Adam B. Kustka

Phosphorus limitation of nitrogen fixation by Trichodesmium in the central Atlantic Ocean

Marine fixation of atmospheric nitrogen is believed to be an important source of biologically useful nitrogen to ocean surface waters, stimulating productivity of phytoplankton and so influencing the global carbon cycle. The majority of nitrogen fixation in tropical waters is carried out by the marine cyanobacterium Trichodesmium, which supplies more than half of the new nitrogen used for primary production. Although the factors controlling marine nitrogen fixation remain poorly understood, it has been thought that nitrogen fixation is limited by iron availability in the ocean. This was inferred from the high iron requirement estimated for growth of nitrogen fixing organisms and the higher apparent densities of Trichodesmium where aeolian iron inputs are plentiful. 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 calculated. 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.

Amazon River enhances diazotrophy and carbon sequestration in the tropical North Atlantic Ocean

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 N2 fixation far from the mouth and provides important pathways for sequestration of atmospheric CO2 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 NO3 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 CO2 and the implied oceanic carbon transport. Global Biogeochem Cycles 21, doi:10.1029/2006GB002751]. The enhancement of N2 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.

Niche of harmful alga Aureococcus anophagefferens revealed through ecogenomics.

Harmful algal blooms (HABs) cause significant economic and ecological damage worldwide. Despite considerable efforts, a comprehensive understanding of the factors that promote these blooms has been lacking, because the biochemical pathways that facilitate their dominance relative to other phytoplankton within specific environments have not been identified. Here, biogeochemical measurements showed that the harmful alga Aureococcus anophagefferens outcompeted co-occurring phytoplankton in estuaries with elevated levels of dissolved organic matter and turbidity and low levels of dissolved inorganic nitrogen. We subsequently sequenced the genome of A. anophagefferens and compared its gene complement with those of six competing phytoplankton species identified through metaproteomics. Using an ecogenomic approach, we specifically focused on gene sets that may facilitate dominance within the environmental conditions present during blooms. A. anophagefferens possesses a larger genome (56 Mbp) and has more genes involved in light harvesting, organic carbon and nitrogen use, and encoding selenium- and metal-requiring enzymes than competing phytoplankton. Genes for the synthesis of microbial deterrents likely permit the proliferation of this species, with reduced mortality losses during blooms. Collectively, these findings suggest that anthropogenic activities resulting in elevated levels of turbidity, organic matter, and metals have opened a niche within coastal ecosystems that ideally suits the unique genetic capacity of A. anophagefferens and thus, has facilitated the proliferation of this and potentially other HABs.

A REVISED ESTIMATE OF THE IRON USE EFFICIENCY OF NITROGEN FIXATION, WITH SPECIAL REFERENCE TO THE MARINE CYANOBACTERIUM TRICHODESMIUM SPP. (CYANOPHYTA)1

Estimates of the iron use efficiency (IUE) for diazotrophic plant growth have been used to suggest iron limitation of marine N2 fixation. However, in the course of these inferences, neither the physiological complexity of these estimates nor the specific physiological parameters of marine diazotrophs were evaluated. Here, a semiempirical prediction of the IUE of diazotrophic growth for Trichodesmium was computed from considerations of the Fe content and reaction rates of the nitrogenase complex and PSI:PSII ratios, as well as field measurements of Mehler activity, cellular Fe‐superoxide dismutase activity, and diel variability in C and N2 fixation. With a PSI:PSII ratio of 1 and 48% Mehler activity, the instantaneous IUE (0.33 mol C fixed·mol cellular Fe− 1 ·s− 1 ) was only 4‐fold lower than that calculated for a phytoplankter growing on reduced N. We computed a range of daily integrated IUE values from 2900 to 7700 mol C·mol Fe− 1 ·d− 1 , accounting for the diel variability in C and N2 fixation as well as the uncertainties in cyanobacterial nitrogenase biochemistry and PSI:II ratios of field‐collected Trichodesmium. The lowest observed Fe‐superoxide dismutase:C quota of 2.9 (μmol:mol) suggests a maintenance requirement for this enzyme. The maintenance Fe:C requirement of 13.5 μmol:mol (derived from cultures of Trichodesmium IMS 101) and values of the IUE yielded an Fe requirement ranging from 27 to 48 Fe:C (μmol:mol) to achieve a diazotrophic growth rate of 0.1 d− 1 . Based on these predicted requirements, the Fe:C contents of Caribbean Sea and most North Atlantic Ocean populations sampled thus far exceed that required to support the observed rates of N2 fixation.

Sequence analysis and transcriptional regulation of iron acquisition genes in two marine diatoms1

The centric diatom Thalassiosira pseudonana Hasle et Heimdal and the pennate diatom Phaeodactylum tricornutum Bohlin possess genes with translated sequences homologous to high‐affinity ferric reductases present in model organisms. Thalassiosira pseudonana also possesses putative genes for membrane‐bound ferroxidase (TpFET3) and two highly similar iron (Fe) permeases (TpFTR1 and TpFTR2), as well as a divalent metal (M2+) transporter belonging to the NRAMP superfamily (TpNRAMP). In baker’s yeast, the ferroxidase–permease complex transports Fe(II) produced by reductases. We investigated transcript abundances of these genes as a function of Fe quota (QFe). Ferric reductase transcripts are abundant in both species (15%–60% of actin) under low QFe and are down‐regulated by 5‐ to 35‐fold at high QFe, suggesting Fe(III) reduction is a common, inducible strategy for Fe acquisition in marine diatoms. Permease transcript abundance was regulated by Fe status in T. pseudonana, but we did not detect significant differences in expression of the copper (Cu)‐containing ferroxidase. TpNRAMP showed the most dramatic regulation by QFe, suggesting a role in cellular Fe transport in either cell‐surface uptake or vacuolar mobilization. We could not identify ferroxidase or permease homologues in the P. tricornutum genome. The up‐regulation of genes in T. pseudonana that appear to be missing altogether from P. tricornutum as well as the finding that P. tricornutum seems to have an efficient system to acquire Fe′, suggest that diverse (and uncharacterized) Fe‐uptake systems may be at play within diatom assemblages. Different uptake systems among diatoms may provide a mechanistic basis for niche differentiation with respect to Fe availability in the ocean.

Morphine- and anadamide-stimulated nitric oxide production inhibits presynaptic dopamine release

Morphine and anandamide stimulate the release of nitric oxide (NO) in diverse tissues. The present study examines the consequences of this action on neurotransmitter release in ganglia from two invertebrates: ventral chain ganglia from the leech Hirudo medicinalis and the pedal ganglion from the mussel Mytilus edulis. In these ganglia, preloaded serotonin (5-HT) and dopamine (DA) can be released by 50 mM KCl. Anandamide, an endogenous cannabinoid substance, suppresses the potassium-stimulated release of [3H]DA (80%), but not 5-HT, in a concentration-dependent manner, from the neural tissues of both. The effect of anandamide can be antagonized by pre-exposing the neural tissues of both animals to SR 141716A, a potent cannabinoid receptor antagonist. Prior treatment of the ganglia with N-omega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, significantly diminishes the inhibitory effect of anandamide. Morphine also inhibits [3H]DA release in a naloxone- and L-NAME-sensitive manner. Anandamide and morphine act through separate mechanisms since the respective antagonists show no cross-reactivity. The NO donor, SNAP, depressed the potassium-stimulated release of preloaded [3H]DA, but not 5-HT, in the neural tissues of both animals. D-Ala2-Met5 enkephalinamide (DAMA) also inhibited the potassium-stimulated release of [3H]DA in a naloxone-sensitive process. However, the effect of DAMA was seen in the presence of L-NAME (10(-4) M), indicating that the opioid peptide inhibition of the presynaptic release of DA is not coupled to NO. We postulate that cannabinoids and their endogenous effectors play a prominent role in the regulation of catecholamine release in invertebrates via NO release as is the case for opiate alkaloids.

Iron and marine nitrogen fixation: progress and future directions

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.

Transcriptional Orchestration of the Global Cellular Response of a Model Pennate Diatom to Diel Light Cycling under Iron Limitation.

Environmental fluctuations affect distribution, growth and abundance of diatoms in nature, with iron (Fe) availability playing a central role. Studies on the response of diatoms to low Fe have either utilized continuous (24 hr) illumination or sampled a single time of day, missing any temporal dynamics. We profiled the physiology, metabolite composition, and global transcripts of the pennate diatom Phaeodactylum tricornutum during steady-state growth at low, intermediate, and high levels of dissolved Fe over light:dark cycles, to better understand fundamental aspects of genetic control of physiological acclimation to growth under Fe-limitation. We greatly expand the catalog of genes involved in the low Fe response, highlighting the importance of intracellular trafficking in Fe-limited diatoms. P. tricornutum exhibited transcriptomic hallmarks of slowed growth leading to prolonged periods of cell division/silica deposition, which could impact biogeochemical carbon sequestration in Fe-limited regions. Light harvesting and ribosome biogenesis transcripts were generally reduced under low Fe while transcript levels for genes putatively involved in the acquisition and recycling of Fe were increased. We also noted shifts in expression towards increased synthesis and catabolism of branched chain amino acids in P. tricornutum grown at low Fe whereas expression of genes involved in central core metabolism were relatively unaffected, indicating that essential cellular function is protected. Beyond the response of P. tricornutum to low Fe, we observed major coordinated shifts in transcript control of primary and intermediate metabolism over light:dark cycles which contribute to a new view of the significance of distinctive diatom pathways, such as mitochondrial glycolysis and the ornithine-urea cycle. This study provides new insight into transcriptional modulation of diatom physiology and metabolism across light:dark cycles in response to Fe availability, providing mechanistic understanding for the ability of diatoms to remain metabolically poised to respond quickly to Fe input and revealing strategies underlying their ecological success.

Carbonate-sensitive phytotransferrin controls high-affinity iron uptake in diatoms

In vast areas of the ocean, the scarcity of iron controls the growth and productivity of phytoplankton. Although most dissolved iron in the marine environment is complexed with organic molecules, picomolar amounts of labile inorganic iron species (labile iron) are maintained within the euphotic zone and serve as an important source of iron for eukaryotic phytoplankton and particularly for diatoms. Genome-enabled studies of labile iron utilization by diatoms have previously revealed novel iron-responsive transcripts, including the ferric iron-concentrating protein ISIP2A, but the mechanism behind the acquisition of picomolar labile iron remains unknown. Here we show that ISIP2A is a phytotransferrin that independently and convergently evolved carbonate ion-coordinated ferric iron binding. Deletion of ISIP2A disrupts high-affinity iron uptake in the diatom Phaeodactylum tricornutum, and uptake is restored by complementation with human transferrin. ISIP2A is internalized by endocytosis, and manipulation of the seawater carbonic acid system reveals a second-order dependence on the concentrations of labile iron and carbonate ions. In P. tricornutum, the synergistic interaction of labile iron and carbonate ions occurs at environmentally relevant concentrations, revealing that carbonate availability co-limits iron uptake. Phytotransferrin sequences have a broad taxonomic distribution and are abundant in marine environmental genomic datasets, suggesting that acidification-driven declines in the concentration of seawater carbonate ions will have a negative effect on this globally important eukaryotic iron acquisition mechanism.

EFFECT OF EDTA ADDITIONS ON NATURAL TRICHODESMIUM SPP. (CYANOPHYTA) POPULATIONS1

Despite nearly two decades of intensive research, many questions regarding the physiology and ecology of the marine, non‐heterocystous cyanobacterium, Trichodesmium, remain unresolved. We note here the effect of EDTA (ethylenediaminetetraacetate) on N2 fixation by Trichodesmium, and the use of EDTA as a means of extending the viability of natural Trichodesmium spp. populations. We examined nitrogenase activity (NA) as a function of EDTA concentration, time of collection, light level, and iron addition. Samples collected early in the day and treated with EDTA maintain a steady rate of activity for hours longer than controls. Furthermore, samples preincubated through the night with EDTA were active the next morning, compared with controls that were inactive. The discovery that (10–50 μM) low concentrations of EDTA prolong the duration of NA of Trichodesmium during experimental manipulations without affecting the rate of acetylene reduction allows for longer term manipulative experiments to be conducted.