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The McMurdo Dry Valleys (MDV) in Antarctica harbor a diverse assemblage of mat-forming diazotrophic cyanobacteria that play a key role in nitrogen cycling. Prior research showed that heterotrophic diazotrophs also make a substantial contribution to nitrogen fixation in MDV. The goals of this study were to survey autotrophic and heterotrophic diazotrophs across the MDV to investigate factors that regulate the distribution and relative ecological roles of each group. Results indicated that diazotrophs were present only in samples with mats, suggesting a metabolic coupling between autotrophic and heterotrophic diazotrophs. Analysis of 16S rRNA and nifH gene sequences also showed that diazotrophs were significantly correlated to the broader bacterial community, while co-occurrence network analysis revealed potential interspecific interactions. Consistent with previous studies, heterotrophic diazotrophs in MDV were diverse, but largely limited to lakes and their outlet streams, or other environments protected from desiccation. Despite the limited distribution, heterotrophic diazotrophs may make a substantial contribution to the nitrogen budget of MDV due to larger surface area and longer residence times of lakes. This work contributes to our understanding of key drivers of bacterial community structure in polar deserts and informs future efforts to investigate the contribution of nitrogen fixation to MDV ecosystems., A survey of N-fixing autotrophic and heterotrophic bacteria in the McMurdo Dry Valleys suggests a substantial role for heterotrophic diazotrophs in N cycling and reveals distinct distributional patterns and potential associations with the broader microbial community.

The extensive loss of tidal marsh habitat surrounding the San Francisco Bay has led to numerous restoration projects to restore ecosystem function. Native cordgrass (Spartina foliosa) is important for sediment accumulation and nesting habitat for endangered California Ridgway’s Rail (Rallus obsoletus obsoletus), and because salt marshes are typically nitrogen-limited ecosystems, previous restoration studies in California have used nitrogen fertilizers to increase plant growth. This study compared the use of two low-nitrogen, high-carbon fertilizers (Macrosystis pyrifera (kelp) and sodium alginate) to stimulate N2-fixing bacteria inhabiting the S. foliosa rhizosphere in a historic and recently restored marsh in San Francisco Bay. Alginate increased N2-fixation, though there was no observed S. foliosa growth response. Kelp suppressed N2-fixation, and increased the foliar nitrogen content of Salicornia pacifica in mixed Spartina-Salicornia stands. The restored marsh had less than 0.5 μM phosphate in porewaters, suggesting phosphorus, not nitrogen, limits S. foliosa growth in this marsh. Alginate increased rates of N2-fixation and may promote plant growth in marshes exhibiting nitrogen limitation. Phosphate limitation was a surprise considering numerous previous studies indicate nitrogen as the primary limiting nutrient of tidal marsh plants. We recommend first assessing nutrient stoichiometry when considering manipulations to promote plant growth in restored marshes.

Phytoplankton are the oceans' principal source of polyunsaturated fatty acids that support the growth and reproduction of consumers such as copepods. Previous studies have demonstrated ocean acidification (OA) can change the availability of polyunsaturated fatty acids to consumer diets which may affect consumer reproduction. Two laboratory experiments were conducted to examine the effects of feeding high-pCO2-reared phytoplankton on copepod egg production, hatching success, and naupliar survival. Marine phytoplankton Rhodomonas salina, Skeletonema marinoi, Prorocentrum micans, and Isochrysis galbana were exponentially grown in semi-continuous cultures at present (control) (400 ppm CO2, pH~8.1) and future (1,000 ppm CO2, pH~7.8) conditions and provided to Acartia tonsa copepods over 4 consecutive days as either nitrogen-limited (Exp. I) or nitrogen-depleted (Exp. II) mixed assemblage of phytoplankton. The composition of FAs in the phytoplankton diet was affected by pCO2 concentration and nitrogen deficiency; the ratio of essential fatty acids to total polyunsaturated fatty acids decreased in phytoplankton grown under high pCO2 and the mass of total fatty acids increased under nitrogen depletion. Additionally, total concentrations of essential fatty acids and polyunsaturated fatty acids in the diet mixtures were less under the high-pCO2 compared to the control-pCO2 treatments. Median egg production, hatching success, and naupliar survival were 48-52%, 4-87%, and 9-100% lower, respectively, in females fed high-pCO2 than females fed low-pCO2 phytoplankton, but this decrease in reproductive success was less severe when fed N-depleted, but fatty acid-rich cells. This study demonstrates that the effects of OA on the nutritional quality of phytoplankton (i.e., their cellular fatty acid composition and quota) were modified by the level of nitrogen deficiency and the resulting negative reproductive response of marine primary consumers.

As part of the ANACONDAS program, we investigated the role of the Amazon plume in stimulating offshore nitrogen fixation and export production during the river's high-discharge period (May–June 2010). Using the shipboard underway system, we performed high-resolution sampling of over 450,000 km2 of surface waters, characterizing the distribution of nutrients, phytoplankton, particulate organic matter (POM), and stable carbon and nitrogen isotopes of POM in the offshore plume. We found distinct regional variations in diazotroph communities, with the Diatom-Diazotroph Associations (DDA) Hemiaulus hauckii – Richelia intracellularis dominating the low N : P mesohaline waters to the northwest of the plume axis and Trichodesmium spp. primarily occupying oceanic waters to the east. Nutrient availability broadly shaped diazotroph distributions along the salinity gradient, but habitat longevity may also play a role in the finer-scale distributions of communities, particularly of DDAs. H. hauckii and Trichodesmium spp. affected the nitrogen and carbon budgets in fundamentally different ways within the plume-influenced regions, with H. hauckii making much greater contributions to the particulate nitrogen pool and to CO2 drawdown than Trichodesmium spp., leading to much higher export fluxes. Our findings provide an important constraint on the role of the Amazon plume in creating distinct niches and roles for diazotrophs in the nutrient and carbon budgets of the western tropical North Atlantic.

The cold deserts of the McMurdo Dry Valleys (MDV), Antarctica, host a high level of microbial diversity. Microbial composition and biomass in arid vs. ephemerally wetted regions are distinctly different, with wetted communities representing hot spots of microbial activity that are important zones for biogeochemical cycling. While climatic change is likely to cause wetting in areas not historically subject to wetting events, the responses of microorganisms inhabiting arid soils to water addition is unknown. The purpose of this study was to observe how an associated, yet non-wetted microbial community responds to an extended addition of water. Water from a stream was diverted to an adjacent area of arid soil with changes in microbial composition and activities monitored via molecular and biochemical methods over seven weeks. The frequency of genetic signatures related to both prokaryotic and eukaryotic organisms adapted to MDV aquatic conditions increased during the limited seven week period, indicating that the soil community was transitioning into a typical “high-productivity” MDV community. This work is consistent with current predictions that MDV microbial communities in arid regions are highly sensitive to climate change, and further supports the notion that changes in community structure and associated biochemical cycling may occur much more rapidly than predicted.

A large percentage of CO2 emitted into the atmosphere is absorbed by the oceans, causing chemical changes in surface waters known as ocean acidification (OA). Despite the high interest and increased pace of OA research to understand the effects of OA on marine organisms, many ecologically important organisms remain unstudied. Calcidiscus is a heavily calcified coccolithophore genus that is widespread and genetically and morphologically diverse. It contributes substantially to global calcium carbonate production, organic carbon production, oceanic carbon burial, and ocean–atmosphere CO2 exchange. Despite the importance of this genus, relatively little work has examined its responses to OA. We examined changes in growth, morphology, and carbon allocation in multiple strains of Calcidiscus leptoporus in response to ocean acidification. We also, for the first time, examined the OA response of Calcidiscus quadriperforatus, a larger and more heavily calcified Calcidiscus congener. All Calcidiscus coccolithophores responded negatively to OA with impaired coccolith morphology and a decreased ratio of particulate inorganic to organic carbon (PIC:POC). However, strains responded variably; C. quadriperforatus showed the most sensitivity, while the most lightly calcified strain of C. leptoporus showed little response to OA. Our findings suggest that calcium carbonate production relative to organic carbon production by Calcidiscus coccolithophores may decrease in future oceans and that Calcidiscus distributions may shift if more resilient strains and species become dominant in assemblages. This study demonstrates that variable responses to OA may be strain or species specific in a way that is closely linked to physiological traits, such as cellular calcite quota.

Diazotrophic cyanobacteria, those capable of fixing di-nitrogen (N2), are considered one of the major sources of new nitrogen (N) in the oligotrophic tropical ocean, but direct incorporation of diazotrophic N into food webs has not been fully examined. In the Amazon River-influenced western tropical North Atlantic (WTNA), diatom diazotroph associations (DDAs) and the filamentous colonial diazotrophs Trichodesmium have seasonally high abundances. We sampled epipelagic mesozooplankton in the Amazon River plume and WTNA in May–June 2010 to investigate direct grazing by mesozooplankton on two DDA populations: Richelia associated with Rhizosolenia diatoms (het-1) and Hemiaulus diatoms (het-2), and on Trichodesmium using highly specific qPCR assays targeting nitrogenase genes (nifH). Both DDAs and Trichodesmium occurred in zooplankton gut contents, with higher detection of het-2 predominantly in calanoid copepods (2.33–16.76 nifH copies organism-1). Abundance of Trichodesmium was low (2.21–4.03 nifH copies organism-1), but they were consistently detected at high salinity stations (>35) in calanoid copepods. This suggests direct grazing on DDAs, Trichodesmium filaments and colonies, or consumption as part of sinking aggregates, is common. In parallel with the qPCR approach, a next generation sequencing analysis of 16S rRNA genes identified that cyanobacterial assemblage associated with zooplankton guts was dominated by the non-diazotrophic unicellular phylotypes Synechococcus (56%) and Prochlorococcus (26%). However, in two separate calanoid copepod samples, two unicellular diazotrophs Candidatus Atelocyanobacterium thalassa (UCYN-A) and Crocosphaera watsonii (UCYN-B) were present, respectively, as a small component of cyanobacterial assemblages (

Salt marsh restoration is hypothesized to provide shoreline stabilization, increased fish habitat, and organic carbon subsidies for estuarine food webs. Organic carbon comes from diverse primary producers that differ in carbon fixation rates and areal extent within wetland systems. This study was designed to obtain some of the first estimates of the relative contribution of different primary producers to total organic carbon production within open water and tidally flooded wetlands of the northern San Francisco Estuary (SFE). Carbon fixation rates of phytoplankton, microphytobenthos, and low marsh emergent vegetation were measured in two natural and four restoring wetlands in 2004. Areal (m2) rates of carbon fixation were greatest for low marsh vegetation, while phytoplankton and microphytobenthos rates were one and two orders of magnitude lower, respectively. However, when areal production rates were scaled to the amount of habitat available for each primary producer group, the relative importance of each group varied by location. Given that each primary producer group supports a different subset of estuarine consumers, the type of food subsidy desired should influence the amount open water channel, mudflat and low marsh area restored. Large-scale wetland restoration activities should consider the types of primary producers likely to occupy restored habitats when estimating future food web impacts.

The last several decades have witnessed dramatic advances in unfolding the diversity and commonality of oceanic diazotrophs and their N2 -fixing potential. More recently, substantial progress in diazotrophic cell biology has provided a wealth of information on processes and mechanisms involved. The substantial contribution by the diazotrophic cyanobacterial genus Trichodesmium to the nitrogen influx of the global marine ecosystem is by now undisputable and of paramount ecological importance, while the underlying cellular and molecular regulatory physiology has only recently started to unfold. Here, we explore and summarize current knowledge, related to the optimization of its diazotrophic capacity, from genomics to ecophysiological processes, via, for example, cellular differentiation (diazocytes) and temporal regulations, and suggest cellular research avenues that now ought to be explored.

Coccolithophores are unicellular phytoplankton that produce calcium carbonate coccoliths as an exoskeleton. Emiliania huxleyi, the most abundant coccolithophore in the world’s ocean, plays a major role in the global carbon cycle by regulating the exchange of CO2 across the ocean-atmosphere interface through photosynthesis and calcium carbonate precipitation. As CO2 concentration is rising in the atmosphere, the ocean is acidifying and ammonium (NH4 + ) concentration of future ocean water is expected to rise. The latter is attributed to increasing anthropogenic nitrogen (N) deposition, increasing rates of cyanobacterial N2 fixation due to warmer and more stratified oceans, and decreased rates of nitrification due to ocean acidification. Thus, future global climate change will cause oceanic phytoplankton to experience changes in multiple environmental parameters including CO2, pH, temperature and nitrogen source. This study reports on the combined effect of elevated pCO2 and increased NH4 + to nitrate (NO3 ) ratio (NH4 + /NO3 ) on E. huxleyi, maintained in continuous cultures for more than 200 generations under two pCO2 levels and two different N sources. Herein, we show that NH4 + assimilation under N-replete conditions depresses calcification at both low and high pCO2, alters coccolith morphology, and increases primary production. We observed that N source and pCO2 synergistically drive growth rates, cell size, and the ratio of inorganic to organic carbon. These responses to N source suggest that, compared to increasing CO2 alone, a greater disruption of the organic carbon pump could be expected in response to the combined effect of increased NH4 + /NO3 ratio and CO2 level in the future acidified ocean. Additional experiments conducted under lower nutrient conditions are needed prior to extrapolating our findings to the global oceans. Nonetheless, our results emphasize the need to assess combined effects of multiple environmental parameters on phytoplankton biology to develop accurate predictions of phytoplankton responses to ocean acidification.

Low nutrient availability in salt marsh ecosystems can potentially limit primary productivity and subsequent carbon export to coastal waters. In temperate marshes with low external nitrogen inputs, nitrogen (N2) fixation may enhance availability of usable nitrogen to marsh plant communities and increase their growth. The effects of sub-surface soil amendment of polysaccharides on rhizosphere N2 fixation, and on plant growth and tissue characteristics of the California cordgrass, Spartina foliosa, were tested in microcosms in a tidal simulator. Polysaccharide was added as alginate extract, and rhizosphere N2 fixation and plant characteristics were measured over 9 weeks. N2 fixation rates increased 10-fold in the alginate treatment compared to controls after 1 week of exposure as a result of increased availability of a readily usable form of carbon. N2 fixation rates in amended sediment were significantly higher than rates in control sediments at weeks 1 and 3. Although plant characteristics did no...

The fresh water discharged by large rivers such as the Amazon is transported hundreds to thousands of kilometers away from the coast by surface plumes. The nutrients delivered by these river plumes contribute to enhanced primary production in the ocean, and the sinking flux of this new production results in carbon sequestration. Here, we report that the Amazon River plume supports N 2 fixation far from the mouth and provides important pathways for sequestration of atmospheric CO 2 in the western tropical North Atlantic (WTNA). We calculate that the sinking of carbon fixed by diazotrophs in the plume sequesters 1.7 Tmol of C annually, in addition to the sequestration of 0.6 Tmol of C yr −1 of the new production supported by NO 3 delivered by the river. These processes revise our current understanding that the tropical North Atlantic is a source of 2.5 Tmol of C to the atmosphere [Mikaloff-Fletcher SE, et al. (2007) Inverse estimates of the oceanic sources and sinks of natural CO 2 and the implied oceanic carbon transport. Global Biogeochem Cycles 21, doi:10.1029/2006GB002751]. The enhancement of N 2 fixation and consequent C sequestration by tropical rivers appears to be a global phenomenon that is likely to be influenced by anthropogenic activity and climate change.

The harpacticoid copepod Macrosetella gracilis uses colonies of the nitrogen-fixing cyanobacterium Trichodesmium spp. as a floating substrate and nursery. This association enables the copepod to lead a pelagic existence. Molecular and morphological data both indicate that M. gracilis is maintained as a single, circumglobal species across the tropical and subtropical Atlantic and Pacific. Mitochondrial DNA sequence data from cytochrome c oxidase subunit I (COI) showed mod- erate to high levels of diversity and limited phylogeographic structure in M. gracilis. Thirty-six differ- ent haplotypes were found among the 149 M. gracilis individuals sequenced; 4 haplotypes were shared between the Atlantic and Pacific. Samples from Japan showed the highest haplotypic and nucleotide diversity levels, suggesting that the Indo-West Pacific may be the center of diversity for M. gracilis. Wide distribution and large population size in M. gracilis may limit divergence through increased gene flow or slowing of the loss of ancestral diversity through lineage sorting. The pelagic existence of M. gracilis has led to less phylogenetic structuring than found in other species of harpacticoid copepods, illustrating the evolutionary significance of specialization on a floating sub- strate in this copepod.

Large areas of the western tropical Atlantic Ocean are influenced by the Amazon River plume, in which dissolved inorganic nitrogen (DIN) concentrations are low and nutrient N:P and N:Si ratios are lower than elemental ratios typical of phytoplankton. Accordingly, nutrient concentrations and ratios measured during winter (January to February 2001) and summer (July to August 2001) cruises in the area 2 to 14° N and 57 to 40° W suggest that DIN may limit phytoplankton growth at the majority of the 40 stations sampled. As diatoms are the dominant plankton in the Amazon plume- waters, short-term nutrient enrichment experiments were conducted to determine whether ambient N, P, or Si limited silica production rates (SiP). Results indicated that SiP were substrate limited both above and below the pycnocline at the majority of stations during both high (summer cruise) and low (winter cruise) discharge conditons. DIN concentrations also limited SiP in plumewater stations, but N additions depressed SiP at other stations, possibly using energy to take up the limiting nutrient. Finally, primary production rates were limited by ambient dissolved silicon concentrations at 9 of 13 summer stations. These results indicate the importance of dissolved silicon availability in controlling phytoplankton growth rates in a diatom-dominated river plume system.

The vertical and horizontal distributions of seven diazotrophic populations in the western tropical north Atlantic (WTNA) Ocean were examined using a nifH DNA quantitative polymerase chain reaction (QPCR) approach. The nifH phylotype abundances were highest near the surface and decreased with depth, with the exception of the cyanobacterial symbiont Calothrix, which was not detected at any station. Richelia associated with the diatoms Rhizosolenia clevei and Hemiaulus hauckii were distributed within the freshwater lens of the Amazon plume. Abundances of H. hauckii-Richelia nifH genes dominated all depths in 6 of 10 vertical profiles and 10 of 20 surface samples. In addition, estimates of Richelia associated with H. hauckii increased northwest (8– 12uN, 56–54uW) from the river mouth, where significantly ( p , 0.001) higher abundances (.105 copies L–1) were found in mesohaline waters (31–34.9). nifH copy abundance for surface populations of the H. hauckii-Richelia symbioses were positively correlated (r2 5 0.59) with salinity. Three unicellular cyanobacterial groups and Trichodesmium had similar horizontal distributions, where the highest nifH copy estimates were at stations with salinity $35 and northeast (6–10u N5 0uW) of the freshwater lens. The abundance of Trichodesmium spp. and unicellular Group B nifH gene copies co-varied (r2 5 0.60). The QPCR study showed the dominance of H. hauckii-Richelia symbioses in the Amazon plume waters, implying that these associations had an ecological advantage over the other diazotrophs. Outside of the plume nutrients were below detection, abundances of freeliving unicellular cyanobacterial phylotypes, including a novel group designated Group C, were abundant (.105 copies L21) and comparable to the abundances of Trichodesmium spp. Thus, there appeared to be a cascade of diazotrophic communities along gradients of salinity and nutrients in the WTNA.

The Amazon River has the largest discharge of all rivers on Earth, and its complex plume system fuels a wide array of biogeochemical processes, across a large area of the western tropical North Atlantic. The plume thus stimulates microbial processes affecting carbon sequestration and nutrient cycles at a global scale. Chromosomal gene expression patterns of the 2.0 to 156 μm size-fraction eukaryotic microbial community were investigated in the Amazon River Plume, generating a robust dataset (more than 100 million mRNA sequences) that depicts the metabolic capabilities and interactions among the eukaryotic microbes. Combining classical oceanographic field measurements with metatranscriptomics yielded characterization of the hydrographic conditions simultaneous with a quantification of transcriptional activity and identity of the community. We highlight the patterns of eukaryotic gene expression for 31 biogeochemically significant gene targets hypothesized to be valuable within forecasting models. An advantage to this targeted approach is that the database of reference sequences used to identify the target genes was selectively constructed and highly curated optimizing taxonomic coverage, throughput, and the accuracy of annotations. A coastal diatom bloom highly expressed nitrate transporters and carbonic anhydrase presumably to support high growth rates and enhance uptake of low levels of dissolved nitrate and CO2. Diatom-diazotroph association (DDA: diatoms with nitrogen fixing symbionts) blooms were common when surface salinity was mesohaline and dissolved nitrate concentrations were below detection, and hence did not show evidence of nitrate utilization, suggesting they relied on ammonium transporters to aquire recently fixed nitrogen. These DDA blooms in the outer plume had rapid turnover of the photosystem D1 protein presumably caused by photodegradation under increased light penetration in clearer waters, and increased expression of silicon transporters as silicon became limiting. Expression of these genes, including carbonic anhydrase and transporters for nitrate and phosphate, were found to reflect the physiological status and biogeochemistry of river plume environments. These relatively stable patterns of eukaryotic transcript abundance occurred over modest spatiotemporal scales, with similarity observed in sample duplicates collected up to 2.45 km in space and 120 minutes in time. These results confirm the use of metatranscriptomics as a valuable tool to understand and predict microbial community function., Gordon and Betty Moore Foundation (River Ocean Continuum of the Amazon Project. Grants GBMF 2293 and 2928), National Science Foundation (U.S.) (Grant NSF-OCE 0934095)

During the summer months, wet (hyporheic) soils associated with ephemeral streams and lake edges in the Antarctic Dry Valleys (DVs) become hotspots of biological activity and are hypothesized to be an important source of carbon and nitrogen for arid DV soils. Recent research in the DV has focused on the geochemistry and microbial ecology of lakes and arid soils, with substantially less information being available on hyporheic soils. Here, we determined the unique properties of hyporheic microbial communities, resolved their relationship to environmental parameters and compared them to archetypal arid DV soils. Generally, pH increased and chlorophyll a concentrations decreased along transects from wet to arid soils (9.0 to ~7.0 for pH and ~0.8 to ~5 μg/cm(3) for chlorophyll a, respectively). Soil water content decreased to below ~3% in the arid soils. Community fingerprinting-based principle component analyses revealed that bacterial communities formed distinct clusters specific to arid and wet soils; however, eukaryotic communities that clustered together did not have similar soil moisture content nor did they group together based on sampling location. Collectively, rRNA pyrosequencing indicated a considerably higher abundance of Cyanobacteria in wet soils and a higher abundance of Acidobacterial, Actinobacterial, Deinococcus/Thermus, Bacteroidetes, Firmicutes, Gemmatimonadetes, Nitrospira, and Planctomycetes in arid soils. The two most significant differences at the genus level were Gillisia signatures present in arid soils and chloroplast signatures related to Streptophyta that were common in wet soils. Fungal dominance was observed in arid soils and Viridiplantae were more common in wet soils. This research represents an in-depth characterization of microbial communities inhabiting wet DV soils. Results indicate that the repeated wetting of hyporheic zones has a profound impact on the bacterial and eukaryotic communities inhabiting in these areas.

Unicellular N 2 -fixing cyanobacteria from tropical marine oligotrophic environments have been proposed to be major contributors to the global N cycle but still remain poorly characterized. These organisms are likely to be limited by phosphorus availability in situ. The aim of this study was to identify growth kinetics of isolates from the tropical North Atlantic and subtropical North Pacific in relation to phosphorus and temperature in continuous cultures. Cells from the Atlantic measured 2.5 μm in diameter (A-2.5). Genetically identical isolates from the Pacific showed 2 diameters depending on P-media concentrations (small: 3 μm, 1 pM PO 4 [P-3] and large: 7 pm, 4 pM PO 4 [P-7]). All 3 isolates were highly stenothermal, and optimal growth temperatures ranged between 26 and 30°C. Small cells (A-2.5 and P-3) had lower half-saturation constants (K s ) for PO 4 than large cells (P-7) (0.06 to 0.21 μM vs. 0.20 to 0.25 μM). Maximum growth rates and N:P ratios increased with temperature for all isolates; N:P ratios were close to Redfield ratios (N:P = 16) when isolates approached maximum growth rates. N 2 -fixation activity did not vary between growth rates, but did increase with temperature; rates were consistently lower than previously published rates for the same isolates under non-P-limiting conditions. From these studies, we conclude that both Atlantic and Pacific unicellular cyanobacteria that have the capacity to fix N 2 have a limited temperature range for growth and that smaller sized isolates could be better adapted for conditions of phosphorus limitation.

A synthesis of the current understanding of potential iron limitation of pelagic nitrogen fixation is given, considering biochemical bases of Fe requirements and empirical observations of growth and Fe quotas of cultures and field populations of Trichodesmium. The potential for iron limitation of heterotrophic diazotrophy in the marine environment is also evaluated.

Large populations (200 to 5,000 cells ml −1 in snowmelt) of bacteria were present in surface snow and firn from the south pole sampled in January 1999 and 2000. DNA isolated from this snow yielded ribosomal DNA sequences similar to those of several psychrophilic bacteria and a bacterium which aligns closely with members of the genus Deinococcus , an ionizing-radiation- and desiccation-resistant genus. We also obtained evidence of low rates of bacterial DNA and protein synthesis which indicates that the organisms were metabolizing at ambient subzero temperatures (−12 to −17°C).

New molecular and microscopic evidence indicates that the open ocean harbors a diverse range of novel free-living and symbiotic nitrogen-fixing microorganisms. Although the extent to which these microorganisms fix nitrogen is currently unclear, ongoing research indicates that they might make a substantial contribution to the open ocean nitrogen budget.

The genetic diversity of Trichodesmium, a marine nitrogen-fixing non-heterocystous cyanobacterium of great ecological importance, was examined using the partial gene sequences of the small subunit ribosomal RNA (16S rDNA) gene and the regulatory gene hetR. Different species and morphotypes (fusiform and spherical colonies) of Trichodesmium were collected in the northern Caribbean Sea, the central Atlantic Ocean and southern Pacific Ocean. The trichome morphologies were observed with light microscopy before DNA extraction and PCR amplification. Phylogenetic analysis of 16S rDNA revealed that all cyanobacterial sequences from the colonies of Trichodesmium spp. were very closely related to the laboratory culture of Trichodesmium sp. NIBB 1067. The overall results from the hetR analysis were congruent with that of 16S rDNA, but the variation in nucleotide sequence between different species was higher within the hetR gene. The sequence data showed that three main clades were represented. One clade comprised sequences from Trichodesmium hildebrandtii Gomont and Trichodesmium thiebautii Gomont (including both its fusiform and spherical colony forms). Sequences from Trichodesmium contortum Wille and Trichodesmium tenue Wille constituted a second clade. The third clade contained Trichodesmium erythraeum Ehrenberg together with the two laboratory strains of this species, Trichodesmium sp. NIBB 1067 and Trichodesmium sp. IMS 101.

We encountered an extensive bloom of the colonial diatom Hemiaulus hauckii along a 2500 km cruise track off the NE coast of South America in autumn 1996. Each diatom cell contained the heterocystous, N 2 -fixing cyanobacterial endosymbiont Richelia intracellularis. Surface Richelia heterocyst (and filament) densities increased from 10 6 heterocyst 1 -1 in the bloom. Total abundance ranged from 10 6 heterocyst m -2 outside the bloom to over 10 10 heterocyst m -2 within the bloom. Rates of primary production averaged 1.2 g C m -2 d -1 , higher than typical for oligotrophic open ocean waters. N 2 fixation during the bloom by the Richelia/Hemiaulus association added an average of 45 mg N m -2 d -1 to the water column. The relative importance of NH 4 + uptake over the course of the bloom increased from 0 to 42% of total N uptake by the Hemiaulus/Richelia association. N 2 fixation by Richelia exceeded estimates of 'new' N flux via NO 3 diffusion from deep water and, together with additional N 2 fixation by the cyanobacterium Trichodesmium, could supply about 25 % of the total N demand through the water column during the bloom. Suspended particles and zooplankton collected within the bloom were depleted in 15 N, reflecting the dominant contribution of N 2 fixation to the planktonic N budget. The bloom was spatially extensive, as revealed by satellite imagery, and is calculated to have contributed about 0.5 Tg N to the euphotic zone. Such blooms may represent an important and previously unrecognized source of new N to support primary production in nutrient-poor tropical waters. Furthermore, this bloom demonstrates that heterocystous cyanobacteria can also make quantitatively important contributions of N in oceanic water column environments.

LVe encountered an extensive surface bloom of the N, fixing cyanobactenum Trichodesrniurn erythraeum in the central basin of the Arabian Sea during the spring ~nter-n~onsoon of 1995. The bloom, which occurred dunng a penod of calm winds and relatively high atmospher~c iron content, was metabollcally active. Carbon fixation by the bloom represented about one-quarter of water column primary productivity while input by h:: flxation could account for a major fraction of the estimated 'new' N demand of pnmary production. Isotopic measurements of the N in surface suspended material confirmed a direct contribution of N, fixation to the organic nltrogen pools of the upper water column. Retrospective analysis of NOAA-12 AVHRR imagery indicated that blooms covered up to 2 X 106 km2, or 20% of the Arabian Sea surface, during the period from 22 to 27 May 1995. In addition to their biogeochemical impact, surface blooms of this extent may have secondary effects on sea surface albedo and light penetration as well as heat and gas exchange across the air-sea interface. A preliminary extrapolation based on our observed, non-bloom rates of N, fixation from our limited sampling in the spring intermonsoon, including a conservative estimate of the input by blooms, suggest N2 fixation may account for an input of about 1 Tg N yr-I This is substantial, but relatively minor compared to current estimates of the removal of N through denitrification in the basin. However, N2 fixation may also occur in the central basin through the mild winter monsoon, be considerably greater during the fall intermonsoon than we observed during the spring intermonsoon, and may also occur at higher levels in the chronically oligotrophic southern basin. Ongoing satellite observations will help to determine more accurately the distribution and density of Trichodesmium in this and other tropical oceanic basins, as well as resolving the actual frequency and duration of bloom occurrence.

Planktonic marine cyanobacteria of the genus Trichodesmium occur throughout the oligotrophic tropical and subtropical oceans. Their unusual adaptations, from the molecular to the macroscopic level, contribute to their ecological success and biogeochemical importance. Trichodesmium fixes nitrogen gas (N 2 ) under fully aerobic conditions while photosynthetically evolving oxygen. Its temporal pattern of N 2 fixation results from an endogenous daily cycle that confines N 2 fixation to daylight hours. Trichodesmium colonies provide a unique pelagic habitat that supports a complex assemblage of consortial organisms. These colonies often represent a large fraction of the plant biomass in tropical, oligotrophic waters and contribute substantially to primary production. N 2 fixation by Trichodesmium is likely a major input to the marine and global nitrogen cycle.

Increased atmospheric p CO 2 is expected to render future oceans warmer and more acidic than they are at present. Calcifying organisms such as coccolithophores that fix and export carbon into the deep sea provide feedbacks to increasing atmospheric p CO 2 . Acclimation experiments suggest negative effects of warming and acidification on coccolithophore calcification, but the ability of these organisms to adapt to future environmental conditions is not well understood. Here, we tested the combined effect of p CO 2 and temperature on the coccolithophore Emiliania huxleyi over more than 700 generations. Cells increased inorganic carbon content and calcification rate under warm and acidified conditions compared with ambient conditions, whereas organic carbon content and primary production did not show any change. In contrast to findings from short-term experiments, our results suggest that long-term acclimation or adaptation could change, or even reverse, negative calcification responses in E. huxleyi and its feedback to the global carbon cycle. Genome-wide profiles of gene expression using RNA-seq revealed that genes thought to be essential for calcification are not those that are most strongly differentially expressed under long-term exposure to future ocean conditions. Rather, differentially expressed genes observed here represent new targets to study responses to ocean acidification and warming.

p34cdc2 and cyclin B are two key proteins in the eukaryotic cell cycle control machinery. They thus could be important cell cycle markers for studies of environmental effects on cell cycle progression and on growth rate of marine phytoplankton. From July 1993 to March 1995, we used commercially available antibodies to examine the presence of their homologs in a marine phytoplankton species, Dunaliella tertiolecta Butcher. A p34cdc2-like protein was detected on the Western blots, with an apparent molecular mass as expected (34 kDa). Anti-cyclin B detected a protein of 63 kDa, a size similar to that of cyclin B in other organisms. The two proteins decreased from the exponential to the stationary growth phase. As determined on the Western blots, their abundance only changed slightly during the cell cycle, being slightly more abundant prior to cell division. Immunofluorescence performed for a partially synchronized culture showed that the fraction of the cell population that was positively stained by anti-p34cdc2 was highest at the time when the culture was mainly in the late G1 or early S phase, and in the late G2 or early M phase, respectively. The fraction was low when the culture was mainly in the S phase. Although further characterization is required to verify their identities, these two growth phase-related proteins appear to be p34cdc2 and cyclin B homologs, which may be useful in studying the cell cycle and growth rates of phytoplankton.

Observations of the dinoflagellate Dinophysis norvegica in the Baltic Sea during the summers of 1991–1993 indicate that maximal abundances (c 40–150 × 103 cells l-1) were found at the thermocline, typically at 12°C. Maximum densities were usually between 12 and 15 m where 2·9% and 1·5% of surface photon irradiances, respectively, were measured. No diel vertical migration was observed, and cell densities in the mixed layer were always low. Photosynthesis versus irradiance measurements with an oxygen electrode indicated that these populations had a P max of 2·47 [coefficient of variation (CV) 7·3%] and 3·4 (CV 4·7%) mg O2 mg Chl a -1 h-1, and compensation values of photon irradiance were 16·5 and 83 μmol m-2 s-1 in 1992 and 1993, respectively. Both oxygen electrode and 14C light/dark bottle measurements indicated that D. norvegica had very little net photosynthesis at the depths where it was most abundant; it would have had about 2·5-fold greater capacity at photon irradiances present closer to the surface....

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 N 2 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.

The growth rate of an oceanic dinoflagellate, Ceratium teres Kofoid, was investigated in the Sargasso and Caribbean Seas from September 1989 to July 1990 using the cell cycle analysis method. Estimated growth rates ranged from 0.29 to 0.58 day−1 and were 1.5–7.2 times higher than generally accepted rates for oceanic dinoflagellates. The higher rates in this report were mainly due to an improvement in techniques that determine the duration of a terminal cell cycle phase in situ. The day-to-day variation in growth rates was surprisingly small, but, from long-term measurements, a weak correlation was found among temperature, daily irradiance, and seasonal growth rate. The calculated species-specific primary production ranged from 0.5 to 1.8 mg C·m−2·day−1, about 1% of the estimated total production. Ceratium teres may be an important carbon source at the base of the grazing food chain.

Eolian transport of biomass from ephemerally wetted soils, associated with summer glacial meltwater runoffs and lake edges, to low-productivity areas of the Antarctic Dry Valleys (DV) has been postulated to be an important source of organic matter (fixed nitrogen and fixed carbon) to the entire DV ecosystem. However, descriptions and identification of the microbial members responsible for N(2) fixation within these wetted sites are limited. In this study, N(2) fixers from wetted soils were identified by direct nifH gene sequencing and their in situ N(2) fixation activities documented via acetylene reduction and RNA-based quantitative PCR assays. Shannon-index nifH diversity levels ranged between 1.8 and 2.6 and included the expected cyanobacterial signatures and a large number of phylotypes related to the gamma-, beta-, alpha-, and delta-proteobacteria. N(2) fixation rates ranged between approximately 0.5 and 6 nmol N cm(-3) h(-1) with measurements indicating that approximately 50% of this activity was linked with sulfate reduction at some sites. Comparisons with proximal dry soils also suggested that these communities are not ubiquitously distributed, and conditions unrelated to moisture content may define the composition, diversity, or habitat suitability of the microbial communities within wetted soils of the DVs.

unicellular diazotrophs were responsible for activity there. Our studies show a geographical variation in the dominant diazotroph in the North Pacific Subtropical Gyre in the summer with Trichodesmium being dominant around the Hawaiian Islands, Richelia associated with diatoms to be found in high numbers to the south of the islands while unicellular diazotrophs dominated to the west, away from the islands and evidence from the literature suggests iron may play a role.

Immunochemical labeling was used to study the subcellular distribution of cytochrome oxidase, a respiratory protein, in Trichodesmium thiebautii . The protein was found associated with both cytoplasmic and thylakoid membranes. About a sixfold variation in the protein content (gold particle count) was found among Trichodesmium cells within a single colony. Double labeling was performed with cytochrome oxidase and nitrogenase antisera. Regression analysis of gold particle counts per unit of cell area of cytochrome oxidase and nitrogenase showed a positive correlation ( r 2 = 0.911); cells with higher nitrogenase levels also had higher levels of cytochrome oxidase. The parallel expression of two proteins suggests that respiratory oxygen uptake may be involved in nitrogenase protection (respiratory protection) in Trichodesmium spp.

Rodeo Lagoon, a low-salinity coastal lagoon in the Golden Gate National Recreation Area, California, United States, has been identified as an important ecosystem due to the presence of the endangered goby (Eucyclogobius newberri). Despite low anthropogenic impacts, the lagoon exhibits eutrophic conditions and supports annual episodes of very high phytoplankton biomass. Weekly assess- ments (February-December 2007) of phytoplankton indicated diatoms, Nodularia spumigena, Chaetocer- os muelleri var. muelleri, flagellated protozoa, a mixed assemblage, and Microcystis aeruginosa dom- inated the algal community in successive waves. Phytoplankton succession was significantly corre- lated (r 2 = 0.37, p \ 0.001) with averaged daily irradiance (max = 29.7 kW m -2 d -1 ), water column light attenuation (max = 14 m -1 ), and orthophos- phate and dissolved inorganic carbon concentrations (max = 1.5 and 2920 lM, respectively). Negative effects of phytoplankton growth and decay included excessive ammonia concentrations (exceeded EPA guidelines on 77% of sampling days), hypoxia (\ 3m g l -1 dissolved oxygen), and introduction of several microcystins, all in the latter half of the year. Our one-year study suggests that this coastal lagoon is a highly seasonal system with strong feedbacks between phytoplankton and geochemical processes.

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.

Three types of filamentous microorganisms were associated with colonies of the marine planktonic cyanobacterium Trichodesmium collected in the Sargasso Sea. The filaments were categorized according...

Publisher Summary This chapter first summarizes the recent advances and insights relative to nitrogen (N2) fixation in benthic marine environments, move on to the water column, then summarizes controls on N2 fixation and comments on the broader, biogeochemical impacts of N2 fixation. In benthic environments, ranging from the rhizosphere of shallow water macrophyte communities such as Zostera, Thalassia, and Spartina hundreds of different diazotrophic strains have been isolated. These diazotrophs make significant contributions to the nitrogen economy of their respective plant communities. In the pelagic zone, iron and phosphorus availability is a major constraint on N2 fixation. In the north Atlantic, positive nitrogen anomalies in the subeuphotic zone, resulting from Aeolian dust deposition of Fe corresponds to areas in which P limits growth of diazotrophs. There is precious little information on the pathways of fixed N from pelagic diazotrophs to other members of the plankton and nekton. Viral lysis may be a major pathway as is the leakage of fixed N from healthy Trichodesmium cells. The spectrum of organisms recognized to contribute to N2 fixation in the sea has been greatly expanded, and there has been a virtual revolution in understanding of its quantitative significance, the interplay between N2 fixation and the C cycle and the major controls on this process.

Natural populations of the nonheterocystous marine cyanobacterium Trichodesmium thiebautii exhibit a diel periodicity in nitrogenase activity (NA). NA “turns on” near dawn and “turns off” near dusk, independent of photic conditions. Chloramphenicol (CAP) and ammonium prevented turn on of NA in T. thiebautii when added to samples collected before dawn but were progressively less effective in inhibiting NA in samples collected later in the morning. In samples collected after turn on, activities declined with time with both CAP and ammonium treatments, with ammonium having a stronger effect. In contrast, CAP added to samples collected in late afternoon prolonged NA, compared with controls, which turned off. Direct analysis of the presence of the Fe protein of nitrogenase in T. thiebautii by using a Western immunoblot procedure found a strong protein band present in samples collected after 0800 h through the late evening but little or no Fe protein in samples collected within the 2 to 4 h preceding dawn. We conclude that the diel cycle of NA in T. thiebautii results from de novo synthesis of nitrogenase each morning and from the inactivation and degradation of nitrogenase in the late afternoon and night.

1. INTRODUCTIONSymbioses between cyanobacteria and marine organisms are abundant and widespreadamong marine plants and animals. Generally, they are most likely to be found inoligotrophic areas in which either fixation or dissolved organic carbon (DOC) releasebenefit the host organism, although a few occur in nutrient rich areas such as mudflats.Research on these symbioses is in its infancy, and generally there is very little knownabout the nature of many of these symbioses. Furthermore, from microscopicobservations, it appears that there are many more symbiotic relationships yet to bediscovered.In the marine environment, symbioses are known to occur between cyanobacteriaand sponges, Ascidians (sea squirts), and Echuroid worms in the benthos, and diatoms,dinoflagellates and a protozoan among the plankton. These symbioses can often besignificant in terms of the biogeochemistry of coastal and open ocean areas. Forexample, the heterocystous cyanobacterium

N 2 -fixing proteobacteria (α and γ) and unicellular cyanobacteria are common in both the tropical North Atlantic and Pacific oceans. In near-surface waters proteobacterial nifH transcripts were present during both night and day while unicellular cyanobacterial nifH transcripts were present during the nighttime only, suggesting separation of N 2 fixation and photosynthesis by unicellular cyanobacteria. Phylogenetic relationships among unicellular cyanobacteria from both oceans were determined after sequencing of a conserved region of 16S ribosomal DNA (rDNA) of cyanobacteria, and results showed that they clustered together, regardless of the ocean of origin. However, sequencing of nifH transcripts of unicellular cyanobacteria from both oceans showed that they clustered separately. This suggests that unicellular cyanobacteria from the tropical North Atlantic and subtropical North Pacific share a common ancestry (16S rDNA) and that potential unicellular N 2 fixers have diverged ( nifH ). N 2 fixation rates for unicellular bacterioplankton (including small cyanobacteria) from both oceans were determined in situ according to the acetylene reduction and 15 N 2 protocols. The results showed that rates of fixation by bacterioplankton can be almost as high as those of fixation by the colonial N 2 -fixing marine cyanobacteria Trichodesmium spp. in the tropical North Atlantic but that rates are much lower in the subtropical North Pacific.

The Redfield ratio of 106 carbon:16 nitrogen:1 phosphorus in marine phytoplankton1 is one of the foundations of ocean biogeochemistry, with applications in algal physiology2, palaeoclimatology3 and global climate change4. However, this ratio varies substantially in response to changes in algal nutrient status5 and taxonomic affiliation6,7. Here we report that Redfield ratios are also strongly affected by partitioning into surface-adsorbed and intracellular phosphorus pools. The C:N:surface-adsorbed P (80-105 C:15-18 N:1 P) and total (71-80 C:13-14 N:1 P) ratios in natural populations and cultures of Trichodesmium were close to Redfield values and not significantly different from each other. In contrast, intracellular ratios consistently exceeded the Redfield ratio (316-434 C:59-83 N:1 intracellular P). These high intracellular ratios were associated with reduced N2 fixation rates, suggestive of phosphorus deficiency. Other algal species also have substantial surface-adsorbed phosphorus pools, suggesting that our Trichodesmium results are generally applicable to all phytoplankton. Measurements of the distinct phytoplankton phosphorus pools may be required to assess nutrient limitation accurately from elemental composition. Deviations from Redfield stoichiometry may be attributable to surface adsorption of phosphorus rather than to biological processes, and this scavenging could affect the interpretation of marine nutrient inventories and ecosystem models., This work was supported by NSF Chemical and Biological Oceanography and by NOAA Ocean Global Carbon Cycle Program

We present data on the genetic diversity and phylogenetic affinities of N 2 -fixing unicellular cyanobacteria in the plankton of the tropical North Atlantic Ocean. Our dinitrogenase gene ( nifH ) sequences grouped together with a group of cyanobacteria from the subtropical North Pacific; another subtropical North Pacific group was only distantly related. Most of the 16S ribosomal DNA sequences from our tropical North Atlantic samples were closely allied with sequences from a symbiont of the diatom Climacodium frauenfeldianum . These findings suggest a complex pattern of evolutionary and ecological divergence among unicellular cyanobacteria within and between ocean basins.

Marine fixation of atmospheric nitrogen is believed to be an important source of biologically useful nitrogen to ocean surface waters1, stimulating productivity of phytoplankton and so influencing the global carbon cycle2. The majority of nitrogen fixation in tropical waters is carried out by the marine cyanobacterium Trichodesmium3, which supplies more than half of the new nitrogen used for primary production4. Although the factors controlling marine nitrogen fixation remain poorly understood, it has been thought that nitrogen fixation is limited by iron availability in the ocean2,5. This was inferred from the high iron requirement estimated for growth of nitrogen fixing organisms6 and the higher apparent densities of Trichodesmium where aeolian iron inputs are plentiful7. Here we report that nitrogen fixation rates in the central Atlantic appear to be independent of both dissolved iron levels in sea water and iron content in Trichodesmium colonies. Nitrogen fixation was, instead, highly correlated to the phosphorus content of Trichodesmium and was enhanced at higher irradiance. Furthermore, our calculations suggest that the structural iron requirement for the growth of nitrogen-fixing organisms is much lower than previously calculated6. Although iron deficiency could still potentially limit growth of nitrogen-fixing organisms in regions of low iron availability—for example, in the subtropical North Pacific Ocean—our observations suggest that marine nitrogen fixation is not solely regulated by iron supply.

The mechanism by which planktonic marine cyanobacteria of the genus Trichodesmium fix N 2 aerobically during photosynthesis without heterocysts is unknown. As an aid in understanding how these species protect nitrogenase, we have developed an immunofluorescence technique coupled to light microscopy (IF-LM) with which intact cyanobacteria can be immunolabeled and the distribution patterns of nitrogenase and other proteins can be described and semiquantified. Chilled ethanol was used to fix the cells, which were subsequently made permeable to antibodies by using dimethyl sulfoxide. Use of this technique demonstrated that about 3 to 20 cells (mean ± standard deviation, 9 ± 4) consecutively arranged in a Trichodesmium trichome were labeled with the nitrogenase antibody. The nitrogenase-containing cells were distributed more frequently around the center of the trichome and were rarely found at the ends. On average 15% of over 300 randomly encountered cells examined contained nitrogenase. The percentage of nitrogenase-containing cells (nitrogenase index [NI]) in an exponential culture was higher early in the light period than during the rest of the light-dark cycle, while that for a stationary culture was somewhat constant at a lower level throughout the light-dark cycle. The NI was not affected by treatment of the cultures with the photosynthetic inhibitor dichloro 1,3′-dimethyl urea or with low concentrations of ammonium (NH 4 Cl). However, incubation of cultures with 0.5 μM NH 4 Cl over 2 days reduced the NI. The IF technique combined with 14 C autoradiography showed that the CO 2 fixation rate was lower in nitrogenase-containing cells. The results of the present study suggest that (i) the IF-LM technique may be a useful tool for in situ protein localization in cyanobacteria, (ii) cell differentiation occurs in Trichodesmium and only a small fraction of cells in a colony have the potential to fix nitrogen, (iii) the photosynthetic activity (CO 2 uptake) is reduced if not absent in N 2 -fixing cells, and (iv) variation in the NI may be a modulator of nitrogen-fixing activity.