies of CO(2) fixation and cellular deposition of the tetrazolium salt 2,4,5-triphenyltetrazolium chloride, revealed that (i) nitrogenase was widely distributed throughout individual filaments during illuminated and dark periods, (ii) CO(2) fixation was most active in intercalary regions, and (iii) daylight 2,4,5-triphenyltetrazolium chloride reduction (formazan deposition) was most intense in terminal regions. Results suggest lateral partitioning of photosynthesis and N(2) fixation during illumination, with N(2) fixation being confined to terminal regions. During darkness, a larger share of the filament appears capable of N(2) fixation.     

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.     

Free-living dinitrogen-fixing bacteria isolated from petroleum refinery oily sludge

Dinitrogen-fixing activity (acetylene reduction and N(2) fixation) was found in an oily sludge originating from a petroleum refinery. Two representative dinitrogen-fixing bacterial strains were isolated from this oily waste. Their nitrogenase activity was effective when they were cultivated on sterilized sludge or simple carbon substrates (organic acid salts, sugars). Using the classical methods, these strains could not be unambiguously related to other diazotrophic taxa. The landfarming process is widely used for oily sludge disposal; this study shows that oily sludges are more than a simple carbon input into the soil but that they must also be considered as real sources of dinitrogen-fixing and probably degradative microorganisms.     

SIGNIFICANCE OF BACTERIAL-ANABAENA (CYANOPHYCEAE) ASSOCIATIONS WITH RESPECT TO N2 FIXATION IN FRESHWATER1, 2

The functional aspects of specific associations between bluegreen algae and bacteria were investigated using both naturally occurring and cultured species of Anabaena. In take waters where bacteria were associated with Anabaena heterocysls, the bacteria exhibited a chemotactic response to a variety of amino acids and glucose. Earlier autoradiographic evidence that bacteria associated with heterocysts incorporate identical substrates indicates that associated bacteria probably benefit by utilizing algal excretion products. In return, the bacteria stimulate algal N₂fixation. The most likely mechanism explaining such stimulation appeared to be bacterial oxygen removal in microzones (< 3 μm diam) bordering heterocysts during periods of high ambient oxygen concentrations. In the presence of bacteria, Anabaena rapidly overcame nitrogenase- inhibiting concentrations of oxygen. Axenic cullures had more extensive nitrogenase inhibition, and took longer to recover in response to oxygenation. Algal-bacterial mutualism aids Anabaena in maintaining concurrent optimal N2 fixation and high photosynthetic rates in highly oxygenated surface waters.     

BACTERIAL ASSOCIATIONS WITH MARINE OSCILLATORIA SP. (TRICHODESMIUM SP.) POPULATIONS: ECOPHYSIOLOGICAL IMPLICATIONS1

On three separate occasions we investigated morphological and physiological aspects of bacterial associations with planktonic aggregates of the ubiquitous marine N₂ fixing cyanobacterium Trichodesmium sp. Close associations generally characterized Trichodesmium blooms; associations were present during day- and night-time. Colonization by both rod-shaped and filamentous heterotrophic bacteria occurred on Trichodesmiun aggregates actively fixing N₂ (acetylene reduction). Scanning electron and optical microscopy showed bacteria located both around and within aggregates. Microautoradiography demonstrated that associated bacteria largely mediated utilization of trace additions of ³H-labeled carbohydrates (fructose, glucose, mannitol) and amino acids, whereas Trichodesmium utilized amino acids only. Oxygen measurements using microelectrodes revealed high localized oxygen consumption among aggregates, with rapid (within a minute) changes from supersaturated to subsaturated oxygen following the transition from photosynthetic illuminated to dark periods. Stab culturing techniques confirmed the presence of heterotrophic N₂ fixers among aggregate-associated bacteria. Parallel deployment of oxygen microelectrodes, the tetrazolium salt 2,3,5 triphenyl tetrazolium chloride (TTC) and acetylene reduction assays demonstrated microaerophilic requirements for expression of nitrogenase activity among cultured bacteria. Trichodesmium aggregates are characterized by dynamic nutrient and oxygen regimes, which promote and maintain simultaneous and contiguous oxygenic photosynthesis and N₂ fixation. In part, the above-mentioned consortial interactions with a variety of heterotrophic bacteria facilitate Trichodesmium biomass production and bloom formation in nitrogen depleted, oligotrophic tropical/subtropical waters.     

Current Nitrogen Fixation Is Involved in the Regulation of Nitrogenase Activity in White Clover (Trifolium repens L.)

Previous studies have shown that nitrogenase activity decreases dramatically after defoliation, presumably because of an increase in the O2 diffusion resistance in the infected nodules. It is not known how this O2 diffusion resistance is regulated. The aim of this study was to test the hypothesis that current N2 fixation (ongoing flux of N2 through nitrogenase) is involved in the regulation of nitrogenase activity in white clover (Trifolium repens L. cv Ladino) nodules. We compared the nitrogenase activity of plants that were prevented from fixing N2 (by continuous exposure of their nodulated root system to an Ar:O2 [80:20] atmosphere) with that of plants allowed to fix N2 (those exposed to N2:O2, 80:20). Nitrogenase activity was determined as the amount of H2 evolved under Ar:O2. An open flow system was used. In experiment I, 6 h after complete defoliation and the continuous prevention of N2 fixation, nitrogenase activity was higher by a factor of 2 compared with that in plants allowed to fix N2 after leaf removal. This higher nitrogenase activity was associated with a lower O2 limitation (measured as the partial pressure of O2 required for highest nitrogenase activity). In experiment II, the nitrogenase activity of plants prevented from fixing N2 for 2 h before leaf removal showed no response to defoliation. The extent to which nitrogenase activity responded to defoliation was different in plants allowed to fix N2 and those that were prevented from doing so in both experiments. This leads to the conclusion that current N2 fixation is directly involved in the regulation of nitrogenase activity. It is suggested that an N feedback mechanism triggers such a response as a result of the loss of the plant's N sink strength after defoliation. This concept offers an alternative to other hypotheses (e.g. interruption of current photosynthesis, carbohydrate deprivation) that have been proposed to explain the immediate decrease in nitrogenase activity after defoliation.     

Evidence for NH4+ switch-off regulation of nitrogenase activity by bacteria in salt marsh sediments and roots of the grass Spartina alterniflora.

The regulatory effect of NH4+ on nitrogen fixation in a Spartina alterniflora salt marsh was examined. Acetylene reduction activity (ARA) measured in situ was only partially inhibited by NH4+ in both the light and dark after 2 h. In vitro analysis of bulk sediment divided into sediment particles, live and dead roots, and rhizomes showed that microbes associated with sediment and dead roots have a great potential for anaerobic C2H2 reduction, but only if amended with a carbon source such as mannose. Only live roots had significant rates of ARA without an added carbon source. In sediment, N2-fixing mannose enrichment cultures could be distinguished from those enriched by lactate in that only the latter were rapidly inhibited by NH4+. Ammonia also inhibited ARA in dead and live roots and in surface-sterilized roots. The rate of this inhibition appeared to be too rapid to be attributed to the repression and subsequent dilution of nitrogenase. The kinetic characteristics of this inhibition and its prevention in root-associated microbes by methionine sulfoximine are consistent with the NH4+ switch-off-switch-on mechanism of nitrogenase regulation.     

Evidence for NH4+ switch-off regulation of nitrogenase activity by bacteria in salt marsh sediments and roots of the grass Spartina alterniflora

The regulatory effect of NH4+ on nitrogen fixation in a Spartina alterniflora salt marsh was examined. Acetylene reduction activity (ARA) measured in situ was only partially inhibited by NH4+ in both the light and dark after 2 h. In vitro analysis of bulk sediment divided into sediment particles, live and dead roots, and rhizomes showed that microbes associated with sediment and dead roots have a great potential for anaerobic C2H2 reduction, but only if amended with a carbon source such as mannose. Only live roots had significant rates of ARA without an added carbon source. In sediment, N2-fixing mannose enrichment cultures could be distinguished from those enriched by lactate in that only the latter were rapidly inhibited by NH4+. Ammonia also inhibited ARA in dead and live roots and in surface-sterilized roots. The rate of this inhibition appeared to be too rapid to be attributed to the repression and subsequent dilution of nitrogenase. The kinetic characteristics of this inhibition and its prevention in root-associated microbes by methionine sulfoximine are consistent with the NH4+ switch-off-switch-on mechanism of nitrogenase regulation.     

Nitrogenase activity in Alnus incana root nodules. Responses to O(2) and short-term N(2) deprivation

O(2) and host-microsymbiont interactions are key factors affecting the physiology of N(2)-fixing symbioses. To determine the relationship among nitrogenase activity of Frankia-Alnus incana root nodules, O(2) concentration, and short-term N(2) deprivation, intact nodulated roots were exposed to various O(2) pressures (pO(2)) and Ar:O(2) in a continuous flow-through system. Nitrogenase activity (H(2) production) occurred at a maximal rate at 20% O(2). Exposure to short-term N(2) deprivation in Ar:O(2) carried out at either 17%, 21%, or 25% O(2) caused a decline in the nitrogenase activity at 21% and 25% O(2) by 12% and 25%, respectively. At 21% O(2), nitrogenase activity recovered to initial activity within 60 min. The decline rate was correlated with the degree of inhibition of N(2) fixation. Respiration (net CO(2) evolution) decreased in response to the N(2) deprivation at all pO(2) values and did not recover during the time in Ar:O(2). Increasing the pO(2) from 21% to 25% and decreasing the pO(2) from 21% to 17% during the decline further decreased rather than stimulated nitrogenase activity, showing that the decline was not due to O(2) limitation. The decline was possibly due to a temporary disturbance in the supply of reductant to nitrogenase with a partial O(2) inhibition of nitrogenase at 25% O(2). These results are consistent with a fixed O(2) diffusion barrier in A. incana root nodules, and show that A. incana nodules differ from legume nodules in the response of the nitrogenase activity to O(2) and N(2) deprivation.     

Diazotrophy in Modern Marine Bahamian Stromatolites

N2 fixation (nitrogenase activity), primary production, and diazotrophic community composition of stromatolite mats from Highborne Cay, Exuma, Bahamas, were examined over a 2-year period (1997-1998). The purpose of the study was to characterize the ecophysiology of N2 fixation in modern marine stromatolites. Microbial mats are an integral surface component of these stromatolites and are hypothesized to have a major role in stromatolite formation and growth. The stromatolite mats contained active photosynthetic and diazotrophic assemblages that exhibited temporal separation of nitrogenase activity (NA) and photosynthesis. Maximal NA was detected at night. Seasonal differences in NA and net O2 production were observed. Photosynthetic activity and the availability of reduced organic carbon appear to be the key determinants of NA. Additions of the de novo protein synthesis inhibitor chloramphenicol did not inhibit NA in March 1998, but greatly inhibited NA in August 1998. Partial sequence analysis of the nifH gene indicates that a broad diversity of diazotrophs may be responsible for NA in the stromatolites.     

Anoxygenic Photosynthesis and Nitrogen Fixation by a Microbial Mat Community in a Bahamian Hypersaline Lagoon

Simultaneous measurements of photosynthesis (both oxygenic and anoxygenic) and N(inf2) fixation were conducted to discern the relationships between photosynthesis, N(inf2) fixation, and environmental factors potentially regulating these processes in microbial mats in a tropical hypersaline lagoon (Salt Pond, San Salvador Island, Bahamas). Major photoautotrophs included cyanobacteria, purple phototrophic bacteria, and diatoms. Chemosystematic photopigments were used as indicators of the relative abundance of mat phototrophs. Experimental manipulations consisted of light and dark incubations of intact mat samples exposed to the photosystem II inhibitor DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea], a dissolved organic carbon source (D-glucose), and normal seawater (37(permil)). Photosynthetic rates were measured by both O(inf2) and (sup14)C methods, and nitrogenase activity (NA) was estimated by the acetylene reduction assay. Moderate reductions in salinity (from 74 to 37(permil)) had no measurable effect on photosynthesis, O(inf2) consumption, or NA. CO(inf2) fixation in DCMU-amended samples was (symbl)25% of that in the control (nonamended) samples and demonstrated photosynthetic activity by anoxygenic phototrophs. NA in DCMU-amended samples, which was consistently higher (by a factor of 2 to 3) than the other (light and dark) treatments, was also attributed to purple phototrophic bacteria. The ecological implication is that N(inf2) fixation by anoxygenic phototrophs (purple phototrophic bacteria and possibly cyanobacteria) may be regulated by the activity of oxygenic phototrophs (cyanobacteria and diatoms). Consortial interactions that enhance the physiological plasticity of the mat community may be a key for optimizing production, N(inf2) fixation, and persistence in these extreme environments.     

Ammonium inhibition of nitrogenase activity in Herbaspirillum seropedicae.

The effect of oxygen, ammonium ion, and amino acids on nitrogenase activity in the root-associated N2-fixing bacterium Herbaspirillum seropedicae was investigated in comparison with Azospirillum spp. and Rhodospirillum rubrum. H. seropedicae is microaerophilic, and its optimal dissolved oxygen level is from 0.04 to 0.2 kPa for dinitrogen fixation but higher when it is supplied with fixed nitrogen. No nitrogenase activity was detected when the dissolved O2 level corresponded to 4.0 kPa. Ammonium, a product of the nitrogenase reaction, reversibly inhibited nitrogenase activity when added to derepressed cell cultures. However, the inhibition of nitrogenase activity was only partial even with concentrations of ammonium chloride as high as 20 mM. Amides such as glutamine and asparagine partially inhibited nitrogenase activity, but glutamate did not. Nitrogenase in crude extracts prepared from ammonium-inhibited cells showed activity as high as in extracts from N2-fixing cells. The pattern of the dinitrogenase and the dinitrogenase reductase revealed by the immunoblotting technique did not change upon ammonium chloride treatment of cells in vivo. No homologous sequences were detected with the draT-draG probe from Azospirillum lipoferum. There is no clear evidence that ADP-ribosylation of the dinitrogenase reductase is involved in the ammonium inhibition of H. seropedicae. The uncoupler carbonyl cyanide m-chlorophenylhydrazone decreased the intracellular ATP concentration and inhibited the nitrogenase activity of whole cells. The ATP pool was not significantly disturbed when cultures were treated with ammonium in vivo. Possible mechanisms for inhibition by ammonium of whole-cell nitrogenase activity in H. seropedicae are discussed.     

Bacterial life and dinitrogen fixation at a gypsum rock

The organisms of a bluish-green layer beneath the shards of a gypsum rock were characterized by molecular techniques. The cyanobacterial consortium consisted almost exclusively of Chroococcidiopsis spp. The organisms of the shards expressed nitrogenase activity (C2H2 reduction) aerobically and in light. After a prolonged period of drought at the rock, the cells were inactive, but they resumed nitrogenase activity 2 to 3 days after the addition of water. In a suspension culture of Chroococcidiopsis sp. strain PCC7203, C2H2 reduction required microaerobic conditions and was strictly dependent on low light intensities. Sequencing of a segment of the nitrogenase reductase gene (nifH) indicated that Chroococcidiopsis possesses the alternative molybdenum nitrogenase 2, expressed in Anabaena variabilis only under reduced O2 tensions, rather than the widespread, common molybdenum nitrogenase. The shards apparently provide microsites with reduced light intensities and reduced O2 tension that allow N2 fixation to proceed in the unicellular Chroococcidiopsis at the gypsum rock, unless the activity is due to minute amounts of other, very active cyanobacteria. Phylogenetic analysis of nifH sequences tends to suggest that molybdenum nitrogenase 2 is characteristic of those unicellular or filamentous, nonheterocystous cyanobacteria fixing N2 under microaerobic conditions only.     

Microbial communities and diazotrophic activity differ in the root‐zone of Alamo and Dacotah switchgrass feedstocks

Nitrogen (N) bioavailability is a primary limiting nutrient for crop and feedstock productivity. Associative nitrogen fixation (ANF) by diazotrophic bacteria in root‐zone soil microbial communities have been shown to provide significant amounts of N to some tropical grasses, but this potential in switchgrass, a warm‐season, temperate, US native, perennial tallgrass has not been widely studied. ‘Alamo’ and ‘Dacotah’ are cultivars of switchgrass, adapted to the southern and northern regions of the United States, respectively, and offer an opportunity to better describe this plant–bacterial association. The nitrogenase enzyme activity, microbial communities, and amino acid profiles in the root‐zones of the two ecotypes were studied at three different plant growth stages. Differences in the nitrogenase enzyme activity and free soluble amino acid profiles indicated the potential for greater nitrogen fixation in the high productivity Alamo compared with the lower productivity Dacotah. Changes in the amino acid profiles and microbial community structure (rRNA genes) of the root‐zone suggest different plant–bacterial interactions can help to explain differences in nitrogenase activity. PICRUSt analysis revealed functional differences, especially nitrogen metabolism, that supported ecotype differences in root‐zone nitrogenase enzyme activity. It is thought that the greater productivity of Alamo increased the belowground flow of carbon into roots and root‐zone habitats, which in turn support the high energy demands needed to support nitrogen fixation. Further research is thus needed to understand plant ecotype and cultivar trait differences that can be used to breed or genetically modify crop plants to support root‐zone associations with diazotrophs.     

Synthesis and activity of nitrogenase in Klebsiella pneumoniae exposed to low concentrations of oxygen

Effects of very low concentrations of dissolved O2 on nitrogenase activity in Klebsiella pneumoniae were studied in a stirred chamber system which enabled simultaneous measurements of steady-state O2 concentrations, O2 consumption and C2H2 reduction. A strain carrying a chromosomal nifH::lac fusion as well as the Nif+ plasmid pRD1, expressed nitrogenase activity with 80 nM-O2, a concentration known to inhibit nifH::lac expression by about 50% Thus nitrogenase activity in vivo was no more sensitive to O2 than expression of nifH::lac. When compared with anaerobic treatments, dissolved O2 near 30 nM apparently stimulated nitrogenase derepression and enhanced the activity of nitrogenase synthesized anaerobically. Thus, in this organism, N2 fixation occurs in microaerobic as well as anaerobic conditions.     

Effect of oxygen level on simultaneous nitrogenase and sMMO expression and activity in Methylosinus trichosporium OB3b and its sMMO(C) mutant, PP319: aerotolerant N2 fixation in PP319

Soluble methane monooxygenase (sMMO) expression and activity were monitored under conditions that either promoted or suppressed the expression of nitrogenase in Methylosinus trichosporium OB3b wild-type (WT) and in its sMMO-constitutive mutant, PP319. Both WT and mutant cultures had reduced sMMO activity and protein levels under elevated O2 conditions (188 microM) compared with low O2 conditions (24 microM). Simultaneous N2 fixation also reduced sMMO activity in both cultures when O2 was low. However, when O2 levels were increased, nitrogenase expression ceased and sMMO activity was reduced by approximately 77% in the WT, whereas sMMO and nitrogenase expression and activity in PP319 were relatively unaffected by the higher O2 levels. Western immunoblot analysis showed that the nitrogenase Fe protein resolved as two components (apparent molecular mass of 30.5 and 32 kDa) in both the WT and PP319 when O2 levels were low. When O2 levels were high, only the 32-kDa form of the Fe protein was present in PP319, whereas neither form was detectable in the WT. Aerotolerant N2 fixation appears to be associated with the 32-kDa Fe protein in M. trichosporium OB3b.     

Nitrogen fixation by the thermophilic green sulfur bacterium Chlorobium tepidum.

The thermophilic green sulfur bacterium Chlorobium tepidum grew with N2, NH4+, or glutamine as the sole nitrogen source under phototrophic (anaerobic-light) conditions. Growth on N2 required increased buffering capacity to stabilize uncharacterized pH changes that occurred during diazotrophic growth. Increased sulfide levels were stimulatory for growth on N2. Levels of nitrogenase activity (acetylene reduction) in N2-grown C. tepidum cells were very high, among the highest ever reported for anoxygenic phototrophic bacteria. Maximal acetylene reduction rates in C. tepidum cells were observed at 48 to 50 degrees C, which is about 15 degrees C higher than the optimum temperature for nitrogenase activity in mesophilic chlorobia, and nitrogenase activity in C. tepidum responded to addition of ammonia by a "switch-off/switch-on" mechanism like that in phototrophic purple bacteria. C. tepidum cells assimilated ammonia mainly via the glutamine synthetase-glutamate synthase pathway, elevated levels of both of these enzymes being present in cells grown on N2. These results show that N2 fixation can occur in green sulfur bacteria up to at least 60 degrees C and that regulatory mechanisms important in control of nitrogenase activity in mesophilic anoxygenic phototrophs also appear to regulate thermally active forms of the enzyme.     

Interaction between hydrogenase, nitrogenase, and respiratory activities in a Frankia isolate from Alnus rubra

H2 uptake and H2-supported O2 uptake were measured in N2-fixing cultures of Frankia strain ArI3 isolated from root nodules of Alnus rubra. H2 uptake by intact cells was O2 dependent and maximum rates were observed at ambient O2 concentrations. No hydrogenase activity could be detected in NH4+-grown, undifferentiated filaments cultured aerobically indicating that uptake hydrogenase activity was associated with the vesicles, the cellular site of nitrogen fixation in Frankia. Hydrogenase activity was inhibited by acetylene but inhibition could be alleviated by pretreatment with H2. H2 stimulated acetylene reduction at supraoptimal but not suboptimal O2 concentrations. These results suggest that uptake hydrogenase activity in ArI3 may play a role in O2 protection of nitrogenase, especially under conditions of carbon limitation.     

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.