Immunochemical Localization of Nitrogenase in Marine Trichodesmium Aggregates: Relationship to N2 Fixation Potential

Colonial aggregation among nonheterocystous filaments of the planktonic marine cyanobacterium Trichodesmium is known to enhance N₂ fixation, mediated by the O₂-sensitive enzyme complex nitrogenase. Expression of nitrogenase appears linked to the formation of O₂-depleted microzones within aggregated bacterium-associated colonies. While this implies a mechanism by which nonheterocystous N₂ fixation can take place in an oxygenated water column, both the location and regulation of the N₂-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 N₂ as an opportunistic response to the dynamic nature of the sea state; during quiescent conditions, aggregation and consequent expression of nitrogenase can proceed rapidly.     

ORGANIZATION OF THE nif GENES OF THE NONHETEROCYSTOUS CYANOBACTERIUM TRICHODESMIUM SP. IMS101

An approximately 16-kb fragment of the Trichodesmium sp. IMS101 (a nonheterocystous filamentous cyanobacterium) "conventional" nif gene cluster was cloned and sequenced. The gene organization of the Trichodesmium and Anabaena variabilis vegetative ( nif 2 ) nitrogenase gene clusters spanning the region from nif B to nif W are similar except for the absence of two open reading frames (ORF3 and ORF1) in Trichodesmium . The Trichodesmium nif EN genes encode a fused Nif EN polypeptide that does not appear to be processed into individual Nif E and Nif N polypeptides. Fused nif  EN genes were previously found in the A. variabilis nif 2 genes, but we have found that fused nif EN genes are widespread in the nonheterocystous cyanobacteria. Although the gene organization of the nonheterocystous filamentous Trichodesmium nif gene cluster is very similar to that of the A. variabilis vegetative nif 2 gene cluster, phylogenetic analysis of nif sequences do not support close relatedness of Trichodesmium and A. variabilis vegetative ( nif 2 ) nitrogenase genes.     

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 limitation in the marine cyanobacterium Trichodesmium reveals new insights into regulation of photosynthesis and nitrogen fixation

* As iron (Fe) deficiency is a main limiting factor of ocean productivity, its effects were investigated on interactions between photosynthesis and nitrogen fixation in the marine nonheterocystous diazotrophic cyanobacterium Trichodesmium IMS101. * Biophysical methods such as fluorescence kinetic microscopy, fast repetition rate (FRR) fluorimetry, and in vivo and in vitro spectroscopy of pigment composition were used, and nitrogenase activity and the abundance of key proteins were measured. * Fe limitation caused a fast down-regulation of nitrogenase activity and protein levels. By contrast, the abundance of Fe-requiring photosystem I (PSI) components remained constant. Total levels of phycobiliproteins remained unchanged according to single-cell in vivo spectra. However, the regular 16-kDa phycoerythrin band decreased and finally disappeared 16-20 d after initiation of Fe limitation, concomitant with the accumulation of a 20-kDa protein cross-reacting with the phycoerythrin antibody. Concurrently, nitrogenase expression and activity increased. Fe limitation dampened the daily cycle of photosystem II (PSII) activity characteristic of diazotrophic Trichodesmium cells. Further, it increased the number and prolonged the time period of occurrence of cells with elevated basic fluorescence (F(0)). Additionally, it increased the effective cross-section of PSII, probably as a result of enhanced coupling of phycobilisomes to PSII, and led to up-regulation of the Fe stress protein IsiA. * Trichodesmium survives short-term Fe limitation by selectively down-regulating nitrogen fixation while maintaining but re-arranging the photosynthetic apparatus.     

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.     

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.     

Localized Tetrazolium Reduction in Relation to N(2) Fixation, CO(2) Fixation, and H(2) Uptake in Aquatic Filamentous Cyanobacteria

The aquatic filamentous cyanobacteria Anabaena oscillarioides and Trichodesmium sp. reveal specific cellular regions of tetrazolium salt reduction. The effects of localized reduction of five tetrazolium salts on N(2) fixation (acetylene reduction), CO(2) fixation, and H(2) utilization were examined. During short-term (within 30 min) exposures in A. oscillarioides, salt reduction in heterocysts occurred simultaneously with inhibition of acetylene reduction. Conversely, when salts failed to either penetrate or be reduced in heterocysts, no inhibition of acetylene reduction occurred. When salts were rapidly reduced in vegetative cells, CO(2) fixation and H(2) utilization rates decreased, whereas salts exclusively reduced in heterocysts were not linked to blockage of these processes. In the nonheterocystous genus Trichodesmium, the deposition of reduced 2,3,5-triphenyl-2-tetrazolium chloride (TTC) in the internal cores of trichomes occurs simultaneously with a lowering of acetylene reduction rates. Since TTC deposition in heterocysts of A. oscillarioides occurs contemporaneously with inhibition of acetylene reduction, we conclude that the cellular reduction of this salt is of use in locating potential N(2)-fixing sites in cyanobacteria. The possible applications and problems associated with interpreting localized reduction of tetrazolium salts in cyanobacteria are presented.     

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.     

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.     

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.     

Amino Acid Cycling in Colonies of the Planktonic Marine Cyanobacterium Trichodesmium thiebautii

We examined diel trends in internal pools and net efflux of free amino acids in colonies of the nonheterocystous, diazotrophic cyanobacterium Trichodesmium thiebautii, freshly collected from waters of the Caribbean and the Bahamas. The kinetics of glutamate uptake by whole colonies were also examined. While intracolonial pools of most free amino acids were relatively constant through the day, pools of glutamate and glutamine varied over the diel cycle, with maxima during the early afternoon. This paralleled the daily cycle of nitrogenase activity. We also observed a large net release of these two amino acids from intact colonies. Glutamate release was typically 100 pmol of N colony^-1 h^-1. This is about one-fourth the concurrent rate of N₂ fixation during the day. However, while nitrogenase activity only occurs during the day, net release of glutamate and glutamine persisted into the night and may therefore account for a greater loss of recently fixed N on a daily basis. This release may be an important route of new N input into tropical, oligotrophic waters. Whole colonies also displayed saturation kinetics with respect to glutamate uptake. The K_s for whole colonies varied from 1.6 to 3.2 μM, or about 100-fold greater than typical ambient concentrations. Thus, uptake systems appear to be adapted to the higher concentrations of glutamate found within the intracellular spaces of the colonies. This suggests that glutamate may be a vehicle for N exchange among trichomes in the colony.     

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.     

Oxygen relations of nitrogen fixation in cyanobacteria.

The enigmatic coexistence of O2-sensitive nitrogenase and O2-evolving photosynthesis in diazotrophic cyanobacteria has fascinated researchers for over two decades. Research efforts in the past 10 years have revealed a range of O2 sensitivity of nitrogenase in different strains of cyanobacteria and a variety of adaptations for the protection of nitrogenase from damage by both atmospheric and photosynthetic sources of O2. The most complex and apparently most efficient mechanisms for the protection of nitrogenase are incorporated in the heterocysts, the N2-fixing cells of cyanobacteria. Genetic studies indicate that the controls of heterocyst development and nitrogenase synthesis are closely interrelated and that the expression of N2 fixation (nif) genes is regulated by pO2.     

KATAGNYMENE: CHARACTERIZATION OF A NOVEL MARINE DIAZOTROPH

The marine planktonic cyanobacterial genus Katagnymene was described by Lemmermann in 1900 and is found in oligotrophic tropical and subtropical oceans. The genus comprises two species, K. pelagica Lemmermann and K. spiralis Lemmermann, and both were observed in most stations sampled during a cruise in the southwest Pacific Ocean in 1998. Katagnymene is nonheterocystous and characterized by single ensheathed trichomes that do not form colonies. Acetylene reduction-GC demonstrated that natural populations of Katagnymene fixed nitrogen and that nitrogenase activity occurred exclusively in the light during a 12:12 light:dark cycle. Whole cell immunolocalization revealed that nitrogenase (the Fe protein) appeared in 7% of the total number of cells and that these were arranged in zones composed of consecutively arranged cells. At least one zone of nitrogenase-containing cells per trichome was found. Nitrogenase was present throughout both the day and night, as shown by Western blotting, and in the same percentage of cells. Ultrastructural immunolocalization on sectioned trichomes also confirmed the presence and localization of nitrogenase in Katagnymene. Cultures of Katagnymene are able to grow on nitrogen-free media and fix nitrogen only in the light. Finally, cloning and sequencing of nifH verified the diazotrophic nature of the genus Katagnymene and demonstrated a close relationship to members of the marine genus Trichodesmium.     

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.     

Molecular cloning and characterization of hetR genes from filamentous cyanobacteria

HetR, a serine type protease, plays an important role in heterocyst differentiation in filamentous cyanobacteria. We isolated and sequenced the hetR genes from different heterocystous and filamentous nonheterocystous cyanobacteria. The hetR gene in the heterocyst forming Anabaena variabilis ATCC 29413 FD was interrupted by interposon mutagenesis (mutant strain WSIII8). This mutant does not form heterocysts and shows no diazotrophic growth under aerobic conditions. However, under anaerobic N(2)-fixing conditions, the WSIII8 cells are able to grow, and high nitrogenase (Nif2) activity is detectable. Nif2 expression was demonstrated in each vegetative cell of the filament by immunolocalization 4 h after nitrogen step-down.     

Oxygen-poor microzones as potential sites of microbial n(2) fixation in nitrogen-depleted aerobic marine waters

The nitrogen-deficient coastal waters of North Carolina contain suspended bacteria potentially able to fix N(2). Bioassays aimed at identifying environmental factors controlling the development and proliferation of N(2) fixation showed that dissolved organic carbon (as simple sugars and sugar alcohols) and particulate organic carbon (derived from Spartina alterniflora) additions elicited and enhanced N(2) fixation (nitrogenase activity) in these waters. Nitrogenase activity occurred in samples containing flocculent, mucilage-covered bacterial aggregates. Cyanobacterium-bacterium aggregates also revealed N(2) fixation. In all cases bacterial N(2) 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 O(2). Microelectrode O(2) profiles revealed that aggregates had lower internal O(2) 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 O(2) was excluded. Triphenyltetrazolium chloride reduction also strongly inhibited nitrogenase activity. These findings suggest that N(2) 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 N(2) fixation responses of these waters.     

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.